Method for detecting carbobenzoxy-L-alanine through high performance liquid chromatography

Abstract

The invention provides a method for analyzing and detecting carbobenzoxy-L-alanine by high performance liquid chromatography, which adopts a chiral chromatographic column, takes an alcohol reagent as a mobile phase A and takes water with the pH value of 1.8-2.2 as a mobile phase B; wherein the chiral chromatographic column is a cellulose chiral chromatographic column. The method provided by the invention can effectively separate benzyloxycarbonyl-L-alanine and optical isomers thereof, and ensures that the quality of a starting material of metaraminol bitartrate is controllable.

Description

Technical Field

[0001] The present invention relates to the field of liquid chromatography analysis, and particularly to a method for high performance liquid chromatography analysis and detection of carbobenzoxy-L-alanine. Background Art

[0002] Metaraminol bitartrate is a β-adrenergic receptor agonist, commonly known as Aramine and metaraminol. Clinically, it is mainly used to prevent and treat acute hypotension occurring during intravenous regional anesthesia, and can also be used to treat hypotension caused by cardiogenic shock or septicemia. As a starting material for the preparation of metaraminol bitartrate, carbobenzoxy-L-alanine contains a chiral carbon, which will affect the quality and yield of metaraminol bitartrate during the production process. Therefore, in order to improve the yield of metaraminol bitartrate, in accordance with the pharmaceutical production specifications and product standards, it is necessary to effectively analyze and control the contents of carbobenzoxy-L-alanine and its isomer (carbobenzoxy-D-alanine). However, there are currently no relatively mature reports on the analysis and detection of the contents of carbobenzoxy-L-alanine and its isomers by high performance liquid chromatography. Therefore, there is an urgent need to provide a method for high performance liquid chromatography analysis and detection of carbobenzoxy-L-alanine. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems in the related art to some extent. For this purpose, the present invention provides a method for high performance liquid chromatography analysis and detection of carbobenzoxy-L-alanine. This method can be used to analyze and detect the content of carbobenzoxy-L-alanine, and can effectively separate carbobenzoxy-L-alanine and its isomers at the same time, so as to realize the quality control of metaraminol bitartrate raw materials.

[0004] For this purpose, the first aspect of the present invention provides a method for high performance liquid chromatography analysis and detection of carbobenzoxy-L-alanine. The method uses a chiral chromatographic column, with an alcohol reagent as the mobile phase A and water with a pH of 1.8 - 2.2 as the mobile phase B;

[0005] Wherein, the chiral chromatographic column is a cellulose-based chiral chromatographic column.

[0006] The method provided by the present invention is based on high performance liquid chromatography. Its technical principle is that when the components dissolved in the mobile phase pass through the stationary phase, due to the different sizes and strengths of their interactions (adsorption, distribution, ion attraction, exclusion, affinity) with the stationary phase, their residence times in the stationary phase are different, and thus they flow out of the stationary phase successively. By using the method provided by the present invention, carbobenzoxy-L-alanine and its optical isomers can be effectively separated, thus ensuring the quality control of the starting material of metaraminol bitartrate.

[0007] According to an embodiment of the present invention, the volume ratio of mobile phase A to mobile phase B is (25 - 35):(65 - 75), preferably 30:70.

[0008] According to an embodiment of the present invention, the alcohol reagent includes ethanol selected from the group.

[0009] According to an embodiment of the present invention, the method includes adjusting the pH value of mobile phase B with phosphoric acid.

[0010] According to an embodiment of the present invention, the pH value of mobile phase B is 2.0.

[0011] According to an embodiment of the present invention, the chiral chromatographic column uses cellulose-3,5-dichlorophenylcarbamate-bonded silica gel as the packing material.

[0012] According to an embodiment of the present invention, the chiral chromatographic column includes CHIRALPAK IC selected from the group.

