Method for measuring content of o-nitrobenzaldehyde genotoxicity in nifedipine

Abstract

The invention belongs to the technical field of drug detection, and provides a method for determining the content of o-nitrobenzaldehyde genotoxicity in nifedipine. The method comprises the following steps: performing high performance liquid chromatography detection on a reference solution, a test solution and a test labeling solution, and calculating the content of o-nitrobenzaldehyde genotoxicity in nifedipine according to the peak area and concentration of an external standard method, the mobile phase A is water, and the mobile phase B is acetonitrile. According to the method, the detection sensitivity of o-nitrobenzaldehyde genotoxicity is improved by optimizing high performance liquid chromatography conditions, using a reasonable gradient elution procedure and combining with a proper diluent, and the determination method is high in specificity, high in sensitivity, good in repeatability and good in durability and can be widely applied to detection of o-nitrobenzaldehyde genotoxicity. The method has important significance on quality control of o-nitrobenzaldehyde genotoxicity in nifedipine, and provides a basis for establishment of nifedipine quality standards.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining the genotoxic content of o-nitrobenzaldehyde in nifedipine. Background Art

[0002] Nifedipine is a dihydropyridine calcium antagonist used to prevent and treat coronary heart disease and angina pectoris, especially variant angina pectoris and angina pectoris caused by coronary artery spasm. Nifedipine has no adverse effects on respiratory function, so it is suitable for patients with angina pectoris who suffer from obstructive respiratory diseases. Its efficacy is better than that of beta-receptor antagonists. It is also suitable for various types of hypertension and has good efficacy for refractory and severe hypertension. Nifedipine also has good efficacy for refractory congestive heart failure because it can reduce afterload, and is suitable for long-term use.

[0003] The chemical name of nifedipine is 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydro-3,5-pyridinedicarboxylic acid dimethyl ester, and its molecular formula is C 17 H 18 N2O6, chemical structure is as follows:

[0004]

[0005] The starting reaction material o-nitrobenzaldehyde in the synthesis process of nifedipine contains an aldehyde structure. This type of impurity is considered to contain a warning structure, or is called a genotoxic impurity. Therefore, careful and in-depth research is required to ensure that it has no effect on the quality of the product. o-nitrobenzaldehyde contains an aldehyde group with a genotoxic warning structure, and the toxicological concern threshold (TTC, 1.5 μg per day) is used to control the risk of medication. Based on the TTC calculation method, the maximum daily dose of nifedipine is 120 mg, and it is calculated that the genotoxic limit of o-nitrobenzaldehyde should not exceed 12.5 ppm. However, the current testing methods have limitations such as low detection sensitivity, non-repeatability, and low detection accuracy.

[0006] Since the limit of genotoxic impurities is extremely low, a very high injection concentration is required during the detection process. When the injection concentration increases, the sample consumption is also large, resulting in high costs. Therefore, it is very important to provide a safe, reliable, sensitive, accurate and durable method for determining the genotoxic content of o-nitrobenzaldehyde in nifedipine. Summary of the invention

[0007] The purpose of the present invention is to provide a method for determining the genotoxic content of o-nitrobenzaldehyde in nifedipine in order to overcome the deficiencies of the prior art.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for determining the genotoxic content of o-nitrobenzaldehyde in nifedipine, comprising the following steps:

[0010] 1) mixing o-nitrobenzaldehyde reference substance and acetonitrile to obtain a reference substance solution;

[0011] Mixing a nifedipine test sample and acetonitrile to obtain a test sample solution;

[0012] The o-nitrobenzaldehyde reference substance, the nifedipine test substance and acetonitrile were mixed to obtain a test substance spiked solution;

[0013] 2) The reference solution, the test solution and the test spiked solution were tested by HPLC, and the genotoxic content of o-nitrobenzaldehyde in nifedipine was calculated by the peak area and concentration of the external standard method;

[0014] In the HPLC detection, mobile phase A was water and mobile phase B was acetonitrile.

[0015] Preferably, the procedure of gradient elution in HPLC detection is:

[0016] 0-8min: 65-40% mobile phase A, 35-60% mobile phase B;

[0017] 8-11 min: 40-10% mobile phase A, 60-90% mobile phase B;

[0018] 11-12 min: 10-65% mobile phase A, 90-35% mobile phase B;

[0019] 12-20 min: 65% mobile phase A, 35% mobile phase B.

