Method for determining residual quantity of petroleum ether in icotinamide benzonate bulk drug by using capillary gas chromatography

Abstract

The invention discloses a method for determining the residual quantity of petroleum ether in an isonicotinamide benzonate bulk drug by using capillary gas chromatography, and belongs to the technical field of drug analysis. According to the method, an external standard method is adopted for quantification, a 6% cyanopropyl phenyl-94% dimethyl polysiloxane capillary column is used as a chromatographic column, and an FID detector is adopted for detection; the residual quantity of the petroleum ether is the total residual quantity of normal hexane, 2-methylpentane and 3-methylpentane in the petroleum ether. The method disclosed by the invention is good in specificity, good in linearity, reliable in accuracy and high in sensitivity; the gas chromatographic method can be used for rapidly, effectively, accurately and reliably separating and detecting the residual amount of petroleum ether in the isopropylamine bulk drug, and is beneficial to improving the product quality of the isopropylamine bulk drug and improving the medication safety of a patient.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and more specifically, relates to a method for determining the residual amount of petroleum ether in prothionamide raw material medicine by using capillary gas chromatography. Background Art

[0002] Prothionamide is a derivative of isonicotinic acid, mainly used as an anti-tuberculosis drug to inhibit the synthesis of mycolic acid in tuberculosis bacteria. This product has partial cross-resistance with ethionamide. The organic solvent petroleum ether is used in the synthesis process of prothionamide API.

[0003] Petroleum ether is considered as a Class IV solvent in the General Chapter 0861 of the 2015 edition of the Chinese Pharmacopoeia, and there is no sufficient toxicological data. Petroleum ether is mainly composed of low molecular weight hydrocarbons, mainly a mixture of pentane and hexane; hexane belongs to the restricted Class II solvent, and if it is not controlled, long-term use may cause great harm to human health; pentane (mainly 2-methylpentane and 3-methylpentane), and the Chinese Pharmacopoeia does not make clear limit requirements for pentane residues. Therefore, a method that can better detect the residual amount of petroleum ether in drugs, that is, the total residual amount of n-hexane, 2-methylpentane and 3-methylpentane, is needed to ensure the safety of human medication. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for determining the residual amount of petroleum ether in the raw material drug of prothionamide by using capillary gas chromatography. The capillary gas chromatography can quickly, effectively, accurately and reliably detect the residual amount of petroleum ether in prothionamide, which is beneficial to improving the product quality of the raw material drug prothionamide.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for determining the residual amount of petroleum ether in a prothionamide raw material drug by using a capillary gas chromatography method, wherein the method adopts an external standard method for quantification, a 6% cyanopropylphenyl-94% dimethylpolysiloxane capillary column is used as a chromatographic column, and an FID detector is used for detection; the residual amount of petroleum ether is the total residual amount of n-hexane, 2-methylpentane and 3-methylpentane in the petroleum ether.

[0007] Preferably, the detection conditions are as follows: the carrier gas is nitrogen; the carrier gas flow rate is 2.0 mL / min; the initial column temperature is 35°C, maintained for 5 min, increased to 220°C at a rate of 20°C / min, maintained for 5 min; the FID detector temperature is 250°C; and the injection port temperature is 200°C.

[0008] Preferably, the split ratio is 15:1, the injection method is headspace injection, and the injection volume is 5 mL.

[0009] Preferably, the equilibrium temperature of the headspace bottle is 85° C. and the equilibrium time is 30 minutes.

[0010] Preferably, the test solution and the reference solution are prepared using the solvent N,N-dimethylformamide.

[0011] Preferably, the preparation process of the test solution is: accurately weigh 0.5 g of prothionamide, place it in an empty bottle, accurately add 5 mL of N,N-dimethylformamide solution, seal it, and dissolve it by ultrasonication to obtain the test solution.

[0012] Preferably, the preparation process of the reference solution is: take appropriate amounts of 2-methylpentane, 3-methylpentane and n-hexane, dissolve and dilute with N,N-dimethylformamide solution to form a solution containing 29 μg of 2-methylpentane, 29 μg of 3-methylpentane and 29 μg of n-hexane per 1 mL, as the reference solution.

