A method for determining the content of proline in captopril bulk drug

Abstract

The invention relates to a method for determining the content of proline in a captopril bulk drug. The method comprises the following steps: step 1, preparation of a test solution: taking an appropriate amount of the captopril bulk drug, accurately weighing it, adding an appropriate amount of water, dissolving it by ultrasonication, adding water for quantitative dilution to prepare a solution containing about 12 mg per 1 ml, which is used as the test solution; step 2, preparation of a reference solution: taking an appropriate amount of a proline reference substance, accurately weighing it, dissolving it by water, and quantitative dilution to prepare a solution containing about 12 μg per 1 ml, which is used as the reference solution; step 3, determination method: injecting 20 μl of each of the reference solution and the test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the proline content by peak area according to the chromatogram using an external standard method.

Description

Technical field:

[0001] The invention relates to an analysis and detection method for impurity components in chemical drugs, in particular to a method for determining the content of proline, an impurity component in an antihypertensive drug, captopril, a raw material drug. Background technology:

[0002] Captopril was first developed and marketed by Bristol-Myers Squibb in 1981. It is suitable for treating various types of hypertension, but not for renal hypertension. Its chemical name is 1-[(2S)-2-methyl-3-mercaptooxypropyl]-L-proline, and its structural formula is as follows:

[0003]

[0004] The preparation method of Captopril is completed by the following steps:

[0005]

[0006] Captopril API contains proline as an impurity. Its source can be analyzed as follows:

[0007]

[0008] Among them, L-proline is the starting material in the preparation of Captopril API, and there is a possibility of residue. In addition, the formed Captopril may also be hydrolyzed at the amide position to produce proline. In order to ensure the quality of Captopril API products, it is necessary to detect the impurity component proline contained in Captopril API.

[0009] Since proline has very weak UV absorption and high polarity, it cannot be directly quantitatively determined, so it is generally determined indirectly by derivatization. During the derivatization reaction, the thiol group in the captopril molecule has a stronger nucleophilicity than the secondary amine group in proline, which greatly interferes with the proline derivatization reaction. Therefore, the content of proline in the captopril raw material cannot be determined by the derivatization method. Summary of the invention:

[0010] The present invention provides a method for detecting proline, an impurity component contained in a captopril bulk drug, the method comprising the following steps:

[0011] Step 1, preparation of test solution:

[0012] Take an appropriate amount of Captopril API, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and add water to quantitatively dilute it to make a solution containing 10-14 mg per 1 ml as the test solution;

[0013] Step 2, preparation of reference solution:

[0014] Take an appropriate amount of proline reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 10-14μg per 1ml as the reference substance solution;

[0015] Step 3, assay:

[0016] 10-30 μl of the reference solution and the test solution were injected into the HPLC, and the chromatogram was recorded. According to the chromatogram, the proline content was calculated by the peak area according to the external standard method;

[0017] The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as a filler (Welch Ultimate XB-C18, 4.6 mm×150 mm, 5 μm); the column temperature is 25-35° C.; the flow rate is 0.8-1.2 ml per minute; the detector is a charged atomization detector (CAD); the atomization temperature is 30-40° C.; 0.04-0.06% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in the following gradient elution mode;

[0018]

[0019] Preferably, the method of the present invention is as follows:

[0020] Step 1, preparation of test solution:

[0021] Take an appropriate amount of Captopril API, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and quantitatively dilute it with water to make a solution containing about 12 mg per 1 ml as the test solution;

[0022] Step 2, preparation of reference solution:

[0023] Take an appropriate amount of proline reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 12 μg per 1 ml as the reference substance solution;

[0024] Step 3, assay:

[0025] 20 μl of each reference solution and test solution were injected into the HPLC, and the chromatogram was recorded. According to the chromatogram, the proline content was calculated by the peak area using the external standard method;

[0026] The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as a filler (Welch Ultimate XB-C18, 4.6 mm×150 mm, 5 μm); the column temperature is 30° C.; the flow rate is 1.0 ml per minute; the detector is a charged atomization detector (CAD); the atomization temperature is 35° C.; 0.05% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in the following gradient elution mode;

[0027]

[0028] Wherein, the proline includes L-proline and D-proline, and its content is the sum of L-proline and D-proline.