[0013] According to an embodiment of the present invention, the method includes the following steps:

[0014] (1) Prepare a test sample solution containing carbobenzoxy-L-alanine;

[0015] (2) Set the mobile phase flow rate to 0.8 - 1.2 mL / min, the detection wavelength to 200 - 210 nm, the column temperature to 20°C - 30°C, and the injection volume to 5 - 20 μL to complete the separation and determination of carbobenzoxy-L-alanine.

[0016] According to an embodiment of the present invention, step (1) further includes taking the test sample and dissolving it with the mobile phase to obtain the test sample solution.

[0017] According to an embodiment of the present invention, the concentration of carbobenzoxy-L-alanine in the test sample solution is 0.5 mg / mL.

[0018] According to an embodiment of the present invention, the mobile phase flow rate is 1.0 ml / min.

[0019] According to an embodiment of the present invention, the detection wavelength is 205 nm.

[0020] According to an embodiment of the present invention, the column temperature is 25°C.

[0021] According to an embodiment of the present invention, the injection volume is 10 μL.

[0022] The second aspect of the present invention provides the application of the method described in the first aspect in the quality control of carbobenzoxy-L-alanine.

[0023] Advantages of the present invention over the prior art:

[0024] The method for analyzing and detecting carbobenzoxy-L-alanine by high performance liquid chromatography provided by the present invention can effectively separate carbobenzoxy-L-alanine and its optical isomers. The peak shapes of the two substances are symmetrical and well separated, with a resolution of greater than or equal to 1.5. Under the optimal method, the quantitative limit concentration is 0.528 μg / mL. At the same time, this method is simple to operate, only requires an ordinary liquid chromatograph, and the mobile phase medium used is easily available, with high feasibility and good applicability, and can be widely used in the quality research of carbobenzoxy-L-alanine.

[0025] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be understood through the practice of the present invention. Brief Description of the Drawings

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and be easily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0027] Figure 1 Shows the high performance liquid chromatogram of the test solution in Example 1 of the present invention;

[0028] Figure 2 Shows the high performance liquid chromatogram of the reference solution in Example 1 of the present invention;

[0029] Figure 3 Shows the high performance liquid chromatogram of the reference solution in Example 2 of the present invention;

[0030] Figure 4 Shows the high performance liquid chromatogram of the reference solution in Example 3 of the present invention;

[0031] Figure 5 Shows the high performance liquid chromatogram of the reference solution in Example 4 of the present invention;

[0032] Figure 6 Shows the high performance liquid chromatogram of the reference solution in Example 5 of the present invention;

[0033] Figure 7 Shows the high performance liquid chromatogram of the reference solution in Example 6 of the present invention;

[0034] Figure 8 Shows the high performance liquid chromatogram of the reference solution in Example 7 of the present invention;

[0035] Figure 9 Shows the high performance liquid chromatogram of the reference solution in Example 8 of the present invention;

[0036] Figure 10 Shows the high performance liquid chromatogram of the reference solution in Example 9 of the present invention;

[0037] Figure 11 shows the high performance liquid chromatography (HPLC) chromatogram of the reference solution in Example 10 of the present invention;

[0038] Figure 12 shows the high performance liquid chromatography (HPLC) chromatogram of the reference solution in Example 11 of the present invention;

[0039] Figure 13 shows the high performance liquid chromatography (HPLC) chromatogram of the reference solution in Comparative Example 1 of the present invention;

[0040] Figure 14 shows the high performance liquid chromatography (HPLC) chromatogram of the reference solution in Comparative Example 2 of the present invention;

[0041] Figure 15 shows the high performance liquid chromatography (HPLC) chromatogram of the reference solution in Comparative Example 3 of the present invention;

[0042] Figure 16 shows the high performance liquid chromatography (HPLC) chromatogram of the reference solution in Comparative Example 4 of the present invention;

[0043] Figure 17 shows the high performance liquid chromatography (HPLC) chromatogram of the test solution in Comparative Example 5 of the present invention;

[0044] Figure 18 shows the high performance liquid chromatography (HPLC) chromatogram of the reference solution in Comparative Example 6 of the present invention. Detailed Description of the Invention

[0045] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0046] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0047] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0048] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein shall have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0049] As used herein, the term "comprising" or "including" is an open-ended expression, meaning it includes the content specified in the present invention but does not exclude other aspects.

