High performance liquid chromatography for separating and detecting citric acid monoethyl ester and citric acid diethyl ester in minoxidil foaming agent

Abstract

The invention discloses a high performance liquid chromatography for separating and detecting monoethyl citrate and diethyl citrate in a minoxidil foaming agent, which is characterized in that a chromatographic column takes octadecyl silane bonded silica gel as a filling agent, a monopotassium phosphate buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and linear gradient elution is carried out; the flow velocity is 0.9 ml / min to 1.1 ml / min, and the column temperature is 35 DEG C to 45 DEG C. The invention provides accurate detection of the citric acid monoethyl ester and the citric acid diethyl ester in the minoxidil foaming agent, and solves the problem that the prior art is lack of detection means of the citric acid monoethyl ester and the citric acid diethyl ester; the method is high in sensitivity and good in accuracy, precision and durability, and accurate detection and quantification can be guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis methods, and particularly relates to a high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam. Background Art

[0002] Minoxidil foam is a new anti-hair loss drug, and its prescription composition includes minoxidil, anhydrous citric acid, lactic acid, octadecanol, cetyl alcohol, polysorbate 60, glycerol, butylated hydroxytoluene, anhydrous ethanol, purified water, propane, butane and isobutane. Its prescription uses pharmaceutical excipients ethanol and citric acid, which can undergo esterification reaction. According to literature search, [1][2] The main degradation products are monoethyl citrate and diethyl citrate. Therefore, it is necessary to conduct quality control on the degradation impurities and detect the amount of monoethyl citrate and diethyl citrate as impurities in minoxidil foam.

[0003] The excipients citric acid and lactic acid and the main component minoxidil contained in the prescription of this product are weakly retained in liquid chromatography. Monoethyl citrate and diethyl citrate are also weakly retained impurities, and the chromatographic peaks are difficult to separate. No method for separating this compound from monoethyl citrate and diethyl citrate was found in the public literature.

[0004] References

[0005] [1]Berdugo CE,Suaza A,Santaella MA,et al.Synthesis and Characterization of Dieth yl Citrate and Phase Equilibria in Mixtures withEthanol and Water[J].Journal of Che mical&Engineering Data.2018,63:2644-52;

[0006] [2]OU Y,HAN J,CHEN B,et al.Synthesis and Characterization andAnticoagulant Properties of Diethyl Citrate[J].Asian Journal ofChemistry.2012,24:4953-4960.

[0007] In order to improve the quality control of minoxidil, it is urgent to develop a method that can effectively separate and detect monoethyl citrate and diethyl citrate in minoxidil foam. Summary of the invention

[0008] In order to overcome the above problems of the prior art, the present method aims to establish a high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate. The technical scheme of the present invention is as follows:

[0009] A high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate, wherein the chromatographic column uses octadecylsilane bonded silica gel as a filler, potassium dihydrogen phosphate buffer as a mobile phase A, acetonitrile as a mobile phase B, and linear gradient elution is performed; the flow rate is 0.9 ml / min to 1.1 ml / min, and the column temperature is 35 to 45°C.

[0010] Preferably, the pH value of the potassium dihydrogen phosphate buffer in the mobile phase A is 2.2 to 2.6; preferably, the pH value is 2.4.

[0011] Preferably, the concentration of potassium dihydrogen phosphate buffer in mobile phase A is in the range of 0.02 mol / L to 0.04 mol / L.

[0012] Preferably, the initial volume ratio of mobile phase A to mobile phase B is 92:8 to 96:4.

[0013] Preferably, the gradient program of the mobile phase is:

[0014] From 0 to 15 minutes, the initial volume ratio of mobile phase A and mobile phase B was maintained at 92:8 to 96:4; from 15 to 15.01 minutes, the volume ratio of mobile phase A and mobile phase B linearly changed from 92:8 to 96:4 to 80:20; from 15.01 to 30 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 80:20; from 30 to 30.01 minutes, the volume ratio of mobile phase A and mobile phase B linearly changed from 80:20 to 92:8 to 96:4; from 30.01 to 40 minutes, the initial volume ratio of mobile phase A and mobile phase B remained unchanged at 92:8 to 96:4.

