Method for simultaneously detecting nine nitrosamines in medicinal infusion bag

Abstract

The invention discloses a method for simultaneously detecting nine nitrosamines in a medicinal infusion bag. Comprising the following steps: weighing standard substances of N-nitrosodimethylamine, N-nitrosodiethyl amine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodiisopropylamine and N-nitrosoethyl isopropylamine, and then preparing a standard solution by using an organic solvent; the method comprises the following steps: weighing standard substances of five internal standard substances, namely N-nitrosodimethylamine-d6, N-nitrosodipropylamine-d14, N-nitrosodiethylamine-d10, N-nitrosopyrrolidine-d4 and N-nitrosomorpholine-d8, and then preparing an internal standard solution by using an organic solvent; the method comprises the following steps: pretreating a medicinal infusion bag sample, extracting to obtain an extracting solution, mixing the extracting solution with an internal standard solution, extracting with a solid-phase extraction column, concentrating, and finally filtering with an organic filter membrane to obtain a sample solution; and detecting the sample solution and the standard solution by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and relates to the detection of nine kinds of nitrosamines in a medicinal infusion bag, and specifically to a method for simultaneously detecting nine kinds of nitrosamines in a medicinal infusion bag. Background Art

[0002] Nitrosamines are a class of organic compounds containing nitroso (-N=O) functional groups. About 90% of the more than 3,000 nitrosamines discovered so far are carcinogenic. Studies have shown that nitrosamines can induce tumors in almost all organs and tissues of animals. Therefore, in the food, tobacco, drinking water and other industries, research on nitrosamines has been quite extensive. However, the detection of nitrosamines in pharmaceutical packaging materials started relatively late, and the detection of nitrosamines in pharmaceutical infusion bags has not been reported.

[0003] Some antioxidants and stabilizers containing amines may be used in the production process of pharmaceutical infusion bags. These amine compounds may react with trace amounts of nitrite in the raw materials under certain conditions to form nitrosamines. High-temperature production processes such as injection molding, extrusion, and molding will also accelerate the formation of nitrosamines. In addition, there is also a risk of introducing nitrosamines during filling, sealing, storage, and transportation. Therefore, there are many types of nitrosamines in pharmaceutical infusion bags, and the more common ones are: N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiisopropylamine (NDIPA) and N-nitrosoethylisopropylamine (NEIPA). Since medicinal infusion bags are in direct contact with drugs during use and are closely related to human health, the detection of nitrosamine content in medicinal infusion bags is extremely important.

[0004] According to the requirements of the "Guidelines for Compatibility Testing of Pharmaceutical Packaging Materials and Drugs (Trial)", the detection limit of nitrosamines in medicinal infusion bags is 50% of the limit concentration. The permissible daily exposure (PDE) of nitrosamines in the result evaluation standard is only 0.0265 μg / D. According to the solvent limit calculation formula: concentration (ppm) = (1000×PDE) / dose, the detection limit of nitrosamines in infusion bags of glucose and sodium chloride solutions with large intake amounts must reach the level of one billionth (0.01 μg / L). However, existing nitrosamine detection methods, such as gas chromatography-mass spectrometry, gas chromatography-triple quadrupole mass spectrometry and high performance liquid chromatography-triple quadrupole mass spectrometry, cannot meet the detection requirements. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for simultaneously detecting 9 kinds of nitrosamines in medicinal infusion bags. The present invention can effectively reduce the detection limit of nitrosamines, improve the sensitivity, and the detection result is highly reliable, thereby effectively solving the problem that the detection of nitrosamines in medicinal infusion bags requires a small detection limit.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag comprises the following steps:

[0008] (1) Preparation of standard solution

[0009] Weigh out the standard samples of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodiisopropylamine and N-nitrosoethylisopropylamine respectively, then prepare a standard solution containing all the standard samples with an organic solvent for later use.

[0010] (2) Preparation of internal standard solution

[0011] Weigh respectively the standard samples of five internal standard substances, namely N-nitrosodimethylamine-d6, N-nitrosodipropylamine-d14, N-nitrosodiethylamine-d10, N-nitrosopyrrolidine-d4 and N-nitrosomorpholine-d8, and then prepare internal standard solutions containing the standard samples of each internal standard substance with an organic solvent for use.

