A method for separation and analysis of chiral isomers of chloroindole hydrazide

Abstract

The present invention relates to the field of chemical analysis technology, and in particular to a separation and analysis method for chloroindole hydrazide chiral isomers. Comprise the following steps: (1) n-hexane, anhydrous ethanol and diethylamine are mixed to obtain a mobile phase; (2) anhydrous ethanol is used as a solvent to dissolve the sample to be tested; (3) the sample is detected by high performance liquid chromatography, the detection wavelength is 260-300nm, the chromatographic column is a cellulose chiral chromatographic column, the flow rate is 0.5-2mL / min, and the chromatographic column temperature is 25-45°C. The inventive method can effectively separate the four chiral isomers of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido [3,4-b] indole-3-formyl hydrazide, with strong specificity, good separation, and high accuracy of detection of the content of each chiral isomer; it is helpful to further study the optical activity of chloroindole hydrazide, which is of great significance for experimental quality control.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for separating and analyzing chloroindole hydrazide chiral isomers. Background Art

[0002] With the development of stereochemistry, the research of chiral drugs has also attracted much attention. Pesticide chiral isomers with the same physical and chemical properties may show completely different biological activities, and may also show completely different properties in terms of toxicity, teratogenicity, carcinogenicity, etc. For example, the R isomer in the technical drug of Quizalofop-Ph-butyl is absorbed by plants at a higher speed and has a higher mobility in the plant body. It not only exerts its efficacy quickly, but also has a relatively stable efficacy and is not easily affected by environmental conditions such as temperature and humidity. However, its inactive S isomer does not have herbicidal activity.

[0003] Chloroindole hydrazide was synthesized by Wang Qingmin's research group at Nankai University using β-carboline alkaloids. It has high anti-tobacco mosaic virus activity and is currently being industrialized and developed by Jingbo Agrochemical Technology Co., Ltd. The IUPAC name of chloroindole hydrazide is N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide.

[0004] There are four configurations of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, namely (1S,3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, (1R,3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, The optical purity of hydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, (1R,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide and (1S,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide directly affects the biological activity of the product.

[0005] Based on this, it is necessary to provide a method for separating and analyzing chiral isomers of chloroindole hydrazide. Summary of the invention

[0006] Aiming at the technical gap in the field of chiral isomer analysis of chloroindole hydrazide in the prior art, the present invention provides a method for separating and analyzing chiral isomers of chloroindole hydrazide, which can separate (1S, 3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, (1R, 3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, and The four stereoisomers of hydrazide, (1R,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide and (1S,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide were effectively separated with strong specificity and good separation degree. The content of each isomer was detected with high accuracy, which is of great significance for experimental quality control.

[0007] The technical solution of the present invention is as follows:

[0008] A method for separating and analyzing chloroindole hydrazide chiral isomers comprises the following steps:

[0009] (1) Preparation of mobile phase: n-hexane, anhydrous ethanol and diethylamine are mixed to obtain a mobile phase;

[0010] (2) Preparation of the test solution: Use anhydrous ethanol as a solvent to dissolve the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide sample to obtain a test solution;

[0011] (3) Detection: The samples were detected by high performance liquid chromatography, with a detection wavelength of 260-300 nm, a cellulose chiral column, a flow rate of 0.5-2 mL / min, and a column temperature of 25-45°C.

[0012] Furthermore, in step (1), the volume ratio of n-hexane, anhydrous ethanol and diethylamine is (80-90):(10-20):(0.05-0.2).

[0013] Furthermore, in step (1), the volume ratio of n-hexane, anhydrous ethanol and diethylamine is 85:15:0.1.

[0014] Furthermore, the concentration of the test solution prepared in step (2) is 0.4-10 mg / mL.

[0015] Furthermore, the injection volume of the test solution in step (3) is 0.1-10 μL.

[0016] Furthermore, in step (3), the detection wavelength is 282 nm, the column flow rate is 1.0 mL / min, the column temperature is 40° C., and the injection volume is 5.0 μL.

[0017] Furthermore, the chromatographic column model in step (3) is Lux R 5 μm i-Cellulose-5.

[0018] Furthermore, the specification of the chromatographic column in step (3) is 250 mm×4.6 mm.

[0019] The beneficial effects of the present invention are:

[0020] The present invention adopts a cellulose chiral chromatographic column, takes n-hexane, anhydrous ethanol and diethylamine as mobile phases, and can carry out (1S, 3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, (1R, 3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole- The four stereoisomers of 3-carboxylic acid hydrazide, (1R,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide and (1S,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide were effectively separated, and the effect was significantly better than that of conventional mobile phase schemes;

[0021] At the same time, choosing anhydrous ethanol to dissolve the sample can not only increase the solubility of the sample and make the sample fully dissolved, but also increase the mutual solubility between the sample and the mobile phase due to the use of mobile phase components to dissolve, reduce the solvent effect in chromatography, and maintain the stability of the sample;

[0022] Selecting the appropriate injection volume (5 μL is optimal) and column temperature (40° C. is optimal) improves the symmetry of the chromatographic peak and improves the chromatographic peak area and peak height.