[0050] As used herein, the terms "optionally", "optional" or "option" generally refer to the subsequent event or condition that may or may not occur, and this description includes the cases where the event or condition occurs and the cases where the event or condition does not occur.

[0051] According to an embodiment of the present invention, the present invention provides a method for analyzing and detecting carbobenzoxy-L-alanine by high performance liquid chromatography.

[0052] According to a specific embodiment of the present invention, the method uses a chiral chromatographic column, with an alcohol reagent as mobile phase A and water with a pH of 1.8 - 2.2 as mobile phase B.

[0053] According to a specific embodiment of the present invention, the chiral chromatographic column is packed with cellulose-3,5-dichlorophenylcarbamate-bonded silica gel, including but not limited to selected from CHIRALPAK IC. The length of the chiral chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the inner diameter of the packing is 5 μm. Thus, a better separation effect is achieved.

[0054] According to a specific embodiment of the present invention, the alcohol reagent includes ethanol selected from. Compared with acetonitrile with too fast a separation speed and methanol with too slow a separation speed, it can achieve better elution efficiency and resolution. Phosphoric acid is used to adjust the pH value of mobile phase B. The pH value of mobile phase B is preferably 2.0. The volume ratio of mobile phase A to mobile phase B is (25 - 35):(65 - 75), preferably 30:70.

[0055] According to a specific embodiment of the present invention, the method specifically includes the following steps:

[0056] (1) Prepare a sample solution to be tested containing carbobenzoxy-L-alanine;

[0057] (2) Set the flow rate of the mobile phase to 0.8 - 1.2 mL / min, the detection wavelength to 200 - 210 nm, the column temperature to 20°C - 30°C, and the injection volume to 5 - 20 μL to complete the separation and determination of carbobenzoxy-L-alanine.

[0058] According to a specific embodiment of the present invention, step (1) further includes taking a sample to be tested, dissolving it with a mobile phase to obtain the sample solution to be tested. The concentration of carbobenzoxy-L-alanine in the sample solution to be tested is 0.5 mg / mL. The flow rate of the mobile phase is preferably 1.0 ml / min, the detection wavelength is preferably 205 nm, the column temperature is preferably 25 °C, and the injection volume is preferably 10 μL.

[0059] According to a specific embodiment of the present invention, the present invention provides a method for analyzing and detecting carbobenzoxy-L-alanine by high performance liquid chromatography, and its chromatographic conditions can be:

[0060] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0061] Mobile phase: aqueous phase (adjust the pH value to 1.8 - 2.2 with phosphoric acid): ethanol = (65 - 75):(25 - 35) (v / v);

[0062] Flow rate: 0.8 - 1.2 mL / min;

[0063] Detection wavelength: 200 - 210 nm;

[0064] Column temperature: 20 °C - 30 °C;

[0065] Injection volume: 10 μL;

[0066] Isocratic elution running time: 30 min.

[0067] The specific experimental steps can be:

[0068] (1) Prepare a reference solution: Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 mL volumetric flask, add the mobile phase to dissolve and dilute to the scale, shake well, and prepare a solution containing 0.5 mg of each of carbobenzoxy-L-alanine reference substance and carbobenzoxy-D-alanine reference substance in 1 mL as the reference solution;

[0069] (2) Take the reference solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram. According to the chromatogram results, observe whether carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are well separated. The specific indicators include: whether there are peaks of the two substances, whether the peak shape is symmetric, and whether the resolution meets the requirements (resolution ≧ 1.5).

[0070] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. Those reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchases.

[0071] Example 1

[0072] Chromatographic conditions:

[0073] High performance liquid chromatograph: Dionex: UltiMate3000;

[0074] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0075] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0076] Flow rate: 1.0 mL / min;

[0077] Detection wavelength: 205 nm;

[0078] Column temperature: 25°C;

[0079] Injection volume: 10 μL;

[0080] Isocratic elution running time: 30 min.

