Method for detecting diazepam, nordiazepam and nordiazepam isomer BP-271 in fish meat

Abstract

The invention provides a method for detecting diazepam, nordiazepam and nordiazepam isomeride BP-271 in fish meat, and belongs to the technical field of analysis and detection. According to the method disclosed by the invention, the diazepam, nordiazepam and BP-271 in the fish meat are subjected to qualitative analysis and quantitative analysis by adopting ultra-high performance liquid chromatography-tandem mass spectrometry, and the situation that the BP-271 is detected in fish bodies when the fish is polluted by the diazepam is revealed; the method is suitable for simultaneous detection of diazepam, BP-271 and nordiazepam in a large batch of fish bodies, the detection accuracy and precision are good, and methodological requirements are met.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting diazepam, nordiazepam and nordiazepam isomer BP-271 in fish meat. Background Art

[0002] Diazepam (DZP, also known as Valium) is a long-acting benzodiazepine with clinical efficacy as a sedative, hypnotic, and anxiolytic. However, human metabolism may cause diazepam to contaminate aquatic environments. Furthermore, diazepam is commonly used in aquaculture, transportation, and fishing bait. For example, if 13.0 to 389.7 mg of diazepam is introduced into water bodies during baiting, this can cause a temporary increase in diazepam concentrations in localized waters, contaminating aquatic products. This can ultimately lead to the accumulation of diazepam and its metabolites in animals, where it can biomagnify and subsequently enter the human body through the food chain or the water cycle, potentially inducing adverse reactions such as neurotoxicity.

[0003] Emerging contaminants (ECs), represented by diazepam, are frequently detected in water environments worldwide. These substances have attracted widespread attention due to their environmental persistence and bioaccumulation. Georg Braun et al. found that the cumulative toxicity of multiple compounds can cause significant neurotoxicity even at concentrations below the effect threshold of individual substances. Existing wastewater treatment technologies are unable to effectively remove ECs, resulting in their entry into the natural environment through the water cycle, posing a threat to human health.

[0004] Conventional tap water disinfection processes mostly use chlorine disinfection to treat water samples to reduce the occurrence of waterborne infectious diseases. However, during the chlorine disinfection process, diazepam in the water will react with the disinfectant to form chlorine disinfection by-products (DBPs). Studies have shown that some DBPs are more toxic and mutagenic than the parent compounds. Carpinteiro et al. speculated through computer simulation that DBPs are more harmful to the human body than the parent compounds. A technology has successfully identified 5 emerging DBPs of diazepam by simulating chlorine disinfection experiments using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, and 4 of them have been detected in actual drinking water, one of which is BP-271 with a molecular formula of C 15 H 11 N2OCl and the diazepam metabolite nordiazepam (NZP) in fish are isomers with structures shown in Formula I. The total concentration of the four diazepam DBPs ranges from 0.6 ng / L to 7.37 ng / L. Existing technologies have only detected the presence of BP-271 in water environments and have not tested its presence in fish. Furthermore, the accuracy of existing technologies for detecting diazepam, nordiazepam, and BP-271 needs to be further improved.

[0005] Summary of the Invention

[0006] The object of the present invention is to provide a method for detecting diazepam, nordiazepam and the nordiazepam isomer BP-271 in fish meat. The present invention reveals that BP-271 is also detected in fish bodies when fish are contaminated with diazepam, and the detection method of the present invention has good accuracy and precision, meeting methodological requirements.

[0007] In order to achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0008] A method for detecting diazepam, nordiazepam, and nordiazepam isomer BP-271 in fish meat comprises the following steps:

[0009] The fish sample is sequentially extracted, purified and concentrated to obtain a test solution; the extraction agent used in the extraction is acetonitrile; the extraction agent used in the extraction is sodium chloride; the purification is a solid phase extraction column purification;

[0010] The test solution is qualitatively analyzed by high resolution mass spectrometry;

[0011] The chromatographic separation conditions in the qualitative analysis include: a T3 column; mobile phase A is a 0.1-0.3% volume fraction of formic acid aqueous solution, and mobile phase B is acetonitrile; the column temperature is 35-40° C., and the flow rate is 0.2-0.4 mL / min; the elution method is gradient elution, and the elution program of the gradient elution is: 0 min, 40% mobile phase B; 0.5 min, 40% mobile phase B; 4 min, 50% mobile phase B; 9 min, 100% mobile phase B; 10 min, 100% mobile phase B; 10.1 min, 40% mobile phase B; 12 min, 40% mobile phase B;