[0020] Preferably, the concentration of the o-nitrobenzaldehyde genotoxicity in the reference solution is 0.02-0.18 μg / mL; and the concentration of the test solution is 6-14 mg / mL.

[0021] Preferably, the concentration of the nifedipine test sample in the test sample spiked solution is 6-14 mg / mL, and the concentration of the o-nitrobenzaldehyde genotoxicant is 0.02-0.18 μg / mL.

[0022] Preferably, the conditions for the HPLC detection are: flow rate of 1.1-1.3 mL / min, column temperature of 26-30° C., injection volume of 18-22 μL, detection wavelength of 210 nm, and detection time of 18-22 min.

[0023] Preferably, the chromatographic column used in the high performance liquid chromatography detection is an octadecylsilane bonded silica gel column with a column length of 100 to 300 mm, an inner diameter of 4 to 5 mm, and a filler particle size of 3 to 5 μm.

[0024] The beneficial effects of the present invention include the following:

[0025] 1) The present invention optimizes the high performance liquid chromatography conditions, uses a reasonable gradient elution program, and combines a suitable diluent to effectively separate and quantitatively detect the o-nitrobenzaldehyde genotoxicity that may exist in nifedipine, thereby improving the detection sensitivity of the o-nitrobenzaldehyde genotoxicity. The detection limit of o-nitrobenzaldehyde is 0.0126 μg / mL, and the quantitative limit is 0.0253 μg / mL.

[0026] 2) The determination method of the present invention has strong specificity, high sensitivity, good repeatability and good durability, which is of great significance for the quality control of o-nitrobenzaldehyde genotoxicity in nifedipine and provides a basis for the establishment of nifedipine quality standards.

[0027] 3) The determination method of the present invention has mild conditions and is applicable to conventional liquid chromatographs, chromatographic columns and analytical reagents; at the same time, the method of the present invention is easy to operate and saves time, manpower and financial costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the HPLC detection chart of the blank solution of Example 1;

[0029] Figure 2 It is the HPLC detection chart of the reference substance solution of Example 1;

[0030] Figure 3 It is the HPLC detection chart of the test solution with batch number SS06-P-2404001 in Example 1;

[0031] Figure 4 This is the HPLC detection chart of the spiked solution of the test sample with batch number SS06-P-2404001 in Example 1;

[0032] Figure 5 This is the curve of concentration and peak area in linear solutions of different concentrations of o-nitrobenzaldehyde. DETAILED DESCRIPTION

[0033] The present invention provides a method for determining the genotoxic content of o-nitrobenzaldehyde in nifedipine, comprising the following steps:

[0034] 1) mixing o-nitrobenzaldehyde reference substance and acetonitrile to obtain a reference substance solution;

[0035] Mixing a nifedipine test sample and acetonitrile to obtain a test sample solution;

[0036] The o-nitrobenzaldehyde reference substance, the nifedipine test substance and acetonitrile were mixed to obtain a test substance spiked solution;

[0037] 2) The reference solution, the test solution and the test spiked solution were tested by HPLC, and the genotoxic content of o-nitrobenzaldehyde in nifedipine was calculated by the peak area and concentration of the external standard method;

[0038] In the HPLC detection, mobile phase A was water and mobile phase B was acetonitrile.

[0039] In the present invention, the o-nitrobenzaldehyde genotoxicity is o-nitrobenzaldehyde, and the o-nitrobenzaldehyde genotoxicity content is the o-nitrobenzaldehyde content.

[0040] In the present invention, the high performance liquid chromatography detection further comprises a blank solution, and the blank solution is preferably acetonitrile.

[0041] In the present invention, the procedure of gradient elution in high performance liquid chromatography detection is preferably:

[0042] 0-8min: 65-40% mobile phase A, 35-60% mobile phase B;

[0043] 8-11 min (excluding 8 min) 40-10% mobile phase A, 60-90% mobile phase B;

[0044] 11-12 min (not including 11 min) 10-65% mobile phase A, 90-35% mobile phase B;

[0045] 12 to 20 min (excluding 12 min) was 65% mobile phase A and 35% mobile phase B.

