Method for simultaneously determining D-cysteine and L-cystine in L-cysteine by high performance liquid chromatography

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for simultaneously determining D-cysteine and L-cystine in L-cysteine by high performance liquid chromatography. According to the method for simultaneously determining D-cysteine and L-cystine in L-cysteine through high performance liquid chromatography, adopted chromatographic conditions are as follows: a chromatographic column takes 18-crown ether-6 coated on the surface of silica gel as a filler, a mobile phase is a perchloric acid aqueous solution (pH is 1.0-2.0), the flow velocity is 0.4-0.6 mL / min, the column temperature is 25-35 DEG C, and the detection wavelength is 220 nm. The method for simultaneously determining D-cysteine and L-cystine in L-cysteine by high performance liquid chromatography provided by the invention is simple, convenient, rapid, high in sensitivity and good in separation degree, fills the blank of drug detection, and effectively controls the quality of bulk drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical detection, and in particular 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 play an important role in the treatment of metabolic diseases, tumors and immune-related diseases due to their high specificity and low toxicity. Etekatide hydrochloride, as a new type of calcimimetic, is used to treat secondary hyperparathyroidism in patients with chronic kidney disease by regulating 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, eterkatide hydrochloride. In particular, the D-cysteine ​​(enantiomeric impurity) and L-cystine (oxidized dimer impurity) that may remain in L-cysteine ​​may cause drug stereoconfiguration deviation, decreased bioavailability and even immunogenicity risks if not effectively controlled. Therefore, it is crucial to establish a highly sensitive and selective impurity detection method for API quality control.

[0003] At present, the traditional method for the detection of amino acid impurities in L-cysteine ​​is mainly thin layer chromatography (TLC) (such as the general rule 0502 of the 2020 edition of the Chinese Pharmacopoeia). However, TLC has significant limitations: ① low sensitivity (detection limit is usually > 0.1%), which makes it difficult to meet the requirements of ICH Q3A for the quantitative limit of impurities; ② insufficient separation efficiency, unable to distinguish D-cysteine ​​from L-cystine (the Rf values ​​of the two are similar); ③ quantitative accuracy depends on the color reaction and is easily affected by 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, which is simple, fast, 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: (1) Setting chromatographic conditions: The detector is a PDA or UV detector; The chromatographic column is filled with 18-crown ether-6 coated on the surface of silica gel; The detection wavelength is 220nm; Column temperature is 25~35℃; The mobile phase was aqueous perchloric acid; Flow rate: 0.4~0.6mL / min; The injection volume is 5~50μL; (2) Prepare system suitability solution: dissolve L-cysteine, D-cysteine ​​and L-cystine reference substances in mobile phase and dilute to obtain a mixed solution; (3) Prepare the test solution: accurately weigh L-cysteine, dissolve and dilute it with mobile phase; (4) Determination: Inject the mobile phase, system suitability solution and test sample 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 correction factor added.

[0006] Preferably, the chromatographic column is CROWNPAK ® CR (+), specification is 4.0mm×150mm, particle size is 5µm.

[0007] 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.

[0008] 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.

[0009] Preferably, the concentration of L-cysteine ​​in the test solution is 2-10 mg / mL.

[0010] 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.

[0011] In the chromatographic separation, the separation degrees of L-cysteine, D-cysteine ​​and L-cystine are all ≥1.5.

[0012] The calculation formula of the area normalization method with the addition of correction factor is: impurity content (%) = (impurity peak area / total peak area) × 100% × impurity correction factor.

[0013] The method is applicable to the determination of D-cysteine ​​and L-cystine in raw materials, and the quality control standard is that the D-cysteine ​​content is ≤0.2%, and the L-cystine content is ≤0.2%.

[0014] The mobile phase has a pH of 1.0-2.0, preferably 1.0.

[0015] Specifically, the method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography comprises the following steps: (1) Chromatographic conditions: Detector: PDA or UV detector; Chromatographic column: 18-crown ether-6 coated on silica gel as filler, CROWNPAK ® CR (+), specification: 4.0mm×150mm, 5µm; Detection wavelength: 220nm; Column temperature: 25~35℃, preferably 30℃; Mobile phase: perchloric acid aqueous solution (pH 1.0-2.0), preferably perchloric acid aqueous solution (pH 1.0); Flow rate: 0.4~0.6mL / min, preferably 0.5mL / min; Injection volume: 5~50μL, preferably 10μL.

