A method for simultaneous determination of glutathione and cysteine

Abstract

This invention discloses a method for simultaneously determining glutathione and cysteine, relating to the field of health food and the detection of functional components in food. The method includes: serially diluting standard solutions of glutathione and cysteine, performing liquid chromatography analysis, and establishing a standard curve for concentration and peak area; preparing a mixed standard solution containing glutathione and cysteine, performing liquid chromatography analysis to establish a correspondence between target components and retention times; performing liquid chromatography analysis on the sample solution, obtaining the peak area of ​​each target component in the sample solution based on the correspondence, and calculating the content based on the peak area and the standard curve. This invention utilizes liquid chromatography technology, enabling the rapid and simultaneous determination of glutathione and cysteine ​​content in a single injection analysis. It is simple to operate, highly efficient, shortens the experimental cycle, reduces experimental costs, and is widely applicable to formulations such as gummies, tablets, and capsules.

Description

Technical Field

[0001] This invention relates to the field of health food and the field of detection technology for the functional components of food, and in particular to a method that can simultaneously, rapidly and accurately determine the content of glutathione and cysteine ​​in food. Background Technology

[0002] Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine, containing a sulfhydryl group. It possesses antioxidant and detoxification properties. The sulfhydryl group on cysteine ​​is the active group of glutathione, readily binding to certain drugs (such as acetaminophen) and toxins (such as free radicals, iodoacetic acid, mustard gas, and heavy metals like lead, mercury, and arsenic), thus exhibiting detoxification effects. Therefore, glutathione (especially glutathione in liver cells) can participate in biotransformation, converting harmful toxins in the body into harmless substances for excretion, and also helping to maintain normal immune system function.

[0003] Cysteine ​​(Cys) is a sulfur-containing non-essential amino acid that plays multiple key roles in the human body: it participates in protein structure stability, antioxidant defense, detoxification metabolism, and supports immune and skin health. It maintains protein spatial structure by forming disulfide bonds and is also an important raw material for the synthesis of glutathione (GSH), helping to scavenge free radicals and neutralize toxins. It also plays a supporting role in collagen and immune function.

[0004] In health supplements, glutathione is often combined with vitamin C, alpha-lipoic acid, or cysteine, primarily to provide antioxidant properties and enhance skin whitening effects. To control product quality, suitable methods need to be developed to accurately determine the content of glutathione and cysteine.

[0005] Currently, the main methods for determining these two substances include high-performance liquid chromatography (HPLC), capillary electrophoresis, and liquid chromatography-mass spectrometry (LC-MS). Among these, HPLC is widely used due to its readily available instruments and good stability. However, directly applying existing HPLC methods to complex formulations such as gummies, tablets, and capsules faces several challenges. First, these formulations have complex matrices, containing large amounts of sugars, starches, excipients, and potentially other active ingredients, which severely interfere with the separation of the target analytes. Second, GSH and Cys (especially their thiol groups) are prone to oxidation during sample processing and analysis, leading to lower measurement results and difficulty in ensuring accuracy. Furthermore, conventional methods often require complex sample pretreatment (such as the derivatization process described in CN105572234B) or lengthy analysis times, making them unsuitable for the rapid quality control requirements of industrial production.

[0006] Therefore, developing a method for the simultaneous determination of glutathione and cysteine ​​that is easy to operate, has strong anti-interference ability, high accuracy, and is applicable to various complex formulation matrices has important practical application value. Summary of the Invention

[0007] The purpose of this invention is to solve the aforementioned technical problems and provide a method for the simultaneous determination of glutathione and cysteine. This method is particularly suitable for complex food preparations such as gummies, tablets, and capsules, as well as health food formulations, and has the advantages of simple pretreatment, rapid analysis, and high accuracy.