[0012] (3) Preparation of sample solution

[0013] The medicinal infusion bag sample is pretreated, then extracted to obtain an extract, which is then mixed with the internal standard solution of step (2), then extracted with a solid phase extraction column and concentrated, and finally filtered with an organic filter membrane to obtain a sample solution for standby use.

[0014] (4) Detection

[0015] The sample solution and the standard solution were detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with those in the standard solution. The 9 nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the abscissa and the peak area as the ordinate, and the content of each analyte was quantitatively calculated by the internal standard method.

[0016] The detection conditions are as follows: the chromatographic column is ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150mm, 3.5μm); the mobile phase A is 0.05%-0.1% formic acid aqueous solution; the mobile phase B is methanol; the column temperature is 35-50℃; the injection volume is 30μL; the atmospheric pressure chemical ionization ion source positive ion mode is adopted; the dynamic multiple reaction ion monitoring acquisition method is adopted; the ion source temperature is 400-500℃; the ionization current is 3μA; the curtain gas is 30psi; the spray gas is 45psi; and the collision gas is 9.

[0017] Furthermore, the organic solvent in step (1) and step (2) is one or both of methanol and acetonitrile.

[0018] Furthermore, the concentration of each standard substance in the standard solution is 0.1-20 μg / L.

[0019] Furthermore, the concentration of each internal standard substance in the internal standard solution is 1 to 10 μg / L.

[0020] Furthermore, in step (3), the pretreatment is to cut the medicinal infusion bag sample into pieces of 2.0 cm×2.0 cm, and wash it with ultrapure water and dry it.

[0021] Furthermore, in step (3), during extraction, the medicinal infusion bag sample is immersed in an acidic solution, an alkaline solution or an alcoholic solution for extraction to obtain an extract.

[0022] Furthermore, the pH of the acidic solution is 3±0.1, the pH of the alkaline solution is 10±0.1, and the extraction temperature is 121°C; the alcoholic solution is a 20% ethanol aqueous solution, and the extraction temperature is 60°C.

[0023] Furthermore, in step (3), the specific steps of extraction and concentration are as follows: after activating the solid phase extraction column, the mixed solution of the extract and the internal standard solution passes through the solid phase extraction column, and then is rinsed and blown dry, and then eluted with dichloromethane to obtain an eluate, and then acetonitrile is added to the eluate, and finally concentrated using a vacuum parallel concentrator.

[0024] Furthermore, the solid phase extraction column is a coconut shell activated carbon solid phase extraction column.

[0025] Furthermore, the organic filter membrane is a 0.22 μm hydrophilic polytetrafluoroethylene filter membrane.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention adopts a coconut shell activated carbon solid phase extraction column to effectively enrich and recover nitrosamines in the extract of the medicinal infusion bag sample, thereby effectively reducing the detection limit of nitrosamines and improving the detection sensitivity. At the same time, the amount of solvent used in the solid phase extraction is small, and it has good reproducibility, which can effectively ensure the reliability and consistency of the analysis results.

[0028] 2. The present invention adopts ultra-high performance liquid chromatography-triple quadrupole mass spectrometry for detection, which has high detection sensitivity and can effectively resist matrix interference. The internal standard method for quantitative analysis can reduce background interference, making the quantitative results more accurate. The 9 nitrosamines in the present invention have good linear relationships within their respective concentration ranges (r 2 >0.99), the average spiked recovery was 84%~114%, the relative standard deviation (RSD) was 0.2%~9.2%, and the limit of detection (LOD) was 0.05~0.1μg / L.

[0029] 3. The present invention can detect 9 kinds of nitrosamines within 12 minutes by optimizing chromatographic conditions such as chromatographic column, column temperature, mobile phase, initial mobile phase ratio, ion source temperature, and mass spectrometry conditions such as quantitative ions, qualitative ions, and optimal collision energy of each target substance. It has high separation and reliability, and effectively improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 -Dynamic multiple reaction monitoring chromatograms of 9 nitrosamines and 5 internal standards.

[0031] Figure 2 - Results of spike recovery experiments with different concentrated protection solvents.

[0032] Figure 3 -Effect of different solid phase extraction columns on the recovery of 9 nitrosamines.

[0033] Figure 4 - Example diagram of positive sample detection. DETAILED DESCRIPTION

[0034] The mass spectrometry scanning parameters of the 9 nitrosamines and 5 internal standard substances in the present invention are shown in Table 1, and the dynamic multiple reaction monitoring chromatograms are shown in Figure 1 As shown, Figure 1 (a) is a dynamic multiple reaction monitoring chromatogram of nine nitrosamines; Figure 1 (b) is the dynamic multiple reaction monitoring chromatogram of 5 internal standard substances.

