Analysis method for measuring purity of L-cysteine bulk drug by HPLC-DAD (High Performance Liquid Chromatography-Diode Array Detector)

Abstract

The invention relates to an analysis method for determining the purity of an L-cysteine bulk drug by HPLC-DAD (High Performance Liquid Chromatography-Diode Array Detector), and aims to provide an analysis method for determining the purity of an L-cysteine bulk drug by only using a conventional octadecyl silica gel bonded chromatographic column. The invention discloses an analysis method for measuring the purity of an L-cysteine bulk drug by HPLC-DAD (High Performance Liquid Chromatography-Differential AAD), which can obtain an L-cysteine chromatographic peak with a good peak shape and a symmetry factor of more than 0.80 and a separation degree of more than 1.5, and comprises the following steps: setting chromatographic conditions: a mobile phase is a 0.1% phosphoric acid aqueous solution and acetonitrile in a ratio of 93: 7-95: 5, and a chromatographic column is an octadecyl silica gel bonded column; the method comprises the following steps: preparing an aqueous solution of an L-cysteine bulk drug test sample: adding ultrapure water into an L-cysteine bulk drug as a solvent to dissolve the L-cysteine bulk drug; and a determination step: injecting the blank solution and the test solution into a liquid chromatograph to obtain the purity of the L-cysteine.

Description

Technical Field

[0001] This invention relates to the field of purity analysis, and more specifically to an analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD. Background Technology

[0002] L-cysteine ​​is a non-essential amino acid for humans, one of the 20 common protein amino acids. The L-cysteine ​​molecule contains a sulfhydryl group, which is the chemical basis for many of its biological activities and applications. L-cysteine ​​is used in cosmetics, pharmaceuticals, and food.

[0003] Currently, methods for determining the purity of L-cysteine ​​using high-performance liquid chromatography (HPLC) have been developed and researched, with a focus on developing more efficient, specific, and sensitive methods. It is well known that conventional octadecyl silica-bonded columns (C18 columns) do not yield satisfactory peak shapes and resolutions when determining the purity of L-cysteine ​​using HPLC. Current research often focuses on optimizing hydrophilic HPLC columns or improving the mobile phase to achieve better peak shapes and resolutions. However, optimizing the column requires modifying the mobile phase, which complicates the detection method and increases costs. Therefore, there is a need to develop an HPLC method that can achieve good peak shapes and resolutions for the analysis of L-cysteine ​​raw materials using only a conventional C18 column. Summary of the Invention

[0004] The problem to be solved by the present invention

[0005] In view of the problems in the prior art, the purpose of this invention is to provide an analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD, which can obtain L-cysteine ​​chromatographic peaks with good peak shape, a symmetry factor of 0.80 or higher, and a resolution of 1.5 or higher, using only a conventional octadecyl silica bonded chromatographic column.

[0006] Methods for solving problems

[0007] The inventors have discovered that when determining the purity of L-cysteine ​​raw material using HPLC-DAD, using ultrapure water as the solvent to prepare the test solvent for L-cysteine ​​raw material, and simultaneously setting the mobile phase for HPLC determination to 0.1% phosphoric acid aqueous solution: acetonitrile (93:7~95:5), the obtained L-cysteine ​​chromatographic peak can be well-shaped with a symmetry factor of 0.80 or higher and a resolution of 1.5 or higher using only a conventional octadecyl silica bonded chromatographic column.

[0008] Specifically, to solve the above-mentioned technical problems, the present invention provides the following analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD.

[0009] [1] An analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD, characterized by comprising the following steps:

[0010] Steps for setting chromatographic conditions:

[0011] The mobile phase is 0.1% phosphoric acid aqueous solution: acetonitrile = 93:7~95:5.

[0012] The chromatographic column is an octadecyl silica bonded column;

[0013] The steps for preparing an aqueous solution of L-cysteine ​​raw material are as follows: Add ultrapure water as a solvent to the L-cysteine ​​raw material to dissolve it;

[0014] Determination procedure: Inject blank solution and test solution into liquid chromatograph to obtain the purity of L-cysteine.

[0015] [2] The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD as described in [1] is characterized in that,

[0016] The mobile phase is 0.1% phosphoric acid aqueous solution: acetonitrile = 94:6~95:5.

