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Title: Method Validation: Analysis, of Ergothioneine in Dried Mushroom Powder
标题：方法验证：干蘑菇粉中麦角硫因的分析

Table of Contents  目录

Title Page …  标题页 …
Table of Contents … ii
目录 … ii
I. Summary … 1
I. 摘要 … 1
II. Introduction … 2
II. 引言 … 2
III. Materials and Methods … 2
III. 材料与方法 … 2
Standard Preparation … 2
标准制备… 2
Sample Preparation … 3
样品制备 … 3
Sample Analysis on the HPLC … 3
样品在 HPLC 上的分析 … 3
IV. Results … 4
IV. 结果 … 4
V. Conclusions … 5
V. 结论 … 5
VI. References … 16
VI. 参考文献 … 16
VII. Approvals. … 17
VII. 批准。… 17
Figure 1. Structure of L-(+)-Ergothioneine … 6
图 1. L-(+)-麦角硫因的结构 … 6
Figure 2. HPLC Chromatogram and Report for Limit of Quantitation Ergothioneine Standard … 7
图 2. 定量限麦角硫因标准品的 HPLC 色谱图和报告 … 7
Figure 3. HPLC Chromatogram and Report for Limit of Detection Ergothioneine Standard … 8
图 3. HPLC 色谱图和检测限麦角硫因标准品的报告…8
Figure 4. Standard Curve and Linear Regression for Ergothioneine quantification … 9
图 4. Ergothioneine 定量的标准曲线和线性回归 … 9
Figure 5. HPLC Chromatogram and Report for low control of Ergothioneine … 10
图 5. Ergothioneine 低浓度对照的 HPLC 色谱图及报告 … 10
Figure 6. HPLC Chromatogram and Report for high control of Ergothioneine … 11
图 6. 高对照 Ergothioneine 的 HPLC 色谱图及报告…11
Figure 7. HPLC Chromatogram and Report for Ergothioneine in mushroom powder sample … 12
图 7. 蘑菇粉样品中麦角硫因的 HPLC 色谱图及报告 … 12
Table 1. Evaluation of the accuracy of Ergothioneine in control samples … 13
表 1. 对照样品中麦角硫因准确性的评估 … 13
Table 2. Amount of Ergothioneine detected in mushroom powder samples … 14
表 2. 蘑菇粉末样品中检测到的 Ergothioneine 含量 … 14
Table 3. Recovery of Ergothioneine in spiked mushroom powder samples … 15
表 3. 蘑菇粉末样品中 Ergothioneine 的回收率 … 15
I. Summary  I. 摘要
Modifications to published methodology (Dubost et., 2006;Chen et al., 2012) for measuring L- ( + ) ( + ) (+)(+)-Ergothioneine in dried mushroom powder using high pressure liquid chromatography (HPLC) were verified and validated in the laboratory. Standards of known concentration and samples were extracted with 10 mM DTT / 100 μ M 10 mM DTT / 100 μ M 10mMDTT//100 muM10 \mathrm{mM} \mathrm{DTT} / 100 \mu \mathrm{M} Betaine / 100 μ M MMI / 100 μ M MMI //100 muMMMI/ 100 \mu \mathrm{M} \mathrm{MMI} in 70 % 70 % 70%70 \% Ethanol and 1 % 1 % 1%1 \% SDS in 100 % 100 % 100%100 \% ethanol. 1 mL of the organic layer was collected, dried and reconstituted in 50 mM Sodium Phosphate / 3 % ACN / 0.1 % / 3 % ACN / 0.1 % //3%ACN//0.1%/ 3 \% \mathrm{ACN} / 0.1 \% TEA, pH = 7.3 pH = 7.3 pH=7.3\mathrm{pH}=7.3 and filtered. Samples were analyzed using reverse-phase HPLC. A linear standard curve ( R 2 = 1 R 2 = 1 R^(2)=1R^{2}=1 ) was generated using the response values for each known standard concentration. The standard curve was calculated using a linear equation over the range of standards used ( 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} to 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL} ). The assay was determined to be sensitive and specific for Ergothioneine, with a detection limit of 0.00195 mg / mL 0.00195 mg / mL 0.00195mg//mL0.00195 \mathrm{mg} / \mathrm{mL}.
对已发表的方法（Dubost 等，2006；Chen 等，2012）进行了修改，用于使用高压液相色谱法（HPLC）测量干燥蘑菇粉中的 L- ( + ) ( + ) (+)(+) -麦角硫因，并在实验室中进行了验证和确认。已知浓度的标准品和样品用 10 mM DTT / 100 μ M 10 mM DTT / 100 μ M 10mMDTT//100 muM10 \mathrm{mM} \mathrm{DTT} / 100 \mu \mathrm{M} 甜菜碱 / 100 μ M MMI / 100 μ M MMI //100 muMMMI/ 100 \mu \mathrm{M} \mathrm{MMI} 在 70 % 70 % 70%70 \% 乙醇和 1 % 1 % 1%1 \% SDS 在 100 % 100 % 100%100 \% 乙醇中提取。收集 1 mL 有机层，干燥并在 50 mM 磷酸钠 / 3 % ACN / 0.1 % / 3 % ACN / 0.1 % //3%ACN//0.1%/ 3 \% \mathrm{ACN} / 0.1 \% TEA pH = 7.3 pH = 7.3 pH=7.3\mathrm{pH}=7.3 中重新溶解并过滤。样品使用反相 HPLC 进行分析。使用每个已知标准浓度的响应值生成了线性标准曲线（ R 2 = 1 R 2 = 1 R^(2)=1R^{2}=1 ）。标准曲线使用线性方程在所用标准品范围内（ 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} 至 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL} ）计算。该测定方法被确定为对麦角硫因具有灵敏性和特异性，检测限为 0.00195 mg / mL 0.00195 mg / mL 0.00195mg//mL0.00195 \mathrm{mg} / \mathrm{mL} 。
The inter-assay variation was determined from replicate assays to be 2.42 % 2.42 % 2.42%2.42 \% based upon the 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} control value. The average intra-assay variation was 1.50 % 1.50 % 1.50%1.50 \%. The inter-assay variation for the mushroom powder sample analyzed was determined from replicate assays to be 2.28 % 2.28 % 2.28%2.28 \%. The average intra-assay variation was 0.90 % 0.90 % 0.90%0.90 \%. Recovery of ergothioneine from the spiked mushroom samples averaged 89.9 ± 5.53 % 89.9 ± 5.53 % 89.9+-5.53%89.9 \pm 5.53 \%. This method was determined to be valid for its intended use.
批次间变异通过重复测定确定为 2.42 % 2.42 % 2.42%2.42 \% ，基于 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} 的对照值。平均批次内变异为 1.50 % 1.50 % 1.50%1.50 \% 。蘑菇粉末样品的批次间变异通过重复测定确定为 2.28 % 2.28 % 2.28%2.28 \% 。平均批次内变异为 0.90 % 0.90 % 0.90%0.90 \% 。从加标蘑菇样品中回收的麦角硫因平均为 89.9 ± 5.53 % 89.9 ± 5.53 % 89.9+-5.53%89.9 \pm 5.53 \% 。该方法被确定为适用于其预期用途。