[0015] Preferably, in the liquid chromatography method, the chromatographic column is Agilent Zorbax Eclipse XDB-C18, 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent performance.

[0016] Preferably, in the liquid chromatography method, the detection wavelength is 210 nm and the injection volume is 20 μl.

[0017] Preferably, the sample is prepared by quantitative dilution with a diluent, wherein the diluent is an acetonitrile-pH 2.4 potassium dihydrogen phosphate solution with a volume ratio of 5:95 to 15:85.

[0018] In one embodiment of the present invention, the diluent is an acetonitrile-pH 2.4 potassium dihydrogen phosphate solution with a volume ratio of 1:9.

[0019] Preferably, the sample preparation method comprises:

[0020] Reference solution: Place monoethyl citrate and diethyl citrate reference substances in the same container and use diluent to prepare solutions with a concentration of 0.1 mg / ml respectively;

[0021] Test solution: Take 1 can of this product, remove the cap, wipe it clean, shake it thoroughly, invert it, press the valve, discard an appropriate amount of foam and weigh it accurately, take an appropriate amount of liquid medicine and put it in a dry and clean beaker, weigh this product again, and the difference between the two weights is the weight of the liquid medicine taken; heat the beaker in a water bath to make the foam completely collapse into solution, transfer it to a measuring bottle, add acetonitrile to the above beaker, rinse it and combine the washing solution into the above measuring bottle, wash the beaker with pH2.4 potassium dihydrogen phosphate solution, transfer it quantitatively to the above measuring bottle and dilute to the scale, shake well, centrifuge, and filter with a 0.45μm Nylon 66 filter membrane to obtain.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention provides a method for accurately detecting monoethyl citrate and diethyl citrate in minoxidil foam, which solves the problem of lack of detection means for monoethyl citrate and diethyl citrate in the prior art;

[0024] 2. The present invention avoids the possible interference problem of the foaming agent diluent, avoids the disadvantages of the product under other chromatographic conditions such as the solvent effect, the impurity peak shape to be measured is good, and the impurity peaks can be well separated, ensuring accurate quantification;

[0025] 3. The method has high sensitivity, with the quantitative limit of monoethyl citrate being 4.7871 μg / ml and the quantitative limit of diethyl citrate being 5.0847 μg / ml; the method has high accuracy, with the recovery rate of monoethyl citrate being between 100.9% and 104.2%, and the RSD value being 1.2%; the recovery rate of diethyl citrate being between 99.3% and 102.9%, and the RSD value being 1.3%; the method has high precision, and the repeatability of the inspection of citric acid degradation products in minoxidil foam is good; the method has good durability, and the method has good stability when fine-tuning the flow rate (0.9 ml / min, 1.1 ml / min), column temperature (35°C, 45°C), pH value (2.2, 2.6), potassium dihydrogen phosphate buffer concentration (0.02mol / L-0.04mol / L), diluent ratio (acetonitrile-pH2.4 potassium dihydrogen phosphate solution is 5:95, 15:85), initial volume ratio of mobile phase A and mobile phase B (92:8, 96:4), replacement of chromatographic columns from different batches and brands, the measured contents of monoethyl citrate and diethyl citrate were compared with the contents measured under standard conditions and the ratio was between 96.2% and 101.0%, which can ensure effective quality control of impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is a specific test chromatogram in the method verification test of Example 2, wherein from bottom to top are diluent, monoethyl citrate solution, diethyl citrate solution, reference solution, test solution and mixed solution.

[0028] Figure 2 This is the chromatogram of Comparative Example 1; wherein, from bottom to top, there are the reference solution and the mixed test solution.

[0029] Figure 3 This is the chromatographic column of comparative example 2.1; wherein, from bottom to top, there are diluent, reference solution, and mixed test solution.

[0030] Figure 4 This is the chromatogram of Comparative Example 2.2; wherein, from bottom to top, there are diluent, monoethyl citrate solution, diethyl citrate solution, test solution and mixed solution.

[0031] Figure 5 This is the chromatogram of comparative example 2.3; wherein, from bottom to top, there are mixed solutions at column temperatures of 35°C, 45°C, 37°C, 39°C and 38°C.

