Detection method for determining nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry

Abstract

The invention provides a detection method for determining nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry. The detection method comprises the following steps: (1) preparing a standard stock solution; (2) preparing a standard series stock solution; (3) injecting the standard series stock solution into a gas chromatograph-mass spectrometer for detection to obtain a standard mass chromatogram of the corresponding nitrosamine compound, and taking the quantitative ion pair peak area of the nitrosamine compound as a vertical coordinate and the concentration of the nitrosamine compound in the standard series stock solution as a horizontal coordinate to obtain a standard working curve; (4) sample extraction; and (5) injecting the purified sample into a gas chromatograph-mass spectrometer to obtain a mass chromatogram of the purified sample, and if a chromatographic peak corresponding to a standard mass chromatogram exists in the mass chromatogram of the purified sample, substituting the chromatographic peak into the standard working curve to obtain the type and content of the nitrosamine compound in the purified sample. The detection method is high in precision, high in speed and accurate in result.

Description

Technical Field

[0001] The invention relates to the technical field of drug chemical component analysis and detection, and specifically to a detection method for determining nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry. Background Art

[0002] N-nitrosamines are a class of nitroso-containing genotoxic substances with the general chemical formula R1(R2)NN=O. Among the known nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) can cause diseases such as liver cancer and sinus cancer, and have been classified as Class 2A carcinogens by the International Agency for Research on Cancer. The United States Environmental Protection Agency Integrated Risk Information System and the International Agency for Research on Cancer have rated NDMA, NDEA, N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosopiperidine (NPIP), N-nitroso-n-propylamine (NDPA), N-nitrosodiphenylamine (NDPhA) and N-nitroso-n-butylamine (NDBA) as possible human carcinogens. The contamination of nitrosamine impurities in drugs has attracted widespread attention from drug regulatory agencies and the public. For example, NDMA impurities have been detected in valsartan raw materials and metformin hydrochloride preparations.

[0003] At present, the quantitative analysis methods for nitrosamine impurities in drugs mainly include GC-MS, GC-MS / MS and LC-MS / MS. However, these current methods have problems such as large sampling volume, multiple reagents used, and poor accuracy. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention provides a method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry, which solves the problems raised in the above-mentioned background technology.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] According to a first aspect of the present invention, a method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry is provided, comprising the following steps:

[0009] (1) Preparation of standard stock solution: Pipette the mixed standard solution of nitrosamine compounds into a volumetric flask and dilute to the mark with methanol to prepare a 1.00 μg / mL standard stock solution;

[0010] (2) Preparation of standard series stock solutions: Pipette the standard stock solution into a volumetric flask, add different volumes of methanol, and adjust the volume to a standard series stock solution with a concentration gradient;

[0011] (3) injecting the standard series stock solutions into a gas chromatography-mass spectrometer for detection, obtaining a standard mass chromatogram of the corresponding nitrosamine compound, taking the quantitative ion pair peak area of ​​the nitrosamine compound as the ordinate and the concentration of the nitrosamine compound in the standard series stock solution as the abscissa, to obtain a standard working curve;

[0012] (4) Sample extraction: Weigh the cold medicine and extract it, vortex, sonicate, shake, centrifuge and filter the extract to obtain a purified sample;

[0013] (5) Injecting the purified sample into a gas chromatograph-mass spectrometer to obtain a mass chromatogram of the purified sample. If there is a chromatographic peak in the mass chromatogram of the purified sample corresponding to the standard mass chromatogram, it is inserted into the standard working curve to obtain the type and content of the nitrosamine compound in the purified sample.

[0014] Preferably, in step (1), the nitrosamine compound includes N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-methyl-N-nitrosoaniline, N-nitroso-N-ethylaniline, N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodiphenylamine, and N-nitrosodibenzylamine.

[0015] Preferably, the chromatographic column of the gas chromatography-mass spectrometry instrument is a DB-WAXETR capillary chromatographic column.

[0016] Preferably, the temperature rising program of the gas chromatography-mass spectrometry is: inject at 50°C and hold for 1 min, heat to 160°C at 10°C / min and hold for 1 min, heat to 250°C at 40°C / min and hold for 7.25 min.

[0017] Preferably, the carrier gas of the gas chromatography-mass spectrometry is helium, and a constant flow mode is adopted with a carrier gas flow rate of 1.2 mL / min.