[0008] To achieve the above-mentioned technical objectives and requirements, the technical solution adopted by this invention is: a method for simultaneously determining glutathione and cysteine, comprising the following steps: (1) Sample solution preparation: Weigh 0.5g~2.5g of sample into a 25ml volumetric flask, add 20mg~100mg of antioxidant and 10~20ml of mobile phase, vortex mix and sonicate to dissolve, then add mobile phase to dilute to the mark and mix to obtain sample solution for high performance liquid chromatography determination; (2) Preparation of mobile phase A: Weigh 5-10g of potassium dihydrogen phosphate and 1-5g of ion-pairing reagent, dissolve and dilute with water to 1000ml to prepare an aqueous solution, and adjust the pH of the aqueous solution to 3.0-3.5 with phosphoric acid to obtain mobile phase A; (3) High performance liquid chromatography experimental conditions: Chromatographic column: Octadecyl bonded silica gel column; Mobile phase: A mixed solution consisting of mobile phase A and an organic phase; Flow rate: 0.8~1.5 ml / min; Detection wavelength: 200~250nm; Column temperature: 30~40℃; Injection volume: 5~20 μl; (4) Preparation of standard solutions: Weigh 30 mg to 70 mg of glutathione and cysteine ​​standards into their respective 25 ml volumetric flasks, add 20 mg to 100 mg of antioxidant, add mobile phase, sonicate to dissolve and dilute to the mark, and mix well to prepare standard stock solutions; pipette 0.1 ml, 0.2 ml, 0.4 ml, 0.8 ml and 1.0 ml of the above standard stock solutions into 20 ml volumetric flasks, add mobile phase to dilute to the mark and mix well to prepare standard solutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL and 100 μg / mL, respectively. (5) Establishment of standard curve: The series of standard solutions obtained in step (4) are subjected to liquid chromatography analysis under the conditions of step (3), and the peak area of ​​each standard solution is measured. The standard curve is established with the peak area of ​​each standard solution as the ordinate and the concentration as the abscissa, and the linear regression equation is obtained. (6) Preparation of mixed standard solutions: Pipette 0.4 ml of glutathione and cysteine ​​standard stock solutions into the same 20 ml volumetric flask, add mobile phase to dilute to the mark and mix well. Perform liquid phase analysis under the conditions of step (3) to establish the correspondence between target components and retention time. (7) Content determination: The sample solution obtained in step (1) is subjected to liquid phase analysis under the conditions of step (3), the peak area is measured, and the concentration is obtained by comparing with the standard curve. Combined with the weighing of the sample, the contents of glutathione and cysteine ​​in the sample are calculated respectively.

[0009] Preferably, in step (1), the antioxidant is one or more of vitamin C and lipoic acid.

[0010] Preferably, in step (2), the ion-pairing reagent is selected from one or more of sodium hexanesulfonate, sodium heptanesulfonate, and sodium octanesulfonate.

[0011] Preferably, in step (3), the organic phase is one or more of methanol and acetonitrile.

[0012] Preferably, in step (3), the mobile phase is isocratic elution, wherein the volume ratio of mobile phase A to organic phase is kept constant, and the volume percentage of mobile phase A is 80%~99%.

[0013] Preferred: Under the chromatographic conditions of step (3), the chromatographic peaks of glutathione and cysteine ​​are baseline separated from the interference peaks generated by sugars, starches and / or excipients in the sample, with a resolution of not less than 1.5.

[0014] Preferably, the sample is a food or health food preparation, selected from tablets, capsules, powders or gummies.

[0015] Preferred formula: In step (7), the formula for calculating the content of glutathione and cysteine ​​is: ; Among them, C 样 The concentration of the sample solution, expressed in μg / mL, is calculated from the peak area of ​​the sample solution using a standard curve; V is the dilution volume of the sample solution, expressed in mL; W 样 The sample weight is expressed in grams.

[0016] Compared with the traditional structure, the beneficial effects of the present invention are as follows: 1. This invention is simple to operate and can simultaneously determine the content of glutathione and cysteine ​​in a single injection analysis, resulting in high analytical efficiency. Direct detection using an HPLC UV detector eliminates the need for derivatization, significantly shortening the experimental cycle and reducing experimental costs.

