Separation and detection method of enantiomer in (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido] tert-butyl propionate hydrochloride

Abstract

The invention belongs to the field of detection and analysis, and relates to a method for separating and detecting enantiomers in (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido] tert-butyl propionate hydrochloride. According to the method, HPLC is adopted for separation and detection, a polysaccharide derivative chiral chromatographic column is selected as a chromatographic column, a mobile phase A is a weak base aqueous solution, and a mobile phase B is acetonitrile and / or methanol. The method is strong in specificity, high in separation degree, high in accuracy, stable, reliable and high in sensitivity, and a simple, accurate, rapid and reliable detection method is provided for control of impurities in a starting material of (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido] tert-butyl propionate hydrochloride.

Description

Technical Field

[0001] The present invention relates to the field of detection and analysis, and in particular to a method for separating and detecting enantiomers in tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride. Background Art

[0002] (S)-tert-Butyl 2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride has a molecular formula of C13H25ClN2O5S and a molecular weight of 324.80. Its structure is shown in Formula I. This compound is often used as a key starting material in the synthesis of chemical drug precursors. It possesses a chiral center, and chiral enantiomeric impurities can affect the quality of the precursor, and thus the quality of the drug. Therefore, the control of chiral impurities is extremely important. In the research of chiral drugs, the study of stereoisomers is a key focus of quality control. In theory, diastereomers can be separated and detected using non-stereospecific methods, while enantiomers require stereospecific methods for separation and detection. Therefore, the development of methods for separating and detecting the enantiomers of key starting materials is particularly important for optimizing synthesis processes and controlling the enantiomer content of key starting materials, thereby laying the foundation for controlling the enantiomer content in the final drug. Its enantiomer is (R)-tert-butyl 2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride, and its structure is shown in Formula III.

[0003]

[0004] At present, there is no report on the separation, detection and analysis method of (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionic acid tert-butyl ester hydrochloride and its enantiomers in relevant literature at home and abroad. Summary of the Invention

[0005] The first object of the present invention is to provide a method for separating and detecting enantiomers in tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride, which has strong specificity, high accuracy, stability, reliability and high sensitivity.

[0006] In order to achieve the above-mentioned purpose of this application, this application adopts the following technical solutions:

[0007] A method for separating and detecting enantiomers in tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride adopts an HPLC separation and detection method. The chromatographic conditions are as follows: a chromatographic column is a chiral chromatographic column of a polysaccharide derivative, mobile phase A is an aqueous solution of a weak base, and mobile phase B is acetonitrile, methanol, or a mixture of the two.

[0008] The present application provides a method for separating and detecting enantiomers in tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride. By selecting a polysaccharide derivative chiral chromatographic column, using an aqueous solution of a weak base as mobile phase A, and using acetonitrile and / or methanol as mobile phase B, tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride and its corresponding isomers, the method has strong specificity, high accuracy, and high sensitivity, and can be effectively used for controlling impurities in the starting raw material drug of tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride.

[0009] In some embodiments, the chromatography column is selected from AS-3R, AY-3R, AZ-3R, OD-3R, OJ-3R, OX-3R, OZ-3R and AD-3R. This chromatographic column has good selectivity and high sensitivity for the separation and detection of tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride and its enantiomers, achieving good separation in a relatively short time and improving the efficiency of separation and detection.

[0010] In some embodiments, the mobile phase A is selected from one or more aqueous solutions of ammonia, butylamine, ethanolamine, diethylamine, ethylenediamine, and triethylamine.

[0011] Optionally, the mobile phase A is an ammonia solution with a volume fraction of 0.01% to 0.1%.

[0012] Optionally, the mobile phase A is an ammonia solution with a volume fraction of 0.02% to 0.08%. For example, the mobile phase A can be an ammonia solution with the following volume fractions: 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, etc.

[0013] In some embodiments, the mobile phase B is acetonitrile.