[0023] The present invention fills the technical gap in the corresponding field of the prior art, can achieve good separation of various isomers, has good chromatographic peak shape, accurate integral calculation results, good repeatability, and high credibility of the obtained results. It is particularly suitable for quality control of pesticide technical products and has an important role and practical significance for ensuring product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is the HPLC chromatogram of the SS configuration monomer of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide in Example 1.

[0026] Figure 2 This is the HPLC chromatogram of the RS-configuration monomer of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide in Example 1.

[0027] Figure 3 This is the high performance liquid chromatogram of the RR configuration monomer of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide in Example 1.

[0028] Figure 4 This is the high performance liquid chromatogram of the SR configuration monomer of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide in Example 1.

[0029] Figure 5 This is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 2.

[0030] Figure 6 It is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 3.

[0031] Figure 7 It is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 4.

[0032] Figure 8 This is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 5.

[0033] Fig. 9This is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 6.

[0034] Fig.10 It is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 7.

[0035] Fig.11 It is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 8.

[0036] Fig.12 It is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Example 9.

[0037] Fig.13 It is the HPLC chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Comparative Example 1.

[0038] Fig.14 It is the high performance liquid chromatogram of the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug in Comparative Example 2. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0040] The following examples and comparative examples were analyzed using Shimadzu LC-20A high performance liquid chromatograph and Lux R The chromatography was performed using a 5 μm i-Cellulose-5 (250×4.6 mm) chiral chromatography column. In other embodiments, Shimadzu SPD-M20A, SIL-20A or DGU-20A3R or other manufacturers and models of high performance liquid chromatographs and other cellulose chiral chromatography columns may also be used.

[0041] Example 1

[0042] The SR, SS, RS, and RR configuration monomers of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide were tested respectively, and the peak positions of the four configuration monomers were determined.

[0043] The chromatographic analysis conditions are as follows:

[0044] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 85:15:0.1;

[0045] Flow rate: 1.0 mL / min;

[0046] Detection wavelength: 282nm;

[0047] Column temperature: 40°C;

[0048] Injection volume: 5.0 μL.

[0049] The test steps are as follows:

[0050] (1) Accurately weigh 0.0216 g of (1S,3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 1.

[0051] Take 5.0 μL of the test solution 1 and inject it into the HPLC. Perform chromatographic analysis under the above conditions and record the chromatogram. The results are shown in Figure 1 .

[0052] (2) Accurately weigh 0.0208 g of (1R,3S)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 2.

[0053] Take 5.0 μL of the test solution 2 and inject it into the HPLC. Perform chromatographic analysis under the above conditions and record the chromatogram. The results are shown in Figure 2 .

[0054] (3) Accurately weigh 0.0221 g of (1R,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 3.

[0055] Take 5.0 μL of the test solution 3 and inject it into the high performance liquid chromatograph. Perform chromatographic analysis under the above conditions and record the chromatogram. The results are shown in Figure 3 .

[0056] (4) Accurately weigh 0.0204 g of (1S,3R)-N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 4.

[0057] Take 5.0 μL of the test solution 4 and inject it into the high performance liquid chromatograph. Perform chromatographic analysis under the above conditions and record the chromatogram. The results are shown in Figure 4 .

[0058] (5) Through Figure 1-Figure 4 Based on comprehensive analysis, it was determined that the peak position of SS configuration was 9.244 min, the peak position of RS configuration was 19.446 min, the peak position of RR configuration was 10.429 min, and the peak position of SR configuration was 16.129 min.

[0059] The above results indicate that under the chromatographic analysis conditions of the present invention, the four chiral isomers of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide achieve good baseline separation.

[0060] Example 2

[0061] The chromatographic analysis conditions are as follows:

[0062] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 85:15:0.1;

[0063] Flow rate: 1.0 mL / min;

[0064] Detection wavelength: 282nm;

[0065] Column temperature: 40°C;

[0066] Injection volume: 5.0 μL.

[0067] The test steps are as follows:

[0068] (1) Accurately weigh 0.0509 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50 mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 5.

[0069] Take 5.0 μL of the test solution 5 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Figure 5 .

[0070] (2) Comparative analysis Figure 1-Figure 5 The chromatographic peaks with retention times of 9.238min, 10.488min, 16.204min and 19.429min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively.

[0071] It can be seen that good baseline separation can be achieved for the four isomers in this example.