[0035] Table 1 Mass spectrometry scanning parameters of 9 nitrosamines and 5 internal standard substances

[0036]

[0037] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0038] Example 1

[0039] A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag comprises the following steps:

[0040] (1) Preparation of standard solution

[0041] Weigh appropriate amounts of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiisopropylamine (NDIPA) and N-nitrosoethylisopropylamine (NEIPA) respectively, and prepare standard solutions containing various standard substances with acetonitrile. The concentration of each standard substance in the standard solution is 0.1-20 μg / L, and set aside.

[0042] (2) Preparation of internal standard solution

[0043] Weigh the standard samples of five internal standard substances, namely N-nitrosodimethylamine-d6 (NDMA-d6), N-nitrosodipropylamine-d14 (NDPA-d14), N-nitrosodiethylamine-d10 (NDEA-d10), N-nitrosopyrrolidine-d4 (NPYR-d4), and N-nitrosomorpholine-d8 (NMOR-d8), respectively, and then prepare internal standard solutions containing the standard samples of each internal standard substance with acetonitrile. The concentration of each internal standard substance in the internal standard solution is 1000 μg / L for later use.

[0044] (3) Preparation of sample solution

[0045] 3.1 Cut the negative medicinal infusion bag sample into 2.0 cm × 2.0 cm pieces and place them in a polytetrafluoroethylene bottle. Wash with ultrapure water and dry them. Then inject 500 mL of pH 3 acidic solution to immerse the material, seal it, and extract it at 121 ° C for 2 h to obtain the extract.

[0046] Acidic solution: Dissolve 14.9g potassium chloride in 1L purified water to prepare a 0.2mol / L potassium chloride solution. Adjust the pH to 3±0.1 with 0.2mol / L hydrochloric acid.

[0047] 3.2 Take an appropriate amount of the above sample extract and divide it into three portions, then add three concentration levels of standard solutions and 10 μg / L internal standard solution (obtained by diluting the internal standard solution with a concentration of 1000 μg / L) to mix to obtain a mixed solution, activate the coconut shell activated carbon solid phase extraction column, pass each mixed solution through the solid phase extraction column, rinse, blow dry, and finally elute with dichloromethane to obtain an eluent, then add acetonitrile protective solvent to the eluent, and then concentrate it with a vacuum parallel concentrator, then make the volume to 1.0 mL with ultrapure water, mix well, and finally filter with a 0.22 μm hydrophilic polytetrafluoroethylene filter membrane to obtain three sample solutions for standby use.

[0048] (4) Detection

[0049] The sample solution and the standard solution were detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry. Each concentration level was detected 6 times in parallel. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with the retention time of each analyte in the standard solution, and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared. The 9 nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the horizontal axis and the peak area as the vertical axis, and the content of each analyte was quantitatively calculated by the internal standard method.

[0050] The detection conditions are as follows: the chromatographic column is ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150mm, 3.5μm); the mobile phase A is 0.05% formic acid aqueous solution; the mobile phase B is methanol; the column temperature is 40℃; the injection volume is 30μL; the atmospheric pressure chemical ionization ion source positive ion mode (APCI+) is adopted; the dynamic multiple reaction ion monitoring (dMRM) acquisition method is adopted; the ion source temperature is 400℃; the ionization current is 3μA; the curtain gas is 30psi; the spray gas is 45psi; and the collision gas is 9.

[0051] The results are shown in Table 2. The average recovery of 9 nitrosamines in pH = 3 extracts ranged from 84% to 108%, and the relative standard deviation (RSD) ranged from 1.2% to 7.5%. This method showed good accuracy and precision in pH = 3 extracts.

[0052] Example 2

[0053] A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag comprises the following steps:

[0054] (1) Preparation of standard solution

[0055] Weigh appropriate amounts of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiisopropylamine (NDIPA) and N-nitrosoethylisopropylamine (NEIPA) respectively, and prepare standard solutions containing various standard substances with acetonitrile. The concentration of each standard substance in the standard solution is 0.1-20 μg / L, and set aside.

