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3.2.P.5Quality Control of Preparations

Quality Standards

Gadopentetate Dimeglumine Injection

This product is a sterile aqueous solution of gadopentetate dimeglumine. It contains gadopentetate dimeglumine (C14H20GdN3O102C7H17NO5)

Should be 95.0% to 105.0% of the labeled amount.

Description】 The product is a clear, colorless to pale yellow or pale yellowish-green liquid.

Identification】 (1) Carry out the test as described in the Thin-layer Chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rule 0502).

Test solution Take an appropriate amount of the product, dilute with water to prepare a solution containing approximately 35mg of gadopentetate dimeglumine per 1ml (equivalent to meglumine14.6mg).

Reference Solution Dissolve an appropriate amount of gadopentetate dimeglumine reference standard in water and dilute to prepare a solution containing approximately 56mg per 1ml (equivalent to 14.6mg of dimeglumine).

Chromatographic Conditions Use a silica gel GF254 thin-layer plate, with a developing solvent system of n-butanol-glacial acetic acid-water (412).

Assay Spot 2μl each of the test solution and the reference solution on the same thin-layer plate, develop, dry, spray with ninhydrin-cadmium acetate solution (prepared by dissolving 0.1g of ninhydrin and 0.25g of cadmium acetate in 1ml of glacial acetic acid, diluting with ethanol to 50ml, and mixing well), and heat at 120°C for 10 minutes to develop the color.

Result Interpretation The position and color of the principal spot in the test solution should be identical to those of the principal spot in the reference solution.

(2) Carry out the test as described in Thin-layer Chromatography (Chinese Pharmacopoeia 2020 Edition, General Rule 0502).

Test solution Dilute an appropriate amount of the product with water to prepare a solution containing about 35mg of gadopentetate dimeglumine per 1ml (equivalent to 20.4mg of gadopentetic acid).

Reference solution Dilute the reference solution from Identification (1) with water to prepare a solution containing about 28mg per 1ml (equivalent to 20.4mg of gadopentetic acid).

Chromatographic conditions Use silica gel GF254 thin-layer plates, with a mobile phase of methanol-acetonitrile-water-glacial acetic acid (522 0.2).Assay Method Draw 2μl each of the test solution and the reference solution, spot them on the same thin-layer plate, develop, and air-dry. Spray with a cerium sulfate-arsenite solution (take 5g of cerium sulfate, place it in a 100ml volumetric flask, set it in an ice bath, add 50ml of 0.5mol/L sulfuric acid solution cooled to 0°C, shake to dissolve, filter, and refrigerate as solution A; separately, take 2.5g of sodium arsenite, dissolve it in 15ml of 1mol/L sodium hydroxide solution, refrigerate at 0°C, carefully add it to 32.5ml of 1mol/L sulfuric acid solution cooled to 0°C, dilute with water to 50ml as solution B; mix equal volumes of solution A and solution B immediately before use, and use within 5 minutes), then spray with a 1% o-phenylenediamine acetone solution.

Result Interpretation The position and color of the principal spot in the test solution should be identical to those of the principal spot in the reference solution.

(3) In the chromatograms obtained in the assay, the retention time of the principal peak in the test solution should correspond to that of the principal peak in the reference

solution.

(4) Take 5 ml of the product, place it in a 25 ml volumetric flask, dilute with water to the mark, mix well, and measure according to the ultraviolet-visible spectrophotometry

(Chinese Pharmacopoeia2020 Edition, Part IV, General Rule0401). The maximum absorption is observed at a wavelength of275 nm.

For the above items (2) and (3), either one may be selected for testing.

[Inspection] pH value: Take the product and inspect it according to the method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rule 0631 pH Value Determination Method). The pH value should be

7.0 to 7.9.

Color Take the sample and examine it according to the method (General Rule 0901, Method I of Part IV, Chinese Pharmacopoeia 2020 Edition), comparing it with yellow No. 4 or yellow-green

No. 4 standard color solution, and it should not be darker.

Meglumine Take the sample and measure the optical rotation at 25°C according to the method (General Rule 0621 of Part IV, Chinese Pharmacopoeia 2020 Edition), calculate using the following formula

The calculated meglumine content should be 39.6%~43.9% of the labeled amount of gadopentetate dimeglumine.

Meglumine content (%)=measured optical rotation*1000

24.9*469 × 100%

Pentetic acid Accurately measure 2ml of the sample, place it in a 100ml conical flask, add 20ml of water, and 10ml of dilute xylenol orange solution.

Shake well, adjust the pH to 5.0 with 0.1mol/L sodium hydroxide solution or glacial acetic acid, and titrate with gadolinium sulfate solution (0.001mol/L).

Titrate until the solution changes from yellow to reddish-purple. Each 1ml of gadolinium sulfate solution (0.001mol/L) is equivalent to 0.7867mg of

pentetic acid (C14H23N3O10). Each 1ml of the preparation should contain 230–450μg of pentetic acid.

Gadolinium Determine by atomic absorption spectrophotometry (Chinese Pharmacopoeia 2020 Edition, General Rule 0406 , Method I).

Cesium Chloride Solution Take an appropriate amount of cesium chloride, dissolve it in water, and dilute to the mark to prepare a solution containing100mg per1ml.

Blank Solution Mix cesium chloride solution, hydrochloric acid (spectral pure), and water in a ratio of20:10:70 (V/V/V) to obtain.

Test Solution Precisely measure5ml of the sample (equivalent to gadopentetate dimeglumine 2.3g), place in a porcelain crucible, add nitric acid

1.0ml, mix well, place on an electric stove, and slowly char until completely carbonized. Incinerate at about800°C until completely ashed (approximately 1 hour),

After cooling, transfer to a desiccator, allow to cool to room temperature, then take out, add 5ml of water and (spectral purity) hydrochloric acid10ml, heat to dissolve

, transfer to a100ml volumetric flask, add 20ml of cesium chloride solution, dilute with water to the mark, and use as the test solution.

Standard stock solution Accurately weigh 5.75g of gadolinium oxide, place in a 100ml volumetric flask, add (spectral purity) hydrochloric acid10ml

and an appropriate amount of water, heat to dissolve, cool to room temperature, then dilute with water to the mark to obtain.

Reference solution Accurately transfer 1ml, 2ml, 3ml, 4ml, and 5ml of the standard stock solution1ml, 2ml, 3ml, 4ml, 5ml, respectively, into separate 50ml

volumetric flasks, add 10ml of cesium chloride solution10ml and 10ml of hydrochloric acid (spectral purity)10ml, dilute with water to the mark, and prepare solutions containing

1ml of gadolinium element1000μg, 2000μg, 3000μg, 4000μg and 5000μg, respectively.

Spectral line search 3000μg/mL reference solution.

AssaySubtract the background with blank solution, accurately measure the reference solution and test solution at368.4nmwavelength

Measure the absorbance and calculate using the standard curve method. (This method is a flame continuous emission spectrometry, requiring the use of a high-temperature burner and

Nitrous oxide-acetylene gas (no gadolinium hollow cathode lamp required)

Limit The gadolinium content of this product is15.9%-17.5% of the labeled amount of gadopentetate dimeglumine.

Related substancesDetermined by high-performance liquid chromatography (Chinese Pharmacopoeia2020 Edition, Part IV General Rule0512).

Copper nitrate solution Take 12.88g of copper nitrate trihydrate, dissolve in water and dilute to1000ml.

Copper nitrate sodium citrate solution Take 12.88g of copper nitrate trihydrate, 38.0g of sodium citrate dihydrate, dissolve in water and dilute

to1000ml.

Test solution Accurately measure1ml of the sample (approximately equivalent to gadopentetate dimeglumine469mg), transfer to a20ml volumetric flask,

Dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Reference solution (1) Take an appropriate amount of nitrilotriacetic acid reference substance and ethylenediaminetetraacetic acid reference substance, accurately weigh, dissolve in copper

nitrate solution and quantitatively dilute to prepare a solution containing approximately0.02mg of nitrilotriacetic acid and0.4mg of ethylenediaminetetraacetic acid per1ml.

Mixed solution.

Reference solution (2) Accurately measure 1 ml of reference solution (1)1ml, transfer to a 20ml volumetric flask, and dilute with copper nitrate solution to

the mark, and mix well.

Mixed solution of reference solution (1) and test solution Accurately measure 1 ml of reference solution (1)1ml, transfer to a 20ml volumetric

Into the flask, dilute to the mark with the test solution, and mix well.

Chromatographic conditions Use octylsilane-bonded silica gel as the filler (MOS-1Hypersil, 125mm×4.6mm, 5μm,

ora chromatographic column with equivalent performance); using10%(W/V) aqueous solution of tetrabutylammonium hydroxide10ml, methanol10ml,

water 170ml, mix well, adjust the pH to 7.5±0.1 with phosphoric acid, then add methanol 90ml, and dilute with water to a final volume of

1000ml as mobile phaseA,using10%(W/V) aqueous solution of tetrabutylammonium hydroxide10ml, methanol 10ml,

water 170ml, mix well and adjustpHto7.5±0.1withphosphoric acid, add methanol450ml, and dilute with water to

1000ml as mobile phase B; perform gradient elution according to the table below at a flow rate of 2ml per minute; the detection wavelength is

254nm; injectionvolume20μl; after injection, elute with mobile phaseAandrecord the chromatogram until20min;

After the recording is completed, elute theB column with mobile phase for 20min, then equilibrate the column with mobile phaseA.

Time (min)

Mobile phaseA (%)

Mobile phaseB (%)

0

100

0

20

100

0

21

0

100

41

0

100

42

100

0

85

100

0

System Suitability RequirementsIn the mixed solution of reference standard solution (1), the nitrilotriacetic acid peak and copper nitrate

The resolution between the peaks of copper nitrate and EDTA should meetthe requirements (for nitrilotriacetic acid and ethylenediaminetetraacetic acid

In thetest solution and reference solution (2), the retention times may vary slightly).

Assay Accurately measure copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, and test solution,

Reference solution (1) and test solution mixture20μl, inject into the chromatograph separately, and record the chromatograms.

Limit In the chromatogram of the test solution, if any impurity peaks appear between the start (excluding the solvent peak) and the copper nitrate peak,

Calculate by the external standard method using the peak area of nitrilotriacetic acid in the reference solution (2), record from the copper nitrate peak to the end of the chromatogram,

If any impurity peaks appear, calculate by the external standard method using the peak area of ethylenediaminetetraacetic acid in the reference solution (2), related substances

The total amount shall not exceed 0.3% of the labeled amount of pentetic acid (each 1ml of this product contains a total of 197mg of pentetic acid).

Heavy metals Take an appropriate amount of the product(equivalent to gadopentetic acid dimeglumine1.0g), add 20ml of water and sodium hydroxide test solution

5ml, shake well, and examine as directed (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rule 0821 , Method III). The heavy metal content shall not

exceed 20 parts per million.

Bacterial Endotoxins Take the product and test according to the method (Chinese Pharmacopoeia 2020 Edition, Part IV General Chapter 1143 Method 1),

The amount of endotoxin in each 1ml should be less than 0.5EU.

Sterility Take the product, process it by membrane filtration, and test according to the method (Chinese Pharmacopoeia 2020 Edition, Part IV General Chapter 1101),

It should comply with the requirements.

Others should comply with the relevant requirements for injections (Chinese Pharmacopoeia2020 Edition, Part IV, General Chapter0102).

Assay】 Determine by high-performance liquid chromatography (Chinese Pharmacopoeia2020 Edition, Part IV, General Chapter0512).

Test solution Pipette accurately2ml of the product, transfer to a200ml volumetric flask, dilute with water to volume, and mix well,

Pipette accurately15ml, transfer to a100ml volumetric flask, add10ml of acetonitrile, dilute with water to volume, and mix well.

Reference Solution Accurately weigh an appropriate amount of gadopentetate meglumine reference standard, dissolve in10%acetonitrile solution, and quantitatively

diluteto prepare a solution containing approximately0.6mgper1ml.

Chromatographic Conditions Use octadecylsilane-bonded silica gel as the stationary phase; the mobile phase is tetrabutylammonium perchlorate solution (dissolve

1.7gof tetrabutylammonium perchlorate in100mlof acetonitrile, dilute with water to1000ml); the detectionwavelength is

195nm; injection volume20μl.

System suitability requirements The number of theoretical plates, calculated based on the gadopentetate dimeglumine peak, should not be less than3000.

Assay Accurately measure the test solution and reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

Calculate using the external standard method based on peak area, multiply the obtained result by1.263.

Category】 Diagnostic agent.

SpecificationCalculated asC14H20GdN3O102C7H17NO5 (1)10ml:4.69g (2)12ml:5.63g(3)15ml:7.04g(4)20ml:9.38g

Storage】 Protect from light, store tightly closed at a temperature not exceeding30°C.

Shelf life36 months

Reviewing authority】Guangdong Institute for Drug Control

Marketing Authorization Holder】 Guangzhou Kangchen Pharmaceutical Co., Ltd.

Analytical Methods

Analytical Methods

Appearance

Inspection method: Visual inspection.

Method: Take the product and observe visually.

Acceptance criteria: Should be a clear, colorless to pale yellow or pale yellowish-green liquid.

Identification 1

Test method: Thin-layer chromatography.

Test conditions: Silica gel GF254 thin-layer plate was used, with n-butanol-glacial acetic acid-water (4:1:2) as the developing solvent.

Procedure:

Test solution: Take an appropriate amount of the sample, dilute with water to prepare a solution containing approximately 35mg of gadopentetate dimeglumine per 1ml

(equivalent to 14.6mg of meglumine).

Reference solution Dissolve an appropriate amount of gadopentetate dimeglumine reference standard in water and dilute to prepare a solution containing approximately 56mg per 1ml (equivalent to 14.6mg of dimeglumine).

Assay method Pipette l each of the test solution and reference solution, spot them on the same thin-layer plate, develop, dry, and spray with ninhydrin-cadmium acetate solution (dissolve 0.1g of ninhydrin, 0.25g of cadmium acetate, and 1ml of glacial acetic acid in ethanol, dilute to 50ml, and mix well). Heat at 120°C for 10 minutes to develop the color.

Standard limit: The position and color of the principal spot in the test solution should be the same as that of the principal spot in the reference solution.

Identification 2

Test method: Thin-layer chromatography.

Test conditions: Silica gel GF254 thin-layer plate was used, with a mobile phase of methanol-acetonitrile-water-glacial acetic acid (5:2:2:

0.2) as the developing solvent.

Operation method:

Test solution Take an appropriate amount of the product, dilute with water to prepare a solution containing approximately 35mg of gadopentetate dimeglumine per 1ml

(equivalent to 20.4mg of gadopentetic acid).

Reference solution Take the reference solution from the identification(1), dilute with water to prepare a solution containing approximately 28mg per

1ml (equivalent to 20.4mg of gadopentetic acid).

Assay Method Draw l each of the test solution and the reference solution, spot them on the same thin-layer plate, develop, dry, spray with cerium sulfate-arsenious acid solution (take 5g of cerium sulfate, place it in a 100ml volumetric flask, place in an ice bath, add cooled to 0°C 0.5mol/L sulfuric acid solution 50ml, shake to dissolve, filter, refrigerate, as solution A; take 2.5g of sodium arsenite, add 1mol/L sodium hydroxide solution 15ml to dissolve, refrigerate at 0°C, carefully add to cooled 0°C 1mol/L sulfuric acid solution 32.5ml , dilute with water to 50ml, as solution B; immediately before use, mix equal volumes of solution A and solution B, use within 5 minutes), then spray with 1%o-phenylenediamine acetone solution.

Standard limit: The position and color of the principal spot in the test solution should be identical to that of the principal spot in the reference solution.

Identification 3

Test method: HPLC (Chinese Pharmacopoeia 2020 Edition, Part IV General Chapter 0512).

Test conditions: See the Assay section.

Procedure: See the Assay section.

Standard limit: The retention time of the main peak in the test solution should match that of the main peak in the reference solution.

Identification 4

Test method: Ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 Edition, Part IV General Chapter 0401).

Test conditions: Sample solution, water, ultraviolet-visible spectrophotometer.

Operating procedure:

Transfer 5ml of the sample to a 25ml volumetric flask, dilute to volume with water, mix well, and determine by ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 Edition, Part IV General Chapter 0401).

Standard limit: Maximum absorption at 275 nm wavelength.

pH value

Test method: pH determination (Chinese Pharmacopoeia 2020 Edition, Part IV General Chapter 0631).

Test conditions: pH meter, freshly boiled and cooled distilled water, potassium hydrogen phthalate buffer solution (pH 4.00), mixed phosphate buffer (pH 6.86), and sodium tetraborate standard buffer solution (pH 9.18).

Procedure: Take 1 vial of the product, immerse the electrode into the test solution, gently shake to equilibrate and stabilize, then take the reading.

Acceptance criteria: pH value should be 7.0~7.9.

Color

Test method: Solution color test method, Chinese Pharmacopoeia 2020 Edition, Part IV General Principles 0901 Method 1.

Test conditions: Standard colorimetric solution.

Procedure: Take 1 vial of the product, observe visually, and compare with yellow 4 or yellow-green 4 standard colorimetric solution.

Acceptance criteria: No deeper color allowed.

Meglumine

Test method: Optical rotation determination (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rule 0621).

Test conditions: Polarimeter.

Procedure: Take the sample and measure the optical rotation at 25°C according to the method, then calculate the meglumine content using the following formula

Meglumine content (%)=Measured optical rotation×1000 × 100%

24.9×469

Standard limit: Should be 39.6%~43.9% of the labeled amount of gadopentetate dimeglumine.

Pentetic Acid

Test method: Titration.

Test conditions: Dilute xylenol orange solution, 0.1mol/L sodium hydroxide solution, glacial acetic acid, gadolinium sulfate titrant (0.001mol/L).

Procedure: Accurately measure 2ml of the sample, transfer to a 100ml conical flask, add 20ml of water and 10ml of dilute xylenol orange solution, mix well. Adjust the pH to 5.0 using 0.1mol/L sodium hydroxide solution or glacial acetic acid. Titrate with gadolinium sulfate titrant (0.001mol/L) until the solution changes from yellow to reddish-purple. Each 1ml of gadolinium sulfate titrant (0.001mol/L) is equivalent to 0.7867mg of pentetic acid (C14H23N3O10).

Standard limit: Each 1ml of the product should contain 230~450μg of pentetic acid.

Elemental impurities (not included in quality standard method)

Test Method: Inductively Coupled Plasma Mass Spectrometry (Chinese Pharmacopoeia 2020 Edition General Chapter 0412).

Test Conditions: ICP-MS.

Operating Procedure:

Test Solution: Accurately measure 215μl of Gadopentetate Dimeglumine Injection into a 100ml volumetric flask, using diluent

(Measure 10ml of hydrochloric acid and 200μl of Au standard solution (1mg/ml), dilute with water to 1000ml, and mix well.) Dissolve and dilute to the mark, mix well to obtain the solution.

Accurately measure each element standard solution and dilute with diluent according to the table below to prepare standard curve solutions:

Element

Standard curve solution concentration ng/ml

Concentration 1

Concentration 2

Concentration 3

Concentration 4

Concentration 5

Hg

0.0540

0.0900

0.1800

0.2160

0.2700

Cd

0.0360

0.0600

0.1200

0.1440

0.1800

Pb

0.0900

0.1500

0.3000

0.3600

0.4500

As

0.2670

0.4450

0.8900

1.0680

1.3350

Co

0.0900

0.1500

0.3000

0.3600

0.4500

V

0.1770

0.2950

0.5900

0.7080

0.8850

Ni

0.3600

0.6000

1.2000

1.4400

1.8000

Li

4.5000

7.5000

15.0000

18.0000

22.5000

Sb

1.5000

2.5000

5.0000

6.0000

7.5000

Cu

5.1000

8.5000

17.0000

20.4000

25.5000

Internal standard solution: Prepare by diluting Ge, In, Re, Bi standard solutions with water to obtain a mixed solution containing 20ng/ml of each.

Standard limits:

Element

Limit μg/ml

Hg

0.125

Cd

0.083

Pb

0.208

As

0.625

Co

0.208

V

0.417

Ni

0.833

Li

10.417

Sb

3.750

Cu

12.500

Perform according to the Atomic Absorption Spectrophotometry method (General Rule 0406, First Method, Chinese Pharmacopoeia 2020 Edition).

Cesium Chloride Solution Take an appropriate amount of cesium chloride, dissolve it in water, and dilute to the mark to prepare a solution containing 100mg per 1ml.

Blank Solution Mix cesium chloride solution, hydrochloric acid (spectral grade), and water in a ratio of 20:10:70 (V/V/V) to obtain the blank solution.

Test Solution Accurately measure 5ml of the sample (equivalent to 2.3g of gadopentetate dimeglumine), place it in a porcelain crucible, add 1.0ml of nitric acid, shake well, and heat gently on an electric stove until completely charred. Ignite at approximately 800°C until completely ashed (about 1 hour). After cooling, transfer to a desiccator, cool to room temperature, then remove, add 5ml of water and 10ml of hydrochloric acid (spectral grade), heat to dissolve, transfer to a 100ml volumetric flask, add 20ml of cesium chloride solution, and dilute with water to the mark to prepare the test solution.

Standard Stock Solution Accurately weigh 5.75g of gadolinium oxide, place it in a 100ml volumetric flask, add 10ml of hydrochloric acid (spectral grade) and an appropriate amount of water, heat to dissolve, cool to room temperature, and dilute with water to the mark to obtain the standard stock solution.

Reference Solution Precisely transfer 1ml, 2ml, 3ml, 4ml, and 5ml of the standard stock solution into separate 50ml volumetric flasks. Add 10ml of cesium chloride solution and 10ml of hydrochloric acid (spectral purity), then dilute with water to the mark to prepare solutions containing 1000μg, 2000μg, 3000μg, 4000μg, and 5000μg of gadolinium per 1ml, respectively.

Spectral Line Search 3000μg/mL reference solution.

Assay Subtract the background with a blank solution. Precisely measure the reference solution and the test solution, and determine the absorbance at a wavelength of 368.4nm. Calculate using the standard curve method. (This method employs flame continuous emission spectrometry, requiring a high-temperature burner and nitrous oxide-acetylene gas, without the need for a gadolinium hollow cathode lamp.)

Limit The gadolinium content in this product is 15.9%-17.5% of the labeled amount of gadopentetate dimeglumine.

Related substances

Test method: High-performance liquid chromatography (Chinese Pharmacopoeia2020 Edition, Part IV, General Chapter 0512).

Test conditions: High-performance liquid chromatograph equipped with an ultraviolet detector or diode array detector, detection wavelength at 254nm; column packed with octylsilane-bonded silica gel (MOS-1 Hypersil, 125mm

×4.6mm, 5μm, or equivalent performance column); flow rate at 2.0ml/min; column temperature at 25°C; injection volume of 20μl, mobile phase 1: take 10% (W/V) tetrabutylammonium hydroxide aqueous solution 10ml, 10ml meth

Alcohol and water 170ml are mixed, adjust the pH to 7.5±0.1 with phosphoric acid, add methanol 90ml, and dilute with water to 1000ml. Mobile phase 2: Take 10%(W/V) tetrabutylammonium hydroxide aqueous solution 10ml, methanol 10ml, and water 170ml, mix, adjust the pH to 7.5±0.1 with phosphoric acid, add methanol 450ml, and dilute with water to 1000ml. Perform gradient elution according to the table below. After injection, elute with mobile phase 1 and record the chromatogram until 20min; after recording is complete, elute the column with mobile phase 2 for 20min , then equilibrate the column with mobile phase 1.

Time min

Mobile phase 1 (%)

Mobile phase 2 (%)

0

100

0

20

100

0

21

0

100

41

0

100

42

100

0

85

100

0

The injection sequence is: copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, mixed solution of reference solution (1) and test solution (1:20), and reference solution (1). The resolution between the nitrilotriacetic acid (NTA) peak and the copper nitrate peak, and between the copper nitrate peak and the EDTA peak, should meet the requirements. The retention times of NTA and EDTA in the test solution and reference solution (2) may slightly differ.

Operation method:

Solvent Copper nitrate solution (take 12.88g of copper nitrate trihydrate 12.88g, dissolve in water and dilute to 1000ml). Copper nitrate sodium citrate solution (take 12.88g of copper nitrate trihydrate 12.88g, 38.0g of sodium citrate dihydrate 38.0g, dissolve in

water and dilute to 1000ml).

Test solution Accurately measure 1ml of the sample (1ml(approximately equivalent to 469mg of gadopentetate dimeglumine 469mg), transfer to a 20ml

volumetric flask, dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Reference Solution (1) Accurately weigh an appropriate amount of nitrilotriacetic acid reference standard and ethylenediaminetetraacetic acid reference standard, dissolve in copper nitrate solution, and quantitatively dilute to prepare a solution containing approximately 0.02mg of nitrilotriacetic acid and 0.4mg of ethylenediaminetetraacetic acid per 1ml.

Reference Solution (2) Accurately measure 1ml of Reference Solution (1), transfer to a 20ml volumetric flask, dilute with copper nitrate solution to the mark, and mix well.

Mixed Solution of Reference Solution (1) and Test SolutionAccurately measure 1ml of Reference Solution (1), transfer to a 20ml volumetric flask, dilute with the test solution to the mark, and mix well.

Assay Accurately measure 20μL each of Reference Solution (2), test solution, and dilution solvent, inject into the liquid chromatograph in the sequence specified in the system suitability test. When calculating the test solution, the corresponding peak of copper nitrate sodium citrate solution should be deducted.

