A method for detecting organic acids in a bulk drug substance

Abstract

The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting organic acid substances in bulk drugs. In the present invention, a tandem reversed-phase chromatographic column and an ion exclusion chromatographic column are used, and an aqueous solution of sulfuric acid, phosphoric acid or p-toluenesulfonic acid is used as the mobile phase, and high performance liquid chromatography equipped with an ultraviolet detector or a diode array detector is used for analysis and detection. The method of the present invention can remove the interference of the matrix on trace organic acids, can quickly and accurately detect the content of residual organic acids in bulk drugs, has a good linear relationship, high precision, high accuracy, good repeatability and durability, high resolution, strong applicability, is not interfered by the raw materials, and fills the blank of the method for determining the content of residual organic acids in bulk drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting organic acid substances in bulk drugs. Background Art

[0002] In the field of pharmaceutical technology, most bulk drugs are obtained through chemical synthesis. In chemical synthesis, most organic acid solvents or organic acid by-products will be used. In order to ensure the quality of bulk drugs, it is necessary to detect the residual amounts of these organic acids in bulk drugs.

[0003] Currently, the main methods for detecting organic acids are gas chromatography, liquid chromatography, and ion chromatography. In gas chromatography, due to the strong activity of organic acid substances, they are prone to adsorption, resulting in low sensitivity and poor precision of the results. When using ion chromatography to detect organic acids, it is easily interfered by the matrix and cannot accurately quantify; moreover, the penetration rate of ion chromatographs is relatively low, further reducing the application of such methods. Liquid chromatography is further divided into reverse-phase chromatography and ion-exclusion chromatography. In reverse-phase chromatography, most organic acid substances have weak retention and elute within 5 minutes, being easily interfered by the matrix and the mobile phase, and the method has poor durability. Although the traditional ion-exclusion chromatography uses an 8% cross-linked resin hydrogen ion chromatographic column for detection, the bulk drug will also enter the chromatographic column and elute. The peak shape of the high-content bulk drug is very wide, which may cover up the organic acids with lower contents, and still cannot avoid the interference of the matrix on the organic acids; in addition, the direct entry of the bulk drug into the ion chromatographic column will seriously reduce the service life of the chromatographic column, cause the peak shape of the organic acid to deteriorate, and reduce the sensitivity. Whether it is ion chromatography or ion-exclusion chromatography, for the interference of the matrix, the common practice at present is to pretreat the test sample with a solid-phase extraction column, so that the raw material is adsorbed in the filler to avoid subsequent entry into the ion chromatographic column or ion-exclusion chromatographic column and thus interfere with the detection of organic acids. However, the process of pretreatment by solid-phase extraction is relatively complex, the method exploration needs to be further developed, the operation is relatively complex, the technical requirements are high, and problems such as low recovery rate and poor reproducibility are likely to occur.

[0004] Therefore, it is very necessary to develop a detection method for organic acids with simple operation, high sensitivity, accuracy, and good durability. Summary of the Invention

[0005] The purpose of the present invention is to provide a high-performance liquid chromatography method for detecting the residual amount of organic acids in bulk drugs. By combining a reverse-phase chromatographic column and an ion-exclusion chromatographic column, the bulk drug and the organic acid can be effectively separated to quickly detect the residual amount of organic acids in the bulk drug, making the quality of the bulk drug stable and controllable.

[0006] The technical solution of the present invention is as follows:

[0007] A method for detecting organic acids in an active pharmaceutical ingredient, which uses a reversed-phase chromatographic column in series with an ion-exclusion chromatographic column, with an aqueous solution of sulfuric acid, phosphoric acid, or p-toluenesulfonic acid as the mobile phase, and analyzes and detects using a high-performance liquid chromatograph equipped with an ultraviolet detector or a diode array detector.

[0008] Preferably, the reversed-phase chromatographic column is a C8 column, a C18 column, a phenyl column, an amino column, a pentafluorophenyl column, a cyano column, or a bare silica gel column, and more preferably a C18 column. The present invention does not particularly limit the type of the reversed-phase chromatographic column. For example, in some embodiments, the reversed-phase chromatographic column is phenomenex onyx monolitnic HD-C18, in some other embodiments, the reversed-phase chromatographic column is YMCTriart C18, and in some other embodiments, the reversed-phase chromatographic column is Welch XB-C18.

