A method for detecting enantiomers of thiamphenicol hydrochloride glycine ester

Abstract

The invention provides a method for detecting enantiomers of thiamphenicol glycine ester hydrochloride or thiamphenicol glycine ester hydrochloride in a preparation thereof. The method comprises the following steps: preparing a test solution: taking thiamphenicol glycine ester hydrochloride or a preparation thereof to prepare a test solution; chromatographic conditions: adopting chiral chromatography with cellulose tris-(4-chloro-3-methylphenylcarbamate) as a stationary phase and n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine as a mobile phase; and a determination method: taking the test solution and injecting it into a liquid chromatograph for determination.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical and chemical detection, and in particular to a method for detecting thiamphenicol hydrochloride glycine ester or thiamphenicol hydrochloride glycine ester enantiomers in its preparation. Background Art

[0002] Thiamphenicol glycinate hydrochloride, chemical name: [R-(R*,R*)]N-[1-(hydroxymethyl)-2-hydroxy-2-[4-(methylsulfonyl)phenyl]ethyl]-2,2-dichloroacetamide glycinate hydrochloride, structural formula is as follows:

[0003]

[0004] Thiamphenicol, also known as thiamphenicol, has a chemical name of 1-p-methylsulfonyl-phenyl-2-dichloroacetamide-1,3-propanediol. Its structure is similar to chloramphenicol, and its antimicrobial spectrum is largely similar. It is also a powerful immunosuppressant, six times more potent than chloramphenicol, primarily through inhibition of protein synthesis and antibody production. Trials in my country began in 1971, but its poor water solubility limited its dosage form. Several companies in China currently produce and sell thiamphenicol preparations, but for the aforementioned reasons, the thiamphenicol preparations currently widely used in clinical practice are solid preparations, such as tablets and capsules.

[0005] In order to expand the clinical application of thiamphenicol and overcome its disadvantage of poor water solubility, Japan Airei Co., Ltd. has developed its derivative thiamphenicol hydrochloride glycine ester, which has good water solubility and quickly releases thiamphenicol in the body to exert its effect.

[0006] As impurities in the thiamphenicol hydrochloride glycinate API, the enantiomers of thiamphenicol hydrochloride glycinate must be controlled within certain limits. Therefore, it is necessary to detect and analyze the enantiomers of thiamphenicol hydrochloride glycinate in the thiamphenicol hydrochloride glycinate API. The structural formula of the enantiomers of thiamphenicol hydrochloride glycinate is as follows:

[0007]

[0008] So far, there is no published technology for the enantiomer detection of thiamphenicol hydrochloride glycinate. Summary of the Invention

[0009] In view of the above problems, the present application provides a method for detecting the enantiomers of thiamphenicol glycine hydrochloride or thiamphenicol glycine hydrochloride in its preparation, the method comprising the following steps:

[0010] Preparation of test solution: Take thiamphenicol hydrochloride glycine ester or its preparation to prepare the test solution;

[0011] Chromatographic conditions: A chiral column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase and n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine as the mobile phase was used;

[0012] Determination method: Take the test solution and inject it into the liquid chromatograph for determination.

[0013] Preferably, the test solution is prepared by taking thiamphenicol glycine hydrochloride or its preparation, dissolving it in an organic solvent, and diluting it with a mobile phase to obtain a test solution with a concentration of 0.1 to 3 mg / ml; the organic solvent is methanol or anhydrous ethanol.

[0014] Further preferably, the test solution is prepared by accurately weighing thiamphenicol hydrochloride glycine ester or its preparation in a volumetric flask, adding an organic solvent at a ratio of 0.5 ml / 10 ml and sonicating to dissolve the solution, followed by dilution with mobile phase to produce a 1 mg / ml solution. The "0.5 ml / 10 ml" refers to the addition of 0.5 ml of organic solvent to dissolve 10 ml of the test solution.

[0015] Preferably, the mobile phase is 74-80:15-20:10-15:0.1-0.3:0.05-0.2 of n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine solution; further preferably, the mobile phase is 75-77:16-18:11-13:0.15-0.25:0.05-0.15 of n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine solution; most preferably, the mobile phase is 76:17:12:0.2:0.1 of n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine solution.

[0016] Preferably, the detection wavelength is 200-240 nm, the column temperature is 20-30°C, the flow rate is 0.5-2 ml / min, and the injection volume is 1-30 μl; further preferably, the detection wavelength is 226 nm, the column temperature is 25°C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0017] The thiamphenicol hydrochloride glycinate preparations described in the present application include but are not limited to injections.