[0017] 2. This invention has high detection accuracy. By optimizing the mobile phase composition and using a phosphate buffer solution with a specific pH combined with ion-pairing reagents, the retention behavior of glutathione and cysteine ​​on the C18 column is effectively improved, enabling them to be well separated from common impurities in the formulation, such as sugars and excipients, and exhibiting strong anti-interference ability.

[0018] 3. This invention exhibits good stability and wide applicability. A strong reducing antioxidant is added during sample processing. This antioxidant forms an antioxidant coupling with glutathione and cysteine ​​in the sample. The synergistic protective effect of this antioxidant coupling inhibits the oxidative degradation of thiol groups during sample processing and analysis, ensuring the accuracy of the measurement results. It is applicable to the content determination of various dosage forms such as tablets, capsules, powders, and gummies. Attached Figure Description

[0019] Figure 1 This is a chromatogram of the standard solutions of glutathione and cysteine ​​in Example 1 of the present invention; Figure 2 This is a chromatogram of the sample solution in Example 1 of the present invention. Detailed Implementation

[0020] The present invention will be further described below.

[0021] Example 1: Determination of glutathione and cysteine ​​in tablets

[0022] (1) Preparation of mobile phase A Weigh out 8g of potassium dihydrogen phosphate and 3g of sodium hexanesulfonate, dissolve and dilute in water to 1000ml, and adjust the pH of the aqueous solution to 3.0 with phosphoric acid.

[0023] (2) High performance liquid chromatography conditions Chromatographic column: Octadecyl bonded silica gel column; Mobile phase: Mobile phase A: Methanol = 95:5 (volume ratio); Flow rate: 0.8 ml / min; Detection wavelength: 200nm; Column temperature: 30℃; Injection volume: 5 μl.

[0024] (3) Preparation of standard solutions Weigh 50 mg of glutathione and 50 mg of cysteine ​​standards into separate 25 ml volumetric flasks. Add 50 mg of vitamin C to each flask, dissolve and dilute to the mark using the prepared mobile phase (mobile phase A: methanol = 95:5) via sonication, and mix well to prepare standard stock solutions. Pipette 0.1 ml, 0.2 ml, 0.4 ml, 0.8 ml, and 1.0 ml of the above standard stock solutions into a series of 20 ml volumetric flasks, dilute to the mark with the mobile phase, and mix well to prepare a series of standard solutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL, respectively.

[0025] (4) Preparation of mixed standard solutions Pipette 0.4 ml of glutathione and cysteine ​​standard stock solutions into the same 20 ml volumetric flask, dilute to the mark with mobile phase and mix well, perform liquid chromatography analysis, and establish the correspondence between target components and retention times.

[0026] (5) Sample solution preparation Take 20 tablets and crush them (Product Name: Women's Multivitamin Tablets, Batch No.: CC202345, Manufacturer: Jiangsu Ailande Nutrition Products Co., Ltd.). Weigh 1g of sample into a 25mL volumetric flask, add 50mg of vitamin C, add about 15mL of mobile phase and vortex to mix, then sonicate to dissolve completely. After cooling to room temperature, dilute to the mark with mobile phase and mix well for high performance liquid chromatography (HPLC) determination.

[0027] (6) Content determination Standard solutions of glutathione and cysteine ​​at different concentrations were injected separately, and the peak areas of each standard solution were measured. A standard curve for glutathione and cysteine ​​was established with peak area as the ordinate and corresponding concentration as the abscissa, and the linear regression equation was calculated. Sample solutions were then injected and analyzed under the same chromatographic conditions to obtain the peak areas of glutathione and cysteine. These peak areas were then substituted into their respective standard curve equations to calculate the concentrations of glutathione and cysteine ​​in the sample solutions.

[0028] (7) Calculation of results The contents of glutathione and cysteine ​​in the sample are calculated using the following formula: ; In the formula: C 样 —The concentration of the sample solution, in μg / mL, is calculated from the peak area of ​​the sample solution using the standard curve. W 样 —Sample weight, g; V — Dilution volume, ml.