[0046] In the present invention, the concentration of the o-nitrobenzaldehyde genotoxicity in the reference solution is preferably 0.02-0.18 μg / mL, more preferably 0.1-0.15 μg / mL, and more preferably 0.125 μg / mL; the concentration of the test solution is preferably 6-14 mg / mL, more preferably 8-12 mg / mL, and more preferably 10 mg / mL.

[0047] In the present invention, the concentration of the nifedipine test sample in the test sample spiked solution is preferably 6-14 mg / mL, more preferably 8-12 mg / mL, and more preferably 10 mg / mL, and the concentration of the o-nitrobenzaldehyde genotoxic agent is preferably 0.02-0.18 μg / mL, more preferably 0.1-0.15 μg / mL, and more preferably 0.125 μg / mL.

[0048] In the present invention, the conditions for the high performance liquid chromatography detection are: the flow rate is preferably 1.1 to 1.3 mL / min, more preferably 1.15 to 1.25 mL / min, more preferably 1.2 mL / min, the column temperature is preferably 26 to 30°C, more preferably 27 to 29°C, more preferably 28°C, the injection volume is preferably 18 to 22 μL, more preferably 19 to 21 μL, more preferably 20 μL, the detection wavelength is preferably 210 nm, the detection time is preferably 18 to 22 min, more preferably 19 to 21 min, more preferably 20 min.

[0049] In the present invention, the chromatographic column used for high performance liquid chromatography detection is preferably an octadecylsilane bonded silica gel column, the column length is preferably 100-300 mm, more preferably 150-250 mm, more preferably 200 mm; the inner diameter is preferably 4-5 mm, more preferably 4.2-4.8 mm, more preferably 4.5-4.6 mm, the filler particle size is preferably 3-5 μm, more preferably 3.5-4.5 μm, more preferably 4 μm; the model of the chromatographic column is preferably Waters Symmetry C18.

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] In the embodiment, the structural formula of the o-nitrobenzaldehyde reference substance is The manufacturer is Fujian Future Pharmaceutical Co., Ltd., the batch number is LXBQ-231229, and the content of o-nitrobenzaldehyde is 99.2%; the manufacturer of the nifedipine test sample is Jiangsu Weiqida Pharmaceutical Co., Ltd., the batch numbers are SS06-P-2404001, SS06-240425, SS06-240426, SS06-240427, SS06-2407002, SS06-2407003 and SS06-2407004; the manufacturer of acetonitrile is Merck KGaA (Merck), the batch number is 20240927-14; the manufacturer of water is Wahaha Group Co., Ltd., the batch number is 20240922.

[0052] The manufacturer of the high performance liquid chromatograph is Agilent Technologies (China) Co., Ltd. (Agilent), and the model is Agilent 1260II; the manufacturer of the chromatographic column is Waters, and the model is Waters Symmetry C18; the manufacturer of the workstation is Agilent, and the model is Openlab CDS2.4.

[0053] Example 1

[0054] The o-nitrobenzaldehyde reference substance and acetonitrile are mixed to obtain a reference solution, wherein the concentration of o-nitrobenzaldehyde in the reference solution is 0.125 μg / mL;

[0055] The nifedipine test samples of batch numbers SS06-P-2404001, SS06-2404025, SS06-2404026, SS06-2404027, SS06-2407002 and SS06-2407003 were respectively mixed with acetonitrile to obtain test sample solutions of different batch numbers, and the concentration of each batch test sample solution was 10 mg / mL;

[0056] The o-nitrobenzaldehyde reference substance, the nifedipine test sample with batch number SS06-P-2404001 and acetonitrile were mixed to obtain a test sample spiked solution. In the test sample spiked solution, the concentration of o-nitrobenzaldehyde was 0.125 μg / mL, and the concentration of the nifedipine test sample was 10 mg / mL.

[0057] The blank solution (acetonitrile), reference solution, test solution and test spiked solution were detected by HPLC, and the content of o-nitrobenzaldehyde in nifedipine was calculated by peak area and concentration using the external standard method.