[0017] [3] The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD as described in [1] is characterized in that,

[0018] The chromatographic conditions also include:

[0019] The detector is a DAD;

[0020] The detection wavelength is 200 nm;

[0021] The column temperature is 25~35℃;

[0022] The flow rate is 0.4~1.5 mL / min;

[0023] The injection volume was 5 μL.

[0024] [4] The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD as described in [1] is characterized in that,

[0025] The concentration of the prepared L-cysteine ​​raw material test sample aqueous solution was 0.0006~7.5 mg / mL.

[0026] [5] The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to [1] is characterized in that,

[0027] The concentration of the prepared L-cysteine ​​raw material test sample aqueous solution was 1.5 mg / mL.

[0028] [6] The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD as described in [3] is characterized in that,

[0029] The column temperature is 30~35℃.

[0030] [7] The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to [1] is characterized in that the purity of L-cysteine ​​raw material is above 95%.

[0031] [8] The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to [1] is characterized in that the purity of L-cysteine ​​is obtained by the area normalization method.

[0032] The effects of the invention

[0033] This invention provides an analytical method for determining the purity of L-cysteine ​​raw material using HPLC-DAD. This method allows for the determination of L-cysteine ​​using a conventional C18 column, yielding a good peak shape and a resolution of 1.5 or higher. It accurately quantifies the purity of L-cysteine ​​with low baseline noise and high responsiveness. This provides a convenient, rapid, accurate, and objective method for evaluating the quality of L-cysteine ​​raw material, significantly improving the efficiency of product sample testing, especially for the release of multiple batches of products, and is of great practical significance for product quality control. Attached Figure Description

[0034] Figure 1A The mobile phase was 0.1% phosphoric acid aqueous solution: acetonitrile = 95:5, the solvent was water, the column temperature was 30℃, and the blank chromatogram was obtained.

[0035] Figure 1B Chromatogram of L-cysteine ​​test solution (concentration 1.5 mg / mL) with mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 95:5, solvent of water, and column temperature of 30℃;

[0036] Figure 2A The mobile phase was 0.1% phosphoric acid aqueous solution: acetonitrile = 95:5, the solvent was water, the column temperature was 35℃, and the blank chromatogram was obtained.

[0037] Figure 2BChromatogram of L-cysteine ​​test solution (concentration 1.5 mg / mL) with mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 95:5, solvent of water, and column temperature of 35℃;

[0038] Figure 3A Chromatogram of a blank solution with a mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 93:7, water as solvent, and column temperature of 30℃.

[0039] Figure 3B Chromatogram of L-cysteine ​​test solution (concentration 1.5 mg / mL) with mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 93:7, solvent of water, and column temperature of 30℃.

[0040] Figure 4A Chromatogram of a blank solution with a mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 94:6, water as solvent, and column temperature of 30℃.

[0041] Figure 4B Chromatogram of L-cysteine ​​test solution (concentration 1.5 mg / mL) with mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 94:6, solvent of water, and column temperature of 30℃.

[0042] Figure 5A Chromatogram of a blank solution with a mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 95: 5, a solvent of hydrochloric acid buffer at pH = 1.2, and a column temperature of 30℃.

[0043] Figure 5B Chromatogram of an L-cysteine ​​test solution (concentration 1.5 mg / mL) with a mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 95:5, a solvent of hydrochloric acid buffer at pH = 1.2, and a column temperature of 30℃.

[0044] Figure 6A Chromatogram of a blank solution with a mobile phase of 0.1% trifluoroacetic acid aqueous solution: acetonitrile = 95:5, water as solvent, and column temperature of 30℃.

[0045] Figure 6B Chromatogram of L-cysteine ​​test solution (concentration 1.5 mg / mL) with mobile phase of 0.1% trifluoroacetic acid aqueous solution: acetonitrile = 95:5, solvent of water, and column temperature of 30℃;

[0046] Figure 7A Blank chromatogram of mobile phase 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20, solvent water, column temperature 30℃;

[0047] Figure 7BChromatogram of L-cysteine ​​test solution (concentration 1.5 mg / mL) with mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20, solvent of water, and column temperature of 30℃;

[0048] Figure 8A Blank chromatogram of mobile phase 0.1% phosphoric acid aqueous solution: acetonitrile = 92:8, solvent water, column temperature 30℃;

[0049] Figure 8B Chromatogram of an L-cysteine ​​test solution (concentration 1.5 mg / mL) with a mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 92:8, solvent of water, and column temperature of 30℃.

