Method for detecting N-nitroso-deethylidocaine genotoxic impurities in lidocaine

Abstract

The invention belongs to the field of medicine quality control, and particularly relates to a method for detecting N-nitroso-deethylidocaine genotoxic impurities in lidocaine. The method is realized by the following steps: firstly, preparing a test solution and a reference solution; and then, detecting by adopting a high performance liquid chromatography-triple quadrupole mass spectrometry method. The detection method provided by the invention can realize good quality control on lidocaine raw material medicines and preparations thereof, is strong in durability and high in detection sensitivity, can effectively avoid interference generated by a solvent, has a good impurity separation effect, can accurately detect toxic impurities in lidocaine, and is suitable for large-scale popularization and application. The method has the advantages of high sensitivity, strong specificity and good reproducibility. The detection method provided by the invention is simple and strong in operability.

Description

Technical Field

[0001] The present invention belongs to the field of drug quality control, and particularly relates to a method for detecting the genotoxic impurity N-nitroso-deethyl lidocaine in lidocaine. Background Art

[0002] Lidocaine was first synthesized by Swedish chemist Nils Löfgren in 1943. It is a medium-acting local anesthetic of the amino amide type. Compared with other local anesthetics, it has the advantages of rapid onset, strong and lasting local anesthetic effect, rare allergic reactions, and the ability to penetrate the placental barrier in pain control, and has a wide range of clinical applications. During its synthesis process, N-nitroso-deethyl lidocaine is easily generated, and N-nitroso impurities are classified as potential genotoxic impurities. Genotoxic impurities refer to compounds that directly or indirectly damage cellular DNA, cause gene mutations or in vivo mutagenesis, and have the potential to be carcinogenic. In recent years, USP-NF, EP, and ChP have all had more explicit requirements for genotoxic impurities, and more and more pharmaceutical production enterprises and drug R & D institutions have increased the control and detection of genotoxic impurities.

[0003] N-nitroso-deethyl lidocaine has a relatively large molecular weight (Mol. 235.28), is difficult to vaporize and has a certain polarity, the limit to be controlled is relatively low, and the response on HPLC is relatively low. In the prior art, there is no reported method for detecting the residual amount of N-nitroso-deethyl lidocaine in lidocaine. Summary of the Invention

[0004] In view of the research gap in the prior art, the present invention provides a method for detecting the genotoxic impurity N-nitroso-deethyl lidocaine in lidocaine.

[0005] The structural formula of N-nitroso-deethyl lidocaine is as follows: The technical solution adopted by the present invention to achieve the above object is as follows: The present invention provides a method for detecting the genotoxic impurity N-nitroso-deethyl lidocaine in lidocaine, comprising the following steps: (1) Test solution: Take lidocaine, dissolve it with a solvent and quantitatively dilute to obtain test solution 1; take lidocaine hydrochloride injection to obtain test solution 2; (2) Reference solution: Accurately weigh the reference substance of N-nitroso-deethyl lidocaine, dissolve it with a solvent and quantitatively dilute to obtain a reference stock solution; then quantitatively dilute it step by step with the solvent to obtain a series of reference solutions; (3) Detection is carried out by high performance liquid chromatography-triple quadrupole mass spectrometry.

[0006] Preferably, in step (1), the solvent is acetonitrile; the concentration of the test solution 1 is 100 mg / mL; the concentration of the test solution 2 is 20 mg / mL.

[0007] Preferably, in step (2), the solvent is acetonitrile; the concentrations of the reference solution are 1, 2, 4, 8, 10, 12, 15, and 20 ng / mL.

[0008] Preferably, in step (3), the conditions of the high performance liquid chromatography are as follows: using a Shiseido C18 chromatographic column, 150 mm × 4.6 mm, 5 μm; using 0.020 mol / L ammonium acetate solution - acetonitrile (90:10) as mobile phase A and acetonitrile as mobile phase B for gradient elution, the flow rate is 1.0 ml / min, the column temperature is 30 °C, and the injection volume is 10 μL.