[0081] Experimental procedure:

[0082] (1) Take an appropriate amount of raw material CBZ-L-alanine, place it in a 20 ml volumetric flask, dissolve it with the mobile phase and dilute it to the scale, shake well, and prepare a test solution with a concentration of 1 mg / mL;

[0083] (2) Take the test solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0084] The results are shown in Figure 1 , Figure 1 In which, the peak 1 is carbobenzoxy-L-alanine, and the peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0085] Chromatographic conditions:

[0086] High performance liquid chromatograph: Dionex: UltiMate3000;

[0087] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0088] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0089] Flow rate: 1.0 mL / min;

[0090] Detection wavelength: 205 nm;

[0091] Column temperature: 25 °C;

[0092] Injection volume: 10 μL;

[0093] Isocratic elution running time: 30 min.

[0094] Experimental procedure:

[0095] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution;

[0096] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0097] The results are shown in Figure 2 , Figure 2 in which peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0098] Example 2

[0099] Chromatographic conditions:

[0100] High performance liquid chromatograph: Dionex: UltiMate3000;

[0101] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0102] Mobile phase: aqueous phase (pH adjusted to 1.8 with phosphoric acid): ethanol = 70:30 (v / v);

[0103] Flow rate: 1.0 mL / min;

[0104] Detection wavelength: 205 nm;

[0105] Column temperature: 25 °C;

[0106] Sample injection volume: 10 μL;

[0107] Isocratic elution running time: 30 min.

[0108] Experimental procedure:

[0109] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference standards, place them in a 20 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference standard solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0110] (2) Take the reference standard solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0111] The results are shown in Figure 3 , Figure 3 In [reference], peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0112] Example 3

[0113] Chromatographic conditions:

[0114] High performance liquid chromatograph: Dionex: UltiMate3000;

[0115] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0116] Mobile phase: aqueous phase (pH adjusted to 2.2 with phosphoric acid): ethanol = 70:30 (v / v);

[0117] Flow rate: 1.0 mL / min;

[0118] Detection wavelength: 205 nm;

[0119] Column temperature: 25 °C;

[0120] Sample injection volume: 10 μL;

[0121] Isocratic elution running time: 30 min.

[0122] Experimental procedure:

[0123] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference standards, place them in a 20 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference standard solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0124] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0125] The results are shown in Figure 4 , Figure 4 In [reference], peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0126] Example 4

[0127] Chromatographic conditions:

[0128] High performance liquid chromatograph: Dionex: UltiMate3000;

[0129] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0130] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 75:25 (v / v);

[0131] Flow rate: 1.0 mL / min;

[0132] Detection wavelength: 205 nm;

[0133] Column temperature: 25 °C;

[0134] Injection volume: 10 μL;

[0135] Isocratic elution running time: 30 min.

[0136] Experimental procedure:

[0137] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances in 1 mL as the reference substance solution;

[0138] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0139] The results are shown in Figure 5 , Figure 5 In [reference], peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0140] Example 5

[0141] Chromatographic conditions:

[0142] High performance liquid chromatograph: Dionex: UltiMate3000;

[0143] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0144] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 65:35 (v / v);

[0145] Flow rate: 1.0 mL / min;

[0146] Detection wavelength: 205 nm;

[0147] Column temperature: 25 °C;

[0148] Injection volume: 10 μL;

[0149] Isocratic elution running time: 30 min.

[0150] Experimental procedure:

[0151] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution;

[0152] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0153] The results are shown in Figure 6 , Figure 6 in which peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0154] Example 6

[0155] Chromatographic conditions:

[0156] High performance liquid chromatograph: Dionex: UltiMate3000;

[0157] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0158] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0159] Flow rate: 0.8 mL / min;

[0160] Detection wavelength: 205 nm;

[0161] Column temperature: 25 °C;

[0162] Injection volume: 10 μL;

[0163] Isocratic elution running time: 30 min.

