Method for separating and determining N-isobutyl acrylamide in rifudine bulk drug

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and determining N-isobutyl acrylamide in a rifudine bulk drug. The method is an LC-MS / MS mass spectrometry method, large diisobutyl bonded monofunctional silane is adopted as a chromatographic column filler, a water phase is adopted as a mobile phase A and an organic solvent is adopted as a mobile phase B for elution, and then a tandem quadrupole mass spectrometry method is adopted for detection. According to the method, multi-reaction detection is carried out in an electrospray ion source positive ion mode, and a detection ion pair of N-isobutyl acrylamide with the mass-to-charge ratio of 128.2 m / z to 72.1 m / z is used. According to the method, a limit method is adopted, a matrix does not interfere with detection of impurity N-isobutyl acrylamide, meanwhile, high reproducibility and high sensitivity of N-isobutyl acrylamide can be achieved through the method, LOD can reach 0.3 ng / ml, and the method has important significance on quality control of rifudine bulk drugs.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and determining N-isobutylacrylamide in rifabutin bulk drug substance. Background Art

[0002] Acrylamide compounds are Class 2A carcinogens certified by the International Agency for Research on Cancer (IARC). A large number of experiments have found that such compounds have significant neurotoxicity, reproductive and genetic toxicity, seriously endangering human health. Therefore, establishing a rapid and sensitive monitoring and analysis method for N-isobutylacrylamide in drugs is of great significance for understanding the quality of drugs, ensuring public safety and population health.

[0003] Existing studies have shown that N-isobutylacrylamide may be present in rifabutin bulk drug substance. The molecular formula of N-isobutylacrylamide is C 6 H 15 NO 3 , and its structural formula is shown in Formula I. The molecular formula of rifabutin is C 46 H 62 N 4 O 11 , and its structural formula is shown in Formula II.

[0004]

[0005] In the process of drug synthesis, it is necessary to strictly control the content of N-isobutylacrylamide impurities in the finished product. N-isobutylacrylamide is an unstable compound, and ordinary gas chromatography detectors do not respond in a timely manner during its detection, so biochemical treatment of N-isobutylacrylamide should be done before loading onto the machine. At present, there are relatively few studies on the content analysis and toxicology of N-isobutylacrylamide in drugs internationally, and there is a lack of systematic research results. Therefore, it is necessary to find a more effective, reliable and simple detection method for N-isobutylacrylamide in drugs, which is of great significance for the toxicity detection and research of N-isobutylacrylamide.

[0006] For N-isobutylacrylamide in rifabutin bulk drug substance, its content needs to be strictly controlled. The detection sensitivities of the currently reported related detection methods cannot meet the requirements of the control limit of rifabutin bulk drug substance. Therefore, it is necessary to further develop a new detection method to achieve the content control of N-isobutylacrylamide in rifabutin bulk drug substance. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a method for separating N-isobutylacrylamide from rifabutin bulk drug based on LC-MS / MS method. This method can effectively separate N-isobutylacrylamide from rifabutin bulk drug, which is of great significance for the impurity research of rifabutin bulk drug.

[0008] To achieve the above objective, the present invention adopts the following technical solutions:

[0009] A method for separating N-isobutylacrylamide from rifabutin bulk drug based on LC-MS / MS method, the structural formula of the N-isobutylacrylamide is shown in Formula I, and the structural formula of the rifabutin is shown in Formula II; the method includes: using a relatively large diisobutyl-bonded monofunctional silane as the filler for the chromatographic column, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is ultrapure water, and the mobile phase B is an organic solvent; separating N-isobutylacrylamide by elution;

[0010]

[0011] Further, the volume ratio of the mobile phase A to the mobile phase B is 40:60.

[0012] Further, the organic solvent is acetonitrile.

[0013] There are many factors affecting the separation of drug impurities. Under the determined stationary phase conditions, for a determined sample, the composition of the mobile phase is the key factor affecting the detection method. The concentration of organic ammonium affects the response and peak shape of N-isobutylacrylamide impurities, so water is selected. And the type of water has a great influence on the impurity response. When using "C'estbon" water as the mobile phase, the impurity response is low; when using ultrapure water as the mobile phase, the impurity response is high, so ultrapure water is selected as the mobile phase A.