[0009] Preferably, the ion-exclusion chromatographic column is a cross-linked resin hydrogen ion chromatographic column, and more preferably a cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatographic column. Further, the ion-exclusion chromatographic column is a cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatographic column with a cross-linking degree of 5%, 8%, or 10%. The present invention does not particularly limit the type of the ion-exclusion chromatographic column. For example, in some embodiments, the ion-exclusion chromatographic column is Rezex ROA-Organic Acid H+, in some other embodiments, the ion-exclusion chromatographic column is Aminex HPX-87H, and in some other embodiments, the ion-exclusion chromatographic column is Shim-pack SCR-102H.

[0010] Preferably, the mobile phase is an aqueous solution of sulfuric acid, phosphoric acid, or p-toluenesulfonic acid with a concentration of 0.1 - 10 mmol / L; preferably an aqueous solution of sulfuric acid with a concentration of 5 - 10 mmol / L; further preferably an aqueous solution of sulfuric acid with a concentration of 8 - 10 mmol / L. In some embodiments, the mobile phase is an aqueous solution of sulfuric acid with a concentration of 8 mmol / L; in some other embodiments, the mobile phase is an aqueous solution of sulfuric acid with a concentration of 9 mmol / L; in some other embodiments, the mobile phase is an aqueous solution of sulfuric acid with a concentration of 10 mmol / L.

[0011] Preferably, the flow rate of the mobile phase is 0.4 - 0.8 ml / min, and more preferably 0.5 - 0.6 ml / min.

[0012] Preferably, the column temperature is 30 - 60 °C, and preferably 40 - 45 °C.

[0013] Preferably, the detection wavelength is 200 - 210 nm.

[0014] Preferably, the injection volume is 10 - 30 μl.

[0015] Preferably, the organic acids include, but are not limited to, formic acid, acetic acid, butyric acid, malonic acid, citric acid, succinic acid, tartaric acid, malic acid, fumaric acid, butanedioic acid, ascorbic acid, oxalic acid, lactic acid, benzoic acid and other organic acids.

[0016] The present invention does not limit the category of the active pharmaceutical ingredient. As long as the synthesis process of the active pharmaceutical ingredient uses an organic acid and the organic acid may be present in the active pharmaceutical ingredient, the technical solution of the present invention can be used for detection.

[0017] The following content takes formic acid, acetic acid, malonic acid, citric acid, tartaric acid, fumaric acid as examples to illustrate the detection method described in the present invention, a method for determining organic acids in an active pharmaceutical ingredient, which can be achieved through the following steps:

[0018] (1) Preparation of solutions

[0019] ① Stock solution of reference substances: Weigh appropriate amounts of formic acid, acetic acid, malonic acid, citric acid, tartaric acid accurately, and dilute them with the diluent to about 500 μg / ml respectively. Weigh an appropriate amount of fumaric acid accurately and dilute it with the diluent to a solution of about 100 μg / ml.

[0020] ② Test solution: Weigh an appropriate amount of the active pharmaceutical ingredient accurately, dissolve and dilute it with the diluent to a solution containing 10 mg / ml of the active pharmaceutical ingredient.

[0021] ③ Mixed reference substance solution: Weigh appropriate amounts of formic acid, acetic acid, malonic acid, citric acid, tartaric acid accurately, and dilute them with the diluent to a solution of about 50 μg / ml; weigh an appropriate amount of fumaric acid accurately and dilute it with the diluent to a solution of about 10 μg / ml;

[0022] ④ Spiked test solution: Weigh the test sample and the reference substance accurately, dissolve and dilute them with the reference substance solution.