[0012] The high-resolution mass spectrometry detection in the qualitative analysis includes a primary analysis in full scan mode and a secondary analysis in product ion mode; the orbitrap resolution in full scan mode is 100,000 to 120,000, the scan range is 150 to 400 m / z, and the gas is nitrogen; the conditions of the product ion mode include: the ion source is an electrospray ion source; the ESI capillary voltage is 1000 to 3000 V; the sheath gas is 30 to 50 Arb, the auxiliary gas is 10 to 30 Arb, and the purge gas is 1 to 5 Arb, and the sheath gas, auxiliary gas, and purge gas are nitrogen; the ion transfer tube temperature is 300 to 325° C., and the vaporization chamber temperature is 330 to 350° C.

[0013] An internal standard is added to the test solution, and quantitative analysis is performed using ultra-performance liquid chromatography-tandem mass spectrometry; the internal standard is diazepam-D5 and nordiazepam-D5;

[0014] The conditions of ultra-high performance liquid chromatography in the quantitative analysis include: the chromatographic column is C 18 Column; mobile phase A is a 0.1-0.3% volume fraction of formic acid aqueous solution, mobile phase B is acetonitrile; column temperature is 30-35°C, flow rate is 0.2-0.3 mL / min; gradient elution, the elution program of the gradient elution is: 0-2.00 min, 30% mobile phase B; 2.00-7.00 min, 30%-70% mobile phase B; 7.00-9.00 min, 70-100% mobile phase B; 9.00 min, 100% mobile phase B; 10.00-10.10 mobile phase min, 100%-30% mobile phase B, hold for 1.9 min;

[0015] The mass spectrometry conditions in the quantitative analysis include: the ion source is an electrospray ion source; the capillary voltage is 0.5-1 kV, the ion source temperature is 100-150°C, the desolvation gas temperature is 300-500°C, the desolvation gas flow rate is 600-800 L / h, the cone hole backflush gas flow rate is 120-150 L / h, and the desolvation gas is nitrogen.

[0016] Preferably, the ratio of the volume of the extractant to the mass of the fish sample is 3-10 mL:1-5 g.

[0017] Preferably, the extraction comprises sequentially performing vortex extraction and ultrasonic extraction;

[0018] The speed of the vortex is 1000-2000 rpm, and the frequency of the ultrasound is 30k-40kHz; the time of the vortex extraction and the ultrasound extraction is independently 10-30 minutes.

[0019] Preferably, the extraction agent used in the extraction is sodium chloride; the mass ratio of the extraction agent to the fish sample is 1:1 to 3.

[0020] Preferably, the filler of the solid phase extraction column in the purification comprises C 18 / PSA, EMR, C 18 / SAX、Silica / PSA、C 18 and one of SLC.

[0021] Preferably, after the concentration, the obtained product is further redissolved and microfiltered; the reagent used for the redissolution is acetonitrile.

[0022] Preferably, the pore size of the filter membrane used for microfiltration is 0.1 to 3 μm; and the filter membrane is a polytetrafluoroethylene membrane.

[0023] Preferably, the contents of diazepam, nordiazepam and nordiazepam isomer BP-271 are calculated using the internal standard method.

[0024] Preferably, the concentration of the mixed standard solution of the internal standard is 100-400 ng / mL.

[0025] Preferably, the injection volume of the chromatographic separation in the qualitative analysis is 1 to 10 μL; the injection volume of the ultra-high performance liquid chromatography in the quantitative analysis is 1 to 5 μL.