[0018] Beneficial effects of this application:

[0019] The enantiomer detection method of thiamphenicol hydrochloride glycine ester of the present invention has strong specificity, good repeatability, precision, and tolerance, and high sensitivity, and can effectively detect the content of thiamphenicol hydrochloride glycine ester enantiomers, further ensuring the safety, effectiveness, and controllability of clinical drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiment below. The accompanying drawings are only for the purpose of illustrating the preferred embodiment and are not to be considered as limiting the present invention. In the accompanying drawings:

[0021] Attachment Figure 1 Chromatogram of blank solution of the present invention

[0022] Attachment Figure 2 Chromatogram of the test solution of the present invention

[0023] Attachment Figure 3 The chromatogram of the solution of the system of the present invention is DETAILED DESCRIPTION

[0024] Exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0025] Example 1

[0026] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0027] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0028] Solubilizing solvent: 0.5ml / 10ml methanol;

[0029] Detection wavelength: 226nm;

[0030] Column temperature: 25°C;

[0031] Flow rate: 1.0 ml / min;

[0032] Preparation of blank solution: Take 1 ml of methanol and place it in a 20 ml volumetric flask, dilute to the mark with mobile phase, and shake well;

[0033] Preparation of test solution: Weigh about 20 mg of the test sample into a 20 ml volumetric flask, accurately weigh, add 1 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase;

[0034] Preparation of system suitability solution: Weigh approximately 5 mg of thiamphenicol glycinate racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0035] Determination method: Accurately measure 10 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. Figure 1-3 .

[0036] The test results showed that the separation between thiamphenicol hydrochloride glycine ester and its enantiomers in the system suitability solution was greater than 1.5, and the blank solution did not interfere with the detection, indicating that the method had good specificity.

[0037] Example 2

[0038] Chromatographic conditions and system suitability experiments

[0039] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0040] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0041] Solubilizing solvent: 0.5ml / 10ml methanol;

[0042] Detection wavelength: 226nm;

[0043] Column temperature: 20°C;

[0044] Flow rate: 1.0 ml / min;

[0045] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0046] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0047] Example 3

[0048] Chromatographic conditions and system suitability experiments

[0049] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0050] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0051] Solubilizing solvent: 0.5ml / 10ml methanol;

[0052] Detection wavelength: 226nm;

[0053] Column temperature: 30°C;

[0054] Flow rate: 1.0 ml / min;

[0055] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0056] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0057] Example 4

[0058] Chromatographic conditions and system suitability experiments

[0059] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0060] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0061] Solubilizing solvent: 0.5ml / 10ml methanol;

[0062] Detection wavelength: 226nm;

[0063] Column temperature: 25°C;

[0064] Flow rate: 0.5 ml / min;

[0065] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0066] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0067] Example 5

[0068] Chromatographic conditions and system suitability experiments

[0069] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0070] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0071] Solubilizing solvent: 0.5ml / 10ml methanol;

[0072] Detection wavelength: 226nm;

[0073] Column temperature: 25°C;

[0074] Flow rate: 2.0 ml / min;

[0075] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0076] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0077] Example 6

[0078] Chromatographic conditions and system suitability experiments

[0079] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0080] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (74:17:12:0.2:0.1);

[0081] Solubilizing solvent: 0.5ml / 10ml methanol;

[0082] Detection wavelength: 226nm;

[0083] Column temperature: 25°C;

[0084] Flow rate: 1.0 ml / min;

[0085] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0086] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0087] Example 7

[0088] Chromatographic conditions and system suitability experiments

[0089] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0090] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (80:17:12:0.2:0.1);

[0091] Solubilizing solvent: 0.5ml / 10ml methanol;

[0092] Detection wavelength: 226nm;

[0093] Column temperature: 25°C;

[0094] Flow rate: 1.0 ml / min;

[0095] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0096] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0097] Example 8

[0098] Chromatographic conditions and system suitability experiments

[0099] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0100] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:15:12:0.2:0.1);

[0101] Solubilizing solvent: 0.5ml / 10ml methanol;

[0102] Detection wavelength: 226nm;

[0103] Column temperature: 25°C;

[0104] Flow rate: 1.0 ml / min;

[0105] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0106] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0107] Example 9

[0108] Chromatographic conditions and system suitability experiments

[0109] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0110] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:20:12:0.2:0.1);

[0111] Solubilizing solvent: 0.5ml / 10ml methanol;

[0112] Detection wavelength: 226nm;

[0113] Column temperature: 25°C;

[0114] Flow rate: 1.0 ml / min;

