Simultaneous determination of D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography

Abstract

The present invention belongs to the technical field of pharmaceutical detection, and in particular to a method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography adopts the following chromatographic conditions: the chromatographic column is filled with 18-crown ether-6 coated on the surface of silica gel, the mobile phase is a perchloric acid aqueous solution (pH1.0~2.0), the flow rate is 0.4~0.6mL / min, the column temperature is 25℃~35℃, and the detection wavelength is 220nm. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography provided by the present invention is simple, fast, highly sensitive, and has good separation, which fills the gap in drug detection and effectively controls the quality of raw materials.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical detection, and particularly relates to a method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography. Background Art

[0002] Peptide drugs, due to their high specificity and low toxicity, play an important role in the treatment of metabolic diseases, tumors, and immune-related disorders. Etekatide hydrochloride, a novel calcimimetic, is used to treat secondary hyperparathyroidism in patients with chronic kidney disease by modulating the parathyroid calcium-sensing receptor. Its synthesis process uses L-cysteine ​​as a key starting material, and the purity of L-cysteine ​​directly affects the structural integrity and pharmacological activity of the final product, etekatide hydrochloride. In particular, residual D-cysteine ​​(enantiomeric impurity) and L-cystine (oxidized dimer impurity) in L-cysteine ​​can, if not effectively controlled, lead to deviations in the drug's stereochemical configuration, decreased bioavailability, and even the risk of immunogenicity. Therefore, the development of highly sensitive and selective impurity detection methods is crucial for the quality control of active pharmaceutical ingredients.

[0003] Currently, the traditional method for detecting amino acid impurities in L-cysteine ​​is primarily thin-layer chromatography (TLC) (e.g., General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia). However, TLC has significant limitations: ① Low sensitivity (detection limit typically >0.1%), making it difficult to meet the ICH Q3A requirements for the limit of quantification of impurities; ② Inadequate separation efficiency, making it impossible to distinguish between D-cysteine ​​and L-cystine (the Rf values ​​of the two are similar); and ③ Quantitative accuracy relies on a colorimetric reaction, which is susceptible to background interference. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography. The method is simple, rapid, highly sensitive and has good separation, thus filling the gap in drug detection and effectively controlling the quality of raw materials.

[0005] The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography of the present invention comprises the following steps:

[0006] (1) Setting chromatographic conditions:

[0007] The detector is a PDA or UV detector;

[0008] The chromatographic column is filled with silica gel coated with 18-crown ether-6;

[0009] The detection wavelength is 220 nm;

[0010] Column temperature is 25~35℃;

[0011] The mobile phase was aqueous perchloric acid;

[0012] Flow rate: 0.4~0.6mL / min;

[0013] The injection volume is 5-50 μL;

[0014] (2) Prepare system suitability solution: Dissolve L-cysteine, D-cysteine, and L-cystine reference in mobile phase and dilute to obtain a mixed solution;

[0015] (3) Prepare the test solution: Accurately weigh L-cysteine, dissolve it in the mobile phase, and dilute it;

[0016] (4) Determination: Inject the mobile phase, system suitability solution and test solution into the liquid chromatograph respectively, record the chromatogram and separate the target components, and calculate the impurity content by the area normalization method with the addition of the correction factor.

[0017] Preferably, the chromatographic column is CROWNPAK ® CR (+), specifications are 4.0mm×150mm, particle size is 5µm.

[0018] Preferably, the column temperature is 30° C., the mobile phase pH is 1.0, the flow rate is 0.5 mL / min, and the injection volume is 10 μL.

[0019] Preferably, the concentration of L-cysteine ​​in the system suitability solution is 2-10 mg / mL, and the concentrations of D-cysteine ​​and L-cystine are both 4-20 μg / mL.

[0020] Preferably, the concentration of L-cysteine ​​in the test solution is 2-10 mg / mL.

[0021] Preferably, the detection limit of D-cysteine ​​and L-cystine is 0.3-0.6 μg / mL, and the quantification limit is 1.0-2.0 μg / mL.

[0022] In the chromatographic separation, the separation degrees of L-cysteine, D-cysteine ​​and L-cystine are all greater than or equal to 1.5.

[0023] The calculation formula of the area normalization method with the addition of the correction factor is: impurity content (%) = (impurity peak area / total peak area) × 100% × impurity correction factor.