[0029] The detection method of the present invention is obtained through screening, and the screening process is as follows:

[0030] HPLC-CAD (high performance liquid chromatograph equipped with CAD detector, a new type of universal detector, its detection does not depend on the molecular structure of the compound, and has been widely used in the analysis of UV non-absorption or weak absorption compounds) was used to directly determine the proline in the captopril raw material. A conventional C18 chromatographic column was used; since proline was weakly retained on the conventional C18 chromatographic column, an aqueous phase containing an inorganic acid (trifluoroacetic acid and formic acid) was used from 0 to 5 minutes to increase the retention of proline on the chromatographic column; other chromatographic conditions had little effect on the retention of proline, so they remained unchanged.

[0031] The results of optimizing the aqueous mobile phase are as follows:

[0032] Table 1 Screening of aqueous mobile phase

[0033]

[0034]

[0035] The CAD detector is easily contaminated by high concentrations of inorganic acids, so low concentration formic acid, i.e., 0.05% formic acid aqueous solution, is used while satisfying the separation degree.

[0036] Therefore, the analytical method for determining the proline content in the captopril bulk drug of this application is:

[0037] Take an appropriate amount of this product, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and quantitatively dilute it with water to make a solution containing about 12 mg per 1 ml as the test solution.

[0038] Take an appropriate amount of proline reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 12 μg per 1 ml as the reference substance solution.

[0039] According to the high performance liquid chromatography method (Chinese Pharmacopoeia 2015 Edition Part IV General Rules 0512), octadecylsilane bonded silica gel was used as the filler (Welch Ultimate XB-C18, 4.6mm×150mm, 5μm); 0.05% formic acid solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the following table; the column temperature was 30°C; the flow rate was 1.0ml per minute; the detector was a charged atomization detector (CAD), and the atomization temperature was 35°C. Accurately measure 20μl of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. If there is a peak in the chromatogram of the test solution that is consistent with the retention time of proline, the peak area calculated by the external standard method shall not exceed 0.10%.

[0040] Table 2 Gradient elution conditions

[0041] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 6 60 40 10 60 40 11 100 0 15 100 0

[0042] Table 3 Test instruments, chromatographic columns and reagents

[0043]

[0044]

[0045] Methodological validation:

[0046] 1. Exclusivity

[0047] (1) Blank solution: water.

[0048] (2) L-proline positioning solution: Take about 12 mg of L-proline, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the L-proline stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the L-proline positioning solution.

[0049] (3) D-proline positioning solution: Take about 12 mg of D-proline, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the D-proline stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the D-proline positioning solution.

[0050] (4) Test solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh it, place it in a 10 ml volumetric flask, add an appropriate amount of water, sonicate to dissolve it, add water to dilute to the scale, shake well, and use this as the test solution.

[0051] (5) Test sample spike solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, and place in a 10-ml volumetric flask. Accurately add 1 ml of L-proline stock solution, add appropriate amount of water, sonicate to dissolve, add water to dilute to the scale, shake well, and use this as the test sample spike solution.

[0052] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram.

[0053] Table 4 Proline peak retention time and separation degree of each positioning solution and test sample spike solution

[0054] Solution Retention time / min Resolution from adjacent peaks L-Proline Positioning Solution 2.032 3.98 D-Proline Positioning Solution 2.033 4.01 Test sample spike solution 2.032 3.85

[0055] The results showed that the retention time of L-proline peak was consistent with that of D-proline peak. L-proline was used as reference substance to study the content of proline (the sum of L-proline and D-proline) in captopril raw materials. The proline peak had good separation from adjacent impurity peaks. The blank solution and the test solution had no interference with the proline determination. The analytical method for the determination of proline content in captopril raw materials had good specificity.

[0056] 2. Linearity and range

[0057] Take about 12 mg of L-proline, weigh it accurately, put it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the reference stock solution.

[0058] Accurately measure 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.2ml and 1.5ml of the reference stock solution, place them in 10ml volumetric flasks respectively, add water to dilute to the scale, shake well, and use them as 20%, 50%, 80%, 100%, 120% and 150% linear solutions respectively.

[0059] Accurately measure 20 μl of each of the above linear solutions, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 5.

[0060] Table 5 Proline linear determination results

[0061] / Concentration (μg / ml) Peak area LOQ 0.7999 1816612 20％ 2.3998 7804764 50％ 5.9994 19107575 80％ 9.5990 29394869 100％ 11.9988 36099353 120％ 14.3986 41245502 150％ 17.9982 49032825

[0062] The results showed that proline was in the range of 0.7999μg / ml to 17.9982μg / ml, regression equation: y=2762143x+1421643, correlation index: R 2 =0.9927, good linear relationship.

