Method for detecting gsh and gssh content in complex solution
Patent Information
- Application Number
- CN202610973409.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-01
- Publication Date
- 2026-08-18
AI Technical Summary
[0005]本申请所要解决的技术问题在于医美产品中谷胱甘肽虽功效明确，但其高反应活性难以避免向GSSH的氧化降解，检测端面临二者在分析过程中相互转化，导致检测结果不准确，难以对产品进行质控等问题
[0025] Preferably, the vitamin is vitamin H.
Smart Images
Figure CN122591842A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for detecting the content of GSH and GSSH in a composite solution. Background Technology
[0002] In the field of cosmetic injections, reduced glutathione (GSH) is often added as a core active ingredient to enhance the overall efficacy of products. Its mechanism of action is mainly reflected in three dimensions: First, powerful antioxidant properties. Due to the reducing properties of its active thiol groups, GSH can directly eliminate excess free radicals induced by ultraviolet radiation and environmental pollutants, effectively resisting oxidative stress damage to the skin and delaying cell aging. Second, inhibition of melanin production. By inhibiting tyrosinase activity, GSH blocks the melanin synthesis pathway at its source and accelerates the metabolism of existing pigments, thereby brightening the skin tone and fading dark spots. Third, repair and anti-inflammation. GSH can synergistically promote the repair of damaged cells, reduce inflammatory responses, strengthen the skin barrier function, and improve skin health.
[0003] However, the highly reactive thiol groups in the GSH molecule make it chemically unstable. During production and storage, factors such as terminal sterilization, dissolved oxygen, and pH fluctuations can induce GSH to oxidize into GSSH (oxidized glutathione), a product defined as a critical degradation impurity. Therefore, the residual level of GSSH directly determines the clinical efficacy and shelf life of the product and is a core benchmark for measuring the maturity of the formulation process; if GSSH levels exceed the limit, it indicates a significant shortcoming in the current antioxidant strategy or manufacturing process.
[0004] Even more challenging is that, during the analysis and detection phase, GSH and GSSH can still undergo valence state interconversion due to detection conditions. This instability severely interferes with the accuracy and reproducibility of content determination. Therefore, establishing a detection system that can both inhibit transformation and accurately and rapidly distinguish between the two has become a bottleneck problem restricting the improvement of product quality. Summary of the Invention
[0005] The technical problem this application aims to solve is that while glutathione in medical aesthetic products has a clear efficacy, its high reactivity makes it difficult to avoid oxidative degradation into GSSH. This leads to problems such as the interconversion of GSH and GSSH during analysis, resulting in inaccurate detection results and difficulties in quality control. Therefore, this application provides a method for detecting the content of GSH and GSSH in a composite solution. This application can achieve simultaneous and accurate quantitative detection of GSH and GSSH without pre-column derivatization, and the detection time is short, providing a reliable technical means for the quality control of glutathione-containing medical aesthetic products. Furthermore, the detection method of this application has high sensitivity, meeting the requirements for accurate detection of trace residues and trace components, effectively ensuring the comprehensiveness and safety of the analytical results.
[0006] This application solves the above-mentioned technical problems through the following technical solutions.
[0007] This application provides a method for detecting the content of GSH and GSSH in a composite solution, comprising the following steps: detecting the composite solution using high performance liquid chromatography combined with external standard method, and calculating the content of GSH and GSSH in the composite solution;
[0008] The chromatographic column is a Kromasil C18 column; the mobile phase includes mobile phase A and mobile phase B; mobile phase A includes phosphate buffer and methanol, the phosphate buffer includes dihydrogen phosphate, heptane sulfonate and water; the pH of the phosphate buffer is 3.0~3.1; in mobile phase A, the volume percentage of the phosphate buffer is 92%~98%, and the volume percentage of the methanol is 2%~8%; mobile phase B is acetonitrile.
[0009] Using a linear gradient elution method, at 0 min, the volume percentage of mobile phase A is 100% and the volume percentage of mobile phase B is 0%; at 12-18 min, the volume percentage of mobile phase A decreases to 75%-85% and the volume percentage of mobile phase B increases to 15%-25%.
[0010] In some embodiments, the volume percentage of the phosphate buffer in mobile phase A is preferably 94% to 96%, for example, 95%.