[0024] The method is applicable to the determination of D-cysteine ​​and L-cystine in raw materials, and the quality control standard is D-cysteine ​​content ≤ 0.2%, and L-cystine content ≤ 0.2%.

[0025] The mobile phase has a pH of 1.0 to 2.0, preferably 1.0.

[0026] Specifically, the method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography comprises the following steps:

[0027] (1) Chromatographic conditions:

[0028] Detector: PDA or UV detector;

[0029] Chromatographic column: 18-crown ether-6 coated on silica gel as filler, CROWNPAK ® CR (+), specifications: 4.0 mm × 150 mm, 5 µm;

[0030] Detection wavelength: 220nm;

[0031] Column temperature: 25~35℃, preferably 30℃;

[0032] Mobile phase: perchloric acid aqueous solution (pH 1.0-2.0), preferably perchloric acid aqueous solution (pH 1.0);

[0033] Flow rate: 0.4~0.6mL / min, preferably 0.5mL / min;

[0034] Injection volume: 5~50μL, preferably 10μL.

[0035] (2) Preparation of system suitability solution:

[0036] Take L-cysteine, D-cysteine ​​and L-cystine reference substances, dissolve them in the mobile phase and dilute them to make a mixed solution containing 5 mg of L-cysteine, 10 μg of D-cysteine ​​and L-cystine per 1 mL, which is used as the system suitability solution.

[0037] (3) Preparation of test solution:

[0038] Accurately weigh L-cysteine, dissolve it in the mobile phase and dilute it to make a solution containing 5 mg per 1 mL as the test solution.

[0039] (4) Determination method:

[0040] Accurately measure the solvent (mobile phase), system suitability solution, and test solution, inject them into the liquid chromatograph, record the chromatogram, and separate L-cysteine, D-cysteine, and L-cystine. In the chromatogram of the test solution, identify the chromatographic peaks with the same retention times as D-cysteine ​​and L-cystine in the test solution, and calculate them by area normalization.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The method of the present invention for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography is simple and convenient, has a fast analysis speed, high sensitivity, and good separation, and can accurately and quantitatively detect the contents of D-cysteine ​​and L-cystine in L-cysteine, thereby objectively and accurately evaluating the quality of L-cysteine, and has important practical significance for product quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 HPLC chromatogram of the solvent in Example 1.

[0044] Figure 2 HPLC chromatogram of the system suitability solution in Example 1.

[0045] Figure 3 The HPLC chromatogram of the test solution in Example 1 is shown.

[0046] Figure 4 This is the HPLC chromatogram of the system suitability solution in Example 2.

[0047] Figure 5 This is the HPLC chromatogram of the system suitability solution in Example 3.

[0048] Figure 6 This is the HPLC chromatogram of the system suitability solution in Example 4.

[0049] Figure 7 This is the HPLC chromatogram of the system suitability solution in Example 5.

[0050] Figure 8 This is the HPLC chromatogram of the system suitability solution in Example 6.

[0051] Figure 9 This is a comparison chart of the HPLC results in Comparative Example 1. DETAILED DESCRIPTION

[0052] The present invention will be further described below with reference to specific embodiments.

[0053] The raw materials and additives used in the following examples and comparative examples are all commercially available products.

[0054] Example 1

[0055] The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography comprises the following steps:

[0056] (1) Chromatographic conditions:

[0057] Detector: PDA detector;

[0058] Chromatographic column: CROWNPAK ® CR (+), specifications: 4.0 mm × 150 mm, 5 µm;

[0059] Detection wavelength: 220nm;

[0060] Column temperature: 30°C;

[0061] Mobile phase: perchloric acid aqueous solution (pH 1.0);

[0062] Flow rate: 0.5 mL / min;

[0063] Injection volume: 10 μL.

[0064] (2) Preparation of system suitability solution:

[0065] Take L-cysteine, D-cysteine ​​and L-cystine reference substances, dissolve them in the mobile phase and dilute them to make a mixed solution containing 5 mg of L-cysteine, 10 μg of D-cysteine ​​and L-cystine per 1 mL, which is used as the system suitability solution.

[0066] (3) Preparation of test solution:

[0067] Accurately weigh L-cysteine, dissolve it in the mobile phase and dilute it to make a solution containing 5 mg per 1 mL as the test solution.