[0072] Example 3

[0073] The chromatographic analysis conditions are as follows:

[0074] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 80:20:0.1;

[0075] Flow rate: 1.0 mL / min;

[0076] Detection wavelength: 282nm;

[0077] Column temperature: 40°C;

[0078] Injection volume: 5.0 μL.

[0079] The test steps are as follows:

[0080] (1) Accurately weigh 0.0516 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 6.

[0081] Take 5.0 μL of the test solution 6 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Figure 6 .

[0082] (2) Comparative analysis Figure 1-Figure 4 and Figure 6 The chromatographic peaks with retention times of 6.617min, 7.463min, 10.804min and 12.754min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively. At this time, the chromatographic analysis time is greatly reduced, and the baseline separation effect is slightly worse than that of Example 2.

[0083] Example 4

[0084] The chromatographic analysis conditions are as follows:

[0085] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 90:10:0.1;

[0086] Flow rate: 1.0 mL / min;

[0087] Detection wavelength: 282nm;

[0088] Column temperature: 40°C;

[0089] Injection volume: 5.0 μL.

[0090] The test steps are as follows:

[0091] (1) Accurately weigh 0.0499 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50 mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 7.

[0092] Take 5.0 μL of the test solution 7 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Figure 7 .

[0093] (2) Comparative analysis Figure 1-Figure 4 and Figure 7 The chromatographic peaks with retention times of 15.813min, 18.763min, 29.764min and 35.597min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively. At this time, the baseline separation effect is better, but the separation time is slightly longer than that of Example 2.

[0094] Example 5

[0095] The chromatographic analysis conditions are as follows:

[0096] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 85:15:0.1;

[0097] Flow rate: 0.5 mL / min;

[0098] Detection wavelength: 282nm;

[0099] Column temperature: 40°C;

[0100] Injection volume: 5.0 μL.

[0101] The test steps are as follows:

[0102] (1) Accurately weigh 0.0503 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug, place it in a 50-mL volumetric flask, add 40 mL of anhydrous ethanol, shake to dissolve, dilute to the scale with anhydrous ethanol, shake well, and use this as the test solution 8.

[0103] Take 5.0 μL of the test solution 8 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Figure 8 .

[0104] (2) Comparative analysis Figure 1-Figure 4 and Figure 8 The chromatographic peaks with retention times of 13.363min, 15.063min, 23.913min and 28.151min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively. At this time, good baseline separation can be achieved, but the separation time is longer than that in Example 2.

[0105] Example 6

[0106] The chromatographic analysis conditions are as follows:

[0107] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 85:15:0.1;

[0108] Flow rate: 2.0 mL / min;

[0109] Detection wavelength: 282nm;

[0110] Column temperature: 40°C;

[0111] Injection volume: 5.0 μL.

[0112] The test steps are as follows:

[0113] (1) Accurately weigh 0.0503 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 9.

[0114] Take 5.0 μL of the test solution 9 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Fig. 9 .

[0115] (2) Comparative analysis Figure 1-Figure 4 and Fig. 9The chromatographic peaks with retention times of 7.306min, 8.271min, 13.079min and 13.988min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively. At this time, the chromatographic analysis time is shortened and the baseline separation effect is slightly worse than that of Example 2.

[0116] Example 7

[0117] The chromatographic analysis conditions are as follows:

[0118] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 85:15:0.1;

[0119] Flow rate: 1.0 mL / min;

[0120] Detection wavelength: 282nm;

[0121] Column temperature: 25°C;

[0122] Injection volume: 5.0 μL.

[0123] The test steps are as follows:

[0124] (1) Accurately weigh 0.0522 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 10.

[0125] Take 5.0 μL of the test solution 10 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Fig.10 .

[0126] (2) Comparative analysis Figure 1-Figure 4 and Fig.10 The chromatographic peaks with retention times of 10.629min, 11.921min, 18.885min and 22.338min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively, and good baseline separation can be achieved at this time.

[0127] Example 8

[0128] The chromatographic analysis conditions are as follows:

[0129] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 85:15:0.1;

[0130] Flow rate: 1.0 mL / min;

[0131] Detection wavelength: 282nm;

[0132] Column temperature: 45°C;

[0133] Injection volume: 5.0 μL.

[0134] The test steps are as follows:

[0135] (1) Accurately weigh 0.0522 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 11.

[0136] Take 5.0 μL of the test solution 11 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Fig.11 .

[0137] (2) Comparative analysis Figure 1-Figure 4 and Fig.11 The chromatographic peaks with retention times of 8.459min, 9.496min, 14.576min and 17.594min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively, and good baseline separation can be achieved at this time.

[0138] Example 9

[0139] The chromatographic analysis conditions are as follows:

[0140] Mobile phase: a mixture of n-hexane, anhydrous ethanol and diethylamine in a volume ratio of 85:15:0.1;

[0141] Flow rate: 1.0 mL / min;

[0142] Detection wavelength: 260nm;

[0143] Column temperature: 40°C;

[0144] Injection volume: 10.0 μL.

