A method for detecting R-isomer in mitiglinide calcium or its preparation

Abstract

The invention provides a method for detecting the R-isomer of mitiglinide calcium in mitiglinide calcium or its preparation. The method comprises the following steps: preparing a test solution: taking mitiglinide calcium or its preparation to prepare a test solution; chromatographic conditions: using a chromatographic column with cellulose tris(4-xylylcarboxylate) coated on a silica gel surface as a filler, and using n-hexane-anhydrous ethanol-trifluoroacetic acid 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 an R-isomer of mitiglinide calcium in mitiglinide calcium or its preparation. Background Art

[0002] Mitiglinide calcium, chemical name: bis{(2S)-2-benzyl-4-[(3aR,7aS)octahydroisoindol-2-yl]-4-oxobutanoic acid} monocalcium salt dihydrate, has the following structural formula:

[0003]

[0004] Mitiglinide calcium was developed by Japan Kikyo Pharmaceutical Co., Ltd. and applied for approval in December 2002 for use in patients with type 2 diabetes to control postprandial blood sugar. It was launched in Japan in April 2004 and is currently sold by several domestic drug manufacturers. Mitiglinide calcium works by shutting down the ATP-dependent K + channels, causing Ca 2+ Influx of intracellular Ca 2+ Increased concentrations cause extracellular insulin-containing vesicles to degranulate, thereby stimulating insulin secretion. Mitiglinide calcium tablets are used to control postprandial blood sugar in patients with type 2 diabetes who have not achieved satisfactory results with diet therapy and exercise therapy.

[0005] The 18th edition of the Japanese Pharmacopoeia includes mitiglinide calcium and its preparations, which have extremely high requirements for impurities, requiring that individual impurities not exceed 0.10% and total impurities not exceed 0.15%. However, there is no control for the R-isomer impurity. As an impurity component in mitiglinide calcium and its preparations, the R-isomer must be strictly controlled within certain limits to ensure drug safety. Its structural formula is as follows:

[0006]

[0007] Patent CN 110568100 B discloses a method for detecting the R-isomer of mitiglinide calcium. Although the mitiglinide calcium peak can be separated from the R-isomer peak, the method suffers from a low number of theoretical plates and poor peak shape, making it difficult to ensure sensitive detection. Furthermore, the method has a long run time and low detection efficiency. Summary of the Invention

[0008] In view of the above problems, the present application provides a method for detecting the R-isomer of mitiglinide calcium or its preparations. The detection method has high separation between the mitiglinide calcium peak and the R-isomer peak, high theoretical plate number, can significantly improve the tailing of the R-isomer, and shortens the analysis run time.

[0009] This application is implemented through the following technical solutions:

[0010] A method for detecting the R-isomer of mitiglinide calcium in mitiglinide calcium or its preparation, comprising the following steps:

[0011] Preparation of test solution: Take mitiglinide calcium or its preparation to prepare test solution;

[0012] Chromatographic conditions: A column filled with cellulose-tris(4-xylylcarboxylate) coated on the silica surface was used, and n-hexane-anhydrous ethanol-trifluoroacetic acid was used as the mobile phase.

[0013] Determination method: Take the test solution and inject it into the liquid chromatograph for determination.

[0014] Preferably, the method for preparing the test solution is: take mitiglinide calcium or its preparation, add an organic solvent to dissolve it, dilute it with a mobile phase, and obtain a test solution with a concentration of 0.1 to 3 mg / ml; further preferably, the method for preparing the test solution is: take an appropriate amount of mitiglinide calcium or its preparation, accurately weigh it, place it in a volumetric flask, add an organic solvent to ultrasonically dissolve it, and dilute it with a mobile phase to make a 1 mg / ml test solution. Even more preferably, the method for preparing the test solution is: take about 25 mg of mitiglinide calcium or its preparation, accurately weigh it, place it in a 25 ml volumetric flask, add 1 ml of organic solvent to ultrasonically dissolve it, and dilute it to the scale with a mobile phase to obtain a test solution. Wherein, the organic solvent is methanol or anhydrous ethanol.

[0015] Preferably, the mobile phase is a n-hexane-anhydrous ethanol-trifluoroacetic acid solution with a ratio of 92-99: 1-8: 0.02-0.10; further preferably, the mobile phase is a n-hexane-anhydrous ethanol-trifluoroacetic acid solution with a ratio of 94-98: 2-6: 0.03-0.07; even more preferably, the mobile phase is a n-hexane-anhydrous ethanol-trifluoroacetic acid solution with a ratio of 95-97: 3-5: 0.04-0.06; most preferably, the mobile phase is a n-hexane-anhydrous ethanol-trifluoroacetic acid solution with a ratio of 96: 4: 0.05.