[0023] (2) Detection by chromatographic system

[0024] Inject the above-mentioned reference substance solution, test solution, mixed reference substance solution, and spiked test solution into the chromatograph for detection respectively, and calculate the acid residue in the test sample by the external standard method based on the peak area. The detection conditions are as follows:

[0025] Chromatographic column: Reverse-phase chromatographic column in series with an ion exclusion chromatographic column;

[0026] Column temperature: 40 - 45 °C;

[0027] Mobile phase: 0.1 - 10 mmol / L sulfuric acid aqueous solution;

[0028] Flow rate of mobile phase: 0.4 - 0.8 ml / min

[0029] Detection wavelength: 210 nm;

[0030] Sample injection volume: 10 - 20 μl.

[0031] Preferably, the diluent in the solution preparation is the mobile phase or purified water.

[0032] Preferably, the mobile phase is a 5 - 10 mmol / L sulfuric acid aqueous solution.

[0033] Preferably, the flow rate of the mobile phase is 0.5 - 0.6 ml / min.

[0034] Preferably, the sample injection volume is 20 μl.

[0035] The detection method of the present invention is applicable to the detection of organic acids used in the synthesis or preparation method of bulk drugs and may cause the residue of organic acids in bulk drugs, such as the detection of organic acids in bulk drugs such as zolpidem tartrate, amlodipine besylate, afatinib maleate, isosorbide mononitrate, ketorolac tromethamine, riluzole, gemcitabine, etc.

[0036] By connecting a reversed-phase chromatographic column and an ion exclusion chromatographic column in series, the present invention can remove the interference of the matrix on trace organic acids, can quickly and accurately detect the content of residual organic acids in bulk drugs, has a good linear relationship, high precision, high accuracy, good repeatability and durability, high resolution, strong applicability, is not interfered by raw materials, and fills the blank of the determination method for the content of residual organic acids in bulk drugs. Description of the Drawings

[0037] Figure 1 : Chromatogram of tartaric acid reference substance in Example 1;

[0038] Figure 2 : Chromatogram of citric acid reference substance in Example 1;

[0039] Figure 3 : Chromatogram of malonic acid reference substance in Example 1;

[0040] Figure 4 : Chromatogram of formic acid reference substance in Example 1;

[0041] Figure 5 : Chromatogram of acetic acid reference substance in Example 1;

[0042] Figure 6 : Chromatogram of fumaric acid reference substance in Example 1;

[0043] Figure 7 : Chromatogram of mixed reference substances in Example 1;

[0044] Figure 8 : Chromatogram of test solution in Example 1;

[0045] Figure 9 : Chromatogram of test solution with standard addition in Example 1;

[0046] Figure 10 : Locating chromatogram of the mixed reference substances in Example 2;

[0047] Figure 11 : Chromatogram of the test solution in Example 2;

[0048] Figure 12 : Chromatogram of the spiked test solution in Example 3;

[0049] Figure 13 : Chromatogram of the spiked test solution in Example 4;

[0050] Figure 14 : Locating chromatogram of the mixed reference substances in Example 5;

[0051] Figure 15 : Chromatogram of the test solution in Example 5;

[0052] Figure 16 : Locating chromatogram of the mixed reference substances in Example 6;

[0053] Figure 17 : Chromatogram of the test solution in Example 6;

[0054] Figure 18 : Locating chromatogram of the mixed reference substances in Example 7;

[0055] Figure 19 : Chromatogram of the test solution in Example 7;

[0056] Figure 20 : Chromatogram of the sensitivity solution. Detailed implementation manners

[0057] The present invention will be further explained below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the technical solution of the present invention and do not limit the protection scope of the present invention. The active pharmaceutical ingredient can be synthesized and prepared by the methods of the prior art or obtained through commercial channels.

[0058] Example 1: Determination of formic acid residue in zolpidem tartrate active pharmaceutical ingredient

[0059] (1) Solution preparation

[0060] Preparation of the reference stock solution: Accurately weigh 13.05 mg of formic acid, 14.61 mg of acetic acid, 12.71 mg of malonic acid, 13.08 mg of tartaric acid, and 12.44 mg of citric acid into a 25-ml volumetric flask respectively. Accurately weigh 5.033 mg of fumaric acid into a 50-ml volumetric flask, and dissolve and make up the volume with the mobile phase as the diluent.