[0016] (2) Preparation of system suitability solution: Take L-cysteine, D-cysteine ​​and L-cystine reference substances, add mobile phase to dissolve and dilute to make a mixed solution containing 5 mg of L-cysteine, 10 μg of D-cysteine ​​and L-cystine per 1 mL as the system suitability solution.

[0017] (3) Preparation of test solution: 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.

[0018] (4) Determination method: Accurately measure the solvent (mobile phase), system suitability solution and test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and separate L-cysteine, D-cysteine ​​and L-cystine. In the chromatogram of the test solution, determine the chromatographic peaks in the obtained test solution that are consistent with the retention time of D-cysteine ​​and L-cystine, and calculate them according to the area normalization method.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography of the present invention 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, which has important practical significance for product quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The HPLC chromatogram of the solvent in Example 1.

[0021] Figure 2 HPLC chromatogram of the system suitability solution in Example 1.

[0022] Figure 3 The HPLC chromatogram of the test solution in Example 1.

[0023] Figure 4 This is the HPLC chromatogram of the system suitability solution in Example 2.

[0024] Figure 5 HPLC chromatogram of the system suitability solution in Example 3.

[0025] Figure 6 This is the HPLC chromatogram of the system suitability solution in Example 4.

[0026] Figure 7 This is the HPLC chromatogram of the system suitability solution in Example 5.

[0027] Figure 8 This is the HPLC chromatogram of the system suitability solution in Example 6.

[0028] Fig. 9 This is a comparison chart of the HPLC results in Comparative Example 1. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments.

[0030] The raw materials and additives used in the following examples and comparative examples are all commercially available products.

[0031] Example 1 The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography comprises the following steps: (1) Chromatographic conditions: Detector: PDA detector; Column: CROWNPAK ® CR (+), specification: 4.0mm×150mm, 5µm; Detection wavelength: 220nm; Column temperature: 30°C; Mobile phase: perchloric acid aqueous solution (pH 1.0); Flow rate: 0.5 mL / min; Injection volume: 10 μL.

[0032] (2) Preparation of system suitability solution: Take L-cysteine, D-cysteine ​​and L-cystine reference substances, add mobile phase to dissolve and dilute to make a mixed solution containing 5 mg of L-cysteine, 10 μg of D-cysteine ​​and L-cystine per 1 mL as the system suitability solution.

[0033] (3) Preparation of test solution: 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.

[0034] (4) Determination method: Accurately measure the solvent (mobile phase), system suitability solution and test solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0035] The HPLC chromatogram of the solvent in this example is as follows Figure 1 As shown, the solvent peak does not interfere with the detection of this method.

[0036] 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 the peaks is greater than 1.5. The peak area and other data are shown in Table 1.

[0037] The HPLC chromatogram of the test solution in this example 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.

[0038] Table 1 Solution peak table for system suitability of Example 1

[0039] Table 2 Peak table of test solution in Example 1

[0040] Example 2 The detection method and chromatographic conditions used in this embodiment are the same as those in embodiment 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.

[0041] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 4 As shown, the separation degree and other data are shown in Table 3. Figure 4 It can be seen from Table 3 that the resolution between the peaks is greater than 1.5, and the resolution is good.

[0042] Table 3 Solution peak table of system suitability of Example 2

[0043] Example 3 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.

[0044] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 5 As shown, the separation degree and other data are shown in Table 4. Figure 5 It can be seen from Table 4 that the resolution between the peaks is greater than 1.5, and the resolution is good.

[0045] Table 4 Solution peak table for system suitability of Example 3

[0046] Example 4 The detection method and chromatographic conditions used in this embodiment are the same as those in embodiment 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.

[0047] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 6 As shown, the separation degree and other data are shown in Table 5. Figure 6 It can be seen from Table 5 that the resolution between the peaks is greater than 1.5, and the resolution is good.

[0048] Table 5 Solution peak table for system suitability of Example 4

[0049] Example 5 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.

[0050] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 7 As shown, the separation degree and other data are shown in Table 6. Figure 7 It can be seen from Table 6 that the resolution between the peaks is greater than 1.5, and the resolution is good.