Standard limits:

In the chromatogram of the test solution, if any impurity peaks appear between the start (excluding the solvent peak) and the copper nitrate peak, calculate them using the external standard method based on the peak area of nitrilotriacetic acid in reference solution (2). From the copper nitrate peak to the end of the chromatogram, if any impurity peaks appear, calculate them using the external standard method based on the peak area of ethylenediaminetetraacetic acid in reference solution (2). The total amount of related substances must not exceed 0.3% of the labeled amount of pentetic acid (each 1 mL of this product contains a total of 197 mg of pentetic acid).

Insoluble particles

Test method: Light obscuration method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0903).

Test conditions: Clean bench, insoluble particle analyzer.

Procedure: Take 4 vials of the test sample, carefully open the containers, add water for testing or a suitable solvent,combine and dissolve to 50ml, place in a sampling cup, and let stand for 2 minutes or an appropriate time to remove bubbles, then place on the sampler. Turn on stirring to mix the solution (avoiding bubble formation),and perform the measurement 4times, sampling 5mleach time.Discard the first measurement data and take the average of the subsequent 3measurements as the result. Based on the sampling volumeand dilution volume, calculate the number of particles per container.

Standard limit: Each test container must contain no more than 10µm and 10µm or larger particles exceeding 6000

particles, and no more than 25µm and 25µm or larger particles exceeding 600 particles.

Visible particles

Inspection method: Visual inspection method, 2020 edition of the Chinese Pharmacopoeia, General Rule 0904.

Test conditions: Clarity inspection apparatus.

Procedure: Take 20 vials of the product, place the test solution just outside the edge of the light shield. During inspection, the illumination at the location of the test sample should be 1000–1500 lx. Hold the container by the neck, gently rotate and invert it to suspend any potential visible particles in the solution. Visually inspect against both black and white backgrounds, repeating the observation.

4)Standard limits: The test sample must not contain metal shavings, glass fragments, fibers longer than 2 mm, particles larger than 2 mm in diameter, or visible smoke-like particulate deposits that appear after standing for a certain period and gentle rotation, uncountable particle clusters or insoluble precipitates, or clearly visible protein flocculates that are difficult to count within the specified time. If fine visible particles such as dots, short fibers less than 2 mm, or small particles are detected in the test sample: if 2 or more vials are found with such particles, the product does not meet the specification. If 1 vial is found, an additional 20 vials should be tested. The total of initial and retest samples is 40 vials. If more than 1 vial is found with particles, the product does not meet the specification, meaning none of the 20 retest vials should show such particles.

Fill volume

Test method: Filling volume according to Chinese Pharmacopoeia 2020 edition, General Chapter 0102

Test conditions: Graduated cylinder.

Procedure: Take 3 vials of the product, open them carefully to avoid loss, and withdraw the contents completely using a dry syringe with an appropriate needle size. Then slowly and continuously inject the contents into a calibrated measuring cylinder (the cylinder size should be such that the volume to be measured occupies at least 40% of its nominal capacity, without emptying the liquid remaining in the needle). Examine at room temperature.

The cylinder size should ensure that the volume to be measured occupies at least 40% of its nominal capacity, without emptying the liquid remaining in the needle. Examine at room temperature.

Standard limit: Each vial (bottle) must contain no less than the labeled amount.

Bacterial endotoxins

Test method: Gel method (Chinese Pharmacopoeia 2020 Edition IV General Chapter 1143)

Experimental conditions: Constant-temperature tube apparatus, vortex mixer, clean bench, pipette, pyrogen-free pipette tips, pyrogen-free tubes, bacterial endotoxin test water, limulus amebocyte lysate (lyophilized product, specification 0.1ml/vial, sensitivity of 0.06EU/ml), bacterial endotoxin working standard, etc.

Operation Method:

First calculate the MVD, dilute the product with bacterial endotoxin test water, the dilution factor must not exceed MVD, and use it as the test solution; dilute the bacterial endotoxin standard with bacterial endotoxin test water to prepare a concentration of endotoxin standard solution as the positive control solution; prepare the bacterial endotoxin standard with the test solution to make a concentration of endotoxin solution as the test positive control solution, and use bacterial endotoxin test water as the negative control solution. Take 8 tubes of reconstituted limulus amebocyte lysate (LAL), of which 2 tubes are added with 0.1ml of test solution as the test tubes, 2 tubes are added with 0.1ml of positive control solution as the positive control tubes (PC), 2 tubes are added with 0.1ml of test water as the negative control (NC), 2 tubes are added with 0.1ml of test positive control solution as the test positive control tubes (PPC).Gently mix the solution in the test tube, then seal the tube opening with sealing film and place it vertically in a suitable constant temperature incubator at 37°C ± 1°C, maintaining the temperature for (60 ± 2) min.

Result judgment: (1) The experiment is considered valid only when all positive controls are positive, all test sample positive controls are positive, and all negative controls are negative.

If both tubes of the test sample are negative, the sample is considered compliant; if both tubes of the test sample are positive, it is considered non-compliant; if one of the two tubes is positive and the other is negative, follow the above method to take an additional

4 test sample tubes for retesting. If 1 out of the 4 tubes is positive, it is considered non-compliant.

If during the first test, the dilution factor of the test sample is less than MVD and the results show 2 tubes as positive or 2 tubes with 1 tube positive, a retest shall be conducted using the same method, requiring dilution to MVD during the retest.

Standard limit: The amount of endotoxin in each 1ml of gadopentetate dimeglumine injection should be less than 0.5EU.

Sterility

Test method: Membrane filtration method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rule 1101).

Experimental conditions: Clean bench, biochemical incubator, alcohol lamp, tweezers, etc.

Test drugs and disinfectants:75% ethanol, 0.1% benzalkonium bromide solution. Control strain: Staphylococcus aureus.

Culture media: Thioglycollate fluid medium, Tryptic Soy Broth.

Operating procedure:

Operating procedure: Take 30 samples of this product, and perform the test according to the membrane filtration method specified in (Chinese Pharmacopoeia 2020 Edition, Part IV 1101 Sterility Test).

Take 30 units of the product, and use the membrane filtration method with 10 units per filter. Rinse with pH7.0 sterilesodium chloride-peptonebuffer solution, 100ml each time, for a total of 3 rinses. Add tryptic soy agar medium to one filter and incubate at 20~25°C for 14 days. To the other filter, add 100ml of thioglycollate fluid medium. For the positive control, add less than 100cfu of Staphylococcus aureus suspension to the final rinse solution, filter, add 100ml of thioglycollate fluid medium, and incubate at 30~35°C for 14 days.

Result judgment: If all test tubes are clear, or if turbidity is observed but confirmed to be sterile, the product can be deemedqualified. If any test tube shows turbidity and microbial growth is confirmed, the product is deemed unqualified unlessit can be sufficiently demonstrated that the test results are invalid, i.e., the growth is not attributable to the product.

Assay

Test method: High-performance liquid chromatography (2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0512).

Test Conditions: Use octylsilane-bonded silica gel as the stationary phase (Symmetry C8, 4.6mm × 150mm, 5μm); tetrabutylammonium perchlorate solution as the mobile phase, with a flow rate of 1.0 ml per minute; injection volume of 20μl; column temperature of 25°C; detection wavelength of 195 nm.

Operating Procedure

Test Solution Accurately measure 2ml of the sample, transfer to a 200ml volumetric flask, dilute with water to the mark, and mix well. Accurately transfer 15ml to a 100ml volumetric flask, add 10ml of acetonitrile, dilute with water to the mark, and mix well.

Reference Solution Accurately weigh gadopentetate dimeglumine reference standard, dissolve in 10% acetonitrile, and quantitatively dilute to prepare a solution containing approximately 0.6mg per 1ml.

System Suitability Requirements The number of theoretical plates, calculated based on the gadopentetate dimeglumine peak, should not be less than 3000.

Assay Method Accurately measure the test solution and reference solution, inject them separately into the liquid chromatograph, and record the chromatograms. Calculate the results using the external standard method based on peak area, then multiply the obtained results by 1.263.

Standard Limit: The content of gadopentetate dimeglumine (C14H20GdN3O102C7H17NO5) should be 95.0%~105.0% of the labeled amount.

Method Screening and Optimization

Description, Identification (1), Identification (2), Identification (3), Identification (4), and the following items under Tests: pH, color, sterility, insoluble particles, visible foreign matter, fill volume, and assay method remain unchanged from the current official standard (Chinese Pharmacopoeia 2020 Edition, Part II).

In response to changes during the study and potential degradation products, the quality standard for Gadopentetate Dimeglumine Injection was screened and optimized based on pharmacopoeial standards from various countries and the product's quality profile. The screening and optimization process for related substances, pentetic acid, elemental impurities, and other test items in the standard are described in detail.

The pentetic acid method was compared between the Chinese Pharmacopoeia 2020 Edition Part II and the import registration standard (consistent with USP43 standards). The import registration standard includes this pentetic acid test method, where in a pH5 buffer solution, dilute xylenol orange is used as an indicator. When titrated with 0.001mol/L gadolinium sulfate titrant, the endpoint changes from yellow to reddish-purple, with a clear and easily discernible transition, demonstrating good accuracy and reliability. The pentetic acid method in the Chinese Pharmacopoeia 2020 Edition Part II uses xylenol orange indicator at a concentration more than 10 times higher than the dilute xylenol orange solution in the import registration standard. The endpoint color change ranges from yellow to orange, which is less distinct compared to the import registration standard method.

Neither the Chinese Pharmacopoeia 2020 Edition nor the USP43 standard includes a method for related substances. Our company referenced the import registration standard to draft and establish the related substances test, without revising the method or limits.

The assay methods in the Chinese Pharmacopoeia 2020 Edition, USP43 standard, and import registration standard are largely consistent in terms of the mobile phase system and chromatographic conditions, with only minor differences in sample preparation and mobile phase ratios. Our company referenced the current assay method for gadopentetate dimeglumine injection in the Chinese Pharmacopoeia 2020 Edition Part II for drafting, without revisions. 3.2.P.5.2.2.1 Related Substances

Method Source

Refer to the test method for "Related Substances" under the quality standard of Gadopentetate Dimeglumine Injection in the imported registration standard (JX20020190).

Related Substances Determine by high-performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). Solvent Copper nitrate solution (dissolve 12.88g of copper nitrate trihydrate in water and dilute to 1000ml). Copper nitrate sodium citrate solution (dissolve 12.88g of copper nitrate trihydrate and 38.0g of sodium citrate dihydrate in water and dilute to

1000ml).

Test solution Accurately measure 1ml of the sample (equivalent to approximately 469mg of gadopentetate dimeglumine) and transfer to a 20ml

Into a volumetric flask, dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Reference Solution (1) Accurately weigh an appropriate amount of nitrilotriacetic acid reference standard and ethylenediaminetetraacetic acid reference standard, dissolve in copper nitrate solution, and quantitatively dilute to prepare a solution containing approximately 0.02mg of nitrilotriacetic acid and 0.4mg of ethylenediaminetetraacetic acid per 1ml.

Reference Solution (2) Accurately measure 1ml of Reference Solution (1), transfer to a 20ml volumetric flask, dilute to the mark with copper nitrate solution, and mix well.

Chromatographic Conditions and System Suitability Use octylsilane-bonded silica gel as the filler (MOS-1Hypersil, 125mm×4.6mm,5μm, or a column with equivalent performance), Mobile Phase 1: Take 10% (W/V) aqueous tetrabutylammonium hydroxide solution 10ml, 10ml methanol and water 170ml, mix, adjust the pH to 7.5±0.1 with phosphoric acid, add 90ml methanol, and dilute with water to 1000ml.Mobile phase 2: Take 10% (W/V) tetrabutylammonium hydroxide aqueous solution 10ml, methanol 10ml, and water 170ml, mix, adjust the pH to 7.5±0.1 with phosphoric acid, add methanol 450ml, and dilute with water to 1000ml. Perform gradient elution according to the table below. After injection, elute with mobile phase 1 and record the chromatogram until 20min; after recording, elute the column with mobile phase 2 for 20min , then equilibrate the column with mobile phase 1.

Time min

Mobile phase 1 (%)

Mobile phase 2 (%)

0

100

0

20

100

0

21

0

100

41

0

100

42

100

0

85

100

0

The flow rate was 2ml per minute, and the detection wavelength was 254nm. The injection sequence was: copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, reference solution (1), and a mixed solution of test solution and reference solution (1:20), followed by reference solution (1). The separation between nitrilotriacetic acid (NTA) peak and copper nitrate peak, as well as between copper nitrate peak and EDTA peak, should meet the requirements. The retention times of NTA and EDTA in the test solution and reference solution (2) may slightly differ.

Assay Accurately inject 20μL each of reference solution (2), test solution, and dilution solvent into the liquid chromatograph following the system suitability test injection sequence. For the test solution calculation, the corresponding peak of copper nitrate sodium citrate solution should be deducted. If any impurity peaks appear between the injection and the copper nitrate peak, calculate them using the external standard method based on the NTA reference peak area. From the copper nitrate peak to the end of the chromatogram, if any impurity peaks appear, calculate them using the external standard method based on the EDTA

Calculated based on the reference standard peak area, the total related substances shall not exceed 0.3% of the labeled amount of pentetic acid (each 1ml of this product contains a total of 197mg of pentetic acid).

Figure 3.2.P.5-1 System suitability and sample overlay chromatogram for related substances method3.2.P.5-2 Results of related substances method screening

Method screening conclusion

Copper nitrate solution and copper citrate nitrate solution do not interfere with the detection of related substances in this product, nitrilotriacetic acid

(NTA) peak and copper nitrate peak, copper nitrate peak and edetate peak meet the resolution requirements. This method can be used for the control of related substances in this product.

Pentetic Acid

Method source

Chinese Pharmacopoeia 2020Edition Part II, Import Registration Standard JX20020190, and USP43 standards all include the pentetic acid test method. Method comparison is shown in Table 3.2.P.5-3

Method screening conclusion

Table3.2.P.5-3Pentetic Acid Test Method Screening

Method Source

Solution Preparation

Buffer Solution

Indicator Solution

Titrant

Endpoint Determination

Conclusion

Chinese Pharmacopoeia 2020Edition Part II Gadopentetate Dimeglumine Injection

Take 5ml of the product, place it in a conical flask, add 25ml of water, and add acetic acid-sodium acetate buffer solution

(pH5.0)10ml

acetic acid-sodium acetate buffer solution

(pH5.0)

Xylenol Orange

0.5ml

Gadolinium Chloride Titration Solution (0.002mol/L)

)

Changes from orange-yellow to orange-red

Imported Registration Standard The method exhibits distinct endpoint color changes, facilitating easy judgment, and demonstrates good accuracy and reliability. Therefore, the Imported Registration Standard is adopted as the proposed method.

Imported Registration Standard

(JX20020190) and USP43

Pipette accurately 2ml of the sample, transfer to a 100ml conical flask, add 20ml of water,

0.1mol sodium hydroxide and acetic acid to adjust the pH value

to 5

dilute xylenol orange 10ml

gadolinium sulfate titration solution (0.001mol/L

)

Yellow turns to red-purple

Content

Method source

Table3.2.P.5-4Content test method comparison table

Product name determination method

Import Registration Standard (JX20020190) and USP43

ChP2020Preparation

HPLCMethod

HPLCMethod

Chromatographic Conditions

Chromatographic Column

Octylsilane chemically bonded silica as filler

Octylsilane chemically bonded silica as filler

Mobile phase

Acetonitrile[containing1.37gtetrabutylammonium perchlorate]-water (120:880)as the mobile phase

Mobile phaseA: water-phosphate buffer (pH7.6) (per1000mloftetrabutylammonium perchlorate solution (take1.7gof tetrabutylammonium perchlorate, add

acetonitrile100mlto dissolve, dilute with water to1000ml)as the mobile phase;

Elution Mode

Isocratic Elution

Isocratic Elution

Detection Wavelength

195nm

195nm

Flow rate

1ml/min

1ml/min

Injection volume

10μl

20μl

Solvent

10%acetonitrile

Sample concentration

1ml200ml

2ml200ml,15ml100ml

Limit

Calculate the amount of gadopentetate dimeglumine in the test sample using the external standard method based on peak area, then multiply by 93802/74280 to obtain the amount of gadopentetate dimeglumine in the test sample. The content of gadopentetate dimeglumine (C14H20GdN3O102C7H17NO5)

should be 95.0%-105.0% of the labeled amount

The result calculated by the external standard method using peak area is multiplied by 1.263, and the content of gadopentetate dimeglumine (C14H20GdN3O10 2C7H17NO5) should be 95.0%-105.0% of the labeled amount.

The content determination method in the Chinese Pharmacopoeia 2020 edition and USP43 standard, as well as the imported registration standard mobile phase system and chromatographic conditions, are basically consistent. Only the sample preparation method and mobile phase ratio differ slightly. Our company referenced the content determination method for gadopentetate dimeglumine injection in Part II of the current Chinese Pharmacopoeia 2020 edition for drafting, with no revisions made.

Refer to the "Content" determination method under the quality standard for gadopentetate dimeglumine injection in Part II of the Chinese Pharmacopoeia 2020 edition. The determination is performed according to the high-performance liquid chromatography method (Chinese Pharmacopoeia 2020 edition, General Chapter 0512).

[Assay] Perform the test as described under High Performance Liquid Chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rule 0512).

Test solution Accurately measure 2ml of the sample, transfer to a 200ml volumetric flask, dilute with water to volume, and mix well. Accurately transfer 15ml to a 100ml volumetric flask, add 10ml of acetonitrile, dilute with water to volume, and mix well.

Reference solution Take gadopentetate dimeglumine reference substance, accurately weigh, dissolve in 10% acetonitrile, and dilute quantitatively to prepare a solution containing approximately 0.6mg per 1ml.

Chromatographic conditions Use octyl-bonded silica gel as the stationary phase; use tetrabutylammonium perchlorate solution (dissolve 1.7g of tetrabutylammonium perchlorate in 100ml of acetonitrile, dilute with water to 1000ml) as the mobile phase; detection wavelength is 195nm; injection volume is 20μl.

System Suitability Requirements The number of theoretical plates, calculated based on the gadopentetate dimeglumine peak, should not be less than 3000.

Assay Procedure Accurately measure the test solution and reference solution, inject them separately into the liquid chromatograph, and record the chromatograms. Calculate the results using the external standard method based on peak area, then multiply the obtained results by 1.263.

Figure 3.2.P.5-2 Overlay Chromatograms of Blank Solvent, Reference Solution, and Test Solution for Content Determination

Method Screening Conclusion

The blank solvent does not interfere with the detection of gadopentetate dimeglumine, and this method is proposed to be adopted as the content determination method.

Validation of Analytical Methods

In accordance with the "Technical Guidelines for Validation of Analytical Methods for Quality Control of Chemical Drugs," "Technical Guidelines for the Standardization Process of Establishing Quality Standards for Chemical Drugs," "Technical Guidelines for the Study of Impurities in Chemical Drugs," and the "Chinese Pharmacopoeia" 2020 Edition, General Chapter 9101 "Guidelines for Validation of Analytical Methods for Drug Quality Standards," the methodology for related substances, elemental impurities, meglumine, pentetic acid, content, bacterial endotoxins, and sterility in the quality standards of gadopentetate dimeglumine injection was validated. Since the methods for meglumine and elemental impurities, as well as their limits, are identical to those for the gadopentetate dimeglumine raw material, the validation content refers to the raw material, and no further validation is conducted for gadopentetate dimeglumine injection. The specific details are as follows:

Pentetic Acid

Test drugs, reagents, and instruments

Table3.2.P.5-5Test Reagent Information Sheet

Name

Batch number

Content(%)/Specification

Source

Pentetic Acid

20210703

99.4

Guangzhou Kangchen Pharmaceutical Co., Ltd.

Gadopentetate Dimeglumine Injection

020210801

20ml

Guangzhou Kangchen Pharmaceutical Co., Ltd

Table3.2.P.5-6Test Reagent Information Sheet

Name

Manufacturer

Batch number

Grade

Xylenol Orange

McLin

C1190944428

Indicator Grade

Glacial Acetic Acid

Tianjin Damao

02170801

AR

Sodium acetate anhydrous

Guangzhou Chemical Reagent Factory

20150701-2

AR

Sodium hydroxide

Tianjin Damao

20180801

AR

Gadolinium Sulfate

Aladdin

A1316011

AR

Table3.2.P.5-7Test Equipment Information List

Name

Manufacturer and Model

Internal Number

Valid Until

One Hundred Thousandth Electronic Balance

CPA225D

VAC07

2022.09.02

Determination Method for Pentetic Acid Content

Pentetic Acid Accurately measure 2ml of the sample, place it in a 100ml conical flask, add 20ml of water, and 10ml of dilute xylenol orange solution. Shake well, adjust the pH to 5.0 using 0.1mol/L sodium hydroxide solution or glacial acetic acid, and titrate with gadolinium sulfate titrant (0.001mol/L) until the solution changes from yellow to reddish-purple. Each 1ml of gadolinium sulfate titrant

(0.001mol/L) is equivalent to 0.7867mg of pentetic acid (C14H23N3O10). Each 1ml of the sample should contain 200~450μg of pentetic acid.

Amount of pentetic acid=(V–V0)×T×F × 1000

2

V:Volume of titrant consumed,ml

V0:Volume of titrant consumed in blank test,ml

TTiter:0.7867 corresponds to 1ml of gadolinium sulfate standard titrant[=0.001000mol/L], expressed in mg units

FTitrant correction factor 2: Volume of solution transferred,ml

Validation Results Table

The specific validation summary is shown in Table 3.2.P.5-8 below:

Table 3.2.P.5-8 Summary of Pentetic Acid Methodology Validation Results

Item

Acceptance Criteria

Verification Results

Specificity

The volume of titrant consumed by the blank solution is less than 0.10ml.

The volume of titrant consumed by the blank solution is 0.01ml, specific

The properties meet the requirements.

Solution stability

At 0h, 2h, 4h, 6h, the content of pentetic acid in the sample was measured, and the ratio to 0h was between 95.0%~105.0%.

Within 6h, the ratio of pentetic acid content in the sample to 0h was within

95.0%~105.0%, and the solution remains stable within 6 hours.

Accuracy (Recovery Rate)

The average recovery rate at each concentration should be within

90.0%-110.0%, and the relative standard deviation (RSD) of recovery data should be ≤10.0%.

50%, 100%, 150% spiked recovery rates were

98.0%, 97.1%, 97.8%; RSD were 1.2%,

0.6%, 0.0%, meeting the accuracy requirements.

Linearity and range

The correlation coefficient of the regression line Rshall not be less than 0.990,and the ratio between the Y-axis intercept and the theoretical response value at 100% concentration should be ≤25.0%.

The linear equation is y=792.2086x+14.0943, with a correlation coefficient of 0.9995,and the ratio of the y-axis intercept to the 100% concentration is 1.8%, meeting the linearity requirements.

Precision

For 6 sample solutions, the RSD of the obtained pentetic acid content shall be ≤

5.0%. The relative standard deviation (RSD) of 12 test sample solutions measured by different analysts was 5.0%.

For a single operator, the RSD of pentetic acid content measured in 6 test samples

was 0.5% and 0.4%, respectively; the RSD for 12 test samples was

0.5%, meeting the precision requirements.

Robustness

Prepare sample solutions and measure the pentetic acid content at 20°C, 25°C, 30°C respectively. The content at each temperature point should be consistent with that at 25°C.

Compared to, the ratio is between 90.0%-110.0%.

20°C, 30°C, the measured pentetic acid content ratio to 25°C was 98.9%, 100.0%, meeting the robustness requirements.

Specificity

Experimental Design and Requirements

Prepare a blank solution, and the volume of titrant consumed by the color change of the blank solution should not exceed 0.1ml. 3.2.P.5.3.1.4.2 Experimental Procedure

Solution Preparation

Blank solution: Take 20ml of water, place it in a 100ml conical flask, add 10ml of dilute xylenol orange solution, and mix well.Sodium acetate buffer solution: Take approximately 50g of sodium acetate and place it in a 1000ml volumetric flask, add about 10ml of glacial acetic acid, and add water

to the mark, mix well, and adjust the 0.1mol/L sodium hydroxide solution or glacial acetic acid to pH 5.0.

Xylenol orange solution: Take 50.8mg of xylenol orange, place it in a 100ml volumetric flask, dissolve in water, and dilute to themark, then mix well.

Dilute xylenol orange solution: Take 30ml of sodium acetate buffer solution and 3ml of xylenol orange solution, place them in the same 200ml

Transfer to a volumetric flask, add water to the mark, and mix well.

Gadolinium sulfate titrant (0.001mol/L): Take 0.7468g of gadolinium sulfate octahydrate, place it in a 1000ml volumetric flask, dissolve in water, and dilute to the mark.

Determination

Using a blank solution as reference, titrate with gadolinium sulfate titrant (0.001mol/L) until the solution changes from yellow to reddish-purple, and record the volume of titrant consumed.

Experimental Results

Table3.2.P.5-9Specificity Test Results

Sample

Titrant Consumption Volume(ml)

Blank solution 1

0.01

Blank solution 2

0.01

Original record index: KY-GP-02-A009P23

Result analysis

Two blank solutions were measured, with titrant consumption volumes of 0.01ml and 0.01ml, both less than 0.10ml, meeting the specificity requirement.