[0013] Preferably, the test solution has a peak in the chromatogram with the same retention time as the reference solution, and the sum of the contents of 2-methylpentane, 3-methylpentane and n-hexane is no more than 0.029% by peak area calculation using the external standard method.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1) The method of the present invention has good specificity, good linearity, reliable accuracy, high sensitivity, and can be applied to most pharmaceutical companies;

[0016] 2) Compared with the method reported in the literature that the residual amount of petroleum ether in the raw material drug is mostly calculated by the peak area of ​​each single component of petroleum ether, the method of the present invention calculates the residual amount of petroleum ether by the sum of the peak areas of each component of petroleum ether, which more accurately represents the residual amount of petroleum ether that may be contained in the drug to be tested, which is beneficial to improving the product quality of the prothionamide raw material drug and improving the safety of patient use. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a chromatogram of the petroleum ether residue in the prothionamide raw material drug determined by capillary gas chromatography; Figure 1 a is the blank spectrum of this method; Figure 1 b Spectrum of the reference solution of this method; Figure 1 c is the sample solution spectrum of this method;

[0018] Figure 2 is the standard curve of 2-methylpentane;

[0019] Figure 3 is the standard curve of 3-methylpentane;

[0020] Figure 4 This is the standard curve of n-hexane. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0022] In the following examples, the instruments, reagents, reference substances and sample information used are as follows:

[0023] The model of the high performance gas chromatograph used is 8890 / 7697A, manufactured by Agilent Technologies.

[0024] The sample selected is prothionamide produced by Jiangsu Cixin Pharmaceutical, batch number 201910001.

[0025] Table 1 Test reagents

[0026] Reagents batch number Manufacturer content% N,N-Dimethylformamide 20190313 Yonghua Chemical / Acetonitrile 19065195 TEDIA / Ethanol C1927061 Aladdin 99.8％ 2-Methylpentane EK533 Jiuding Chemical 98％ 3-Methylpentane OX214 Jiuding Chemical 99％ n-Hexane 20190605 Sinopharm Group 97.05 Chloroform 1802022 Xilong Science 99.0％ benzene 20181123 Yonghua Chemical 99.8％ Toluene 1811232 Xilong Science 99.8％

[0027] Example 1

[0028] A method for determining the residual amount of petroleum ether in prothionamide raw material medicine by capillary gas chromatography comprises the following steps:

[0029] 1) Prepare the test solution and control solution:

[0030] The preparation method of the test solution is as follows: accurately weigh 0.5 g of prothionamide, place it in an empty bottle, accurately add 5 mL of N,N-dimethylformamide solution, seal it, and dissolve it by ultrasonication to serve as the test solution;

[0031] The reference solution was prepared as follows: appropriate amounts of 2-methylpentane, 3-methylpentane, and n-hexane were dissolved and diluted with N,N-dimethylformamide solution to form a solution containing 29 μg of 2-methylpentane, 29 μg of 3-methylpentane, and 29 μg of n-hexane per 1 mL, as the reference solution;

[0032] 2) Set the gas chromatography detection conditions: the carrier gas is nitrogen; the carrier gas flow rate is 2.0 mL / min; the initial column temperature is 35°C, maintained for 5 min, and then increased to 220°C at a rate of 20°C / min and maintained for 5 min; the FID detector temperature is 250°C; the injection port temperature is 200°C; the split ratio is 15:1, the injection method is headspace injection, and the injection volume is 5 mL; the equilibrium temperature of the headspace bottle is 85°C, and the equilibrium time is 30 minutes;

[0033] 3) Accurately pipette the test solution and the control solution respectively, inject them into the gas chromatograph, detect and record the chromatogram;

[0034] 4) According to the chromatogram, a linear regression equation was made with the peak area A as the ordinate and the linear solution concentration C as the abscissa. The linear equation of 2-methylpentane was A=35.644C+2.0206, the linear equation of 3-methylpentane was A=31.901C-4.7971, and the linear equation of n-hexane was A=29.086C+2.5853. The linear concentration range of 2-methylpentane was 0.029 μg / mL to 42.39 μg / mL, and the limit of quantification was 0.029 μg / mL; the linear concentration range of 3-methylpentane was 0.029 μg / mL to 43.15 μg / mL, and the limit of quantification was 0.029 μg / mL; the linear concentration range of n-hexane was 0.029 μg / mL to 42.43 μg / mL, and the limit of quantification was 0.029 μg / mL.

[0035] Example 2

[0036] The organic solvents used in the synthesis process of the raw material drug prothionamide include chloroform, petroleum ether and ethanol, and toluene is used in the synthesis process of the starting material 4-cyanopyridine, among which toluene and ethanol will introduce a type of solvent, benzene.