[0068] (4) Determination method:

[0069] Accurately measure the solvent (mobile phase), system suitability solution and test solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0070] The HPLC chromatogram of the solvent in this example is as follows Figure 1 As shown in the figure, the solvent peak does not interfere with the detection of this method.

[0071] The HPLC chromatogram of the system suitability solution of this example is as follows Figure 2 As shown by Figure 2 It can be seen that D-cysteine, L-cysteine ​​and L-cystine appear in sequence, and the separation between each peak is greater than 1.5. The data such as the peak area are shown in Table 1.

[0072] The HPLC chromatogram of the test solution in this embodiment is as follows Figure 3 As shown by Figure 3 It can be seen that the test solution contains L-cystine, and the peak area and other data are shown in Table 2.

[0073] Table 1 Solution peaks for system suitability in Example 1

[0074]

[0075] Table 2 Peak table of test solution in Example 1

[0076]

[0077] Example 2

[0078] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the column temperature is adjusted to 25° C., the system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded.

[0079] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 4 As shown, the separation data shown in Table 3 is shown. Figure 4 As can be seen from Table 3, the separation between each peak is greater than 1.5, and the separation is good.

[0080] Table 3 Solution peaks for system suitability in Example 2

[0081]

[0082] Example 3

[0083] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the column temperature is adjusted to 35° C., the system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded.

[0084] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 5 As shown, the separation data shown in Table 4 is shown. Figure 5 As can be seen from Table 4, the separation between each peak is greater than 1.5, and the separation is good.

[0085] Table 4. Solution peaks for system suitability of Example 3

[0086]

[0087] Example 4

[0088] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the pH of the mobile phase is adjusted to 2.0, the system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded.

[0089] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 6 As shown, the separation data shown in Table 5 is shown. Figure 6 As can be seen from Table 5, the separation between each peak is greater than 1.5, and the separation is good.

[0090] Table 5 Solution peaks for system suitability of Example 4

[0091]

[0092] Example 5

[0093] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the flow rate is adjusted to 0.4 mL / min, the system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded.

[0094] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 7 As shown, the separation data shown in Table 6 is shown. Figure 7 As can be seen from Table 6, the separation between each peak is greater than 1.5, and the separation is good.

[0095] Table 6 Solution peaks for system suitability of Example 5

[0096]

[0097] Example 6

[0098] The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the flow rate is adjusted to 0.6 mL / min, the system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded.

[0099] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 8 As shown, the separation data shown in Table 7 is shown. Figure 8 As can be seen from Table 7, the separation between each peak is greater than 1.5, and the separation is good.

[0100] Table 7 Solution peaks for system suitability of Example 6

[0101]

[0102] Comparative Example 1

[0103] The chromatographic conditions of this comparative example are basically the same as those of Example 4, except that the chromatographic column is changed to CHIRALPAK® AD-RH with specifications of 4.6 mm × 150 mm and 5 μm.

[0104] Using the mobile phase as solvent, L-cysteine, D-cysteine ​​and L-cystine positioning solutions were prepared and injected for analysis. The HPLC comparison chart is as follows: Figure 9 As shown, from top to bottom are L-cysteine, D-cysteine, L-cystine, Figure 9L-cysteine, D-cysteine ​​and L-cystine are basically overlapped, and there is no separation effect, which shows that this comparative example is not suitable for the detection of the compounds in the present invention.

[0105] Methodological verification of the present invention:

[0106] (1) Limit of detection and limit of quantification

[0107] Use the solvent (mobile phase) as the blank, adjust the instrument sensitivity, inject samples three times continuously, record the instrument noise level within the peak time range of the analyte, and calculate the average noise.

[0108] Detection limit: Accurately prepare a reference solution of the analyte, gradually dilute it to a specific concentration, and inject it for three consecutive measurements. Calculate the ratio of peak height to noise (signal-to-noise ratio). The detection limit is the sample concentration with a signal-to-noise ratio (S / N) greater than 3. The detection limit is the ratio of the sample concentration to the theoretical sample concentration.

[0109] Limit of quantitation: Accurately prepare a reference solution of the analyte, gradually dilute it to a specific concentration, and inject it for six consecutive measurements. Calculate the ratio of peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) greater than 10 is the limit of quantitation. The ratio relative to the theoretical sample concentration is the limit of quantitation.