[0014] In some embodiments, the volume ratio of the mobile phase is: mobile phase A:mobile phase B=58:42 to 70:30; alternatively, mobile phase A:mobile phase B=58:42 to 62:38. For example, the volume ratio of mobile phase A to mobile phase B can be 58:42, 59:41, 60:40, 61:39, 62:38, 65:35, 68:32, 70:30, etc.

[0015] In some embodiments, the diluent is a 40%-60% volume fraction acetonitrile solution, for example, 40%, 43%, 45%, 48%, 49%, 50%, 51%, 52%, 53%, 55%, 58%, 60%, etc.

[0016] In some embodiments, the flow rate of the mobile phase is 0.4-0.8 ml / min, for example, 0.4 ml / min, 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, or 0.8 ml / min.

[0017] In some embodiments, the flow rate of the mobile phase is 0.6 ml / min.

[0018] In some embodiments, the concentration of the test solution is 1.0 to 3.5 mg / ml, for example, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, or 3.5 mg / ml.

[0019] In some embodiments, the concentration of the test solution is 3.0 mg / ml.

[0020] In some embodiments, the column temperature of the chromatographic column is 35-45° C. Alternatively, the column temperature of the chromatographic column is 38-42° C. For example, it can be 38° C., 39° C., 40° C., 41° C., or 42° C.

[0021] In some embodiments, the detection wavelength is 210 nm.

[0022] In some embodiments, the detector is selected from the group consisting of: an ultraviolet detector, a differential detector, an evaporative light scattering detector, an electrospray detector, or a diode array detector.

[0023] Optionally, the detector is a diode array detector.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present application establishes a method for separating and detecting enantiomers in (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionic acid tert-butyl ester hydrochloride by using a polysaccharide derivative chiral chromatographic column, an aqueous solution of a weak base as mobile phase A, and acetonitrile and / or methanol as mobile phase B. The method has strong specificity, high resolution, high accuracy, stability, reliability, and high sensitivity, and provides a simple, accurate, rapid, and reliable detection method for controlling impurities in the starting raw material drug of (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionic acid tert-butyl ester hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0027] Figure 1 This is the liquid chromatogram of the blank solution of Example 8.

[0028] Figure 2 The figure is the separation liquid chromatogram.

[0029] Figure 3 This is an enlarged diagram of the liquid chromatogram of the separation solution.

[0030] Figure 4 The figure is the liquid chromatogram of the test solution of the starting material.

[0031] Figure 5 The linear relationship diagram of enantiomers. DETAILED DESCRIPTION

[0032] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0033] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0034] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0035] Example 1

[0036] 1) Experimental conditions:

[0037] Agilent 1260 high-performance liquid chromatograph;

[0038] Chromatographic column: AD-3R, 4.6 × 150 mm, 3 μm;

[0039] Flow rate: 0.6 mL / min;

[0040] Column temperature: 40°C;

[0041] Injection volume: 5 μL;

[0042] Sample concentration: 3 mg / ml;

[0043] Diluent: 50% acetonitrile aqueous solution (volume fraction);

[0044] Detector: DAD detector;

[0045] Detection wavelength: 210nm;

[0046] Mobile phase A: aqueous solution containing 0.05% (volume fraction) ammonia

[0047] Mobile phase B: methanol

[0048] Mobile phase A: mobile phase B = 70:30 (volume ratio)

[0049] 2) Solution preparation

[0050] Blank solution: 50% acetonitrile aqueous solution.

[0051] Stock solution of the starting material enantiomer reference substance: Take an appropriate amount of the starting material enantiomer reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to a solution containing approximately 3 mg of the starting material enantiomer per 1 ml.

[0052] Separation solution: Take about 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve it (if necessary, briefly assist with ultrasonication until it is completely dissolved), accurately transfer 100 uL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well.

[0053] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml.

[0054] 3) Determination method

[0055] Accurately measure the blank solution and inject it into the liquid chromatograph for 1 injection, and inject the separation solution into the liquid chromatograph for 6 injections continuously, and record the chromatogram.

[0056] 4) Experimental results

[0057] The blank solution showed no interference, demonstrating good method specificity. Six consecutive injections of the resolution solution revealed RSDs for the starting material peak retention time of 0.14% and the enantiomer peak retention time of 0.28%. The RSDs for the starting material peak area were 1.8% and 1.9%, respectively, both less than 5%, demonstrating good method reproducibility. The average resolution for the six injections of resolution solution was 1.7.