[0016] Preferably, the detection wavelength is 200-250 nm, the column temperature is 25-40 ° C, the flow rate is 0.2-2 ml / min, and the injection volume is 1-30 μl; further preferably, the detection wavelength is 200-220 nm, the column temperature is 30-37 ° C, the flow rate is 0.5-2 ml / min, and the injection volume is 10-20 μl; most preferably, the detection wavelength is 210 nm, the column temperature is 35 ° C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0017] The mitiglinide calcium preparations described herein include but are not limited to tablets and capsules.

[0018] Beneficial effects of this application:

[0019] Compared with the prior art, the present application has the following advantages: ① better separation of mitiglinide calcium and R isomer; ② significantly improved tailing of the R isomer; ③ significantly increased the theoretical plate number of the R isomer and improved detection sensitivity; ④ significantly shortened detection time.

[0020] The remarkable effect of this application is demonstrated in detail by the following comparative tests:

[0021] Comparative test 1

[0022] Method 1: The detection method is the method of Example 1 of the present application; the solution preparation method is: take an appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, add an appropriate amount of anhydrous ethanol and ultrasonically dissolve, and add the mobile phase "n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05)" to make a mixed solution containing 0.2 mg per 1 ml.

[0023] Method 2: The detection method is the method of Example 1 of patent CN110568100 B; the solution preparation method is: take appropriate amounts of mitiglinide calcium and R-isomer reference substance, accurately weigh them, and add the mobile phase "acetonitrile-trifluoroacetic acid (100:0.1)" to make a mixed solution containing 0.2 mg per 1 ml.

[0024] Method 3: The detection method is the method described in patent CN100441570 C "Chromatographic Conditions and System Suitability Test"; the solution preparation method is as follows: take appropriate amounts of mitiglinide calcium and R-isomer reference substance, accurately weigh them, and add the mobile phase "n-hexane-chloroform-methanol-trifluoroacetic acid (80:160:7.5:0.5)" to make a mixed solution containing 0.2 mg per 1 ml.

[0025] Test results are shown in Figure 1-3 and Table 1.

[0026] Table 1 Comparative test 1 test results

[0027] method Method 1 Method 2 Method 3 Separation 3.28 2.63 0.92 Mitiglinide calcium theoretical plate number 6061 2530 4528 Theoretical plate number of R isomer 10792 1154 3684 R isomer tailing factor 1.09 1.35 —— Runtime 15min 30min 30min Corresponding chromatogram Figure 1 Figure 2 Figure 3

[0028] The above test results show that: the method of patent CN 110568100 B (i.e., method 2) can achieve the separation of mitiglinide calcium and R isomer under the detection environment described in its specification, but the resolution and theoretical plate number are significantly inferior to the detection method of the present invention (i.e., method 1). In addition, the tailing of the R isomer is more serious than that of the present invention, and the detection time is significantly longer than that of the present invention; and the method of patent CN100441570 C (i.e., method 3) cannot achieve the separation of mitiglinide calcium and R isomer under the detection environment described in its specification.

[0029] Comparative Test 2

[0030] Method 1: The mobile phase used was acetonitrile-trifluoroacetic acid (100:0.1), and the rest was the same as in Example 1 of the present application.

[0031] Method 2: The mobile phase used was n-hexane-chloroform-methanol-trifluoroacetic acid (80:160:7.5:0.5), and the rest was the same as Example 2 of the present application.

[0032] Test results are shown in Figure 4 、 5 and Table 2.

[0033] Table 2 Comparative test 2 test results

[0034] method Method 1 Method 2 Separation 0.549 0.803 Mitiglinide calcium theoretical plate number 6222 1394 Theoretical plate number of R isomer 5766 226 R isomer tailing factor —— —— Runtime —— 15min Corresponding chromatogram Figure 4 Figure 5

[0035] The above test results show that under the detection environment and detection conditions of this application, the existing mobile phase acetonitrile-trifluoroacetic acid (100:0.1) and the mobile phase n-hexane-chloroform-methanol-trifluoroacetic acid (80:160:7.5:0.5) are simply unable to achieve the separation of mitiglinide calcium and R isomer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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:

[0037] Figure 1 The present invention detects the chromatogram of the same concentration experimental solution in the prior art

[0038] Figure 2 Chromatogram for patent CN110568100 B method

[0039] Figure 3 Chromatogram for patent CN100441570 C method

[0040] Figure 4 Reproduce the mobile phase detection chromatogram of patent CN110568100 B for the method of this invention

[0041] Figure 5 Reproduce the mobile phase detection chromatogram of patent CN100441570 C for the method of the present invention

[0042] Figure 6 Chromatogram of blank solution of the present invention

[0043] Figure 7 Chromatogram of the test solution of the present invention

[0044] Figure 8 The chromatogram of the solution of the system of the present invention is DETAILED DESCRIPTION

[0045] 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.