II. Introduction  II. 引言

The amino acid, L-(+)-ergothioneine, is a naturally occurring antioxidant that is found in dietary sources, such as mushrooms and meat and is also water soluble. The presence of anti-oxidants in a diet can serve as protective agents against oxidative stress and damage. Ergothioneine is not naturally synthesized by plants, animals or humans. The amino acid is transported from soil to the roots of plants consumed by animals and humans. Soil conditions strongly influence the amount of ergothioneine found in various plants. 1 , 2 1 , 2 ^(1,2){ }^{1,2} Previous methods for extracting ergothioneine have been developed, however, require a sizeable amount of sample ( 1 g ). Our laboratory was able to validate and verify that ergothioneine levels are detectable in 100 mg of sample. The following report details the method validation and verification performed in our laboratory.
氨基酸 L-(+)-麦角硫因是一种天然存在的抗氧化剂，存在于蘑菇和肉类等膳食来源中，并且也是水溶性的。饮食中的抗氧化剂可以作为氧化应激和损伤的保护剂。麦角硫因不能由植物、动物或人类自然合成。这种氨基酸从土壤运输到被动物和人类食用的植物根部。土壤条件强烈影响各种植物中麦角硫因的含量。 1 , 2 1 , 2 ^(1,2){ }^{1,2} 以前提取麦角硫因的方法已经开发出来，但需要大量的样品（1 克）。我们的实验室能够验证并确认在 100 毫克的样品中可以检测到麦角硫因的水平。以下报告详细介绍了我们实验室进行的方法验证和确认。

III. Materials and Methods
III. 材料与方法

Standard/Control Preparation (F-162; P-143):
标准/对照品制备 (F-162; P-143):

Using form F-162, ergothioneine standards were prepared using 1.5 N perchloric acid (Fisher Scientific, Waltham, MA) cleaned glassware. Roughly 5 mg of ergothioneine standard (SigmaAldrich, St. Louis, MO) was weighed into a 1.5 N perchloric acid cleaned 10 mL amber volumetric flask and the flask filled to the 10 mL mark with reverse osmosis water (Metabolic Technologies, Inc., Ames, IA). A stir bar and stir plate were used to stir the mixture for 5 minutes. The standard stock was aliquoted into 1.5 mL cryovials and stored in − 20 ∘ C − 20 ∘ C -20^(@)C-20^{\circ} \mathrm{C}.
使用 F-162 表格，使用 1.5 N 高氯酸（Fisher Scientific, Waltham, MA）清洗的玻璃器皿制备了麦角硫因标准品。将大约 5 mg 的麦角硫因标准品（SigmaAldrich, St. Louis, MO）称入一个用 1.5 N 高氯酸清洗的 10 mL 琥珀色容量瓶中，并用反渗透水（Metabolic Technologies, Inc., Ames, IA）将瓶子加至 10 mL 刻度。使用搅拌棒和搅拌板将混合物搅拌 5 分钟。将标准品母液分装到 1.5 mL 的冷冻管中，并储存在 − 20 ∘ C − 20 ∘ C -20^(@)C-20^{\circ} \mathrm{C} 。
Ergothioneine standards of the following concentrations were prepared directly in the 25 × 150 25 × 150 25 xx15025 \times 150 mm glass cultutre tubes containing 20 mL of ethanolic extraction buffer: 0.0039 mg / mL , 0.0078 0.0039 mg / mL , 0.0078 0.0039mg//mL,0.00780.0039 \mathrm{mg} / \mathrm{mL}, 0.0078 mg / mL , 0.03125 mg / mL , 0.0625 mg / mL mg / mL , 0.03125 mg / mL , 0.0625 mg / mL mg//mL,0.03125mg//mL,0.0625mg//mL\mathrm{mg} / \mathrm{mL}, 0.03125 \mathrm{mg} / \mathrm{mL}, 0.0625 \mathrm{mg} / \mathrm{mL} and 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL}. To determine the volume of ergothioneine stock needed to prepare the above concentrations, the following equation was used: C 1 V 1 = C 2 V 2 C 1 V 1 = C 2 V 2 C_(1)V_(1)=C_(2)V_(2)C_{1} V_{1}=C_{2} V_{2}, where C 1 C 1 C_(1)C_{1} is the concentration of the stock solution, C 2 C 2 C_(2)C_{2} is the concentration of the desired standard solution, V 2 V 2 V_(2)\mathrm{V}_{2} is the total volume of the desired standard solution and V 1 V 1 V_(1)\mathrm{V}_{1} is the volume of stock solution added. To ensure normalization of all standards, V 2 V 2 V_(2)\mathrm{V}_{2} was equal to 1 mL . RO H2O was used to make up for the difference between 1 mL total volume and the amount of ergothioneine stock added. Standards were vortexed briefly after this addition. Additionally, a 0.0078 mg / mL 0.0078 mg / mL 0.0078mg//mL0.0078 \mathrm{mg} / \mathrm{mL} and 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} ergothioneine standards were prepared to serve as low and high controls for the assay.
以下浓度的 Ergothioneine 标准品直接在含有 20 mL 乙醇提取缓冲液的 25 × 150 25 × 150 25 xx15025 \times 150 mm 玻璃培养管中制备： 0.0039 mg / mL , 0.0078 0.0039 mg / mL , 0.0078 0.0039mg//mL,0.00780.0039 \mathrm{mg} / \mathrm{mL}, 0.0078 、 mg / mL , 0.03125 mg / mL , 0.0625 mg / mL mg / mL , 0.03125 mg / mL , 0.0625 mg / mL mg//mL,0.03125mg//mL,0.0625mg//mL\mathrm{mg} / \mathrm{mL}, 0.03125 \mathrm{mg} / \mathrm{mL}, 0.0625 \mathrm{mg} / \mathrm{mL} 和 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL} 。为了确定制备上述浓度所需的 Ergothioneine 原液体积，使用了以下方程： C 1 V 1 = C 2 V 2 C 1 V 1 = C 2 V 2 C_(1)V_(1)=C_(2)V_(2)C_{1} V_{1}=C_{2} V_{2} ，其中 C 1 C 1 C_(1)C_{1} 是原液浓度， C 2 C 2 C_(2)C_{2} 是所需标准溶液的浓度， V 2 V 2 V_(2)\mathrm{V}_{2} 是所需标准溶液的总体积， V 1 V 1 V_(1)\mathrm{V}_{1} 是添加的原液体积。为了确保所有标准品的归一化， V 2 V 2 V_(2)\mathrm{V}_{2} 等于 1 mL。使用 RO H2O 来弥补 1 mL 总体积与添加的 Ergothioneine 原液体积之间的差异。添加后，标准品短暂涡旋混合。此外，还制备了 0.0078 mg / mL 0.0078 mg / mL 0.0078mg//mL0.0078 \mathrm{mg} / \mathrm{mL} 和 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} 的 Ergothioneine 标准品，作为测定的低和高对照。
One liter of ethanolic extraction buffer ( 10 mM 1,4-dithiothreitol (DTT)/ 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} betaine/100 μ M μ M muM\mu \mathrm{M} 2-Mercapto-1-methylimidazole (MMI) in 70% ethanol) was prepared according to procedure P-143, section 5.1.2. To a 1 L volumetric flask wrapped in aluminum foil (MMI-light sensitive compound), 700 mL of 200 proof ethanol was added. Using weigh paper, the following amounts of each chemical were weighed out to achieve their desired concentrations and then added to the flask: 10 mM DTT (Fisher Scientific, Waltham, MA): 1.5425 g , 100 μ M 1.5425 g , 100 μ M 1.5425g,100 muM1.5425 \mathrm{~g}, 100 \mu \mathrm{M} betaine (Sigma-Aldrich, St. Louis, MO): 11.715 mg and 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} MMI (Fisher Scientific, Waltham, MA): 11.417 mg . The following equation was used to determine the amount of each compound required to achieve its desired concentration:
根据程序 P-143 第 5.1.2 节，制备了一升乙醇提取缓冲液（10 mM 1,4-二硫苏糖醇（DTT）/ 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} 甜菜碱/100 μ M μ M muM\mu \mathrm{M} 2-巯基-1-甲基咪唑（MMI）于 70%乙醇中）。向一个用铝箔包裹的 1 升容量瓶（MMI 为光敏感化合物）中，加入 700 毫升 200 proof 乙醇。使用称量纸，称取以下每种化学品的量以达到其所需浓度，然后加入瓶中：10 mM DTT（Fisher Scientific，Waltham，MA）： 1.5425 g , 100 μ M 1.5425 g , 100 μ M 1.5425g,100 muM1.5425 \mathrm{~g}, 100 \mu \mathrm{M} 甜菜碱（Sigma-Aldrich，St. Louis，MO）：11.715 mg 和 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} MMI（Fisher Scientific，Waltham，MA）：11.417 mg。使用以下公式确定每种化合物达到其所需浓度所需的量：
Amount of compound required ( g ) = ( g ) = (g)=(\mathrm{g})= Desired concentration ( mol / L mol / L mol//L\mathrm{mol} / \mathrm{L} )*compound’s molecular weight ( g / mol g / mol g//mol\mathrm{g} / \mathrm{mol} )*total volume of solution being prepared ( L )
所需化合物量 ( g ) = ( g ) = (g)=(\mathrm{g})= = 所需浓度 ( mol / L mol / L mol//L\mathrm{mol} / \mathrm{L} ) * 化合物分子量 ( g / mol g / mol g//mol\mathrm{g} / \mathrm{mol} ) * 溶液总体积 ( L )
The 1 L volumetric flask was then brought to volume with RO H 2 O . A stir bar and stir plate were used to mix the ethanolic extraction buffer thoroughly until all dry ingredients were completely dissolved. The solution was then transferred to a 1 L foil-wrapped glass screwcap bottle.
将 1 L 容量瓶用 RO H2O 加至刻度。使用搅拌棒和搅拌板彻底混合乙醇提取缓冲液，直到所有干成分完全溶解。然后将溶液转移至 1 L 铝箔包裹的玻璃螺口瓶中。
A 10 mL glass seriological pipette and dispenser were used to pipette 20 mL of extraction buffer directly into 1.5 N perchloric acid cleaned 25 × 150 mm 25 × 150 mm 25 xx150mm25 \times 150 \mathrm{~mm} glass screwcap culture tubes.
使用 10 mL 玻璃血清移液管和分配器将 20 mL 提取缓冲液直接移入用 1.5 N 高氯酸清洗的 25 × 150 mm 25 × 150 mm 25 xx150mm25 \times 150 \mathrm{~mm} 玻璃螺旋盖培养管中。