[0058] The calculation formula of the o-nitrobenzaldehyde content in nifedipine is shown in Formula 1:

[0059] o-nitrobenzaldehyde content (ppm) = (A 样 ×C 对 ×P) / (A 对 ×C 样 )×10 6 Formula 1; where: C 对 is the concentration of the reference solution, mg / mL; A 样 A is the peak area of ​​o-nitrobenzaldehyde in the test solution; 对 is the peak area of ​​o-nitrobenzaldehyde in the reference solution; C 样 is the concentration of the test solution, mg / mL; P is the content of the o-nitrobenzaldehyde reference substance.

[0060] The conditions for HPLC detection were as follows: the chromatographic column was Waters Symmetry C18, 4.6 mm × 250 mm × 5 μm, the filler particle size was 4 μm; the column temperature was 28 °C, the detection wavelength was 210 nm, the flow rate was 1.2 mL / min, the injection volume was 20 μL, and the running time was 20 min;

[0061] The gradient elution program was:

[0062] 0 min: 65% mobile phase A, 35% mobile phase B;

[0063] 0-8min: 65-40% mobile phase A, 35-60% mobile phase B;

[0064] 8-11 min (excluding 8 min) 40-10% mobile phase A, 60-90% mobile phase B;

[0065] 11-12 min (not including 11 min) 10-65% mobile phase A, 90-35% mobile phase B;

[0066] 12-20 min (excluding 12 min): 65% mobile phase A, 35% mobile phase B;

[0067] Mobile phase A was water and mobile phase B was acetonitrile.

[0068] The results of the determination of the content of o-nitrobenzaldehyde in the test solution of Example 1 are shown in Table 1.

[0069] Table 1 Determination results of o-nitrobenzaldehyde content in the test solution

[0070] Test sample batch number o-Nitrobenzaldehyde content SS06-P-2404001 Not detected SS06-2404025 Not detected SS06-2404026 Not detected SS06-2404027 Not detected SS06-2407002 Not detected SS06-2407003 Not detected

[0071] According to the external standard method, the o-nitrobenzaldehyde in the test solution shall not exceed 12.5 ppm. As can be seen from Table 1, o-nitrobenzaldehyde was not detected in the test solution of the present invention, and the test results meet the requirements.

[0072] Figure 1 It is the HPLC detection chart of the blank solution of Example 1; Figure 2 It is the HPLC detection chart of the reference substance solution of Example 1; Figure 3 It is the HPLC detection chart of the test solution with batch number SS06-P-2404001 in Example 1; Figure 4 This is the HPLC detection chart of the spiked solution of the test sample with batch number SS06-P-2404001 in Example 1.

[0073] The determination method of Example 1 was subjected to specificity testing, system suitability testing, linearity and range testing, quantitative limit and detection limit testing, accuracy testing, precision testing, and solution stability testing.

[0074] (1) Specificity detection: The blank solution (acetonitrile), the test solution, the test spiked solution, and the reference solution of Example 1 were tested according to the chromatographic conditions in Example 1, with one injection per solution. The test results are shown in Table 2.

[0075] Table 2 Specificity test results

[0076]

[0077] As can be seen from Table 2, acetonitrile has no interference with the detection of o-nitrobenzaldehyde. The absolute difference in the retention time of o-nitrobenzaldehyde in the reference solution and the test solution is 0.002 min, which is less than 0.3 min, indicating that the determination method of the present invention has good specificity.

[0078] (2) System suitability test: The blank solution and the reference solution of Example 1 were tested according to the chromatographic conditions in Example 1. The blank solution was injected once and the reference solution was injected five times. The test results are shown in Table 3.

[0079] Table 3 System suitability test results

[0080]

[0081] As shown in Table 3, the RSD value of the peak area of ​​o-nitrobenzaldehyde in the reference solution for 5 consecutive times was 0.83%, which was less than 5%, indicating that the system applicability of the determination method of the present invention met the requirements.

[0082] (3) Linearity and range test: Linear solutions of different concentrations (from the limit of quantitation to 150% of the limit) were prepared with o-nitrobenzaldehyde reference substance and injected separately. The concentration range of o-nitrobenzaldehyde in the linear solution was 0.0253-0.1895 μg / mL. Figure 5 The curves of concentration and peak area in linear solutions of different concentrations of o-nitrobenzaldehyde are shown in Table 4. The standard curve is drawn with concentration as the abscissa and peak area as the ordinate, and the linear equation of concentration and peak area is obtained.