[0050] Figure 9A Blank chromatogram of mobile phase 0.1% phosphoric acid aqueous solution: acetonitrile = 96:4, solvent water, column temperature 30℃;

[0051] Figure 9B Chromatogram of L-cysteine ​​test solution (concentration 1.5 mg / mL) with mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 96:4, solvent of water, and column temperature of 30℃;

[0052] Figure 10 Linear relationship between L-cysteine ​​peak area and concentration. Detailed Implementation

[0053] The specific embodiments of the present invention will be further explained and described below with reference to specific examples; however, such explanation and description do not constitute a limitation on the technical solution of the present invention.

[0054] The present invention provides an analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD, characterized by comprising the following steps.

[0055] Steps for setting chromatographic conditions:

[0056] The mobile phase is a 0.1% phosphoric acid aqueous solution (1.0 mL of phosphoric acid is mixed with 1000 mL of water and sonicated):acetonitrile = 93:7~95:5. From the perspective of superior resolution, a 0.1% phosphoric acid aqueous solution:acetonitrile ratio of 94:6~95:5 is preferred.

[0057] The chromatographic column is an octadecyl silica bonded column.

[0058] The detector is a DAD.

[0059] The detection wavelength is 200 nm.

[0060] The column temperature is 25~35℃, and 30~35℃ is preferred from the perspective of excellent symmetry factor and resolution.

[0061] The flow rate is 0.4~1.5 mL / min.

[0062] The injection volume was 5 μL.

[0063] The steps for preparing an aqueous solution of L-cysteine ​​raw material for testing are as follows:

[0064] In the L-cysteine ​​raw material, ultrapure water is added as a solvent, and the mixture is shaken to dissolve. The solution is then diluted to the mark with water and shaken well. The ultrapure water can be degassed by sonication for 10–20 min to improve the accuracy of the results. The concentration of the prepared L-cysteine ​​raw material test solution is 0.0006–7.5 mg / mL. Due to the instability of L-cysteine, the L-cysteine ​​raw material test solution should be analyzed within 4 hours after preparation.

[0065] Measurement steps:

[0066] The blank solution and the test solution were injected into the liquid chromatograph, and the chromatograms were recorded. The purity of L-cysteine ​​was obtained using the area normalization method. The formula for calculating the content of a component i (%) is: [peak area of ​​component i / sum of the areas of all peaks] × 100%.

[0067] This invention provides an analytical method for determining the purity of L-cysteine ​​raw material using HPLC-DAD. The method uses ultrapure water as the solvent to prepare the test solvent for L-cysteine ​​raw material. Simultaneously, when the mobile phase for HPLC determination is set to 0.1% phosphoric acid aqueous solution: acetonitrile (93:7~95:5), it can obtain good peak shape using a conventional C18 column with a symmetry factor above 0.80 and a resolution above 1.5. This method accurately quantifies the purity of L-cysteine ​​with very low linear noise and high responsiveness. It provides a convenient, rapid, accurate, and objective method for evaluating the quality of L-cysteine ​​raw material, improving the efficiency of product sample testing, especially for the release of multiple batches of products. This has significant practical implications for product quality control, providing scientific analytical results for subsequent production activities and playing a positive role in improving production efficiency.

[0068] Example

[0069] The instruments and reagents used in the examples are as follows:

[0070] Agilent HPLC 1260-DAD;

[0071] Octadecyl silica bonded column (Waters XBridge C18, 250 mm * 4.6 mm * 5 μm);

[0072] Laboratory-grade water (prepared using a Millipore ultrapure water system, resistivity 18.2 MΩ);

[0073] L-cysteine ​​sample, sourced from Maclean's, purity 99.4%;

[0074] L-cysteine ​​reference standard, sourced from Aladdin, purity 99.0%;

[0075] Water blank solution and solvent water: Measure 1000 mL of ultrapure water into a solvent bottle and sonicate until ready for use.

[0076] Example 1

[0077] (1) Chromatographic conditions:

[0078] Column: Octadecyl silica bonded column;

[0079] Mobile phase: 0.1% phosphoric acid aqueous solution: acetonitrile = 95:5;

[0080] Solvent: Water

[0081] Column temperature: 30℃;

[0082] Flow rate: 1.0 mL / min;

[0083] Detection wavelength: 200 nm (190-400 nm full wavelength scan is enabled).