Solution Stability

Experimental Design and Requirements

Sample solutions were prepared and measured at 0h, 2h, 4h, and 6h. The requirement is that the ratio of the tiaprofenic acid content at each time point compared to 0h should be within 95.0%-105.0%.

Test Procedure

Sample Solution

Blank Solution: Take 20ml of water, place it in a 100ml conical flask, add 10ml of dilute xylenol orange solution, and mix well. Sample Solution: Accurately measure 2ml of the sample, place it in a 100ml conical flask, add 20ml of water, and add dilute xylenol

orange solution 10ml, mix well, adjust the pH to 5.0 using 0.1mol/L sodium hydroxide solution or glacial acetic acid, and titrate with gadolinium sulfate titrant (0.001mol/L) until the solution changes from yellow to reddish-purple.

Sample Determination

Prepare sample solutions and perform measurements at 0h, 2h, 4h, 6h respectively, recording the volume of titrant consumed.

Experimental Results

Table 3.2.P.5-10 Stability Test Results of Pentetic Acid Solution

Titer(mg/ml)

0.7867

Correction factor

0.995

Time point(h)

Volume transferred(ml)

Volume of titrant consumed

(ml)

Amount of pentetic acid

(μg/ml)

Content to 0h ratio

(%)

Blank

0

0.01

367.9

n/a

0

2.00

0.95

367.9

n/a

2

0.95

367.9

100.0

4

0.95

367.9

100.0

6

0.96

371.8

101.1

Original Record Index

KY-GP-02-A009 P24

Result Analysis

6h The ratio of pentetic acid measured at 0h compared to 95.0%~105.0%, indicating the solution is stable within 6h .

Linearity and Range

Experimental Design and Requirements

The linearity range is set at 50%-150%. Acceptance criterion: the correlation coefficient R of the regression line must not be less than

0.990,Y-axis intercept and the ratio between the 100% theoretical concentration should be ≤25.0%.

Experimental procedure

Solution Preparation

Blank solution: Take 20ml of water, place it in a 100ml conical flask, add 10ml of dilute xylenol orange solution, and mix well. Pentetic acid solution (400μg/ml): Take 40mg of pentetic acid, weigh accurately, and place in a 100ml volumetric flask, add

water to dissolve and dilute to the mark. (LSS)

Linear Solution Preparation

Use a pipette to transfer the following solutions

Table3.2.P.5-11Linear Solution Preparation Table

Serial Number

Concentration Level

Volume transferred

Dilution volume (ml)

Amount of pentetic acid, μg

S1

50%

1.0ml of LSS

30

400

S2

80%

1.6ml of LSS

30

640

S3

100%

2.0ml of LSS

30

800

S4

120%

2.4ml of LSS

30

960

S5

150%

3.0ml of LSS

30

1200

Prepare linear solutions according to Table 3.2.P.5-11, perform titration as per the proposed method, and record the volume of titrant consumed.

Experimental results

Table 3.2.P.5-12Results of linear solutions

Sample weight (mg)

40.16

Purity (%)

99.4

Linearity level

Volume transferred (ml)

Titrant consumption volume (ml)

Average volume consumed (ml)

Amount of pentetic acid (μg)

50%

1.0

0.49

0.50

0.50

399.2

80%

1.6

0.77

0.77

0.77

638.7

100%

2.0

1.00

1.00

1.00

798.4

120%

2.4

1.19

1.19

1.19

958.1

150%

3.0

1.50

1.49

1.50

1197.6

Linear equation

y=792.2086x+14.0943

Correlation coefficient r

0.9995

100%concentration response

800.00

y-intercept

14.0943

y-intercept to 100%concentration ratio

1.8%

Spray1400.0Series1

Substitute1200.0LineLinear(Series1)

of 1000.0

Quantity800.0y= 792.2086x+ 14.0943

(= 0.9991

600.0

)400.0

200.0

0.0

0.000.501.001.502.00

Titrant consumption volume (ml)

Original Record Index

KY-GP-02-A009 P25

Result Analysis

The linear equation is y=792.2086x+14.0943, with a correlation coefficient of 0.9995 , which is greater than 0.990;the y-axis intercept ratio to the 100% concentration is 1.8%, less than 25%, indicating good linearity of the solution.

Accuracy

Experimental Design and Requirements

Prepare spiked solutions of pentetic acid at concentrations of 50%, 100%, and 150%. The recovery rate of pentetic acid at each concentration should be within 90.0%-110.0%, and the relative standard deviation (RSD) of the recovery data should be ≤10.0%.

Experimental Procedure

Solution Preparation

Pentetic Acid Solution (400μg/ml):Weigh accurately 40mg of pentetic acid, transfer it to a 100ml volumetric flask, and dissolve and dilute to volume with water.

Accuracy Solution

The preparation and transfer volumes of pentetic acid solutions at various concentrations are shown in Table 3.2.P.5-13

Table3.2.P.5-13Preparation of Accuracy Test Solutions

Name

Spiking Level

Volume of Pentetic Acid Solution Transferred(ml)

Dilution volume (ml)

Sample 1

50%

1.0

30

Sample 2

100%

2.0

30

Sample 3

150%

3.0

30

Prepare sample solution and spiked solution, determine using the proposed method, and record the volume of titrant consumed.

Experimental results

Table3.2.P.5-14Accuracy Results

Sample weight (mg)

40.16

Purity(%)

99.4

Titer(mg/ml)

0.7867

Correction factor

0.995

Name

Amount of pentetic acid added (μg)

Volume of titrant consumed (ml)

Measured amount of pentetic acid (μg)

Recovery rate

(%)

Average recovery rate

(%)

RSD(%)

Blank

-

0.01

N/A

N/A

N/A

N/A

Test

0

0.95

735.8

N/A

N/A

N/A

0.95

735.8

N/A

N/A

N/A

0.96

743.6

N/A

N/A

N/A

50%Spiked Test

399.2

1.46

1135.0

99.4

98.0

1.2

1.45

1127.2

97.4

1.45

1127.2

97.4

100%Spiked Test

798.4

1.94

1510.7

96.7

97.1

0.6

1.94

1510.7

96.7

1.95

1518.6

97.7

150%Spiked Sample

1197.6

2.45

1910.0

97.8

97.8

0.0

2.45

1910.0

97.8

2.45

1910.0

97.8

Original Record Index

KY-GP-02-A009 P26

Result Analysis

50%, 100%, 150% spike recovery rates were 98.0%, 97.1%, 97.8%, respectively, within the range of 90.0%~110.0%

; RSD values were 1.2%, 0.6%, 0.0%, all below 10.0%, meeting the accuracy requirements.

Precision

Experimental Design and Requirements

Repeatability: Prepare 6 sample solutions, analyze using the proposed method, calculate the 6 sample RSD values, with the requirement RSD not exceeding 5.0%;

Intermediate precision: Another analyst prepares 6 sample solutions, analyzes using the proposed method, calculates 12

sample RSD values, with the requirement not exceeding 5.0%.

Experimental procedure

Solution Preparation:

Blank solution: Take 20ml of water, place it in a 100ml conical flask, add 10ml of dilute xylenol orange solution, and mix well. Sample solution: Precisely measure 2ml of the sample, place it in a 100ml conical flask, add 20ml of water, and add dilute xylenol

orange solution 10ml, mix well, adjust the pH to 5.0 using 0.1mol/L sodium hydroxide solution or glacial acetic acid, and titrate with gadolinium sulfate titrant (0.001mol/L) until the solution changes from yellow to reddish-purple.

The sample solution was prepared and measured using the proposed method, and the volume of titrant consumed was recorded.

Experimental Results

Table3.2.P.5-15Precision Measurement Results

Titer(mg/ml)

0.7867

Correction Factor

0.995

Blank consumption volume (ml)

0.01

Volume transferred (ml)

2

Sample

Titrant consumption volume (ml)

Amount of pentetic acid

g/ml)

RSD(%)

Repeatability

0.95

367.9

0.5

0.5

0.95

367.9

0.95

367.9

0.95

367.9

0.96

371.8

0.96

371.8

Intermediate Precision

0.95

367.9

0.4

0.95

367.9

0.96

371.8

0.95

367.9

0.95

367.9

0.95

367.9

Original Record Index

KY-GP-02-A009 P24

Result Analysis

6The RSD of pentetic acid content measured in 6 test sample solutions was 0.5%; another analyst measured the 6 test sample solutions with an RSD of pentetic acid content of 0.4%, and the RSD of pentetic acid content in 12 test sample solutions was 0.5%, meeting the precision requirements.

Robustness

Experimental Design and Requirements

Prepare sample solutions and place them at 20°C, 25°C, and 30°C respectively. The measured content at each temperature point should have a ratio of 25°C within the range of 90.0%-110.0%.

Experimental Procedure

Solution Preparation:

Blank solution: Take 20ml of water, place it in a 100ml conical flask, add 10ml of dilute xylenol orange solution, and mix well. Sample solution: Precisely measure 2ml of the product, place it in a 100ml conical flask, add 20ml of water, and add dilute xylenol

orange solution 10ml, mix well, adjust the pH to 5.0 using 0.1mol/L sodium hydroxide solution or glacial acetic acid, and titrate with gadolinium sulfate titrant (0.001mol/L) until the solution changes from yellow to reddish-purple.

Prepare the sample solution, place it at 20°C, 25°C, and 30°C, respectively, and measure according to the proposed method, recording the volume of titrant consumed.

Experimental results

Table3.2.P.5-16Robustness Verification Results

Pipetting Volume (ml)

2.0

Blank Consumption Volume

(ml)

0.01

Titer (mg/ml)

0.7867

Correction factor

0.995

Temperature(℃)

Titrant consumption volume

(ml)

Amount of pentetic acid (μg)

Ratio to 25°C (%)

20

0.95

367.9

98.9

25

0.96

371.8

100.0

30

0.96

371.8

100.0

Original record index

KY-GP-02-A009 P26

Result Analysis

20℃, 30℃ measured pentetic acid content ratio compared to 25℃ is 98.9%, 100.0%, within 90.0%~110.0%

range. Minor temperature variations have essentially no impact on pentetic acid detection.

Sample Testing

Table3.2.P.5-17Determination Results of Pentetic Acid in Trial Production Batches and Reference Preparations

Name

Specification

Batch

Containing pentetic acid as

200-450 μg/ml

Gadopentetate Dimeglumine Injection (manufacturing validation batch)

20 ml:9.38 g

020210801

393

020210802

365

020210803

347

10ml:4.69g

020210804

361

020210805

361

020210806

385

12ml:5.63g

020210807

393

15ml:7.04g

020210808

388

Gadopentetate Dimeglumine Injection Reference

Preparation

20ml:9.38g

KT045S32

391.4

KT05TA1

378.0

Gadopentetate Dimeglumine Injection ReferencePreparation

15ml:7.04g

KT051T2

379.3

KT07B25

384.5

Gadopentetate Dimeglumine Injection Reference

Preparation

10ml:4.69g

KT05B9J

383.6

Original Record Traceability:KY-GP-02-A009P33;KY-GP-02-A010P29,P30;

Related substances

The chromatographic conditions for the related substances method of Gadopentetate Dimeglumine Injection are consistent with those of the raw material, therefore the linearity, limit of quantitation, limit of detection, and accuracy data refer to the methodological validation section for related substances of the raw material.

Test drugs, reagents, and instruments

Table3.2.P.5-18Test Drug Information Table

Sample name

Specification/Purity%

Batch number

Manufacturer

Gadopentetate Dimeglumine Injection

10ml:4.69g

20210401-S

Guangzhou Kangchen Pharmaceutical Co., Ltd.

Nitrilotriacetic acid

100%

101123-201001

National Institutes for Food and Drug Control

Disodium edetate

100%

190190-201701

National Institutes for Food and Drug Control

Table3.2.P.5-19Test Reagent Information Sheet

Name

Manufacturer

Batch number

Grade

Methanol

Shanghai Xingke High Purity Reagent Co., Ltd.

A21T0213

HPLC

10%(W/V)sodium hydroxide

Tetrabutylammonium aqueous solution

Aladdin

G2027227

1L

Phosphoric Acid

Guangzhou Chemical Reagent Factory

20150501-1

500ml

Copper(II) nitrate

Macklin

C11971072

25g

Sodium citrate dihydrate

Macklin

S818273

1000g

Sodium hydroxide

Macklin

C10819538

500g

Hydrochloric Acid

Guangzhou Chemical Reagent Factory

20200604

500ml

3%Hydrogen Peroxide

Guilin Lifeng Pharmaceutical

20191007

500ml

Ultrapure Water

Self-made

Table3.2.P.5-20Test Equipment Information Sheet

Name

Manufacturer and Model

Internal Number

Valid Until

Analytical Balance (0.1 mg precision)

Sartorius CPA225D

VAC06

2021.10.28

One-millionth electronic balance

Sartorius MSA116P

VAC87

2021.11.10

Electrothermal constant temperature water bath

Shanghai Yiheng HWS-12

VAC92

2022.06.29

High Performance Liquid Chromatograph

Thermo Fisher U3000

VAC73

2022.10.28

High Performance Liquid Chromatograph

Thermo Fisher U3000

VAC88

2021.11.21

High Performance Liquid Chromatograph

Thermo Fisher U3000

VAC99

2023.01.19

C8 column

Thermo Hypersil

H-C8-T-04

4.6×150mm,5μm

C8 column

Thermo Hypersil

H-C8-T-05

4.6×150mm,5μm

C8 column

Thermo Hypersil

H-C8-T-06

4.6×150mm, 5μm

Water purification system

Rephile Genie G10

VAC02

/

Pre-validation method for related substance determination

Related substances Determined by high-performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV General Chapter 0512). Chromatographic conditions and system suitability The column is packed with octylsilane-bonded silica gel (MOS-1 Hypersil, 5μm,125*4.6mm), mobile phase 1: take 10% (W/V) aqueous solution of tetrabutylammonium hydroxide 10ml,

Methanol 10ml and water 170ml are mixed, adjusted to pH 7.5±0.1 with phosphoric acid, followed by the addition of methanol 90ml, and water is added to 1000ml. Mobile phase 2: Take 10% (W/V) aqueous tetrabutylammonium hydroxide solution 10ml, methanol 10ml, and water 170ml are mixed, adjusted to pH 7.5±0.1 with phosphoric acid, followed by the addition of methanol 450ml, and water is added to 1000ml. Perform gradient elution according to the table below. After injection, elute with mobile phase 1 and record the chromatogram until 20min; after recording is completed, elute the column with mobile phase 2 for 20min , then equilibrate the column with mobile phase 1 .

Time min

Mobile phase 1 (%)

Mobile phase 2 (%)

0

100

0

20

100

0

21

0

100

41

0

100

42

100

0

85

100

0

The flow rate was 2ml per minute, and the detection wavelength was 254nm; the injection sequence was: copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, reference solution (1), and a mixed solution of test solution and reference solution (1:20), reference solution (1). The resolution between the nitrilotriacetic acid peak and the copper nitrate peak, and between the copper nitrate

peak and the EDTA peak should meet the requirements. The retention times of nitrilotriacetic acid and EDTA in the test solution and reference solution (2) may slightly differ.

Preparation of reference solution (1): Weigh approximately 10mg of nitrilotriacetic acid reference standard, place it in a 20ml volumetric flask, add 15ml of copper nitrate solution, sonicate to dissolve, cool to room temperature, dilute to volume with copper nitrate solution, to prepare the nitrilotriacetic acid reference stock solution. Weigh approximately 20mg of EDTA reference standard, accurately weigh, place in a 50ml volumetric flask, accurately measure 2ml of the nitrilotriacetic acid reference stock solution, place in the same 50ml volumetric flask, add 40ml of copper nitrate solution, sonicate to dissolve, cool to room temperature, dilute to volume with copper nitrate solution, and mix well.

Preparation of reference solution (2): Accurately measure 1ml of reference solution (1), transfer to a 20ml volumetric flask, dilute to the mark with copper nitrate solution, and mix well.

Preparation of the test solution: Accurately measure 1ml of the sample, transfer it to a 20ml volumetric flask, dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Assay method: Accurately measure 20μL each of the reference solution (2), test solution, and dilution solvent, and inject them into the liquid chromatograph in the order specified in the system suitability test. When calculating the test solution, the corresponding peak of the copper nitrate sodium citrate solution should be deducted. If impurity peaks appear between the injection and the copper nitrate peak, calculate them using the external standard method based on the peak area of the nitrilotriacetic acid reference standard. Record the chromatogram from injection until 20min after the copper nitrate peak. If impurity peaks appear, calculate them using the external standard method based on the peak area of the edetate reference standard. The total amount of related substances should not exceed 0.3%.

Note: Copper nitrate solution: Dissolve 12.88g of copper nitrate trihydrate in water and dilute to 1000ml. Copper nitrate sodium citrate solution: Dissolve 12.88g of copper nitrate trihydrate and 38.0g of sodium citrate dihydrate in water and dilute to

1000ml.Method validation solution preparation:

Copper nitrate solution: Take 12.88g of copper nitrate trihydrate,12.88g dissolve in water and dilute to 1000ml.

Copper nitrate sodium citrate solution: Take 12.88g of copper nitrate trihydrate,12.88g 38.0g of sodium citrate dihydrate,38.0g, dissolve in water and dilute to 1000ml.

Preparation of reference solution (1): Take about 10mg of nitrilotriacetic acid reference standard,10mg place in a 20ml volumetric flask, add 15ml of copper nitrate solution,15ml ultrasonicate to dissolve, cool to room temperature, dilute to the mark with copper nitrate solution, as the nitrilotriacetic acid reference stock solution. Take about 20mg of edetate reference standard,20mg accurately weighed, place in a 50ml volumetric flask, accurately measure 2ml of the nitrilotriacetic acid reference stock solution,2ml place in the same 50ml volumetric flask, add 40ml of copper nitrate solution,40ml ultrasonicate to dissolve, cool to room temperature, dilute to the mark with copper nitrate solution, and mix well.

Preparation of reference solution (2): Accurately measure 1ml of reference solution (1),1ml place in a 20ml volumetric flask, dilute to the mark with copper nitrate solution, and mix well.

Preparation of the test solution: Accurately measure 1ml of the sample, transfer it to a 20ml volumetric flask, dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Reference solution (1) and mixed solution of test solution (1:20): Measure

1ml of the reference solution and 20ml of the test solution into the same volumetric flask, mix well to obtain the solution.Injection and system suitability requirements:

Injection sequence: Copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, mixed solution of reference solution and test solution (1:20), reference solution (1).

System suitability requirements: The resolution between nitrilotriacetic acid peak and copper nitrate peak, and between copper nitrate peak and EDTA peak should meet the requirements. The retention times of nitrilotriacetic acid and EDTA in the test solution and reference solution (2) may slightly differ.

Summary of validation results

Table3.2.P.5-21Summary table of methodology validation results for related substances

Item

Acceptance criteria

Verification Results

Specificity and SystemSuitability

Blank InterferenceTest

Copper nitrate solution and copper nitrate sodium citrate solution showed no interference with the detection of nitrilotriacetic acid and edetic acid peaks..The resolution between the nitrilotriacetic acid peak and the copper nitrate peak, as well as between the copper nitrate peak and the edetic acid peak, should meet the requirements. Therelative standard deviation (RSD) of peak areas for 5 consecutive reference standard injections should be ≤5%.

Copper nitrate solution and copper nitrate sodium citrate solution showed no interference with the detection of nitrilotriacetic acid and EDTA peaks.The separation between nitrilotriacetic acid peak and copper nitrate peak, as well as between copper nitrate peak and EDTA peak,met the requirements. For five consecutive5reference standard nitrilotriacetic acid peak areas, theRSDwas1.6%, which is less than5%. For five consecutive5reference standard EDTA peaks,

Area RSD is 0.4%, less than 5%.

Forced degradation studies

Under acidic, alkaline, high temperature, oxidative, and light exposure conditions, the test sample solutions should exhibit the required resolution between the nitrilotriacetic acid peak and the copper nitrate peak, as well as between the copper nitrate peak and the EDTA peak.

Degradation occurs under acidic, alkaline, high-temperature, oxidative, and light conditions. In the test solutions under various degradation conditions, the resolution between the nitrilotriacetic acid peak and the copper nitrate peak, as well as between the copper nitrate peak and the EDTA peak, meets the requirements.

Precision

Repeatability

6 replicate test solutions showed a very small range in total impurity content.

0.05%.

6 test solution samples showed a maximum deviation

of 0.031% in total impurities, which is less than 0.05%.

Intermediate precision

Different analysts, at different times, using different equipment, with single operator preparing 6 test sample solutions, the range of total impurity content obtained should be ≤0.05%.12 test sample solutions, the range of total impurity content obtained should be ≤0.05%.

For a single operator processing 6 test sample solutions, the range of total impurity content obtained was 0.031%, which is less than 0.05%. For 12 test sample solutions, the range of total impurity content obtained was 0.047%, which is less than

0.05%.

Solution stability

Room temperature stability study of test solution and reference solution at 0, 4, 8, 12, 24h. The peak area of nitrilotriacetic acid reference solution compared with 0h.

The ratio should be between 80%~120%.

The peak area of nitrilotriacetic acid reference solution compared to 0h was within 97.3%~104.0%,and the peak area of edetic acid reference solution compared to 0h

was within 100.1%~101.8%, indicating

The peak area of edetic acid reference solution compared to 0h should be between 90%~110%.

Test solution: The total impurity content should remain within ±0h compared to the initial value.

0.05%.

No new impurities exceeding 0.05% should appear.

The reference solution remains relatively stable at room temperature for 24h;

Test solution: The total impurity content compared to 0h was -0.023%, within ±0.05%, with no new impurities exceeding 0.05%, indicating the test

solution remained relatively stable at room temperature for 24h.

Robustness

Column temperature variation of ±2°C, flow rate variation (±5%),

pH value (±0.2), mobile phase 1 methanol ratio

±10%, three chromatographic columns. Under all conditions, copper nitrate solution and copper nitrate sodium citrate solution showed no interference with the detection of nitrilotriacetic acid and EDTA peaks.The resolution between nitrilotriacetic acid peak and copper nitrate peak, as well as between copper nitrate peak and EDTA peak, should meet the requirements. The total impurity content should be within ±0.05% compared to standard conditions.

Conditions

Content

(%)

Difference from standard conditions(%)

Standard conditions

0.123

N/A

Flow rate

2.1ml/min

0.136

0.013

Flow rate

1.9ml/min

0.120

-0.003

Column temperature23

0.124

0.001

Column temperature27

0.161

0.038

pH7.7

0.142

0.019

pH7.3

0.155

0.032

Methanol

80ml

0.167

0.044

Methanol

100ml

0.159

0.036

Column 2

0.123

0.000

Column 3

0.135

0.012

The total impurity content compared with standard conditions is within ±

0.05% or less.

Specificity Test

System Suitability Test

Test Design and Requirements: Copper nitrate solution and copper nitrate sodium citrate solution should show no interference. In the test solution, the resolution between the nitrilotriacetic acid peak and copper nitrate peak, as well as between the copper nitrate peak and EDTA peak, should meet the requirements. The RSD of 5 consecutive injections of the reference solution should be within 5%.

Experimental Procedure

(1) Blank solvent(diluent): See 3.2.P.5.3.2.2.

(2)Reference standard solution: See 3.2.P.5.3.2.2.

(3Test solution: see 3.2.P.5.3.2.2.

Injection: Injection sequence: copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, mixed solution of reference solution and test solution (1:20), reference solution (1).

System suitability requirements: The resolution between nitrilotriacetic acid peak and copper nitrate peak, and between copper nitrate peak and edetate peakshould meet the requirements. The retention times of NTA and EDTA in the test solution and reference solution (2) may slightlydiffer.