Sample Preparation (F-162; F-163):
样品制备（F-162；F-163）：

Roughly 100 mg of dried mushroom powder was weighed out onto weigh paper and transferred to a 1.5 N perchloric acid cleaned 25 × 150 mm 25 × 150 mm 25 xx150mm25 \times 150 \mathrm{~mm} glass screwcap culture tube and the weight recorded using F-163, followed by the addition of 20 mL ethanolic extraction buffer directly to the sample using a 10 mL glass seriological pipette and dispenser. Samples were vortexed thoroughly. Additionally, 4 mL of 1 % 1 % 1%1 \% SDS in 100 % 100 % 100%100 \% EtOH was added to the standards, controls and samples. All tubes were thoroughly vortexed to homogenize samples.
大约 100 毫克干燥蘑菇粉末称重到称重纸上，并转移到 1.5 N 高氯酸清洗的 25 × 150 mm 25 × 150 mm 25 xx150mm25 \times 150 \mathrm{~mm} 玻璃螺旋盖培养管中，使用 F-163 记录重量，然后使用 10 mL 玻璃血清移液管和分配器直接将 20 mL 乙醇提取缓冲液加入样品中。样品充分涡旋。此外，向标准品、对照品和样品中加入 4 mL 的 1 % 1 % 1%1 \% SDS 在 100 % 100 % 100%100 \% EtOH 中的溶液。所有管子充分涡旋以使样品均质化。
All standards, controls and samples were centrifuged at 3000 rpm for 20 minutes at 25 ∘ C 25 ∘ C 25^(@)C25^{\circ} \mathrm{C}. Following centrifugation, 1 mL of supernatant was collected from the top layer of each standard, control and sample and transferred to its respective new 12 × 75 mm 12 × 75 mm 12 xx75mm12 \times 75 \mathrm{~mm} disposable glass test tube. The supernatant was dried using a constant stream of N 2 N 2 N_(2)\mathrm{N}_{2} gas at 40 ± 4 ∘ C 40 ± 4 ∘ C 40+-4^(@)C40 \pm 4^{\circ} \mathrm{C}. Standards, controls and samples were reconstituted with 0.5 mL of 50 mM Sodium Phosphate / 3 % / 3 % //3%/ 3 \% ACN / 0.1 % / 0.1 % //0.1%/ 0.1 \% TEA, pH = 7.3 pH = 7.3 pH=7.3\mathrm{pH}=7.3 buffer solution (see P − 143 P − 143 P-143\mathrm{P}-143, section 5.1 .4 for preparing solution). All tubes were capped, briefly vortexed and then transferred to 0.45 μ m 0.45 μ m 0.45 mum0.45 \mu \mathrm{~m} spin filter microcentrifuge tubes using a glass Pasteur pipette. The microcentrifuge tubes were centrifuged for 1 minute at 10 , 000 rpm .150 10 , 000 rpm .150 10,000rpm.15010,000 \mathrm{rpm} .150 μ L μ L muL\mu \mathrm{L} of each filtered standard, control and sample was pipetted into injection inserts in 2 mL Amber HPLC vials for HPLC analysis.
所有标准品、对照品和样品在 25 ∘ C 25 ∘ C 25^(@)C25^{\circ} \mathrm{C} 下以 3000 rpm 离心 20 分钟。离心后，从每个标准品、对照品和样品的顶层收集 1 mL 上清液，并转移到各自的新 12 × 75 mm 12 × 75 mm 12 xx75mm12 \times 75 \mathrm{~mm} 一次性玻璃试管中。上清液在 40 ± 4 ∘ C 40 ± 4 ∘ C 40+-4^(@)C40 \pm 4^{\circ} \mathrm{C} 下使用恒定气流 N 2 N 2 N_(2)\mathrm{N}_{2} 进行干燥。标准品、对照品和样品用 0.5 mL 的 50 mM 磷酸钠 / 3 % / 3 % //3%/ 3 \% ACN / 0.1 % / 0.1 % //0.1%/ 0.1 \% TEA, pH = 7.3 pH = 7.3 pH=7.3\mathrm{pH}=7.3 缓冲溶液重新配制（参见 P − 143 P − 143 P-143\mathrm{P}-143 ，第 5.1.4 节以准备溶液）。所有试管加盖，短暂涡旋，然后使用玻璃巴斯德吸管转移到 0.45 μ m 0.45 μ m 0.45 mum0.45 \mu \mathrm{~m} 旋转过滤微量离心管中。微量离心管在 10 , 000 rpm .150 10 , 000 rpm .150 10,000rpm.15010,000 \mathrm{rpm} .150 下离心 1 分钟，每个过滤后的标准品、对照品和样品的 μ L μ L muL\mu \mathrm{L} 被移入 2 mL 琥珀色 HPLC 小瓶的进样插管中，用于 HPLC 分析。

