A method for rapid determination of diazepam, chlorpromazine and ribavirin in animal-derived foods by accelerated solvent extraction-LC-MS

Abstract

The present invention provides a method for determining the content of diazepam, chlorpromazine and ribavirin in animal-derived food by accelerated solvent extraction-liquid chromatography-mass spectrometry, which belongs to the technical field of residual chemical substance analysis and detection. The present invention uses an accelerated solvent extractor, optimizes the pretreatment extraction and purification methods, and then uses high-performance liquid chromatography-tandem mass spectrometry for analysis, so that the operation process is fast and simple, the detection accuracy is high, and it can provide good detection technology support, and save a lot of time and economic costs. Compared with traditional methods, the experimental time can be reduced from two days to half a day.

Description

Technical Field

[0001] The invention belongs to the technical field of residual chemical substance analysis and detection, and specifically relates to a method for rapidly determining the contents of diazepam, chlorpromazine and ribavirin in animal-derived food by accelerated solvent extraction-liquid chromatography-mass spectrometry. Background Art

[0002] The related technical solutions involving the detection of diazepam, chlorpromazine or ribavirin in the existing technology include: SN / T3235-2012 Detection method of multiple types of banned drug residues in exported animal-derived foods by liquid chromatography-mass spectrometry / mass spectrometry, SN / T 2624-2010 Detection method of multiple basic drug residues in animal-derived foods, GB / T20763-2006 Determination of acepromazine, chlorpromazine, haloperidol, propionyldimethylaminoprophenothiazine, xylazine, azaperone, azaperol, carbazole residues in pig kidney and muscle tissue by liquid chromatography-tandem mass spectrometry, SN / T 2113-2008 Detection method of sedative drug residues in imported and exported animal-derived foods, SN / T 4519-2016 Determination of ribavirin residues in exported animal-derived foods by liquid chromatography-tandem mass spectrometry. The comparison of the existing technical solutions is as follows:

[0003]

[0004]

[0005] At present, there are no joint testing standards, patents and research literature for the three projects in China. The pre-treatment process of the national standard testing method alone is complicated, the testing time is long, the recovery rate is not very ideal, and the testing cost is high. Therefore, the purpose of this patent is to improve the above existing methods and use accelerated solvent extraction instrument-liquid chromatography-mass spectrometry to quickly determine the content of diazepam, chlorpromazine and ribavirin in animal-derived foods, so as to facilitate the rapid and accurate determination of the content of diazepam, chlorpromazine and ribavirin in animal-derived foods and save costs. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for rapidly determining the contents of diazepam, chlorpromazine and ribavirin in animal-derived foods by using an accelerated solvent extractor-liquid chromatography-mass spectrometry. By optimizing the pretreatment extraction and purification methods and then using high performance liquid chromatography-tandem mass spectrometry for analysis, diazepam, chlorpromazine and ribavirin can be detected simultaneously while saving time and testing costs.

[0007] To achieve the above-mentioned purpose, the present application provides a method for rapidly determining the content of diazepam, chlorpromazine and ribavirin in animal-derived food by accelerated solvent extraction instrument-liquid chromatography-mass spectrometry, comprising the following steps:

[0008] S1, weigh the homogenized sample, add diatomaceous earth and grind evenly until the sample is in the form of dry sand;

[0009] S2, transfer to the extraction cell of the accelerated solvent extractor, replace the mobile phase with the extraction solvent, and start extraction;

[0010] S3, take out the extract, blow it almost dry with nitrogen, reconstitute it with the initial mobile phase, vortex it, pass it through the membrane for liquid chromatography-mass spectrometry detection.

[0011] Animal muscle tissue contains a lot of impurities such as protein and fat. The use of accelerated solvent extraction to extract and purify the raw materials can achieve efficient extraction and purification of diazepam, chlorpromazine and ribavirin, thereby avoiding contamination of chromatographic columns and instruments, reducing the appearance of interference peaks, and achieving high-precision and high-efficiency determination of diazepam, chlorpromazine and ribavirin.

[0012] Preferably, in step S1, 5-10 g of diatomaceous earth is added to 5 g of the homogenized sample and ground and mixed.