Experimental results

Table3.2.P.5-22Specificity Test Results

Reference standard content:

100.0%

Nitrilotriacetic acid

Reference standard number

RTmin

Peak area

Sample weight

mg

Dilution Factor

1

Response Factor F

F Average

Peak Area

RSD(%)

Reference solution2-1

2.477

7.9006

5.719

5000

1.44774E-04

1.4838E-04

1.6

Reference solution2-2

2.490

7.5665

1.51166E-04

Reference solution2-3

2.497

7.6668

1.49189E-04

Reference solution2-4

2.503

7.6964

1.48615E-04

Reference solution2-5

2.507

7.7209

1.48143E-04

Reference content:

100.0%

Edetate

Reference standard number

RT (min)

Peak area

Sample weight

(mg)

Dilutionfactor

1

Response factor F

Faverage

Peak area

RSD(%)

Reference solution2-1

9.807

121.9278

10.140

5000

1.66328E-05

1.6687E-05

0.4

Reference solution2-2

9.813

120.7211

1.67991E-05

Reference Solution2-3

9.820

121.3136

1.67170E-05

Reference Solution2-4

9.827

121.8733

1.66402E-05

Reference Solution2-5

9.830

121.8494

1.66435E-05

Original Record Traceability:KY-GP-02-A006P18

Table3.2.P.5-23System Suitability Test Results

Reference Standard Content:

100.0%

Nitrilotriacetic Acid

Reference Standard Number

RT(min)

Peak area

Sample weight

(mg)

Dilution factor

1

Response Factor F

FAverage

Control 2-1

2.500

7.9024

5.719

5000

1.44741E-04

1.4421E-04

Control 2-2

2.503

7.9605

1.43684E-04

Reference Standard Content:

100.0%

Edetate

Reference Standard Number

RT(min)

Peak area

Sample weight

(mg)

Dilution factor

1

Response Factor F

FAverage

Control 2-1

9.867

120.8632

10.140

500

1.67793E-04

1.6696E-04

Control 2-2

9.877

122.0691

1.66135E-04

Sample

Sampling amount

(ml)

Peak assignment

RT(min)

Peak area

Content(%)

Total impurities%

Test1

1

Unknown Impurity1

1.560

16.4695

0.024

0.123

Unknown Impurity2

1.937

18.9258

0.028

Copper nitrate

3.777

489.2621

/

Unknown Impurity3

6.130

14.0904

0.024

Unknown Impurity4

7.960

22.0497

0.037

Unknown Impurity5

9.957

2.4544

0.004

Unknown Impurity6

10.497

3.3964

0.006

Sample

Sampling amount

(ml)

Peak assignment

RT (min)

Peak area

Resolution

Reference substance 1-Test

1

amino triethyl

2.160

14.6191

1.84

(1:20)

Copper nitrate

3.447

444.4366

5.96

Edetate

8.727

91.0155

n.a

Source of original records:KY-GP-02-A005P23

Figure 3.2.P.5- 3System Suitability and Specificity Overlay Chromatogram

Experimental Conclusion

Copper nitrate solution and copper nitrate sodium citrate solution showed no interference with the detection of nitrilotriacetic acid and edetate peaks. The resolution between nitrilotriacetic acid peak and copper nitrate peak, as well as between copper nitrate peak and edetate peak in the reference solution 1-test solution (1:20) met the requirements. The peak area RSD of nitrilotriacetic acid in the reference solution for 5 consecutive injections was 1.6%, which is less than 5%. The peak area RSD of edetate in the reference solution for 5 consecutive injections was 0.4%, which is less than 5%, meeting the validation requirements. 3.2.P.5.3.2.5Forced Degradation Test

Experimental Design and Requirements

The resolution between nitrilotriacetic acid peak and copper nitrate peak, as well as between copper nitrate peak and edetic acidpeak in the test solution under various stress conditions should meet the requirements; calculate the total impurities under each stress condition using the external standard method. 3.2.P.5.3.2.5.2Experimental Procedure

(1)Blank solvent and diluent: see 3.2.P.5.3.2.2

(2)Preparation of reference standard solution: see 3.2.P.5.3.2.2

Undamaged test solution: Accurately measure 1ml of the sample, transfer to a 20ml volumetric flask, dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Alkaline degradation

Accurately measure 1ml of the sample, transfer to a 20ml volumetric flask, dissolve with copper nitrate sodium citrate solution, add 1mol/L sodium hydroxide solution 1ml, allow to stand at room temperature for 24h, then add 1mol/L hydrochloric acid solution 1ml, dilute to the mark with copper nitrate sodium citrate solution, and mix well to obtain the alkaline degradation test solution. Prepare a blank solvent simultaneously.

Acid Degradation

Acid degradation test solution: Accurately measure 1ml of the sample, place it in a 20ml volumetric flask, dissolve with copper nitrate sodium citrate solution, add 1mol/L hydrochloric acid solution 1ml, allow to stand at room temperature for 24h, then add 1mol/L sodium hydroxide solution 1ml, dilute to volume with copper nitrate sodium citrate solution, mix well to obtain the acid degradation test solution. Prepare a blank solvent simultaneously.

High-Temperature Degradation

High-temperature degradation test solution: Accurately measure 1ml of the sample, place it in a 20ml volumetric flask, dilute to volume with copper nitrate sodium citrate solution, mix well, place in an 80°C water bath and heat for 2h to obtain the high-temperature test solution. Prepare a blank solvent simultaneously.

Oxidative Degradation

Oxidative Blank Solution: Measure 1ml of 3% hydrogen peroxide, transfer to a 20ml volumetric flask, dilute to volume with copper nitrate sodium citrate solution, and mix well to prepare the oxidative blank solution.

Oxidative Degradation Test Solution: Accurately measure 1ml of the sample, transfer to a 20ml volumetric flask, dissolve with copper nitrate sodium citrate solution, add 1ml of 3% hydrogen peroxide, allow to stand at room temperature for 10min, then dilute to volume with copper nitrate sodium citrate solution, and mix well to prepare the oxidative degradation test solution.

Note: The forced degradation conditions are to be determined. Adjust the reagent amount, concentration, or degradation time based on the actual degradation rate observed.

Photodegradation

Photodegradation test solution: Accurately measure 1ml of the sample, place it in a 20ml volumetric flask, dissolve with copper nitrate sodium citrate solution, expose in a light chamber at 5000lx for 2 days, then remove, dilute to volume with water, mix well, and use as the photodegradation test solution. Prepare a dark control solution simultaneously.

Sample injection and analysis

Injection sequence: Copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, mixed solution of reference solution and test solution (1:20), and reference solution (1).

System suitability requirements: The resolution between nitrilotriacetic acid peak and copper nitrate peak, and between copper nitrate peak and edetate peak shall meet the specified criteria. The retention times of nitrilotriacetic acid and edetate in the test solution and reference solution (2) may slightly differ.

For test solutions under other stress conditions, calculate the total amount of degradation impurities using the external standard method.

3.2.P.5.3.2.5.3Experimental Results

The main degradation impurities in samples subjected to acid/base stress, thermal stress, oxidative stress, and photolytic stress are as follows:

Table3.2.P.5-24Major degradation impurities in destructive testing of self-prepared preparations and raw materials

Sample

Sampling volume

(ml)

Peak Assignment

RT(min)

Peak Area

Resolution

Content (%)

Undamaged sample

1

Unknown impurity 1

1.563

17.2154

3.81

0.027

Unknown Impurity 2

2.067

7.2669

6.15

0.011

Copper nitrate

3.867

469.5910

3.10

/

Unknown Impurity 3

6.537

14.1344

1.59

0.024

Unknown Impurity 4

8.173

22.0262

5.83

0.037

Unknown Impurity 5

10.250

2.6471

n.a

0.004

Total impurities%

0.103

Sample

Sampling amount

(ml)

Peak Identification

RT(min)

Peak Area

Resolution

Content(%)

Acid degradation

1

Unknown impurity 1

1.563

7.9454

1.05

0.012

Unknown Impurity 2

1.723

5.9097

3.31

0.009

Unknown Impurity 3

2.213

14.335

8.10

0.022

Copper nitrate

3.867

469.5910

2.69

/

Unknown Impurity 4

6.117

23.1529

1.88

0.039

Unknown Impurity 5

8.197

22.5288

n.a

0.038

Total impurities%

0.120

Sample

Sample size

(ml)

Peak assignment

RT(min)

Peak area

Resolution

Content(%)

Alkali degradation

1

Unknown impurity 1

1.353

7.0615

1.61

0.011

Unknown Impurity 2

1.733

64.1534

2.10

0.099

Unknown Impurity 3

2.233

14.3550

3.98

0.022

Copper nitrate

3.867

469.5910

3.39

/

Unknown Impurity 4

6.347

7.1844

0.82

0.012

Unknown Impurity 5

7.017

3.0633

1.70

0.005

Unknown Impurity 6

7.843

138.5155

0.89

0.232

Unknown Impurity 7

8.267

3.5209

5.02

0.006

Unknown Impurity8

10.330

3.2139

n.a

0.005

Total impurities%

0.392

Sample

Sample quantity

(ml)

Peak assignment

RT(min)

Peak area

Resolution

Content(%)

light-induced degradation

1

Unknown impurity 1

1.543

20.1754

2.70

0.031

Unknown Impurity 2

1.947

5.5891

1.48

0.009

Unknown Impurity 3

2.167

31.4541

1.80

0.048

Unknown Impurity 4

2.3334.8466

6.64

0.007

Copper nitrate

3.867

469.5910

6.14

/

Unknown Impurity 5

6.687

13.3296

1.76

0.022

Unknown Impurity 6

7.627

37.4034

1.09

0.063

Unknown Impurity 7

8.047

12.0329

3.62

0.020

Unknown Impurity8

10.010

5.0821

n.a

0.008

Total impurities%

0.208

Sample

Sample quantity

(ml)

Peak assignment

RT(min)

Peak area

Resolution

Content (%)

Light Degradation - Dark Control

1

Unknown impurity 1

1.547

15.2325

2.61

0.023

Unknown Impurity 2

1.943

6.1508

1.62

0.009

Unknown Impurity 3

2.177

15.5222

1.82

0.024

Unknown Impurity 4

2.330

4.1631

6.59

0.006

Copper nitrate

3.867

469.5910

6.18

/

Unknown Impurity 5

6.737

13.0582

2.33

0.022

Unknown Impurity 6

8.067

21.7403

3.89

0.036

Unknown Impurity 7

9.990

3.5865

n.a

0.006

Total impurities%

0.126

Sample

Sampling amount

(ml)

Peak Identification

RT(min)

Peak Area

Resolution

Content(%)

High-temperature destruction

1

Unknown impurity 1

1.547

14.8891

2.71

0.023

Unknown Impurity 2

1.950

5.9722

1.57

0.009

Unknown Impurity 3

2.173

16.1704

1.79

0.025

Unknown Impurity 4

2.327

4.226

6.50

0.007

Copper nitrate

3.867

469.5910

1.24

/

Unknown Impurity 5

3.880

2.7810

5.13

0.005

Unknown Impurity 6

6.773

12.9724

1.59

0.022

Unknown Impurity 7

7.640

6.847

1.08

0.011

Unknown Impurity8

8.063

18.6531

3.25

0.031

Unknown Impurity9

10.020

3.1934

n.a

0.005

Total Impurities%

0.138

Sample

Sample size

(ml)

Peak assignment

RT(min)

Peak area

Resolution

Content(%)

Oxidative degradation

1

Unknown impurity 1

1.637

25.2602

0.81

0.032

Unknown Impurity 2

1.763

10.4857

7.54

0.013

Copper nitrate

4.007

352.2179

1.87

/

Unknown Impurity 3

4.917

18.2491

0.79

0.029

Unknown Impurity 4

5.363

9.7178

0.41

0.015

Unknown Impurity 5

5.553

28.9175

1.78

0.046

Unknown Impurity 6

6.193

18.8227

0.43

0.030

Unknown Impurity 7

6.360

8.9128

2.64

0.014

Unknown Impurity8

7.467

4.0368

1.72

0.006

Unknown Impurity9

8.330

6.5592

n.a

0.010

Total Impurities%

0.195

Source of Original Record:KY-GP-02-A006P20,P37

The typical chromatograms under various forced degradation conditions are as follows:

Figure 3.2.P.5-4 Overlaid chromatograms of acid degradation solution samples

Figure 3.2.P.5-5 Overlaid chromatograms of base degradation solution samples

Figure 3.2.P.5-6 Overlaid chromatograms of light degradation solution samples

Figure 3.2.P.5-7Light Degradation-Dark Control Solution Test Overlay Chromatogram

Figure 3.2.P.5-8High Temperature Degradation Solution Test Overlay Chromatogram

Figure 3.2.P.5-9Oxidative Degradation Solution Test Overlay Chromatogram

3.2.P.5.3.2.5.4Result Analysis

Degradation occurs under acidic, alkaline, high-temperature, oxidative, and light exposure conditions. In the test solutions under each degradation condition, the resolution between the nitrilotriacetic acid peak and the copper nitrate peak, as well as between the copper nitrate peak and the EDTA peak, meets the requirements. No significant interference is observed in the blanks under each degradation condition. The impurity content in the sample solution increases significantly under alkaline, light exposure, and oxidative conditions, while no significant change is observed under other conditions, indicating that the sample is unstable under alkaline, light exposure, and oxidative conditions.

Precision

Experimental Design and Requirements

Repeatability: Prepare 6 samples of the test solution at the same concentration, tested by one analyst under as identicalconditions as possible. The range of total impurity content obtained should be ≤0.05%.

Intermediate Precision: Prepare 6samples of the test solution at the same concentration, tested separately by another analyst under

Testing conducted on different equipment and different dates showed that the 6 test solution impurity content results had a total impurity content range ≤

0.05%.

Experimental Procedure

(1) Blank solvent and diluent: See 3.2.P.5.3.2.2.

(2)Reference standard solution preparation: See 3.2.P.5.3.2.2.

Test solution: Preparation of test solution: Accurately measure 1ml of the sample, transfer to a 20ml volumetric flask, dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Mixed solution of reference standard solution (1) and test solution (1:20):Measure 1ml

of reference standard solution and 20ml of test solution into the same volumetric flask, mix well to obtain.

Sample Injection and Measurement

Injection sequence: Copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, mixed solution of reference solution and test solution (1:20), reference solution (1).

System suitability requirements: The resolution between nitrilotriacetic acid peak and copper nitrate peak, and between copper nitrate peak and edetate peak should meet the requirements. The retention times of nitrilotriacetic acid and edetate in the test solution and reference solution (2) may slightly differ.

Intermediate precision: Prepare solutions according to the method under "Repeatability", and perform measurements using different instruments by different analysts.

Experimental Results

Table3.2.P.5-25Results of Precision Determination for Related Substances Methodology

Analyst 1

(Repeatability)

Test 1

Test 2

Test 3

Test 4

Test 5

Test 6

Average

(n=6)

Total impuritiespoor,

%(n=6)

Total impuritiespoor,

%(n=12)

Total impurities (%)

0.123

0.103

0.103

0.101

0.126

0.132

0.115

0.031

0.047

Analyst 2

(Intermediate precision)

Test 1

Test 2

Test 3

Test 4

Test 5

Test 6

Average value

(n=12)

Total impurity deviation,

%(n=6)

Total impurities (%)

0.123

0.118

0.117

0.127

0.141

0.148

0.129

0.031

Original record traceability: Repeatability: KY-GP-02-A006P19; Intermediate precision: KY-GP-02-A005P25

Result Analysis

Repeatability: Six test solutions were prepared, and the range of total impurities content was 0.031%, which is less than 0.05%, meeting the validation requirements.

Intermediate Precision: Six test solutions were prepared by different analysts at different times using different equipment, and the range of total impurities content was 0.031%, which is less than 0.05%; Twelve test solutions were prepared, and the range of total impurities content was 0.047%, which is less than 0.05%, meeting the validation requirements.

Solution Stability

Experimental Design and Requirements

Measure the test solution at 0, 4, 8, 12, 16, 20, and 24 hours: the total impurity content should be within ±0.05% compared to 0h.

Measure the reference solution at 0, 4, 8, 12, 16, 20, and 24 hours. The peak area of nitrilotriacetic acid in the reference solution should be between 80%~120% compared to 0h; the peak area of the EDTA reference solution should be between 90%~110% compared to 0h. No new impurities greater than 0.05% should appear.

Experimental Procedure

(1)Blank solvent and diluent: See 3.2.P.5.3.2.2.

(2)Reference standard solution: See 3.2.P.5.3.2.2.

Test solution: Preparation of test solution: Accurately measure 1ml of the sample, transfer to a 20ml volumetric flask, dilute to volume with copper nitrate sodium citrate solution, and mix well.

Mixed solution of reference standard solution (1) and test solution (1:20): Measure 1ml

and the test solution 20mlinto the same volumetric flask, mix well to obtain.

Injection and measurement

Injection sequence: copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, mixed solution of reference solution and test solution (1:20), reference solution (1).

System suitability requirements: resolution between nitrilotriacetic acid peak and copper nitrate peak, and between copper nitrate peak and edetate peakshall meet the requirements. The retention times of nitrilotriacetic acid and edetate in the test solution and reference solution (2) may slightly differ.

Experimental Results

Table3.2.P.5-26Methodology for Related Substances-Stability of Impurity Solution (Room Temperature 25°C)Validation Results

Stability of Test Solution

Time

Total Number of Impurities

Total impurity content(%)

Difference from 0h(%)

0h

6

0.163

N/A

4h

7

0.161

-0.002

8h

7

0.159

-0.004

12h

7

0.150

-0.013

24h

7

0.140

-0.023

Reference solution 2Nitrilotriacetic acid solution stability

Time

RTmin

Peak area

Relative 0hrecovery%

0h

2.310

7.6538

100.0

4h

2.290

7.8430

102.5

8h

2.287

7.9514

103.9

12h

2.283

7.9633

104.0

24h

2.323

7.4454

97.3

Reference Solution 2EDTA Solution Stability

Time

RTmin

Peak area

Relative 0hrecovery%

0h

5.630

119.1736

100.0

4h

5.637

120.8067

101.4

8h

5.640

121.0268

101.6

12h

5.637

121.3614

101.8

24h

5.617

119.3404

100.1

Original record traceability:KY-GP-02-A005P42,P45

Result Analysis

Test solution: The total impurity content varies within ±0.05% compared to 0h, with no new impurities exceeding 0.05%, meeting the validation requirements. Reference solution: The peak area of nitrilotriacetic acid reference solution remains within 80%~120% compared to 0h. The peak area of edetate reference solution remains within 90%~110% compared to 0h, meeting the validation requirements.

Robustness Test

Test Design and Requirements

Under all conditions, copper nitrate solution and copper nitrate sodium citrate solution show no interference with the detection of nitrilotriacetic acid and edetate peaks. The resolution between nitrilotriacetic acid peak and copper nitrate peak, as well as between copper nitrate peak and edetate peak, should meet the requirements. The total impurity content varies within ±0.05% compared to standard conditions.

Experimental Procedure

(1)Blank solvent and diluent: See 3.2.P.5.3.2.2.

(2)Reference standard solution: See 3.2.P.5.3.2.2.

Test solution: Preparation of test solution: Accurately measure 1ml of the sample, transfer to a 20ml volumetric flask, dilute to the mark with copper nitrate sodium citrate solution, and mix well.

Reference solution (1) and test solution mixture (1:20): Measure 1ml of the reference solution

and 20ml of the test solution into the same volumetric flask, mix well to obtain.

Injection and determination

Injection sequence: Copper nitrate solution, reference solution (2), copper nitrate sodium citrate solution, test solution, mixture of reference solution and test solution (1:20), reference solution (1).

System suitability requirements: The resolution between nitrilotriacetic acid peak and copper nitrate peak, and between copper nitrate peak and edetate peak shall meet the requirements. The retention times of nitrilotriacetic acid and edetate in the test solution and reference solution (2) may slightly differ.

Factors affecting chromatographic conditions

Table3.2.P.5-27Parameters for chromatographic condition variations

Chromatographic conditions

Standard conditions

Variable conditions

Flow rate(±20%)

2.0 ml/min

1.9ml/min, 2.1ml/min

Column temperature variation ±2

25

23℃, 27

Mobile phase pH value±0.2

7.5

7.3,7.7

Mobile phase I methanol ratio

Methanol 90ml

Methanol 80ml, methanol 100ml

Chromatographic column

MOS-1Hypersil, 5μm,

125*4.6mmor equivalent performance

Column 2, Column 3

Experimental results

Table3.2.P.5-28Parameters for chromatographic condition variations

Conditions

Total number of impurities

Total impurity content(%)

Deviation from standard conditions(%)

Standard conditions

6

0.123

N/A

Flow rate 2.1ml/min

7

0.136

0.013

Flow rate 1.9ml/min

6

0.120

-0.003

Column temperature 23

9

0.124

0.001

Column temperature 27

6

0.161

0.038

pH7.7

7

0.142

0.019

pH7.3

9

0.155

0.032

Methanol 80ml

8

0.167

0.044

Methanol 100ml

9

0.159

0.036

Chromatographic column 2

6

0.123

0.000

Chromatographic column 3

6

0.135

0.012

Result Analysis:

Under various conditions, copper nitrate solution and copper nitrate sodium citrate solution showed no interference with the detection of nitrilotriacetic acid and edetate peaks. The resolution between the nitrilotriacetic acid peak and copper nitrate peak, as well as between the copper nitrate peak and edetate peak, met the requirements. The total impurity content under all conditions was within ±0.05% compared to the standard conditions, meeting the validation requirements. 3.2.P.5.3.2.9Sample Testing

Table3.2.P.5-29Sample Testing

Gadopentetate Dimeglumine Injection

Batch number

Specification

Total impurities%

Reference Preparation

KT045S3

20ml:9.38g

0.055

KT05TA1

0.045

KT02JB1

15ml:7.04g

0.067

KT051T2

0.069

KT07B25

0.054

KT05B9J

10ml:4.69g

0.051

Process validation batch

020210801

20ml:9.38g

0.03

020210802

20ml:9.38g

0.03

020210803

20ml:9.38g

0.03

020210804

10ml:4.69g

0.02

020210805

10ml:4.69g

0.03

020210806

10ml:4.69g

0.03

020210807

15ml:7.04g

0.03

020210808

12ml:5.63g

0.03

Original Record Traceability:KY-GP-02-A008P29;

Content

The chromatographic conditions for the gadopentetate dimeglumine injection content method are consistent with those of the raw material, hence the linearity and accuracy data refer tothe raw material content method validation section.

Test Drugs, Reagents, and Equipment

Table3.2.P.5-30Test Drug Information Table

Name

Batch number

Purity%

Manufacturer

Gadopentetate Dimeglumine Injection

20210401-S

10mlSpecification

Guangzhou Kangchen Pharmaceutical Co., Ltd

Gadopentetate Monomeglumine Reference Standard

100541-201801

97.1%

National Institutes for Food and Drug Control

Table3.2.P.5-31Test Reagent Information

Name

Manufacturer

Batch number

Grade

Tetrabutylammonium perchlorate

Shanghai Yuanye Biotechnology Co., Ltd.

A02D11L132999

AR

Acetonitrile

Shanghai Xingke High Purity Reagent Co., Ltd

0114210501

HPLC

Ultrapure water

Self-prepared

N/A

N/A

Table3.2.P.5-32Test Equipment Information

Equipment Name

Model

Manufacturer

Number

Expiration Date

One Hundred Thousandth Electronic Balance

CPA225D

SARTORIUS

VAC06

2021.10.28

One Millionth Electronic Balance

MSA116P

METTLERT

VAC87

2021.11.10

pHmeter

FE20

SARTORIUS

VAC16

2022.06.21

High Performance Liquid Chromatograph

U3000

Thermo Fisher

VAC88

2021.11.21

High Performance Liquid Chromatograph

U3000

Thermo Fisher Scientific

VAC52

2023.04.22

Pre-validation method

[Content]Determined by high-performance liquid chromatography (Chinese Pharmacopoeia 2020Edition General Chapter 0512).

Test solution Accurately measure 2ml of the sample, transfer to a 200ml volumetric flask, dilute with water to volume, and mix well. Accurately measure 15ml, transfer to a 100ml volumetric flask, add 10ml of acetonitrile, dilute with water to volume, mix well, and use as the test solution.

Reference solution Accurately weigh an appropriate amount of gadopentetate dimeglumine reference standard, dissolve it in 10% acetonitrile solution, and quantitatively dilute to prepare a solution containing approximately 0.6mg per 1ml, which serves as the reference solution.

Chromatographic conditions Use octylsilane-bonded silica gel as the stationary phase; use tetrabutylammonium perchlorate solution (dissolve 1.7g of tetrabutylammonium perchlorate in 100ml of acetonitrile, then dilute with water to 1000ml) as the mobile phase, with a flow rate of 1.0ml per minute, a detection wavelength of 195nm, a column temperature of 25°C, and an injection volume of 20μl.

System SuitabilityThe number of theoretical plates, calculated based on the gadopentetate dimeglumine peak, should not be less than 3000.

Assay Method Accurately measure the test solution and the reference solution, and inject them separately into the liquid chromatograph. Record the chromatogram, and calculate the amount of gadopentetate meglumine in the test solution using the external standard method based on peak area. Multiply the obtained result by 1.263,to obtain the final value.

Preparation of validation process solution:

Blank solvent:

10%acetonitrile water (take 900mlof water, add 100mlof acetonitrile, mix well to obtain).

Preparation of blank excipient solution:

Take meglumine 20mg,pentetic acid 8mg,weigh accurately, and transfer to a 20mlvolumetric flask. Dissolve in water and diluteto the mark. Precisely measure 1mland transfer to a 200mlvolumetric flask. Dissolve in water and dilute to the mark. Precisely measure 15ml.Transfer to a 100ml volumetric flask, add acetonitrile 10ml,dilute with water to the mark, and mix well to prepare the blank excipient solution.

Preparation of reference solution

Take gadopentetate dimeglumine reference standard 30mg, weigh accurately, and transfer to a 50ml volumetric flask. Dilute with blank solventto the mark, mix well to obtain.

Test solution

Accurately measure 2ml of the sample, transfer it to a 200ml volumetric flask, dilute to volume with water, and mix well. Then accurately measure 15ml, transfer it to a 100ml volumetric flask, add 10ml of acetonitrile, dilute to volume with water, and mix well to prepare the test solution.

Validation Results Table

The validation includes system suitability and specificity, solution stability, precision repeatability, and robustness. Linearity and range, as well as recovery, refer to the validation of the active pharmaceutical ingredient content.

Table3.2.P.5-33Summary of Content Methodology Validation Results

Item

Acceptance criteria

Verification results

Specificity

The blank solvent and blank excipients do not interfere with gadopentetate dimeglumine

Detection of amines.

The blank solvent and blank excipients do not interfere with the detection of gadopentetate mono.

glucamine.

System suitability

For 6 consecutive injections of the reference solution, RSD2.0%. The theoretical plate number for gadopentetate glucamine should not be less than 3000.

Continuous 6 reference solution peak area RSD was 0.15%, less than 2.0%, meeting the requirements. The theoretical plate count calculated for the gadopentetate dimeglumine peak was not less than

3000.

Solution stability

At 0, 4, 6, 8, 12, 16, 24h, the peak areas of the reference and test samples were measured, and the peak area change rate

Within±2.0%.