Sample Analysis on the HPLC (P-143):
HPLC (P-143) 上的样品分析：

The filtered standards, controls and samples were analyzed with an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA), using a Kinetex 5 μ m 5 μ m 5mum5 \mu \mathrm{~m} C18 100 Å ̊ 100 Å ̊ 100"Å"̊100 \AA ̊Å column, 250 × 4.6 mm 250 × 4.6 mm 250 xx4.6mm250 \times 4.6 \mathrm{~mm} (Phenomenex, Torrance, CA). The mobile phase used was a 50 mM Sodium Phosphate/3% ACN / 0.1 % ACN / 0.1 % ACN//0.1%\mathrm{ACN} / 0.1 \% TEA, pH = 7.3 pH = 7.3 pH=7.3\mathrm{pH}=7.3 buffer solution. The following HPLC parameters were used for analysis: flow rate of 0.4 mL / min 0.4 mL / min 0.4mL//min0.4 \mathrm{~mL} / \mathrm{min}, column temperature of 40 ∘ C 40 ∘ C 40^(@)C40^{\circ} \mathrm{C}, UV detection limit set at 254 nm , injection volume of 5 μ L 5 μ L 5muL5 \mu \mathrm{~L} and a run time of 7 minutes and a 23 minute post-run. The retention time for ergothioneine is ∼ 6.4 ∼ 6.4 ∼6.4\sim 6.4 minutes.
过滤后的标准品、对照品和样品使用 Agilent 1100 系列 HPLC（Agilent Technologies，Santa Clara, CA）进行分析，采用 Kinetex 5 μ m 5 μ m 5mum5 \mu \mathrm{~m} C18 100 Å ̊ 100 Å ̊ 100"Å"̊100 \AA ̊Å 色谱柱（Phenomenex, Torrance, CA）。流动相为 50 mM 磷酸钠/3% ACN / 0.1 % ACN / 0.1 % ACN//0.1%\mathrm{ACN} / 0.1 \% TEA, pH = 7.3 pH = 7.3 pH=7.3\mathrm{pH}=7.3 缓冲溶液。分析时采用以下 HPLC 参数：流速为 0.4 mL / min 0.4 mL / min 0.4mL//min0.4 \mathrm{~mL} / \mathrm{min} ，柱温为 40 ∘ C 40 ∘ C 40^(@)C40^{\circ} \mathrm{C} ，紫外检测限设定为 254 nm，进样体积为 5 μ L 5 μ L 5muL5 \mu \mathrm{~L} ，运行时间为 7 分钟，后运行时间为 23 分钟。麦角硫因的保留时间为 ∼ 6.4 ∼ 6.4 ∼6.4\sim 6.4 分钟。
A six point external standard curve was created by averaging the peak area for each standard run prior to samples being run and after samples had been run. Peak areas for the standards, controls and samples were entered into an Excel spreadsheet. Quantification of the unknown amount of ergothioneine in the samples was determined by a linear equation determined by linear regression analysis of the average ergothioneine peak areas used to generate the standard curve. To determine the amount of ergothioneine assayed per sample (mg), the
通过在每个标准品运行前和样品运行后对每个标准品的峰面积进行平均，创建了一个六点外部标准曲线。标准品、对照品和样品的峰面积被输入到 Excel 电子表格中。样品中未知量的麦角硫因的定量是通过线性回归分析生成标准曲线的平均麦角硫因峰面积所确定的线性方程来确定的。为了确定每个样品中测定的麦角硫因的量（mg），
ergothioneine peak areas for controls and samples were divided by the slope of regression. The amount of ergothioneine assayed per g of dry weight for each sample was calculated by dividing the assayed amount ( mg ) by the initial dry weight of the sample.
对照和样品的 ergothioneine 峰面积除以回归斜率。每个样品中每克干重的 ergothioneine 含量通过将测定的量（mg）除以样品的初始干重计算得出。

IV. Results  IV. 结果

The structure for L-(+)-Ergothioneine is shown in Figure 1. The standard curve for quantitation of Ergothioneine in unknown samples is shown in Figure 2. The average peak area of ergothioneine was plotted for each of the 6 known concentrations. The dynamic range of the curve was 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} to 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL} ergothionenine. The linear regression for ergothioneine was: 3789075.61 ∗ 3789075.61 ∗ 3789075.61^(**)3789075.61^{*} (observed response)- 5924.90 with an R 2 = 1 R 2 = 1 R^(2)=1R^{2}=1 as shown in Figure 2. The limit of quantitation was determined to be 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} as shown in Figure 3. The limit of detection was determined to be 0.00195 mg / mL 0.00195 mg / mL 0.00195mg//mL0.00195 \mathrm{mg} / \mathrm{mL} as shown in Figure 4.
L-(+)-麦角硫因的结构如图 1 所示。未知样品中麦角硫因的定量标准曲线如图 2 所示。麦角硫因的平均峰面积绘制了 6 个已知浓度中的每一个。曲线的动态范围为 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} 到 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL} 麦角硫因。麦角硫因的线性回归为： 3789075.61 ∗ 3789075.61 ∗ 3789075.61^(**)3789075.61^{*} （观察到的响应）- 5924.90， R 2 = 1 R 2 = 1 R^(2)=1R^{2}=1 如图 2 所示。定量限确定为 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} ，如图 3 所示。检测限确定为 0.00195 mg / mL 0.00195 mg / mL 0.00195mg//mL0.00195 \mathrm{mg} / \mathrm{mL} ，如图 4 所示。