[0061] Mixed reference substance stock solution: Accurately weigh 13.01 mg of formic acid, 14.66 mg of acetic acid, 12.73 mg of malonic acid, 13.05 mg of tartaric acid, and 12.47 mg of citric acid into a 25-ml volumetric flask, dissolve and make up the volume with the above-mentioned fumaric acid reference substance solution to obtain a 10-fold reference substance stock solution.

[0062] Reference substance positioning solution: Take 1 ml of each of the above reference substance stock solutions into a 10-ml volumetric flask, dilute and make up the volume with the mobile phase.

[0063] Mixed reference substance solution: Take 5 ml of the above mixed reference substance stock solution, dilute and make up the volume to 50 ml with the diluent.

[0064] Test solution: Accurately weigh 100.70 mg of zolpidem tartrate into a 10-ml volumetric flask, dilute and make up the volume with the mobile phase.

[0065] Test solution spiked with reference substance: Accurately weigh 100.40 mg of zolpidem tartrate into a 10-ml volumetric flask, dilute and make up the volume with the reference substance stock solution.

[0066] (2) Chromatographic system detection

[0067] Inject the above reference substance positioning solution, mixed reference substance solution, test solution, and test solution spiked with reference substance into the chromatographic system respectively, record the chromatogram, identify by the retention time of the organic acids in the reference substance positioning solution, and calculate the residual content of formic acid in the zolpidem tartrate test sample by the external standard method based on the peak area.

[0068] Instrument: Waters e2695 liquid chromatographic system;

[0069] Chromatographic column: phenomenex onyx monolitnic HD-C18, 100×4.6 mm in series with Rezex ROA-Organic Acid H+(8%), 300×7.8 mm;

[0070] Mobile phase: 9 mmol / L sulfuric acid aqueous solution;

[0071] Flow rate: 0.6 ml / min;

[0072] Detection wavelength: 210 nm;

[0073] Column temperature: 40 °C;

[0074] Injection volume: 20 μl

[0075] The reference substance positioning chromatogram is as shown in Figure 1-6 , the mixed reference substance chromatogram is as shown in Figure 7 , the test sample chromatogram is as shown in Figure 8 , the test sample spiked with reference substance chromatogram is as shown in Figure 9 . FromFigure 1-6 It can be seen that the retention time of tartaric acid is 12.130 min, the retention time of citric acid is 13.079 min, the retention time of malonic acid is 14.251 min, the retention time of formic acid is 17.385 min, the retention time of acetic acid is 19.289 min, and the retention time of fumaric acid is 20.975 min; from Figure 7 It can be seen that tartaric acid, citric acid, malonic acid, formic acid, acetic acid, and fumaric acid elute in sequence, and the chromatographic peaks of each organic acid can be effectively separated with good resolution; from Figure 8 It can be seen that the zolpidem tartrate raw material drug does not contain formic acid residue.

[0076] Determination of formic acid residue in zolpidem tartrate raw material drug in Example 2

[0077] Inject the mixed reference solution and the test solution prepared in Example 1 into the following chromatographic system, record the chromatogram, identify the organic acids by the retention time of the reference positioning solution, and calculate the formic acid residue content in the zolpidem tartrate test sample by the external standard method based on the peak area.

[0078] Chromatographic system detection:

[0079] Instrument: Waters e2695 liquid chromatography system;

[0080] Chromatographic column: Phenomenex onyx monolitnic HD-C18, 100×4.6 mm in series with Rezex ROA-Organic Acid H+(8%), 300×7.8 mm;

[0081] Mobile phase: 10 mmol / L sulfuric acid aqueous solution;

[0082] Flow rate: 0.5 ml / min;

[0083] Detection wavelength: 210 nm;

[0084] Column temperature: 50 °C;

[0085] Injection volume: 20 μl

[0086] The reference positioning chromatogram is as Figure 10 , and the test sample chromatogram is as Figure 11 , from Figure 10 It can be seen that the chromatographic peaks of each organic acid can be effectively separated with good resolution; from Figure 11 It can be seen that the zolpidem tartrate raw material drug does not contain formic acid residue.