[0014] Further, the flow rate of the mobile phase is 0.9 - 1.1 ml / min, preferably 1.0 ml / min; the column oven temperature of the chromatographic column is 35 - 45 °C, preferably 40 °C.

[0015] Further, the specification of the chromatographic column is 150×4.6 mm, 5 μm.

[0016] For this specification of reversed-phase analytical column, the optimal mobile phase flow rate is generally selected as 1 ml / min. At high flow rates, the peak broadening caused by the solute transfer effect will lead to a decrease in column efficiency and resolution.

[0017] Preferably, the chromatographic column adopts an Agilent ZORBAX SB-C18 column.

[0018] The chromatographic column of the present invention uses a relatively large diisobutyl-bonded monofunctional silane as the filler

[0019] The chromatographic column has high selectivity and sensitivity for N-isobutylacrylamide. The chromatographic column has high selectivity and sensitivity for N-isobutylacrylamide.

[0020] The second object of the present invention is to provide a method for determining N-isobutylacrylamide in rifabutin bulk drugs, which can effectively determine N-isobutylacrylamide in rifabutin bulk drugs, and the method has high sensitivity and good reproducibility.

[0021] To achieve the above object, the present invention adopts the following technical solutions:

[0022] The method for determining N-isobutylacrylamide in rifabutin bulk drugs is to separate N-isobutylacrylamide in rifabutin bulk drugs by the aforementioned separation method; then enter the detector for detection; the detector is a tandem quadrupole mass spectrometer.

[0023] The method of the present invention is an LC-MS / MS mass spectrometry method, adopting the limit method, and the matrix does not interfere with the detection of the impurity N-isobutylacrylamide.

[0024] Further, the detector is MS / MS.

[0025] Further, the ion source of the tandem quadrupole mass spectrometer is an ESI source; the method is multiple reaction monitoring (MRM) in the positive ion mode.

[0026] Further, the selected detection ion pair of N-isobutylacrylamide is a mass-to-charge ratio of 128.2 m / z → 72.1 m / z.

[0027] Further, before separation, an acetonitrile diluent is used to prepare the sample to be measured.

[0028] As a preferred method of the present invention, it can be specifically realized according to the following method:

[0029] (1) The selected detection ion pair of N-isobutylacrylamide is a mass-to-charge ratio of 128.2 m / z → 72.1 m / z;

[0030] (2) Take an appropriate amount of N-isobutylacrylamide, dissolve the sample with a 60% acetonitrile aqueous solution (acetonitrile V: water V = 60:40) to prepare a sample solution containing 3 ng of N-isobutylacrylamide per 1 ml;

[0031] (3) Select a mass spectrometer with model AB4500, a chromatographic column with model Agilent ZORBAX SB-C18 (150×4.6 mm, 5 μm), mobile phase A as ultrapure water; mobile phase B as acetonitrile; the ratio of mobile phase A to mobile phase B is 40:60. Inject 5 μl of the sample solution from step (2) into the liquid chromatograph, set the flow rate of the mobile phase to 1 ml / min, and the column oven temperature of the chromatographic column to 40 °C to complete the detection of N-isobutylacrylamide.

[0032] Furthermore, the retention time of the N-isobutylacrylamide is 1.77 ± 0.5 min.

[0033] Furthermore, when using the method of the present invention for detection, the detection limit concentration of the N-isobutylacrylamide is 0.30 ng / ml, and the quantification limit concentration is 0.97 ng / ml.

[0034] The present invention also aims to provide an application of a composition in the detection of N-isobutylacrylamide, where the detection is based on a liquid chromatography-mass spectrometry method; the composition includes a larger diisobutyl-bonded monofunctional silane, ultrapure water, and an organic solvent; the larger diisobutyl-bonded monofunctional silane serves as the stationary phase, and the ultrapure water and the organic solvent serve as the mobile phase.

[0035] Preferably, the organic solvent is acetonitrile; the liquid chromatography-mass spectrometry method is multiple reaction monitoring (MRM) in the positive ion mode under an ESI ion source.

