A method for determining impurities in sodium stearyl fumarate

Abstract

The invention discloses a method for determining impurities in sodium stearyl fumarate. The method adopts high performance liquid chromatography for detection, the chromatographic column is a C1-C18 alkylsilane bonded silica gel, the mobile phase is a mixed solution consisting of C1-C5 primary alcohol, water, acetonitrile, trifluoroacetic acid or tetrahydrofuran, isocratic elution is adopted, and the detector is a mass detector. The method has good specificity, high sensitivity, high accuracy, simple operation and good repeatability.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical analytical chemistry, and particularly relates to a method for determining impurities in sodium stearyl fumarate. Background Art

[0002] Sodium stearyl fumarate is an important pharmaceutical excipient and is widely used as a lubricant in pharmaceutical preparations. In addition to being chemically inert, pharmaceutical excipients also need to control the trace impurities they contain to ensure the quality, safety and effectiveness of the drug.

[0003] Sodium stearyl maleate and stearyl alcohol are specific impurities in sodium stearyl fumarate and need to be accurately measured and controlled to ensure the quality of the auxiliary materials is safe and reliable.

[0004] Among the existing detection methods for sodium stearyl fumarate impurities, the "Chinese Pharmacopoeia" only controls sodium stearyl maleate and uses gas chromatography with pre-column derivatization for determination. Experiments have found that it is poorly separated from the main peak, the recovery rate is low, and the derivatization operation steps are complicated, which places extremely high demands on personnel and is prone to deviations. It is necessary to use a silanized solution, and improper operation can easily cause safety accidents. The "United States Pharmacopoeia" uses thin layer chromatography for determination, but this method has poor reproducibility, poor separation of impurity spots, and cannot achieve accurate quantification, and can only perform limit checks.

[0005] Therefore, it is still necessary to develop an analytical detection method with good specificity, high sensitivity and convenient operation to accurately separate and detect impurities in sodium stearyl fumarate, so as to ensure the quality, safety and effectiveness of the drug. Summary of the invention

[0006] In view of the shortcomings of the determination methods of sodium stearyl fumarate impurities in the prior art, the purpose of the present invention is to provide a detection method with good specificity, high sensitivity, simple operation, high accuracy and good repeatability. The method adopts high performance liquid chromatography for determination, has good separation and quantitative effects, overcomes the problems of complex sample preparation, cumbersome operation, poor separation, inaccurate quantification and the like in the existing determination methods, and can conveniently and accurately separate and determine the impurities of sodium stearyl fumarate.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0008] In the implementation scheme, the detection method of the present invention is used to detect sodium stearyl fumarate impurities, including determination by high performance liquid chromatography, wherein the chromatographic column is a C1~C18 alkylsilane bonded silica gel chromatographic column, the mobile phase is a mixed solution of C1~C5 primary alcohol and water or acetonitrile and trifluoroacetic acid or tetrahydrofuran, isocratic elution is used, and the detector is a mass detector.

[0009] Preferably, in the above method of the present invention, the impurities are sodium stearyl maleate and stearyl alcohol.

[0010] Preferably, in the above method of the present invention, the injection volume is 5~20μl, the flow rate is 0.5~2.0ml / min, the column temperature is 20~40°C, the mobile phase is methanol-water-trifluoroacetic acid, and the detector is an evaporative light scattering detector, an electrospray detector or a differential shading detector.

[0011] Preferably, in the above method of the present invention, the mobile phase is methanol-water-trifluoroacetic acid in a volume ratio of 90:10:0.2 to 93:7:0.5, preferably 90:10:0.2.

[0012] Preferably, in the above method of the present invention, the injection volume is 10 μl, the flow rate is 1.0 ml / min, the column temperature is 30° C., and the detector is an evaporative light scattering detector (ELSD).

[0013] Preferably, in the above method of the present invention, the chromatographic column is an octylsilane bonded silica gel chromatographic column.

[0014] Preferably, in the above method of the present invention, the related substances of sodium stearyl fumarate are sodium stearyl maleate and stearyl alcohol impurities.