[0084] Injection volume: 5 μL;

[0085] (2) Preparation of L-cysteine ​​test solution:

[0086] Weigh 22.5 mg of L-cysteine ​​sample into a 15 mL volumetric flask, add pure water (laboratory grade I water), shake to dissolve, dilute to the mark with solvent water and shake well to obtain an L-cysteine ​​test solution with a concentration of 1.5 mg / mL.

[0087] (3) Measurement method:

[0088] Accurately measure pure water (blank) and the test solution and inject them into the HPLC-DAD chromatograph, and record the chromatograms.

[0089] The HPLC chromatogram of the L-cysteine ​​test solution in this embodiment is shown in Figure 1B. Figure 1B The L-cysteine ​​peak was observed at a retention time of 2.64 min, indicating a good peak shape. The separation between the L-cysteine ​​peak and adjacent impurity peaks was greater than 1.5, demonstrating good separation, and the symmetry factor was above 0.8. The chromatographic conditions and data of the L-cysteine ​​peaks measured in the following examples and comparative examples are summarized in Table 1 below.

[0090] Example 2

[0091] Example 2 was identical to Example 1 in terms of detection method and chromatographic conditions, except that the column temperature was adjusted to 35°C. Chromatograms were recorded.

[0092] The HPLC chromatogram of the test solution in this embodiment is as follows: Figure 2B As shown. Figure 2B The peak of L-cysteine ​​was observed at a retention time of 2.62 min, indicating that the peak shape of L-cysteine ​​was good. The separation degree between the L-cysteine ​​peak and the adjacent impurity peak was greater than 1.5, indicating good separation, and the symmetry factor was above 0.8.

[0093] Example 3

[0094] Example 3 was identical to Example 1 in terms of detection method and chromatographic conditions, except that the ratio of the mobile phase 0.1% phosphoric acid aqueous solution to acetonitrile was adjusted to 93:7. The chromatogram was recorded.

[0095] The HPLC chromatogram of the test solution in this embodiment is as follows: Figure 3B As shown. Figure 3B The peak of L-cysteine ​​was observed at a retention time of 2.55 min, indicating that the peak shape of L-cysteine ​​was good. The separation degree between the L-cysteine ​​peak and the adjacent impurity peak was greater than 1.5, indicating good separation, and the symmetry factor was above 0.8.

[0096] Example 4

[0097] Example 4 was identical to Example 1 in terms of detection method and chromatographic conditions, except that the ratio of the mobile phase 0.1% phosphoric acid aqueous solution to acetonitrile was adjusted to 94:6. The chromatogram was recorded.

[0098] The HPLC chromatogram of the test solution in this embodiment is as follows: Figure 4B As shown. Figure 4B The peak of L-cysteine ​​was observed at a retention time of 2.59 min, indicating that the peak shape of L-cysteine ​​was good. The separation degree between the L-cysteine ​​peak and the adjacent impurity peak was greater than 1.5, indicating good separation, and the symmetry factor was above 0.8.

[0099] In the above embodiments of the present invention, when determining the purity of L-cysteine ​​raw material using HPLC-DAD, ultrapure water is used as the solvent to prepare the test solvent for L-cysteine ​​raw material. Simultaneously, when the mobile phase for HPLC determination is set to 0.1% phosphoric acid aqueous solution: acetonitrile (93:7~95:5), even with only a conventional octadecyl silica bonded column, the obtained chromatographic peaks have good peak shapes, a resolution of 1.5 or higher, and a symmetry factor of 0.8 or higher. Furthermore, as can be seen from the chromatograms of the embodiments, the signal generated by the instrument (peak height or peak area) is very strong, while the baseline noise is very low, indicating high responsiveness of the method of the present invention.

[0100] Comparative Example 1

[0101] Comparative Example 1 was identical to Example 1 in both detection method and chromatographic conditions, except that the solvent for preparing the test sample was changed from water to pH 1.2 buffer (22.5 mg of L-cysteine ​​sample was accurately weighed into a 15 mL volumetric flask, 10 mL of pH 1.2 buffer was added, shaken to dissolve, diluted to the mark with pH 1.2 buffer, and shaken well). The chromatogram was recorded. Figure 5B As shown, the L-cysteine ​​peak and the impurity peaks were not completely separated.