[0036] The beneficial effects of the present invention are as follows:

[0037] 1. The present invention uses a combination of reversed-phase high-performance liquid chromatography and mass spectrometry to detect N-isobutylacrylamide in rifabutin bulk drugs, solving the problem of determining N-isobutylacrylamide, and the method has good reproducibility and high sensitivity.

[0038] 2. The present invention uses a larger diisobutyl-bonded monofunctional silane as the filler and a method for rapidly detecting N-isobutylacrylamide by multiple reaction monitoring (MRM) in the positive ion mode under an ESI ion source, which is a very meaningful work for the study of amide impurities.

[0039] 3. For the detection method proposed by the present invention, the detection limit concentration of N-isobutylacrylamide is as low as 0.30 ng / ml, and the quantification limit concentration is as low as 0.97 ng / ml, with high sensitivity, simple operation, and rapid detection; it ensures the quality control of rifabutin bulk drugs and the safety and effectiveness of the final products, and is of great significance for the quality control of rifabutin bulk drugs. Description of the Drawings

[0040] Figure 1It is the secondary mass spectrum of impurity SM2h;

[0041] Figure 2 It is the chromatogram of the blank solution;

[0042] Figure 3 It is the chromatogram of the test solution;

[0043] Figure 4 It is the chromatogram of the system suitability solution;

[0044] Figure 5 It is the chromatogram of the detection limit solution;

[0045] Figure 6 It is the chromatogram of the quantitation limit solution;

[0046] Figure 7 It is the chromatogram of impurity SM2h impurity channel - test sample spiked solution - 1;

[0047] Figure 8 It is the chromatogram of impurity SM2h impurity channel - RIFB - Z4 - 220911 test sample solution;

[0048] Figure 9 It is the chromatogram of impurity SM2h impurity channel - under the condition that "C'estbon" purified water is mobile phase A;

[0049] Figure 10 It is the chromatogram of impurity SM2h impurity channel - under the condition that 0 mM ammonium acetate aqueous solution is mobile phase A;

[0050] Figure 11 It is the chromatogram of impurity SM2h impurity channel - under the condition that 10 mM ammonium acetate aqueous solution is mobile phase A. Detailed implementation manners

[0051] Next, the technical solutions of the present invention will be further clearly and completely described in conjunction with specific embodiments. For the experimental methods without specific conditions indicated in the preferred embodiments, they are usually carried out under conventional conditions. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0052] In the embodiments of the present invention, the instruments and chromatographic conditions used are as follows: High-performance liquid chromatograph: Shimadzu LC-40B X3; Mass spectrometer: AB4500; Chromatographic column: Agilent ZORBAX SB-C18 (150×4.6mm, 5μm); Ultra-pure water is used as mobile phase A, and acetonitrile is used as mobile phase B. The ratio of mobile phase A to mobile phase B is 40:60; The flow rate of the mobile phase is 1 ml / min; The column temperature of the chromatographic column oven is 40°C; The injection volume is 5 μl.

[0053] In the embodiments of the present invention, the blank solution / diluent (the solvent for dissolving the reference substance and the sample to be tested) is acetonitrile.

[0054] In the embodiments of the present invention:

[0055] (1) Take a reference substance of N-isobutylacrylamide, dissolve it with the diluent to prepare a sample to be tested, inject the sample to be tested, and perform mass spectrometry analysis to determine the exact molecular weight and the maximum response fragment of N-isobutylacrylamide;

[0056] (2) Take the test sample of rifabutin raw material drug, add the diluent to prepare a test solution, then take the diluent as the blank solution, inject the test solution and the blank solution respectively, perform mass spectrometry analysis, record the chromatogram, and complete the determination of the detection of matrix and blank without interfering impurities;

[0057] (3) Take a reference substance of N-isobutylacrylamide, dissolve and dilute it with the diluent to prepare a sample to be tested, inject the sample to be tested, and perform mass spectrometry analysis to determine the sensitivity of N-isobutylacrylamide.

[0058] In the embodiments of the present invention, the impurity SM2h is N-isobutylacrylamide.