[0018] Preferably, the injection port temperature of the gas chromatography-mass spectrometry instrument is 250° C., the transmission line temperature is 260° C., the injection volume is 1 μL, and a splitless injection mode is adopted.

[0019] Preferably, the mass spectrometry conditions of the gas chromatography-mass spectrometer are: the ion source is an EI source, the ion source temperature is 230°C, the quadrupole temperature is 150°C, the EM voltage is 70eV, the solvent delay is 5min, the collision gas is nitrogen, and the scanning mode is DMRM mode.

[0020] Preferably, the solvent is selected from dichloromethane.

[0021] Preferably, the standard working curve is as follows:

[0022] NDMA: y = 11349x-299, correlation coefficient is 0.999;

[0023] NMEA: y = 10050x + 154, the correlation coefficient is 0.999;

[0024] NDEA: y = 5890x + 22.3, correlation coefficient is 0.998;

[0025] NDPA: y = 7372x + 107, correlation coefficient is 0.999;

[0026] NDBA: y = 6280x + 436, correlation coefficient is 0.999;

[0027] NMPhA: y = 83099x + 5291, correlation coefficient is 0.999;

[0028] NEPhA: y = 27340x + 2033, correlation coefficient is 0.999;

[0029] NPIP: y = 8437x + 339, correlation coefficient is 0.999;

[0030] NPYR: y = 4269x + 93.8, correlation coefficient is 0.999;

[0031] NMOR: y = 7167x + 283, correlation coefficient is 0.999;

[0032] NDPhA: y = 33230x + 1905, correlation coefficient is 0.999;

[0033] NDBzA: y=3778x+428, the correlation coefficient is 0.999.

[0034] Preferably, the detection limit of the nitrosamine compound is 0.3-0.6 μg / kg, and the quantification limit of the nitrosamine compound is 1.0-2.0 μg / kg.

[0035] (III) Beneficial effects

[0036] The present invention provides a method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry. It has the following beneficial effects:

[0037] (1) This scheme provides a method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry. By using a gas chromatography-mass spectrometer to detect nitrosamine compounds in cold medicines, the detection has high precision, fast speed, accurate results, and effectively reduces false positives.

[0038] (2) This scheme provides a method for determining nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry, which requires a small amount of sampling, uses fewer types of organic reagents, has simple pretreatment, and has high reproducibility.

[0039] (3) This protocol provides a method for the determination of nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry, with a concentration of 0.2 to 4.0 ng·mL -1 Within the range, 12 nitrosamine compounds can be quantitatively determined. The ion pair peak area of ​​the 12 nitrosamine compounds has a good linear relationship with the concentration, and the correlation coefficient is greater than 0.99. The detection limit of the detection method of the present application is 0.3-0.6 μg / kg, and the quantitative limit is 1.0-2.0 μg / kg. The spiked recovery test of cold medicines is carried out, and the average spiked recovery rate is 81-102%, and the relative standard deviation of the determination result is 1.7-4.9% (n=6). DETAILED DESCRIPTION

[0040] In order to better illustrate the content of the present invention, a detailed description is given below in conjunction with specific embodiments.

[0041] Example 1

[0042] This example detects 12 nitrosamine compounds in cold medicine samples:

[0043] raw material:

[0044] 8 batches of cold medicines: Zhenkang Children's Paracetamol and Phenylephrine Granules, Baiyun Dongtai Shangqiu Pharmaceutical Co., Ltd., batch number 220914131; China Resources Sanjiu (Huangshi) Pharmaceutical Co., Ltd., batch numbers 2305127Z, 2307168Z; Paracetamol and Phenylephrine Three-flavor Extract Capsules, Wuhu Luye Pharmaceutical Co., Ltd., batch number 2302121; Huluba Compound Ganmao Ling Tablets; Kuake Compound Amino Amine Capsules, Zhejiang Yafeng Pharmaceutical Co., Ltd., batch number 01230108;

[0045] Reagents: methanol, chromatographic grade, produced by TEDIA, 16105085; dichloromethane, chromatographic grade, produced by Shanghai Aladdin Biochemical Technology Co., Ltd., batch number H2315016; ethyl acetate, produced by Sigma-Aldrich, batch number STBB9905V;

[0046] The 12 nitrosamine standard mixed solutions, as shown in Table 1, all had a concentration of 200 μg / mL and were purchased from Anpu; methanol, ethyl acetate and dichloromethane were all chromatographically pure.