[0032] Figure 6 This is the chromatogram of Comparative Example 3; wherein, from bottom to top, there are diluent, mixed reference solution, and mixed test solution.

[0033] Figure 7 This is the chromatogram of Comparative Example 4; wherein, from bottom to top, there are diluent, mixed reference solution, and mixed test solution. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0035] Example 1, a high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate, chromatographic conditions:

[0036] Chromatographic column: Agilent Zorbax Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm;

[0037] Detection wavelength: 210nm; flow rate: 1.0ml / min;

[0038] Column temperature: 40°C; injection volume: 20 μl;

[0039] Mobile phase A: pH 2.4 potassium dihydrogen phosphate solution (10.8 g potassium dihydrogen phosphate, add 3000 ml water to dissolve, and adjust the pH to 2.4 with phosphoric acid);

[0040] Mobile phase B: acetonitrile;

[0041] The gradient program is as follows:

[0042]

[0043] Diluent: acetonitrile-pH 2.4 potassium dihydrogen phosphate solution (1:9).

[0044] Reference solution: Take about 10 mg of monoethyl citrate and diethyl citrate reference substances, accurately weigh them, place them in the same 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0045] Test solution: Take 1 can of this product, remove the cap, wipe it clean, shake it thoroughly, invert it, press the valve, discard an appropriate amount of foam and weigh it accurately (recorded as W1), take about 2.0g of the liquid, put it in a dry and clean beaker, weigh this product again (recorded as W2), and the difference between the two weights is the weight of the liquid taken. Heat the beaker in a 40℃ water bath for 2 minutes to completely collapse the foam into a solution, transfer it to a 20ml volumetric flask, add 2ml of acetonitrile to the above beaker, rinse it and combine the washing liquid to the above volumetric flask, wash the beaker with pH2.4 potassium dihydrogen phosphate solution, transfer it quantitatively to the above volumetric flask and dilute it to the scale, shake it well, centrifuge it at 10000rpm for 5 minutes, and filter it with a 0.45μm Nylon 66 filter membrane to obtain it.

[0046] Determination method: Accurately measure 20 μl of each of the above solutions, inject into liquid chromatograph, record the chromatogram, and calculate the content of monoethyl citrate and diethyl citrate by peak area according to the external standard method.

[0047] Calculation formula:

[0048]

[0049] In the formula,

[0050] A i is the peak area of ​​monoethyl citrate / diethyl citrate in the chromatogram of the test solution;

[0051] It is the average peak area of ​​monoethyl citrate / diethyl citrate in the chromatogram of the reference solution;

[0052] m i is the mass of the test sample, m i＝ W1-W2, g;

[0053] m r is the mass of monoethyl citrate / diethyl citrate reference substance, mg;

[0054] B r is the content of monoethyl citrate / diethyl citrate reference substance, %;

[0055] C r is the concentration of the reference substance, mg / ml.

[0056] The test results of three batches of samples using this method are shown in Table 1:

[0057] Table 1 Test results of three batches of samples

[0058] batch number Monoethyl citrate (mg / g) Diethyl citrate (mg / g) 2401933 0.05 ND 2402901 0.05 ND 2402902 0.06 ND

[0059] Example 2, method verification

[0060] The chromatographic conditions are:

[0061] Instrument: Agilent liquid chromatograph;

[0062] Chromatographic column: Agilent Zorbax Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm;

[0063] Detection wavelength: 210nm;

[0064] Flow rate: 1.0ml / min;

[0065] Column temperature: 40°C;

[0066] Injection volume: 20 μl;

[0067] Mobile phase A: pH 2.4 potassium dihydrogen phosphate solution (10.8 g potassium dihydrogen phosphate, add 3000 ml water to dissolve, and adjust the pH to 2.4 with phosphoric acid);

[0068] Mobile phase B: acetonitrile;

[0069] The gradient program is as follows:

[0070]

[0071] Diluent: acetonitrile-pH 2.4 potassium dihydrogen phosphate solution (1:9).

[0072] ①Specificity:

[0073] Monoethyl citrate solution: Take about 10 mg of monoethyl citrate reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well; accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the scale with pH 2.4 potassium dihydrogen phosphate solution, shake well, and obtain.