[0037] 1. Exclusivity

[0038] Take 0.5 g of ethanol, accurately weigh it, put it into a 50 mL volumetric flask, dissolve it with N,N-dimethylformamide (DMF) and dilute it to the mark as the ethanol positioning solution;

[0039] Take 0.18 g of chloroform, accurately weigh it, put it into a 50 mL volumetric flask, dissolve it with DMF and dilute it to the mark as the chloroform positioning solution;

[0040] Take 0.145 g of n-hexane, 0.145 g of 2-methylpentane, and 0.145 g of 3-methylpentane, accurately weigh them, put them into different 50 mL volumetric bottles, dissolve them with DMF and dilute to the scale as n-hexane, 2-methylpentane and 3-methylpentane positioning solutions;

[0041] Take 0.145 g of 2-methylpentane, weigh accurately, and transfer to the same 50 mL volumetric flask, dissolve with DMF and dilute to the mark as the 2-methylpentane positioning solution;

[0042] Take 0.145 g of 3-methylpentane, weigh accurately, put it into a 50 mL volumetric flask, dissolve it with DMF and dilute it to the mark as the 3-methylpentane positioning solution;

[0043] Take 0.445 g of toluene, weigh accurately, put it into a 50 mL volumetric flask, dissolve and dilute to the mark as the toluene positioning solution;

[0044] Take 0.2 g of benzene, weigh accurately, put it into a 100 mL volumetric flask, dissolve and dilute to the mark as the benzene positioning solution;

[0045] Accurately measure 1 mL each of n-hexane, 2-methylpentane, and 3-methylpentane solution into a 100 mL volumetric flask, and dilute to the mark with DMF as the reference solution.

[0046] Accurately measure 5 mL of solvent, each positioning solution, and reference solution, place them in a headspace bottle, seal, inject the sample into the headspace, and record the chromatogram. Investigate the peak time and separation of each component. The results are shown in Table 2 and Figure 1 .

[0047] Table 2 Impurity location and separation

[0048]

[0049] From Table 2 and Figure 1 It can be seen that the method of the present invention has good specificity. When detecting the content of petroleum ether in prothionamide, N,N-dimethylformamide and other residual solvents of prothionamide do not interfere with the determination of 2-methylpentane, 3-methylpentane and n-hexane.

[0050] 2. Limit of Quantitation

[0051] Take appropriate amount of n-hexane, 2-methylpentane and 3-methylpentane reference substances, dissolve them in N, N-dimethylformamide, and dilute them step by step until the signal-to-noise ratio of each solvent to be tested is 10, which is the quantitative limit solution. The quantitative limit concentration does not exceed half of the limit, and the method has good sensitivity, as shown in Table 3.

[0052] Table 3 Quantitation limit test results

[0053]

[0054]

[0055] As shown in Table 3, the quantitative limit concentrations were all less than half of the limit, and the method of the present invention had good sensitivity.

[0056] 3. Linearity and range

[0057] Take appropriate amount of n-hexane, 2-methylpentane, 3-methylpentane reference substances, dissolve and dilute with N, N-dimethylformamide to prepare a series of linear solutions (take 6 concentration points in the range of 150% of the limit of quantification), accurately measure 5mL of each linear solution, put it into a headspace bottle, inject it into the gas chromatograph, record the chromatogram, measure the peak area, use the linear solution concentration C as the abscissa and the peak area A as the ordinate, perform linear regression by the least square method, and calculate the linear regression equation and correlation coefficient of each compound. The results are shown in Table 4 and Figures 2 to 4.

[0058] Table 4 Linearity determination results

[0059]

[0060] From Table 4 and Figures 2 to 4 It can be seen that each solvent to be tested has good linearity within the range of quantification limit to 150% of the limit, the correlation coefficient r is greater than 0.99, and the absolute value of the Y-axis intercept at 100% response value is no more than 10%, indicating good linearity.

[0061] 4. Accuracy

[0062] Take an appropriate amount of each solvent reference substance to be tested, add it to the prothionamide raw material at a ratio of 50% to 150% of the limit of each solvent, and prepare 3 portions for each level. The preparation process is as follows:

[0063] Take 0.145 g each of n-hexane, 2-methylpentane and 3-methylpentane, accurately weigh them, put them into the same 100 mL volumetric flask, dissolve them with N,N-dimethylformamide and dilute to the scale, shake well, and use them as the reference substance stock solution.