[0015] In a specific embodiment, the method of the present invention comprises the following steps:

[0016] (1) Preparation of system suitability solution: Take appropriate amounts of sodium stearyl fumarate, sodium stearyl maleate and stearyl alcohol respectively, and use diluent to prepare a solution containing about 0.1-2 mg of sodium stearyl fumarate and about 0.1-15 μg of sodium stearyl maleate and stearyl alcohol per 1 ml;

[0017] (2) Preparation of reference solution: Take appropriate amounts of sodium stearyl maleate and stearyl alcohol and use a diluent to prepare a solution containing approximately 0.1-15 μg of sodium stearyl maleate and stearyl alcohol per 1 ml;

[0018] (3) Preparation of test solution: Take an appropriate amount of sodium stearyl fumarate and use a diluent to prepare a solution containing approximately 0.1-2 mg of sodium stearyl fumarate per 1 ml;

[0019] (4) Chromatographic conditions: A chromatographic column with octylsilane bonded silica as filler was used; the column temperature was set to 30 °C; the mobile phase was methanol-water-trifluoroacetic acid, with isocratic elution and an injection volume of 1-100 μl; the flow rate was 1.0 ml / min; the detector was an evaporative light scattering detector (ELSD) from Alltech.

[0020] (5) Sample determination: Inject the system suitability solution, reference solution, and test solution in steps (1), (2), and (3) into a high performance liquid chromatograph, record the spectrum, and calculate the content of each impurity using the standard curve method.

[0021] In the above specific embodiment, in the method of the present invention, preferably, the diluent in step (1) is a primary alcohol having a carbon number of C1 to C5, acetonitrile, diethyl ether or a mixture thereof, more preferably methanol.

[0022] The detection method of the present invention realizes the determination of specific impurities of sodium stearyl fumarate. The method has good specificity and no interference in the determination; high sensitivity, and the limit of quantification is as low as 0.5 μg / ml; simple operation, simple sample preparation, and no derivatization; high accuracy, the recovery rate measured by the method is in the range of 98.0% to 101.6%, and the RSD is not greater than 5.0%; good repeatability, and the RSD of continuous injection of the method is not greater than 5.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Blank solution spectrum of Example 1;

[0024] Figure 2 System suitability solution profile of Example 1;

[0025] Figure 3 The sample solution spectrum of Example 1;

[0026] Figure 4 Blank solution spectrum of Example 2;

[0027] Figure 5 System suitability solution profile of Example 2;

[0028] Figure 6 The sample solution spectrum of Example 2;

[0029] Figure 7 Blank solution spectrum of Example 7;

[0030] Figure 8 System suitability solution profile for Example 7;

[0031] Fig. 9 Spectrum of the test solution of Example 7;

[0032] Fig.10 Comparative Example 1 Typical spectrum determined by the method of "Chinese Pharmacopoeia";

[0033] Fig.11 Comparative Example 2 Typical spectrum determined by the United States Pharmacopoeia method. DETAILED DESCRIPTION

[0034] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way.

[0035] Specificity test

[0036] Example 1

[0037] Chromatographic conditions:

[0038] Column: Agilent ZORBAX-SB-C8, 4.6×150 mm, 5 μm

[0039] Mobile phase: methanol-water-trifluoroacetic acid (90:10:0.2, v / v / v)

[0040] Flow rate: 1.0ml / min

[0041] Column temperature: 25°C

[0042] Injection volume: 10 μl

[0043] Detector: Evaporative Light Scattering Detector (ELSD)

[0044] Solution preparation:

[0045] Diluent (blank solution): methanol

[0046] Preparation of system suitability solution: Take appropriate amounts of sodium stearyl fumarate, sodium stearyl maleate and stearyl alcohol respectively, and use diluent to prepare a solution containing approximately 1 mg of sodium stearyl fumarate, approximately 5 μg of sodium stearyl maleate and stearyl alcohol per 1 ml.

[0047] Preparation of test solution: Take an appropriate amount of sodium stearyl fumarate and use a diluent to prepare a solution containing approximately 1 mg of sodium stearyl fumarate per 1 ml.

[0048] Determination method:

[0049] Accurately measure 10 μl of blank solution, system suitability solution, and test solution, inject them into the HPLC, and record the spectrum. The experimental results are shown in Figures 1 to 3 .