[0026] The present invention provides a method for detecting diazepam, nordiazepam and nordiazepam isomer BP-271 in fish meat, comprising the following steps: extracting, purifying and concentrating a fish meat sample in sequence to obtain a test solution; the extractant used in the extraction is acetonitrile; the extractant used in the extraction is sodium chloride; the purification is a solid phase extraction column purification; the test solution is qualitatively analyzed by high-resolution mass spectrometry; the chromatographic separation conditions in the qualitative analysis include: chromatographic column The column was a T3 column; the mobile phase A was a 0.1-0.3% volume fraction of formic acid in water, and the mobile phase B was acetonitrile; the column temperature was 35-40°C, and the flow rate was 0.2-0.4 mL / min; the elution method was gradient elution, and the elution program of the gradient elution was as follows: 0 min, 40% mobile phase B; 0.5 min, 40% mobile phase B; 4 min, 50% mobile phase B; 9 min, 100% mobile phase B; 10 min, 100% mobile phase B; 10. 1 min, 40% mobile phase B; 12 min, 40% mobile phase B; the high-resolution mass spectrometry detection in the qualitative analysis includes preliminary analysis in full scan mode and secondary analysis in product ion mode; the orbitrap resolution in full scan mode is 100,000 to 120,000, the scan range is 150 to 400 m / z, and the gas is nitrogen; the conditions of the product ion mode include: the ion source is an electrospray ionization source; the ESI capillary voltage is 1000 to 3000 V ; Sheath gas 30-50Arb, auxiliary gas 10-30Arb, purge gas 1-5Arb, sheath gas, auxiliary gas and purge gas are nitrogen; ion transfer tube temperature 300-325 ° C, vaporization chamber temperature 330-350 ° C; internal standard is added to the test solution, and ultra-performance liquid chromatography-tandem mass spectrometry is used for quantitative analysis; the internal standard is diazepam-D5 and nordiazepam-D5; the ultra-performance liquid chromatography conditions in the quantitative analysis include: the chromatographic column is C 18Column; mobile phase A is a 0.1-0.3% volume fraction of formic acid aqueous solution, mobile phase B is acetonitrile; column temperature is 30-35 ° C, flow rate is 0.2-0.3 mL / min; gradient elution, the elution program of the gradient elution is: 0-2.00 min, 30% mobile phase B; 2.00-7.00 min, 30%-70% mobile phase B; 7.00-9.00 min, 70-100% mobile phase B; 9.00 min, 10 0% mobile phase B; 10.00-10.10 min of mobile phase, 100%-30% mobile phase B, hold for 1.9 min; mass spectrometry conditions for the quantitative analysis include: an electrospray ion source; a capillary voltage of 0.5-1 kV, an ion source temperature of 100-150°C, a desolvation gas temperature of 300-500°C, a desolvation gas flow rate of 600-800 L / h, a cone backflush gas flow rate of 120-150 L / h, and nitrogen as the desolvation gas. The present invention discloses the detection of BP-271 in fish contaminated with diazepam; this method is suitable for the simultaneous detection of diazepam, BP-271, and nordiazepam in fish. Furthermore, the present invention is time-saving and highly efficient in the pretreatment of fish samples, and is suitable for large-scale sample testing. At the same time, according to the results of the embodiments of the present invention, diazepam, BP-271 and nordiazepam have good linearity in the range of 0.2 to 10.0 μg / kg, and the correlation coefficients are all higher than 0.999; the average spiked recovery rate is 100.2% to 119.9%, the relative standard deviation is 1.7% to 9.5%, the method detection limit is 0.2 μg / kg, the quantification limit is 0.6 μg / kg, the accuracy and precision are good, and the methodological requirements are met. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the scan spectrum of a diazepam-positive fish meat sample after screening in high-resolution mass spectrometry full scan mode;

[0029] Figure 2 This is the MS / MS spectrum of BP-271 in the diazepam-positive fish sample (corresponding to the RT 3.19 min peak);

[0030] Figure 3 The MRM spectra of diazepam, nordiazepam, BP-271 mixed standard solution and two internal standard mixed standard solutions;

[0031] Figure 4The chromatograms of the mixed standard solution of nordiazepam and BP-271 separated by different mobile phases are shown in Figure 2. Figure 4 Acetonitrile was used as mobile phase B on the left, and methanol was used as mobile phase B on the right;

[0032] Figure 5 The sample chromatogram shows the detection of BP-271 in fish samples in the application example. DETAILED DESCRIPTION

[0033] The present invention provides a method for detecting diazepam, nordiazepam and nordiazepam isomer BP-271 in fish meat, comprising the following steps:

[0034] The fish sample is sequentially extracted, purified and concentrated to obtain a test solution; the extraction agent used in the extraction is acetonitrile; the extraction agent used in the extraction is sodium chloride; the purification is a solid phase extraction column purification;

[0035] The test solution is qualitatively analyzed by high resolution mass spectrometry;

[0036] The chromatographic separation conditions in the qualitative analysis include: a T3 column; mobile phase A is a 0.1-0.3% volume fraction of formic acid aqueous solution, and mobile phase B is acetonitrile; the column temperature is 35-40° C., and the flow rate is 0.2-0.4 mL / min; the elution method is gradient elution, and the elution program of the gradient elution is: 0 min, 40% mobile phase B; 0.5 min, 40% mobile phase B; 4 min, 50% mobile phase B; 9 min, 100% mobile phase B; 10 min, 100% mobile phase B; 10.1 min, 40% mobile phase B; 12 min, 40% mobile phase B;