[0009] The conditions of the above gradient elution are as follows: Preferably, the conditions of the mass spectrometry are as follows: using positive ion electrospray (ESI + ) mode for ionization, multiple reaction monitoring (MRM) mode, Fragmentor voltage: 42 V. Select the monitored ion pairs m / z 236.2→105.1 (quantitative ion pair, collision energy: 38 V) and m / z 236.2→58.1 (qualitative ion pair, collision energy: 28 V).

[0010] The present invention extrapolates using the data of the known toxic impurity N - diethylnitrosamine. The maximum daily intake of known N - diethylnitrosamine is about 26.5 ng / day, while the maximum daily dosage of lidocaine is 300 mg. Based on this, the limit value of N - nitroso - deethyl lidocaine in lidocaine can be calculated as = 26.5 ng / 300 mg = 0.00001% (0.1 μg / g) The beneficial effects of the present invention are as follows: (1) The detection method provided by the present invention can achieve good quality control of lidocaine raw materials and its preparations, has strong durability, high detection sensitivity, can effectively avoid the interference caused by solvents, has good impurity separation effect, and can accurately detect the toxic impurities in lidocaine, with the advantages of high sensitivity, strong specificity, and good reproducibility.

[0011] (2) The detection method provided by the present invention is simple and has strong operability. Description of the Drawings

[0012] Figure 1 is the chromatogram of the blank solvent; Figure 2Chromatogram of the spiked solution of the test sample; Figure 3 Precision chromatogram; Figure 4 Precision mass spectrum; Figure 5 Chromatogram of the mixed solution of the ultraviolet detector; Figure 6 Chromatogram of the mixed solution eluted with methanol by the ultraviolet detector. Detailed implementation manners

[0013] The technical solutions of the present invention will be further explained and illustrated below through specific embodiments.

[0014] Unless otherwise specified, the relevant reagents used in the present invention are all commercially available.

[0015] Example 1 1 Experimental part 1.1 Instruments and reagents Agilent 1290 high performance liquid chromatograph, Agilent 6470B triple quadrupole mass spectrometer (Agilent Technologies, USA), CP225D one in a hundred thousandth electronic balance (Sartorius, Germany) N-nitroso-deethyl lidocaine reference substance (content 98.42%, Sinco Pharmachem, USA); 3 batches of lidocaine raw materials were provided by domestic production enterprises.

[0016] Ammonium acetate (content 100%, Anaqua Chemicals Supply, USA), acetonitrile (purity 99.999%, Honeywell, USA), ultrapure water was prepared by Milli-Q Advantage A10 ultrapure water machine (Merck Millipore, USA).

[0017] 1.2 Experimental methods 1.2.1 Chromatography and mass spectrometry conditions Using Shiseido C 18 Chromatographic column (150 mm × 4.6 mm, 5 μm); 0.020 mol / L ammonium acetate solution - acetonitrile (90:10) was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution (see Table 1), the flow rate was 1.0 ml / min, the column temperature was 30 °C, and the injection volume was 10 μL.

[0018] Table 1 Gradient elution table Using positive ion electrospray (ESI +)(Mode ionization, multiple reaction monitoring (MRM) mode, Fragmentor voltage: 42 V. Select to monitor the ion pairs m / z 236.2→105.1 (quantitative ion pair, collision energy: 38 V) and m / z 236.2→58.1 (qualitative ion pair, collision energy: 28 V).

[0019] 1.2.2 Solution preparation Test solution: Take an appropriate amount of lidocaine, dissolve it with a solvent (acetonitrile) and quantitatively dilute it to prepare a solution containing about 100 mg per 1 ml.

[0020] Stock solution of N-nitroso-deethyl lidocaine reference substance: Weigh accurately about 10 mg of N-nitroso-deethyl lidocaine reference substance, dissolve it with a solvent and quantitatively dilute it to prepare a solution containing about 100 ng per 1 ml. Then quantitatively dilute it step by step with the solvent to prepare a series of reference substance solutions with concentrations of 1, 2, 4, 8, 10, 12, 15, and 20 ng / ml.

[0021] 2. Methodological verification 2.1 Specificity test Inject the solvent and the reference substance solution at the limit concentration respectively for determination, and record the chromatogram. As Figure 1 and Figure 2 shown. N-nitroso-deethyl lidocaine shows a double-peak state, and the chromatographic peak emergence times are 4.6 min and 4.8 min. When quantifying, it is calculated based on the sum of the areas of the two chromatographic peaks. There is no interference peak for the solvent at the same retention time, and the method has good specificity. The results are shown in the typical chromatogram.