The amount of ergothioneine assayed, percent accuracy, absolute error, inter-assay variation and intra-assay variation were calculated for the ergothioneine quality controls over 4 assays. All values were calculated for both low and high concentration quality controls: 0.0078 mg / mL 0.0078 mg / mL 0.0078mg//mL0.0078 \mathrm{mg} / \mathrm{mL} and 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} (Figures 5,6; Table 1). The average amount of ergothioneine assayed was 0.0079 mg for the low control and 0.0609 mg for the high control. Percent accuracy and absolute error results were 0.95 % 0.95 % 0.95%0.95 \% and 0.0001 mg for the low control and − 2.59 % − 2.59 % -2.59%-2.59 \% and -0.0016 mg for the high control. For the low control, intra-assay variation ranged from 0.24 % 0.24 % 0.24%0.24 \% to 4.95 % 4.95 % 4.95%4.95 \% and had an average of 2.08 % 2.08 % 2.08%2.08 \%. The inter-assay variation was calculated to be 11.95 % 11.95 % 11.95%11.95 \%. For the high control, intra-assay variation ranged from 0.06 % 0.06 % 0.06%0.06 \% to 4.59 % 4.59 % 4.59%4.59 \% and had an average of 1.65 % 1.65 % 1.65%1.65 \%. The inter-assay variation was calculated to be 2.42 % 2.42 % 2.42%2.42 \%.
在 4 次测定中，计算了麦角硫因质量控制品的麦角硫因测定量、准确度百分比、绝对误差、测定间变异和测定内变异。所有值均针对低浓度和高浓度质量控制品计算： 0.0078 mg / mL 0.0078 mg / mL 0.0078mg//mL0.0078 \mathrm{mg} / \mathrm{mL} 和 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} （图 5,6；表 1）。低浓度对照品的麦角硫因平均测定量为 0.0079 mg，高浓度对照品为 0.0609 mg。低浓度对照品的准确度百分比和绝对误差结果分别为 0.95 % 0.95 % 0.95%0.95 \% 和 0.0001 mg，高浓度对照品为 − 2.59 % − 2.59 % -2.59%-2.59 \% 和-0.0016 mg。对于低浓度对照品，测定内变异范围为 0.24 % 0.24 % 0.24%0.24 \% 至 4.95 % 4.95 % 4.95%4.95 \% ，平均为 2.08 % 2.08 % 2.08%2.08 \% 。测定间变异计算为 11.95 % 11.95 % 11.95%11.95 \% 。对于高浓度对照品，测定内变异范围为 0.06 % 0.06 % 0.06%0.06 \% 至 4.59 % 4.59 % 4.59%4.59 \% ，平均为 1.65 % 1.65 % 1.65%1.65 \% 。测定间变异计算为 2.42 % 2.42 % 2.42%2.42 \% 。
The average amount of ergothioneine assayed from dried mushroom powder across 4 assays (Table 2) was 0.092 mg , with a standard error of 0.0006 mg . The intra-assay variation ranged from 0.24 % 0.24 % 0.24%0.24 \% to 2.18 % 2.18 % 2.18%2.18 \% and had an average of 0.90 % 0.90 % 0.90%0.90 \%. The inter-assay variation was calculated to be 2.28 % 2.28 % 2.28%2.28 \%. Results were reported as mg of ergothioneine assayed per g of sample’s dry weight, therefore, the average amount of ergothioneine detected was 0.92 ± 0.006 mg / g 0.92 ± 0.006 mg / g 0.92+-0.006mg//g0.92 \pm 0.006 \mathrm{mg} / \mathrm{g} with a CV of 2.28%.
从干燥蘑菇粉末中测得的麦角硫因平均含量（表 2）为 0.092 mg，标准误差为 0.0006 mg。测定内变异范围为 0.24 % 0.24 % 0.24%0.24 \% 至 2.18 % 2.18 % 2.18%2.18 \% ，平均值为 0.90 % 0.90 % 0.90%0.90 \% 。测定间变异计算为 2.28 % 2.28 % 2.28%2.28 \% 。结果以每克样品干重中测得的麦角硫因毫克数报告，因此，检测到的麦角硫因平均含量为 0.92 ± 0.006 mg / g 0.92 ± 0.006 mg / g 0.92+-0.006mg//g0.92 \pm 0.006 \mathrm{mg} / \mathrm{g} ，变异系数为 2.28%。
Percentage recovery was calculated by spiking 0.0078 mg and 0.0625 mg of ergothioneine into dried mushroom powder samples, in duplicate, for each assay (Table 3). Percentage recovery was determined by subtracting the average amount of ergothioneine assayed in non-spiked samples (Figure 7) from the amount of ergothioneine assayed in each spiked sample, then dividing by the original amount of ergothioneine spiked into the sample. The average percentage recovery for samples spiked with 0.0078 mg of ergothioneine was 80.1 ± 4.3 % 80.1 ± 4.3 % 80.1+-4.3%80.1 \pm 4.3 \%, an intra-assay variation ranging from 1.47 % 1.47 % 1.47%1.47 \% to 8.85 % 8.85 % 8.85%8.85 \%, with an average intra-assay variation of 5.2 % 5.2 % 5.2%5.2 \%. The inter-assay variation was calculated to be 10.9 % 10.9 % 10.9%10.9 \%, respectively. For the samples spiked with 0.0625 mg of ergothioneine, the average percent recovery was 103.0 ± 8.45 % 103.0 ± 8.45 % 103.0+-8.45%103.0 \pm 8.45 \% and an average intra-assay variation of 9.79 % 9.79 % 9.79%9.79 \%. The inter-assay variation was calculated to be 14.2 % 14.2 % 14.2%14.2 \%, respectively.
通过向干燥蘑菇粉末样品中分别加入 0.0078 mg 和 0.0625 mg 的 ergothioneine（每个测定重复两次），计算了回收率（表 3）。回收率是通过从每个加标样品中测定的 ergothioneine 量中减去未加标样品中测定的 ergothioneine 平均量，然后除以加入样品中的原始 ergothioneine 量来确定的。对于加入 0.0078 mg ergothioneine 的样品，平均回收率为 80.1 ± 4.3 % 80.1 ± 4.3 % 80.1+-4.3%80.1 \pm 4.3 \% ，测定内变异范围为 1.47 % 1.47 % 1.47%1.47 \% 至 8.85 % 8.85 % 8.85%8.85 \% ，平均测定内变异为 5.2 % 5.2 % 5.2%5.2 \% 。测定间变异计算为 10.9 % 10.9 % 10.9%10.9 \% 。对于加入 0.0625 mg ergothioneine 的样品，平均回收率为 103.0 ± 8.45 % 103.0 ± 8.45 % 103.0+-8.45%103.0 \pm 8.45 \% ，平均测定内变异为 9.79 % 9.79 % 9.79%9.79 \% 。测定间变异计算为 14.2 % 14.2 % 14.2%14.2 \% 。