[0013] Preferably, in step S2, the extraction solvent is selected from acetonitrile, methanol, ethyl acetate, and 0.1% formic acid acetonitrile, more preferably acetonitrile.

[0014] Preferably, in step S3, the nitrogen blowing temperature is 35-50°C, the initial mobile phase is acetonitrile / 0.1% formic acid water (volume ratio is 1:19), and the membrane is a 0.22 μm organic filter membrane.

[0015] Preferably, the accelerated solvent extraction conditions are:

[0016] Heating temperature 35-50℃ Extraction pressure 7-12Mpa Heating time 150-200s Static time 240-360s Eluent volume 20-60％ Purge time 80-120s Cycle times 1-4 times

[0017] More preferably:

[0018] Heating temperature 40℃ Extraction pressure 10Mpa Heating time 180s Static time 300s Eluent volume 40％ Purge time 100s Cycle times 2 times

[0019] Preferably, the liquid phase conditions are:

[0020] Mobile phase: A: acetonitrile; B: 0.1% formic acid water

[0021] Chromatographic column: C18 (1.7μm, 21mm*100mm) Injection volume: 2uL Column temperature: 40℃

[0022] Time / min Flow rate mL / min Mobile phase A (%) Mobile phase B (%) 0 0.2 5 95 2 0.2 5 95 2.1 0.3 95 5 5 0.3 95 5 5.1 0.2 5 95 7 0.2 5 95

[0023] Preferably, the mass spectrometry conditions are:

[0024] Ion source: electrospray source ESI; scanning mode: multiple reaction monitoring (MRM); spray voltage: 0.5 kV; ion source temperature: 500 °C.

[0025]

[0026] The present invention has the following beneficial effects:

[0027] 1. Step S1 of the present invention saves complicated sample processing processes such as freeze-drying, has the effect of saving energy consumption, and meets the requirements of the "dual carbon" environmental protection trend.

[0028] 2. The recovery rate of the present invention is between 80% and 100%, which is much better than that of the prior art.

[0029] 3. The present invention is suitable for the detection of ribavirin, diazepam and chlorpromazine residues in animal muscle and liver. The accelerated solvent extractor is used to make the operation process fast and simple, with high detection accuracy. It can provide good detection technical support and save a lot of time and economic costs. Compared with traditional methods, the experimental time can be reduced from two days to half a day. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 . When eluting in liquid phase, 5mmol·L -1 The elution curve was that formic acid-ammonium formate aqueous solution was used as mobile phase A and methanol:acetonitrile (volume ratio was 1:1) was used as mobile phase B.

[0031] Figure 2 . Elution curve when acetonitrile formic acid water is used as the mobile phase in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technical solution of the present invention is described below through specific examples.

[0033] Example 1

[0034] Reagents: acetonitrile (chromatographic grade); formic acid (chromatographic grade); ultrapure water (first-class water quality); diatomaceous earth, particle size 60 mesh; anhydrous ethanol (chromatographic grade);

[0035] Equipment and consumables: High performance liquid chromatography mass spectrometer (Waters TQS-micro); vortex oscillator; electronic balance (0.0001g); accelerated solvent extractor; nitrogen blowdown apparatus; 0.22μm organic filter membrane.

[0036] Standard solution preparation

[0037] Standard stock solutions: diazepam standard solution (100 mg / L, methanol solvent), ribavirin standard solution (100 mg / L, methanol solvent), chlorpromazine standard solution (100 mg / L, methanol solvent)

[0038] Test steps:

[0039] Weigh 5g of the homogenized sample, add 5g of diatomaceous earth and grind and mix until the sample becomes dry sand without lumps. Transfer all samples to the extraction pool of the 22mL accelerated solvent extractor, cover with filter paper and tighten the nut, and place it on the accelerated solvent extractor for extraction: turn on the power of the equipment, open the nitrogen to 0.8Mpa, replace the mobile phase with acetonitrile, rinse the equipment 3 times, and start extraction (extraction conditions see instrument conditions).