[0056] (2) Preparation of internal standard solution

[0057] Weigh the standard samples of five internal standard substances, namely N-nitrosodimethylamine-d6 (NDMA-d6), N-nitrosodipropylamine-d14 (NDPA-d14), N-nitrosodiethylamine-d10 (NDEA-d10), N-nitrosopyrrolidine-d4 (NPYR-d4), and N-nitrosomorpholine-d8 (NMOR-d8), respectively, and then prepare internal standard solutions containing the standard samples of each internal standard substance with acetonitrile. The concentration of each internal standard substance in the internal standard solution is 1000 μg / L for later use.

[0058] (3) Preparation of sample solution

[0059] 3.1 Cut the negative medicinal infusion bag sample into 2.0 cm × 2.0 cm pieces and place them in a polytetrafluoroethylene bottle. Wash with ultrapure water and dry them. Then inject 500 mL of alkaline solution with a pH of 10 to immerse the material, seal it, and extract it at 121°C for 2 hours to obtain the extract.

[0060] Alkaline solution: dissolve 14.2 g of disodium hydrogen phosphate in 1 L of purified water, and adjust the pH to 10 ± 0.1 with 0.1 mol / L hydrochloric acid solution or sodium hydroxide solution.

[0061] 3.2 Take an appropriate amount of the above sample extract and divide it into three portions, then add three concentration levels of standard solutions and 10 μg / L internal standard solution (obtained by diluting the internal standard solution with a concentration of 1000 μg / L) to mix to obtain a mixed solution, activate the coconut shell activated carbon solid phase extraction column, pass each mixed solution through the solid phase extraction column, rinse, blow dry, and finally elute with dichloromethane to obtain an eluent, then add acetonitrile protective solvent to the eluent, and then concentrate it with a vacuum parallel concentrator, then make the volume to 1.0 mL with ultrapure water, mix well, and finally filter with a 0.22 μm hydrophilic polytetrafluoroethylene filter membrane to obtain three sample solutions for standby use.

[0062] (4) Detection

[0063] The sample solution and the standard solution were detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry. Each concentration level was detected 6 times in parallel. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with the retention time of each analyte in the standard solution, and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared. The 9 nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the horizontal axis and the peak area as the vertical axis, and the content of each analyte was quantitatively calculated by the internal standard method.

[0064] The detection conditions are as follows: the chromatographic column is ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150mm, 3.5μm); the mobile phase A is 0.05% formic acid aqueous solution; the mobile phase B is methanol; the column temperature is 40℃; the injection volume is 30μL; the atmospheric pressure chemical ionization ion source positive ion mode (APCI+) is adopted; the dynamic multiple reaction ion monitoring (dMRM) acquisition method is adopted; the ion source temperature is 400℃; the ionization current is 3μA; the curtain gas is 30psi; the spray gas is 45psi; and the collision gas is 9.

[0065] The results are shown in Table 2. The average spiked recoveries of the nine nitrosamines in the pH = 10 extract ranged from 88% to 114%, and the relative standard deviations (RSDs) ranged from 1.1% to 9.2%. The method showed good accuracy and precision in the pH = 10 extract.

[0066] Example 3

[0067] A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag comprises the following steps:

[0068] (1) Preparation of standard solution

[0069] Weigh appropriate amounts of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiisopropylamine (NDIPA) and N-nitrosoethylisopropylamine (NEIPA) respectively, and prepare standard solutions containing various standard substances with acetonitrile. The concentration of each standard substance in the standard solution is 0.1-20 μg / L, and set aside.

[0070] (2) Preparation of internal standard solution

[0071] Weigh the standard samples of five internal standard substances, namely N-nitrosodimethylamine-d6 (NDMA-d6), N-nitrosodipropylamine-d14 (NDPA-d14), N-nitrosodiethylamine-d10 (NDEA-d10), N-nitrosopyrrolidine-d4 (NPYR-d4), and N-nitrosomorpholine-d8 (NMOR-d8), respectively, and then prepare an internal standard solution containing the standard samples of each internal standard substance with acetonitrile. The concentration of each internal standard substance in the internal standard solution is 1000 μg / L for later use.

[0072] (3) Preparation of sample solution

[0073] 3.1 Cut the negative medicinal infusion bag sample into 2.0 cm × 2.0 cm pieces and place them in a polytetrafluoroethylene bottle. Wash with ultrapure water and dry them. Then inject 500 mL of 20% ethanol solution to immerse the material, seal it, and extract it at 60 ° C for 8 h to obtain the extract.

[0074] 20% ethanol solution: Mix ultrapure water and ethanol in a ratio of 8:2 (V / V).