[0046] Example 1

[0047] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0048] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05)

[0049] Detection wavelength: 210nm

[0050] Column temperature: 35°C

[0051] Flow rate: 1.0ml / min

[0052] Preparation of blank solution: Take 1 ml of anhydrous ethanol and place it in a 25 ml volumetric flask, dilute to the mark with mobile phase, and shake well;

[0053] Preparation of test solution: Take about 25 mg of mitiglinide calcium, accurately weigh it, place it in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve it, dilute it to the scale with mobile phase, and shake well;

[0054] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0055] Determination method: Accurately measure 10 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. Figure 6-8 .

[0056] The test results showed that the separation between the mitiglinide calcium peak and the R isomer peak in the system suitability solution was greater than 2.0, the blank solution did not interfere with the detection, and the method had good specificity.

[0057] Example 2

[0058] Chromatographic conditions and system suitability experiments

[0059] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0060] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05)

[0061] Detection wavelength: 210nm

[0062] Column temperature: 35°C

[0063] Flow rate: 1.0ml / min

[0064] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of methanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0065] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0066] Example 3

[0067] Chromatographic conditions and system suitability experiments

[0068] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0069] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (92:8:0.05)

[0070] Detection wavelength: 210nm

[0071] Column temperature: 35°C

[0072] Flow rate: 1.0ml / min

[0073] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of methanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0074] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0075] Example 4

[0076] Chromatographic conditions and system suitability experiments

[0077] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0078] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (99:1:0.05)

[0079] Detection wavelength: 210nm

[0080] Column temperature: 35°C

[0081] Flow rate: 1.0ml / min

[0082] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0083] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0084] Example 5

[0085] Chromatographic conditions and system suitability experiments

[0086] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0087] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.02)

[0088] Detection wavelength: 210nm

[0089] Column temperature: 35°C

[0090] Flow rate: 1.0ml / min

[0091] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of methanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0092] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0093] Example 6

[0094] Chromatographic conditions and system suitability experiments

[0095] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0096] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.10)

[0097] Detection wavelength: 210nm

[0098] Column temperature: 30°C

[0099] Flow rate: 1.0ml / min

[0100] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0101] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0102] Example 7

[0103] Chromatographic conditions and system suitability experiments

[0104] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0105] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05)

[0106] Detection wavelength: 210nm

[0107] Column temperature: 40°C

[0108] Flow rate: 1.0ml / min

[0109] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of methanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0110] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0111] Example 8

[0112] Chromatographic conditions and system suitability experiments

[0113] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0114] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05)

[0115] Detection wavelength: 210nm

[0116] Column temperature: 25°C

[0117] Flow rate: 1.0ml / min

[0118] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0119] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0120] Example 9

[0121] Chromatographic conditions and system suitability experiments

[0122] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0123] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0124] Detection wavelength: 210nm;

[0125] Column temperature: 35°C;

[0126] Flow rate: 0.2 ml / min;

[0127] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0128] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0129] Example 10

[0130] Chromatographic conditions and system suitability experiments

[0131] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0132] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0133] Detection wavelength: 210nm;

[0134] Column temperature: 35°C;

[0135] Flow rate: 2.0 ml / min;

[0136] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0137] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0138] Example 11

[0139] Chromatographic conditions and system suitability experiments

[0140] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0141] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0142] Detection wavelength: 210nm;

[0143] Column temperature: 35°C;

[0144] Flow rate: 1.0 ml / min;

[0145] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0146] Determination method: Accurately measure 1 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0147] Example 12

[0148] Chromatographic conditions and system suitability experiments

[0149] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0150] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0151] Detection wavelength: 210nm;

[0152] Column temperature: 35°C;

[0153] Flow rate: 1.0 ml / min;

[0154] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0155] Determination method: Accurately measure 30 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0156] Example 13

[0157] Chromatographic conditions and system suitability experiments

[0158] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0159] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0160] Detection wavelength: 200nm;

[0161] Column temperature: 35°C;

[0162] Flow rate: 1.0 ml / min;

[0163] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0164] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0165] Example 14

[0166] Chromatographic conditions and system suitability experiments

[0167] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0168] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0169] Detection wavelength: 220nm;

[0170] Column temperature: 35°C;

[0171] Flow rate: 1.0 ml / min;

[0172] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0173] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0174] Example 15