[0040] The extract was taken out and transferred into a 50 mL centrifuge tube, blown almost dry with nitrogen at 40°C, re-dissolved with 2 mL of acetonitrile + 0.1% formic acid water (volume ratio of 1:19), vortexed for 30 seconds, and passed through a 0.22 μm organic filter membrane for instrument detection.

[0041] The accelerated solvent extraction conditions were:

[0042] Heating temperature 40℃ Extraction pressure 10Mpa Heating time 180s Static time 300s Eluent volume 40％ Purge time 100s Cycle times 2 times

[0043] The liquid phase conditions are:

[0044] Mobile phase: A: acetonitrile; B: 0.1% formic acid water

[0045] Chromatographic column: C18 (1.7μm, 21mm*100mm) Injection volume: 2uL Column temperature: 40℃

[0046] Time / min <![CDATA[Flow rate mL / min]]> Mobile phase A (%) Mobile phase B (%) 0 0.2 5 95 2 0.2 5 95 2.1 0.3 95 5 5 0.3 95 5 5.1 0.2 5 95 7 0.2 5 95

[0047] The mass spectrometry conditions were:

[0048] Ion source: electrospray source ESI; scanning mode: multiple reaction monitoring (MRM); spray voltage: 0.5 kV; ion source temperature: 500 °C.

[0049]

[0050] Test samples: blank pork front trough meat; blank chicken liver; pork front trough meat and chicken liver samples added with ribavirin, diazepam and chlorpromazine at three different concentrations, namely, high, medium and low. Among them, the blank pork front trough meat and blank chicken liver samples were tested three times, and the pork front trough meat and chicken liver samples added with ribavirin, diazepam and chlorpromazine at three different concentrations, namely, high, medium and low, were tested six times. The test results are shown in Table 1.

[0051] Table 1 Experimental data of precision and recovery

[0052]

[0053] The invention utilizes liquid chromatography-tandem mass spectrometry to qualitatively determine the retention time, quantitatively determine the peak area, and quantitatively determine the ribavirin, diazepam, and chlorpromazine residues in animal-derived foods by external standard method. After accelerated solvent extraction, the recovery rate of the target ribavirin in pig front trough meat reached 88.2-96.0%, with a precision RSD of 1.3-2.4%, the recovery rate of diazepam reached 86.8-96.3%, with a precision RSD of 0.6-2.1%, and the recovery rate of chlorpromazine reached 87.2-99.4%, with a precision RSD of 1.4-3.2%; in chicken liver, the recovery rate of the target ribavirin reached 85.2-94.5%, with a precision RSD of 1.0-2.5%, the recovery rate of diazepam reached 83.2-96.1%, with a precision RSD of 0.7-2.1%, and the recovery rate of chlorpromazine reached 86.2-95.4%, with a precision RSD of 1.2-1.8%.

[0054] Example 2

[0055] Accelerated Solvent Extraction - Selection of Extraction Solvents and Optimization of Extraction Conditions

[0056] 1. Selection of extraction solvent

[0057] For drug residues in animal-derived foods, organic solvents are generally used as extraction solvents. This experiment also compared the extraction effects of three veterinary drugs by adding 15mL of acetonitrile, methanol, 0.1% formic acid acetonitrile, and ethyl acetate, using 5g of pork front trough meat as a sample. A spike recovery experiment was conducted at 25μg / kg (the extraction solution was not subjected to accelerated solvent extraction and purification), and the extraction efficiency of the three veterinary drugs by different extraction solvents was compared. It was found that acetonitrile had the best average extraction effect. The results are shown in Table 2.

[0058] Table 2 Extraction effects of different extraction solvents

[0059]

[0060] 2. Optimization of extraction reagent elution volume

[0061] When the sample weight is 5g of pork front trough meat sample, acetonitrile is used as the extraction solvent, the spiked amount is 25ug / kg for recovery experiment, and the elution volume is set to 10%, 20%, 30%, 40%, 50%, and the extraction time is 300s. Comparing the extraction rates of the three veterinary drugs with the five extraction solvent volumes, it is found that the average extraction effect of 40% is the best under the premise of saving reagent costs. The results are shown in Table 3.