[0075] 3.2 Take an appropriate amount of the above sample extract and divide it into three portions, then add three concentration levels of standard solutions and 10 μg / L internal standard solution (obtained by diluting the internal standard solution with a concentration of 1000 μg / L) to mix to obtain a mixed solution, activate the coconut shell activated carbon solid phase extraction column, pass each mixed solution through the solid phase extraction column, rinse, blow dry, and finally elute with dichloromethane to obtain an eluent, then add acetonitrile protective solvent to the eluent, and then concentrate it with a vacuum parallel concentrator, then make the volume to 1.0 mL with ultrapure water, mix well, and finally filter with a 0.22 μm hydrophilic polytetrafluoroethylene filter membrane to obtain three sample solutions for standby use.

[0076] (4) Detection

[0077] The sample solution and the standard solution were detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry. Each concentration level was detected 6 times in parallel. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with the retention time of each analyte in the standard solution, and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared. The 9 nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the horizontal axis and the peak area as the vertical axis, and the content of each analyte was quantitatively calculated by the internal standard method.

[0078] The detection conditions are as follows: the chromatographic column is ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150mm, 3.5μm); the mobile phase A is 0.05% formic acid aqueous solution; the mobile phase B is methanol; the column temperature is 40℃; the injection volume is 30μL; the atmospheric pressure chemical ionization ion source positive ion mode (APCI+) is adopted; the dynamic multiple reaction ion monitoring (dMRM) acquisition method is adopted; the ion source temperature is 400℃; the ionization current is 3μA; the curtain gas is 30psi; the spray gas is 45psi; and the collision gas is 9.

[0079] The results are shown in Table 2. The average recovery of the nine nitrosamines in 20% ethanol solution extracts ranged from 86% to 107%, and the relative standard deviation (RSD) ranged from 0.2% to 6.3%. The method showed good accuracy and precision in 20% ethanol solution extracts.

[0080] Table 2. Experimental results of Examples 1 to 3

[0081]

[0082] Example 4

[0083] A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag comprises the following steps:

[0084] (1) Preparation of standard solution

[0085] Weigh appropriate amounts of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiisopropylamine (NDIPA) and N-nitrosoethylisopropylamine (NEIPA) respectively, and prepare standard solutions containing various standard substances with acetonitrile. The concentration of each standard substance in the standard solution is 0.1-20 μg / L, and set aside.

[0086] (2) Preparation of internal standard solution

[0087] Weigh the standard samples of five internal standard substances, namely N-nitrosodimethylamine-d6 (NDMA-d6), N-nitrosodipropylamine-d14 (NDPA-d14), N-nitrosodiethylamine-d10 (NDEA-d10), N-nitrosopyrrolidine-d4 (NPYR-d4), and N-nitrosomorpholine-d8 (NMOR-d8), respectively, and then prepare internal standard solutions containing the standard samples of each internal standard substance with acetonitrile. The concentration of each internal standard substance in the internal standard solution is 1000 μg / L for later use.

[0088] (3) Preparation of sample solution

[0089] 3.1 Cut the negative medicinal infusion bag sample into 2.0 cm × 2.0 cm pieces and place them in a polytetrafluoroethylene bottle. Wash with ultrapure water and dry them. Then inject 500 mL of pH 3 acidic solution to immerse the material, seal it, and extract it at 121 ° C for 2 h to obtain the extract.

[0090] Acidic solution: Dissolve 14.9g potassium chloride in 1L purified water to prepare a 0.2mol / L potassium chloride solution. Adjust the pH to 3±0.1 with 0.2mol / L hydrochloric acid.

[0091] 3.2 Take 500mL of the above extract, add the standard solution and 10μg / L internal standard solution (obtained by diluting the internal standard solution with a concentration of 1000μg / L) to the extract to obtain a mixed solution. After activating the coconut shell activated carbon solid phase extraction column, the mixed solution is passed through the solid phase extraction column, rinsed, and blown dry, and finally eluted with dichloromethane to obtain an eluent. The eluent is divided into five parts, and then 50uL of five protective solvents, including water, acetonitrile, methanol, ethyl acetate, and n-hexane, are added thereto respectively, concentrated with a vacuum parallel concentrator, and then fixed to 1.0mL with ultrapure water, mixed, and finally filtered with a 0.22μm hydrophilic polytetrafluoroethylene filter membrane to obtain a sample solution for standby use.