[0058] Example 2

[0059] 1) Experimental conditions:

[0060] Agilent 1260 high-performance liquid chromatograph;

[0061] Chromatographic column: AD-3R, 4.6 × 150 mm, 3 μm;

[0062] Flow rate: 0.6 mL / min;

[0063] Column temperature: 40°C;

[0064] Injection volume: 5 μL;

[0065] Sample concentration: 3 mg / ml;

[0066] Diluent: 50% acetonitrile aqueous solution (volume fraction);

[0067] Detector: DAD detector;

[0068] Detection wavelength: 210nm;

[0069] Mobile phase A: aqueous solution containing 0.05% (volume fraction) diethylamine

[0070] Mobile phase B: acetonitrile

[0071] Mobile phase A: mobile phase B = 60:40 (volume ratio)

[0072] 2) Solution preparation

[0073] Blank solution: 50% acetonitrile aqueous solution.

[0074] Stock solution of the starting material enantiomer reference substance: Take an appropriate amount of the starting material enantiomer reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to a solution containing approximately 3 mg of the starting material enantiomer per 1 ml.

[0075] Separation solution: Take about 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve it (if necessary, briefly assist with ultrasonication until it is completely dissolved), accurately transfer 100 uL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well.

[0076] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml.

[0077] 3) Determination method

[0078] Accurately measure the blank solution and inject it into the liquid chromatograph for 1 injection, and inject the separation solution into the liquid chromatograph for 6 injections continuously, and record the chromatogram.

[0079] 4) Experimental results

[0080] The blank solution showed no interference, demonstrating good method specificity. Six consecutive injections of the resolution solution revealed RSDs for the retention time of the starting material peak and the enantiomer peak, respectively, of 0.19% and 0.48%, respectively. The RSDs for the starting material peak area and enantiomer peak areas were both less than 5%, respectively, at 2.4% and 2.7%, respectively, demonstrating good method reproducibility. The average resolution for the six injections of resolution solution was 1.9.

[0081] Example 3

[0082] 1) Experimental conditions:

[0083] Agilent 1260 high-performance liquid chromatograph;

[0084] Chromatographic column: AD-3R, 4.6 × 150 mm, 3 μm;

[0085] Flow rate: 0.6 mL / min;

[0086] Column temperature: 38°C;

[0087] Injection volume: 5 μL;

[0088] Sample concentration: 3 mg / ml;

[0089] Diluent: 50% acetonitrile aqueous solution (volume fraction);

[0090] Detector: DAD detector;

[0091] Detection wavelength: 210nm;

[0092] Mobile phase A: aqueous solution containing 0.05% (volume fraction) ammonia

[0093] Mobile phase B: acetonitrile

[0094] Mobile phase A: mobile phase B = 60:40 (volume ratio)

[0095] 2) Solution preparation

[0096] Blank solution: 50% acetonitrile aqueous solution.

[0097] Stock solution of the starting material enantiomer reference substance: Take an appropriate amount of the starting material enantiomer reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to a solution containing approximately 3 mg of the starting material enantiomer per 1 ml.

[0098] Separation solution: Take about 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve it (if necessary, briefly assist with ultrasonication until it is completely dissolved), accurately transfer 100 uL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well.

[0099] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml.

[0100] 3) Determination method

[0101] Accurately measure the blank solution and inject it into the liquid chromatograph for 1 injection, and inject the separation solution into the liquid chromatograph for 6 injections continuously, and record the chromatogram.

[0102] 4) Experimental results

[0103] The blank solution showed no interference, demonstrating good method specificity. Six consecutive injections of the resolution solution revealed RSDs for the retention time of the starting material peak and the enantiomer peak, respectively, of 0.05% and 0.11%, respectively. The RSDs for the starting material peak area and enantiomer peak areas were both less than 5%, respectively, at 1.2% and 1.5%, respectively, demonstrating good method reproducibility. The average resolution for the six injections of resolution solution was 2.1.