[0059] Example 1

[0060] 1. Specificity

[0061] Reference solution of N-isobutylacrylamide: Take about 15 mg of the reference substance of N-isobutylacrylamide, place it in a 50-ml volumetric flask, dissolve and dilute it to the mark with the diluent, and shake well; Accurately pipette 1 ml of the above solution into a 100-ml volumetric flask, dilute it to the mark with the diluent, and shake well; Then accurately pipette 1 ml of the above solution into a 100-ml volumetric flask, dilute it to the mark with the diluent, and shake well to obtain the reference stock solution of N-isobutylacrylamide. Then accurately pipette 1 ml of the above solution into a 10-ml volumetric flask, dilute it to the mark with the diluent, and shake well to obtain the reference solution (limit solution) with a concentration of 3 ng / ml of N-isobutylacrylamide.

[0062] Test solution: Weigh accurately about 10 mg of the drug substance test sample, place it in a 10-ml volumetric flask, dissolve it with the diluent and make up the volume to the mark, shake well to obtain the test solution (concentration 1 mg / ml).

[0063] Spiked test solution: Take 10 mg of the drug substance test sample and accurately measure 1 ml of the stock solution of N-isobutylacrylamide reference substance, place it in a 10-ml volumetric flask, mix well to obtain the spiked test solution (spiked at the 3-ppm limit).

[0064] Take an appropriate amount of the reference substance of impurity SM2h, dissolve it with the diluent and dilute to a solution containing about 1 μg per 1 ml. Take an appropriate amount of the solution, inject it into the mass spectrometer, record the mass spectrum, and confirm that the parent ion of impurity SM2h is 128.2 and the daughter ion is 72.1. The results are shown in Figure 1 .

[0065] According to the above chromatographic conditions, inject 5 μl of the blank solvent, reference solution and test solution respectively, and record the chromatogram. The results are as Figure 2 - Figure 3 shown. The blank solvent and the sample matrix do not interfere with the determination of impurity SM2h.

[0066] 2. Detection of reference solution

[0067] Inject the reference solution according to the above chromatographic conditions, inject continuously for 6 times, record the chromatogram, and calculate the RSD of the main peak area.

[0068] Results: The test results are shown in Table 1 Figure 4 . The results show that the blank diluent does not interfere with the sample determination; the reproducibility of N-isobutylacrylamide is good, and the RSD% of the peak areas of 6 injections is less than 10%.

[0069] Table 1. Determination results of reference solution

[0070]

[0071] 3. Detection of spiked test solution

[0072] Inject the spiked test solution according to the above chromatographic conditions respectively, record the chromatogram, and use the average value of the peak areas of the reference solution in "Table 1" to calculate the recovery rate of the spiking test.

[0073] Results: The test results are shown in Table 2 Figure 7 . The results show that the recovery rates of the two spiked test solutions are good, both between 80% and 120%.

[0074] Table 2. Determination results of spiked test solution

[0075]

[0076] 4. Detection of test solution

[0077] Inject the test solution respectively under the above chromatographic conditions, record the chromatogram, and calculate the peak area.

[0078] Results: The test results are shown in Table 3. Figure 8 . The results showed that except for RIFB-Z4-220911, none was detected in the four batches of samples.

[0079] Table 3. Detection Results of Samples

[0080] Batch number Result RIFB - Z4 - 220911 Greater than the limit RIFB - Z4 - 220916 Not detected RIFB - Z4 - 221201 Not detected RIFB - Z4 - 221202 Not detected

[0081] Comparative Example 1

[0082] In this comparative example, except that the mobile phase A used was different, the other conditions were the same as those in Example 1. The mobile phase A in this example was the water output from different pure water machines and "C'estbon" purified water. Inject the reference solution under the above chromatographic conditions and record the chromatogram.

[0083] Results: The measurement results are shown in Table 4. Figure 9 . In the separation experiment where other conditions were the same as those in the above example except for the different mobile phase A, the peak height of N-isobutylacrylamide with ultrapure water was much higher than that with purified water, and the ultrapure water from different pure water machines had no significant effect on the peak height of N-isobutylacrylamide.