[0087] Determination of formic acid residue in zolpidem tartrate raw material drug in Example 3

[0088] Configure the test sample spiked solution according to the method of Example 1, inject the test sample spiked solution into the chromatographic system respectively, and record the chromatogram.

[0089] Chromatographic system:

[0090] Instrument: Ultimate 3000 liquid chromatographic system;

[0091] Chromatographic column: phenomenex onyx monolitnic HD-C18, 100×4.6 mm;

[0092] Mobile phase: 9 mmol / L sulfuric acid aqueous solution;

[0093] Flow rate: 0.6 ml / min;

[0094] Detection wavelength: 210 nm;

[0095] Column temperature: 40 °C;

[0096] Injection volume: 20 μl

[0097] The chromatogram of the test sample spiked solution is shown in Figure 12 as follows. It can be seen from Figure 12 that the organic acid has weak retention and an early elution time, causing matrix interference and unable to achieve effective separation.

[0098] Determination of formic acid residue in zolpidem tartrate raw material drug in Example 4

[0099] Inject the test sample spiked solution configured by the method of Example 1 into the chromatographic system respectively, and record the chromatogram.

[0100] Chromatographic system:

[0101] Instrument: Waters e2695 liquid chromatographic system;

[0102] Chromatographic column: Rezex ROA-Organic Acid H+(8%), 300×7.8 mm;

[0103] Mobile phase: 9 mmol / L sulfuric acid aqueous solution;

[0104] Flow rate: 0.6 ml / min;

[0105] Detection wavelength: 210 nm;

[0106] Column temperature: 40 °C;

[0107] Injection volume: 20 μl

[0108] The chromatogram of the test sample spiked solution is shown in Figure 13 as follows. It can be seen from Figure 13It can be seen that a high concentration of the test sample results in a high response, which can easily interfere with the detection of organic acids.

[0109] Determination of acetic acid residue in isosorbide mononitrate raw material drug in Example 5

[0110] Prepare the mixed solution of organic acid reference substances according to the method in Example 1.

[0111] Test sample solution: Accurately weigh 100.27 mg of isosorbide mononitrate and dilute it to 10 ml with the mobile phase.

[0112] Inject the above mixed solution of reference substances and the test sample solution into the chromatographic system respectively, record the chromatogram, and calculate the acetic acid residue content in the isosorbide mononitrate test sample by the external standard method based on the peak area.

[0113] Instrument: Ultimate 3000 liquid chromatographic system;

[0114] Chromatographic column: YMC Triart C18, 4.6x150 mm, 5 μm in series with Rezex ROA - Organic Acid H+ (8%), 300×7.8 mm;

[0115] Mobile phase: 1 mmol / L sulfuric acid aqueous solution;

[0116] Flow rate: 0.4 ml / min;

[0117] Detection wavelength: 210 nm;

[0118] Column temperature: 40 °C;

[0119] Injection volume: 20 μL

[0120] The chromatogram of the mixed reference substances is shown in Figure 14, and the chromatogram of the test sample is as Figure 15 , from Figure 14 it can be seen that the chromatographic peaks of each organic acid can be effectively separated with good resolution; from Figure 15 it can be seen that there is no acetic acid residue in the isosorbide mononitrate raw material drug.

[0121] Determination of acetic acid residue in gemcitabine raw material drug in Example 6

[0122] Prepare the mixed solution of reference substances according to the method in Example 1.

[0123] Test sample solution: Accurately weigh 100.13 mg of gemcitabine in a 10 ml volumetric flask, dilute it with the mobile phase and make up to the mark.

[0124] Inject the above reference substance positioning solutions, test sample solutions, and test sample spiked solutions into the chromatographic system respectively, record the chromatograms, identify by the retention times of the organic acids in the reference substance mixed solution, and calculate the acetic acid residue content in the test sample of gemcitabine by the external standard method based on the peak areas.