[0063] 3. Limit of detection and limit of quantification

[0064] (1) Limit of quantification

[0065] The quantification limit was determined by the signal-to-noise ratio method, and the quantification limit was determined by the corresponding concentration when the signal-to-noise ratio was about 10:1. Accurately measure 1 ml of the 100% linear solution under the linear item, place it in a 15 ml volumetric bottle, add water to dilute to the scale, shake well, and use it as the quantification limit solution. The concentration of the proline quantification limit solution is 0.7999 μg / ml, which is 6.67% of the limit.

[0066] Accurately measure 20 μl of the quantitative limit solution, repeat the injection 6 times, record the chromatogram, and examine the peak area RSD. The results are shown in Table 6.

[0067] Table 6 Proline quantitative limit solution determination results

[0068]

[0069]

[0070] The results showed that the quantitative limit precision of proline was good.

[0071] (2) Detection limit

[0072] The detection limit was determined by the signal-to-noise ratio method, and the detection limit was determined by the corresponding concentration when the signal-to-noise ratio was about 3:1. Accurately measure 3 ml of the proline quantitative limit solution, place it in a 10 ml volumetric flask, dilute it to the scale with water, shake it well, and use it as the proline detection limit solution (see the chromatogram for details). Figure 3 ).

[0073] The detection limit of proline solution concentration is 0.2400 μg / ml, which is 2.00% of the limit.

[0074] 4. Precision

[0075] (1) Instrument precision

[0076] Take about 12 mg of L-proline, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the reference stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the reference solution.

[0077] Accurately measure 20 μl of the reference solution and repeat the injection 6 times to examine the RSD of the proline peak area and retention time. The results are shown in Table 7.

[0078] Table 7 Instrument precision test results

[0079] Reference solution Retention time / min Peak area 1 2.033 36540462 2 2.033 36779409 3 2.033 36707081 4 2.033 35687852 5 2.032 35258853 6 2.032 35796583 average value 2.033 36128373 SD 0.0005 630678 RSD% 0.03 1.75

[0080] The results showed that the instrumental precision of the analytical method for determining the proline content in captopril bulk drug was good.

[0081] (2) Repeatability

[0082] Reference solution: Take about 12 mg of L-proline, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the reference stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the reference solution.

[0083] Test solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, add appropriate amount of water, sonicate to dissolve, add water to dilute to the scale, shake well, and use as the test solution.

[0084] Test sample spiked solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, accurately add 1 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use as the test sample spiked solution (prepare 6 portions in parallel).

[0085] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, record the chromatogram, and examine the RSD of the proline content in the test sample spiked solution. The results are shown in Table 8.

[0086] Table 8 Repeatability test results

[0087] Test sample spike solution Proline content% 1 0.0998 2 0.0991 3 0.0998 4 0.0978 5 0.0973 6 0.0982 average value 0.0987 SD 0.001 RSD% 1.08

[0088] The results showed that the analytical method for determining the proline content in captopril raw materials had good repeatability.

[0089] (3) Intermediate precision

[0090] Reference solution: Take about 12 mg of L-proline, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the reference stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the reference solution.

[0091] Test solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, add appropriate amount of water, sonicate to dissolve, add water to dilute to the scale, shake well, and use as the test solution.

[0092] Test sample spiked solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, accurately add 1 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use as the test sample spiked solution (prepare 6 portions in parallel).

[0093] Different personnel, on different dates, accurately measured 20 μl of each of the above solutions, injected them into the liquid chromatograph, and recorded the chromatogram. The RSD of the proline content of 12 test sample spiked solutions (including 6 test sample spiked solutions under repeatability test) was investigated. The results are shown in Table 9.

[0094] Table 9 Intermediate precision determination results

[0095]

[0096] The results showed that the intermediate precision of the analytical method for the determination of proline content in captopril bulk drug was good.

[0097] 5. Accuracy

[0098] Reference substance stock solution: Take about 12 mg of L-proline, weigh accurately, place in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use as the reference substance stock solution.

[0099] Reference substance solution: Accurately measure 1 ml of reference substance stock solution, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the reference substance solution.

[0100] Test solution: Test solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, add appropriate amount of water, sonicate to dissolve, add water to dilute to the scale, shake well, and use as the test solution.

[0101] 10% spiked recovery solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, accurately add 0.1 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use this as the 10% spiked recovery solution. Prepare three copies in parallel.

[0102] 80% spiked recovery solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, put into a 10 ml volumetric flask, accurately add 0.8 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use this as the 80% spiked recovery solution. Prepare three copies in parallel.

[0103] 100% spiked recovery solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, accurately add 1.0 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use this as the 100% spiked recovery solution. Prepare three copies in parallel.