[0022] The chromatogram of N-nitroso-deethyl lidocaine shows a double-peak state, and its possible mechanism is as follows: First, the reason is dipole resonance. The lone pair electrons on the nitrogen connected to the nitroso group flow to the nitroso group due to the strong electron-withdrawing effect of the nitroso group, resulting in the formation of a structure similar to a double bond between the two nitrogens, which is similar to the cis-trans isomers formed by carbon-carbon double bonds, and double peaks appear in the chromatogram and ion current diagram. Second, the nitrosoamine group and the hydrogen on the adjacent nitrogen can form a hydrogen bond, resulting in a difference in the environment of the hydrogen near the double bond, so the sizes of the double peaks are different. The two isomers can be effectively separated in the selected liquid chromatographic column, and they have the same parent ion m / z 236.2. Therefore, chromatographic peaks of the two isomers can be extracted in both of the two ion channels of m / z 236.2→105.1 and m / z 236.2→58.1 set, so N-nitroso-deethyl lidocaine shows double peaks in the chromatogram.

[0023] 2.2 Precision test Take the reference substance solution and inject it continuously for 6 times, and record the chromatogram. The results are as Figure 2 and Figure 3As shown, the RSD of the peak area of N-nitroso-deethyl lidocaine ( n n = 6) was 0.7%, indicating good precision.

[0024] 2.3 Linear range Series of reference substance solutions with concentrations of approximately 1, 2, 4, 8, 10, 12, 15, and 20 ng / ml were respectively injected into the liquid chromatograph, and the chromatograms were recorded. Using the peak area as y and the concentration of the N-nitroso-deethyl lidocaine reference substance solution (ng / ml) as x, a regression curve of the area was plotted. The regression linear equation of N-nitroso-deethyl lidocaine was y = 96.9522x + 9.6877, with r = 0.9997.

[0025] The results showed that N-nitroso-deethyl lidocaine had a good linear relationship within the range of 1.058 - 21.160 ng / ml.

[0026] 2.4 Stability The reference substance solution was taken and placed in a 5°C injector, and samples were injected and measured at 0, 2, 4, 8, 12, and 24 hours respectively. The RSD of the peak area (n = 6) was 1.9%, indicating good stability of the reference substance solution within 24 hours.

[0027] 2.5 Quantitation limit and detection limit The reference substance solution was taken, diluted successively, and injected respectively. The mass concentration of the quantitation limit was determined with a signal-to-noise ratio not less than 10. The mass concentration of the detection limit was determined with a signal-to-noise ratio not less than 3. The results showed that the quantitation limit was 1.058 ng / ml and the detection limit was 0.349 ng / ml. According to the daily dosage of lidocaine, the quantitation limit of N-nitroso-deethyl lidocaine in lidocaine was 10.58 ng / g, and the detection limit was 3.49 ng / g.

[0028] 2.6 Recovery investigation A total of 9 portions of the bulk drug of this product, each approximately 2000 mg, were placed in 20-ml volumetric flasks. 1 ml, 2 ml, and 3 ml of the N-nitroso-deethyl lidocaine reference substance stock solution were respectively added, and the solvent was added to dissolve and diluted to the mark to prepare recovery solutions at 3 concentration levels of 50%, 100%, and 150%. They were diluted to the mark with the solvent, shaken well, and measured. As Figure 4 shown. The recovery rate and relative standard deviation of N-nitroso-deethyl lidocaine were calculated by the external standard method. The recovery rate of N-nitroso-deethyl lidocaine in lidocaine was between 97.1% and 101.4%, the average recovery rate was 99.1%, and the RSD was 1.5%. The results are shown in Table 2.

[0029] Table 2 Results of the recovery test 2.7 Determination of Samples Take 3 batches of bulk drugs, prepare the reference solution and test solution 1 according to the method under "1.2.2", inject and determine according to the chromatographic conditions under "1.2.1", and calculate by the standard curve method. The detection amounts of the 3 batches of samples are 12 ng / g, 9 ng / g, and 12 ng / g respectively.