[0050] The results showed that the blank solution had no interference at the peaks of sodium stearyl fumarate and various impurities, and the detection method of the present invention had good specificity.

[0051] Example 2

[0052] Chromatographic conditions:

[0053] Column: Agilent ZORBAX-SB-C8, 4.6×150 mm, 5 μm

[0054] Mobile phase: methanol-water-trifluoroacetic acid (93:7:0.5, v / v / v)

[0055] Flow rate: 0.5ml / min

[0056] Column temperature: 40°C

[0057] Injection volume: 15 μl

[0058] Detector: Evaporative Light Scattering Detector (ELSD)

[0059] Solution preparation:

[0060] Diluent (blank solution): methanol

[0061] Preparation of system suitability solution: Take appropriate amounts of sodium stearyl fumarate, sodium stearyl maleate and stearyl alcohol respectively, and use diluent to prepare a solution containing approximately 1 mg of sodium stearyl fumarate, approximately 5 μg of sodium stearyl maleate and stearyl alcohol per 1 ml.

[0062] Preparation of test solution: Take an appropriate amount of sodium stearyl fumarate and use a diluent to prepare a solution containing approximately 1 mg of sodium stearyl fumarate per 1 ml.

[0063] Determination method:

[0064] Accurately measure 10 μl of blank solution, system suitability solution, and test solution, inject them into the HPLC, and record the spectrum. The experimental results are shown in Figures 4 to 6 .

[0065] The results showed that the blank solution had no interference at the peaks of sodium stearyl fumarate and various impurities, and the detection method of the present invention had good specificity.

[0066] Linearity test

[0067] Example 3

[0068] Chromatographic conditions:

[0069] Column: Waters Symmetry C8, 4.6×150 mm, 5 μm

[0070] Mobile phase: methanol-water-trifluoroacetic acid (90:10:0.5, v / v / v)

[0071] Flow rate: 1.0ml / min

[0072] Column temperature: 30°C

[0073] Injection volume: 5 μl

[0074] Detector: Evaporative Light Scattering Detector (ELSD)

[0075] Solution preparation:

[0076] Diluent: Methanol

[0077] Linear solution: Accurately weigh an appropriate amount of sodium stearyl fumarate, and use a diluent to prepare solutions containing approximately 250μg, 500μg, 1000μg, 1500μg, and 2000μg of sodium stearyl fumarate per 1ml; Accurately weigh an appropriate amount of sodium hard maleate and stearyl alcohol, and use a diluent to prepare solutions containing approximately 0.5μg, 1μg, 2.5μg, 5μg, and 10μg of sodium hard maleate and stearyl alcohol per 1ml.

[0078] Determination method:

[0079] Accurately measure 5 μl of each of the above linear solutions, inject into high performance liquid chromatograph, and record the spectrum. Linear regression is performed with the logarithmic value of the concentration of sodium stearyl fumarate and the logarithmic value of the corresponding peak area. The results are shown in Tables 1 to 3.

[0080] Table 1 Linearity test results - sodium stearyl fumarate

[0081]

[0082] Note: x represents concentration, μg / ml; y represents peak area.

[0083] The results show that the detection method of the present invention has a good linear relationship in the range of 265 μg / ml to 2120 μg / ml of sodium stearyl fumarate.

[0084] Table 2 Linearity test results - Sodium stearyl maleate

[0085]

[0086] Note: x represents concentration, μg / ml; y represents peak area.

[0087] The results show that the detection method of the present invention has a good linear relationship in the range of 0.5 μg / ml to 10.08 μg / ml of sodium stearyl maleate.

[0088] Table 3 Linearity test results - Stearyl alcohol

[0089]

[0090] Note: x represents concentration, μg / ml; y represents peak area.

[0091] The results show that the detection method of the present invention has a good linear relationship in the range of 0.5 μg / ml to 9.92 μg / ml of stearyl alcohol.