[0104] 120% spiked recovery solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, accurately add 1.2 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use this as the 120% spiked recovery solution. Prepare three copies in parallel.

[0105] Accurately measure 20 μl of each of the reference solution, test solution, 10% spiked recovery solution, 80% spiked recovery solution, 100% spiked recovery solution, and 120% spiked recovery solution, respectively, and inject them into the liquid chromatograph, record the chromatogram, and examine the average proline recovery rate and RSD of each spiked recovery solution. The results are shown in Table 10.

[0106] Table 10 Accuracy test results

[0107]

[0108] The results showed that the analytical method for determining the proline content in captopril raw materials had good accuracy.

[0109] 6. Durability

[0110] Reference solution: Take about 12 mg of L-proline, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the reference stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the reference solution.

[0111] Test solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, add appropriate amount of water, sonicate to dissolve, add water to dilute to the scale, shake well, and use as the test solution.

[0112] Test sample spiked solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, accurately add 1 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use as the test sample spiked solution.

[0113] The RSD% of the proline content of the test sample spiked solution was investigated at different flow rates of 1.0±0.1ml / min, different column temperatures of 35±2℃, and different batches of chromatographic columns. The results are shown in Table 11.

[0114] Table 11 Durability test results

[0115]

[0116] The results showed that the analytical method for determining the proline content in captopril raw materials had good durability.

[0117] 7. Solution stability

[0118] Reference solution: Take about 12 mg of L-proline, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the reference stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and use it as the reference solution.

[0119] Test sample spiked solution: Take about 120 mg of Captopril API (Batch No.: 1810081), accurately weigh, place in a 10 ml volumetric flask, accurately add 1 ml of reference stock solution, add appropriate amount of water, sonicate to dissolve, dilute with water to the scale, shake well, and use as the test sample spiked solution.

[0120] Accurately measure 20 μl of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram and examine the 24h proline peak area RSD. The results are shown in Table 12.

[0121] Table 12 Solution stability test results

[0122]

[0123]

[0124] The results showed that the solution of the analytical method for determining the proline content in captopril raw materials had good stability for 24 hours.

[0125] 8. Summary of validation of analytical method for determination of proline content in Captopril API

[0126] Table 13 Summary of validation of analytical method for determination of proline content in Captopril API

[0127]

[0128] 9. Determination of proline content in Captopril API

[0129] According to the analytical method for determining the proline content in captopril raw materials, the proline content in 8 batches of captopril raw materials was determined. The results are shown in Table 14.

[0130] Table 14 Proline determination results

[0131]

[0132]

[0133] The results showed that proline (the sum of L-proline and D-proline) was not detected in 8 batches of capril raw materials.

[0134] The method provided by the present invention has the following advantages:

[0135] 1. Based on the fact that proline has very weak ultraviolet absorption and high polarity, the present invention adopts a derivatization method to determine proline, wherein:

[0136] According to the sensitivity, inorganic acid was selected. In order to avoid the CAD detector being contaminated by high concentration of inorganic acid, 0.05% formic acid aqueous solution was finally selected.

[0137] 2. The method provided by the present invention has good specificity, linear relationship, precision of quantitative limit, precision of analytical method and instrument, repeatability, intermediate precision, accuracy, durability and 24h stability of solution. Description of the drawings:

[0138] Figure 1 :Chromatogram when 0.5% trifluoroacetic acid was used as the mobile phase;

[0139] Figure 2 :Chromatogram when 0.05% formic acid is used as mobile phase;

[0140] Figure 3 : Linear relationship diagram of proline;

[0141] Figure 4 : Chromatogram of reference substance;

[0142] Figure 5 : Chromatogram of test sample. Specific implementation method:

[0143] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0144] Example 1: A method for detecting proline as an impurity in a captopril bulk drug

[0145] Step 1, preparation of test solution:

[0146] Take an appropriate amount of Captopril API, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and quantitatively dilute it with water to make a solution containing about 12 mg per 1 ml as the test solution;

[0147] Step 2, preparation of reference solution:

[0148] Take an appropriate amount of proline reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 12 μg per 1 ml as the reference substance solution;

[0149] Step 3, assay:

[0150] Inject 20 μl of each reference solution and test solution into a high performance liquid chromatograph and record the chromatograms (the reference solution chromatogram and the test solution chromatogram are shown in Figure 4 , Figure 5), according to the chromatogram, the proline content was calculated by the peak area according to the external standard method;

[0151] The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as a filler (Welch Ultimate XB-C18, 4.6 mm×150 mm, 5 μm); the column temperature is 30° C.; the flow rate is 1.0 ml per minute; the detector is a charged atomization detector (CAD); the atomization temperature is 35° C.; 0.05% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in the following gradient elution mode;

[0152]

[0153] Wherein, the proline includes L-proline and D-proline, and its content is the sum of L-proline and D-proline.