[0037] The high-resolution mass spectrometry detection in the qualitative analysis includes a primary analysis in full scan mode and a secondary analysis in product ion mode; the orbitrap resolution in full scan mode is 100,000 to 120,000, the scan range is 150 to 400 m / z, and the gas is nitrogen; the conditions of the product ion mode include: the ion source is an electrospray ion source; the ESI capillary voltage is 1000 to 3000 V; the sheath gas is 30 to 50 Arb, the auxiliary gas is 10 to 30 Arb, and the purge gas is 1 to 5 Arb, and the sheath gas, auxiliary gas, and purge gas are nitrogen; the ion transfer tube temperature is 300 to 325° C., and the vaporization chamber temperature is 330 to 350° C.

[0038] An internal standard is added to the test solution, and quantitative analysis is performed using ultra-performance liquid chromatography-tandem mass spectrometry; the internal standard is diazepam-D5 and nordiazepam-D5;

[0039] The conditions of ultra-high performance liquid chromatography in the quantitative analysis include: the chromatographic column is C 18Column; mobile phase A is a 0.1-0.3% volume fraction of formic acid aqueous solution, mobile phase B is acetonitrile; column temperature is 30-35°C, flow rate is 0.2-0.3 mL / min; gradient elution, the elution program of the gradient elution is: 0-2.00 min, 30% mobile phase B; 2.00-7.00 min, 30%-70% mobile phase B; 7.00-9.00 min, 70-100% mobile phase B; 9.00 min, 100% mobile phase B; 10.00-10.10 mobile phase min, 100%-30% mobile phase B, hold for 1.9 min;

[0040] The mass spectrometry conditions in the quantitative analysis include: the ion source is an electrospray ion source; the capillary voltage is 0.5-1 kV, the ion source temperature is 100-150°C, the desolvation gas temperature is 300-500°C, the desolvation gas flow rate is 600-800 L / h, the cone hole backflush gas flow rate is 120-150 L / h, and the desolvation gas is nitrogen.

[0041] In the present invention, unless otherwise specified, all raw materials are commercially available products well known to those skilled in the art.

[0042] In the present invention, the extraction agent used is acetonitrile, and the ratio of the volume of the extraction agent to the mass of the fish sample is 3-10 mL:1-5 g. In a specific embodiment, it can be 5 mL:1 g or 10 mL:2 g. The extraction includes sequentially performing vortex extraction and ultrasonic extraction. The time of the vortex extraction can be 20 min, and the time of the ultrasonic extraction can be 30 min.

[0043] In the present invention, the extraction agent used is sodium chloride, and the mass ratio of the extraction agent to the fish sample is 1:1 to 3. In a specific embodiment, it can be 1:1. The extraction is vortex extraction, and the time is 5 to 10 minutes. After the extraction, solid-liquid separation is further included, and the obtained supernatant is prepared into a 90% acetonitrile aqueous solution and then purified. The solid-liquid separation is centrifugal at a rate of 7000 to 9000 r / min and a time of 3 to 5 minutes. The purification is solid phase extraction column purification; the filler of the solid phase extraction column (SPE column) includes C 18 / PSA, EMR, C 18 / SAX、Silica / PSA、C 18 and one of the SLC columns, in a specific embodiment, C 18 / PSA; the solid phase extraction column was activated with 90% acetonitrile aqueous solution before use.

[0044] The present invention has no special restrictions on the conditions for concentration, and the concentration can be performed to near dryness. In a specific embodiment, the concentration can be performed to a water content of less than 36%. After the concentration, the obtained product is further dissolved and filtered. The reagent used for the dissolution is acetonitrile. The pore size of the filter membrane used for the filtration is 0.1 to 3 μm, and in a specific embodiment, it can be 0.22 or 1 μm. The filter membrane is a polytetrafluoroethylene membrane.

[0045] In the present invention, a spectrum is obtained after quantitative analysis, and the contents of diazepam, nordiazepam and nordiazepam isomer BP-271 are calculated using the internal standard method; the internal standard is nordiazepam-D5.

[0046] In the present invention, the concentration of the mixed standard solution of the internal standard is 100-400 ng / mL.

[0047] To further illustrate the present invention, the following detailed description of the method for detecting diazepam, nordiazepam and nordiazepam isomer BP-271 in fish provided by the present invention is provided in conjunction with the accompanying drawings and examples. However, these should not be construed as limiting the scope of protection of the present invention.