[0092] Limit of Quantitation and Limit of Detection Tests

[0093] Example 4

[0094] Chromatographic conditions:

[0095] Column: Agilent ZORBAX-SB-C8, 4.6×150 mm, 5 μm

[0096] Mobile phase: methanol-water-trifluoroacetic acid (90:10:0.2, v / v / v)

[0097] Flow rate: 1.0ml / min

[0098] Column temperature: 30°C

[0099] Injection volume: 10 μl

[0100] Detector: Evaporative Light Scattering Detector (ELSD)

[0101] Solution preparation:

[0102] Diluent: Methanol

[0103] Quantitative limit solution: Take appropriate amount of sodium stearyl maleate and stearyl alcohol, and use diluent to prepare a solution containing about 0.5 μg of sodium stearyl maleate and stearyl alcohol per 1 ml. (Equivalent to 0.05% of the test sample concentration)

[0104] Detection limit solution: Accurately measure an appropriate amount of quantitative limit solution and use a diluent to prepare a solution containing approximately 0.25 μg of sodium stearyl maleate and stearyl alcohol per 1 ml. (Equivalent to 0.025% of the test sample concentration)

[0105] Determination method:

[0106] Accurately measure 10 μl of the quantitative limit solution and the detection limit solution, inject them into the high performance liquid chromatograph, and record the spectrum. The results are shown in Tables 4 and 5.

[0107] Table 4 Quantitation limit test results - Sodium stearyl maleate

[0108]

[0109] Table 5 Quantitation limit test results - stearyl alcohol

[0110]

[0111] The results show that the signal-to-noise ratios of sodium stearyl maleate and stearyl alcohol at a test sample concentration of 0.05% are greater than 10, and at a test sample concentration of 0.025%, the signal-to-noise ratios are greater than 3. The detection method of the present invention has good sensitivity.

[0112] Accuracy test Example 5

[0113] Chromatographic conditions:

[0114] Column: Agilent ZORBAX-SB-C8, 4.6×150 mm, 5 μm

[0115] Mobile phase: methanol-water-trifluoroacetic acid (90:10:0.2, v / v / v)

[0116] Flow rate: 1.0ml / min

[0117] Column temperature: 35°C

[0118] Injection volume: 20 μl

[0119] Detector: Evaporative Light Scattering Detector (ELSD)

[0120] Solution preparation:

[0121] Diluent: Methanol

[0122] Accuracy solution: Take appropriate amounts of sodium stearyl fumarate, sodium stearyl maleate, and stearyl alcohol, and use diluent to prepare solutions containing about 1 mg of sodium stearyl fumarate, about 0.5 μg of sodium stearyl maleate, and about 0.5 μg of stearyl maleate and 0.5 μg of stearyl alcohol per ml as 0.05%, 0.25%, and 0.5% accuracy solutions, respectively. Prepare three copies of each concentration level accuracy solution in parallel.

[0123] Determination method:

[0124] Accurately measure 20 μl of each of the above accuracy solutions, inject them into the high performance liquid chromatograph, and record the spectrum. The results are shown in Tables 6 and 7.

[0125] Table 6 Accuracy test results - Sodium stearyl maleate

[0126]

[0127] Table 7 Accuracy test results - Stearyl alcohol

[0128]

[0129] The results showed that the recovery of sodium stearyl maleate in the accuracy solution of each concentration level was 98.4%-101.2%, with an RSD of 0.9%, and the recovery of stearyl alcohol was 98.0%-101.6%, with an RSD of 1.1%. The detection method of the present invention can accurately quantify the related substances of sodium stearyl fumarate.

[0130] Repeatability test

[0131] Example 6

[0132] Chromatographic conditions:

[0133] Column: Agilent Eclipse XDB-C8, 4.6×150 mm, 5 μm

[0134] Mobile phase: methanol-water-trifluoroacetic acid (93:7:0.2, v / v / v)

[0135] Flow rate: 2.0ml / min

[0136] Column temperature: 40°C

[0137] Injection volume: 10 μl

[0138] Detector: Evaporative Light Scattering Detector (ELSD)

[0139] Solution preparation:

[0140] Diluent: Methanol

[0141] Repeatable solution: Take appropriate amounts of sodium stearyl fumarate, sodium stearyl maleate, and stearyl alcohol, and use a diluent to prepare a solution containing about 1 mg of sodium stearyl fumarate, about 2.5 μg of sodium stearyl maleate, and about 2.5 μg of stearyl alcohol per ml. Prepare 6 portions in parallel.