[0125] Instrument: Ultimate 3000 liquid chromatographic system;

[0126] Chromatographic column: Welch XB-C18, 150x4.6mm, 5μm in series with Aminex HPX-87H(8%), 300×7.8mm;

[0127] Mobile phase: 5mmol / L phosphoric acid aqueous solution;

[0128] Flow rate: 0.8ml / min;

[0129] Detection wavelength: 210nm;

[0130] Column temperature: 40°C;

[0131] Injection volume: 20μl

[0132] The reference substance mixed solution chromatogram is as shown in Figure 16, and the test sample chromatogram is as Figure 17 , from Figure 16 it can be seen that the chromatographic peaks of each organic acid can be effectively separated with good resolution; from Figure 17 it can be seen that there is no acetic acid residue in the gemcitabine raw material drug.

[0133] Determination of acetic acid residue in riluzole raw material drug in Example 7

[0134] Prepare the reference substance mixed solution according to the method of Example 1.

[0135] Test sample solution: Weigh accurately 100.13mg of riluzole into a 10ml volumetric flask, dilute with the mobile phase and make up to the mark.

[0136] Inject the above reference substance positioning solutions, test sample solutions, and test sample spiked solutions into the chromatographic system respectively, record the chromatograms, identify by the retention times of the organic acids in the reference substance mixed solution, and calculate the acetic acid residue content in the test sample of riluzole by the external standard method based on the peak areas.

[0137] Instrument: Waters e2695 liquid chromatographic system;

[0138] Chromatographic column: phenomenex onyx monolitnic HD-C18, 100×4.6mm in series with Rezex ROA-Organic Acid H+(8%), 300×7.8mm;

[0139] Mobile phase: 8 mmol / L sulfuric acid aqueous solution;

[0140] Flow rate: 0.5 ml / min;

[0141] Detection wavelength: 210 nm;

[0142] Column temperature: 40 °C;

[0143] Sample injection volume: 20 μl

[0144] The chromatogram of the mixed reference substance is shown in Figure 18, and the chromatogram of the test sample is as Figure 19 , from Figure 18 it can be seen that the chromatographic peaks of each organic acid can be effectively separated with good resolution; from Figure 19 it can be seen that the riluzole bulk drug does not contain acetic acid residue.

[0145] Example 8 Verification Example

[0146] (1) System precision test

[0147] The preparation method of the spiked solution of zolpidem tartrate test sample is the same as that in Example 1. The spiked solution of the test sample is used as the system suitability solution. Accurately measure 20 μl of the system suitability solution and inject it into the liquid chromatograph. Inject samples continuously for 6 times and perform determination according to the chromatographic conditions in Example 1. Record the chromatogram and calculate the RSD of the peak areas of the organic acids in each chromatogram respectively. The results of the retention time and peak area of formic acid are shown in Table 1.

[0148] Table 1

[0149]

[0150]

[0151] It can be seen from Table 1 that the RSD values of the peak areas of each organic acid are all less than 1%, indicating that the system precision is good

[0152] (2) Linear test of organic acid substances

[0153] The preparation method of the mixed reference stock solution is the same as that in Example 1. Accurately measure 500 μl, 800 μl, 1 ml, 1.2 ml, 1.5 ml, and 2 ml of the mixed reference stock solution respectively, place them in 10-ml volumetric flasks, dilute to the mark with the mobile phase, and prepare linear solutions of fumaric acid at 5 μg / L, 8 μg / L, 10 μg / L, 12 μg / L, 15 μg / L, and 20 μg / L; and five organic acids, namely formic acid, acetic acid, malonic acid, tartaric acid, and citric acid, at 25 μg / L, 40 μg / L, 50 μg / L, 60 μg / L, 75 μg / L, and 100 μg / L. Accurately measure 20 μl of each linear solution, inject them into the liquid chromatograph respectively, and perform detection according to the chromatographic conditions in Example 1. Record the chromatograms, perform linear regression with the concentration in mg / ml as the abscissa and the peak area (mAu*min) as the ordinate, and the linear regression equations of each organic acid are shown in Table 2.