[0064] Accurately measure 2 mL of the reference stock solution into a 100 mL volumetric flask, dilute to the mark with N,N-dimethylformamide, and shake well to prepare the reference solution.

[0065] Accurately measure 1 mL of the reference stock solution into a 100 mL volumetric flask, dilute to the mark with N,N-dimethylformamide, and shake well to prepare the 50% impurity limit solution.

[0066] Accurately measure 3 mL of the reference stock solution into a 100 mL volumetric flask, dilute to the mark with N,N-dimethylformamide, and shake well to prepare the impurity limit 150% solution.

[0067] Take 0.5 g of prothionamide, weigh it accurately, put it into a headspace bottle, accurately add 5 mL of 50% impurity limit solution, seal it, and dissolve it by ultrasonication to obtain the 50% recovery rate sample solution.

[0068] Take 0.5 g of prothionamide, accurately weigh it, put it into a headspace bottle, accurately add 5 mL of the reference solution, seal it, and dissolve it by ultrasonication to prepare the sample solution with a 100% recovery rate.

[0069] Take 0.5 g of prothionamide, weigh it accurately, put it into a headspace bottle, accurately add 5 mL of 150% impurity limit solution, seal it, and dissolve it by ultrasonication to obtain the 150% recovery rate sample solution.

[0070] Unspecified test solution: Take 0.5 g of prothionamide, accurately weigh it, put it into a headspace bottle, accurately add 5 mL of N,N-dimethylformamide, seal it, and dissolve it by ultrasonication as the test solution.

[0071] Accurately measure 5 mL of solvent, reference solution, test solution, and recovery sample solution into different headspace bottles, record the peak area of ​​each known impurity, calculate the measured amount by peak area according to the external standard method, deduct the background amount of the test sample, and calculate the recovery rate and RSD. The results are shown in Tables 5 to 7.

[0072] Table 5 2-methylpentane recovery test results

[0073]

[0074] Table 6 3-methylpentane recovery test results

[0075]

[0076]

[0077] Table 7 Recovery test results of n-hexane

[0078]

[0079] It can be seen from Tables 5 to 7 that the recoveries of 2-methylpentane, 3-methylpentane and n-hexane at three concentration levels of high (150% of the limit), medium (100% of the limit) and low (50% of the limit) are all between 90% and 110%, and the recovery RSD (n=9) is no more than 10.0%, indicating that the method has good accuracy.

[0080] 5. Repeatability

[0081] Since n-hexane, 2-methylpentane and 3-methylpentane were not detected in the sample, the addition method was adopted (taking an appropriate amount of solvent, adding it to the prothionamide raw material according to the limit 100%, and preparing 6 portions in parallel), and the content of each solvent to be tested was calculated by peak area according to the external standard method. The results are shown in Table 8.

[0082] Table 8 Results of the residual solvent content in the repeatability test

[0083]

[0084]

[0085] It can be seen from Table 8 that the RSD of each solvent content to be tested is no more than 10.0%, and the method of the present invention has good repeatability.

[0086] 6. Solution stability

[0087] Accurately measure 1 mL each of n-hexane, 2-methylpentane, and 3-methylpentane fixing solutions into a 100 mL volumetric flask, and add DMF to dilute to the scale as the reference solution.

[0088] 5 mL of the reference solution was accurately measured and dispensed into headspace bottles. The headspace samples were injected at different times to examine the stability of the solution at different times based on the change in peak area. The results are shown in Table 9.

[0089] Table 9 Stability results of reference solution

[0090] time 2-Methylpentane 3-Methylpentane n-Hexane 0h 1007.92 943.54 867.59 3h40min 992.77 929.48 854.72 8h40min 1023.56 958.03 881.03 11h30min 961.67 909.52 841.32 16h 1006.91 942.99 867.55 19h 996.89 934.19 859.23 22h 1035.55 970.08 892.53 26h50min 1034.00 968.89 891.62 RSD 2.5％ 2.2％ 2.1％

[0091] 7. Durability

[0092] Adjust the chromatographic conditions (initial column temperature ±5°C, split ratio ±5, headspace bottle equilibrium temperature ±5°C) to investigate the effect of slight changes in chromatographic conditions on the measurement results. The solution preparation process is as follows:

[0093] Take 0.145 g each of n-hexane, 2-methylpentane and 3-methylpentane, accurately weigh them, put them into the same 100 mL volumetric flask, dilute to the scale with DMF, shake well, and use them as the reference substance stock solution.