[0142] Determination method:

[0143] Accurately measure 10 μl of each of the above reproducible solutions, inject them into the high performance liquid chromatograph, and record the spectrum. The results are shown in Table 8.

[0144] Table 8 Repeatability test results

[0145]

[0146] The results showed that the RSDs of sodium stearyl maleate and stearyl alcohol in 6 replicate solutions were 2.5% and 3.2%, respectively, with good repeatability.

[0147] Determination of Related Substances in Sodium Stearyl Fumarate

[0148] Example 7

[0149] Chromatographic conditions:

[0150] Column: Agilent Eclipse XDB-C8, 4.6×150 mm, 5 μm

[0151] Mobile phase: methanol-water-trifluoroacetic acid (93:7:0.5, v / v / v)

[0152] Flow rate: 1.0ml / min

[0153] Column temperature: 30°C

[0154] Injection volume: 10 μl

[0155] Detector: Evaporative Light Scattering Detector (ELSD)

[0156] Solution preparation:

[0157] Diluent (blank solution): methanol

[0158] Preparation of system suitability solution: Take appropriate amounts of sodium stearyl fumarate, sodium stearyl maleate and stearyl alcohol respectively, and use diluent to prepare a solution containing approximately 1 mg of sodium stearyl fumarate, approximately 5 μg of sodium stearyl maleate and stearyl alcohol per 1 ml.

[0159] Preparation of reference solution: Take appropriate amount of sodium stearyl maleate and stearyl alcohol, and use diluent to prepare a solution containing about 2.5 μg of sodium stearyl maleate and stearyl alcohol per 1 ml.

[0160] Preparation of test solution: Take appropriate amount of sodium stearyl fumarate from different batches and use diluent to prepare a solution containing approximately 1 mg of sodium stearyl fumarate per 1 ml.

[0161] Determination method:

[0162] Accurately measure 10 μl of blank solution, system suitability solution, reference solution, and test solution, inject into high performance liquid chromatograph, and record the spectrum. In the spectrum of system suitability solution, the separation degree of sodium stearyl fumarate, sodium stearyl maleate, and stearyl alcohol should meet the requirements; calculate the content of each impurity by standard curve method, and the results are shown in Table 9. The spectrum of blank solution, system suitability solution, and test solution is shown in Table 9. Figures 7-9 .

[0163] Table 9 Determination results of impurities of sodium stearyl fumarate

[0164]

[0165] Comparative Example 1 Determination of Related Substances of Sodium Stearyl Fumarate (Chinese Pharmacopoeia Method)

[0166] Chromatographic conditions:

[0167] Chromatographic column: HP-1, 0.25mm×30m, 0.25μm

[0168] Carrier gas: Nitrogen

[0169] Inlet temperature: 250℃

[0170] Detector temperature: 320℃

[0171] Heating program: Initial column temperature is 180°C, maintained for 1 minute, then increased from 180°C to 320°C at a heating rate of 7°C per minute, maintained for 15 minutes

[0172] Injection volume: 2 μl

[0173] Detector: Flame Ion Detector (FID)

[0174] Solution preparation:

[0175] Diluent (blank solution): N,O-bis(trimethylsilyl)trifluoroacetamide (containing 1% trimethylchlorosilane)

[0176] Preparation of test solution: Take an appropriate amount of sodium stearyl fumarate and an appropriate amount of sodium stearyl fumarate reference substance, accurately add 1 ml of diluent, seal, heat at 70°C for 1 hour, filter, and take the filtrate as the test solution.

[0177] Determination method:

[0178] Accurately measure 2 μl of blank solution and test solution, inject into gas chromatograph, and record the spectrum. Fig.10 .

[0179] Comparative Example 2 Determination of Related Substances of Sodium Stearyl Fumarate (United States Pharmacopoeia Method)

[0180] Solution preparation:

[0181] Diluent: Measure chloroform and glacial acetic acid in a ratio of 4:1, mix well, and you have the product.

[0182] Color developer: measure sulfuric acid and ethanol in a ratio of 1:9, mix well.