[0104] Example 4

[0105] 1) Experimental conditions:

[0106] Agilent 1260 high-performance liquid chromatograph;

[0107] Chromatographic column: AD-3R, 4.6 × 150 mm, 3 μm;

[0108] Flow rate: 0.6 mL / min;

[0109] Column temperature: 42°C;

[0110] Injection volume: 5 μL;

[0111] Sample concentration: 3 mg / ml;

[0112] Diluent: 50% acetonitrile aqueous solution (volume fraction);

[0113] Detector: DAD detector;

[0114] Detection wavelength: 210nm;

[0115] Mobile phase A: aqueous solution containing 0.05% (volume fraction) ammonia

[0116] Mobile phase B: acetonitrile

[0117] Mobile phase A: mobile phase B = 60:40 (volume ratio)

[0118] 2) Solution preparation

[0119] Blank solution: 50% acetonitrile aqueous solution (volume fraction).

[0120] Stock solution of the starting material enantiomer reference substance: Take an appropriate amount of the starting material enantiomer reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to a solution containing approximately 3 mg of the starting material enantiomer per 1 ml.

[0121] Separation solution: Take about 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve it (if necessary, briefly assist with ultrasonication until it is completely dissolved), accurately transfer 100 uL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well.

[0122] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml.

[0123] 3) Determination method

[0124] Accurately measure the blank solution and inject it into the liquid chromatograph for 1 injection, and inject the separation solution into the liquid chromatograph for 6 injections continuously, and record the chromatogram.

[0125] 4) Experimental results

[0126] The blank solution showed no interference, demonstrating good method specificity. Six consecutive injections of the resolution solution revealed RSDs for the starting material peak retention time of 0.06% and the enantiomer peak retention time of 0.15%. The RSDs for the starting material peak area were 1.3% and 1.4%, respectively, both less than 5%, demonstrating good method reproducibility. The average resolution for the six injections of resolution solution was 2.0.

[0127] Example 5

[0128] 1) Experimental conditions:

[0129] Agilent 1260 high-performance liquid chromatograph;

[0130] Chromatographic column: AD-3R, 4.6 × 150 mm, 3 μm;

[0131] Flow rate: 0.6 mL / min;

[0132] Column temperature: 40°C;

[0133] Injection volume: 5 μL;

[0134] Sample concentration: 3 mg / ml;

[0135] Diluent: 50% acetonitrile aqueous solution (volume fraction);

[0136] Detector: DAD detector;

[0137] Detection wavelength: 210nm;

[0138] Mobile phase A: aqueous solution containing 0.05% (volume fraction) ammonia

[0139] Mobile phase B: acetonitrile

[0140] Mobile phase A: mobile phase B = 58:42 (volume ratio)

[0141] 2) Solution preparation

[0142] Blank solution: 50% acetonitrile aqueous solution (volume fraction).

[0143] Stock solution of the starting material enantiomer reference substance: Take an appropriate amount of the starting material enantiomer reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to a solution containing approximately 3 mg of the starting material enantiomer per 1 ml.

[0144] Separation solution: Take about 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve it (if necessary, briefly assist with ultrasonication until it is completely dissolved), accurately transfer 100 uL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well.

[0145] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml.

[0146] 3) Determination method

[0147] Accurately measure the blank solution and inject it into the liquid chromatograph for 1 injection, and inject the separation solution into the liquid chromatograph for 6 injections continuously, and record the chromatogram.

[0148] 4) Experimental results

[0149] The blank solution showed no interference, demonstrating good method specificity. Six consecutive injections of the resolution solution revealed RSDs for the starting material peak retention time of 0.04% and the enantiomer peak retention time of 0.13%. The RSDs for the starting material peak area were 1.1% and 1.6%, respectively, both less than 5%, demonstrating good method reproducibility. The average resolution for the six injections of resolution solution was 2.0.