[0051] Table 6 Solution peak table for system suitability of Example 5

[0052] Example 6 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.

[0053] In this embodiment, the HPLC chromatogram of the system suitability solution is as follows: Figure 8 As shown, the separation degree and other data are shown in Table 7. Figure 8It can be seen from Table 7 that the resolution between the peaks is greater than 1.5, and the resolution is good.

[0054] Table 7 Solution peak table for system suitability of Example 6

[0055] Comparative Example 1 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 a specification of 4.6 mm×150 mm and 5 μm.

[0056] 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 obtained is as follows: Fig. 9 As shown, from top to bottom, they are L-cysteine, D-cysteine, and L-cystine. Fig. 9 L-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.

[0057] Methodological verification of the present invention: 1. Limit of detection and limit of quantification Use the solvent (mobile phase) as blank, adjust the instrument sensitivity, inject samples three times continuously, record the noise level of the instrument within the peak time range of the analyte, and calculate the average noise.

[0058] Detection limit: Accurately prepare the reference solution of the test object, gradually dilute it to a certain concentration and inject it, and measure it three times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 3 is the detection limit concentration, and its ratio to the theoretical sample concentration is the detection limit.

[0059] Limit of quantitation: Accurately prepare the reference solution of the substance to be measured, gradually dilute it to a certain concentration and inject it, and measure it 6 times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 10 is the limit of quantitation concentration, and its ratio to the theoretical sample concentration is the limit of quantitation.

[0060] Quantitation limit solution: Take D-cysteine ​​and L-cystine reference substances, add mobile phase to dissolve and dilute to make a mixed solution containing 1.0 μg of D-cysteine ​​and 2.0 μg of L-cystine per 1 mL.

[0061] Detection limit solution: Accurately measure 3 mL of quantitation limit solution, place in a 10 mL volumetric flask, dilute to the mark with mobile phase, and shake well.

[0062] 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 quantitative limit solution was injected continuously for 6 times. The chromatogram was recorded and the signal-to-noise ratio was calculated. The results are shown in Tables 8, 9 and 10.

[0063] Table 8 Baseline noise detection results

[0064] Table 9 Detection limit determination results

[0065] Table 10 Results of quantitation limit determination

[0066] Conclusion: Under this method, the sensitivity of D-cysteine ​​and L-cystine meets the requirements.

[0067] (II) Calculation of linearity and correction factors Select sample test points at appropriate intervals within the pre-set range, with a minimum of 5 points for impurities and a minimum of 10 points for main components, and the lowest and highest points of the range should be included.

[0068] 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. The calculated correction factors are shown in Table 14.

[0069] Table 11 D-cysteine ​​linear results

[0070] Table 12 L-cystine linearity results

[0071] Table 13 L-cysteine ​​linear results

[0072] Table 14 Correction factor calculation results

[0073] Conclusion: The linear correlation coefficients of L-cysteine, D-cysteine ​​and L-cystine are all greater than 0.99, and the linear relationship is good. According to the ratio of the linear slope of the main component to the linear slope of the impurity, the correction factor of D-cysteine ​​is 1.0, and the correction factor of L-cystine is 0.3.

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 18-crown ether-6 coated on the surface of silica gel; The detection wavelength is 220nm; Column temperature is 25~35℃; The mobile phase was aqueous perchloric acid; Flow rate: 0.4~0.6mL / min; The injection volume is 5~50μL; (2) Prepare system suitability solution: dissolve L-cysteine, D-cysteine ​​and L-cystine reference substances in mobile phase and dilute to obtain a mixed solution; (3) Prepare the test solution: accurately weigh L-cysteine, dissolve and dilute it with the mobile phase; (4) Determination: Inject the mobile phase, system suitability solution and test sample 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 correction factor added.

2. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, characterized in that: Chromatographic column: CROWNPAK ® CR (+), specification is 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: In the chromatographic separation, the separation degrees of L-cysteine, D-cysteine ​​and L-cystine are all ≥1.

5.

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

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

10. The method for simultaneously determining D-cysteine ​​and L-cystine in L-cysteine ​​by high performance liquid chromatography according to claim 1, characterized in that: The mobile phase pH is 1.0-2.0.

Images