[0183] Developing agent: Measure n-hexane, toluene and glacial acetic acid, mix them evenly in the ratio of 5:5:1, and mix well to obtain.

[0184] Sodium stearoyl maleate reference solution: Take an appropriate amount of sodium stearoyl maleate, dissolve it in diluent and dilute it to make a solution containing approximately 0.1 mg of sodium stearoyl maleate per 1 ml, shake well, and obtain.

[0185] Stearyl alcohol reference solution: Take an appropriate amount of stearyl alcohol, dissolve it in diluent and dilute it to make a solution containing about 0.1 mg of stearyl alcohol per 1 ml, shake well, and the solution is ready.

[0186] Test solution: Take an appropriate amount of sodium stearyl fumarate, dissolve it in diluent and dilute it to make a solution containing about 20 mg of sodium stearyl fumarate per 1 ml, shake well, and you have it.

[0187] Determination method:

[0188] Take 5μl of sodium stearoyl maleate reference solution, 10μl of stearyl alcohol reference solution and test solution, respectively, and spot them on the same activated silica gel thin layer plate, evaporate the solvent, put the sample spotted thin layer plate into a chromatography cylinder with a 10mm thick layer of chloroform at the bottom, let the solvent front reach the upper edge of the spot, take out the thin layer plate, and dry it in a cold air flow; repeat the above operation once, so that the shape of the spot can be linear; put the thin layer plate into a chromatography cylinder saturated with a developing agent, when the solvent front reaches 15cm on the thin layer plate, take out the thin layer plate, dry it for 10min, and then dry it at 90℃ for 2min, and let it cool to room temperature; put the thin layer plate back into the chromatography cylinder for development, when the solvent front reaches 15cm on the thin layer plate, take out the thin layer plate, dry it at room temperature for 15min, spray the color developer, dry it at 150℃ for 15min, dark spots appear, and let it cool. See the thin layer map of the test solution and the reference solution. Fig.11 .

Claims

1. A method for determining impurities in sodium stearyl fumarate, comprising the step of using high performance liquid chromatography for determination, wherein: The chromatographic column is an octylsilane bonded silica gel chromatographic column, the mobile phase is methanol-water-trifluoroacetic acid, the volume ratio is 90:10:0.2~93:7:0.5, isocratic elution is adopted, the detector is a mass detector, and the impurities are sodium stearyl maleate and stearyl alcohol.

2. The assay method according to claim 1, wherein the volume ratio of the mobile phase methanol-water-trifluoroacetic acid is 90:10:0.

2.

3. The determination method according to claim 1, wherein the injection volume is 5-20 μl, the flow rate is 0.5-2.0 ml / min, the column temperature is 20-40°C, and the detector is an evaporative light scattering detector, an electrospray detector or a differential shading detector.

4. The determination method according to claim 3, wherein the injection volume is 10 μl, the flow rate is 1.0 ml / min, the column temperature is 30°C, and the detector is an evaporative light scattering detector (ELSD).

5. The assay method according to any one of claims 1 to 4, comprising the following steps: (1) Preparation of system suitability solution: Take appropriate amounts of sodium stearyl fumarate, sodium stearyl maleate and stearyl alcohol, and use diluent to prepare a solution containing approximately 0.1-2 mg sodium stearyl fumarate and 0.1-15 μg sodium stearyl maleate and stearyl alcohol per 1 ml; (2) Preparation of reference solution: Take appropriate amounts of sodium stearyl maleate and stearyl alcohol and use a diluent to prepare a solution containing 0.1-15 μg of sodium stearyl maleate and stearyl alcohol per 1 ml; (3) Preparation of test solution: Take an appropriate amount of sodium stearyl fumarate and use a diluent to prepare a solution containing 0.1-2 mg of sodium stearyl fumarate per 1 ml; (4) Sample determination: Inject the system suitability solution, reference solution, and test solution into the HPLC, record the spectrum, and calculate the content of each impurity using the standard curve method.

6. The assay method according to claim 5, wherein the diluent is selected from a C1-C5 primary alcohol, acetonitrile, ether or a mixture thereof. The assay method according to claim 6 , wherein the diluent is methanol.

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