[0062] Table 3 Extraction effect of different extraction volumes

[0063]

[0064]

[0065] 3. Optimization of static extraction time

[0066] When the sample weight was 5g of pork front trough meat sample, acetonitrile was used as the extraction solvent, and the spiked amount was 25ug / kg for recovery experiment, and the static extraction time was set as: 60s, 120s, 180s, 240s, 300s. Comparing the extraction rates of the three veterinary drugs with the five extraction times, it was found that the average extraction effect at 300s was the best, and the results are shown in Table 4.

[0067] Table 4 Extraction effect of different static extraction time

[0068]

[0069] Comparative Example 1

[0070] The blank pork front trough meat and blank chicken liver in Example 1 were used as test samples, and the recovery rates of ribavirin, diazepam, and chlorpromazine were determined according to the method described in Section 2.2 of the journal literature (High Performance Liquid Chromatography-Tandem Mass Spectrometry for Rapid Determination of Multiple Veterinary Drug Residues in Chicken, Feng Huihui et al., Modern Food, 2021, pp. 180-185, 189), where the spiked amounts of ribavirin, diazepam, and chlorpromazine were all 10 μg / kg, and the results are shown in Table 5. It can be seen that the recovery rates and precisions of ribavirin, diazepam, and chlorpromazine were greatly reduced by the method of the above-mentioned journal literature, and ribavirin, diazepam, and chlorpromazine could not be recovered with high recovery rate and high precision at the same time.

[0071] Table 5

[0072]

[0073] Comparative Example 2

[0074] When eluting in liquid phase, 5 mmol·L -1 Formic acid-ammonium formate aqueous solution was used as mobile phase A; methanol:acetonitrile (volume ratio of 1:1) was used as mobile phase B. A mixed standard solution of ribavirin, diazepam, and chlorpromazine with a concentration of 100 ng / mL was prepared and injected for analysis. The spectral results are shown in the attached Figure 1 When using ammonium formate solution and methanol + acetonitrile as the mobile phase, the elution time of ribavirin is about 0.44min, and there is almost no retention in the chromatographic column and it is directly eluted. In the sample detection process, there is a matrix interference peak at 0.44min, which interferes with the determination of the quantitative results. When acetonitrile formic acid water is used as the mobile phase in Example 1 of the present invention, the elution time of ribavirin is about 1.20min. Figure 2 , which is more suitable for eliminating interference in large-scale detection of samples.

[0075] The solutions of the present invention are not limited to the technical means disclosed by the above technical means, but also include technical solutions composed of any combination of the above technical features. The above is a specific implementation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A method for rapidly determining the content of diazepam, chlorpromazine and ribavirin in animal-derived food by accelerated solvent extraction instrument-liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: S1, weigh the homogenized sample, add diatomaceous earth and grind evenly until the sample is in the form of dry sand; S2, transfer to the extraction cell of the accelerated solvent extractor, replace the mobile phase with the extraction solvent, and start extraction; S3, taking out the extract, blowing it to near dryness with nitrogen, re-dissolving it with the initial mobile phase, vortexing it, and passing it through the membrane for LC-MS detection; Wherein, in step S1, 5 g of the homogenized sample is added with 5-10 g of diatomaceous earth for grinding and mixing; in step S2, the extraction solvent is selected from one of acetonitrile, methanol, ethyl acetate, and 0.1% formic acid acetonitrile; In step S2, the accelerated solvent extraction conditions are: 。 2. The method according to claim 1, characterized in that In step S2, the extraction solvent is acetonitrile.

3. The method according to claim 1, characterized in that In step S3, the nitrogen blowing temperature is 35-50°C, and the membrane is a 0.22 μm organic filter membrane.

4. The method according to claim 1, characterized in that In step S3, the initial mobile phase is acetonitrile / 0.1% formic acid water, and the volume ratio of acetonitrile to 0.1% formic acid water is 1:

19.

5. The method according to claim 1, characterized in that The liquid phase conditions are: Mobile phase: A: acetonitrile; B: 0.1% formic acid water; Chromatographic column: C18; 。 6. The method according to claim 1, characterized in that The mass spectrometry conditions were: Ion source: electrospray source ESI; scanning mode: multiple reaction monitoring (MRM); spray voltage: 0.5 kV; Ion source temperature: 500°C.

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