[0150] Example 6

[0151] 1) Experimental conditions:

[0152] Agilent 1260 high-performance liquid chromatograph;

[0153] Chromatographic column: AD-3R, 4.6 × 150 mm, 3 μm;

[0154] Flow rate: 0.6 mL / min;

[0155] Column temperature: 40°C;

[0156] Injection volume: 5 μL;

[0157] Sample concentration: 3 mg / ml;

[0158] Diluent: 50% acetonitrile aqueous solution (volume fraction);

[0159] Detector: DAD detector;

[0160] Detection wavelength: 210nm;

[0161] Mobile phase A: aqueous solution containing 0.05% (volume fraction) ammonia

[0162] Mobile phase B: acetonitrile

[0163] Mobile phase A: mobile phase B = 62:38 (volume ratio)

[0164] 2) Solution preparation

[0165] Blank solution: 50% acetonitrile aqueous solution (volume fraction).

[0166] Stock solution of the starting material enantiomer reference substance: Take an appropriate amount of the starting material enantiomer reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to a solution containing approximately 3 mg of the starting material enantiomer per 1 ml.

[0167] Separation solution: Take about 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve it (if necessary, briefly assist with ultrasonication until it is completely dissolved), accurately transfer 100 uL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well.

[0168] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml.

[0169] 3) Determination method

[0170] Accurately measure the blank solution and inject it into the liquid chromatograph for 1 injection, and inject the separation solution into the liquid chromatograph for 6 injections continuously, and record the chromatogram.

[0171] 4) Experimental results

[0172] The blank solution showed no interference, demonstrating good method specificity. Six consecutive injections of the resolution solution revealed RSDs for the retention time of the starting material peak and the enantiomer peak, respectively, of 0.08% and 0.17%, respectively. The RSDs for the starting material peak area and enantiomer peak areas were both less than 5%, respectively, at 1.5% and 1.7%, respectively, demonstrating good method reproducibility. The average resolution for the six injections of resolution solution was 1.9.

[0173] Example 7

[0174] 1) Experimental conditions:

[0175] Agilent 1260 high-performance liquid chromatograph;

[0176] Chromatographic column: AD-3R, 4.6 × 150 mm, 3 μm;

[0177] Flow rate: 0.6 mL / min;

[0178] Column temperature: 40°C;

[0179] Injection volume: 5 μL;

[0180] Sample concentration: 3 mg / ml;

[0181] Diluent: 50% acetonitrile aqueous solution (volume fraction);

[0182] Detector: DAD detector;

[0183] Detection wavelength: 210nm;

[0184] Mobile phase A: aqueous solution containing 0.05% (volume fraction) ammonia

[0185] Mobile phase B: acetonitrile

[0186] Mobile phase A: mobile phase B = 60:40 (volume ratio)

[0187] 2) Solution preparation

[0188] Blank solution: 50% acetonitrile aqueous solution (volume fraction).

[0189] Stock solution of the starting material enantiomer reference substance: Take an appropriate amount of the starting material enantiomer reference substance, accurately weigh it, dissolve it with a diluent and quantitatively dilute it to a solution containing approximately 3 mg of the starting material enantiomer per 1 ml.

[0190] Separation solution: Take about 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve it (if necessary, briefly assist with ultrasonication until it is completely dissolved), accurately transfer 100 uL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well.

[0191] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml.

[0192] 3) Determination method

[0193] Accurately measure the blank solution and inject it into the liquid chromatograph for 1 injection, and inject the separation solution into the liquid chromatograph for 6 injections continuously, and record the chromatogram.

[0194] 4) Experimental results

[0195] The blank solution showed no interference, demonstrating good method specificity. Six consecutive injections of the resolution solution revealed RSDs for the starting material peak retention time of 0.03% and the enantiomer peak retention time of 0.11%. The RSDs for the starting material peak area were 1.0% and 1.2%, respectively, both less than 5%, demonstrating good method reproducibility. The average resolution for the six injections of resolution solution was 2.2.