At 0, 2, 4, 6, 8, 12, 16, 24h, the peak areas of the reference and test samples were measured, and the variation rate of peak areas

was within±2.0%, meeting the requirements.

Precision

6 test sample solutions, the obtained content RSD%

1.0%. Twelve test solutions were analyzed by different analysts on different dates and different instruments, with an RSD% 2.0%.

6 replicate samples showed an RSD of 0.59%, which is less than

1.0%, meeting the requirement. Six intermediate precision samples showed an RSD of 0.59%, which is less than 2.0%, meeting the requirement. Twelve precision samples showed an RSD of 0.68%, which is less than

2.0%, meeting the requirement.

Robustness

Investigate the effects of column temperature variations of±5℃, detection wavelength variations of

±5nm, flow rate variations of±20%, and the use of threedifferent brands or batches of chromatographic columns on the chromatographic behavior of the instrument. The theoretical plate number of the main peakshould be greater than 3000, and the difference in content between robustness conditions and normal conditions should be within

±2%.

Investigate the effects of column temperature variation by±5°C and detection wavelength variation by

±5nm, flow rate variation by±20%, and measurements usingthree different brands/lots of chromatographic columns. The theoretical plate number of the main peak remained above 3000, meetingrequirements. The difference in content between robustness conditions and normal conditions

All are within±2%, meeting the requirements.

System Suitability and Specificity

Experimental Design and Requirements

Prepare solutions for analysis; blank solvent and blank excipients should not interfere with sample determination. The RSD of 6 consecutive reference standard solutions should be ≤2.0%.

Testing process

(1)Blank solvent: Same as 3.2.P.5.3.3.2.

(2)Blank excipients: Same as 3.2.P.5.3.3.2.

(3)Preparation of reference standard solution: Same as 3.2.P.5.3.3.2.

(4)Preparation of test solution: Same as 3.2.P.5.3.3.2.

Sample injection and analysis

Precisely inject 20μl each of blank solvent, test solution, and reference standard solution (6 injections) into the chromatograph, and record the chromatograms.

Test results

Table3.2.P.5-34System Suitability and Specificity Test Results

Specificity Test Results

Test Samples

Retention Time,min

Peak area

Theoretical Plate Number

Blank Solvent

Not detected

N/A

N/A

Blank Excipient

Not detected

N/A

N/A

Reference Solution

14.840

2723.3602

4721

Test solution

15.013

2519.1877

4904

System precision test

Name

Retention time min

Peak Area

Peak area RSD%

System Precision Test 1-1

14.840

2723.3602

0.15

System Precision Test 1-2

14.840

2725.8833

System Precision Test 1-3

14.833

2730.1291

System Precision Test 1-4

14.827

2730.5485

System Precision Test 1-5

14.830

2725.8551

System Precision Test 1-6

14.830

2734.9930

Original Record Traceability:KY-GP-02-A006P27

Figure 3.2.P.5- 10Content Specificity Chromatogram

Result Analysis

The blank solvent and blank excipients do not interfere with the detection of gadopentetate dimeglumine; the 6 consecutive injections of the reference solution showed a peak area RSD of 0.15%, which is less than 2.0%, meeting the requirements. The theoretical plate number calculated for the gadopentetate dimeglumine peak was not less than 3000, complying with the validation requirements.

Solution Stability

Experimental Design and Requirements

At 0, 2, 4, 6, 8, 12, 16, and 24h, the peak areas of the reference and test samples were measured, with the peak area variation rate within ±2.0%.

Test Procedure

(1)Blank solvent: Same as 3.2.P.5.3.3.2.

(2)Reference solution preparation: Same as 3.2.P.5.3.3.2.

(3)Preparation of test solution: Same as 3.2.P.5.3.3.2.

(4)Injection and measurement

At 0, 2, 4, 6, 8, 12, 16, and 24h, accurately measure 20µl of the solution and inject it into the chromatograph, recording the chromatogram.

Experimental results

Table3.2.P.5-35Stability Test Results of Reference Solution

Time(h)

Peak Area

Peak Area Change Rate(%)

0

2743.7539

N/A

2

2744.9501

0.0

4

2740.8660

-0.1

6

2750.8804

0.3

8

2742.9456

0.0

12

2753.7728

0.4

16

2749.5956

0.2

24

2750.7694

0.3

Original Record Traceability:KY-GP-02-A006P28

Table3.2.P.5-36Test Solution Stability Results

Time(h)

Peak Area

Peak Area Change Rate(%)

2251.5326

N/A

2

2258.7290

0.3

4

2257.3496

0.3

6

2255.6642

0.2

8

2259.8955

0.4

12

2261.9642

0.5

16

2264.5469

0.6

24

2274.6328

1.0

Original Record Traceability:KY-GP-02-A006P28

Result Analysis

At room temperature, the peak areas of the reference standard and test samples were measured at 0, 2, 4, 6, 8, 12, 16, 24h, respectively. The variation rate of peak areas was within ±2.0%, meeting the requirements.

Precision

The experimental design and requirements ensure that the blank solvent does not interfere with the detection.

Repeatability: 6 test sample solutions were prepared and tested by one analyst under as identical conditions as possible. The RSD of impurity content in the 6 test sample solutions was ≤1.0%.

Intermediate precision: Another analyst prepares 6 test solutions with the same impurity concentration, using different instruments and reagents for testing. The 12 sample content data obtained should have an RSD2.0%. 3.2.P.5.3.3.6.2Test Procedure

(1)Blank solvent: Same as 3.2.P.5.3.3.2.

(2)Preparation of reference standard solution: Same as 3.2.P.5.3.3.2.

(3)Preparation of test solution: Same as 3.2.P.5.3.3.2.

(4)Injection and determination: Precisely measure 20μl each of blank solvent, reference standard solution, and test solution, inject them sequentially, and record the chromatograms.

Test results

Table3.2.P.5-37Precision Test Results

Condition

Repeatability

RSD%

Intermediate Precision

RSD%

Tester 1:

(n=6)

Technician 2:

(n=6)

Serial Number

Content%

0.59

Content%

0.59

1

97.08

97.33

2

98.39

96.18

3

97.22

97.02

4

97.97

97.91

5

98.38

96.81

6

97.51

97.01

6 average content%

97.76

97.04

12 average content%

97.4

RSD%n=12

0.68

Original record traceability:KY-GP-02-A006P26P29

Result Analysis

The blank solvent shows no interference. The RSD% of content obtained from 6 replicate sample solutions is RSD% 0.59%, which is less than 1.0%; for 12 sample solutions analyzed by different analysts on different days and instruments, the RSD% is 0.68%, less than 2.0%, meeting the requirements.

Robustness

Experimental Design and Requirements

Investigate the changes in chromatographic behavior of the instrument when the column temperature varies by±5℃, the detection wavelength varies by±5nm, the flow rate varies by±20% relative to the set value, and when three chromatographic columns from different batches are used for measurement. The acceptance criteria are: the theoretical plate number calculated based on the gadopentetate dimeglumine peak should not be less than 3000. The difference in content between robustness conditions and normal conditions should be within±2%.

Test Procedure

(1) Blank solvent: same as 3.2.P.5.3.3.2.

(2)Reference solution: Same as 3.2.P.5.3.3.2.

(3)Test solution: Same as 3.2.P.5.3.3.2.

(4)Injection and determination: Accurately measure 20μl each of the blank solvent, reference solution, and test solution, and inject them sequentially, recording the chromatograms.

Table3.2.P.5-38Robustness Condition Ranges

Robustness Study Items

Test Method Conditions

Confirmation of Applicability Range

Detection Wavelength

195nm

190nm~200nm

Flow rate

1.0ml/min

0.8ml/min~1.2ml/min

Column temperature

25°C

20°C~30°C

Chromatographic Column

C8

Chromatographic Column 1:HypersilGOLDC8, 4.6×150mm,3μm, SN:10145979

Chromatographic Column 2:X‐peonyxC8, 4.6×150mm,5μm, SN:0008020114

Column 3:BDSHypersilC8, 4.6*150mm,5μm,S/N:

20193110

Test results

Table3.2.P.5-39Robustness Test Results

Testing Items

Conditions

Theoretical plate number

Content(%)

Compared to normal

Difference value,%

Test

1-1

Test 1-2

Test 2-1

Test 2-2

Detection wavelength

190nm

4940

4968

4903

4930

97.84

0.1

195nm

4904

4936

4897

4881

97.74

N/A

200nm

4941

4949

4922

4923

97.80

0.1

Flow rate

0.8ml/min

4932

4933

4964

4978

95.96

-1.8

1.0ml/min

4904

4936

4897

4881

97.74

N/A

1.2ml/min

4741

4775

4790

4749

96.36

-1.3

Column Temperature

20°C

3615

3611

3634

3632

96.60

-1.1

25°C

4904

4936

4897

4881

97.74

N/A

30°C

3427

3425

3451

3416

96.48

-1.2

Chromatographic column

Chromatographic column 1

4904

4936

4897

4881

97.74

N/A

Chromatographic column 2

3504

3495

3517

3514

96.75

-1.0

Chromatographic column 3

3422

3427

3418

3421

96.58

-1.2

Original Record Traceability:KY-GP-02-A006

P30P35

Result Analysis

Detection wavelength variation±5nm, the theoretical plate number of the main peak is greater than 3000;190nmmeasured content differs from the normalcondition content by 0.1%;200nmmeasured content differs from the normal condition content by 0.1%. Meetsrequirements.

Flow rate variation test±0.2ml/min, the theoretical plate number of the main peak is greater than 3000;0.8ml/min the difference between the measured content and the normal condition content is-1.8%;1.2ml/min the difference between the measured content and the normal condition content is-1.3%. Meets the requirements.

Column temperature variation test±5℃, the theoretical plate number of the main peak is greater than 3000;20℃ the difference between the measured content and the normal condition content is-1.1%;30℃ the difference between the measured content and the normal condition content is-1.2%. Meets the requirements.

Using different batches of columns, the theoretical plate number of the main peak is greater than 3000; column 2 the difference between the measured content and the normal condition content is-1.0%; column 3 the difference between the measured content and the normal condition content is

-1.2%. Meets the requirements.

Sample Testing

Table3.2.P.5-40Assay Results of Trial Production Batches and Reference Preparations

Name

Specification

Batch

Content,%

Gadopentetate Dimeglumine Injection (manufacturing validation batch)

20ml:9.38g

020210801

99.7

020210802

101.0

020210803

101.4

10ml:4.69g

020210804

99.8

020210805

99.1

020210806

99.8

12ml:5.63g

020210807

101.1

15ml:7.04g

020210808

99.0

Gadopentetate Dimeglumine Injection Reference

Preparation

20ml:9.38g

KT045S32

103.30

KT05TA1

102.97

Gadopentetate Dimeglumine Injection Reference Preparation

15ml:7.04g

KT02JB1

102.58

KT051T2

102.71

KT07B25

102.39

Gadopentetate Dimeglumine Injection Reference

Preparation

10ml:4.69g

KT05B9J

102.64

Bacterial Endotoxin Test Method Validation

Methodology validation was conducted using 02021080120ml)、02021080220ml)、02021080320ml) batch samples; bacterial endotoxin testing was performed on 4 specifications 8 batch samples from the process validation.

The results of the bacterial endotoxin test methodology validation are shown in Table 3.2.P.5-41.

Table3.2.P.5-41Methodology Validation Results for Bacterial Endotoxin Testing

Item

Verification Results

Limit

Determined as 0.5 EU/ml

Interference Preliminary Test

When diluted to 8 times the test solution concentration, all A series solutions showed negative results, while B series solutions diluted 2

times, 4 times showed negative results, but showed positive when diluted 8 times. No interference was observed, and the endotoxin interference test was planned with a dilution factor of 8 times.

Interference Test

This product uses a limulus reagent with a sensitivity of 0.06EU/ml, and the test solution is an 8-fold dilution, which shows no interference with bacterial

endotoxin determination and meets the test requirements.

Instrument: Tube Thermostat (Model: TAL-96G, Serial No. CZL-181)

1000µl pipette, 200µl pipette, pyrogen-free tips

Limulus Amebocyte Lysate (LAL): (1) labeled sensitivity 0.06EU/vial; specification 0.1ml/vial; batch number 2107084; Zhanjiang Bokang Marine Biological Co., Ltd.

(2) labeled sensitivity 0.06EU/vial; specification 0.1ml/vial; batch number 21061034; Xiamen Horseshoe Crab Reagent Biotechnology Co., Ltd.

Bacterial Endotoxin Working Standard: Potency 10EU/vial; Batch No. 2004240; Zhanjiang Andes BiologicalCo., Ltd.

Bacterial Endotoxin Test Water: Specification 5ml/vial; Batch No. 2010220, Zhanjiang Andes Biotechnology Co., Ltd.

Endotoxin Limit Determination

This product ChP2020 edition specifies an endotoxin limit of less than 3.0EU per 1ml; the imported registration (J20020190) standard sets an endotoxin limit of less than 0.5EU per 1ml.

0.5EU; to ensure the safety of this product and maintain consistency with the quality of the originator, the imported registration (J20020190) standard is referenced, setting the limit at less than 0.5EU per 1ml.

Determination of Minimum Valid Dilution Concentration and Maximum Valid Dilution Factor

Since the original process for gadopentetate dimeglumine injection used a limulus amebocyte lysate (LAL) reagent with a sensitivity (λ) of 0.06EU/ml, this study also selected an LAL reagent with a sensitivity (λ) of 0.06 EU/ml for the interference test. Therefore, the maximum valid dilution (MVD) is calculated as follows:

MVD=1.0ml/ml×0.5EU/ml÷0.06EU/ml=8.3-fold

Interference Preliminary Test

Based on the calculated maximum valid dilution factor of 8-fold, three dilution gradients of 2-fold, 4-fold, and 8-fold were selected.

These dilution factors were used for the preliminary interference test: Take 1 batch of Gadopentetate Dimeglumine Injection, and prepare 2-fold, 4-fold, and 8-fold diluted test solutions (Solution A) using bacterial endotoxin test water according to the 2-fold dilution gradient method. Simultaneously, prepare corresponding test-positive solutions containing concentration of bacterial endotoxin standard solution (Solution B). Using Limulus Amebocyte Lysate (LAL) with a sensitivity (λ) of 0.06EU/ml, react with the above Solution A and Solution B. Perform 2 parallel tests for each concentration, along with positive (Solution C) and negative (Solution D) controls. The test results are shown in Table 3.2.P.5-42.

Table3.2.P.5-42Interference Pretest Results

Sample Batch Number

Test Solution

Dilution factor

Control

2 times

4 times

8 times

Solution C

Solution D

020210801

Solution A

- -

- -

- -

+ +

- -

Solution B

- -

- -

+ +

N/A

"+"indicates a positive result;"-"indicates a negative result

From the above results, it can be seen that when the test solution is diluted 8-fold, the detection results meet the requirements, and this concentration is used for the formal interference test.

Interference Test

To ultimately confirm the suitability of this method for the product, two manufacturers' Limulus Amebocyte Lysate (LAL) reagents were selected to conduct formal interference tests on 3 batches of the product. The geometric mean of the reaction endpoint concentrations of the reference series solutions (Es) at the labeled sensitivity of the LAL reagent, and the geometric mean of the reaction endpoint concentrations of the endotoxin solutions prepared from the test sample (Et).

The results were calculated using the formulas Es=lg-1(∑Xs/2), Et=lg-1(∑Xt/4), where Xs and Xt are the logarithmic values (lg) of the bacterial endotoxin concentrations at each reaction endpoint for solution series C and solution series B, respectively. Es and Et should be within 0.5-2λ (including 0.5λ and ).

Select 3 batches of Gadopentetate Dimeglumine Injection (consistency evaluation), and prepare solutions A, B, C, and D according to the table below. The test solutions used should be free of detectable endotoxins and not exceed the maximum valid dilution (MVD). Perform the operations as described in the Limulus Amebocyte Lysate (LAL) sensitivity verification test.

The results are shown in Table 3.2.P.5-43, 3.2.P.5-44.

Table3.2.P.5-43Interference Test Results(λ=0.06EU/ml)

Horseshoe crab reagent manufacturer

Zhanjiang Bokang Marine Biology Co., Ltd.

Sample batch number

Test solution

Bacterial endotoxin concentration (EU/ml)

Solution A

(2 vials)

Solution D

(2 pieces)

Et or

Es

2λ

1λ

0.5λ

0.25λ

02021080

1

Solution B (4

Branch)

++++

----

----

----

--

--

0.12

Solution C (2

Branch)

+ +

- -

- -

- -

0.12

02021080

2

Solution B(4

vials)

++++

--

--

--

--

--

0.12

Solution C (2

branch)

+ +

+ -

- -

- -

0.08

02021080

3

Solution B (4

vials)

++++

----

----

----

--

--

0.12

Solution C (2

vials)

+ +

+ -

- -

- -

0.08

"+"" indicates a positive result; "-" indicates a negative result.

Table3.2.P.5-44Interference Test Results(λ=0.06EU/ml)

Limulus Amebocyte Lysate Manufacturer

Xiamen Horseshoe Crab Reagent Biological Technology Co., Ltd.

Sample Batch Number

Test Solution

Bacterial Endotoxin Concentration (EU/ml)

Solution A

Solution D

Et or

2λ

1λ

0.5λ

0.25λ

(2

pieces)

(2 pieces)

Es

02021080

1

Solution B (4

vials)

++++

--

--

--

--

--

0.12

Solution C (2

vials)

+ +

0.12

02021080

2

Solution B4

Branch)

++++

----

----

----

--

--

0.12

Solution C (2

vials)

+ +

- -

- -

- -

0.12

02021080

3

Solution B (4

vials)

++++

----

----

----

--

--

0.12

Solution C (2

vials)

+ +

- -

- -

- -

0.12

"+" indicates a positive result; "-" indicates a negative result.

Based on the results from the above table and the calculation formulas Es=lg-1(∑Xs/2), Et=lg-1(∑Xt/2), the calculated Es and Et both fall within the range of 0.5λ to 2.0λ (including 0.5λ and 2.0λ). When using a limulus amebocyte lysate (LAL) reagent with a sensitivity of 0.06EU/ml and the test solution is diluted 8-fold, there is no interference with the bacterial endotoxin test,

meeting the experimental requirements.

Test Results

Take the product, examine it according to the regulations, and observe the results, as shown in Table 3.2.P.5-45.

Table3.2.P.5-45Bacterial Endotoxin Test Results

Name

Specification

Batch

Results

Gadopentetate Dimeglumine Injection

(Production validation batch)

20ml:9.38g

020210801

Complies with specifications

020210802

Complies with specifications

020210803

Complies with specifications

10ml:4.69g

020210804

Complies with specifications

020210805

Complies with specifications

020210806

Complies with specifications

12ml:5.63g

020210807

Complies with specifications

15ml:7.04g

020210808

Complies with specifications

Conclusion: The bacterial endotoxin test results of 4 specifications 8 batch samples of this product all comply with the regulations.

Sterility test methodology validation

The results of the sterility test methodology validation are shown in Table 3.2.P.5-46.

Table3.2.P.5-46Results of Sterility Test Method Validation

Thioglycollate Fluid

Membrane Filtration Method, sample size is 30 vials,pH7.0 sodium chloride-peptone buffer rinse 3 times,

Each filter cartridge rinse volume is 300ml (100ml*3 times),compared with control tubes, containing test samples in each container

Culture medium

All test microorganisms in the vessel showed good growth, indicating that the test quantity of the sample under these test conditions has no antimicrobial effect

or its antimicrobial effect is negligible

Tryptic Soy BrothCulture Medium

Membrane filtration method, with a sample size of 30 units,pH7.0 sodium chloride-peptone buffer solution 3 times, and a rinsevolume of 300ml(100ml*3times). In the final rinse solution, add no more than 100cfuofCandida albicans, Aspergillus niger, and Bacillus subtilis, respectively, and transfer into 100ml soybean-casein digest broth. Compared with the control tube, the test organisms in all containers containing the test sample showed good growth, indicating that the

test sample quantity under these test conditions has no antimicrobial effect or its antimicrobial effect is negligible.

Determine the testing method

Take 30 sample units and filter them evenly into three sterile collection vessels using a fully enclosed membrane filtration method, with 10 sample units filtered per filter cartridge. After filtration, rinse with pH7.0 sterile sodium chloride-peptone buffer solution 3 times, each time with 300ml (i.e., 100ml*3 rinses per cartridge). Into 2 of the cartridges, add 100ml of thioglycollate fluid medium (to 1 cartridge, add 1ml of Staphylococcus aureus suspension with a bacterial count not exceeding 100cfu as a positive control), and into 1 cartridge, add 100ml of tryptic soy broth. Incubate the thioglycollate fluid medium at 30-35°C for 14 days, and the tryptic soy broth at

Incubate at 20-25°C for 14 days; positive controls should be incubated at 30-35°C for no more than 5 days.

Experimental Materials

The experimental samples, culture media, strains, and instruments used are listed in the table below.

Table3.2.P.5-47Experimental Samples

Specification

Batch number

20ml:9.38g

020210801

020210802

020210803

Table3.2.P.5-48Culture Media and Rinse Solutions

Test materials

Batch number

Source

Valid Until

Thioglycollate Fluid Medium

1086552

Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.

2023 11

06

Soybean-Casein Digest Broth

1090201

Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.

2023 01

04

Tryptic Soy Agar Medium

1090411

Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.

2023 01

16

Sabouraud Dextrose Agar Medium

1090021

Guangdong Huankai Microbial Sci-Tech Co., Ltd.

2023 01

Month 01Day

Sabouraud Dextrose Broth

1091021

Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.

2023 03

17

pH7.0 sterile sodium chloride- peptone buffer

1087661

Guangdong Huankai Microbial Sci. & Tech. Co., Ltd.

2022 12

30

Table3.2.P.5-49Strain

Strain name

Strain Code

Strain Batch Number

Generation

E. coli

Escherichia coli

[CMCC(B)44 102]

44102-21814-3

Generation 3

Staphylococcus aureus

Staphylococcus

[CMCC(B)26 003]

26003-210814-4

Generation 4

Clostridium sporogenes

[ CMCC ( B ) 64 941]

64941-210818-2

Generation 2

Bacillus subtilis

Bacillus

[CMCC(B)63 501]

63501-210814-4

Generation 4

Candida albicans

Candida

[CMCC(F)98 001]

98001-210814-4

Generation 4

Aspergillus niger

[CMCC(F) 98 003]

98003-210820-2

Generation 2

Table3.2.P.5-50Instruments

Serial Number

Instrument Name

Model

Equipment ID

Calibration Expiry Date

1

Electronic balance

JJ500

CZL-091

2021.10.25

2

Constant temperature and humidity incubator

LRH-250-S

CZL-106

2021.10.25

3

Constant temperature and humidity incubator

LRH-250-S

CZL-049

2021.10.25

4

Clean bench

SW-CJ-IFD

CZL-059

N/A

5

Biosafety cabinet

BHC-1300A

CZL-072

N/A

6

Autoclave

G254TW

CZL-147

2021.12.06

7

Dry heat sterilizer

B0S-150

CZL-142

2021.10.25

8

Bacterial collector

ZW-808A

CZL-025

N/A

Experimental operations

Preparation of bacterial suspension

Inoculate fresh cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis into Tryptic Soy Broth or Tryptic Soy Agar medium. Inoculate fresh cultures of Clostridium sporogenes into Fluid Thioglycollate Medium,3035°C incubation for 1824 hours. Inoculate fresh cultures of Candida albicans into Sabouraud Dextrose Broth or Sabouraud Dextrose Agar medium,2025°C incubation for 23 days. The above cultures are prepared with pH7.0 sterile saline-peptone buffer solution to contain no more than 1ml of bacteria per

100cfu (colony-forming units) bacterial suspension. Inoculate fresh cultures of Aspergillus niger onto Sabouraud Dextrose Agar slant medium, incubate at 2025°C for 57 days, then add 35ml of sterile sodium chloride-peptone buffer solution (pH7.0) containing 0.05% (ml/ml) polysorbate 80 to elute the spores. Subsequently, use an appropriate method to aspirate the spore suspension into a sterile test tube, and prepare a spore suspension with 100cfu spores per 1ml using sterile sodium chloride-peptone buffer solution (pH7.0) containing 0.05% (ml/ml) polysorbate 80.

Medium preparation: Commercially available dehydrated fluid thioglycollate medium and tryptic soy agar medium are prepared according to the instructions, then filled into clean infusion bottles. After stoppering, sterilize using a validated sterilization procedure, followed by sterility and sensitivity checks of the medium, all of which meet the requirements.

Diluent: pH7.0 sterile sodium chloride-peptone buffer,preparation method(100ml): Take potassium dihydrogen phosphate 3.56g, anhydrous disodium hydrogen phosphate 5.77g, sodium chloride 4.30g, peptone 1.00g, add water 1000ml, dissolve with slight warming, filter, package, and sterilize.

Method Validation

Blank group: Take pH7.0 sterile sodium chloride-peptone buffer solution 300ml, use the fully enclosed membrane filtration methodto evenly filter into a triple collector(i.e., 100ml per tube),rinse repeatedly 3 times, then pour 100ml of thioglycollate fluid medium into 2 tubes, and 100ml of tryptic soy broth into 1 tube. The thioglycollate fluid medium is incubated at 3035℃ for 14 days, and the tryptic soy broth is incubated at 2025℃ for 14 days.