[0048] The parameters and sources of the experimental instruments and reagents in the following examples of the present invention are as follows:

[0049] Orbitrap high-resolution mass spectrometer (Thermo Scientific Orbitrap Exploris 240), Thermo Fisher Scientific, USA;

[0050] Ultra-high performance liquid chromatography-tandem mass spectrometry (ACQUITY I-Class UPLC, XEVO TQ-S), Waters, USA;

[0051] Automatic vortex mixer, Heidolph, Germany;

[0052] Allegra 64R refrigerated centrifuge, Beckman Coulter, USA;

[0053] Solid phase extraction instrument, Waters Company, USA;

[0054] Ultrapure water preparation instrument, Millipore, USA;

[0055] Acetonitrile, chromatographic grade, Thermo Fisher Scientific (China) Co., Ltd.;

[0056] Sodium chloride, analytical grade, Sinopharm Chemical Reagent Co., Ltd.

[0057] Diazepam, nordiazepam, diazepam-D5, and nordiazepam-D5 standard solutions (all 100 μg / mL) were purchased from Tianjin Alta Co., Ltd.

[0058] BP-271 standard solution, 100 μg / mL, Beijing Center for Disease Control and Prevention;

[0059] C 18 / PSA column was 500 mg / mL and purchased from Kangyuan Technology Co., Ltd.;

[0060] Fish samples were collected from the market;

[0061] 100 ng / mL mixed standard solution: Pipette 10 μL each of diazepam, nordiazepam, and BP-271 standard solutions into a volumetric flask, add acetonitrile to make up to 10 mL, mix well, transfer to a 10 mL brown bottle, and store at 4°C until use;

[0062] 400 ng / mL internal standard mixed solution: Pipette 40 μL each of diazepam-D5 and nordiazepam-D5 into a 10 mL volumetric flask, dilute to volume with acetonitrile, transfer to a brown bottle, and store at 4°C until use.

[0063] Example 1

[0064] Fish muscles were homogenized, and the surimi samples were placed in 50 mL centrifuge tubes and frozen at −20 °C until analysis;

[0065] Accurately weigh 2.00 g of the thawed fish meat, let it stand for 5 minutes, then add 10 mL of acetonitrile, quickly shake the fish meat and extract it by vortexing for 20 minutes, and then ultrasonically extract it for 30 minutes; then add 2 g of NaCl and vortex for 5 minutes, centrifuge at 9000 r / min for 4 minutes, take the supernatant and prepare 5 mL of 90% acetonitrile aqueous solution, and pass it through C 18 / PSA column, discard 1 mL, and collect the remaining 4 mL of purified liquid; concentrate the purified liquid to near dryness, add 0.3 mL of acetonitrile to redissolve it, and make up to 1 mL with ultrapure water. Filter through a 0.22 μm PTFE filter membrane to obtain the test solution;

[0066] The high-resolution mass spectrometer was calibrated with sodium formate, and the metabolites of the test solution were preliminarily screened in full-scan mode. The orbitrap resolution in full-scan mode was 120,000, the scanning range was 150-400 m / z, and the gas was nitrogen. To further verify the presence of metabolites, qualitative analysis was performed in product ion mode. Chromatographic separation was performed using a T3 column (specifications: 100 mm × 2.1 mm, 1.7 μm) at 40°C, 0.1% formic acid water as mobile phase A, acetonitrile as mobile phase B, and the gradient elution program was: 0 mi n, 40% B; 0.5 min, 40% B; 4 min, 50% B; 9 min, 100% B; 10 min, 100% B; 10.1 min, 40% B; 12 min, 40% B, flow rate was 0.4 mL / min, injection volume was 5 μL; mass spectrometry conditions were: electrospray ion source, ESI capillary voltage 3000 V, sheath gas 50 Arb, auxiliary gas 10 Arb, sweep gas 1 Arb, ion transfer tube temperature 325 ° C, vaporization chamber temperature 350 ° C.