[0084] Table 4. Measurement Results of Different Mobile Phase A

[0085] Category Source Peak height Ultra - pure water Water purifier - 1 30000 Ultra - pure water Water purifier - 2 32000 Ultra - pure water Water purifier - 3 28000 Purified water C'estbon 5870

[0086] Comparative Example 2

[0087] In this comparative example, except that the salt concentration of the mobile phase A used was different, the other conditions were the same as those in Example 1 (the impurity concentration was 10 ng / ml). The mobile phase A in this comparative example was aqueous ammonium acetate solutions with different concentrations. Inject the reference solution under the above chromatographic conditions and record the chromatogram.

[0088] Results: The measurement results are shown in Table 5. Figure 10 and Figure 11 . In the separation experiment where other conditions were the same except for the different ammonium salt concentration in the mobile phase, the peak height of N-isobutylacrylamide decreased with the increase of the salt concentration.

[0089] Table 5. Measurement Results of Mobile Phase A with Different Ammonium Salt Concentrations

[0090] Ammonium salt concentration mM in mobile phase A Peak height 0 120000 1 34000 2 20000 10 7980

[0091] Example 2. Basic Research on the Detection Limit and Quantitation Limit of N-Isobutylacrylamide by the Method of the Present Invention

[0092] 1. Prepare the solution to be tested

[0093] Detection limit solution: Accurately pipette 1.0 ml of the N-isobutylacrylamide reference solution into a 10-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain the detection limit solution.

[0094] Quantitation limit solution: Accurately pipette 3.3 ml of the N-isobutylacrylamide reference solution into a 10-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain the quantitation limit solution.

[0095] 2. Determination method:

[0096] Inject the above detection limit solution continuously for 3 times and the quantitation limit solution continuously for 3 times, calculate the ratio of the peak height of the main peak to the noise (signal-to-noise ratio), and record the chromatogram.

[0097] The calculation formulas for the quantitation limit and detection limit are as follows:

[0098]

[0099]

[0100] Results: The test results are shown in Table 6, Table 7, Figure 5 and Figure 6 . The results show that under this method, the detection limit and quantitation limit of N-isobutylacrylamide meet the requirements of S / N = 3(10):1 respectively.

[0101] Table 6. Results of detection limit determination

[0102]

[0103] Table 7. Results of quantitation limit determination

[0104]

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. Method for separating N-isobutylacrylamide from rifabutin raw material by LC-MS / MS method, Characterized in that, The structural formula of the N-isobutylacrylamide is shown in Formula I, and the structural formula of the rifabutin is shown in Formula II; the method includes: using a larger diisobutyl-bonded monofunctional silane as the filler for the chromatographic column, and the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is ultrapure water, and the mobile phase B is an organic solvent; separating the N-isobutylacrylamide by elution; 2. The method according to claim 1, Characterized in that, The volume ratio of the mobile phase A to the mobile phase B is 40:

60.

3. The method according to claim 1, Characterized in that, The organic solvent is acetonitrile.

4. The method according to claim 1, Characterized in that, The flow rate of the mobile phase is 0.9 - 1.1 ml / min, and the column oven temperature of the chromatographic column is 35 - 45 °C.

5. The method according to claim 1, Characterized in that, The specification of the chromatographic column is 150×4.6 mm, 5 μm.

6. Method for determining N-isobutylacrylamide in rifabutin raw material, Characterized in that, Using the method according to any one of claims 1 - 5 to separate the N-isobutylacrylamide in the rifabutin raw material; then entering the detector for detection; the detector is a tandem quadrupole mass spectrometer.

7. The method according to claim 6, Characterized in that, The ion source of the tandem quadrupole mass spectrometer is an ESI source; the method is multiple reaction monitoring in the positive ion mode.

8. The method according to claim 6, Characterized in that, The selected detection ion pair of the N-isobutylacrylamide is a mass-to-charge ratio of 128.2 m / z → 72.1 m / z.

9. The method according to claim 6, Characterized in that, Before separation, the sample to be tested is prepared with acetonitrile as the diluent.

10. The method according to claim 6, Characterized in that, The retention time of the N-isobutylacrylamide is 1.77 ± 0.5 min.

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