Sample group: Take 30 samples and filter them evenly into three sterile containers using the fully enclosed membrane filtration method, with each filter cartridge filtering 10 test samples. After filtration, rinse with pH7.0 sterile sodium chloride-peptone buffer solution 3 times, each time with 300ml (i.e., each cartridge is rinsed with 100ml*3 times). Into two of the containers, pour 100ml of thioglycollate fluid medium, and into one container, pour 100ml of tryptic soy broth. The thioglycollate fluid medium is incubated at 3035°C for 14 days, and the tryptic soy broth is incubated at 2025°C for 14 days.

Sample control group: Take 60 samples, evenly filter them into 2 sets of triple-bottle sterility test assemblies using the fully enclosed membrane filtration method. Each filter cartridge filters 10 test samples. After filtration, rinse with pH7.0 sodium chloride-peptone buffer solution 3 times, each time with 300ml (i.e., each cartridge is rinsed 100ml*3 times). In the final rinse solution, add no more than 100cfu of Staphylococcus aureus, Escherichia coli, Clostridium sporogenes, Candida albicans, Aspergillus niger, and Bacillus subtilis. For the 3 filter cartridges inoculated with Staphylococcus aureus, Escherichia coli, and Clostridium sporogenes, fill each with 100ml of fluid thioglycollate medium. For the 3 filter cartridges inoculated with Candida albicans, Aspergillus niger, and Bacillus subtilis, fill each with 100ml of soybean-casein digest broth. Incubate the fluid thioglycollate medium at 3035℃ for no more than 5 days, and the soybean-casein digest broth at 2025℃ for no more than 5 days.

Bacterial suspension control group: Take pH 7.0 sodium chloride-peptone buffer solution 300 ml, and evenly filter it into a triple collector using a fully enclosed membrane filtration method. During the final filtration of the buffer solution, add no more than 100 cfu of Staphylococcus aureus, Escherichia coli, Clostridium sporogenes, Candida albicans, Aspergillus niger, and Bacillus subtilis into each filter cartridge. For the three filter cartridges inoculated with Staphylococcus aureus, Escherichia coli, and Clostridium sporogenes,3 filter cartridges, respectively, fill each with 100 ml of thioglycollate fluid medium. For the three filter cartridges inoculated with Candida albicans, Aspergillus niger, and Bacillus subtilis,3 filter cartridges, respectively, fill each with 100 ml of tryptic soy broth. The thioglycollate fluid medium is incubated at 3035°C for no more than 5 days, and the tryptic soy broth is incubated at 2025°C for no more than 5 days.

Result interpretation: Compared with the bacterial suspension control group, if the test organisms in the test group all grow well, it indicates that the test sample at the specified quantity under the given conditions has no antimicrobial effect or its antimicrobial effect is negligible. Proceed with the sterility test of the sample using this method and conditions. If the test organisms in any container containing the sample show growth,

If the growth is weak, slow, or absent, it indicates that the test sample at this quantity has antimicrobial activity under the given conditions. Measures such as increasing the rinse volume, increasing the amount of culture medium, using neutralizers or inactivators, or changing the type of filter membrane should be taken to eliminate the antimicrobial effect of the test sample, and the method suitability test should be repeated.

Inspection results

Table3.2.P.5-51Results of Thioglycollate Fluid Medium

Culture Medium

Thioglycollate Fluid Medium

Specification

Batch number

Volume of Rinse Solution

Staphylococcus aureus

Escherichia coli

Clostridium sporogenes

20ml:9.38g

Sample control group

300ml

Bacterial suspension control group

300ml

Blank group

300ml

020210801

300ml

Sample control group

300ml

Bacterial solution control group

300ml

Blank control group

300ml

020210802

300ml

Sample control group

300ml

Bacterial suspension control group

300ml

Blank control group

300ml

020210803

300ml

Note:"+" indicates positive,"-" indicates negative, the same below.

Table3.2.P.5-52Verification and inspection results of Tryptic Soy Agar medium

Culture Medium

Soybean-Casein Digest Agar Medium

Specification

Batch number

Rinse Solution Volume

Bacillus subtilis var. natto

Candida albicans

Aspergillus niger

20ml:9.38g

Sample control group

300ml

Bacterial solution control group

300ml

Blank control group

300ml

020210801

300ml

Sample control group

300ml

Bacterial suspension control group

300ml

Blank group

300ml

020210802

300ml

Sample control group

300ml

Bacterial suspension control group

300ml

Blank control group

300ml

020210803

300ml

Conclusion

Through three parallel tests, no microbial growth was observed in either the negative control or the test sample. Compared with the control tube, the test bacteria in all containers containing the test sample grew well, indicating that the test quantity of the sample under these conditions has no antimicrobial effect or the effect is negligible. The test method is determined as follows:

Take 30 vials of the product and filter them evenly into a triple-collector using the fully enclosed membrane filtration method according to the membrane filtration method.

In the process, each filter cartridge filters 10 test samples, followed by rinsing with pH7.0 sterile sodium chloride-peptone buffer solution 3 times, each time with 300ml (i.e., each cartridge is rinsed 100ml*3 times). Among them, 2 cartridges are filled with 100ml fluid thioglycollate medium (for 1 cartridge, add 1ml of Staphylococcus aureus suspension with no more than 100cfu as a positive control), and 1 cartridge is filled with 100ml tryptic soy broth. The fluid thioglycollate medium is incubated at 3035°C for 14 days, and the tryptic soy broth is incubated at 2025°C for 14 days. The positive control is

30~35°C incubation for no more than 5 days.

Batch Test Report

Table3.2.P.5-53Batch Test Report(20ml:9.38g)

Test Items

020210801

020210802

020210803

Appearance

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Identification

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

pH value

7.0-7.9

7.5

7.5

7.5

Color

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Meglumine

39.6%-43.9%

41.4%

41.5%

41.7%

Pentetic Acid

230-450μg/ml

393μg/ml

365μg/ml

347μg/ml

Insoluble particles

10μm and 10μm or

Upper ≤6000

25tablets/bottle

25tablets/bottle

3 pellets/bottle

25μm and 25μm or

above ≤600

0 pellets/bottle

6 pills/bottle

1 pill/bottle

Visible particles

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Elementalimpurities

Hg

not more than 0.125ppm

0.001 ppm

0.001 ppm

0.001 ppm

Cd

Not more than 0.083ppm

0.0002 ppm

Not detected

0.0001 ppm

Pb

Not more than 0.208ppm

0.006 ppm

0.005 ppm

0.011 ppm

As

Not more than 0.625ppm

0.001 ppm

0.004 ppm

0.009 ppm

Co

Not more than 0.208ppm

0.003 ppm

0.003 ppm

0.004 ppm

V

Not more than 0.417ppm

Not detected

Not detected

Not detected

Ni

Not more than 0.833ppm

0.383 ppm

0.399 ppm

0.569 ppm

Li

Not more than 10.417ppm

0.016 ppm

0.008 ppm

0.009 ppm

Sb

Not more than 3.750ppm

0.0002 ppm

0.001 ppm

0.001 ppm

Cu

Not more than 12.500ppm

0.009 ppm

0.008 ppm

0.014 ppm

Total related substances%

0.3%

0.03%

0.03%

0.03%

Bacterial endotoxins

0.5EU/ml

Complies with specifications

Complies with specifications

Complies with specifications

Sterility

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Content

96.0%-104.0%

99.7%

101.0%

101.4%

Table3.2.P.5-54Batch Test Report(10ml:4.69g)

Test Items

020210804

020210805

020210806

Appearance

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Identification

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

pH value

7.0-7.9

7.5

7.5

7.5

Color

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Meglumine

39.6%-43.9%

41.0%

40.8%

41.1%

Pentetic Acid

230-450μg/ml

361μg/ml

361μg/ml

385μg/ml

Insoluble particles

10μm and 10μm or above ≤6000

1 piece/bottle

0 piece/bottle

1 piece/bottle

25μm and 25μm or

above ≤600

1 piece/bottle

0 tablets/bottle

0 tablets/bottle

Visible particles

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Elementalimpurities

Hg

Not more than 0.125ppm

0.001 ppm

0.001 ppm

0.001 ppm

Cd

Not more than 0.083ppm

0.0001 ppm

0.0001 ppm

Not detected

Pb

Not more than 0.208ppm

0.008 ppm

0.005 ppm

0.009 ppm

As

Not more than 0.625ppm

0.010 ppm

0.011 ppm

0.012 ppm

Co

Not more than 0.208ppm

0.003 ppm

0.003 ppm

0.004 ppm

V

Not more than 0.417ppm

0.003 ppm

0.005 ppm

0.004 ppm

Ni

Not more than 0.833ppm

0.426 ppm

0.405 ppm

0.459 ppm

Li

Not more than 10.417ppm

0.007 ppm

0.008 ppm

0.004 ppm

Sb

Not more than 3.750ppm

0.0005 ppm

0.0004 ppm

0.0002 ppm

Cu

Not more than 12.500ppm

0.008 ppm

0.008 ppm

Not detected

Total related substances%

0.3%

0.02%

0.03%

0.03%

Bacterial endotoxins

0.5EU/ml

Complies with specifications

Complies with specifications

Complies with specifications

Sterility

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

Content

96.0%-104.0%

99.8%

99.1%

99.8%

Table3.2.P.5-55Batch Test Report (15ml:7.04g,12ml:5.63g)

Test Items

020210807

(15ml:7.04g)

020210808

(12ml:5.63g)

Appearance

Should comply with the specification

Complies with specifications

Complies with specifications

Identification

Should comply with the specification

Complies with specifications

Complies with specifications

pH value

7.0-7.9

7.5

7.4

Color

Should comply with the specification

Complies with specifications

Complies with specifications

Meglumine

39.6%-43.9%

41.5%

40.9%

Pentetic Acid

230-450μg/ml

393μg/ml

388μg/ml

Insoluble particles

10μm and 10μm or above ≤

6000

1 pill/bottle

7 pills/bottle

25μm and 25μm or larger ≤600

1 piece/bottle

0 piece/bottle

Visible particles

Should comply with the specification

Complies with specifications

Complies with specifications

Elemental impurities

Hg

Not more than 0.125ppm

0.001 ppm

Not detected

Cd

Not more than 0.083ppm

0.0003 ppm

0.0002 ppm

Pb

Not more than 0.208ppm

0.012 ppm

0.009 ppm

As

Not more than 0.625ppm

0.006 ppm

0.007 ppm

Co

Not more than 0.208ppm

0.003 ppm

0.004 ppm

V

Not more than 0.417ppm

0.003 ppm

Not detected

Ni

Not more than 0.833ppm

0.485 ppm

0.494 ppm

Li

Not more than 10.417ppm

0.012 ppm

0.005 ppm

Sb

Not more than 3.750ppm

0.0007 ppm

0.001 ppm

Cu

Not more than 12.500ppm

Not detected

0.008 ppm

Total related substances%

0.3%

0.03%

0.03%

Bacterial Endotoxins

0.5EU/ml

Complies with specifications

Complies with specifications

Sterility

Should comply with the specification

Complies with specifications

Complies with specifications

Content

96.0%-104.0%

101.1%

99.0%

Impurity Profile Analysis and Control

This product is an injectable preparation, where the impurity profile analysis and control of the formulation are closely related to the active pharmaceutical ingredient (API). By analyzing and controlling the impurity profile of the API, it facilitates the impurity profile analysis and control of the final drug product. Based on the synthetic route of the API and the production process of the formulation, impurity profile analysis and control are conducted on the starting materials for API synthesis (gadolinium oxide, pentetic acid, meglumine), the API itself,and the formulation samples.

Impurity profile analysis and control of the formulation: Combined with the impurity profile analysis of the active pharmaceutical ingredient (API) and the impurity degradation under accelerated and stress conditions of the formulation, the main degradation products were analyzed and controlled. The test method was referenced from the "Related Substances" section of the imported registration standard (JX20020190) for Gadopentetate Dimeglumine Injection. The total impurity limit was set as: not more than 0.3% of the labeled amount of pentetic acid (197mg).

API manufacturing route and impurity profile analysis

Impurity profile analysis of the starting material pentetic acid (diethylenetriaminepentaacetic acid)

Table3.2.P.5-56Impurity profile analysis of pentetic acid

Impurity Name

Source

Control Measures

Residual Allowable

Functionality

Control Strategy

Negative Inclusion

Quality Standards

Chloroacetic Acid

Starting material

Chloroacetic acid is dissolved in water, and after acidification with hydrochloric acid to induce crystallization, the product precipitates while chloroacetic acid remains in the aqueous layer. Additionally, impurities in chloroacetic acid are highly soluble in water, so they can also be removed by this measure.

This approach can be used for elimination.

According to general impurity limits set at 0.1%; relevant methodological validation was conducted, and no impurities were detected in multiple batches of samples, hence they are not specified.

Quality Standards

Sodium Chloroacetate

Starting Material

Same as Chloroacetic Acid

Low risk, no control needed

Diethylenetriamine

Starting material

Second step reaction: Chloroacetic acid is in excess, so the residual diethylenetriamine is minimal. Additionally, diethylenetriamine has good water solubility, and even if there is a small amount of residue, it can

be removed through crystallization.

Low, not addedcontrolleddetected

Low risk, no controlrequired

Nitrilotriaceticacid

Potential by-product

A small amount of residual ammonia in diethylenetriamine participates in the reaction to form; it can

be removed through crystallization;

According to the general impurity limit set at 0.1%, relevant methodological validation was conducted, and no detection was found in multiple batches of samples.

Edetate

(EDTA)

Potential byproducts

Small amounts of ethylenediamine may remain in diethylenetriamine,which can form ethylenediamine derivatives after the reaction.

local acid

The impurity profile of the starting material pentetic acid includes process-related starting materials chloroacetic acid, sodium chloroacetate, diethylenetriamine, as well as degradation byproducts of pentetic acid such as nitrilotriacetic acid and edetic acid. Chloroacetic acid is controlled as a general impurity with a specified limit of 0.1%. Method validation was performed using a limit test, demonstrating good specificity and satisfactory sensitivity. Based on multiple batch testing results, no detection was observed. After research and evaluation, it was not designated as a specified impurity in the quality standard. Nitrilotriacetic acid and edetic acid are potential byproducts of pentetic acid. USP43 includes a control method for pentetic acid nitrilotriacetic acid. Our company referenced this method to control pentetic acid and edetic acid, conducting method validation with good specificity and satisfactory sensitivity. Post-validation, referencing USP43, nitrilotriacetic acid was included in the quality standard with a limit of 0.1%.

Starting material meglumine (1-deoxy-1-(methylamino)-1-D-sorbitol) impurity profile analysis

Process flow: (1) Glucose + methylamine + ethanol (or 92% or higher recycled ethanol) to form methylamine solution; (2) Methylamine solution + hydrogen gas produces meglumine solution under nickel catalysis, followed by cold filtration, cooling crystallization, and centrifugation to obtain crude meglumine; (3) Crude meglumine + ethanol dissolution and drying to yield meglumine.

Table 3.2.P.5-57Meglumine Impurity Profile Analysis

Name

Source

Control measures

Residual solvent

functionality

Control Strategy

Rejected for quality

Standard

Ethanol

Reaction solvent

Removed during drying process

Developed and validated relevant testing methods, with detection levels significantly lowerthan 30% of the limit, thus no control is implemented

Methylamine

Starting material

Removed during crystallization process,

Retained in ethanol

Dimethylamine

(Impurity in methylamine)

Introduced in methylamine

Removed during crystallization, retained in ethanol

Nickel(Ni)

Catalyst

Filter and remove

2.5ppm

Glucose

Reaction excipients

Excess methylamine, minimal residue;

Removed during crystallization process

Not more than 0.2%

5-Hydroxymethylfurfural

Introduction of Glucose

Impurities may be introduced in glucose, removed by crystallization

Study requirement: not to exceed 0.1%, no detection in results, no control needed

The impurity profile of the starting material meglumine includes the starting material methylamine, ethanol, reaction catalyst Ni, dimethylamine, and potential degradation impurity 5-hydroxymethylfurfural. A gas chromatography method was used to control methylamine, dimethylamine, and ethanol, with limits set at 0.15% for methylamine and dimethylamine, and 0.5% for ethanol. Method validation for specificity, system suitability, solution stability, detection limit, and robustness was conducted using the limit method. Multiple batches of production validation samples were tested, and the results showed that the detected levels of methylamine, dimethylamine, and ethanol were all less than 30% of the limits; therefore, no further control was applied.

Control specific impurities without including them in the quality standards.5-Hydroxymethylfurfural is a degradation impurity of meglumine and is controlled as a general impurity with a limit of 0.1%. Specificity, detection limit, and robustness were verified using the limit method. Testing of the meglumine used in validation batches detected no 5-hydroxymethylfurfural, so this item is not included in the quality standards.Niis a reaction catalyst and a key elemental impurity of meglumine, controlled according to the limit

2.5ppm The limit is specified in the quality standard.

Table3.2.P.5-58Test results of process-related impurities in meglumine production validation batches

Name

Methylamine

Dimethylamine

Ethanol

Retention Time (min)

Methylamine Peak Area

Retention Time (min)

Dimethylamine peak area

RT(min)

Ethanol peak area

30%Reference solution

7.280

30.4097

7.631

172.3968

8.143

170.5852

Meglumine 2103044

6.884

8.0843

7.651

7.9180

8.058

8.3429

Meglumine 2104026

N/A

Not detected

7.601

17.2852

8.028

9.4275

Original record traceability: KY-GP-02-A009P17-21

Starting material gadolinium oxide (gadolinium(III) oxide) impurity profile analysis

Gadolinium oxide is derived from rare earth elements, produced through precipitation, filtration, and calcination. The main impurities in this product are elemental impurities. Our company, in accordance with the source of gadolinium oxide and the requirements of ICH Q3D, has established control methods and limits for elemental impurities in gadolinium oxide based on the maximum daily dose of gadopentetate dimeglumine. The maximum daily intake of gadolinium oxide is calculated as 3.26g. The ICP-MS method was employed, with complete method validation conducted. Testing of multiple batches of production validation samples showed compliance with requirements.

Table3.2.P.5-59 Gadolinium Oxide Impurity Profile Analysis

Evaluation object

Research Content

Research Methods

Whether controlled in the standard

Rare Earth Elements

According to the lanthanide series in the periodic table:La, Ce,Pr,Nd,Sm,Eu,Tb,Dy, Ho,Er,Tm,Y,Sc,Yb,Lu,not exceeding 5ppm

Conduct method validation and test samples

Routine elemental impurities

1Class,2AClass,3 Class

Conduct method validation and test samples

Process-specific added elements

No specific additions according to the process route

/

Elements potentially introduced during the reaction process

The first step of chelation reaction and post-chelated salt formation are both conducted in enamel reactors; the product pH is neutral, with later-stage conditions at room temperature, using stainless steel storage tanks and stainless steel freeze-drying trays, introducing minimal risk of elemental impurities, and Ni element belongs to Class 2A.

Risk Assessment

Packaging Materials

Solid-to-solid contact, low introduction risk

L.O.I(Loss on Ignition)

Less than 1%

Test sample

Turbidimetric method for chloride limit(100ppm)

Test Sample

Ca,Fe,Si

Ca not more than 5ppm, Fe not more than

2ppm,Si not more than 10ppm

Ca,Fe,SiThe above 3 elements are harmless to the human body and are non-specific

Addition, no control required

No control is performed

Content determination

Gadolinium Content

Content Determination

Active Pharmaceutical Ingredient Impurity Profile Analysis

Figure 3.2.P.5- 12Gadopentetate Dimeglumine Synthesis Route

Step 1: Gadolinium oxide undergoes condensation reaction with pentetic acid. The reactants, byproducts, and control strategies are listed in Table 3.2.S.4-97.

Step 2: Gadopentetic acid reacts with meglumine. The materials involved in the reaction, by-products, and control strategies are listed in Table 3.2.S.4-97.

Step 3: Freeze-drying of gadopentetic acid meglumine solution.

Table 3.2.P.5-60Analysis and Control Strategy for Drug Substance Impurities

Serial Number

Structural formula

Impurity Name

Source of Impurity

Control Strategy

Included in Raw Material Quality Standards (Yes/No)

1

Nitrilotriacetic acid

(Aminotriethylacid)

Pentetic acid processimpurities

Pentetic acid quality standard controls asknown impurities 0.1%. The raw material quality standard follows non-speci

fied impurity control 0.05%

2

Edetate

Pentetic Acid ProcessImpurities

Raw material quality standards are controlled as unspecified impurities 0.05%

3

Chloroacetic Acid

Pentetic Acid ProcessImpurities

Process impurities, not detected in multiple batches of samples, are not included in the quality standards

Standard

4

Cl

Chloride ion

Pentetic Acid Process

Impurities

Control of general impurities in pentetic acid

Limit 0.1%

5

Methylamine

Synthesis of Meglumine

Starting Material

Process impurities, multiple batches of megluminewere detected below the limit of30%, therefore not included in the quality standard

6

Dimethylamine

Methylglucamine synthesis starting material impurity

7

Ethanol

Methylglucamine synthesis

Starting materials

8

5-hydroxymethylfurfural

Methylglucamine degradationproducts

Methylglucamine may degrade impurities, none were detected in multiple batches, so it is not included

Quality Standards

9

Ni

Methylglucamine synthesis catalyst

Using ICP-MS, according to ICHQ3D to provide PDE values for research, so it is not included in quality standards

10

Elemental Impurities

Gadolinium oxide introduction

Using ICP-MS , in accordance with ICHQ3D, the PDE values were studied. Multiple batch tests showed no exceedance, thus it was not included in the quality standard.

Standard.

Element

Limit

μg/g

(ppm)

Hg

0.27

Cd

0.18

Pb

0.44

As

1.33

Co

0.44

V

0.89

Ni

1.78

Li

22.21

Sb

8.00

Cu

26.65

11

Free pentetic acid

Unreacted starting material

Referring to the imported registration standard for gadopentetate dimeglumine injection, the amount of pentetic acid is 200~450μg/ml. Based on the upper limit of 450μg/ml, after deducting the excipient pentetic acid added to the formulation at 400μg/ml, the upper limit for pentetic acid in the formulation is 50μg/ml. According to 1ml containing 0.469g gadopentetate dimeglumine, this converts to an upper limit for the raw material of

106.61μg/g, which is strictly set as 105μg/g

12

Gd

Free gadolinium

Unreacted starting material,degradation products

According to the limit of pentetic acidin gadopentetate dimeglumine injection

(200~450μg/ml)Lower limit200μg/ml. After adding 400μgpentetic acid, the pentetic acid content must not be less than200μg. Calculated at a molar ratio of 2:1, gadolinium oxide must not exceed 92μg, and free gadolinium must not exceed

80μg/469mg, which converts to

170μg/g. To better controlproduct quality, the limit is tightened to 50μg/g

Analysis of formulation impurity profile and basis for limit setting

Table3.2.P.5-61Impurity Control Strategy for Formulations

Serial Number

Structural Formula

Impurity Name

Source of Impurity

Control Strategy

Inclusion StatusDrug Product Quality

Standard

1

Nitrilotriacetic acid

(Aminotriacetic Acid)

Pentetic Acid Process Impurities

Control as non-specified impurities. Total impurities controlled at 0.3%.

2

Edetate

Pentetic Acid Process Impurities

Control as non-specified impurities. Total impurities controlled at 0.3%.

3

Ni

Synthesis of Meglumine Catalyst

Using ICP-MS, providing PDE values according to ICH Q3D

Multiple batch test results showed no exceedances. Therefore, control at the starting material stage is sufficient. Not included in the drug product

specification.

4

Elemental Impurities

Introduction of Gadolinium Oxide

Using ICP-MS, according to

ICH Q3D provided PDE value

Element

Hg

Conduct the study. Multiple batch tests

Results were all within limits

Cd

Pb

Element

Limit

(ppm)

As

Co

Hg

0.27

V

Cd

0.18

Ni

Pb

0.44

Li

As

1.33

Sb

Co

0.44

Cu

V

0.89

Ni

1.78

Li

22.21

Sb

8.00

Cu

26.65

Possible Mechanism of Impurity Formation

(1)Nitrilotriacetic Acid (NTA), Edetic Acid

Nitrilotriacetic acid (NTA) and ethylenediaminetetraacetic acid (EDTA) are by-products formed during the synthesis of pentetic acid and are introduced in the manufacturing process of the active pharmaceutical ingredient.

Elemental Impurity Profile Analysis

According to the ICH guideline Q3D on elemental impurities, the potential elemental impurities of this product were evaluated. This product has 4 specifications, 10ml:4.69 The specification limits are stricter, so the 10ml:4.69g specification and the 20ml:9.38g specification, each with 3 batches, were evaluated through accelerated 6 -month stability studies. The potential elemental impurities of this product were all within the specified limits, and the results are shown in the table below.