[0067] Qualitative analysis results and analysis

[0068] The fish matrix diazepam-positive samples were preliminarily screened by high-resolution mass spectrometry full scan mode. Figure 1 As shown in the figure, when extracting the chromatographic peak of nordiazepam, it was found that the characteristic fragment ion of m / z 271.0632 existed at the retention time (RT) of 2.71 and 3.19 min. The secondary mass spectrum was further obtained using the product ion mode. The nordiazepam standard showed that its quasi-molecular ion peak in the positive ion mode was m / z 271.0632 ([M+H] + ), further fragmentation yielded highly abundant characteristic fragment ions at m / z 140.0262, m / z 208.0996, and m / z 165.0214. The primary fragment ions and abundances in the nordiazepam standard matched those in the corresponding mass spectrometry spectrum of the compound at RT 2.71 min, although another peak was not completely identical. In addition to the matching fragments, the mass spectrometry spectrum at RT 3.19 min contained other relatively abundant fragment ions, primarily m / z 221.0709 and m / z 193.0163. Furthermore, under collision energy conditions of 70, the fragment 271.0632 at RT 2.71 min was completely fragmented, but remained abundant at RT 3.19 min. In summary, this compound is hypothesized to be a new compound with a similar ionic structure to nordiazepam.

[0069] The secondary mass spectrum was obtained by running in product ion mode under positive ion conditions. The secondary mass spectrum of the chromatographic peak at RT 3.19min is shown in the figure below. Figure 2 As shown, 271.0632 is the quasi-molecular ion peak ([M+H] +), it is speculated that m / z 193.0163 is generated by the loss of C6H6, that is, [M+H-C6H6] + m / z 165.0214 is the loss of C6H6 followed by the loss of CO, [M+H-C6H6-CO] + ; m / z 221.0708 is [M+H-CH3-Cl] + , that is, removing CH3 and Cl, it was believed that there was another unknown compound related to diazepam in the fish meat. It was subsequently found that the compound was consistent with the secondary mass spectrum of diazepam chlorine disinfection by-products, and was named BP-271.

[0070] Example 2

[0071] After qualitative analysis, the recovery was calculated by the internal standard method using diazepam-D5 as the internal standard for diazepam, and nordiazepam-D5 as the internal standard for nordiazepam-D5 and BP-271;

[0072] 2.00 g of the thawed fish meat was accurately weighed, and 20 μL (400 ng / mL) of the mixed internal standard solution was added to obtain a diazepam-positive fish meat sample; after standing for 5 minutes, 10 mL of acetonitrile was added, the fish meat was quickly shaken and vortexed for 20 minutes, and ultrasonically extracted for 30 minutes; 2 g of NaCl was then added and vortexed for 5 minutes, centrifuged at 9000 r / min for 4 minutes, and the supernatant was prepared into 5 mL of 90% acetonitrile aqueous solution, and the mixture was filtered through a C 18 / PSA column, discard 1 mL, and collect the remaining 4 mL of purified liquid; after concentrating the purified liquid to near dryness, add 0.3 mL of acetonitrile to re-dissolve it, and make up to 1 mL with ultrapure water. Filter through a 0.22 μm PTFE filter membrane to obtain the test solution for quantitative analysis.

[0073] For the quantitative analysis of the test solution, the mass spectrometry conditions were as follows: electrospray ionization source, operating in positive ion mode under multiple reaction monitoring (MRM); capillary voltage 0.5 kV, ion source temperature 150 ° C, desolvation temperature 500 ° C, desolvation gas flow rate 800 L / h, cone hole backflush gas flow rate 150 L / h. Ultra-high performance liquid chromatography conditions were: using ACQUITY UPLC BEH C 18Chromatographic separation was performed on a chromatographic column (specifications: 100 mm × 2.1 mm, 1.7 μm) at 35°C, with 0.1% formic acid water as mobile phase A, acetonitrile as mobile phase B, and a gradient elution program: 0-2.00 min, 30% B; 2.00-7.00 min, 30%-70% B; 7.00-9.00 min, 70-100% B; 9.00 min, 100% B; 10.00-10.10 min, 100%-30% B, held for 1.9 min; flow rate: 0.3 mL / min, injection volume: 2 μL; MassLynx software was used for quantitative analysis of diazepam, desmethyldiazepam, and BP-271.

[0074] The retention times and mass spectrometric parameters of the three compounds and the corresponding internal standard solutions are shown in Table 1. The MRM chromatograms are shown in Figure 3 shown.

[0075] Table 1 Retention times and mass spectrometric parameters of diazepam, nordiazepam, BP-271, and internal standard

[0076]

[0077] Example 3

[0078] Quantitative detection was performed according to the method described in Example 2, except that mobile phase B was replaced with methanol; the chromatograms of the mixed standard solution of nordiazepam and BP-271 separated by different mobile phases were as follows: Figure 4 shown.