[0092] (4) Detection

[0093] The sample solution and the standard solution were detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with those in the standard solution. The nine nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the horizontal axis and the peak area as the vertical axis. The content of each analyte was quantitatively calculated by the external standard method, and the loss of the target compound during vacuum concentration was investigated by the response intensity.

[0094] The detection conditions are as follows: the chromatographic column is ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150mm, 3.5μm); the mobile phase A is 0.05% formic acid aqueous solution; the mobile phase B is methanol; the column temperature is 40℃; the injection volume is 30μL; the atmospheric pressure chemical ionization ion source positive ion mode (APCI+) is adopted; the dynamic multiple reaction ion monitoring (dMRM) acquisition method is adopted; the ion source temperature is 400℃; the ionization current is 3μA; the curtain gas is 30psi; the spray gas is 45psi; and the collision gas is 9.

[0095] Water, acetonitrile, methanol, ethyl acetate, and n-hexane were used as protective solvents to perform spike recovery experiments on blank extracts at known concentration levels. Figure 2 As shown, the results showed that when acetonitrile was used as a protective solvent, the response of 9 nitrosamines was the highest, which could effectively reduce the loss of target compounds during vacuum concentration. Therefore, acetonitrile was finally determined as the concentrated protective solvent.

[0096] Example 5

[0097] A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag comprises the following steps:

[0098] (1) Preparation of standard solution

[0099] Weigh appropriate amounts of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiisopropylamine (NDIPA) and N-nitrosoethylisopropylamine (NEIPA) respectively, and prepare standard solutions containing various standard substances with acetonitrile. The concentration of each standard substance in the standard solution is 0.1-20 μg / L, and set aside.

[0100] (2) Preparation of internal standard solution

[0101] Weigh the standard samples of five internal standard substances, namely N-nitrosodimethylamine-d6 (NDMA-d6), N-nitrosodipropylamine-d14 (NDPA-d14), N-nitrosodiethylamine-d10 (NDEA-d10), N-nitrosopyrrolidine-d4 (NPYR-d4), and N-nitrosomorpholine-d8 (NMOR-d8), respectively, and then prepare internal standard solutions containing the standard samples of each internal standard substance with acetonitrile. The concentration of each internal standard substance in the internal standard solution is 1000 μg / L for later use.

[0102] (3) Preparation of sample solution

[0103] 3.1 Cut the negative medicinal infusion bag sample into 2.0 cm × 2.0 cm pieces and place them in a polytetrafluoroethylene bottle. Wash with ultrapure water and dry them. Then inject 500 mL of pH 3 acidic solution to immerse the material, seal it, and extract it at 121 ° C for 2 h to obtain the extract.

[0104] Acidic solution: Dissolve 14.9g potassium chloride in 1L purified water to prepare a 0.2mol / L potassium chloride solution. Adjust the pH to 3±0.1 with 0.2mol / L hydrochloric acid.

[0105] 3.2 Take 500 mL of the above extract, add the standard solution and 10 μg / L internal standard solution (obtained by diluting the internal standard solution with a concentration of 1000 μg / L) to the extract, mix and divide into 5 portions, activate 5 solid phase extraction columns including coconut shell activated carbon, Oasis HLB, OasisPRiME HLB, SHIMSEN Styra GCB / NH2 and macroporous adsorption resin respectively, pass each mixed solution through the solid phase extraction column, rinse, blow dry, and finally elute with dichloromethane to obtain an eluent, then add acetonitrile protective solvent to the eluent, concentrate with a vacuum parallel concentrator, and then make the volume to 1.0 mL with ultrapure water, mix well, and finally filter with a 0.22 μm hydrophilic polytetrafluoroethylene filter membrane to obtain a sample solution for standby use.

[0106] (4) Detection

[0107] The sample solution and the standard solution were detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with the retention time of each analyte in the standard solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion. The 9 nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the abscissa and the peak area as the ordinate, and the content of each analyte was quantitatively calculated by the internal standard method.

[0108] The detection conditions are as follows: the chromatographic column is ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150mm, 3.5μm); the mobile phase A is 0.05% formic acid aqueous solution; the mobile phase B is methanol; the column temperature is 40℃; the injection volume is 30μL; the atmospheric pressure chemical ionization ion source positive ion mode (APCI+) is adopted; the dynamic multiple reaction ion monitoring (dMRM) acquisition method is adopted; the ion source temperature is 400℃; the ionization current is 3μA; the curtain gas is 30psi; the spray gas is 45psi; and the collision gas is 9.