V. Conclusions  V. 结论

This report demonstrates a sensitive and specific method for the quantitation of ergothioneine in dry mushroom powder. Recovery of ergothioneine from the spiked mushroom samples averaged 89.9 ± 5.53 % 89.9 ± 5.53 % 89.9+-5.53%89.9 \pm 5.53 \%, an average intra-assay variation of 6.7 % 6.7 % 6.7%6.7 \% and inter-assay variation of 16.3 % 16.3 % 16.3%16.3 \%. Accuracy of evaluating ergothioneine in all control samples across repeated assays had an average of − 0.82 % − 0.82 % -0.82%-0.82 \%. The average intra-assay variation across all samples was 0.90 % 0.90 % 0.90%0.90 \% and the inter-variation was calculated to be 2.28 % 2.28 % 2.28%2.28 \%. The overall CV between assays was determined in repeated assays to be 1.71 % 1.71 % 1.71%1.71 \% based upon the 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} control value. This method was determined to be appropriate for its intended use.
本报告展示了一种灵敏且特异的方法，用于定量干蘑菇粉中的麦角硫因。从加标蘑菇样品中回收麦角硫因的平均值为 89.9 ± 5.53 % 89.9 ± 5.53 % 89.9+-5.53%89.9 \pm 5.53 \% ，平均批内变异为 6.7 % 6.7 % 6.7%6.7 \% ，批间变异为 16.3 % 16.3 % 16.3%16.3 \% 。在所有对照样品中评估麦角硫因的准确度在重复测定中的平均值为 − 0.82 % − 0.82 % -0.82%-0.82 \% 。所有样品的平均批内变异为 0.90 % 0.90 % 0.90%0.90 \% ，批间变异计算为 2.28 % 2.28 % 2.28%2.28 \% 。基于 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} 对照值，重复测定中确定的测定间总体变异系数为 1.71 % 1.71 % 1.71%1.71 \% 。该方法被确定为适合其预期用途。
Figure 1. Structure of L-(+)-Ergothioneine.
图 1. L-(+)-麦角硫因的结构。
SUMMARY OUTPUT
Regression Statistics  回归统计
Multiple R  多重 R 0.999926656
R Square 0.999853318
Adjusted R Square  调整后的 R 方 0.999816647
Standard Error  标准误差 4942.984995
Observations  观察结果 6
Regression Statistics Multiple R 0.999926656 R Square 0.999853318 Adjusted R Square 0.999816647 Standard Error 4942.984995 Observations 6| Regression Statistics | | | :--- | ---: | | Multiple R | 0.999926656 | | R Square | 0.999853318 | | Adjusted R Square | 0.999816647 | | Standard Error | 4942.984995 | | Observations | 6 |
ANOVA
d f d f dfd f SS MS F F FF Significance F F FF  重要性 F F FF
Regression  回归 1 6.66189 E + 11 6.66189 E + 11 6.66189E+116.66189 \mathrm{E}+11 6.66189 E + 11 6.66189 E + 11 6.66189E+116.66189 \mathrm{E}+11 27265.8572 8.06876 E − 09 8.06876 E − 09 8.06876E-098.06876 \mathrm{E}-09
Residual  残留 4 97732402.64 24433100.66
Total  总计 5 6.66287 E + 11 6.66287 E + 11 6.66287E+116.66287 \mathrm{E}+11
df SS MS F Significance F Regression 1 6.66189E+11 6.66189E+11 27265.8572 8.06876E-09 Residual 4 97732402.64 24433100.66 Total 5 6.66287E+11 | | $d f$ | | SS | MS | $F$ | Significance $F$ | | :--- | :---: | :---: | :---: | :---: | :---: | ---: | | Regression | | 1 | $6.66189 \mathrm{E}+11$ | $6.66189 \mathrm{E}+11$ | 27265.8572 | $8.06876 \mathrm{E}-09$ | | Residual | 4 | 97732402.64 | 24433100.66 | | | | | Total | 5 | $6.66287 \mathrm{E}+11$ | | | | |
Coefficients  系数 Standard Error  标准误差 tStat P-value  P 值 Lower 95%  下限 95% Upper 95%  上限 95% Lower 95.0%  下限 95.0% Upper 95.0%  上界 95.0%
Intercept  截距 -5924.90438 2433.142671 -2.43508301 0.0715871 -12680.39144 830.5826787 -12680.39144 830.5826787
concentration (mg)  浓度 (mg) 3789075.612 22946.88259 165.1237634 8.0688 E − 09 8.0688 E − 09 8.0688E-098.0688 \mathrm{E}-09 3725364.852 3852786.372 3725364.852 3852786.372
Coefficients Standard Error tStat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0% Intercept -5924.90438 2433.142671 -2.43508301 0.0715871 -12680.39144 830.5826787 -12680.39144 830.5826787 concentration (mg) 3789075.612 22946.88259 165.1237634 8.0688E-09 3725364.852 3852786.372 3725364.852 3852786.372| | Coefficients | Standard Error | tStat | P-value | Lower 95% | Upper 95% | Lower 95.0% | Upper 95.0% | | :--- | :--- | :--- | :--- | :--- | :--- | :--- | :--- | :--- | :--- | | Intercept | -5924.90438 | 2433.142671 | -2.43508301 | 0.0715871 | -12680.39144 | 830.5826787 | -12680.39144 | 830.5826787 | | concentration (mg) | 3789075.612 | 22946.88259 | 165.1237634 | $8.0688 \mathrm{E}-09$ | 3725364.852 | 3852786.372 | 3725364.852 | 3852786.372 |
Figure 2. Standard Curve and Linear Regression results for Ergothioneine quantification. The dynamic range of the curve is 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} to 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL}.
图 2. Ergothioneine 定量的标准曲线和线性回归结果。曲线的动态范围为 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} 到 0.25 mg / mL 0.25 mg / mL 0.25mg//mL0.25 \mathrm{mg} / \mathrm{mL} 。
date C:\CHEM32\I\DATA \MA263_1才\MA26317 2017-04-24 14-09-48\MA263 170000002.D
日期 C:\CHEM32\I\DATA\MA263_1 才\MA26317 2017-04-24 14-09-48\MA263 170000002.D
Sample Name: Ergothioneine Std 0.0039
样品名称: Ergothioneine Std 0.0039
Area Percent Report  面积百分比报告
Sorted By  按排序 : Signal  信号
Multiplier:  乘数： : 1.0000
Dilution:  稀释： : 1.0000
Area Percent Report Sorted By : Signal Multiplier: : 1.0000 Dilution: : 1.0000| | Area Percent Report | | | | :---: | :---: | :---: | :---: | | Sorted By | : | Signal | | | Multiplier: | | : | 1.0000 | | Dilution: | | : | 1.0000 |
Use Multiplier & Dilution Factor with ISTDs
使用乘数和稀释因子与 ISTDs
Siqnai 1 : 1 : 1:1: DAD1 B, Sig=254, 16 Ref = 360 , 100 = 360 , 100 =360,100=360,100

** End of Report **
** 报告结束 **
Figure 3. HPLC Chromatogram and Report for LOQ Ergothioneine standard, 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL}.
图 3. LOQ Ergothioneine 标准的 HPLC 色谱图及报告， 0.0039 mg / mL 0.0039 mg / mL 0.0039mg//mL0.0039 \mathrm{mg} / \mathrm{mL} 。
No peaks found  未发现峰
** End of Report ***
** 报告结束 ***
Figure 4. HPLC Chromatogram and Report for LOD Ergothioneine standard, 0.00195 mg / mL 0.00195 mg / mL 0.00195mg//mL0.00195 \mathrm{mg} / \mathrm{mL}.
图 4. LOD Ergothioneine 标准的 HPLC 色谱图及报告， 0.00195 mg / mL 0.00195 mg / mL 0.00195mg//mL0.00195 \mathrm{mg} / \mathrm{mL} 。
Sample Name: Ergothioneine Control 0.0078 Std B
样品名称: Ergothioneine Control 0.0078 Std B
Area Percent Report  面积百分比报告
Sorted By  按排序 : Signal  信号
Multiplier:  乘数： 1.0000
Dilution:  稀释： 1.0000
Use Multiplier Dilution Factor with ISTDs
使用 ISTDs 的乘数稀释因子
Signal 1: DAD1 B, Sig-254, 16 Ref=360, 100
信号 1：DAD1 B，Sig-254，16 Ref=360，100
Peak RetTime Type | [min] Width [min]  宽度 [分钟] Area [ m A U ∗ s ]  Area  [ m A U ∗ s ] {:[" Area "],[[mAU**s]]:}\begin{gathered} \text { Area } \\ {[m A U * s]} \end{gathered} Height [mAU]  高度 [mAU] Area !  区域 !
16.364 MM 0.1053 18.21606 2.883 100.000
Totals:  总计： 18.21606 2.883
Area Percent Report Sorted By : Signal Multiplier: 1.0000 Dilution: 1.0000 Use Multiplier Dilution Factor with ISTDs Signal 1: DAD1 B, Sig-254, 16 Ref=360, 100 Peak RetTime Type | [min] Width [min] " Area [mAU**s]" Height [mAU] Area ! 16.364 MM 0.1053 18.21606 2.883 100.000 Totals: 18.21606 2.883 | Area Percent Report | | | | | | :---: | :---: | :---: | :---: | :---: | | Sorted By | : Signal | | | | | Multiplier: | 1.0000 | | | | | Dilution: | 1.0000 | | | | | Use Multiplier Dilution Factor with ISTDs | | | | | | Signal 1: DAD1 B, Sig-254, 16 Ref=360, 100 | | | | | | ```Peak RetTime Type \| [min]``` | Width [min] | $\begin{gathered} \text { Area } \\ {[m A U * s]} \end{gathered}$ | Height [mAU] | Area ! | | 16.364 MM | 0.1053 | 18.21606 | 2.883 | 100.000 | | Totals: | | 18.21606 | 2.883 | |
Figure 5. HPLC Chromatogram and Report for low control of Ergothioneine, 0.0078 mg / mL 0.0078 mg / mL 0.0078mg//mL0.0078 \mathrm{mg} / \mathrm{mL}.
图 5. Ergothioneine 低浓度对照的 HPLC 色谱图及报告， 0.0078 mg / mL 0.0078 mg / mL 0.0078mg//mL0.0078 \mathrm{mg} / \mathrm{mL} 。
Data fille C:\CHEM32\1\DATA\MA263_17\MA263_17 2017-04-24 14-09-48\MA263 170000014.D
Sample Name: Ergothionaine Control 0.0625 Std B