[0154] Table 2 Results of the linear test for organic acids

[0155]

[0156]

[0157] It can be seen from the results in Table 2 that in the concentration range of 0.005 mg / ml to 0.02 mg / ml for fumaric acid and 0.025 mg / ml to 0.1 mg / ml for other various organic acids, the linear relationship between the peak area of each component and the concentration is good.

[0158] (3) Sensitivity detection of organic acid substances

[0159] The preparation method of the organic acid reference stock solution is the same as that in Example 1. Accurately measure 500 μl of the fumaric acid stock solution into a 10-ml volumetric flask, dilute and make up the volume with the mobile phase to obtain the fumaric acid mother liquor. Respectively take 100 μl of the tartaric acid stock solution, 200 μl each of the citric acid stock solution and the malonic acid stock solution, 250 μl of the formic acid stock solution, 500 μl of the acetic acid stock solution and 200 μl of the fumaric acid mother liquor into a 10-ml volumetric flask, dilute and make up the volume with the mobile phase to obtain the sensitivity solution. Accurately measure 20 μl of the sensitivity solution, inject it into the liquid chromatograph, and perform detection according to the chromatographic conditions in Example 1. Record the chromatogram. The chromatogram of the sensitivity solution is as Figure 20 shown. At this time, the S / N of each type of organic acid is about 15, indicating that the detection method of the present invention has high sensitivity.

[0160] (4) Accuracy detection of the content of organic acid substances

[0161] The preparation methods of the reference stock solution, the test solution, and the mixed reference solution are the same as those in Example 1.

[0162] 50% accuracy test solution: Weigh 100.0 mg of zolpidem tartrate precisely into a 10-ml volumetric flask, add 0.5 ml of the reference stock solution, dilute to the mark with the mobile phase, and shake well to obtain the solution. Prepare 3 portions in the same manner.

[0163] 100% accuracy test solution: Weigh 100.1 mg of zolpidem tartrate precisely into a 10-ml volumetric flask, add 1 ml of the reference stock solution, dilute to the mark with the mobile phase, and shake well to obtain the solution. Prepare 3 portions in the same manner.

[0164] 150% accuracy test solution: Weigh 100.0 mg of zolpidem tartrate precisely into a 10-ml volumetric flask, add 1.5 ml of the reference stock solution, dilute to the mark with the mobile phase, and shake well to obtain the solution. Prepare 3 portions in the same manner.

[0165] Precisely measure 20 μl each of the test solution, the mixed reference solution, the 50% accuracy test solution, the 100% accuracy test solution, and the 150% accuracy test solution, inject them into the liquid chromatograph, and carry out the determination according to the chromatographic conditions of Example 1. Record the chromatogram, and calculate the recovery rate, average recovery rate (n = 9), and RSD of each organic acid in the three concentration test solutions by the external standard method based on the peak area. The results are shown in Table 3.

[0166] Table 3 Results of the accuracy test for the determination of the content of organic acids

[0167]

[0168]

[0169]

[0170] As can be seen from Table 3, the recovery rates of the organic acids are all greater than 99%, and the RSD is less than 3%, indicating that the detection method of the present invention has good accuracy.

Claims

1. A method for detecting organic acids in an active pharmaceutical ingredient, characterized in that, a reversed-phase chromatographic column is connected in series with an ion-exclusion chromatographic column, an aqueous sulfuric acid solution with a concentration of 5-10 mmol / L is used as the mobile phase, and analysis and detection are carried out with a high-performance liquid chromatograph equipped with an ultraviolet detector or a diode array detector; the reversed-phase chromatographic column is Phenomenex Onyx Monolitnic HD-C18, YMC Triart C18 or Welch XB-C18; the ion-exclusion chromatographic column is Rezex ROA-Organic Acid H+, Aminex HPX-87H or Shim-pack SCR-102H; the organic acids are formic acid, acetic acid, malonic acid, citric acid, tartaric acid, fumaric acid; the flow rate of the mobile phase is 0.4-0.8 ml / min; the temperature of the chromatographic column is 30-60 °C; the detection wavelength is 200-210 nm; the injection volume is 10-30 μl.

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