[0083] Table 4 Linearity and range test results

[0084] name Linear equations Correlation coefficient scope o-Nitrobenzaldehyde y=57.47x-0.1901 1.000 0.0253~0.1895μg / mL

[0085] Depend on Figure 5 As shown in Table 4, the determination method of the present invention has a good linear relationship for o-nitrobenzaldehyde.

[0086] (4) Quantitation limit and detection limit detection: The reference substance solution of Example 1 was diluted to a certain concentration with acetonitrile. The concentration at a peak signal-to-noise ratio of 10:1 was taken as the quantitation limit concentration, and the quantitation limit solution was injected 6 times continuously. The concentration at a peak signal-to-noise ratio of 3:1 was taken as the detection limit concentration, and the detection limit solution was injected 2 times continuously. The detection results are shown in Table 5.

[0087] Table 5 Quantitation limit and detection limit test results

[0088]

[0089] As can be seen from Table 5, the detection limit and quantification limit of o-nitrobenzaldehyde are relatively low, indicating that the detection sensitivity of the method of the present invention is high and can meet the determination requirements.

[0090] (5) Accuracy test: The nifedipine test sample, o-nitrobenzaldehyde reference substance and acetonitrile were prepared into recovery solutions. The mass fraction of o-nitrobenzaldehyde in the recovery solutions was 80%, 100% and 120%, respectively. Three parallel preparations were made for each of the high, medium and low concentrations. The accuracy test results are shown in Table 6.

[0091] Table 6 Accuracy test results

[0092]

[0093] As shown in Table 6, the recoveries of o-nitrobenzaldehyde in the determination method of the present invention are all within the range of 80-120%, and the RSDs of the 9 recoveries are all less than 10%, indicating that the determination method of the present invention has good accuracy.

[0094] (6) Precision test: According to the preparation method of the reference solution and the test sample spiked solution in Example 1, prepare 1 portion of the reference solution and 6 portions of the test sample spiked solution in parallel, and test them separately as the repeatability result.

[0095] On different dates, another HPLC of the same brand and model and another chromatographic column of the same model and specification were used to prepare 1 portion of the reference solution and 6 portions of the test sample spiked solution in parallel according to the preparation method of the reference solution and the test sample spiked solution in Example 1. The samples were tested separately and taken as intermediate precision results. The RSD of the 12 results was calculated. The precision results are shown in Table 7.

[0096] Table 7 Precision test results

[0097]

[0098] As shown in Table 7, in the 12 results, the RSD of the o-nitrobenzaldehyde content was less than 10%, indicating that the determination method of the present invention has good precision.

[0099] (7) Solution stability test: A reference solution and a test solution were prepared according to the method of Example 1 and placed at room temperature. The RSD of the peak area of ​​o-nitrobenzaldehyde in the reference solution and the RSD of the o-nitrobenzaldehyde content in the test solution at each time point of 0 h, 10 h, 18 h, 31 h, and 44 h were examined. The results of the solution stability test are shown in Table 8.

[0100] Table 8 Solution stability test results

[0101] name Reference solution Test solution Time o-Nitrobenzaldehyde peak area o-Nitrobenzaldehyde content 0h 7.06 Not detected 10h 6.98 Not detected 18h 7.10 Not detected 31h 7.15 Not detected 44h 7.19 Not detected RSD 1.15％ N / A

[0102] As can be seen from Table 8, when the solution was placed at room temperature for 44 hours, the RSD value of the peak area of ​​o-nitrobenzaldehyde in the reference solution at each time point was 1.15%, which was less than 5%. No o-nitrobenzaldehyde was detected in the test solution at each time point. The reference solution and the test solution had good stability when placed at room temperature for 44 hours.

[0103] Example 2

[0104] Compared with Example 1, the only difference is that the flow rate of the chromatographic detection is 1.1 mL / min.

[0105] Example 3

[0106] Compared with Example 1, the only difference is that the flow rate of the chromatographic detection is 1.3 mL / min.

[0107] Example 4

[0108] Compared with Example 1, the only difference is that the injection volume for chromatographic detection is 18 μL.

[0109] Example 5

[0110] Compared with Example 1, the only difference is that the injection volume for chromatographic detection is 22 μL.