Signal 1: DAD1 B, Sig-254,16 Ref-360,100
\begin{tabular}{|c|c|c|c|c|c|c|}
\hline Peak * & \[
\begin{gathered}
\text { RetTime } \\
\text { [min] }
\end{gathered}
\] & тype & \begin{tabular}{l}
Width \\
[min]
\end{tabular} & \[
\begin{gathered}
\text { Area } \\
{[\text { mAU*s }]}
\end{gathered}
\] & Height [taAU] & Area \(\$\) \\
\hline 1 & 6.113 & MM & 0.0993 & 226.43752 & 37.9967 & 100.0000 \\
\hline
\end{tabular}
qquad\qquad
    *** End of Report ***
Figure 6. HPLC Chromatogram and Report for high control of Ergothioneine, 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL}.
图 6. Ergothioneine 高浓度对照的 HPLC 色谱图及报告， 0.0625 mg / mL 0.0625 mg / mL 0.0625mg//mL0.0625 \mathrm{mg} / \mathrm{mL} 。
Data Eile C:\CHEM32\1\DATA\MA263 17\MA26317 2017-04-24 14-09-48\MA263 170000008.D Sample Name: RE170327-005 083 A06 17005170 B
数据文件 C:\CHEM32\1\DATA\MA263 17\MA26317 2017-04-24 14-09-48\MA263 170000008.D 样品名称: RE170327-005 083 A06 17005170 B

Sorted By  按排序 : Signal  信号
Multiplier:  乘数： : 1.0000
Dilution:  稀释： ⋮ ⋮ vdots\vdots 1.0000
Use Multiplier  使用 Multiplier Dilution Factor with  稀释因子与 ISTDs
Sorted By : Signal Multiplier: : 1.0000 Dilution: vdots 1.0000 Use Multiplier Dilution Factor with ISTDs| Sorted By | : | Signal | | :--- | :---: | :---: | | Multiplier: | : | 1.0000 | | Dilution: | $\vdots$ | 1.0000 | | Use Multiplier | Dilution Factor with | ISTDs |
Signal 1: DAD1 B, Sig=254, 16 Ref=360, 100
信号 1: DAD1 B, Sig=254, 16 Ref=360, 100
Peak *  峰值 * RetTime [ m i n ) [ m i n ) [min)[m i n) Type  类型
  宽度 [分钟]
Width
[min]
Width [min]| Width | | :--- | | [min] |
 Area [ m A U ∗ s ]  Area  [ m A U ∗ s  ]  {:[" Area "],[[mAU**s" ] "]:}\begin{aligned} & \text { Area } \\ & {[m A U * s \text { ] }} \end{aligned}
  高度 [mAU]
Height
[mAU]
Height [mAU]| Height | | :--- | | [mAU] |
1 6.413 MM 0.1235 336.61398 45.42683 100.0000
Total  总计 3 : 336.61398 45.42683
Peak * RetTime [min) Type "Width [min]" " Area [mAU**s ] " "Height [mAU]" 1 6.413 MM 0.1235 336.61398 45.42683 100.0000 Total 3 : 336.61398 45.42683 | Peak * | RetTime $[m i n)$ | Type | Width <br> [min] | $\begin{aligned} & \text { Area } \\ & {[m A U * s \text { ] }} \end{aligned}$ | Height <br> [mAU] | | | :---: | :---: | :---: | :---: | :---: | :---: | :---: | | 1 | 6.413 | MM | 0.1235 | 336.61398 | 45.42683 | 100.0000 | | Total | 3 : | | | 336.61398 | 45.42683 | |
*** End of Report ***
*** 报告结束 ***
Figure 7. HPLC Chromatogram and Report for Ergothioneine in mushroom powder sample.
图 7. 蘑菇粉样品中 Ergothioneine 的 HPLC 色谱图及报告。
Table 1. Evaluation of the accuracy of Ergothioneine in control samples
表 1. 对照样品中 Ergothioneine 的准确性评估
Master Assay # Ergothioneine Added (mg)
添加的麦角硫因 (mg)

麦角硫因测定量（mg）
Ergothioneine
Assayed (mg)
Ergothioneine Assayed (mg)| Ergothioneine | | :--- | | Assayed (mg) |
 Average Ergothioneine Assayed (mg)
平均麦角硫因测定量 (mg)
  准确率 (%)
Accuracy
(%)
Accuracy (%)| Accuracy | | :--- | | (%) |
 Absolute Error (mg)  绝对误差（mg） Intraassay Variation (%)
批内变异 (%)		 Interassay Variation (%)
批间变异 (%)
253.17 0.0078 0.0084
253.17 0.0078 0.0082 0.0083 5.88% 0.0005 1.75%
257.17 0.0078 0.0092
257.17 0.0078 0.0090 0.0091 16.57% 0.0013 1.36%
258.17 0.0078 0.0070
258.17 0.0078 0.0075 0.0073 -6.50% -0.0005 4.95%
263.17 0.0078 0.0068
263.17 0.0078 0.0069 0.0069 -12.16% -0.0009 0.24%
0.0079 0.95% 0.0001 11.95%
253.17 0.0625 0.0593
253.17 0.0625 0.0594 0.0594 -5.01% -0.0031 0.06%
257.17 0.0625 0.0621
257.17 0.0625 0.0610 0.0616 -1.50% -0.0009 1.27%
258.17 0.0625 0.0596
258.17 0.0625 0.0636 0.0616 -1.49% -0.0009 4.59%
263.17 0.0625 0.0607
263.17 0.0625 0.0613 0.0610 -2.36% -0.0015 0.70%
0.0609 -2.59% -0.0016 2.42%
Master Assay # Ergothioneine Added (mg) "Ergothioneine Assayed (mg)" Average Ergothioneine Assayed (mg) "Accuracy (%)" Absolute Error (mg) Intraassay Variation (%) Interassay Variation (%) 253.17 0.0078 0.0084 253.17 0.0078 0.0082 0.0083 5.88% 0.0005 1.75% 257.17 0.0078 0.0092 257.17 0.0078 0.0090 0.0091 16.57% 0.0013 1.36% 258.17 0.0078 0.0070 258.17 0.0078 0.0075 0.0073 -6.50% -0.0005 4.95% 263.17 0.0078 0.0068 263.17 0.0078 0.0069 0.0069 -12.16% -0.0009 0.24% 0.0079 0.95% 0.0001 11.95% 253.17 0.0625 0.0593 253.17 0.0625 0.0594 0.0594 -5.01% -0.0031 0.06% 257.17 0.0625 0.0621 257.17 0.0625 0.0610 0.0616 -1.50% -0.0009 1.27% 258.17 0.0625 0.0596 258.17 0.0625 0.0636 0.0616 -1.49% -0.0009 4.59% 263.17 0.0625 0.0607 263.17 0.0625 0.0613 0.0610 -2.36% -0.0015 0.70% 0.0609 -2.59% -0.0016 2.42%| Master Assay # | Ergothioneine Added (mg) | Ergothioneine <br> Assayed (mg) | Average Ergothioneine Assayed (mg) | Accuracy <br> (%) | Absolute Error (mg) | Intraassay Variation (%) | Interassay Variation (%) | | :---: | :---: | :---: | :---: | :---: | :---: | :---: | :---: | | 253.17 | 0.0078 | 0.0084 | | | | | | | 253.17 | 0.0078 | 0.0082 | 0.0083 | 5.88% | 0.0005 | 1.75% | | | 257.17 | 0.0078 | 0.0092 | | | | | | | 257.17 | 0.0078 | 0.0090 | 0.0091 | 16.57% | 0.0013 | 1.36% | | | 258.17 | 0.0078 | 0.0070 | | | | | | | 258.17 | 0.0078 | 0.0075 | 0.0073 | -6.50% | -0.0005 | 4.95% | | | 263.17 | 0.0078 | 0.0068 | | | | | | | 263.17 | 0.0078 | 0.0069 | 0.0069 | -12.16% | -0.0009 | 0.24% | | | | | | 0.0079 | 0.95% | 0.0001 | | 11.95% | | 253.17 | 0.0625 | 0.0593 | | | | | | | 253.17 | 0.0625 | 0.0594 | 0.0594 | -5.01% | -0.0031 | 0.06% | | | 257.17 | 0.0625 | 0.0621 | | | | | | | 257.17 | 0.0625 | 0.0610 | 0.0616 | -1.50% | -0.0009 | 1.27% | | | 258.17 | 0.0625 | 0.0596 | | | | | | | 258.17 | 0.0625 | 0.0636 | 0.0616 | -1.49% | -0.0009 | 4.59% | | | 263.17 | 0.0625 | 0.0607 | | | | | | | 263.17 | 0.0625 | 0.0613 | 0.0610 | -2.36% | -0.0015 | 0.70% | | | | | | 0.0609 | -2.59% | -0.0016 | | 2.42% |
Table 2. Amount of Ergothioneine detected in mushroom powder samples
表 2. 蘑菇粉样品中检测到的 Ergothioneine 含量