[0074] Diethyl citrate solution: Take about 10 mg of diethyl citrate reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well; accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the scale with pH 2.4 potassium dihydrogen phosphate solution, shake well, and obtain.

[0075] Reference substance solution: Take about 10 mg each of monoethyl citrate and diethyl citrate reference substances, accurately weigh, place in the same 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use as the reference substance mixed stock solution; accurately measure 2 ml, place in a 20 ml volumetric flask, dilute to the scale with pH 2.4 potassium dihydrogen phosphate solution, shake well, and obtain.

[0076] Test solution: Take 1 can of this product, remove the cap, wipe it clean, shake it thoroughly, invert it, press the valve, discard an appropriate amount of foam and weigh it accurately (recorded as W1), take about 2.0g of the liquid, put it in a dry and clean beaker, weigh this product again (recorded as W2), and the difference between the two weights is the weight of the liquid taken. Heat the beaker in a 40℃ water bath for 2 minutes to completely collapse the foam into a solution, transfer it to a 20ml volumetric flask, add 2ml of acetonitrile to the above beaker, rinse it and combine the washing liquid to the above volumetric flask, wash the beaker with pH2.4 potassium dihydrogen phosphate solution, transfer it quantitatively to the above volumetric flask and dilute it to the scale, shake it well, centrifuge it at 10000rpm for 5 minutes, and filter it with a 0.45μm Nylon 66 filter membrane to obtain it.

[0077] Mixed solution: Take 1 can of this product, remove the cap, wipe it clean, shake it thoroughly, invert it, press the valve, discard an appropriate amount of foam and weigh it accurately (recorded as W1), take about 2.0g of the liquid medicine, put it in a dry and clean beaker, weigh this product again (recorded as W2), and the difference between the two weights is the weight of the liquid medicine taken. Heat the beaker in a 40℃ water bath for 2 minutes to make the foam completely collapse into a solution, transfer it to a 20ml volumetric flask, accurately measure 2ml of the reference substance mixed stock solution and put it in the above beaker, rinse it and combine the washing solution into the above volumetric flask, wash the beaker with pH2.4 potassium dihydrogen phosphate solution, transfer it quantitatively to the above volumetric flask and dilute it to the scale, shake it well, centrifuge it at 10000rpm for 5 minutes, and filter it with a 0.45μm Nylon 66 filter membrane to obtain it.

[0078] Accurately measure 20 μl of each of the above solutions and inject them into the liquid chromatograph to record the chromatogram.

[0079] Results: Figure 1As shown, the main peak in the chromatograms of the test solution and the mixed solution is the minoxidil peak; the components of the diluent and the test solution (minoxidil foam) do not have peaks at the positions of monoethyl citrate and diethyl citrate, and do not interfere with the detection of monoethyl citrate and diethyl citrate; in the chromatogram of the mixed solution, the minimum separation between monoethyl citrate, diethyl citrate and adjacent peaks is 3.17; the retention time of monoethyl citrate and diethyl citrate in the mixed solution is consistent with that in the reference solution, indicating that this method has good specificity for the inspection of citric acid degradation products of this product.

[0080] ②Limit of quantification: The limit of quantification concentration of monoethyl citrate is 4.7871μg / ml.

[0081] The quantitative limit of diethyl citrate is 5.0847 μg / ml. The sensitivity of this method for detecting the citric acid degradation products of this product meets the requirements.

[0082] ④Linear:

[0083] For monoethyl citrate, in the range of 4.7871 μg / ml to 143.6140 μg / ml (5% to 150%), r is 0.9999;

[0084] Diethyl citrate is in the range of 5.0847 μg / ml to 152.5399 μg / ml (5% to 150%), and r is 0.9999;

[0085] In the verified concentration range, the peak area showed a good linear relationship with the concentration.

[0086] ⑤Accuracy:

[0087] The recovery rate of monoethyl citrate was between 100.9% and 104.2%, and the RSD value was 1.2%;

[0088] The recovery of diethyl citrate was between 99.3% and 102.9%, and the RSD value was 1.3%;

[0089] The accuracy of this method used to examine the citric acid degradation products of this product meets the requirements.

[0090] ⑥Precision:

[0091] The RSD values ​​of the contents of monoethyl citrate and diethyl citrate in the six sample solutions were measured to be 1.9% and 1.5%, respectively.