[0011] In some embodiments of the phosphate buffer solution, the dihydrogen phosphate includes sodium dihydrogen phosphate and / or potassium dihydrogen phosphate.
[0012] In some embodiments of the phosphate buffer, the heptane sulfonate comprises sodium heptane sulfonate and / or potassium heptane sulfonate.
[0013] In some embodiments of the phosphate buffer solution, the concentration of dihydrogen phosphate is 5-10 mg / mL, preferably 6-8 mg / mL, for example 6.8 mg / mL.
[0014] In some embodiments of the phosphate buffer, the concentration of heptane sulfonate is 1-5 mg / mL, preferably 1.5-3 mg / mL, for example 2.2 mg / mL.
[0015] In a preferred embodiment, the elution process includes the following steps: using a linear gradient elution method, at 0 min, the volume percentage of mobile phase A is 100% and the volume percentage of mobile phase B is 0%; at 14-16 min, the volume percentage of mobile phase A decreases to 78%-82% and the volume percentage of mobile phase B increases to 18%-22%.
[0016] In a preferred embodiment, the elution process includes the following steps: using a linear gradient elution method, at 0 min, the volume percentage of mobile phase A is 100% and the volume percentage of mobile phase B is 0%; at 15 min, the volume percentage of mobile phase A decreases to 80% and the volume percentage of mobile phase B increases to 20%.
[0017] In some embodiments, the linear gradient elution may further include a column cleaning operation, wherein the mobile phase is mobile phase A, and the column cleaning time is 5-15 minutes.
[0018] In some embodiments, the flow rate of the mobile phase is 0.5 to 1.5 mL / min, preferably 0.8 to 1.2 mL / min, for example 1 mL / min.
[0019] In some embodiments, the temperature of the chromatographic column is 25-35°C, for example, 30°C.
[0020] In some embodiments, the injection volume is 40-80 μL, preferably 50-70 μL, for example 60 μL.
[0021] In some embodiments, the detection wavelength is 200-220 nm, preferably 205-215 nm, for example 210 nm.
[0022] In some embodiments, the composite solution also includes at least one of hyaluronic acid, amino acids, vitamins, and sodium ascorbate phosphate.
[0023] Preferably, the hyaluronic acid substance is selected from at least one of sodium hyaluronate, potassium hyaluronate, and calcium hyaluronate.
[0024] Preferably, the amino acid is selected from at least one of glycine, proline, valine, cysteine hydrochloride, and lysine hydrochloride.
[0025] Preferably, the vitamin is vitamin H.
[0026] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.
[0027] All reagents and raw materials used in this application are commercially available.
[0028] The positive advancements of this application are as follows: It enables simultaneous and accurate quantitative detection of GSH and GSSH without the need for pre-column derivatization. The method is simple and the detection time is short, providing a reliable technical means for the quality control of glutathione-containing medical aesthetic products. Furthermore, the detection method of this application has high sensitivity, meeting the requirements for accurate detection of trace residues and micro-components, effectively ensuring the comprehensiveness and safety of the analytical results. Attached Figure Description
[0029] Figure 1 In Example 1, the method of Example 1 was used to detect the chromatogram of the blank solvent;
[0030] Figure 2 To demonstrate the effectiveness, the method of Example 1 was used to detect the chromatogram of the reference solution;
[0031] Figure 3 In Example 1, the method of Example 2 was used to detect the chromatogram of the blank solvent;
[0032] Figure 4 To demonstrate the effectiveness, the method of Example 2 was used in Example 1 to detect the chromatogram of the reference solution;
[0033] Figure 5 In Example 1, the method of Comparative Example 1 was used to detect the chromatogram of the blank solvent;
[0034] Figure 6 To demonstrate the effectiveness, the method of Comparative Example 1 was used to detect the chromatogram of the reference solution;
[0035] Figure 7 In Example 1, the method of Comparative Example 2 was used to detect the chromatogram of the blank solvent;
[0036] Figure 8 To demonstrate the effectiveness, the method of Comparative Example 2 was used in Example 1 to detect the chromatogram of the reference solution;
[0037] Figure 9 To demonstrate the effectiveness, Example 2 uses the method from Example 1 to detect the chromatogram of the limit-of-quantitation solution. Detailed Implementation
[0038] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.