[0175] Chromatographic conditions and system suitability experiments

[0176] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0177] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0178] Detection wavelength: 210nm;

[0179] Column temperature: 35°C;

[0180] Flow rate: 1.0 ml / min;

[0181] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium tablets and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0182] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0183] Example 16

[0184] Chromatographic conditions and system suitability experiments

[0185] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0186] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0187] Detection wavelength: 210nm;

[0188] Column temperature: 35°C;

[0189] Flow rate: 1.0 ml / min;

[0190] Preparation of system suitability solution: Take appropriate amount of mitiglinide calcium capsules and mitiglinide calcium R-isomer, accurately weigh, place in a 25 ml volumetric flask, add 1 ml of anhydrous ethanol and ultrasonically dissolve, dilute to the scale with mobile phase to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0191] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0192] The system suitability solution was tested according to the above example, and the separation degree, theoretical plate number and tailing factor of mitiglinide calcium and R isomer were read. The results are shown in Table 3:

[0193] Table 3 Test results of Examples 1-16

[0194]

[0195]

[0196] The test results show that the system suitability solution was tested according to the above embodiment, and the separation degree of mitiglinide calcium and R isomer was good, the theoretical plate number was high, and the tailing factor of R isomer was small. This result shows that Examples 1-16 of the present application can achieve the technical effect of the present application.

[0197] Comparative Example

[0198] Chromatographic conditions and system suitability experiments

[0199] Chromatographic column: Silica gel coated with cellulose-tris (4-xylene carboxylate) as filler;

[0200] Mobile phase: n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) was used as the mobile phase;

[0201] Detection wavelength: 210nm;

[0202] Column temperature: 35°C;

[0203] Flow rate: 1.0 ml / min;

[0204] Preparation of system suitability solution: Take appropriate amounts of mitiglinide calcium and mitiglinide calcium R-isomer, dissolve and configure the sample according to the solvents listed in Table 4 to prepare a mixed solution containing approximately 1 mg / ml of mitiglinide calcium and 15 μg / ml of mitiglinide calcium R-isomer.

[0205] Determination method: Accurately measure 10 μl of the above solution, inject it into liquid chromatograph, and record the chromatogram.

[0206] System suitability solutions were prepared according to the methods of the control example and Examples 1 and 2. The properties of the solutions were observed and tested. The results are shown in Table 4:

[0207] Table 4 Comparative Example 1-3, Example 1-2 Sampling Method and Solution Properties, Test Results

[0208]

[0209] The above results show that when the system suitability solutions were prepared using the methods of Comparative Examples 1 and 2 (i.e., no solubilization or direct solubilization with isopropanol and sample preparation as the mobile phase), the sample did not dissolve during ultrasound, making detection impossible. Furthermore, when acetonitrile was used as the solubilizer in Comparative Example 3, the baseline fluctuated significantly. However, the solutions prepared in this application using anhydrous ethanol or methanol as the solubilizer and a mobile phase of n-hexane-anhydrous ethanol-trifluoroacetic acid (96:4:0.05) to volume were clear, with minimal baseline fluctuation and no blank interference.

Claims

1. A method for detecting the R-isomer of mitiglinide calcium in mitiglinide calcium or its preparation, characterized in that: The method comprises the following steps: Preparation of test solution: Take mitiglinide calcium or its preparation, dissolve it in an organic solvent, and dilute it 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 chromatographic column filled with cellulose-tris(4-xylylcarboxylate) coated on the silica surface was used, and a 92-99:1-8:0.02-0.10 n-hexane-anhydrous ethanol-trifluoroacetic acid solution was used as the mobile phase; The detection wavelength is 200-220 nm, the column temperature is 25-40 ° C, the flow rate is 0.2-2 ml / min, and the injection volume is 1-30 μl; Determination method: Take the test sample 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 mitiglinide calcium or its preparation, accurately weigh it, put it into a volumetric flask, add an organic solvent and ultrasonically dissolve it, and dilute it with a mobile phase to prepare a 0.1-3 mg / ml test solution.

3. The detection method according to claim 1, wherein The mobile phase is a 94-98:2-6:0.03-0.07 n-hexane-anhydrous ethanol-trifluoroacetic acid solution.

4. The detection method according to claim 3, characterized in that The mobile phase is a 95-97:3-5:0.04-0.06 solution of n-hexane-anhydrous ethanol-trifluoroacetic acid.

5. The detection method according to claim 4, characterized in that The mobile phase is a 96:4:0.05 n-hexane-anhydrous ethanol-trifluoroacetic acid solution.

6. The detection method according to any one of claims 1 to 5, wherein The mitiglinide calcium preparation is in the form of tablets or capsules.

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