[0047] Table 1

[0048]

[0049]

[0050] In Table 1, * indicates quantitative ion pairs;

[0051] Gas chromatography-mass spectrometry conditions:

[0052] Chromatographic conditions: DB-WAXETR capillary column;

[0053] Heating program: 50℃ for 1 min, increase the temperature to 160℃ at 10℃ / min, maintain for 1 min, increase the temperature to 250℃ at 40℃ / min, maintain for 7.25 min;

[0054] Carrier gas: Helium with a concentration greater than 99.999%, constant flow mode, flow rate of 1.2mL / min; splitless injection mode, injection port temperature of 250℃, transfer line temperature of 260℃, injection volume of 1μL;

[0055] Mass spectrometry conditions: the ion source was EI source, the ion source temperature was 230 °C, the quadrupole temperature was 150 °C, the EM voltage was 70 eV, the solvent delay was 5 min, the collision gas was nitrogen, and the scanning mode was DMRM mode;

[0056] The solvent was dichloromethane.

[0057] The specific steps are as follows:

[0058] (1) Preparation of standard stock solution:

[0059] Pipette 50 μL of 200 μg / mL nitrosamine mixed standard solution into a 10 mL volumetric flask and dilute to the mark with methanol to obtain a 1.00 μg / mL standard stock solution.

[0060] (2) Preparation of standard series stock solutions:

[0061] Pipette 200 μL of the standard stock solution into a 10 mL volumetric flask and dilute to the mark with methanol to adjust the concentration to 0.02 μg / mL.

[0062] 10 μL, 25 μL, 50 μL, 100 μL, and 200 μL of the working solution were respectively drawn into the injection bottle, and then methanol was added to make up to 1000 μL, and mixed to obtain a series of standard stock solutions with concentrations of 0.2 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2 ng / mL, and 4 ng / mL, respectively;

[0063] (3) The standard series stock solutions were respectively injected into a gas chromatography-mass spectrometer for detection to obtain the standard mass chromatograms of the corresponding nitrosamine compounds, with the quantitative ion pair peak area of ​​the nitrosamine compound as the ordinate and the concentration of the nitrosamine compound in the standard series stock solution as the abscissa to obtain the standard working curve. In the range of 0.2 to 4 ng / mL, the linear relationship between the quantitative ion pair peak area and the concentration of the 12 nitrosamine compounds is shown in Table 2;

[0064] Table 2

[0065]

[0066] (4) Sample extraction:

[0067] Weigh 0.2 g of cold medicine, grind it, add 5 mL of pure water, vortex for 1 min, ultrasonically treat it at room temperature for 10 min, shake it for 15 min, centrifuge it at 6000 r / min for 5 min, discard the aqueous phase, take 1 mL of the organic phase and filter it through a 0.22 μm organic microporous membrane to obtain a purified sample;

[0068] (5) The purified samples were injected into a gas chromatography-mass spectrometer to obtain mass chromatograms of the purified samples. Among them, 5 samples were negative samples and 3 were positive samples. In the 3 positive samples, only NMPhA and NDPhA were detected, and the other 10 nitrosamine compounds were not detected. When put into the corresponding linear relationship curve, the detection concentration of NMPhA was 16.8-58.6 μg / kg, and the detection concentration of NDPhA was 14.6-22.6 μg / kg.

[0069] A spike recovery test was performed on one of the positive samples, with spike masses of 0.500 ng, 1.00 ng and 2.00 ng, and spike concentrations of 2.5 μg / kg, 5.00 μg / kg and 10.0 μg / kg, as shown in Table 3. The results showed that the average spike recovery of nitrosamines was 81.0-102%, which met the detection requirements, and the relative standard deviation of 6 determinations was 1.7-4.9% (n=6);

[0070] A reference solution with a mass concentration of 1.0 ng / mL was taken for stability test and stored in an injection bottle at room temperature. The sample was injected and measured at 0, 2, 4, 8, 12, 18, and 24 h, respectively. The chromatographic peak area of ​​the target substance was recorded and the RSD value of the 7 measurements was calculated. As shown in Table 3, the RSD results were 2.2-5.2%, which had good stability within 24 h.

[0071] Table 3

[0072]

[0073]

[0074] Comparative Example 1

[0075] The detection method of this comparative example is the same as that of Example 1, except that the chromatographic condition of the gas chromatography-mass spectrometry is an HP-5 capillary chromatographic column, and the peak shape and separation degree of nitrosamines are not thorough.