[0039] Example 1
[0040] Test solution: Accurately measure 3 mL of the sample to be tested into a 10 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution;
[0041] Reference solution: Weigh approximately 5 mg each of reduced glutathione and oxidized glutathione accurately, place them in a 50 mL volumetric flask, dissolve and dilute to the mark with water, and shake well; accurately measure 3 mL, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain the reference solution;
[0042] Chromatographic conditions: Kromasil C18 column (250×4.6mm, 5μm); mobile phase A consisted of phosphate buffer and methanol in a volume ratio of 96:4; the phosphate buffer consisted of 6.8g sodium dihydrogen phosphate, 2.2g sodium heptanesulfonate, and 1000mL, with a pH of 3.0; mobile phase B was acetonitrile; column temperature was 30℃; detection wavelength was 210nm; mobile phase flow rate was 1.0mL / min; injection volume was 60µL; a linear gradient elution method was used. At 0 min, the volume percentage of mobile phase A was 100%, and the volume percentage of mobile phase B was 0%; at 15 min, the volume percentage of mobile phase A decreased to 80%, and the volume percentage of mobile phase B increased to 20%; after 15 min, the column was washed with mobile phase A for 10 min.
[0043] Determination method: Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, record the chromatograms, and calculate the content of reduced glutathione and oxidized glutathione by peak area according to the external standard method.
[0044] Reduced / oxidized glutathione content (µg / mL) = A 样 C 对 A 对 N 样 N;
[0045] in:
[0046] A 样 The peak area of reduced glutathione or oxidized glutathione in the test solution;
[0047] A 对 The peak area of reduced or oxidized glutathione in the reference solution;
[0048] C 对 The concentration of reduced glutathione or oxidized glutathione in the reference standard (µg / mL).
[0049] N 样 The volume (mL) of the test solution was diluted.
[0050] N is the volume (mL) of this product measured.
[0051] Example 2
[0052] Compared with Example 1, the only difference is the pH value of the phosphate buffer in mobile phase A, which is adjusted to 3.1. Other conditions and parameters are the same as in Example 1.
[0053] Comparative Example 1
[0054] Compared with Example 1, the only difference is the pH value of the phosphate buffer in mobile phase A, which is adjusted to 2.9. Other conditions and parameters are the same as in Example 1.
[0055] Comparative Example 2
[0056] Compared with Example 1, the difference lies in the chromatographic column, which is replaced with a Waters Atlantis T3 C18 column, while other conditions and parameters are the same as in Example 1.
[0057] Example 1
[0058] Reference solution: Weigh approximately 15 mg each of GSH and GSSH reference standards, accurately weigh them, place them in a 50 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, accurately measure 1 mL, place it in a 100 mL volumetric flask, add water to dilute to the mark, and shake well.
[0059] The blank solvent is water;
[0060] The blank solvent and reference solution were tested using the methods of Examples 1-2 and Comparative Examples 1-2, respectively. See the chromatograms below. Figures 1-8 .
[0061] Figure 1 and Figure 2 The chromatograms are those of the blank solvent and the reference solution, respectively, obtained using the method of Example 1. Figure 3 and Figure 4 The images show the chromatograms of the blank solvent and reference solution detected using the method of Example 2. The results indicate that the detection method of this application effectively separates GSH and GSSH from the solvent peaks, with a short detection time of less than 15 minutes. This effectively avoids the risk of interconversion between the two substances due to excessively long detection times.
[0062] Figure 5 and Figure 6 The chromatograms are shown for the blank solvent and reference solution detected using the method of Comparative Example 1. The results indicate that neither GSH nor GSSH can be effectively separated from the solvent peaks, suggesting that the solvent significantly affects the accuracy of the detection results for both substances.
[0063] Figure 7 and Figure 8 The chromatograms are for the blank solvent and reference solution, respectively, obtained using the method of Comparative Example 2. The results show that the GSSH peak at retention time 11.582 cannot be effectively separated from the solvent peak, which affects the accuracy of the quantitative detection results of GSSH.
[0064] Example 2: Limit of Quantification
[0065] (1) Solution preparation
[0066] Reference solution: Weigh approximately 15 mg each of GSH and GSSH reference standards, accurately weigh them, place them in a 50 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, accurately measure 1 mL, place it in a 100 mL volumetric flask, add water to dilute to the mark, and shake well.