[0110] Quantitation limit solution: Take D-cysteine ​​and L-cystine reference substances, dissolve them in the mobile phase and dilute them to make a mixed solution containing 1.0 μg of D-cysteine ​​and 2.0 μg of L-cystine per 1 mL.

[0111] Detection limit solution: Accurately measure 3 mL of quantification limit solution, place it in a 10 mL volumetric flask, dilute to the scale with mobile phase, and shake well.

[0112] The solvent was accurately measured and injected continuously for 3 times according to the chromatographic conditions of Example 1, the detection limit solution was injected continuously for 3 times, and the quantification limit solution was injected continuously for 6 times. The chromatograms were recorded and the signal-to-noise ratio was calculated. The results are shown in Tables 8, 9, and 10.

[0113] Table 8 Baseline noise detection results

[0114]

[0115] Table 9 Detection limit determination results

[0116]

[0117] Table 10 Quantitation limit determination results

[0118]

[0119] Conclusion: Under this method, the sensitivity of D-cysteine ​​and L-cystine meets the requirements.

[0120] (2) Calculation of linearity and correction factors

[0121] Select sample test points at appropriate intervals within the pre-set range, with at least 5 points for impurities and at least 10 points for main components, and the lowest and highest points of the range should be included.

[0122] A series of linear solutions of D-cysteine, L-cystine, and L-cysteine ​​were prepared according to the relative concentrations in Tables 11, 12, and 13 below. The results are as follows, and the calculated correction factors are shown in Table 14.

[0123] Table 11 D-cysteine ​​linearity results

[0124]

[0125] Table 12 L-cystine linearity results

[0126]

[0127] Table 13 L-cysteine ​​linearity results

[0128]

[0129] Table 14 Correction factor calculation results

[0130]

[0131] Conclusion: The linear correlation coefficients for L-cysteine, D-cysteine, and L-cystine were all greater than 0.99, indicating a good linear relationship. Based on the ratio of the linear slope of the principal component to the linear slope of the impurity, the correction factors for D-cysteine ​​and L-cystine were calculated to be 1.0 and 0.3, respectively.

Claims

1. A method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography, characterized in that: The following steps are involved: (1) Setting chromatographic conditions: The detector is a PDA or UV detector; The chromatographic column is filled with silica gel coated with 18-crown ether-6; The detection wavelength is 220 nm; Column temperature is 25~35℃; The mobile phase was aqueous perchloric acid with a pH of 1.0–2.0; Flow rate: 0.4~0.6mL / min; The injection volume is 5-50 μL; (2) Prepare system suitability solution: Dissolve L-cysteine, D-cysteine, and L-cystine reference in mobile phase and dilute to obtain a mixed solution; (3) Prepare the test solution: Accurately weigh L-cysteine, dissolve it in the mobile phase, and dilute it; (4) Determination: The mobile phase, system suitability solution and test solution are respectively injected into the liquid chromatograph, the chromatogram is recorded and the target components are separated, and the impurity content is calculated by the area normalization method with the addition of a correction factor; in the chromatographic separation, the separation degree of L-cysteine, D-cysteine ​​and L-cystine is ≥1.

5.

2. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, wherein: Chromatographic column is CROWNPAK ® CR (+), specifications are 4.0mm×150mm, particle size is 5µm.

3. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, wherein: The column temperature was 30 °C, the flow rate was 0.5 mL / min, and the injection volume was 10 μL.

4. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, wherein: The concentration of L-cysteine ​​in the system suitability solution is 2-10 mg / mL, and the concentrations of D-cysteine ​​and L-cystine are both 4-20 μg / mL.

5. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, wherein: The concentration of L-cysteine ​​in the test solution is 2-10 mg / mL.

6. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, wherein: The detection limits of D-cysteine ​​and L-cystine were 0.3~0.6μg / mL, and the quantification limits were 1.0~2.0μg / mL.

7. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, wherein: The calculation formula of the area normalization method with the addition of the correction factor is: impurity content (%) = (impurity peak area / total peak area) × 100% × impurity correction factor.

8. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, wherein: The method is applicable to the determination of D-cysteine ​​and L-cystine in raw materials, and the quality control standard is D-cysteine ​​content ≤ 0.2%, and L-cystine content ≤ 0.2%.

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