[0079] Figure 4 The left side of the figure is acetonitrile as mobile phase B, and the right side is methanol as mobile phase B. Figure 4 It can be seen that when methanol is used as the organic phase mobile phase B, the chromatographic peaks of nordiazepam and BP-271 in the mixed standard overlap and have the same retention time, making it impossible to separate them. After adjusting the elution gradient, column temperature, flow rate and other parameters, no separation was achieved. It is speculated that the polarity and hydrophobicity of nordiazepam and BP-271 are similar, resulting in the difference between the two in C 18 The retention difference on the column is small, making it difficult to separate; however, when acetonitrile is used as mobile phase B, the two substances can be effectively separated, meeting the requirements of quantitative analysis. It is speculated that the reason is that acetonitrile has stronger elution and solubility than methanol in reversed-phase liquid chromatography.

[0080] In addition, after comparing the secondary mass spectrometry information of nordiazepam and BP-271, it was found that the secondary fragments of the two were highly consistent, and the separation required the help of chromatography. + In this mode, using an aqueous phase containing formic acid can improve the protonation efficiency of the compound and enhance the chromatographic response value, so 0.1% formic acid water was selected as the aqueous phase.

[0081] Example 4

[0082] Since the fish matrix is ​​complex and contains a large amount of protein, fat, etc., the present invention uses solid-phase extraction for pretreatment to eliminate interference and enhance substance response. The characteristics of solid-phase extraction (SPE) fillers significantly affect their adsorption effect on matrix interfering substances and the recovery rate of target compounds.

[0083] To evaluate the cleanup effects of different SPE columns on the target compounds during quantitative analysis, diazepam, nordiazepam, and BP-271 (2.0 μg / kg) were added to blank fish meat, and the purification performance of six commercially available SPE columns (C 18 / PSA, C 18 、EMR、C 18 / SAX, Silica / PSA and SLC columns) and the results are shown below.

[0084] Table 2 Comparison of the recovery rates of diazepam, nordiazepam and BP-271 using different solid phase extraction columns

[0085]

[0086] As shown in Table 2, except for the Silica / PSA column, the recovery rates of the target compounds of the five SPE columns ranged from 94.45% to 133.60%, and there was no significant difference in the recovery rates of the six SPE columns.

[0087] 2 ng / mL standard solutions of diazepam, nordiazepam, and BP-271 were prepared in acetonitrile to verify the adsorption of the compounds by the solid phase extraction column packing. The recoveries are shown below.

[0088] Table 3 Column recovery rates of diazepam, nordiazepam, and BP-271 using different solid phase extraction column fillers

[0089] Compound <![CDATA[C 18 / PSA(%)]]> SLC (%) EMR (%) <![CDATA[C 18 / SAX(%)]]> Diazepam 77.1 76.1 80.9 78.2 nordiazepam 84.1 82.5 79.0 81.8 BP-271 73.1 51.8 52.2 52.7

[0090] From the results in Table 3, we can see that C 18 The nonspecific adsorption of the target compound by the PSA column was weak, and the recovery rate was above 70%. 18 The / SAX column showed strong adsorption for BP-271, with a low recovery rate of 51.8-52.7%, which was speculated to be related to the charge interaction of the sulfonic acid group or the anion exchange group.

[0091] Although the recovery rates of the above SPE columns in fish matrix were not significantly different, the C 18 and PSA are often used to purify fish extracts in different proportions to detect diazepam and its metabolites, C 18It is a reverse silica gel adsorbent with strong hydrophobicity, which can effectively adsorb non-polar components; PSA filler is a weak anion exchange, which can effectively purify strong polar impurities, pigments and lipids in fish samples. In summary, based on the recovery stability and matrix purification requirements, C 18 / PSA column.

[0092] Example 5

[0093] During the quantitative analysis process, the fish matrix affects the ionization efficiency of the target compounds, resulting in inaccurate quantification. To assess the extent of the matrix effect during sample quantification, blank fish was used to establish matrix standard curves with concentrations of 0.2, 0.5, 1, 2, 5, and 10 ng / mL (with a fixed mixed internal standard concentration of 2 ng / mL). The slope of the curve was compared with that of the pure solution to quantify the matrix effect.

[0094] The matrix effect quantification procedure was as follows: a linear equation was derived using the external standard method. The slope of the matrix standard curve was defined as k1, and the slope of the pure solvent standard curve was defined as k2. ME (%) was calculated using the formula: k1 / k2 × 100%. Accuracy and precision were assessed using six replicates at three spiking levels and expressed as mean recovery and relative standard deviation (RSD). The limit of detection (LOD) and limit of quantification (LOQ) were defined as the concentration at which the signal-to-noise ratio (S / N) of the spiked sample was greater than 3 or 10, respectively.