[0154] Example 2: A method for detecting proline as an impurity component in a captopril bulk drug

[0155] Step 1, preparation of test solution:

[0156] Take an appropriate amount of Captopril API, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and quantitatively dilute it with water to make a solution containing about 14 mg per 1 ml as the test solution;

[0157] Step 2, preparation of reference solution:

[0158] Take an appropriate amount of proline reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 14 μg per 1 ml as the reference substance solution;

[0159] Step 3, assay:

[0160] 30 μl of each reference solution and test solution were injected into the HPLC, and the chromatogram was recorded. According to the chromatogram, the proline content was calculated by the peak area using the external standard method;

[0161] The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as a filler (Welch Ultimate XB-C18, 4.6 mm×150 mm, 5 μm); the column temperature is 35° C.; the flow rate is 1.2 ml per minute; the detector is a charged atomization detector (CAD); the atomization temperature is 40° C.; 0.06% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in the following gradient elution mode;

[0162]

[0163] Wherein, the proline includes L-proline and D-proline, and its content is the sum of L-proline and D-proline.

[0164] Example 3: A method for detecting proline as an impurity in a captopril bulk drug

[0165] Step 1, preparation of test solution:

[0166] Take an appropriate amount of Captopril API, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and quantitatively dilute it with water to make a solution containing about 10 mg per 1 ml as the test solution;

[0167] Step 2, preparation of reference solution:

[0168] Take an appropriate amount of proline reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 10 μg per 1 ml as the reference substance solution;

[0169] Step 3, assay:

[0170] 10 μl of each reference solution and test solution were injected into the HPLC, and the chromatogram was recorded. According to the chromatogram, the proline content was calculated by the peak area using the external standard method;

[0171] The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as a filler (Welch Ultimate XB-C18, 4.6 mm×150 mm, 5 μm); the column temperature is 25° C.; the flow rate is 0.8 ml per minute; the detector is a charged atomization detector (CAD); the atomization temperature is 30° C.; 0.04% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in the following gradient elution mode;

[0172]

[0173]

[0174] Wherein, the proline includes L-proline and D-proline, and its content is the sum of L-proline and D-proline.

Claims

1. A method for determining the content of proline as an impurity component in a Captopril bulk drug, the method being a high performance liquid chromatography method, specifically comprising the following steps: Step 1, preparation of test solution: Take an appropriate amount of Captopril API, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and add water to quantitatively dilute it to make a solution containing 10-14 mg per 1 ml as the test solution; Step 2, preparation of reference solution: Take an appropriate amount of proline reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing 10-14 μg per 1 ml as the reference substance solution; Step 3, assay: Inject 10-30 μl of the reference solution and the test solution into a high performance liquid chromatograph, record the chromatogram, and calculate the proline content by peak area using the external standard method according to the chromatogram; The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as a filler, the chromatographic column is: Welch Ultimate XB-C18, 4.6mm×150mm, 5μm; the column temperature is 25-35°C; the flow rate is 0.8-1.2ml per minute; the detector is a charged atomization detector (CAD); the atomization temperature is 30-40°C; 0.04-0.06% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in the following gradient elution mode; 2. The method according to claim 1, characterized in that The method comprises the following steps: Step 1, preparation of test solution: Take an appropriate amount of Captopril API, weigh it accurately, add an appropriate amount of water, dissolve it by ultrasonication, and quantitatively dilute it with water to make a solution containing 12 mg per 1 ml as the test solution; Step 2, preparation of reference solution: Take an appropriate amount of proline reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing 12 μg per 1 ml as the reference substance solution; Step 3, assay: 20 μl of each reference solution and test solution were injected into the HPLC, and the chromatogram was recorded. According to the chromatogram, the proline content was calculated by the peak area using the external standard method; The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel is used as a filler, the chromatographic column is: Welch Ultimate XB-C18, 4.6mm×150mm, 5μm; the column temperature is 30°C; the flow rate is 1.0ml per minute; the detector is a charged atomization detector (CAD); the atomization temperature is 35°C; 0.05% formic acid solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in the following gradient elution mode; 3. The method according to claim 1, characterized in that: in, The proline content is the sum of L-proline and D-proline.

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