Table3.2.P.5-62 Potential Elemental Impurities (Batch 020210804, 10ml:4.69g)

Element

Chinese Name

Limit

(μg/bottle)

0 Month

Accelerated 6 months

(Upright)

Accelerated 6 months

(Inverted)

Source

Li

83.00

<20

<20

<20

ICHQ3D&Pharmaceutical Glass Guidance

B

500.00

<117

<117

<117

ICH Q3D

Al

83.00

<20

35

40

U.S. Code of Federal Regulations 21CFR201.323

Si

1666.00

391

391

391

ECHA

Ti

500.00

117

117

<117

ICH Q3D

V

3.30

<0.8

<0.8

<0.8

ICH Q3D

Cr

366.00

<86

<86

<86

ICHQ3D&Pharmaceutical Glass Guidelines

Mn

500.00

<117

<117

<117

ICH Q3D

Fe

433.00

102

102

102

Guideline for Pharmaceutical Glass

Co

1.60

<0.4

<0.4

<0.4

ICHQ3&Pharmaceutical Glass Guidance

Ni

6.6

3.6

2.8

3.7

ICH Q3D

Cu

100.00

<23

<23

<23

ICH Q3D

Zn

433.00

<102

102

102

Guidelines for Pharmaceutical Glass

As

5.00

1.2

<1.2

<1.2

ICHQ3D&Pharmaceutical Glass Guidance

Zr

500.00

<117

<117

<117

ICH Q3 D

Mo

500.00

117

117

117

ICH Q3D D

Cd

0.66

<0.16

<0.16

<0.16

ICHQ3D&Pharmaceutical Glass Guidelines

Sb

30.00

<7

<7

<7

ICHQ3D&Pharmaceutical Glass Guidance

Ba

233.00

55

55

55

ICHQ3D&Guideline for Pharmaceutical Glass

W

500.00

<12

<12

<12

ICH Q3D

pt

3.30

<0.8

<0.8

<0.8

ICH Q3 D

Hg

1.00

<0.2

<0.2

0.2

ICH Q3D

Pb

1.60

0.4

0.4

<0.4

ICHQ3D&Guideline for Pharmaceutical Glass

Table3.2.P.5-63 Potential Elemental Impurities (Batch 020210805, 10ml: 4.69g)

Element

Chinese Name

Limit

(μg/bottle)

0 Month

Accelerated 6 months

(Upright)

Accelerated 6 months

(Inverted)

Source

Li

83.00

<20

<20

<20

ICHQ3D&Pharmaceutical Glass Guidance

B

500.00

<117

<117

<117

ICH Q3D

Al

83.00

<20

36

35

U.S. Code of Federal Regulations 21CFR201.323

Yes

1666.00

391

391

391

ECHA

Ti

500.00

<117

<117

<117

ICH Q3D

V

3.30

<0.8

<0.8

<0.8

ICH Q3D

Cr

366.00

<86

<86

<86

ICHQ3D&Pharmaceutical Glass Guidance

Mn

500.00

117

117

117

ICH Q3D

Fe

433.00

<102

<102

<102

Guidelines for Medicinal Glass

Co

1.60

0.4

0.4

0.4

ICHQ3&Pharmaceutical Glass Guidance

Ni

6.6

3.3

2.5

3.5

ICH Q3D

Cu

100.00

<23

<23

<23

ICH Q3D

Zn

433.00

102

102

102

Guideline for Pharmaceutical Glass

As

5.00

1.2

1.2

<1.2

ICHQ3D&Pharmaceutical Glass Guidance

Zr

500.00

<117

<117

<117

ICH Q3 D

Mo

500.00

<117

<117

<117

ICH Q3D D

Cd

0.66

<0.16

<0.16

<0.16

ICHQ3D&Pharmaceutical Glass Guidance

Sb

30.00

<7

<7

<7

ICHQ3D&Pharmaceutical Glass Guidance

Ba

233.00

55

55

55

ICHQ3D&Guideline for Pharmaceutical Glass

W

500.00

<12

<12

<12

ICH Q3D

pt

3.30

<0.8

<0.8

<0.8

ICH Q3 D

Hg

1.00

<0.2

<0.2

<0.2

ICH Q3D

Pb

1.60

<0.4

<0.4

<0.4

ICHQ3D&Guideline for Pharmaceutical Glass

Table3.2.P.5-64 Potential Elemental Impurities (Batch 020210806, 10ml: 4.69g)

Element

Chinese Name

Limit

(μg/bottle)

0 Month

Accelerated 6 months

(Upright)

Accelerated 6 months

(Inverted)

Source

Li

83.00

<20

<20

<20

ICHQ3D&Pharmaceutical Glass Guidance

B

500.00

<117

<117

<117

ICH Q3D

Al

83.00

<20

35

32

U.S. Code of Federal Regulations 21CFR201.323

Si

1666.00

<391

<391

<391

ECHA

Ti

500.00

<117

<117

<117

ICH Q3D

V

3.30

<0.8

<0.8

<0.8

ICH Q3D

Cr

366.00

<86

<86

<86

ICHQ3D&Pharmaceutical Glass Guidelines

Mn

500.00

<117

<117

<117

ICH Q3D

Fe

433.00

<102

<102

<102

Pharmaceutical Glass Guidance

Co

1.60

<0.4

<0.4

<0.4

ICHQ3&Pharmaceutical Glass Guidance

Ni

6.6

3.4

3.0

2.9

ICH Q3D

Cu

100.00

<23

<23

<23

ICH Q3D

Zn

433.00

<102

<102

102

Guidelines for Pharmaceutical Glass

As

5.00

1.2

1.2

<1.2

ICHQ3D&Pharmaceutical Glass Guidance

Zr

500.00

<117

<117

<117

ICH Q3 D

Mo

500.00

<117

<117

<117

ICH Q3D D

Cd

0.66

<0.16

<0.16

<0.16

ICHQ3D&Pharmaceutical Glass Guidance

Sb

30.00

<7

<7

<7

ICHQ3D&Pharmaceutical Glass Guidance

Ba

233.00

55

55

55

ICHQ3D&Guideline for Pharmaceutical Glass

W

500.00

<12

<12

<12

ICH Q3D

pt

3.30

<0.8

<0.8

<0.8

ICH Q3 D

Hg

1.00

<0.2

<0.2

0.2

ICH Q3D

Pb

1.60

0.4

0.4

<0.4

ICHQ3D&Guideline for Pharmaceutical Glass

Table3.2.P.5-65 Potential Elemental Impurities (Batch 020210801, 20ml: 9.38g)

Element

Chinese name

Limit

(μg/unit)

0 Month

Accelerated 6 months

(Upright)

Accelerated 6 months

(Inverted)

Source

Li

166.00

<39

<39

<39

ICHQ3D&Pharmaceutical Glass Guidance

B

1000

<234

<234

<234

ICH Q3D

Al

166.00

Less than39

67

62

U.S. Code of Federal Regulations 21CFR201.323

Si

3333.00

<781

<781

<781

ECHA

Ti

1000.00

<234

<234

<234

ICH Q3D

V

6.60

<1.6

<1.6

<1.6

ICH Q3D

Cr

733.00

<172

<172

<172

ICHQ3D&Guidelines for Pharmaceutical Glass

Mn

1000.00

<234

<234

<234

ICH Q3D

Fe

866.00

203

203

203

Pharmaceutical Glass Guidance

Co

3.30

<0.8

<0.8

<0.8

ICHQ3&Pharmaceutical Glass Guidance

Ni

13.00

7

6

6

ICH Q3D

Cu

200.00

<47

<47

<47

ICH Q3D

Zn

866.00

203

203

203

Pharmaceutical Glass Guidance Guidelines

As

10.00

2

2

2

ICHQ3D&Guideline for Pharmaceutical Glass

Zr

1000.00

<234

<234

<234

ICH Q3 D

Mo

1000.00

234

234

234

ICH Q3D D

Cd

1.30

0.3

0.3

0.3

ICHQ3D&Guideline for Pharmaceutical Glass

Sb

60.00

14

14

14

ICHQ3D&Guideline for Pharmaceutical Glass

Ba

466.00

109

109

109

ICHQ3D&Guideline for Pharmaceutical Glass

W

1000.00

23

<23

<23

ICH Q3D

pt

6.60

<1.6

<1.6

<1.6

ICH Q3 D

Hg

2.00

<0.5

<0.5

<0.5

ICH Q3D

Pb

3.30

<0.8

<0.8

<0.8

ICHQ3D&Guideline for Pharmaceutical Glass

Table3.2.P.5-66 Potential Elemental Impurities (Batch 020210802 20ml:9.38g)

Element

Chinese Name

Limit

(μg/unit)

0 Month

Accelerated 6 months

(Upright)

Accelerated 6 months

(Inverted)

Source

Li

166.00

<39

<39

<39

ICHQ3D&Pharmaceutical Glass Guidance

B

1000

234

234

234

ICH Q3D

Al

166.00

<39

63

66

U.S. Code of Federal Regulations 21CFR201.323

Yes

3333.00

<781

<781

<781

ECHA

Ti

1000.00

<234

<234

<234

ICH Q3D

V

6.60

1.6

1.6

1.6

ICH Q3D

Cr

733.00

<172

<172

<172

ICHQ3D&Pharmaceutical Glass Guidelines

Mn

1000.00

234

234

234

ICH Q3D

Fe

866.00

203

203

203

Pharmaceutical Glass Guidance Guidelines

Co

3.30

<0.8

<0.8

<0.8

ICHQ3&Pharmaceutical Glass Guidance

Ni

13.00

9

7

6

ICH Q3D

Cu

200.00

47

47

47

ICH Q3D

Zn

866.00

203

203

203

Guidelines for Medicinal Glass

As

10.00

2

2

2

ICHQ3D&Guideline for Pharmaceutical Glass

Zr

1000.00

<234

<234

<234

ICH Q3 D

Mo

1000.00

234

234

234

ICH Q3D D

Cd

1.30

<0.3

<0.3

<0.3

ICHQ3D&Guideline for Pharmaceutical Glass

Sb

60.00

14

14

14

ICHQ3D&Guideline for Pharmaceutical Glass

Ba

466.00

<109

109

109

ICHQ3D&Guideline for Pharmaceutical Glass

W

1000.00

<23

<23

<23

ICH Q3D

pt

6.60

1.6

1.6

<1.6

ICH Q3 D

Hg

2.00

<0.5

<0.5

<0.5

ICH Q3D

Pb

3.30

<0.8

<0.8

<0.8

ICHQ3D&Guideline for Pharmaceutical Glass

Table3.2.P.5-67 Potential Elemental Impurities (Batch 020210803, 20ml: 9.38g)

Element

Chinese name

Limit

(μg/unit)

0 Month

Accelerated 6 months

(Upright)

Accelerated 6 months

(Inverted)

Source

Li

166.00

<39

<39

<39

ICHQ3D&Pharmaceutical Glass Guidelines

B

1000

<234

234

234

ICH Q3D

Al

166.00

39

62

66

United States Code of Federal Regulations 21CFR201.323

Yes

3333.00

781

781

781

ECHA

Ti

1000.00

234

234

234

ICH Q3D

V

6.60

1.6

1.6

1.6

ICH Q3D

Cr

733.00

172

172

172

ICHQ3D&Guideline for Pharmaceutical Glass

Mn

1000.00

<234

<234

<234

ICH Q3D

Fe

866.00

<203

<203

203

Guidelines for Medicinal Glass

Co

3.30

<0.8

<0.8

<0.8

ICHQ3&Pharmaceutical Glass Guidance

Ni

13.00

8

6

6

ICH Q3D

Cu

200.00

47

47

<47

ICH Q3D

Zn

866.00

<203

<203

<203

Guidelines for Medicinal Glass

As

10.00

<2

<2

<2

ICHQ3D&Guideline for Pharmaceutical Glass

Zr

1000.00

<234

<234

<234

ICH Q3 D

Mo

1000.00

<234

<234

<234

ICH Q3D D

Cd

1.30

<0.3

<0.3

<0.3

ICHQ3D&Guideline for Pharmaceutical Glass

Sb

60.00

<14

<14

<14

ICHQ3D&Guideline for Pharmaceutical Glass

Ba

466.00

<109

109

109

ICHQ3D&Guideline for Pharmaceutical Glass

W

1000.00

<23

<23

<23

ICH Q3D

pt

6.60

<1.6

<1.6

<1.6

ICH Q3 D

Hg

2.00

<0.5

<0.5

<0.5

ICH Q3D

Pb

3.30

<0.8

<0.8

<0.8

ICHQ3D&Guideline for Pharmaceutical Glass

Conclusion: The elemental impurities of this product comply with the regulations.

Genotoxic Impurity Analysis and Control

The starting materials for this product are meglumine, pentetic acid, and gadolinium oxide; neither the starting material intermediates, synthesis byproducts, nordegradation products contain alert structures.

Potential mutagenic impurity analysis of the synthetic route for the starting material pentetic acid (diethylenetriaminepentaacetic acid)

Table3.2.P.5-68Potential mutagenic impurity analysis of the synthetic route for starting material pentetic acid

Serial Number

Structural formula

Impurity Name

Source of Impurity

Does it contain alertstructures

Are potential mutagenic impurities investigated?

Research control

1

Chloroacetic Acid

Pentetic acid processStarting material

2

Na2CO3

Sodium carbonate (soda ash)

Pentetic Acid ProcessStarting Material

3

N/A

Sodium Chloroacetate

Pentetic Acid ProcessIntermediate

4

Diethylenetriamine

Pentetic Acid ProcessStarting Material

5

Nitrilotriacetic Acid (NTA)

Pentetic Acid ProcessImpurities

6

Edetate

Pentetic Acid ProcessImpurities

7

Cl

Chloride Ion

Pentetic Acid Process

Impurities

8

NaCl

Sodium Chloride

Reaction reagents

Conclusion: The starting materials, reagents, reaction intermediates,and by-products used in the synthesis process of pentetic acid in the above table do not contain any alert structures.

Potential mutagenic impurity analysis of the starting material meglumine (1-deoxy-1-(methylamino)-1-D-sorbitol)synthetic route

Table3.2.P.5-69Potential mutagenic impurity analysis of the starting material meglumine synthetic route

Serial Number

Structural formula

Impurity Name

Source of Impurity

Is there a warning structure

Potential mutagenic impurities

Has research control been conducted on the impurities

1

Methylamine

Methylglucamine synthesis

Starting materials

2

95% ethanol

Methylglucamine synthesis

Starting materials

3

Anhydrous Glucose

Meglumine synthesisStarting material

4

Methylamine hydrochloride

Meglumine synthesis reaction intermediate

5

Dimethylamine

Methylglucamine synthesis starting material impurity

6

95%Ethanol

Methylglucamine synthesis

Starting materials

7

Activated Charcoal

Activated Charcoal

Meglumine Purification

Materials

8

5-Hydroxymethylfurfural

Methylglucamine degradationproducts

9

Ni

Methylglucamine synthesis

Catalyst

Conclusion: The starting materials, reagents, reaction intermediates, and by-products used in the synthesis process of meglumine listed in the above table do not contain any alert structures.

Starting material gadolinium oxide (gadolinium(III) oxide)Potential mutagenic impurity analysis of the synthetic route

Table3.2.P.5-70Analysis of Potential Mutagenic Impurities in the Synthesis Process Route of Starting Material Gadolinium Oxide

Serial Number

Structural formula

Name

Source of Impurity

Is there a warning structure

Potential mutagenic impurities

Has research control been conducted on the substance

1

Gd2O3

Gadolinium solution

Material

2

Oxalic acid

Material

3

N/A

Gadolinium oxalate

Intermediate drug solution

Conclusion: The starting materials, reagents, reaction intermediates, and by-products used in the synthesis process of gadolinium oxide listed in the above table do not contain any alert structures.

Analysis of potential mutagenic impurities in the synthesis route of gadopentetate dimeglumine

Table3.2.P.5-71Analysis of Potential Mutagenic Impurities in the Synthesis Process of Gadopentetate Meglumine API

Serial Number

Structural formula

Name

Source of Impurity

Warning structure

Whether potential mutagenic impurities have been studied

and controlled

1

Pentetic Acid

Synthetic starting materials

2

Meglumine

Synthetic starting materials

3

Gadolinium oxide

Synthetic starting material

4

Gadopentetic acid

Reaction intermediate

5

Free pentetic acid

Synthetic by-products

6

Gd

Free gadolinium

Synthetic by-products

Conclusion: The starting materials, reagents, reactionintermediates, and by-products used in the synthesis process of gadopentetate dimeglumine listed in the above table do not contain any alert structures.

Analysis of Potential Mutagenic Impurities in Formulation Production Process Routes

The formulation of this product is as follows: each 1ml of Gadopentetate Dimeglumine Injection contains 469.01mg of Gadopentetate Dimeglumine, Meglumine 0.99mg, and 0.40mg of Pentetic Acid, with water for injection as the solvent, and does not contain any preservatives or bacteriostatic agents. None of the above raw materials or excipients contain alerting structures.

Basis for Quality Standard Establishment

Based on the systematic research above and with reference to the quality standards for Gadopentetate Dimeglumine Injection (ChP2020), Gadopentetate Dimeglumine USP43, import registration standard JX20020190 , and the Chinese Pharmacopoeia 2020 Edition Part IV, the detailed formulation and revision are explained as follows:

The quality comparison between self-prepared samples and the reference preparation is as follows:

Table3.2.P.5-72Quality Comparison Between Self-Developed Preparation and Reference Preparation(1)

Sample

Self-prepared samples

Reference Preparation

Batch number

020210801

020210802

020210803

KT05TA1

Specification

20ml:9.38g

469.01mg/ml× 20ml

Testing Items

Limit requirement

Test result

Appearance

Colorless to pale yellow or light yellow

clear green liquid

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

Identification

(1) The position and color of the principal spot in the test solution should correspond to those of the principal spot in the reference solution.

The spots are identical

Complies with specifications

Complies with specifications

Complies with specifications

/

(2) The position and color of the main spot in the test solution should correspond to those of the main spot in the reference solution.

The spots are identical

Complies with specifications

Complies with specifications

Complies with specifications

/

(3) In the chromatogram recorded under the assay, the retention time of the principal peak in the test solution should correspond to that of the principal peak in the reference solution

The retention time is consistent.

Complies with specifications

Complies with specifications

Complies with specifications

/

(4)at a wavelength of 275nm

exhibits maximum absorption at this wavelength.

Complies with specifications

Complies with specifications

Complies with specifications

/

pH value

6.7-7.8

7.5

7.5

7.5

7.2

Color

Compared with the yellow 4 or yellowish-green 4

standard color solution, it must not bedarker

Complies with specifications

Complies with specifications

Complies with specifications

/

Meglumine

39.6%~43.9%

41.4

41.5

41.7

43.1

Pentetic Acid

200~450μg

393

365

347

387

Elemental Impurities

Hg

0.125μg/ml

0.001

0.001

0.001

Not detected

Cd

0.083μg/ml

0.0002

Not detected

0.0001

0.00001

Pb

0.208μg/ml

0.006

0.005

0.011

0.007

As

0.625μg/ml

0.001

0.004

0.009

0.010

Co

0.208μg/ml

0.003

0.003

0.004

0.001

V

0.417μg/ml

Not detected

Not detected

Not detected

0.009

Ni

0.833μg/ml

0.383

0.399

0.569

0.448

Li

10.417μg/ml

0.016

0.008

0.009

0.003

Sb

3.750μg/ml

0.0002

0.001

0.001

0.001

Cu

12.500μg/ml

0.009

0.008

0.014

0.002

Related substances

Total impurities

Amount (%)

0.3%

0.03

0.03

0.03

0.03

Insoluble particles

10µm

600particles/bottle

25

25

3

/

25µm

60particles/bottle

0

6

1

/

Bacterial endotoxin

Each 1ml contains endotoxin

Should be less than 0.5EU

Complies with specifications

Complies with specifications

Complies with specifications

/

Sterility

Should be sterile

Complies with specifications

Complies with specifications

Complies with specifications

/

Visible particles

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

/

Content

96.0%~104.0%

99.7

101.0

101.4

105.1

Table3.2.P.5-73Quality Comparison Between Self-Developed Preparation and Reference Preparation(2)

Sample

In-house samples

Reference Preparation

Batch number

020210804

020210805

020210806

KT05B9J

Specification

10ml:4.69g

469.01mg/ml× 10ml

Testing Items

Limit requirements

Test results

Appearance

Colorless to pale yellow or light yellow

Green clear liquid

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

Identification

(1) The position and color of the principal spot in the test solution should correspond to those of the principal spot in the reference solution.

The spots are identical

Complies with specifications

Complies with specifications

Complies with specifications

/

(2) The position and color of the principal spot in the test solution should correspond to those of the principal spot in the reference solution.

The spots are identical

Complies with specifications

Complies with specifications

Complies with specifications

/

(3) In the chromatogram recorded under the assay, the retention time of the main peak in the test solution should be consistent with that of the main peak in the reference

solution.

Complies with specifications

Complies with specifications

Complies with specifications

/

(4)At 275nm wavelength

it shows maximum absorption.

Complies with specifications

Complies with specifications

Complies with specifications

/

pH value

6.7-7.8

7.5

7.5

7.5

7.29

Color

Compared with yellow 4 or yellowish green 4

standard color solution, it should not be darker

Complies with specifications

Complies with specifications

Complies with specifications

/

Meglumine

39.6%~43.9%

41.0

40.8

41.1

42.32

Pentetic Acid

200~450μg

361

361

385

383.6

Elemental Impurities

Hg

0.125μg/ml

0.001

0.001

0.001

Not detected🎧

Cd

0.083μg/ml

0.0001

0.0001

Not detected🎧

0.00004

Pb

0.208μg/ml

0.008

0.005

0.009

0.006

As

0.625μg/ml

0.010

0.011

0.012

0.002

Co

0.208μg/ml

0.003

0.003

0.004

0.001

V

0.417μg/ml

0.003

0.005

0.004

Not detected🎧

Ni

0.833μg/ml

0.426

0.405

0.459

0.398

Li

10.417μg/ml

0.007

0.008

0.004

0.002

Sb

3.750μg/ml

0.0005

0.0004

0.0002

0.003

Cu

12.500μg/ml

0.008

0.008

Not detected🎧

0.002

Related substances

Total impurities

Quantity (%)

0.3%

0.02

0.03

0.03

0.051

Insoluble particles

10µm

600 particles/bottle

1

0

1

/

25µm

60 particles/bottle

1

0

0

/

Bacterial endotoxin

Endotoxin content per 1ml

should be less than 0.5EU

Complies with specifications

Complies with specifications

Complies with specifications

/

Sterility

Should be sterile

Complies with specifications

Complies with specifications

Complies with specifications

/

Visible particles

Should comply with the specification

Complies with specifications

Complies with specifications

Complies with specifications

/

Content

96.0%-104.0%

99.8

99.1

99.8

102.6

Table3.2.P.5-74Quality Comparison Between Self-Developed Preparation and Reference Preparation(3)

Sample

Self-prepared formulation

Reference Preparation

Batch number

020210807

020210808

KT07B54

Specification

15ml:7.04g

12ml:5.63g

469.01mg/ml× 15ml

Testing Items

Limit requirement

Test result

Appearance

Colorless to pale yellow or light yellow

Green, clear liquid

Complies with specifications

Complies with specifications

Complies with specifications

Identification

(1) The position and color of the principal spot in the test solution should be the same as those of the principal spot

in the reference solution

Complies with specifications

Complies with specifications

/

(2) The position and color of the principal spot in the test solution should be the same as those of the principal spot in the reference solution.

The spots should be identical

Complies with specifications

Complies with specifications

/

(3) In the chromatogram recorded under the assay, the retention time of the principal peak in the test solution should correspond to that of the principal peak in the reference solution

The retention time is consistent.

Complies with specifications

Complies with specifications

/

(4)At 275nm wavelength

it exhibits maximum absorption.

Complies with specifications

Complies with specifications

Complies with specifications

pH value

6.7-7.8

7.5

7.4

7.3

Color

Compared with yellow No. 4 or yellowish-green No. 4

standard colorimetric solution, it should not be darker

Complies with specifications

Complies with specifications

/

Meglumine

39.6%~43.9%

41.5

40.9

43.0

Pentetic Acid

200~450μg

393

388

396

Elemental Impurities

Hg

0.125μg/ml

0.001

Not detected

Not detected🎧

Cd

0.083μg/ml

0.0003

0.0002

0.00001

Pb

0.208μg/ml

0.012

0.009

0.009

As

0.625μg/ml

0.006

0.007

Not detected🎧

Co

0.208μg/ml

0.003

0.004

0.0003

V

0.417 μg/mL

0.003

Not detected

0.011

Ni

0.833μg/ml

0.485

0.494

0.431

Li

10.417μg/ml

0.012

0.005

0.003

Sb

3.750μg/ml

0.0007

0.001

0.003

Cu

12.500μg/ml

Not detected

0.008

0.001

Related substances

Total impurities

Content (%)

0.3%

0.03

0.03

0.03

Insoluble particles

10µm

600particles/bottle

1

7

/

25µm

60particles/bottle

1

0

/

Bacterial endotoxin

Each 1mlcontains endotoxin

Should be less than 0.5EU

Complies with specifications

Complies with specifications

/

Sterility

Should be sterile

Complies with specifications

Complies with specifications

/

Visible particles

Should comply with the specification

Complies with specifications

Complies with specifications

/

Content

96.0%-104.0%

101.1

99.0

104.2

Based on the comparative studies above, it can be concluded that the self-prepared samples are comparable in quality to the reference preparation. This product meets the established quality standards, and the basis for setting these standards is as follows:

Appearance

With reference to the quality standards for Gadopentetate Dimeglumine Injection in Part II of the Chinese Pharmacopoeia 2020 edition, combined with inspections and stability studies of 4 specifications totaling 8 batches of self-prepared samples and 2 batches of reference preparations, the description of this product is proposed as: It should be a clear, colorless to pale yellow or pale yellow-green liquid, which is included in the quality standards.

Table3.2.P.5-75Appearance Inspection of Stability Samples

Specification

Batch number

0 Month

Accelerated 6 months

40±2

75%±5%

Intermediate 6 months

30±2

65%±5%

Long-term 6 months

25±2

60%±5%

20ml:9.