[0102] Comparative Example 2

[0103] Comparative Example 2 was identical to Example 1 in both detection method and chromatographic conditions, except that the mobile phase was changed to a 0.1% trifluoroacetic acid aqueous solution: acetonitrile = 95:5. Chromatograms were recorded. Figure 6B The retention time of L-cysteine ​​shown is 3.00 min. There is a significant interfering solvent peak next to L-cysteine, which makes it impossible to accurately quantify the purity.

[0104] Comparative Example 3

[0105] Comparative Example 3 was identical to Example 1 in both detection method and chromatographic conditions, except that the mobile phase was changed to 0.1% phosphoric acid aqueous solution:acetonitrile = 80:20. The HPLC chromatogram of the test solution in Comparative Example 3 is shown below. Figure 7B As shown. Figure 7B The separation degree between the L-cysteine ​​peak and the adjacent impurity peak is less than 1.5, indicating poor separation.

[0106] Comparative Example 4

[0107] Comparative Example 4 was identical to Example 1 in both detection method and chromatographic conditions, except that the mobile phase was changed to 0.1% phosphoric acid aqueous solution:acetonitrile = 92:8. The HPLC chromatogram of the test solution in Comparative Example 4 is shown below. Figure 8B As shown. Figure 8BThe peak of L-cysteine ​​was observed at a retention time of 2.55 min. The separation between the L-cysteine ​​peak and the adjacent impurity peak was less than 1.5, indicating poor separation.

[0108] Comparative Example 5

[0109] Comparative Example 5 was identical to Example 1 in terms of detection method and chromatographic conditions, except that the ratio of the mobile phase 0.1% phosphoric acid aqueous solution to acetonitrile was adjusted to 96:4. Chromatograms were recorded.

[0110] The HPLC chromatogram of the test solution in this embodiment is as follows: Figure 9B As shown. Figure 9B The peak of L-cysteine ​​was observed at a retention time of 2.64 min. It can be seen that although the separation degree between the L-cysteine ​​peak and the adjacent impurity peak is greater than 1.5, indicating good separation, the symmetry factor is less than 0.80, which is worse than that of the embodiment of the present invention.

[0111]

[0112] As can be seen from the above examples and comparative examples, when determining the purity of L-cysteine ​​raw material using HPLC-DAD, if only a conventional octadecyl silica bonded column is used, in order to obtain a good peak shape, it is necessary to use ultrapure water as a solvent to prepare the test solvent for L-cysteine ​​raw material, and at the same time, the mobile phase for HPLC determination should be set to 0.1% phosphoric acid aqueous solution: acetonitrile (93:7~95:5) to obtain a good peak shape, a resolution of 1.5 or higher, a symmetry factor of 0.80 or higher, and high responsiveness, thus achieving the effects of the present invention.

[0113] Methodological verification of the present invention:

[0114] The system suitability evaluation method refers to the "9101 Analytical Method Validation Guidelines" in the Chinese Pharmacopoeia, Part IV. Resolution: The resolution between the L-cysteine ​​peak and adjacent impurity peaks ≥ 1.5; Theoretical Plate Number: The theoretical plate number of the L-cysteine ​​peak is not less than 5000; Symmetry Factor: The symmetry factor of the L-cysteine ​​peak is in the range of 0.8 to 1.5; Repeatability: The retention time repeatability RSD of the L-cysteine ​​peak is ≤ 1%, and the response factor (A / C) repeatability RSD is ≤ 1%.

[0115] 1. System applicability methodology verification

[0116] Chromatographic conditions:

[0117] The chromatographic conditions were the same as in Example 1.

[0118] Preparation of test solution:

[0119] Blank solution: Measure 1000 mL of ultrapure water into a solvent bottle and sonicate until ready for use;

[0120] Reference solutions 1 and 2: Accurately weigh 22.5 mg of L-cysteine ​​reference standard into a 15 mL volumetric flask, add 10 mL of water, shake to dissolve, dilute to the mark with solvent and shake well. Prepare two parallel solutions.

[0121] Measurement method:

[0122] Accurately measure the test solution and inject it into the HPLC-DAD chromatograph.

[0123] The results of the system suitability analysis are shown in Tables 2 and 3 below.

[0124]

[0125]

[0126] System suitability conclusions: In the blank solution, there were no interfering peaks at the position of the main peak of L-cysteine. In the test solution, the symmetry factor of the main peak was 0.80, the resolution was 2.02, and the theoretical plate number was 10037. The system had good repeatability, with a retention time RSD of 0.02% and a response factor (A / C) RSD of 0.28%.

[0127] All meet the requirements.