[0115] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0116] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0117] Example 10

[0118] Chromatographic conditions and system suitability experiments

[0119] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0120] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:10:0.2:0.1);

[0121] Solubilizing solvent: 0.5ml / 10ml methanol;

[0122] Detection wavelength: 226nm;

[0123] Column temperature: 25°C;

[0124] Flow rate: 1.0 ml / min;

[0125] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0126] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0127] Example 11

[0128] Chromatographic conditions and system suitability experiments

[0129] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0130] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:15:0.2:0.1);

[0131] Solubilizing solvent: 0.5ml / 10ml methanol;

[0132] Detection wavelength: 226nm;

[0133] Column temperature: 25°C;

[0134] Flow rate: 1.0 ml / min;

[0135] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0136] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0137] Example 12

[0138] Chromatographic conditions and system suitability experiments

[0139] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0140] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.1:0.05);

[0141] Solubilizing solvent: 0.5ml / 10ml methanol;

[0142] Detection wavelength: 226nm;

[0143] Column temperature: 25°C;

[0144] Flow rate: 1.0 ml / min;

[0145] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0146] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0147] Example 13

[0148] Chromatographic conditions and system suitability experiments

[0149] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0150] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.3:0.2);

[0151] Solubilizing solvent: 0.5ml / 10ml methanol;

[0152] Detection wavelength: 226nm;

[0153] Column temperature: 25°C;

[0154] Flow rate: 1.0 ml / min;

[0155] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0156] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0157] Example 14

[0158] Chromatographic conditions and system suitability experiments

[0159] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0160] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0161] Solubilizing solvent: 0.5ml / 10ml anhydrous ethanol;

[0162] Detection wavelength: 226nm;

[0163] Column temperature: 25°C;

[0164] Flow rate: 1.0 ml / min;

[0165] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine hydrochloride racemate into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of anhydrous ethanol and dissolve by ultrasonication, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0166] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0167] Example 15

[0168] Chromatographic conditions and system suitability experiments

[0169] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0170] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0171] Solubilizing solvent: 0.5ml / 10ml methanol;

[0172] Detection wavelength: 226nm;

[0173] Column temperature: 25°C;

[0174] Flow rate: 1.0 ml / min;

[0175] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0176] Determination method: Accurately measure 1 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0177] Example 16

[0178] Chromatographic conditions and system suitability experiments

[0179] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0180] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0181] Solubilizing solvent: 0.5ml / 10ml methanol;

[0182] Detection wavelength: 226nm;

[0183] Column temperature: 25°C;

[0184] Flow rate: 1.0 ml / min;

[0185] System suitability solution: Weigh approximately 5 mg of thiamphenicol glycine ester racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask, and dilute to the mark with mobile phase;

[0186] Determination method: Accurately measure 30 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0187] Example 17

[0188] Chromatographic conditions and system suitability experiments

[0189] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0190] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0191] Solubilizing solvent: 0.5ml / 10ml methanol;

[0192] Detection wavelength: 226nm;

[0193] Column temperature: 25°C;

[0194] Flow rate: 1.0 ml / min;

[0195] System suitability solution: Weigh approximately 5 mg each of thiamphenicol hydrochloride glycine ester and thiamphenicol hydrochloride enantiomer for injection into a 100 ml volumetric flask, accurately weigh, add 5 ml of methanol, sonicate to dissolve, and dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0196] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0197] The system suitability solutions were tested according to the above 17 examples, and the separation and theoretical plate number were recorded. The results are shown in Table 1.

[0198] Table 1 Test results of Examples 1-17

[0199]

[0200]

[0201] The above results indicate that the above embodiments 1-17 can all achieve the technical effects of the present application.

[0202] Comparative Examples 1-5

[0203] Chromatographic conditions and system suitability experiments

[0204] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0205] Mobile phase: n-hexane: methanol: anhydrous ethanol: trifluoroacetic acid: ethanolamine. The mobile phase ratio is shown in Table 2.

[0206] Solubilizing solvent: 0.5ml / 10ml methanol;

[0207] Detection wavelength: 226nm;

[0208] Column temperature: 25°C;

[0209] Flow rate: 1.0 ml / min;

[0210] Preparation of system suitability solution: Weigh approximately 5 mg of thiamphenicol glycinate racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0211] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0212] The system suitability solution was tested according to the mobile phases described in the above-mentioned comparative examples 1-5, and the separation, theoretical plate number of enantiomers, and tailing factor were recorded. The results are shown in Table 2.