38g

020210801(Upright)

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green cle

ar liquid

020210801(inverted)

Pale yellow-green and clear

clear liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellow-green clear

liquid

020210802(Upright)

Pale yellow-green clear

clear liquid

pale yellow-green clear

clear liquid

pale yellow-green clear

Liquid

Pale yellow-green clear

liquid

020210802(inverted)

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green cle

ar liquid

020210803(Upright)

Pale yellow-green clear

liquid

Pale yellow-green clear

Clear liquid

Pale yellow-green clear

liquid

Pale yellow-green clear

Clear liquid

020210803(inverted)

Pale yellow-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellow-green clear

liquid

10ml:4.

69g

020210804(Upright)

Pale yellow-green clear

liquid

Pale yellow-green clear

Clear liquid

Pale yellow-green clear

liquid

Pale yellow-green clear

clear liquid

020210804(inverted)

pale yellow-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellow-green clear

liquid

020210805(inverted)

Pale yellow-green clear

clear liquid

pale yellow-green clear

clear liquid

pale yellow-green clear

Liquid

Pale yellow-green clear

liquid

020210805(inverted)

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green cle

ar liquid

020210806(Upright)

Pale yellow-green clear

liquid

Pale yellow-green clear

Clear liquid

Pale yellow-green clear

liquid

Pale yellow-green clear

clear liquid

020210806(inverted)

pale yellow-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

Liquid

Clear, pale yellow-green

liquid

12ml:5.

63g

020210808(Upright)

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellow-green clear

liquid

020210808(inverted)

Pale yellow-green clearliquid

Pale yellowish-green clear liquid

Pale yellowish-green transparent liquid

Pale yellowish-green clear liquid

15ml:7.

04g

020210807(Upright)

Pale yellow-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellowish-green clear

liquid

Pale yellow-green clear

liquid

020210807(inverted)

Pale yellow-green clear

clear liquid

pale yellow-green clear

clear liquid

pale yellow-green clear

Liquid

Pale yellow-green clear

liquid

469.01m

g/ml× 15ml

Reference Preparation

(KT01B54)

Pale yellow-green clear liquid

Pale yellow-green clear liquid

clearliquid with pale yellow-green color

Pale yellow-green clear liquid

469.01m

g/ml× 20ml

Reference Preparation

(KT05TA1)

Pale yellowish-green clear liquid

Pale yellowish-green clear liquid

Pale yellowish-green clear liquid

Pale yellowish-green clear liquid

Identification

Refer to the Gadopentetate Meglumine Injection quality standard in Part II of the Chinese Pharmacopoeia 2020 edition, using gadopentetate meglumine as the reference substance. Thin-layer chromatography was employed to identify meglumine, and the test results showed that the position and color of the main spot in the test solution should be the same as those of the main spot in the reference solution, meeting the requirements and thus being included in the quality standard.

Refer to the Gadopentetate Meglumine Injection quality standard in Part II of the Chinese Pharmacopoeia 2020 edition, using gadopentetate meglumine as the reference substance. Thin-layer chromatography was employed to identify gadopentetate, and the test results showed that the position and color of the main spot in the test solution should be the same as those of the main spot in the reference solution, meeting the requirements and thus being included in the quality standard.

With reference to the quality standards for Gadopentetate Dimeglumine Injection in Part II of the Chinese Pharmacopoeia 2020 edition, the retention times of the main peaks of the test sample and reference standard were compared using the HPLC method under the assay item. The results met the requirements and were included in the quality standards.

With reference to the quality standards for Gadopentetate Dimeglumine Injection in Part II of the Chinese Pharmacopoeia 2020 edition, UV-Vis spectrophotometry was employed to scan at 200nm-400nm. Maximum absorption was observed at the wavelength of 275nm, and the results met the requirements, which were included in the quality standards.

pH value

With reference to the quality standards for Gadopentetate Dimeglumine Injection in Part II of the Chinese Pharmacopoeia 2020 edition, the assay method for this product was established

. A total of 8 batches of in-house samples across 4 specifications and 2 batches of reference preparations were tested, and all results complied with the requirements. Therefore, the pH of this product was determined to be: 7.0~7.9 (Chinese Pharmacopoeia 2020 edition, General Chapter 0631). The method was also evaluated for stability, and the test results were consistently stable, meeting the assay requirements. This was included in the quality standards.

Table3.2.P.5-76Stability Sample pHValue Test

Specification

Batch number

0 Month

Accelerated 6 Months

40±2

75%±5%

Intermediate6months

30±2

65%±5%

Long-term 6 months

25±2

60%±5%

20ml:9.38g

020210801(Upright)

7.5

7.4

7.5

7.5

020210801(Inverted)

7.4

7.5

7.5

020210802(upright)

7.5

7.6

7.5

7.6

020210802(inverted)

7.6

7.5

7.6

020210803(upright)

7.5

7.6

7.6

7.6

020210803(inverted)

7.6

7.6

7.6

10ml:4.69g

020210804(upright)

7.5

7.4

7.5

7.6

020210804(inverted)

7.4

7.5

7.5

020210805(upright)

7.5

7.6

7.6

7.6

020210805(inverted)

7.6

7.5

7.6

020210806(upright)

7.5

7.6

7.6

7.6

020210806(inverted)

7.6

7.6

7.6

15ml:7.04g

020210807(Upright)

7.5

7.6

7.5

7.6

020210807(inverted)

7.6

7.6

7.5

12ml:5.63g

020210808(upright)

7.4

7.6

7.5

7.5

020210808(inverted)

7.6

7.4

7.5

469.01mg/ml×15ml

Reference Preparation

(KT01B54)

7.3

7.4

7.4

7.4

469.01mg/ml×20ml

Reference Preparation

(KT05TA1)

7.2

7.3

7.3

7.4

Color

Refer to the quality standards of Gadopentetate Dimeglumine Injection in Part II of the Chinese Pharmacopoeia 2020 edition to establish the solution specifications for this product

The color determination method is proposed as follows: Take the product and compare it with Yellow 4 or Yellow-Green 4standard color solution (Chinese

Pharmacopoeia 2020Edition, General Rule 0901, Method 1). The color should not be darker. Through inspection and stability studies of 4 specifications and a total of

8 batches of in-house samples, the results all met the requirements and were included in the quality standard.

Table3.2.P.5-77Color Inspection of Stability Sample Solutions

Specification

Batch number

0 Month

Accelerated 6 months

40±2

75%±5%

Intermediate 6months

30±2

65%±5%

Long-term 6 pieces

25°C±2°C

60%±5%

20ml:9.38g

020210801(Upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210801(inverted)

Complies with specifications

Complies with specifications

Complies with specifications

020210802(upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210802(inverted)

Complies with specifications

Complies with specifications

Complies with specifications

020210803(upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210803(inverted)

Complies with specifications

Complies with specifications

Complies with specifications

10ml: 4.69g

020210804(upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210804(Inverted)

Complies with specifications

Complies with specifications

Complies with specifications

020210805(Upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210805(Inverted)

Complies with specifications

Complies with specifications

Complies with specifications

020210806(Upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210806(inverted)

Complies with specifications

Complies with specifications

Complies with specifications

15ml: 7.04g

020210807(upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210807(inverted)

Complies with specifications

Complies with specifications

Complies with specifications

12ml: 5.63g

020210808(Upright)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

020210808(Inverted)

Complies with specifications

Complies with specifications

Complies with specifications

469.01mg/ml×15ml

Reference Preparation

(KT01B54)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

469.01mg/ml×20ml

Reference Preparation

(KT05TA1)

Complies with specifications

Complies with specifications

Complies with specifications

Complies with specifications

Meglumine

This product is a complex of gadopentetate and meglumine, where meglumine exhibits optical rotation. The content of meglumine is determined using the optical rotation method to control the content of gadopentetate meglumine. Based on the molecular formula of gadopentetate meglumine,C14H20GdN3O10·2C7H17NO5, the theoretical proportion of meglumine is 41.6%. The acceptance range is set as ±5% deviation from the theoretical content, resulting in a limit range of 39.5%-43.7%, which is consistent with the imported registration standard JX20020190 quality standard range of 39.6%~43.9%.

Therefore, referencing the imported registration standard JX20020190 quality standard, the meglumine determination method for this product is established as follows: Take the product and measure its optical rotation at25°C according to the method (Chinese Pharmacopoeia2020 Edition, Part IV, General Chapter0621), and calculate the meglumine content using the following formula. The result should be within 39.6%~43.9% of the labeled amount of gadopentetate meglumine.

Content of Meglumine (%=Measured optical rotation×1000 × 100%

24.9×469

Through inspection and stability studies of4specifications totaling8batches of self-prepared samples, all results met the specifications and wereincorporated into the draft quality standard.

Table3.2.P.5-78Stability Sample Meglumine Inspection

Specification

Batch number

0 Month

Accelerated 6 months

40±2

75%±5%

Intermediate 6 months

30±2

65%±5%

Long-term 6 months

25±2

60%±5%

20ml:9.38g

020210801(Upright)

41.4%

40.8%

41.4%

40.9%

020210801(inverted)

40.9%

40.8%

40.8%

020210802(upright)

41.5%

40.9%

40.9%

40.7%

020210802(inverted)

40.9%

40.9%

40.9%

020210803(upright)

41.7%

41.1%

41.1%

41.1%

020210803(inverted)

41.1%

41.1%

41.1%

10ml: 4.69g

020210804(Upright)

41.0%

41.1%

40.4%

40.3%

020210804(Inverted)

40.4%

40.4%

40.4%

020210805(Upright)

40.8%

40.3%

40.4%

40.4%

020210805(Inverted)

40.3%

40.4%

40.4%

020210806(Upright)

41.1%

40.5%

40.5%

40.6%

020210806(Inverted)

40.5%

40.5%

40.2%

15ml: 7.04g

020210807(Upright)

41.5%

40.5%

40.9%

41.0%

020210807(inverted)

40.8%

40.7%

40.9%

12ml: 5.63g

020210808(upright)

40.9%

40.4%

40.3%

40.3%

020210808(inverted)

40.1%

40.4%

40.0%

469.01mg/ml×15ml

Reference Preparation

(KT01B54)

43.0%

42.4%

42.4%

42.4%

469.01mg/ml×20ml

Reference Preparation

(KT05TA1)

43.1%

42.5%

42.5%

42.5%

Pentetic Acid

The specification for pentetic acid was drafted with reference to USP43 and the imported drug registration standards. The concentration of xylenol orange indicator was revised, and gadolinium sulfate titrant (0.001mol/L) was used. The Chinese Pharmacopoeia 2020 Edition, Part II Appendix, and corporate standards employ xylenol orange solution as an indicator, which is more than 10 times more dilute than the xylenol orange solution specified in USP43 and the imported drug registration standards. The endpoint color change ranges from orange-yellow to orange-red, which is less distinct than the yellow-to-red-purple transition observed in USP and the imported drug registration standards. The revised method underwent validation for specificity, precision, linearity and range, accuracy, solution stability, and other methodological parameters. The validation results confirmed that the proposed method meets the requirements for pentetic acid testing in this product. Furthermore, referencing the imported drug registration standards, the limit for pentetic acid was tightened to 230~450μg/ml.

Testing and stability studies were conducted on 8 batches of in-house samples across 4 specifications of this product. All results complied with the requirements and were incorporated into the quality standards.

Table 3.2.P.5-79 Stability Sample Pentetic Acid Testing

Specification

Batch number

0 Month

Accelerated 6 months

40±2

75%±5%

Intermediate 6 months

30±2

65%±5%

Long-term 6 months

25°C±2°C

60%±5%

20ml:9.38g

020210801(Upright)

393μg/ml

378μg/ml

392μg/ml

392μg/ml

020210801(inverted)

369μg/ml

392μg/ml

392μg/ml

020210802(Upright)

365μg/ml

353μg/ml

373μg/ml

380μg/ml

020210802(inverted)

349μg/ml

376μg/ml

375μg/ml

020210803(Upright)

347μg/ml

328μg/ml

342μg/ml

351μg/ml

020210803(inverted)

337μg/ml

345μg/ml

345μg/ml

10ml: 4.69g

020210804(upright)

361μg/ml

337μg/ml

334μg/ml

359μg/ml

020210804(inverted)

337μg/ml

334μg/ml

363μg/ml

020210805(Upright)

361μg/ml

333μg/ml

351μg/ml

367μg/ml

020210805(inverted)

333μg/ml

351μg/ml

366μg/ml

020210806(Upright)

385μg/ml

349μg/ml

366μg/ml

375μg/ml

020210806(inverted)

345μg/ml

362μg/ml

371μg/ml

15ml: 7.04g

020210807(upright)

393μg/ml

357μg/ml

384μg/ml

386μg/ml

020210807(inverted)

365μg/ml

384μg/ml

384μg/ml

12ml: 5.63g

020210808(Upright)

388μg/ml

361μg/ml

385μg/ml

387μg/ml

020210808(inverted)

361μg/ml

377μg/ml

385μg/ml

469.01mg/ml×15ml

Reference Preparation

(KT01B54)

396μg/ml

406μg/ml

392μg/ml

390μg/ml

469.01mg/

Reference Preparation

387μg/ml

406μg/ml

369μg/ml

416μg/ml

ml×20ml

(KT05TA1)

Elemental Impurities

Elemental impurities refer to the Chinese Pharmacopoeia2020 Edition, General Chapter0412, using inductively coupled plasma mass spectrometry (ICP-MS) for drafting. The limits are based on the established ICH Q3D Appendix 2 PDE values for elemental impurities in injectable products. According to the instructions, the conventional maximum dosage is 0.4 ml/kg, with an adult body weight factor of 60 kg. The limits for elemental impurities in the injection were calculated, and the method for determining elemental impurities in this product was established. Through methodological validation including specificity, detection limit, quantitation limit, repeatability, linearity, and accuracy, the validation results demonstrate that the proposed analytical method meets the requirements for determining elemental impurities in this product.

0month and accelerated long-term 6month results show that the elemental impurities of this product meet the requirements, with no increase observed during the stability period. Therefore, elemental impurities will not be included in the final proposed quality standards.

Element

Classification

PDE μg/day

Maximum daily intake

ml

Limit μg/ml

Hg

1 type

3

24ml

0.125

Cd

2

0.083

Pb

5

0.208

As

15

0.625

Co

2A

5

0.208

V

10

0.417

Ni

20

0.833

Li

Class 3

250

10.417

Sb

90

3.750

Cu

300

12.500

Table 3.2.P.5-80Elemental Impurity Determination in Stability Samples

Specification

Batch number

Element

(μg/ml)

0 Month

Accelerated 6 months

40℃±275%±5%

Upright

Upside down

20ml:9.38

g

020210801

Hg

0.001

Not detected

Not detected

Cd

0.0002

Not detected

Not detected

Pb

0.006

0.003

0.007

As

0.001

Not detected

0.002

Co

0.003

0.003

0.004

V

Not detected

Not detected

Not detected

Ni

0.383

0.324

0.384

Li

0.016

0.001

0.005

Sb

0.0002

Not detected

0.001

Cu

0.009

0.008

0.009

20ml:9.38

g

020210802

Hg

0.001

Not detected

Not detected

Cd

Not detected

Not detected

Not detected

Pb

0.005

0.004

0.004

As

0.004

0.004

Not detected

Co

0.003

0.004

0.004

V

Not detected

Not detected

Not detected

Ni

0.399

0.408

0.394

Li

0.008

0.005

0.001

Sb

0.001

0.001

Not detected

Cu

0.008

0.010

0.009

20ml:9.38

g

020210803

Hg

0.001

Not detected

Not detected

Cd

0.0001

Not detected

Not detected

Pb

0.011

0.009

0.012

As

0.009

0.001

0.004

Co

0.004

0.004

0.004

V

Not detected

Not detected

Not detected

Ni

0.569

0.516

0.528

Li

0.009

0.004

0.008

Sb

0.001

0.001

0.001

Cu

0.014

0.008

0.012

10ml: 4.69g

020210804

Hg

0.001

Not detected

Not detected

Cd

0.0001

Not detected

Not detected

Pb

0.008

0.008

0.009

As

0.010

0.025

0.014

Co

0.003

0.004

0.004

V

0.003

Not detected

Not detected

Ni

0.426

0.482

0.484

Li

0.007

0.015

0.005

Sb

0.0005

0.001

0.001

Cu

0.008

0.008

0.011

10ml: 4.69g

020210805

Hg

0.001

Not detected

Not detected

Cd

0.0001

Not detected

Not detected

Pb

0.005

0.007

0.007

As

0.011

0.021

0.027

Co

0.003

0.004

0.004

V

0.005

Not detected

Not detected

Ni

0.405

0.438

0.428

Li

0.008

0.001

0.005

Sb

0.0004

0.001

Not detected

Cu

0.008

0.009

0.009

10ml: 4.69g

020210806

Hg

0.001

Not detected

Not detected

Cd

Not detected

Not detected

0.001

Pb

0.009

0.010

0.010

As

0.012

0.018

0.013

Co

0.004

0.005

0.004

V

0.004

Not detected

Not detected

Ni

0.459

0.473

0.482

Li

0.004

0.012

0.005

Sb

0.0002

Not detected

0.001

Cu

Not detected

0.007

0.009

15ml: 7.04g

020210807

Hg

0.001

Not detected

Not detected

Cd

0.0003

Not detected

Not detected

Pb

0.012

0.010

0.011

As

0.006

0.005

0.010

Co

0.003

0.003

0.004

V

0.003

Not detected

Not detected

Ni

0.485

0.415

0.469

Li

0.012

0.002

0.014

Sb

0.0007

0.001

Not detected

Cu

Not detected

0.005

0.005

12ml: 5.63g

020210808

Hg

Not detected

Not detected

Not detected

Cd

0.0002

Not detected

Not detected

Pb

0.009

0.008

0.008

As

0.007

0.019

0.025

Co

0.004

0.003

0.004

V

Not detected

Not detected

Not detected

Ni

0.494

0.500

0.516

Li

0.005

0.008

0.012

Sb

0.001

0.001

0.001

Cu

0.008

0.009

0.007

469.01mg

/ml× 15ml

Reference Preparation

(KT01B54)

Hg

Not detected

Not detected

Cd

0.00001

Not detected

Pb

0.009

0.009

As

Not detected

Not detected

Co

0.0003

0.001

V

0.011

0.010

Ni

0.431

0.440

Li

0.003

0.001

Sb

0.003

0.005

Cu

0.001

0.001

469.01mg

/ml×

Reference Preparation

(KT05TA1)

Hg

Not detected

Not detected

Cd

0.00001

Not detected

20ml

Pb

0.007

0.007

As

0.010

0.017

Co

0.001

0.001

V

0.009

0.011

Ni

0.448

0.460

Li

0.003

Not detected

Sb

0.001

0.002

Cu

0.002

0.004

Related substances

The current editions of the Chinese Pharmacopoeia and USP43 do not specify a method for related substances of this product. Our company has established a method for determining related substances of this product by referencing the import registration standard JX20020190. Through methodological validation, including specificity, robustness, limit of detection and limit of quantitation, precision, solution stability, repeatability, and linearity, the validation results indicate that the proposed method meets the requirements for determining related substances of this product.

Under the established chromatographic conditions, 4 specifications of this product, totaling 8 batches of self-made samples and 2 batches of reference preparations, were tested, along with stability studies. The results are shown in Table 3.2.P.5-78. The proposed limits are as follows: From injection to the copper nitrate peak, if any impurity peaks appear, they should be calculated using the external standard method based on the peak area of the NTA reference standard. From the copper nitrate peak to the end of the chromatogram, if any impurity peaks appear, they should be calculated using the external standard method based on the peak area of the EDTA reference standard. The total amount of related substances must not exceed 0.3% of the labeled amount of pentetic acid (each 1ml of this product contains a total of 197mg of pentetic acid).

Table3.2.P.5-81Determination of Related Substances in Stability Samples

Specification

Batch number

Impurities

(%)

0 Month

Accelerated 6 months

40±2

75%±5%

Intermediate 6 months

30±2

65%±5%

Long-term 6 months

25±2

60%±5%

Upright

Inverted

Upright

Inverted

Upright

Inverted

20ml: 9.38g

020210801

Total Impurities

0.03%

0.09

0.09%

0.04%

0.05%

0.06%

0.05%

020210802

Total Impurities

0.03%

0.08%

0.08%

0.06%

0.05%

0.04%

0.05%

020210803

Total Impurities

0.03%

0.06%

0.1%

0.04%

0.04%

0.04%

0.05%

10ml: 4.69g

020210804

Total Impurities

0.02%

0.08%

0.08%

0.04%

0.04%

0.04%

0.05%

020210805

Total Impurities

0.03%

0.08%

0.08%

0.04%

0.04%

0.04%

0.05%

020210806

Total Impurities

0.03%

0.08%

0.08%

0.04%

0.04%

0.05%

0.05%

15ml: 7.04g

020210807

Total Impurities

0.03%

0.07%

0.08%

0.05%

0.05%

0.05%

0.04%

12ml: 5.63g

020210808

Total Impurities

0.03%

0.07%

0.07%

0.05%

0.04%

0.04%

0.04%

469.0

1mg/

ml× 15ml

Reference Preparation

(KT01B5

4)

Total Impurities

0.03%

0.09%

0.05%

0.04%

469.0

1mg/ ml×

Reference Preparation

(KT05T

A1)

Total Impurities

0.03%

0.07%

0.04%

0.04%

20ml

Bacterial endotoxins

With reference to the quality standards of Gadopentetate Dimeglumine Injection in Part II of the Chinese Pharmacopoeia 2020 edition and the imported drug registration standard JX20020190, the bacterial endotoxin limit for this product is proposed as follows: the amount of endotoxin per 1ml should be less than 0.5EU. Based on the proposed limit, the Limulus amebocyte lysate (LAL) reagent was selected, and preliminary screening and interference tests were conducted on this product to determine the feasibility of the method and the limit. According to the proposed method, 8 batches of self-prepared samples across 4 specifications were tested, and all results complied with the requirements, which were then incorporated into the quality standard.

Sterility

With reference to the sterility test method in General Chapter 1101 of Part IV of the Chinese Pharmacopoeia 2020 edition, the sterility test method for this product was proposed after validation according to the prescribed validation method.

According to the proposed method, 8 batches of self-prepared samples across 4 specifications were tested, and all results complied with the requirements, which were then incorporated into the quality standard.

Others

Refer to the relevant provisions in 2020 Edition of the Chinese Pharmacopoeia, General Rules Part IV, 0102. Other items include insoluble particles and visible foreign matter. Inspection of 8 self-prepared samples across 4 specifications showed compliance with the requirements, which were incorporated into the quality standards.

Content determination

Refer to the quality standard for Gadopentetate Meglumine Injection in the 2020 Edition of the Chinese Pharmacopoeia, Part II. The HPLC method was used to determine this item. A preliminary method for content determination was established. Method validation included durability, precision, solution stability, repeatability, linearity, and specificity. The retention time and theoretical plate number of the gadopentetate meglumine peak met the requirements for content determination. Solution stability studies showed that the solution remained stable at room temperature.

Under the established chromatographic conditions, 8 batches of self-prepared samples for each of the 4 specifications and 2 batches of reference preparations were inspected, along with stability studies, all of which met the requirements.

Table 3.2.P.5-82 Content Determination of Stability Samples

Specification

Batch number

0 Month

Accelerated 6 months

40°C±2°C75%±5%

Intermediate 6 months

30°C±2°C65%±5%

Long-term 6 months

25°C±2°C60%±5%

20ml:9.38g

020210801(Upright)

99.7%

99.6%

100.1%

100.0%

020210801(inverted)

100.9%

98.9%

99.8%

020210802(Upright)

101.0%

100.4%

100.0%

100.3%

020210802(inverted)

100.4%

99.2%

100.1%

020210803(upright)

101.4%

100.6%

100.5%

100.8%

020210803(inverted)

101.5%

99.8%

100.7%

10ml:4.69g

020210804(upright)

99.8%

98.7%

99.2%

99.6%

020210804(inverted)

98.4%

97.8%

99.4%

020210805(Upright)

99.1%

98.6%

98.2%

99.0%

020210805(inverted)

99.1%

97.4%

98.5%

020210806(Upright)

99.8%

98.6%

98.2%

99.4%

020210806(inverted)

99.3%

97.3%

98.8%

15ml:7.04g

020210807(Upright)

101.1%

99.8%

99.7%

100.3%

020210807(inverted)

100.1%

98.0%

100.4%

12ml:5.63g

020210808(upright)

99.0%

98.2%

98.9%

99.0%

020210808(inverted)

98.7%

97.0%

99.0%

469.01mg/ml

×15ml

Reference Preparation

(KT01B54)

104.2%

104.0%

104.4%

103.9%

469.01mg/ml

×20ml

Reference Preparation

(KT05TA1)

105.1%

103.6%

104.7%

104.7%

With reference to the quality standards for Gadopentetate Meglumine Injection in Part II of the Chinese Pharmacopoeia 2020 edition and based on the stability data of this product, the content limit is proposed as follows: the content of gadopentetate dimeglumine (C14H20GdN3O10 2C7H17NO5) should be 95.0%~105.0% of the labeled amount.

【Storage】

Based on the results of influence factor tests and stability studies, it has been demonstrated that light and temperature significantly affect the quality of this product. Therefore, the storage conditions for this product are defined as protection from light, sealed storage at temperatures not exceeding 30°C, and incorporated into the quality standards.

[Shelf Life]

The approved shelf life for this product is 36 months, and is incorporated into the quality standards.