[0111] Example 6

[0112] Compared with Example 1, the only difference is that the column temperature of the HPLC detection is 26°C.

[0113] Example 7

[0114] Compared with Example 1, the only difference is that the column temperature of the HPLC detection is 30°C.

[0115] Example 8

[0116] Compared with Example 1, the only difference is that the initial proportions of the mobile phases for HPLC detection are 67% of mobile phase A and 33% of mobile phase B.

[0117] Example 9

[0118] Compared with Example 1, the only difference is that the initial proportions of the mobile phases for HPLC detection are 63% of mobile phase A and 37% of mobile phase B.

[0119] Example 10

[0120] Compared with Example 1, the difference is that the chromatographic column for HPLC detection is another chromatographic column of the same model and specification, and the instrument is another HPLC instrument of the same brand and model.

[0121] The durability test was carried out on Examples 1 to 10, and the test results of the RSD of the o-nitrobenzaldehyde peak area in the reference solution and the RSD value of the o-nitrobenzaldehyde content in the test solution are shown in Table 9.

[0122] Table 9 Durability test results

[0123]

[0124]

[0125] As can be seen from Table 9, when the flow rate, injection volume, column temperature, initial ratio of mobile phase and chromatographic column are changed, the RSD of the peak area of ​​o-nitrobenzaldehyde in the reference solution is less than 5%, and the RSD of the o-nitrobenzaldehyde content is 2.00%, which is less than 10%, indicating that the determination method of the present invention has good durability.

[0126] As can be seen from the above embodiments, the present invention provides a method for determining the genotoxic content of o-nitrobenzaldehyde in nifedipine, which can well detect the content of o-nitrobenzaldehyde under specific chromatographic columns and detection conditions, and control the o-nitrobenzaldehyde genotoxic residue in nifedipine. The present invention improves the sensitivity of o-nitrobenzaldehyde genotoxic detection by using high performance liquid chromatography, using a gradient elution program combined with a suitable diluent. The determination method of the present invention has strong specificity, good repeatability, good durability, and can accurately detect the o-nitrobenzaldehyde genotoxic residue content in the product.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the genotoxic content of o-nitrobenzaldehyde in nifedipine, characterized in that: The following steps are involved: 1) mixing o-nitrobenzaldehyde reference substance and acetonitrile to obtain a reference substance solution; Mixing a nifedipine test sample and acetonitrile to obtain a test sample solution; The o-nitrobenzaldehyde reference substance, the nifedipine test substance and acetonitrile were mixed to obtain a test substance spiked solution; 2) The reference solution, the test solution and the test spiked solution were tested by HPLC, and the genotoxic content of o-nitrobenzaldehyde in nifedipine was calculated by the peak area and concentration of the external standard method; In the HPLC detection, mobile phase A was water and mobile phase B was acetonitrile.

2. The measuring method according to claim 1, characterized in that The procedure for gradient elution in HPLC detection is: 0-8min: 65-40% mobile phase A, 35-60% mobile phase B; 8-11 min: 40-10% mobile phase A, 60-90% mobile phase B; 11-12 min: 10-65% mobile phase A, 90-35% mobile phase B; 12-20 min: 65% mobile phase A, 35% mobile phase B.

3. The measuring method according to claim 1 or 2, characterized in that The concentration of the o-nitrobenzaldehyde genotoxicity in the reference solution is 0.02-0.18 μg / mL; the concentration of the test solution is 6-14 mg / mL.

4. The measuring method according to claim 3, characterized in that The concentration of the nifedipine test sample in the test sample spiked solution is 6-14 mg / mL, and the concentration of the o-nitrobenzaldehyde genotoxicant is 0.02-0.18 μg / mL.

5. The measuring method according to claim 3, characterized in that The conditions for the HPLC detection are: flow rate of 1.1-1.3 mL / min, column temperature of 26-30° C., injection volume of 18-22 μL, detection wavelength of 210 nm, and detection time of 18-22 min.

6. The measuring method according to claim 4 or 5, characterized in that The chromatographic column used in the high performance liquid chromatography detection is an octadecylsilane bonded silica gel column with a column length of 100 to 300 mm, an inner diameter of 4 to 5 mm, and a filler particle size of 3 to 5 μm.

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