[0164] Experimental procedure:

[0165] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0166] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0167] The results are shown in Figure 7 , Figure 7 in which peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0168] Example 7

[0169] Chromatographic conditions:

[0170] High performance liquid chromatograph: Dionex: UltiMate3000;

[0171] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0172] Mobile phase: aqueous phase (adjusted to pH 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0173] Flow rate: 1.2 mL / min;

[0174] Detection wavelength: 205 nm;

[0175] Column temperature: 25 °C;

[0176] Injection volume: 10 μL;

[0177] Isocratic elution running time: 30 min.

[0178] Experimental procedure:

[0179] (1) Weigh appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference standards, place them in a 20 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference standard solution containing 0.5 mg each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0180] (2) Take the reference standard solution and perform high-performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0181] The results are shown in Figure 8 , Figure 8 where the peak at No. 1 is carbobenzoxy-L-alanine and the peak at No. 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0182] Example 8

[0183] Chromatographic conditions:

[0184] High-performance liquid chromatograph: Dionex: UltiMate3000;

[0185] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0186] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0187] Flow rate: 1.0 mL / min;

[0188] Detection wavelength: 205 nm;

[0189] Column temperature: 20 °C;

[0190] Injection volume: 10 μL;

[0191] Isocratic elution running time: 30 min.

[0192] Experimental procedure:

[0193] (1) Weigh appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference standards, place them in a 20 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference standard solution containing 0.5 mg each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0194] (2) Take the reference standard solution and perform high-performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0195] The results are shown in Figure 9 , Figure 9Peak 1 is carbobenzoxy-L-alanine, and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well-separated.

[0196] Example 9

[0197] Chromatographic conditions:

[0198] High performance liquid chromatograph: Dionex: UltiMate3000;

[0199] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0200] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0201] Flow rate: 1.0 mL / min;

[0202] Detection wavelength: 205 nm;

[0203] Column temperature: 30 °C;

[0204] Injection volume: 10 μL;

[0205] Isocratic elution running time: 30 min.

[0206] Experimental procedure:

[0207] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution;

[0208] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0209] The results are shown in Figure 10 , Figure 10 Peak 1 is carbobenzoxy-L-alanine, and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well-separated.

[0210] Example 10

[0211] Chromatographic conditions:

[0212] High performance liquid chromatograph: Dionex: UltiMate3000;

[0213] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0214] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0215] Flow rate: 1.0 mL / min;

[0216] Detection wavelength: 200 nm;

[0217] Column temperature: 25 °C;

[0218] Injection volume: 10 μL;

[0219] Isocratic elution running time: 30 min.

[0220] Experimental procedure:

[0221] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution;

[0222] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0223] The results are shown in Figure 11 , Figure 11 in which the peak at No. 1 is carbobenzoxy-L-alanine and the peak at No. 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0224] Example 11

[0225] Chromatographic conditions:

[0226] High performance liquid chromatograph: Dionex: UltiMate3000;

[0227] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0228] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): ethanol = 70:30 (v / v);

[0229] Flow rate: 1.0 mL / min;

[0230] Detection wavelength: 210 nm;

[0231] Column temperature: 25 °C;

[0232] Injection volume: 10 μL;

[0233] Isocratic elution running time: 30 min.

[0234] Experimental procedure:

[0235] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference standards, place them in a 20 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference standard solution containing 0.5 mg each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0236] (2) Take the reference standard solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0237] The results are shown in Figure 12 , Figure 12 Peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated.

[0238] Comparative Example 1

[0239] Chromatographic conditions:

[0240] High performance liquid chromatograph: Dionex: UltiMate3000;

[0241] Chromatographic column: CHIRALPAK IC (250 mm × 4.6 mm, 5 μm);

[0242] Mobile phase: water: ethanol = 70:30 (v / v);

[0243] Flow rate: 1.0 mL / min;

[0244] Detection wavelength: 205 nm;

[0245] Column temperature: 25 °C;

[0246] Injection volume: 10 μL;

[0247] Isocratic elution running time: 30 min.