[0067] Limit of Quantitation Solution: Accurately measure 1 mL of the reference solution, place it in a 10 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution.
[0068] (2) Determination method
[0069] Take a solution with a limit of quantitation (LOQ) and inject it into the liquid chromatograph. Detect the solution using the method in Example 1 and record the chromatogram. See [link to example]. Figure 9 .
[0070] (3) Acceptance criteria
[0071] The concentration at the limit of quantitation should not exceed 20% of the limit concentration, and the S / N value of the peaks of reduced glutathione and oxidized glutathione in the limit of quantitation solution should not be less than 10.
[0072] (4) Verification results
[0073] The test results are shown in Table 1.
[0074] Table 1
[0075]
[0076] Verification results: The limit of quantitation (LOQ) for GSH was 0.30 μg / mL, equivalent to 10.16% of the limit concentration, less than 20% of the limit concentration, with an S / N of 26.21, greater than 10; the LQ for GSSH was 0.31 μg / mL, equivalent to 10.24% of the limit concentration, less than 20% of the limit concentration, with an S / N of 46.52, greater than 10. Therefore, the detection method of this application has high sensitivity, meeting the requirements for accurate detection of trace residues and trace components, and effectively ensuring the comprehensiveness and safety of the analytical results.
[0077] Example 3: Accuracy
[0078] (1) Solution preparation
[0079] ① Reference stock solution: Accurately weigh approximately 15 mg each of reduced glutathione and oxidized glutathione, place them in a 50 mL volumetric flask, dissolve and dilute to the mark with water, and shake well; accurately measure 5 mL, place it in a 50 mL volumetric flask, dilute to the mark with water, and shake well.
[0080] Reference solution: Weigh approximately 15 mg each of reduced glutathione and oxidized glutathione reference standards, place them in a 50 mL volumetric flask, dissolve and dilute to the mark with water, shake well, accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well.
[0081] Other component solutions: prepared from sodium hyaluronate, glycine, lysine hydrochloride, proline, valine, cysteine hydrochloride, sodium ascorbate phosphate, vitamin H, phosphate buffer system and water for injection in the prescribed proportions.
[0082] ②Accu-17%: Accurately measure 3 mL of other component solutions and place them in a 10 mL volumetric flask. Accurately add 1.7 mL of the reference solution and place it in the same 10 mL volumetric flask. Dilute with water to the mark and shake well. Prepare 3 parallel aliquots.
[0083] ③Accu-100%: Accurately measure 3 mL of other component solutions and place them in a 10 mL volumetric flask. Accurately add 1 mL of the reference stock solution and place it in the same 10 mL volumetric flask. Dilute with water to the mark and shake well. Prepare 3 parallel batches.
[0084] ④Accu-300%: Accurately measure 3 mL of other component solutions and place them in a 10 mL volumetric flask. Accurately add 3 mL of the reference stock solution and place it in the same 10 mL volumetric flask. Dilute with water to the mark and shake well. Prepare 3 parallel batches.
[0085] (2) Determination method
[0086] Take the reference solution and the recovery solutions of each proportion, inject them into the liquid chromatograph, and detect the peak areas of GSH and GSSH using the method described in Example 1 above. Calculate the recovery rate using the following formula.
[0087] Calculation formula: Recovery rate (%) = (Test volume - Background volume) / Added volume 100.
[0088] (3) Acceptance criteria
[0089] The average recoveries of reduced glutathione and oxidized glutathione at 17%, 100%, and 300% of the limit concentrations should be between 85% and 115%, with an RSD of no more than 5%. Report the confidence interval range.
[0090] (4) Verification results
[0091] The accuracy results for GSH are shown in Table 2, and the accuracy results for GSSH are shown in Table 3.
[0092] Table 2
[0093]
[0094] Table 3
[0095]
[0096] Validation results: GSH showed an average recovery rate of 99.4% at 17%, 100%, and 300% of the limit concentration, ranging from 85% to 115%, with an RSD of 2.02% (less than 5%) and a confidence interval of 97.88% to 100.96%. GSSH showed an average recovery rate of 102.1% at 17%, 100%, and 300% of the limit concentration, ranging from 85% to 115%, with an RSD of 2.34% (less than 5%) and a confidence interval of 100.24% to 103.9%. This demonstrates that the established analytical method can accurately and reliably reflect the actual content of the analyte.