[0076] Comparative Example 2

[0077] The detection method of this comparative example is the same as that of Example 1, except that ethyl acetate is selected as the solvent, and the extraction efficiency of nitrosamines is shown in Table 4. According to Table 4, the extraction efficiency of ethyl acetate for NDMA and NMEA is relatively low, while the extraction efficiency of dichloromethane used in Example 1 for nitrosamine compounds is above 80%.

[0078] Table 4

[0079] Compound Extraction efficiency of ethyl acetate Extraction efficiency of dichloromethane NDMA 58 90 NMEA 56 95 NDEA 99 91 NDPA 78 92 NDBA 82 99 NMh 99 92 NAE 99 92 NPIP 93 95 NPYR 92 96 NMOR 95 90 NDPh 95 95 NbZ 101 95

[0080] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: (1) Preparation of standard stock solution: Pipette the mixed standard solution of nitrosamine compounds into a volumetric flask and dilute to the mark with methanol to prepare a 1.00 μg / mL standard stock solution; (2) Preparation of standard series stock solutions: Pipette the standard stock solution into a volumetric flask, add different volumes of methanol, and adjust the volume to a standard series stock solution with a concentration gradient; (3) injecting the standard series stock solutions into a gas chromatography-mass spectrometer for detection, obtaining a standard mass chromatogram of the corresponding nitrosamine compound, taking the quantitative ion pair peak area of ​​the nitrosamine compound as the ordinate and the concentration of the nitrosamine compound in the standard series stock solution as the abscissa, to obtain a standard working curve; (4) Sample extraction: Weigh the cold medicine and extract it, vortex, sonicate, shake, centrifuge and filter the extract to obtain a purified sample; (5) Injecting the purified sample into a gas chromatograph-mass spectrometer to obtain a mass chromatogram of the purified sample. If there is a chromatographic peak in the mass chromatogram of the purified sample corresponding to the standard mass chromatogram, it is inserted into the standard working curve to obtain the type and content of the nitrosamine compound in the purified sample.

2. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step (1), the nitrosamine compound includes N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-methyl-N-nitrosoaniline, N-nitroso-N-ethylaniline, N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodiphenylamine, and N-nitrosodibenzylamine.

3. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The chromatographic column of the gas chromatography-mass spectrometry instrument is a DB-WAXETR capillary chromatographic column.

4. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The temperature program of the gas chromatography-mass spectrometry instrument is: inject at 50°C and hold for 1 min, heat to 160°C at 10°C / min and hold for 1 min, heat to 250°C at 40°C / min and hold for 7.25 min.

5. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The carrier gas of the gas chromatography-mass spectrometer is helium, and the constant flow mode is adopted, and the carrier gas flow rate is 1.2 mL / min.

6. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The injection port temperature of the gas chromatography-mass spectrometer was 250° C., the transmission line temperature was 260° C., the injection volume was 1 μL, and a splitless injection mode was adopted.

7. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions of the gas chromatography-mass spectrometer are as follows: the ion source is an EI source, the ion source temperature is 230° C., the quadrupole temperature is 150° C., the EM voltage is 70 eV, the solvent delay is 5 min, the collision gas is nitrogen, and the scanning mode is DMRM mode.

8. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 7, characterized in that: The solvent is selected from dichloromethane.

9. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The standard working curve is as follows: NDMA: y = 11349x-299, correlation coefficient is 0.999; NMEA: y = 10050x + 154, the correlation coefficient is 0.999; NDEA: y = 5890x + 22.3, correlation coefficient is 0.998; NDPA: y = 7372x + 107, correlation coefficient is 0.999; NDBA: y = 6280x + 436, correlation coefficient is 0.999; NMPhA: y = 83099x + 5291, correlation coefficient is 0.999; NEPhA: y = 27340x + 2033, correlation coefficient is 0.999; NPIP: y = 8437x + 339, correlation coefficient is 0.999; NPYR: y = 4269x + 93.8, correlation coefficient is 0.999; NMOR: y = 7167x + 283, correlation coefficient is 0.999; NDPhA: y = 33230x + 1905, correlation coefficient is 0.999; NDBzA: y=3778x+428, the correlation coefficient is 0.

999.

10. The method for detecting nitrosamine compounds in cold medicines based on gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The detection limit of the nitrosamine compound is 0.3-0.6 μg / kg, and the quantitative limit of the nitrosamine compound is 1.0-2.0 μg / kg.

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