[0029] Calculations show that the average glutathione content in this batch of tablets is 5.4 mg / g, and the average cysteine ​​content is 10.8 mg / g.

[0030] Example 2: Determination of glutathione and cysteine ​​in capsule contents

[0031] (1) Preparation of mobile phase A Weigh 6.3g of potassium dihydrogen phosphate and 4.5g of sodium hexanesulfonate, dissolve and dilute in water to 1000ml, and adjust the pH of the aqueous solution to 3.5 with phosphoric acid.

[0032] (2) High performance liquid chromatography conditions Chromatographic column: Octadecyl bonded silica gel column; Mobile phase: Mobile phase A: Acetonitrile = 98:2 (volume ratio); Flow rate: 1.0 ml / min; Detection wavelength: 210nm; Column temperature: 30℃; Injection volume: 10 μl.

[0033] (3) Preparation of standard solutions Weigh 50 mg of glutathione and 50 mg of cysteine ​​standards into separate 25 ml volumetric flasks. Add 50 mg of vitamin C to each flask, dissolve by sonication with mobile phase, and dilute to the mark. Mix well to prepare standard stock solutions. Pipette 0.1 ml, 0.2 ml, 0.4 ml, 0.8 ml, and 1.0 ml of the above standard stock solutions into a series of 20 ml volumetric flasks, dilute to the mark with mobile phase, and mix well to prepare a series of standard solutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL, respectively.

[0034] (4) Preparation of mixed standard solutions Pipette 0.4 ml of glutathione and cysteine ​​standard stock solutions into the same 20 ml volumetric flask, dilute to the mark with mobile phase and mix well, perform liquid chromatography analysis, and establish the correspondence between target components and retention times.

[0035] (5) Sample solution preparation Take the contents of 20 capsules (product name: Milk Thistle Capsules, batch number: ZA202338, manufacturer: Jiangsu Ailande Nutrition Products Co., Ltd.), weigh 2g of the contents sample into a 25mL volumetric flask, add 100mg of lipoic acid as an antioxidant, add 15mL of mobile phase and vortex mix, then sonicate to completely dissolve. After cooling to room temperature, dilute to the mark with mobile phase and mix well for high performance liquid chromatography (HPLC) determination.

[0036] (6) Content determination Standard solutions of glutathione and cysteine ​​at different concentrations were injected separately, and the peak areas of each standard solution were measured. A standard curve was established with the peak area as the ordinate and the corresponding concentration as the abscissa, and the linear regression equation was calculated. Sample solutions were injected and analyzed under the above chromatographic conditions to obtain the peak areas of glutathione and cysteine. The concentrations of glutathione and cysteine ​​in the sample solutions were calculated based on the standard curve.

[0037] (7) Calculation of results The contents of glutathione and cysteine ​​in the sample are calculated using the following formula: ; In the formula: C 样 —The concentration of the sample solution, in μg / mL, is calculated from the peak area of ​​the sample solution using the standard curve. W 样 —Sample weight, g; V — Dilution volume, ml; Calculations show that the average content of glutathione in the contents of this batch of capsules is 8.1 mg / g, and the average content of cysteine ​​is 6.8 mg / g.

[0038] To verify the reliability of the method of the present invention, the tablet sample of Example 1 was used as a representative matrix, and a systematic analytical methodology validation was performed under the above chromatographic conditions, including resolution, linearity, precision, accuracy (recovery), limit of detection, limit of quantitation, and solution stability. The validation results are also applicable to Example 2 and other dosage forms.

[0039] 1. Resolution

[0040] Take blank solvent (mobile phase), mixed standard solution (glutathione and cysteine ​​concentrations are both 40 μg / mL), and test solution (tablet sample from Example 1), inject them into the liquid chromatograph, and record the chromatograms.