[0213] Table 2 Comparative Examples 1-5 Mobile Phases and Test Results

[0214]

[0215] The above results indicate that the above control examples cannot achieve the technical effects of the present application.

[0216] Comparative Examples 6-10

[0217] Chromatographic conditions and system suitability experiments

[0218] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0219] Mobile phase: see Table 3;

[0220] Solubilizing solvent: 0.5ml / 10ml methanol;

[0221] Detection wavelength: 226nm;

[0222] Column temperature: 25°C;

[0223] Flow rate: 1.0 ml / min;

[0224] Preparation of system suitability solution: Weigh approximately 5 mg of thiamphenicol glycinate racemate hydrochloride into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with mobile phase; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with mobile phase;

[0225] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0226] The system suitability solution was tested according to the mobile phase described in Control Examples 6-10, and the separation, theoretical plate number of enantiomers, and tailing factor were recorded. The results are shown in Table 3.

[0227] Table 3 Comparative Examples 6-10 Mobile Phases and Test Results

[0228]

[0229]

[0230] The above results indicate that the above control examples cannot achieve the technical effects of the present application.

[0231] Comparative Examples 11-15

[0232] Chromatographic conditions and system suitability experiments

[0233] Chromatographic column: Chiral chromatographic column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase;

[0234] Mobile phase: n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1);

[0235] Sample preparation solvent and dissolution solvent: see Table 4;

[0236] Detection wavelength: 226nm;

[0237] Column temperature: 25°C;

[0238] Flow rate: 1.0 ml / min;

[0239] Preparation of system suitability solution: Weigh approximately 5 mg of thiamphenicol glycinate hydrochloride racemate into a 50 ml volumetric flask, accurately weigh, add 2.5 ml of methanol and sonicate to dissolve, then dilute to the mark with sample preparation solvent; accurately measure 2 ml into a 10 ml volumetric flask and dilute to the mark with sample preparation solvent;

[0240] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0241] System suitability solutions were prepared according to the method of Control Examples 11-15, and the properties of the solutions were observed. The solutions were injected into a chromatograph for detection. The results are shown in Table 4.

[0242] Table 4 Comparative Examples 11-15 and Examples 1, 14 Sampling Methods and Solution Properties and Test Results

[0243]

[0244] The above results show that the above control examples cannot achieve the technical effects of the present application. Examples 1 and 14 were dissolved in 0.5 ml / 10 ml methanol and anhydrous ethanol, respectively, and then fixed to volume with n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine (76:17:12:0.2:0.1). The sample solution was clear, the blank had no interference, and the separation effect of thiamphenicol hydrochloride glycine ester and the enantiomers was good.

Claims

1. A method for detecting the enantiomers of thiamphenicol hydrochloride glycine ester, characterized in that: The method comprises the following steps: Preparation of test solution: Dissolve thiamphenicol hydrochloride glycine ester or its preparation in an organic solvent and dilute with mobile phase to obtain a test solution with a concentration of 0.1-3 mg / ml; the organic solvent is methanol or anhydrous ethanol. Chromatographic conditions: A chiral column with cellulose-tris-(4-chloro-3-methylphenylcarbamate) as the stationary phase was used, and a mobile phase of n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine solution (74-80:15-20:10-15:0.1-0.3:0.05-0.2) was used; the detection wavelength was 200-240 nm, the column temperature was 20-30°C, the flow rate was 0.5-2 ml / min, and the injection volume was 1-30 μl. Determination method: Take the test solution and inject it into the liquid chromatograph for determination.

2. The detection method according to claim 1, wherein The preparation method of the test solution is as follows: take an appropriate amount of thiamphenicol hydrochloride glycine ester or its preparation, accurately weigh it, put it into a volumetric flask, add an organic solvent at a ratio of 0.5 ml / 10 ml, ultrasonically dissolve it, and dilute it with mobile phase to make a 1 mg / ml solution.

3. The detection method according to claim 1, wherein The mobile phase is a solution of n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine at a ratio of 75-77:16-18:11-13:0.15-0.25:0.05-0.

15.

4. The detection method according to claim 3, wherein The mobile phase is a solution of n-hexane:methanol:anhydrous ethanol:trifluoroacetic acid:ethanolamine in a ratio of 76:17:12:0.2:0.

1.

5. The detection method according to claim 1, wherein The chromatographic conditions are as follows: detection wavelength of 226 nm, column temperature of 25° C., flow rate of 1.0 ml / min, and injection volume of 10 μl.

6. The detection method according to any one of claims 1 to 5, wherein The thiamphenicol hydrochloride glycine ester preparation is an injection.

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