[0094] Accurately measure 2 mL of the reference stock solution into a 100 mL volumetric flask, dilute to the mark with DMF, shake well, and use this as the reference solution. Prepare 2 portions in the same way.

[0095] Take 0.5 g of prothionamide, put it into the headspace bottle, accurately add 5 mL of the reference solution, seal it, and dissolve it by ultrasonication to prepare the test solution.

[0096] Accurately measure 5 mL of solvent and reference solution into different headspace bottles, seal them, and inject the sample into the headspace. Dissolve the test solution by ultrasonic and inject the sample into the headspace. Calculate the content of each solvent to be tested by peak area according to the external standard method. The results are shown in Tables 10 to 13.

[0097] Table 10 Effect of different starting column temperatures on system adaptability

[0098]

[0099] Table 11 Effect of different split ratios on system adaptability

[0100]

[0101] Table 12 Effect of different headspace equilibrium temperatures on system adaptability

[0102]

[0103] Table 13 Results of determination of residual solvent content in test solution with slight changes in chromatographic conditions

[0104]

[0105]

[0106] It can be seen from Tables 10 to 13 that the chromatographic conditions undergo slight changes (initial column temperature ±5°C, split ratio ±5, headspace bottle equilibrium temperature ±5°C), the RSD (n=6) of the peak area of ​​the chromatogram of the reference solution is no more than 10.0%, and the separation degree of each solvent is greater than 1.5; under different chromatographic conditions, the RSD of the content of each solvent in the test solution does not exceed 10.0%, and the method of the present invention has good durability.

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the embodiments. The embodiments in this application and the features in the embodiments can be combined with each other in any way without conflict. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. In addition, the technical details not described in detail in this specification belong to the common technology of those skilled in the art, so this specification will not be repeated.

Claims

1. A method for determining the residual amount of petroleum ether in the raw material of prothionamide by capillary gas chromatography, characterized in that: The external standard method is used for quantification, a 6% cyanopropylphenyl-94% dimethylpolysiloxane capillary column is used as the chromatographic column, and a FID detector is used for detection; the petroleum ether residue is the total residue of n-hexane, 2-methylpentane and 3-methylpentane in the petroleum ether.

2. The method for determining the residual amount of petroleum ether in the prothionamide bulk drug by capillary gas chromatography according to claim 1, characterized in that: The test conditions are: The carrier gas is nitrogen; The carrier gas flow rate was 2.0 mL / min; The initial column temperature was 35 °C, maintained for 5 min, then raised to 220 °C at a rate of 20 °C / min and maintained for 5 min; The FID detector temperature is 250°C; The injection port temperature was 200 °C.

3. The method for determining the residual amount of petroleum ether in the prothionamide bulk drug by capillary gas chromatography according to claim 1, characterized in that: The split ratio was 15:1, the injection method was headspace injection, and the injection volume was 5 mL.

4. The method for determining the residual amount of petroleum ether in the prothionamide bulk drug by capillary gas chromatography according to claim 1, characterized in that: The equilibration temperature of the headspace vial was 85°C and the equilibration time was 30 minutes.

5. The method for determining the residual amount of petroleum ether in the prothionamide bulk drug by capillary gas chromatography according to claim 1, characterized in that: The test solution and the reference solution were prepared using the solvent N,N-dimethylformamide.

6. The method for determining the residual amount of petroleum ether in the prothionamide bulk drug by capillary gas chromatography according to claim 5, characterized in that: The preparation process of the test solution is as follows: accurately weigh 0.5 g of prothionamide, place it in an empty bottle, accurately add 5 mL of N,N-dimethylformamide solution, seal it, and dissolve it by ultrasonication to obtain the test solution.

7. The method for determining the residual amount of petroleum ether in the prothionamide bulk drug by capillary gas chromatography according to claim 5, characterized in that: The preparation process of the reference solution is as follows: take appropriate amounts of 2-methylpentane, 3-methylpentane, and n-hexane, dissolve and dilute with N,N-dimethylformamide solution to form a solution containing 29 μg of 2-methylpentane, 29 μg of 3-methylpentane, and 29 μg of n-hexane per 1 mL, as the reference solution.

8. The method for determining the residual amount of petroleum ether in the prothionamide bulk drug by capillary gas chromatography according to claim 1, characterized in that: There is a peak in the chromatogram of the test solution with the same retention time as the reference solution. Calculated by peak area using the external standard method, the sum of the contents of 2-methylpentane, 3-methylpentane and n-hexane is not greater than 0.029%.

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