[0109] Effects of different solid phase extraction columns on the recovery rates of nine nitrosamines Figure 3 As shown by Figure 3 It can be seen that the recovery rate of coconut shell activated carbon column for 9 nitrosamines is between 91% and 110%. It not only has the best enrichment effect on the target compounds, but also has a purification effect on the extract, which can effectively reduce background interference. The enrichment effect of the other four solid phase extraction columns on NDMA is not ideal, and the recovery rate is less than 20%. In addition, HLB, PRiME HLB and GCB / NH2 solid phase extraction columns have matrix interference on compounds such as NMOR and NPYR, resulting in their spiked recovery rates being high.

[0110] Example 6

[0111] A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag comprises the following steps:

[0112] (1) Preparation of standard solution

[0113] Weigh appropriate amounts of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosodiisopropylamine (NDIPA) and N-nitrosoethylisopropylamine (NEIPA) respectively, and prepare standard solutions containing various standard substances with acetonitrile. The concentration of each standard substance in the standard solution is 0.1-20 μg / L, and set aside.

[0114] (2) Preparation of internal standard solution

[0115] Weigh the standard samples of five internal standard substances, namely N-nitrosodimethylamine-d6 (NDMA-d6), N-nitrosodipropylamine-d14 (NDPA-d14), N-nitrosodiethylamine-d10 (NDEA-d10), N-nitrosopyrrolidine-d4 (NPYR-d4), and N-nitrosomorpholine-d8 (NMOR-d8), respectively, and then prepare an internal standard solution containing the standard samples of each internal standard substance with acetonitrile. The concentration of each internal standard substance in the internal standard solution is 1000 μg / L for later use.

[0116] (3) Preparation of sample solution

[0117] 3.1 Cut the medicinal infusion bag samples into 2.0 cm × 2.0 cm pieces in a polytetrafluoroethylene bottle, wash with ultrapure water and dry, divide into three groups, and then inject 500 mL of pH 3 acidic solution, pH 10 alkaline solution and 20% ethanol aqueous solution into the materials, seal them, extract the medicinal infusion bag samples with acidic solution and alkaline solution at 121 ° C for 2 h to obtain the extract, and extract the medicinal infusion bag samples with 20% ethanol solution at 60 ° C for 8 h.

[0118] Acidic solution: Dissolve 14.9g potassium chloride in 1L purified water to prepare a 0.2mol / L potassium chloride solution. Adjust the pH to 3±0.1 with 0.2mol / L hydrochloric acid.

[0119] Alkaline solution: Dissolve 14.2 g of disodium hydrogen phosphate in 1 L of purified water, and adjust the pH to 10 ± 0.1 with 0.1 mol / L hydrochloric acid solution or sodium hydroxide solution.

[0120] 20% ethanol solution: Mix ultrapure water and ethanol in a ratio of 8:2 (V / V).

[0121] 3.2 Take 500mL of the above three extracts, add 10μg / L of internal standard solution (obtained by diluting the internal standard solution with a concentration of 1000μg / L) to each of the three extracts and mix them. After activating the coconut shell activated carbon solid phase extraction column, the mixed solution of each extract and the internal standard solution is passed through the solid phase extraction column, rinsed, and blown dry, and finally eluted with dichloromethane to obtain an eluent, and then acetonitrile protective solvent is added to the eluent, and then concentrated with a vacuum parallel concentrator, and then fixed to 1.0mL with ultrapure water, mixed, and finally filtered with a 0.22μm hydrophilic polytetrafluoroethylene filter membrane to obtain three sample solutions for standby use.

[0122] (4) Detection

[0123] The sample solution and the standard solution were detected by ultra performance liquid chromatography-triple quadrupole mass spectrometry. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with those in the standard solution. The 9 nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the abscissa and the peak area as the ordinate, and the content of each analyte was quantitatively calculated by the internal standard method.

[0124] The detection conditions are as follows: the chromatographic column is ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150mm, 3.5μm); the mobile phase A is 0.05% formic acid aqueous solution; the mobile phase B is methanol; the column temperature is 40℃; the injection volume is 30μL; the atmospheric pressure chemical ionization ion source positive ion mode (APCI+) is adopted; the dynamic multiple reaction ion monitoring (dMRM) acquisition method is adopted; the ion source temperature is 400℃; the ionization current is 3μA; the curtain gas is 30psi; the spray gas is 45psi; and the collision gas is 9.