麦角硫因分析（mg）
Ergothioneine
Analyzed (mg)
Ergothioneine Analyzed (mg)| Ergothioneine | | :---: | | Analyzed (mg) |

平均麦角硫因测定量 (mg)
Average
Ergothioneine
Assayed (mg)
Average Ergothioneine Assayed (mg)| Average | | :---: | | Ergothioneine | | Assayed (mg) |
Intra-assay
Variation (%)
Intra-assay Variation (%)| Intra-assay | | :---: | | Variation (%) |

批间变异 (%)
Inter-assay
Variation (%)
Inter-assay Variation (%)| Inter-assay | | :---: | | Variation (%) |
253.17 1 0.089
253.17 2 0.090
253.17 3 0.094 0.091 2.18 % 2.18 % 2.18%2.18 \%
253.17 4 0.092
257.17 5 0.094
257.17 6 0.094 0.095 0.68 % 0.68 % 0.68%0.68 \%
257.17 7 0.095
257.17 8 0.095 0.092 0.50 % 0.50 % 0.50%0.50 \%
258.17 9 0.092
258.17 10 0.092 0.090 0.24 % 0.24 % 0.24%0.24 \%
263.17 11 0.090 0.092 2.28 % 2.28 % 2.28%2.28 \%
263.17 12 0.090
"Ergothioneine Analyzed (mg)" "Average Ergothioneine Assayed (mg)" "Intra-assay Variation (%)" "Inter-assay Variation (%)" 253.17 1 0.089 253.17 2 0.090 253.17 3 0.094 0.091 2.18% 253.17 4 0.092 257.17 5 0.094 257.17 6 0.094 0.095 0.68% 257.17 7 0.095 257.17 8 0.095 0.092 0.50% 258.17 9 0.092 258.17 10 0.092 0.090 0.24% 263.17 11 0.090 0.092 2.28% 263.17 12 0.090 | | | Ergothioneine <br> Analyzed (mg) | Average <br> Ergothioneine <br> Assayed (mg) | Intra-assay <br> Variation (%) | Inter-assay <br> Variation (%) | | :---: | :---: | :---: | :---: | :---: | :---: | | 253.17 | 1 | 0.089 | | | | | 253.17 | 2 | 0.090 | | | | | 253.17 | 3 | 0.094 | 0.091 | $2.18 \%$ | | | 253.17 | 4 | 0.092 | | | | | 257.17 | 5 | 0.094 | | | | | 257.17 | 6 | 0.094 | 0.095 | $0.68 \%$ | | | 257.17 | 7 | 0.095 | | | | | 257.17 | 8 | 0.095 | 0.092 | $0.50 \%$ | | | 258.17 | 9 | 0.092 | | | | | 258.17 | 10 | 0.092 | 0.090 | $0.24 \%$ | | | 263.17 | 11 | 0.090 | 0.092 | | $2.28 \%$ | | 263.17 | 12 | 0.090 | | | |
Table 3. Recovery of Ergothioneine in spiked mushroom powder samples
表 3. 蘑菇粉样品中麦角硫因的回收率
Master Assay # Samples  样品 Additional Ergothioneine Spiked (mg)
额外添加的麦角硫因（mg）		 Ergothioneine Analyzed (mg)
麦角硫因分析（mg）		 Average Ergothioneine Assayed (mg)
平均麦角硫因测定量 (mg)		 Percent Recovery  回收率

平均回收率
Average
Percent Recovery
Average Percent Recovery| Average | | :--- | | Percent Recovery |
 Intraassay Variation (%)
批内变异 (%)		 Interassay Variation (%)
批间变异 (%)
253.17 13 0.0078 0.099 91.7%
253.17 14 0.0078 0.098 0.099 80.9% 86.3% 8.85%
263.17 15 0.0078 0.098 74.7%
263.17 16 0.0078 0.098 0.098 73.2% 73.9% 1.47%
0.098 80.1% 10.9%
253.17 17 0.0625 0.152 0.152 96.2% 96.2%
257.17 18 0.0625 0.163 113.7%
257.17 19 0.0625 0.154 0.158 99.0% 106.4% 9.79%
0.156 103.0% 14.2%
Master Assay # Samples Additional Ergothioneine Spiked (mg) Ergothioneine Analyzed (mg) Average Ergothioneine Assayed (mg) Percent Recovery "Average Percent Recovery" Intraassay Variation (%) Interassay Variation (%) 253.17 13 0.0078 0.099 91.7% 253.17 14 0.0078 0.098 0.099 80.9% 86.3% 8.85% 263.17 15 0.0078 0.098 74.7% 263.17 16 0.0078 0.098 0.098 73.2% 73.9% 1.47% 0.098 80.1% 10.9% 253.17 17 0.0625 0.152 0.152 96.2% 96.2% 257.17 18 0.0625 0.163 113.7% 257.17 19 0.0625 0.154 0.158 99.0% 106.4% 9.79% 0.156 103.0% 14.2%| Master Assay # | Samples | Additional Ergothioneine Spiked (mg) | Ergothioneine Analyzed (mg) | Average Ergothioneine Assayed (mg) | Percent Recovery | Average <br> Percent Recovery | Intraassay Variation (%) | Interassay Variation (%) | | :---: | :---: | :---: | :---: | :---: | :---: | :---: | :---: | :---: | | 253.17 | 13 | 0.0078 | 0.099 | | 91.7% | | | | | 253.17 | 14 | 0.0078 | 0.098 | 0.099 | 80.9% | 86.3% | 8.85% | | | 263.17 | 15 | 0.0078 | 0.098 | | 74.7% | | | | | 263.17 | 16 | 0.0078 | 0.098 | 0.098 | 73.2% | 73.9% | 1.47% | | | | | | | 0.098 | | 80.1% | | 10.9% | | 253.17 | 17 | 0.0625 | 0.152 | 0.152 | 96.2% | 96.2% | | | | 257.17 | 18 | 0.0625 | 0.163 | | 113.7% | | | | | 257.17 | 19 | 0.0625 | 0.154 | 0.158 | 99.0% | 106.4% | 9.79% | | | | | | | 0.156 | | 103.0% | | 14.2% |

VI. References  VI. 参考文献

  1. Dubost J, Beelman R, Peterson D et al. Identification and Quantification of Ergothioneine in Cultivated Mushrooms by Liquid Chromatography-Mass Spectroscopy. International Journal of Medicinal Mushrooms 2006; 8:215-222.
    Dubost J, Beelman R, Peterson D 等. 通过液相色谱-质谱法鉴定和定量栽培蘑菇中的麦角硫因. International Journal of Medicinal Mushrooms 2006; 8:215-222.
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    陈 S, 何 K, 谢 Y 等。蘑菇子实体和菌丝体中洛伐他汀、 γ γ gamma\gamma -氨基丁酸和麦角硫因的含量。LWT-食品科学与技术 2012; 47:274278。