[0196] Example 8

[0197] 1. Instruments and Conditions

[0198] Agilent 1260 high performance liquid chromatograph (equipped with DAD detector); chromatographic column: AD-3R, 4.6×150mm, 3μm; mobile phase A: aqueous solution containing 0.05% (volume fraction) ammonia; mobile phase B: acetonitrile; mobile phase A: mobile phase B = 60:40 (volume ratio), isocratic elution; flow rate: 0.6mL / min; detection wavelength: 210nm; column temperature: 40℃; injection volume: 5μL; sample concentration: 3.0mg / ml; diluent: 50% acetonitrile aqueous solution.

[0199] 2. Experimental steps

[0200] 1. Preparation of solution:

[0201] Blank solution: 50% acetonitrile aqueous solution (volume fraction).

[0202] Stock solution of starting material enantiomer reference substance: Take an appropriate amount of starting material enantiomer reference substance, accurately weigh it, dissolve it in diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material enantiomer per 1 ml. (Starting material enantiomer concentration 3 mg / ml)

[0203] Separation solution: Take approximately 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve (if necessary, briefly sonicate until completely dissolved), accurately pipette 100 μL of the starting material enantiomer reference substance stock solution into it, dilute to 10 mL with diluent, and shake well. (Starting material concentration 3 mg / mL, starting material enantiomer concentration 30 μg / mL)

[0204] Starting material enantiomer reference solution: Accurately pipette 100uL of the starting material enantiomer reference stock solution into a 100mL volumetric flask, add diluent to dissolve, dilute to 100mL, and shake well. (Starting material enantiomer concentration 3ug / ml)

[0205] 80% accuracy solution: Take approximately 30 mg of the starting material reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve (if necessary, briefly sonicate until completely dissolved), accurately pipette 8 μL of the starting material enantiomer reference substance stock solution into the flask, dilute to 10 mL with diluent, and shake well. Prepare 3 replicates (starting material concentration 3 mg / mL, starting material enantiomer concentration 2.4 μg / mL).

[0206] 100% accuracy solution: Take approximately 30 mg of the starting material, accurately weigh it, place it in a 10 mL volumetric flask, add diluent to dissolve (if necessary, briefly sonicate until completely dissolved), accurately pipette 10 μL of the starting material enantiomer reference stock solution into the flask, dilute to 10 mL with diluent, and shake well. Prepare 3 replicates (starting material concentration 3 mg / mL, starting material enantiomer concentration 3 μg / mL).

[0207] 120% accuracy solution: Accurately weigh approximately 30 mg of the starting material reference substance and place it in a 10 mL volumetric flask. Dissolve with diluent (if necessary, briefly sonicate until completely dissolved). Accurately pipette 12 μL of the starting material enantiomer reference substance stock solution into the flask, dilute to 10 mL with diluent, and shake well. Prepare three replicates (starting material concentration 3 mg / mL, starting material enantiomer concentration 3.6 μg / mL).

[0208] Test solution: Take an appropriate amount of starting material, accurately weigh it, dissolve it with diluent and quantitatively dilute it to a solution containing approximately 3 mg of starting material per 1 ml. (Starting material concentration 3 mg / ml)

[0209] Linear solution: Take the stock solution of the starting material enantiomer reference substance separately and use diluent to make a mixed solution with a concentration of 1.5μg / ml to 6.0μg / ml.

[0210] 2. Methodological Validation

[0211] 2.1 Specificity test

[0212] Take 5 μl of diluent, separation solution and test solution respectively, inject them into liquid chromatograph in sequence, and record the chromatogram. Figures 1 to 4 . Figure 2-4 MS057-G is (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionic acid tert-butyl ester hydrochloride, and Z57MS057 is (R)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionic acid tert-butyl ester hydrochloride.

[0213] Take 5μl of the linear solution and inject it into the liquid chromatograph, record the chromatogram. Perform linear regression with the peak area (Y) versus the concentration (X). The linear regression equation is y=14,936.2008x+519.4356, and the correlation coefficient (R) is 1.0000. The linear relationship is as follows Figure 5 shown.