[0092] This method has good repeatability when used to examine the citric acid degradation products of this product.

[0093] The RSD values ​​of the contents of monoethyl citrate and diethyl citrate in 12 sample solutions were measured to be 1.5% and 1.9%, respectively.

[0094] This method was used to examine the citric acid degradation products of this product with good intermediate precision.

[0095] ⑦ Solution stability: The mixed solution and the reference solution sample bottles were placed at room temperature for 48 hours. The ratio of the peak area of ​​monoethyl citrate and diethyl citrate measured at each time point to the peak area at time 0 was between 99.7% and 101.7%.

[0096] The mixed solution and reference solution sample bottles were placed at room temperature for 48 hours and the solution had good stability.

[0097] ⑧Durability: fine-tuning the flow rate (0.9 ml / min, 1.1 ml / min), column temperature (35°C, 45°C), pH value (2.2, 2.6), potassium dihydrogen phosphate buffer concentration (0.02 mol / L~0.04 mol / L), diluent ratio (acetonitrile-pH2.4 potassium dihydrogen phosphate solution is 5:95, 15:85), initial volume ratio of mobile phase A and mobile phase B (92:8, 96:4), replacing different batches and different brands of chromatographic columns, the measured contents of monoethyl citrate and diethyl citrate were compared with the contents measured under standard conditions, and the ratio was between 96.2% and 101.0%.

[0098] Table 2 Durability test results

[0099]

[0100] It can be seen that the chromatographic conditions of this method for examining the citric acid degradation products of this product are durable.

[0101] Comparative Example 1: Other C18 columns

[0102] Other chromatographic conditions were the same as in Example 1. Waters symmetry C18, 4.6 mm × 250 mm, 5 μm was used to inject the reference solution and the mixed test solution. The results were as follows: Figure 2 As shown, the peak shape of diethyl citrate is poor.

[0103] Comparative Example 2: Other mobile phases

[0104] 2.1, Sodium Perchlorate System

[0105] Detection wavelength: 210nm; flow rate: 1.0ml / min;

[0106] Column temperature: 40°C; injection volume: 20 μl;

[0107] Mobile phase A: pH 3.0 sodium perchlorate solution (take 28.10 g of perchloric acid monohydrate, add 2000 ml of water to dissolve, and adjust the pH value to 3.0 with 10% perchloric acid)

[0108] Mobile phase B: acetonitrile

[0109] The gradient program is as follows:

[0110]

[0111] Under this condition, the diluent, reference solution and mixed test solution were injected. The results were as follows Figure 3 As shown, monoethyl citrate is separated into two chromatographic peaks.

[0112] 2.2, 0.1% phosphoric acid aqueous solution as mobile phase

[0113] Chromatographic column: Agilent zorbax Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm

[0114] Detection wavelength: 230nm;

[0115] Flow rate: 1.0ml / min;

[0116] Column temperature: 40°C;

[0117] Injection volume: 10 μl;

[0118] Mobile phase A: 0.1% phosphoric acid in water;

[0119] Mobile phase B: acetonitrile;

[0120] Elution was performed using the following gradient:

[0121]

[0122] Diluent: 0.1% phosphoric acid aqueous solution-acetonitrile (85:15)

[0123] Monoethyl citrate solution: Take about 10 mg of monoethyl citrate reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the reference substance stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0124] Diethyl citrate solution: Take about 10 mg of diethyl citrate reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the reference substance stock solution; accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0125] Test solution: Take about 2.0 g of the drug solution, place it in a 20 ml volumetric flask, add 14 ml of acetonitrile to dissolve, dilute to the scale with 0.1% phosphoric acid aqueous solution, shake well, filter, and obtain.

[0126] Under this condition, the diluent, reference solution and mixed test solution were injected. The results were as follows Figure 4 As shown in the figure, monoethyl citrate cannot be baseline separated from the nearby peaks, and diethyl citrate cannot be separated from the minoxidil peak. The test solution is not clear after filtration, which damages the chromatographic column. The gradient elution and sample preparation methods were optimized.