[0097] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0098] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.
Claims
1. A method for detecting the content of GSH and GSSH in a composite solution, characterized in that, The process includes the following steps: using high performance liquid chromatography combined with external standard method to detect the composite solution, and calculating the contents of GSH and GSSH in the composite solution; The chromatographic column is a Kromasil C18 column; the mobile phase includes mobile phase A and mobile phase B; mobile phase A includes phosphate buffer and methanol, the phosphate buffer includes dihydrogen phosphate, heptane sulfonate and water; the pH of the phosphate buffer is 3.0~3.1; in mobile phase A, the volume percentage of the phosphate buffer is 92%~98%, and the volume percentage of the methanol is 2%~8%; mobile phase B is acetonitrile. Using a linear gradient elution method, at 0 min, the volume percentage of mobile phase A is 100% and the volume percentage of mobile phase B is 0%; at 12-18 min, the volume percentage of mobile phase A decreases to 75%-85% and the volume percentage of mobile phase B increases to 15%-25%.
2. The method for detecting the content of GSH and GSSH in the composite solution as described in claim 1, characterized in that, The detection method satisfies at least one of the following conditions (1) to (5): (1) The volume percentage of the phosphate buffer solution is 94%~96%; (2) In the phosphate buffer solution, the dihydrogen phosphate includes sodium dihydrogen phosphate and / or potassium dihydrogen phosphate; (3) In the phosphate buffer solution, the heptane sulfonate includes sodium heptane sulfonate and / or potassium heptane sulfonate; (4) In the phosphate buffer solution, the concentration of dihydrogen phosphate is 5~10 mg / mL; (5) In the phosphate buffer solution, the concentration of heptane sulfonate is 1~5 mg / mL.
3. The method for detecting the content of GSH and GSSH in the composite solution as described in claim 2, characterized in that, The detection method satisfies the following conditions (1) and / or (2): (1) In the phosphate buffer solution, the concentration of dihydrogen phosphate is 6~8 mg / mL; (2) In the phosphate buffer solution, the concentration of heptane sulfonate is 1.5~3 mg / mL.
4. The method for detecting the content of GSH and GSSH in the composite solution as described in any one of claims 1 to 3, characterized in that, The detection method satisfies at least one of the following conditions (1) to (6): (1) The elution procedure includes the following steps: using a linear gradient elution method, at 0 min, the volume percentage of mobile phase A is 100% and the volume percentage of mobile phase B is 0%; at 14~16 min, the volume percentage of mobile phase A decreases to 78%~82% and the volume percentage of mobile phase B increases to 18%~22%; (2) The linear gradient elution further includes the operation of cleaning the column, wherein the mobile phase is the mobile phase A, and the cleaning time of the column is 5~15 min; (3) The flow rate of the mobile phase is 0.5~1.5 mL / min; (4) The temperature of the chromatographic column is 25~35℃; (5) The injection volume is 40~80μL; (6) The detection wavelength is 200~220nm.
5. The method for detecting the content of GSH and GSSH in the composite solution as described in claim 4, characterized in that, The detection method satisfies the following conditions (1) to (4): (1) The elution procedure includes the following steps: using a linear gradient elution method, at 0 min, the volume percentage of mobile phase A is 100% and the volume percentage of mobile phase B is 0%; at 15 min, the volume percentage of mobile phase A decreases to 80% and the volume percentage of mobile phase B increases to 20%; (2) The flow rate of the mobile phase is 0.8~1.2 mL / min; (3) The injection volume is 50~70μL; (4) The detection wavelength is 205~215nm.
6. The method for detecting the content of GSH and GSSH in the composite solution as described in any one of claims 1 to 5, characterized in that, The composite solution also includes at least one of the following: hyaluronic acid, amino acids, vitamins, and sodium ascorbate phosphate.
7. The method for detecting the content of GSH and GSSH in the composite solution as described in claim 6, characterized in that, The composite solution satisfies at least one of the following conditions (1) to (3): (1) The hyaluronic acid substance is selected from at least one of sodium hyaluronate, potassium hyaluronate and calcium hyaluronate; (2) The amino acid is selected from at least one of glycine, proline, valine, cysteine hydrochloride and lysine hydrochloride; (3) The vitamin mentioned is vitamin H.