[0041] The results show: No chromatographic peaks appeared at the retention times of glutathione and cysteine ​​in the blank solvent; In the mixed standard solution, the retention time of glutathione was 5.8 min, and the retention time of cysteine ​​was 3.4 min. The resolution was calculated according to the method specified in General Chapter 0512 of Part IV of the Chinese Pharmacopoeia. The results showed that the resolution between the two peaks was greater than 1.5, achieving complete baseline separation. Chromatogram of the test solution (see) Figure 2 The results showed that the chromatographic peaks of glutathione and cysteine ​​were well separated from the interfering peaks generated by sugars, starches, excipients, etc. in the sample, with no overlap or interference.

[0042] The above results indicate that this method is highly specific and can accurately determine target components in complex matrices.

[0043] 2. Linear relationship

[0044] A series of standard solutions of glutathione and cysteine ​​with concentrations of 10, 20, 40, 80, and 100 μg / mL were prepared and analyzed by injection under the chromatographic conditions of Example 1. Linear regression was performed with concentration (x, μg / mL) on the x-axis and peak area (y) on the y-axis.

[0045] The linear regression equation for glutathione is: y = 1856.3x + 127.5, with a correlation coefficient R0. 2 =0.9998; The linear regression equation for cysteine ​​is: y = 1721.6x + 98.3, with a correlation coefficient R0. 2 =0.9996.

[0046] The results showed that glutathione and cysteine ​​had a good linear relationship in the concentration range of 10–100 μg / mL.

[0047] 3. Precision

[0048] Six test solutions were prepared in parallel using the same batch of tablet samples (Example 1) according to the sample solution preparation method, and each solution was injected and analyzed. The relative standard deviations (RSDs) of glutathione and cysteine ​​content were calculated. The results showed that the RSD of glutathione content was 1.2% and the RSD of cysteine ​​content was 1.5%, indicating that the method has good repeatability.

[0049] 4. Accuracy

[0050] Samples with known content were prepared, and three concentration levels (equivalent to 80%, 100%, and 120% of the sample content) of GSH and Cys mixed standards were added, with three replicates for each level. The determination was performed according to this method. Results showed that the average recovery rate of GSH was 99.2%–99.5%, with an RSD of 0.9%–1.4%; the average recovery rate of Cys was 99.5%–100.2%, with an RSD of 0.8%–1.2%. This indicates that the method has good accuracy and meets the requirements for content determination.

[0051] 5. Solution stability

[0052] The test solution (prepared according to the method of Example 1, containing 50 mg of vitamin C) was left at room temperature for 0, 2, 4, 6, 8, and 12 hours, respectively, and then injected for analysis under the chromatographic conditions of Example 1. Within 12 hours, the RSD of the GSH peak area was 1.8%, and the RSD of the Cys peak area was 1.9%, indicating that under the sample pretreatment conditions described in this invention (an acidic mobile phase environment containing antioxidants), the test solution was stable at room temperature for 12 hours, meeting the time requirements for routine analysis.

[0053] Using tablet matrix as the subject, three groups of sample solutions (three replicates per group) were prepared according to the method in Example 1: Group A: No antioxidants added; Group B: Add 50mg of Vitamin C; Group C: Add 50mg of vitamin C + 50mg of lipoic acid.

[0054] After the three sample solutions were left at room temperature for 4 hours, the peak areas were measured under the chromatographic conditions of Example 1 and compared with the initial peak areas (0 hours). The results showed that without the addition of antioxidants, the peak areas of GSH and Cys decreased by 28.8% and 24.6%, respectively, within 4 hours under the same conditions; in group B, glutathione decreased by 5.2% and cysteine ​​by 4.5%; in group C, glutathione decreased by 2.8% and cysteine ​​by 2.5%. This further confirms the role of the pretreatment conditions of the present invention in ensuring the stability of the target analytes.

[0055] The above embodiments of the present invention are merely examples to clearly illustrate the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent technical solutions also fall within the scope of the present invention, and the patent protection scope of the present invention should be defined by the claims.