[0127] 2.3, based on comparative example 2.2, adjust the gradient program to optimize the separation; explore the effect of different diluents on the filtration of the test solution, and the adjustment method is as follows:

[0128] Injection volume: 20 μl

[0129] Flow rate: 1.0ml / min

[0130] Column temperature: 40°C

[0131] Mobile phase A: 0.1% phosphoric acid in water

[0132] Mobile phase B: acetonitrile;

[0133] Elution was performed using the following gradient:

[0134]

[0135] Diluent①: 0.1% phosphoric acid water-acetonitrile (70:30)

[0136] Diluent ②: 0.05 mol / L potassium dihydrogen phosphate buffer (pH 2.5)-acetonitrile (80:20)

[0137] Mixed solution ①: Take 1 can of this product, remove the cap, wipe it clean, shake it thoroughly, invert it, press the valve, discard the appropriate amount of foam and weigh it accurately (recorded as W1), take about 2.0g of the liquid medicine, put it in a dry and clean beaker, weigh this product again (recorded as W2), and the difference between the two weights is the weight of the liquid medicine taken. Add 4ml of acetonitrile to the beaker to dissolve it and transfer it to a 20ml volumetric bottle, accurately measure 1ml of monoethyl citrate stock solution and 1ml of diethyl citrate stock solution respectively, put them in the above volumetric bottle, dilute to the scale with 0.1% phosphoric acid water, shake well, filter with 0.45μm PTFE and 0.45μm Nylon filter membranes respectively, take the subsequent filtrate, and the solution is not clear after filtering with two filter membranes.

[0138] Mixed solution ②: Take 1 can of this product, remove the cap, wipe it clean, shake it thoroughly, invert it, press the valve, discard the appropriate amount of foam and weigh it accurately (recorded as W1), take about 2.0g of the liquid, put it in a dry and clean beaker, weigh this product again (recorded as W2), and the difference between the two weights is the weight of the liquid taken. Heat the beaker in a 40℃ water bath for 5 minutes to defoam it, then transfer it to a 20ml volumetric flask, add 2ml of monoethyl citrate stock solution and 2ml of diethyl citrate stock solution to the beaker to rinse the beaker and combine the washing liquid into the above volumetric flask, dilute to the scale with potassium dihydrogen phosphate buffer, shake well, centrifuge at 10000rpm for 5 minutes, filter with 0.45μm PTFE and 0.45μm Nylon filter membranes respectively, the solution is not clear after filtering with 0.45μm PTFE filter membrane, and the solution is clear after filtering with 0.45μm Nylon filter membrane.

[0139] Take ②20 μl of the mixed solution filtered through a 0.45 μm Nylon filter membrane and inject it into a liquid chromatograph to record the chromatogram.

[0140] The chromatogram of the mixed solution ② showed that monoethyl citrate and diethyl citrate were well separated from the adjacent peaks and had good peak shapes. The durability of the chromatographic conditions was investigated.

[0141] Accurately measure 20 μl of the mixed solution ② at column temperatures of (±5°C), (±3°C), (±2°C), and (±1°C), respectively, and inject it into the liquid chromatograph to record the chromatogram.

[0142] The results are as follows Figure 5 As shown in the figure: the peak of monoethyl citrate in the mixed solution splits at column temperatures of 35°C, 45°C, 37°C, 39°C and 38°C, and the durability of the chromatographic conditions is poor. It can be seen that under this chromatographic condition, the solvent effect has a significant impact on this product, so the mobile phase of 0.1% phosphoric acid aqueous solution cannot achieve reliable detection of this impurity.

[0143] Comparative Example 3, mobile phase pH 2.1

[0144] Mobile phase A: pH 2.1 potassium dihydrogen phosphate solution (10.8 g potassium dihydrogen phosphate, add 3000 ml water to dissolve, and adjust the pH to 2.1 with phosphoric acid);

[0145] The rest is the same as in Example 1.

[0146] The results are as follows Figure 6 As shown: When the pH value is 2.1, the separation of monoethyl citrate and the adjacent peak in the mixed solution is poor.

[0147] The pH of the diluent was adjusted to 2.1 with potassium dihydrogen phosphate and the sample was reinjected, but there was no improvement.