[0248] Experimental procedure:

[0249] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference standards, place them in a 20 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and prepare a reference standard solution containing 0.5 mg each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0250] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0251] The results are shown in Figure 13 , Figure 13 in which peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peak shapes of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are poor and do not meet the separation requirements.

[0252] Comparative Example 2

[0253] Chromatographic conditions:

[0254] High performance liquid chromatograph: Dionex: UltiMate3000;

[0255] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0256] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): methanol = 40:60 (v / v);

[0257] Flow rate: 1.0 ml / min;

[0258] Detection wavelength: 205 nm;

[0259] Column temperature: 25°C;

[0260] Injection volume: 10 μL;

[0261] Isocratic elution running time: 30 min.

[0262] Experimental procedure:

[0263] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve them with the mobile phase and dilute to the mark, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution;

[0264] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0265] The results are shown in Figure 14 , Figure 14 in which peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that by increasing the proportion of methanol, carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are not completely eluted within 30 min.

[0266] Comparative Example 3

[0267] Chromatographic conditions:

[0268] High performance liquid chromatograph: Dionex: UltiMate3000;

[0269] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0270] Mobile phase: aqueous phase (pH adjusted to 2.0 with formic acid): ethanol = 70:30 (v / v);

[0271] Flow rate: 1.0 ml / min;

[0272] Detection wavelength: 205 nm;

[0273] Column temperature: 25 °C;

[0274] Sample injection volume: 10 μL;

[0275] Isocratic elution running time: 30 min.

[0276] Experimental procedure:

[0277] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve with the mobile phase and dilute to the mark, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution;

[0278] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0279] The results are shown in Figure 15 , Figure 15 in which the 1st peak is carbobenzoxy-L-alanine and the 2nd peak is carbobenzoxy-D-alanine. It can be seen that under these conditions, the baseline fluctuates greatly, and the responses of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are relatively low.

[0280] Comparative Example 4

[0281] Chromatographic conditions:

[0282] High performance liquid chromatograph: Dionex: UltiMate3000;

[0283] Chromatographic column: CHIRALPAK IC (250mm×4.6mm, 5μm);

[0284] Mobile phase: aqueous phase (pH adjusted to 2.0 with phosphoric acid): acetonitrile = 95:5 (v / v);

[0285] Flow rate: 1.0 ml / min;

[0286] Detection wavelength: 205 nm;

[0287] Column temperature: 25 °C;

[0288] Sample injection volume: 10 μL;

[0289] Isocratic elution running time: 10 min.

[0290] Experimental procedure:

[0291] (1) Take appropriate amounts of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve with the mobile phase and dilute to the mark, shake well, and prepare a reference substance solution containing 0.5 mg of each of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine in 1 mL of solution.

[0292] (2) Take the reference substance solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0293] The results are shown in Figure 16 , Figure 16 In which, the peak No. 1 is carbobenzoxy-L-alanine and the peak No. 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are not completely separated.

[0294] Comparative Example 5

[0295] Chromatographic conditions:

[0296] High performance liquid chromatograph: Dionex: UltiMate3000;

[0297] Chromatographic column: CHIRALPAK AD-H (250 mm × 4.6 mm, 5 μm);

[0298] Mobile phase: n-hexane: isopropanol: trifluoroacetic acid = 70:30:0.1 (v / v / v);

[0299] Flow rate: 1.0 ml / min;

[0300] Detection wavelength: 205 nm;

[0301] Column temperature: 25 °C;

[0302] Sample injection volume: 10 μL;

[0303] Isocratic elution running time: 20 min.