[0145] The test steps are as follows:

[0146] (1) Accurately weigh 0.0552 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 12.

[0147] Take 5.0 μL of the test solution 12 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Fig.12 .

[0148] (2) Comparative analysis Figure 1-Figure 4 and Fig.12 The chromatographic peaks with retention times of 9.580min, 10.421min, 16.129min and 19.813min are the chromatographic peaks of SS configuration, RR configuration, SR configuration and RS configuration, respectively, and good baseline separation can be achieved at this time.

[0149] Comparative Example 1

[0150] The chromatographic analysis conditions are as follows:

[0151] Mobile phase: a mixture of n-hexane, isopropanol, and diethylamine in a volume ratio of 85:15:0.1;

[0152] Flow rate: 1.0 mL / min;

[0153] Detection wavelength: 282nm;

[0154] Column temperature: 40°C;

[0155] Injection volume: 5.0 μL.

[0156] The test steps are as follows:

[0157] (1) Accurately weigh 0.0526 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 13.

[0158] Take 5.0 μL of the test solution 13 and inject it into the high performance liquid chromatograph, perform chromatographic separation and analysis under the above conditions, record the chromatogram, and the results are shown in Fig.13 .

[0159] (2) Comparative analysis Figure 1-Figure 4 and Fig.13 , only two chromatographic peaks appeared for the four chiral isomers of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide, indicating that after the organic modifier was replaced with isopropanol, the separation degree of the four chiral isomers decreased and the peak shape became worse, which was not conducive to the separation of the four chiral isomers of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide.

[0160] Comparative Example 2

[0161] The chromatographic analysis conditions are as follows:

[0162] Mobile phase: a mixture of n-hexane, n-propanol, and diethylamine in a volume ratio of 85:15:0.15;

[0163] Flow rate: 1.0 mL / min;

[0164] Detection wavelength: 282nm;

[0165] Column temperature: 40°C;

[0166] Injection volume: 5.0 μL.

[0167] The test steps are as follows:

[0168] (1) Accurately weigh 0.0531 g of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide original drug and place it in a 50-mL volumetric flask. Add 40 mL of anhydrous ethanol and shake to dissolve. Then dilute to the mark with anhydrous ethanol and shake well to prepare the test solution 14.

[0169] Take 5.0 μL of the test solution 14 and inject it into the high performance liquid chromatograph. Perform chromatographic separation and analysis under the above conditions and record the chromatogram. The results are shown in Fig.14 .

[0170] (2) Comparative analysis Figure 1-Figure 4 and Fig.14 The separation between RS isomer and SR isomer becomes worse than that in Example 2-9, indicating that after the organic modifier is replaced with n-propanol, the separation between SR configuration and RS configuration, SS configuration and RR configuration decreases, and the peak shape becomes worse, which is not conducive to the separation of the four chiral isomers of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide.

[0171] It can be seen from the above embodiments and comparative examples that the selection of mobile phase is crucial to the separation of the four chiral isomers of N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide. The separation and analysis method of chloroindole hydrazide chiral isomers provided by the present invention has high accuracy, strong specificity, good operability, and can be widely applied to the analysis and detection of chloroindole hydrazide chiral isomers.

[0172] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A method for separation and analysis of chiral isomers of chloroindole hydrazide, characterized in that: The steps include: (1) Preparation of mobile phase: Mix n-hexane, anhydrous ethanol and diethylamine in a volume ratio of (80-90): (10-20): (0.05-0.2) to obtain a mobile phase; (2) Preparation of the test solution: Use anhydrous ethanol as solvent to dissolve the N'-(4-chlorobenzylidene)-1-methyl-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylic acid hydrazide sample to obtain the test solution; (3) Detection: The samples were detected by high performance liquid chromatography, with a detection wavelength of 260-300 nm, a cellulose chiral column, and a Lux column model. R 5µm i-Cellulose-5, flow rate 0.5-2mL / min, column temperature 25-45℃.

2. The separation and analysis method according to claim 1, characterized in that: In step (1), the volume ratio of n-hexane, anhydrous ethanol and diethylamine is 85:15:0.

1.

3. The separation and analysis method according to claim 1, characterized in that The concentration of the test solution prepared in step (2) is 0.4-10 mg / mL.

4. The separation and analysis method according to claim 1, characterized in that: The injection volume of the test solution in step (3) is 0.1-10 μL.

5. The separation and analysis method according to claim 1, characterized in that: In step (3), the detection wavelength was 282 nm, the column flow rate was 1.0 mL / min, the column temperature was 40 °C, and the injection volume was 5.0 μL.

6. The separation and analysis method according to claim 1, characterized in that: Step (3) The chromatographic column specification is 250 mm × 4.6 mm.

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