[0148] Comparative Example 4, adjust the mobile phase pH to 2.7

[0149] Mobile phase A: pH 2.7 potassium dihydrogen phosphate solution (10.8 g potassium dihydrogen phosphate, add 3000 ml water to dissolve, and adjust the pH to 2.7 with phosphoric acid);

[0150] The rest is the same as in Example 1.

[0151] The results are as follows Figure 7 As shown: When the pH value is 2.7, the separation of monoethyl citrate and the adjacent peak in the mixed solution is poor.

[0152] The pH of the diluent was adjusted to 2.7 with potassium dihydrogen phosphate and the sample was reinjected, but there was no improvement.

[0153] Summary: Each component has strong polarity and is prone to solvent effect. The chromatographic conditions and solvents provided by the present invention can avoid the solvent effect of the components of this project. Using Zorbax Eclipse XDB-C18, different mobile phases and gradient experiments were adopted to explore the chromatographic conditions that can separate monoethyl citrate and diethyl citrate from minoxidil and its excipients well. Finally, the above method was determined, with good specificity.

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam, characterized in that: The chromatographic column uses octadecylsilane bonded silica gel as filler, potassium dihydrogen phosphate buffer as mobile phase A, acetonitrile as mobile phase B, and linear gradient elution; the flow rate is 0.9 ml / min to 1.1 ml / min, and the column temperature is 35 to 45°C; the pH value of the potassium dihydrogen phosphate buffer in the mobile phase A is 2.2 to 2.

6.

2. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The pH value of potassium dihydrogen phosphate buffer in mobile phase A was 2.

4.

3. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The concentration range of potassium dihydrogen phosphate buffer in mobile phase A is 0.02 mol / L to 0.04 mol / L.

4. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The initial volume ratio of mobile phase A to mobile phase B is 92:8 to 96:

4.

5. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The gradient program of the mobile phase is as follows: within 0 to 15 minutes, the initial volume ratio of mobile phase A and mobile phase B is maintained at 92:8 to 96:4; within 15 to 15.01 minutes, the volume ratio of mobile phase A and mobile phase B is linearly changed from 92:8 to 96:4 to 80:20; within 15.01 to 30 minutes, the volume ratio of mobile phase A and mobile phase B is maintained at 80:20; within 30 to 30.01 minutes, the volume ratio of mobile phase A and mobile phase B is linearly changed from 80:20 to 92:8 to 96:4; within 30.01 to 40 minutes, the initial volume ratio of mobile phase A and mobile phase B is maintained at 92:8 to 96:

4.

6. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The chromatographic column used is Agilent Zorbax Eclipse XDB-C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance.

7. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The detection wavelength was 210 nm and the injection volume was 20 μl.

8. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The sample was prepared by quantitative dilution with a diluent of acetonitrile-pH 2.4 potassium dihydrogen phosphate solution in a volume ratio of 5:95 to 15:

85.

9. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 8, characterized in that: The diluent is acetonitrile-pH 2.4 potassium dihydrogen phosphate solution with a volume ratio of 1:

9.

10. The high performance liquid chromatography method for separating and detecting monoethyl citrate and diethyl citrate in minoxidil foam according to claim 1, characterized in that: The sample preparation method includes: Reference solution: Place monoethyl citrate and diethyl citrate reference substances in the same container and use diluent to prepare solutions with a concentration of 0.1 mg / ml respectively; Test solution: Take 1 can of this product, remove the cap, wipe it clean, shake it thoroughly, invert it, press the valve, discard an appropriate amount of foam and weigh it accurately, take an appropriate amount of liquid medicine and put it in a dry and clean beaker, weigh this product again, and the difference between the two weights is the weight of the liquid medicine taken; heat the beaker in a water bath to make the foam completely collapse into solution, transfer it to a volumetric flask, add acetonitrile to the above beaker, rinse it and combine the washing liquid into the above volumetric flask, wash the beaker with pH2.4 potassium dihydrogen phosphate solution, transfer it quantitatively to the above volumetric flask and dilute to the scale, shake well, centrifuge, and filter with a 0.45μm Nylon 66 filter membrane to obtain; Determination method: Accurately measure the reference solution and the test solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the contents of monoethyl citrate and diethyl citrate by the peak area according to the external standard method.

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