[0214] 2.2 Linear range, detection limit and quantification limit

[0215] Table 7 Linearity and sensitivity test results

[0216] name Correlation coefficient Detection limit (μg / ml) Limit of quantification (μg / ml) Starting materials 1.0 0.58 1.74 Enantiomers 1.0 0.51 1.54

[0217] 2.3 Accuracy test

[0218] 5 μl of each of the diluent, starting material enantiomer reference solution, 80% accuracy solution, 100% accuracy solution, and 120% accuracy solution were injected into the liquid chromatograph in sequence, and the chromatograms were recorded. The accuracy of an analytical method refers to the degree of closeness of the test results obtained using the method to the true value. The accuracy of an analytical method should be determined within a certain range. The accuracy of the analytical method is demonstrated by the recoveries and RSDs of the isomer-spiked test samples (80% accuracy solution, 100% accuracy solution, and 120% accuracy solution). The specific results are shown in the table below.

[0219]

[0220] The results showed that the average recovery rate of each level was within the acceptable range of 80-120%. The RSD of the recovery rate at each concentration was within the acceptable range of no more than 5%. Therefore, the analytical method of the present invention is accurate.

[0221] 2.4 Stability test

[0222] Take 5 μl of diluent, separation solution and test solution respectively, and inject them into liquid chromatograph in sequence at 2h, 4h, 6h, 8h, 12h, 18h and 24h, and record the chromatogram.

[0223]

[0224]

[0225] This method can ensure that the solution stability is maintained within 24 hours, ensuring that the percentage change rate of the peak area of the starting material during the stability investigation period is ≤0.1%, and the percentage change rate of the peak area of the enantiomer during the stability investigation period is ≤3.0%, meeting the detection time limit requirements.

[0226] 2.5 Sample determination: Enantiomeric impurities are determined using the external standard method.

[0227] 2.6 Conclusion

[0228] The method can accurately determine the content of enantiomers in the key starting material (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionic acid tert-butyl ester hydrochloride, has the characteristics of high efficiency, rapidity and accuracy, and can achieve the purpose of quality control.

[0229] The contents not described in detail in this specification belong to the prior art known to those skilled in the art. The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included within the scope of the claims of the present application.

Claims

1. A method for separating and detecting enantiomers in tert-butyl (S)-2-amino-3-[2-(tert-butoxy)-2-oxoacetamido]propionate hydrochloride, characterized in that: HPLC separation and detection method is adopted, and the chromatographic conditions are as follows: the chromatographic column is a polysaccharide derivative chiral chromatographic column, the mobile phase A is a weak base aqueous solution, and the mobile phase B is acetonitrile, methanol or a mixture of the two.

2. The separation and detection method according to claim 1, wherein The chromatographic column is selected from AS-3R, AY-3R, AZ-3R, OD-3R, OJ-3R, OX-3R, OZ-3R and AD-3R.

3. The separation and detection method according to claim 1, wherein The mobile phase A is selected from one or more aqueous solutions of ammonia, butylamine, ethanolamine, diethylamine, ethylenediamine and triethylamine; Optionally, the mobile phase A is an ammonia solution with a volume fraction of 0.01% to 0.1%; Optionally, the mobile phase A is an aqueous ammonia solution with a volume fraction of 0.02% to 0.08%.

4. The separation and detection method according to claim 1, wherein The mobile phase B is acetonitrile.

5. The separation and detection method according to claim 1, wherein The mobile phase volume ratio is: Mobile phase A:mobile phase B=58:42~70:30; Optionally, mobile phase A:mobile phase B=58:42~62:

38.

6. The separation and detection method according to claim 1, characterized in that The diluent is an acetonitrile solution with a volume fraction of 40%-60%.

7. The separation and detection method according to claim 1, wherein The flow rate of the mobile phase is 0.4-0.8 ml / min.

8. The separation and detection method according to claim 1, wherein The concentration of the test solution is 1.0-3.5 mg / ml.

9. The separation and detection method according to claim 1, wherein The column temperature of the chromatographic column is 35-45°C; Optionally, the column temperature of the chromatographic column is 38-42°C.

10. The separation and detection method according to claim 1, characterized in that The detector is selected from the group consisting of: an ultraviolet detector, a differential detector, an evaporative light scattering detector, an electrospray detector, and a diode array detector; Optionally, the detector is a diode array detector.

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