[0128] (2) Results of repeatability analysis of the test sample

[0129] Chromatographic conditions:

[0130] The chromatographic conditions were the same as in Example 1.

[0131] Preparation of test solution:

[0132] Test solutions 1-6: Accurately weigh 22.5 mg of L-cysteine ​​sample into a 15 mL volumetric flask, add 10 mL of water, shake to dissolve, dilute to the mark with solvent and shake well, and prepare six parallel solutions.

[0133] Measurement method:

[0134] Accurately measure the test solution and inject it into the HPLC-DAD chromatograph.

[0135] The repeatability results are shown in Table 4 below.

[0136]

[0137] Repeatability conclusion: The average purity of the 6 test solutions was 99.480%, and the RSD was 0.05%, which met the requirements.

[0138] 3. Linearity Validation

[0139] Chromatographic conditions:

[0140] The chromatographic conditions were the same as in Example 1.

[0141] Solution preparation:

[0142] Stock solution: Weigh 150 mg of L-cysteine ​​sample into a 20 mL volumetric flask, add pure water and shake to dissolve, dilute to the mark with solvent water and shake well to prepare stock solution (500%, 7.5 mg / mL).

[0143] Take an appropriate amount of the above stock solution, dilute it with water and shake well to obtain linear L-cysteine ​​samples 1-7 with concentrations of 7.5 mg / mL, 2.25 mg / mL, 1.8 mg / mL, 1.5 mg / mL, 1.2 mg / mL, 0.75 mg / mL, 0.3 mg / mL and 0.6 μg / mL.

[0144] Measurement method:

[0145] Accurately measure the test solution and inject it into the HPLC-DAD chromatograph.

[0146] See results Figure 10 The horizontal axis of the figure represents the concentration of L-cysteine, and the vertical axis represents the peak area of ​​L-cysteine ​​in the liquid chromatogram. It can be seen that within the concentration range of 0.6 μg / mL to 7.5 mg / mL, the concentration of L-cysteine ​​shows a good linear relationship with the peak area.

[0147] This invention provides an analytical method for determining the purity of L-cysteine ​​raw material using HPLC-DAD. This method allows for determination using a conventional C18 column with good peak shape, a resolution of ≥1.5, and high responsiveness, accurately quantifying the purity of L-cysteine. This provides a convenient, rapid, accurate, and objective method for evaluating the quality of L-cysteine ​​raw material, significantly improving the efficiency of product sample testing, especially for the release of multiple batches of products, and playing a crucial role in product quality control.

[0148] It should also be noted that the embodiments listed above are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and similar changes or modifications made thereto are those that can be directly derived or easily conceived by those skilled in the art from the content disclosed in the present invention, and should all fall within the protection scope of the present invention.

Claims

1. An analytical method for determining the purity of L-cysteine ​​raw material using HPLC-DAD, characterized in that, Includes the following steps: Steps for setting chromatographic conditions: The mobile phase is 0.1% phosphoric acid aqueous solution: acetonitrile = 93:7~95:

5. The chromatographic column is an octadecyl silica bonded column; The steps for preparing an aqueous solution of L-cysteine ​​raw material are as follows: Add ultrapure water as a solvent to the L-cysteine ​​raw material to dissolve it; Determination procedure: Inject blank solution and test solution into liquid chromatograph to obtain the purity of L-cysteine.

2. The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to claim 1, characterized in that, The mobile phase is 0.1% phosphoric acid aqueous solution: acetonitrile = 94:6~95:

5.

3. The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to claim 1, characterized in that, The chromatographic conditions also include: The detector is a DAD; The detection wavelength is 200 nm; The column temperature is 25~35℃; The flow rate is 0.4~1.5 mL / min; The injection volume was 5 μL.

4. The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to claim 1, characterized in that, The concentration of the prepared L-cysteine ​​raw material test sample aqueous solution was 0.0006~7.5 mg / mL.

5. The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to claim 1, characterized in that, The concentration of the prepared L-cysteine ​​raw material test sample aqueous solution was 1.5 mg / mL.

6. The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to claim 3, characterized in that, The column temperature is 30~35℃.

7. The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to claim 1, characterized in that, The purity of L-cysteine ​​raw material is over 95%.

8. The analytical method for determining the purity of L-cysteine ​​raw material by HPLC-DAD according to claim 1, characterized in that, The purity of L-cysteine ​​was obtained using the area normalization method.

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