Claims

1. A method for simultaneously determining glutathione and cysteine, characterized in that: Includes the following steps: (1) Sample solution preparation: Weigh 0.5g~2.5g of sample into a volumetric flask, add 20mg~100mg of antioxidant and 10~20ml of mobile phase, vortex mix and sonicate to dissolve, then add mobile phase to dilute to the mark and mix to obtain sample solution for high performance liquid chromatography determination; (2) Preparation of mobile phase A: Weigh 5-10g of potassium dihydrogen phosphate and 1-5g of ion-pairing reagent, dissolve and dilute with water to 1000ml to prepare an aqueous solution, and adjust the pH of the aqueous solution to 3.0-3.5 with phosphoric acid to obtain mobile phase A; (3) High performance liquid chromatography experimental conditions: Chromatographic column: Octadecyl bonded silica gel column; Mobile phase: A mixed solution consisting of mobile phase A and an organic phase; Flow rate: 0.8~1.5 ml / min; Detection wavelength: 200~250nm; Column temperature: 30~40℃; Injection volume: 5~20 μl; (4) Preparation of standard solutions: Weigh 30 mg to 70 mg of glutathione and cysteine ​​standards into their respective volumetric flasks, add 20 mg to 100 mg of antioxidant, add mobile phase, sonicate to dissolve and dilute to the mark, and mix well to prepare standard stock solutions; pipette 0.1 ml, 0.2 ml, 0.4 ml, 0.8 ml and 1.0 ml of the above standard stock solutions into 20 ml volumetric flasks, add mobile phase to dilute to the mark and mix well to prepare standard solutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL and 100 μg / mL, respectively. (5) Establishment of standard curve: The series of standard solutions obtained in step (4) are subjected to liquid chromatography analysis under the conditions of step (3), and the peak area of ​​each standard solution is measured. The peak area of ​​each standard solution is used as the vertical axis and the concentration is used as the horizontal axis to establish a standard curve. (6) Preparation of mixed standard solutions: Pipette 0.4 ml of glutathione and cysteine ​​standard stock solutions into the same 20 ml volumetric flask, add mobile phase to dilute to the mark and mix well. Perform liquid phase analysis under the conditions of step (3) to establish the correspondence between target components and retention time. (7) Content determination: The sample solution obtained in step (1) is subjected to liquid phase analysis under the conditions of step (3), the peak area is measured, and the concentration is obtained by comparing with the standard curve. Combined with the weighing of the sample, the contents of glutathione and cysteine ​​in the sample are calculated respectively.

2. The method for simultaneously determining glutathione and cysteine ​​according to claim 1, characterized in that: In step (1), the antioxidant is one or more of vitamin C and lipoic acid.

3. The method for simultaneously determining glutathione and cysteine ​​according to claim 1, characterized in that: In step (2), the ion-pairing reagent is selected from one or more of sodium hexanesulfonate, sodium heptanesulfonate, and sodium octanesulfonate.

4. The method for simultaneously determining glutathione and cysteine ​​according to claim 1, characterized in that: In step (3), the organic phase is one or more of methanol and acetonitrile.

5. The method for simultaneously determining glutathione and cysteine ​​according to claim 1, characterized in that: In step (3), the mobile phase is isocratic elution, and the volume percentage of mobile phase A is 80%~99%.

6. The method for simultaneously determining glutathione and cysteine ​​according to claim 1, characterized in that: Under the chromatographic conditions of step (3), the chromatographic peaks of glutathione and cysteine ​​are baseline separated from the interfering peaks generated by sugars, starches and / or excipients in the sample, with a resolution of not less than 1.

5.

7. The method for simultaneously determining glutathione and cysteine ​​according to claim 1, characterized in that: The sample is a food or health food preparation, selected from tablets, capsules, powders, or gummies.

8. The method for simultaneously determining glutathione and cysteine ​​according to claim 1, characterized in that: In step (7), the formulas for calculating the glutathione and cysteine ​​content are as follows: ； Among them, C 样 The concentration of the sample solution, expressed in μg / mL, is calculated from the peak area of ​​the sample solution using a standard curve; V is the dilution volume of the sample solution, expressed in mL; W 样 The sample weight is expressed in grams.

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