[0304] Experimental procedure

[0305] (1) Take an appropriate amount of raw material CBZ-L-alanine, place it in a 20 ml volumetric flask, dissolve it with the mobile phase and dilute to the mark, shake well to prepare a test solution with a concentration of 1 mg / mL;

[0306] (2) Take the test solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0307] The results are shown in Figure 17 , Figure 17 Peak 1 is carbobenzoxy-L-alanine and peak 2 is carbobenzoxy-D-alanine. It can be seen that under these conditions, the peaks of carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are symmetric and well separated, but the baseline fluctuates greatly and the quantitative limit concentration of carbobenzoxy-D-alanine cannot be reached.

[0308] Comparative Example 6

[0309] Chromatographic conditions:

[0310] High performance liquid chromatograph: Dionex: UltiMate3000;

[0311] Chromatographic column: Elite NH2 (250 mm × 4.6 mm, 5 μm);

[0312] Mobile phase: 0.05 mol / L potassium dihydrogen phosphate aqueous solution (adjusted to pH 4.0 with phosphoric acid): acetonitrile = 35:65;

[0313] Flow rate: 0.7 ml / min;

[0314] Detection wavelength: 215 nm;

[0315] Column temperature: 30 °C;

[0316] Injection volume: 10 μL;

[0317] Isocratic elution running time: 15 min.

[0318] Experimental procedure

[0319] (1) Take appropriate amounts of CBZ-L-alanine and CBZ-D-alanine reference substances, place them in a 20 ml volumetric flask, dissolve them with the mobile phase and dilute to the mark, shake well to prepare a solution containing 0.5 mg each of CBZ-L-alanine and CBZ-D-alanine reference substances in 1 mL as the reference solution;

[0320] (2) Take the reference solution and perform high performance liquid chromatography analysis under the above conditions, and record the chromatogram.

[0321] The results are shown in Figure 18 , Figure 18Peak 1 is carbobenzoxy-L-alanine, and peak 2 is carbobenzoxy-D-alanine. It can be seen that under this condition, carbobenzoxy-L-alanine and carbobenzoxy-D-alanine are not completely separated.

[0322] In the description of this specification, the descriptions with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0323] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for analyzing and detecting carbobenzoxy-L-alanine by high performance liquid chromatography, characterized in that, the method uses a chiral chromatographic column, with an alcohol reagent as mobile phase A and water with a pH of 1.8 - 2.2 as mobile phase B; wherein, the chiral chromatographic column is a cellulose-based chiral chromatographic column.

2. The method according to claim 1, characterized in that, the volume ratio of mobile phase A to mobile phase B is (25 - 35):(65 - 75), preferably 30:

70.

3. The method according to claim 1, characterized in that, the alcohol reagent includes ethanol selected from.

4. The method according to claim 1, characterized in that, the method includes adjusting the pH value of mobile phase B with phosphoric acid.

5. The method according to claim 1, characterized in that, the pH value of mobile phase B is 2.

0.

6. The method according to claim 1, characterized in that, the chiral chromatographic column is packed with cellulose-3,5-dichlorophenylcarbamate bonded silica gel; optionally, the chiral chromatographic column includes CHIRALPAK IC selected from.

7. The method according to claim 1, characterized in that, includes the following steps: (1) Prepare a sample solution to be measured containing carbobenzoxy-L-alanine; (2) Set the mobile phase flow rate to 0.8 - 1.2 mL / min, the detection wavelength to 200 - 210 nm, the column temperature to 20°C - 30°C, and the injection volume to 5 - 20 μL to complete the separation and determination of carbobenzoxy-L-alanine.

8. The method according to claim 7, characterized in that, step (1) further includes taking the sample to be measured and dissolving it with the mobile phase to obtain the sample solution to be measured; optionally, the concentration of carbobenzoxy-L-alanine in the sample solution to be measured is 0.5 mg / mL.

9. The method according to claim 7, characterized in that, the mobile phase flow rate is 1.0 ml / min; optionally, the detection wavelength is 205 nm; optionally, the column temperature is 25°C; optionally, the injection volume is 10 μL.

10. Use of the method according to any one of claims 1 - 9 in the quality control of carbobenzoxy-L-alanine.

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