[0030] Example 2 According to the method described in Example 1, take 3 batches of lidocaine hydrochloride injection as test solution 2, inject and determine according to the chromatographic conditions under "1.2.1", and calculate by the external standard method. The results show that none of the 3 batches of samples are detected.

[0031] Comparative Example 1 Use acetonitrile as the solvent, the concentration of the test solution is 10 mg / ml, the concentration of the impurity reference solution is 10 μg / ml, according to the chromatographic conditions under 1.2.1 (Example 1), use an ultraviolet-visible spectrophotometric detector, and the detection wavelength is 230 nm.

[0032] As Figure 5 shown, the signal-to-noise ratio S / N of the impurity reference is 218. The quantitative limit mass concentration is determined to be 0.48 μg / ml based on the concentration with a signal-to-noise ratio not less than 10, and the quantitative limit mass concentration is determined to be 0.14 μg / m based on the concentration with a signal-to-noise ratio not less than 10; according to the daily dosage of lidocaine, the quantitative limit of N-nitroso-deethyl lidocaine in lidocaine is 4.8 μg / g, and the detection limit is 1.4 μg / g, which is higher than the acceptable limit of 0.1 μg / g. This impurity cannot be accurately detected.

[0033] Comparative Example 2 Use acetonitrile as the solvent, the concentration of the test solution is 10 mg / ml, the concentration of the impurity reference solution is 10 μg / ml, use an ultraviolet-visible spectrophotometric detector, and the detection wavelength is 230 nm. The conditions for high performance liquid chromatography are as follows: use a Shiseido C18 chromatographic column, 150 mm × 4.6 mm, 5 μm; use 0.020 mol / L ammonium acetate solution - methanol (90:10) as mobile phase A, and methanol as mobile phase B, gradient elution, the flow rate is 1.0 ml / min, the column temperature is 30 °C, and the injection volume is 10 μL.

[0034] The impurity elutes after lidocaine, and the peak shape has serious tailing and cannot be accurately integrated. As Figure 6 shown.

[0035] Comparative Example 3 Prepare the test solution using the initial ratio of the mobile phase, and it is impossible to prepare a 100 mg / ml test solution; When preparing the test solution with methanol, it is impossible to prepare a 100 mg / ml test solution.

Claims

1. A method for detecting genotoxic impurities of N-nitroso-desethyl lidocaine in lidocaine, characterized in that: The following steps are involved: (1) Test solution: Take lidocaine bulk drug, add solvent to dissolve and quantitatively dilute to obtain test solution 1; take lidocaine hydrochloride injection to obtain test solution 2; (2) Reference substance solution: Accurately weigh N-nitroso-desethyl lidocaine reference substance, dissolve it in solvent and quantitatively dilute it to obtain reference substance stock solution; then quantitatively dilute it step by step with solvent to obtain a series of reference substance solutions; (3) Detection was performed using high performance liquid chromatography-triple quadrupole mass spectrometry.

2. The method according to claim 1, characterized in that In step (1), the solvent is acetonitrile; the concentration of the test solution 1 is 100 mg / mL; and the concentration of the test solution 2 is 20 mg / mL.

3. The method according to claim 1 or 2, characterized in that: In step (2), the solvent is acetonitrile; the concentration of the reference solution is 1, 2, 4, 8, 10, 12, 15, 20 ng / mL.

4. The method according to any one of claims 1 to 3, characterized in that: In step (3), the conditions of the HPLC are as follows: using a Shiseido C18 column, 150 mm × 4.6 mm, 5 μm; using 0.020 mol / L ammonium acetate solution-acetonitrile (90:10) as mobile phase A, acetonitrile as mobile phase B, gradient elution, flow rate of 1.0 ml / min, column temperature of 30 °C, and injection volume of 10 μL.

5. The method according to claim 4, characterized in that The conditions of the gradient elution are: 。 6. The method according to claim 4 or 5, characterized in that: The mass spectrometry conditions were as follows: positive ion electrospray ionization, multiple reaction ion monitoring mode, fragmentor voltage: 42 V; selected monitoring ion pairs m / z 236.2→105.1, quantitative ion pair, collision energy: 38 V; m / z 236.2→58.1, qualitative ion pair, collision energy: 28 V.

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