[0125] The test results of the three sample solutions are as follows Figure 4 As shown, the contents of NMOR in the pH=3 extract, pH=10 extract and 20% ethanol extract of the sample were 0.302 μg / L, 0.289 μg / L and 0.201 μg / L, respectively.

[0126] Finally, it should be noted that the above embodiments of the present invention are only examples for illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For those skilled in the art, other different forms of changes and modifications can be made based on the above description. It is impossible to list all implementation methods here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting 9 kinds of nitrosamines in a medicinal infusion bag, characterized in that: The specific steps include: (1) Preparation of standard solution Weigh the standard products of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosodiisopropylamine and N-nitrosoethylisopropylamine respectively, and then prepare a standard solution containing each standard product with an organic solvent for later use; (2) Preparation of internal standard solution Weigh the standard products of five internal standard substances, namely, N-nitrosodimethylamine-d6, N-nitrosodipropylamine-d14, N-nitrosodiethylamine-d10, N-nitrosopyrrolidine-d4 and N-nitrosomorpholine-d8, respectively, and then prepare an internal standard solution containing the standard products of each internal standard substance with an organic solvent for later use; (3) Preparation of sample solution Pre-treat the sample of the medicinal infusion bag, then extract to obtain an extract, mix it with the internal standard solution of step (2), extract it with a solid phase extraction column and concentrate it, and finally filter it with an organic filter membrane to obtain a sample solution for standby use; (4) Detection The sample solution and the standard solution were detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The retention time of each analyte in the sample solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion were compared with the retention time of each analyte in the standard solution and the mass-to-charge ratio of the qualitative ion to the quantitative ion. The 9 nitrosamines in the sample solution were qualitatively analyzed. The standard curve was fitted with the concentration of each analyte as the abscissa and the peak area as the ordinate, and the content of each analyte was quantitatively calculated by the internal standard method. The detection conditions were as follows: the chromatographic column was ZORBAX Eclipse XDB~C18 Rapid Resolution (4.6×150 mm, 3.5 μm); the mobile phase A was 0.05%-0.1% formic acid in water; the mobile phase B was methanol; the column temperature was 35-50 ℃; the injection volume was 30 μL; the atmospheric pressure chemical ionization ion source was in positive ion mode; the dynamic multiple reaction ion monitoring acquisition method was adopted; the ion source temperature was 400-500 ℃; the ionization current was 3 μA; the curtain gas was 30 psi; and the spray gas was 45 psi. The collision gas is 9.

2. A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 1, characterized in that: The organic solvent in step (1) and step (2) is one or both of methanol and acetonitrile.

3. The method for simultaneously detecting 9 kinds of nitrosamines in a medicinal infusion bag according to claim 1, characterized in that: The concentration of each standard in the standard solution is 0.1~20μg / L.

4. The method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 1, characterized in that: The concentration of each internal standard substance in the internal standard solution is 1-10 μg / L.

5. The method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 1, characterized in that: In step (3), the pretreatment is to cut the medicinal infusion bag sample into pieces of 2.0 cm×2.0 cm, and wash it with ultrapure water and dry it.

6. The method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 1, characterized in that: In step (3), during extraction, the medicinal infusion bag sample is immersed in an acidic solution, an alkaline solution or an alcoholic solution for extraction to obtain an extract.

7. The method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 6, characterized in that: The pH of the acidic solution is 3±0.1, the pH of the alkaline solution is 10±0.1, and the extraction temperature is 121°C; the alcoholic solution is a 20% ethanol aqueous solution, and the extraction temperature is 60°C.

8. The method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 1, characterized in that: In step (3), the specific steps of extraction and concentration are as follows: after activating the solid phase extraction column, the mixed solution of the extract and the internal standard solution passes through the solid phase extraction column, and then is rinsed and blown dry, and then eluted with dichloromethane to obtain an eluent, and then acetonitrile is added to the eluent, and finally concentrated using a vacuum parallel concentrator.

9. A method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 1 or 8, characterized in that: The solid phase extraction column is a coconut shell activated carbon solid phase extraction column.

10. The method for simultaneously detecting nine nitrosamines in a medicinal infusion bag according to claim 1, characterized in that: The organic filter membrane is a 0.22 μm hydrophilic polytetrafluoroethylene filter membrane.

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