[0095] Matrix effects were calculated using the above formula. The results showed that the ME values ​​for diazepam, nordiazepam, and BP-271 in fish meat were 94.7%, 98.0%, and 91.6%, respectively. These three compounds exhibited matrix inhibition, but the matrix effect was not significant within the 80% to 120% range. The use of matrix standards in conjunction with internal standard quantification can reduce matrix effects. Linear regression was performed based on the ratio of the peak area of ​​the target compound to the corresponding internal standard peak area. Correlation coefficients (r) for all three compounds were not less than 0.999 within the range of 0.2 to 10.0 ng / mL. The results are shown in Table 4. Considering that diazepam should not be detected in aquatic products, three concentration levels of 0.2 μg / kg, 0.5 μg / kg, and 5.0 μg / kg were selected within the linear range for spike recovery experiments (n=6). The average spike recovery rates ranged from 100.2% to 119.9%, with relative standard deviations (RSDs) ranging from 1.7% to 9.5%. The specific results are shown in Table 5. After testing fish samples spiked with 0.2 μg / kg, the signal-to-noise ratios (S / N) for the three compounds were all greater than 10. Therefore, the limits of detection for the three compounds of the present invention were determined to be 0.2 μg / kg, and the limits of quantification were determined to be 0.6 μg / kg.

[0096] Table 4 Linear range and correlation coefficient of diazepam, nordiazepam and BP-271

[0097] Serial number Compound Internal standard Linear equations Correlation coefficient 1 Diazepam <![CDATA[Diazepam-D5]]> y=0.8902x+0.0302 0.9994 2 nordiazepam <![CDATA[Desmethyldiazepam-D5]]> y=1.5645x+0.0409 0.9995 3 BP-271 <![CDATA[Desmethyldiazepam-D5]]> y=0.1674x+0.0027 0.9991

[0098] Table 5 Spiked recovery, precision, and detection limit of this method (n=6)

[0099]

[0100] Application Examples

[0101] The method established in the above example was used to detect diazepam, nordiazepam and BP-271 in 5 fish samples from the market. The detection results of diazepam, nordiazepam and BP-271 in the fish samples are shown below.

[0102] Table 6 Detection values ​​of diazepam, nordiazepam and BP-271 in fish samples

[0103]

[0104]

[0105] Figure 5 This is the sample chromatogram of the detection of BP-271 in the fish sample in the application example, where Figure 5 BP-271 was detected in the sample on the left. Figure 5 No BP-271 was detected in the samples in the right figure. Combined with the results in Table 6, it can be seen that in the 5 diazepam-positive fish samples, nordiazepam was also detected at the same time, with a consistency of 100%. The detection ranges were 1.53-362.94 μg / kg and 0.47-575.41 μg / kg, respectively. BP-271 was detected in 2 samples with a detection range of 1.30-2.61 μg / kg, and the concentration was lower than that of diazepam and its metabolite nordiazepam.

[0106] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for detecting diazepam, nordiazepam and nordiazepam isomer BP-271 in fish, characterized in that: The following steps are involved: The fish sample is sequentially subjected to extraction, purification and concentration to obtain a test solution; the extraction agent used in the extraction is acetonitrile; the extraction agent used in the extraction is sodium chloride; The purification is solid phase extraction column purification; The test solution is qualitatively analyzed by high resolution mass spectrometry; The chromatographic separation conditions in the qualitative analysis include: a T3 column; mobile phase A is a 0.1-0.3% volume fraction of formic acid aqueous solution, and mobile phase B is acetonitrile; the column temperature is 35-40° C., and the flow rate is 0.2-0.4 mL / min; the elution method is gradient elution, and the elution program of the gradient elution is: 0 min, 40% mobile phase B; 0.5 min, 40% mobile phase B; 4 min, 50% mobile phase B; 9 min, 100% mobile phase B; 10 min, 100% mobile phase B; 10.1 min, 40% mobile phase B; 12 min, 40% mobile phase B; The high-resolution mass spectrometry detection in the qualitative analysis includes a primary analysis in full scan mode and a secondary analysis in product ion mode; the orbitrap resolution in full scan mode is 100,000 to 120,000, the scan range is 150 to 400 m / z, and the gas is nitrogen; the conditions of the product ion mode include: the ion source is an electrospray ion source; the ESI capillary voltage is 1000 to 3000 V; the sheath gas is 30 to 50 Arb, the auxiliary gas is 10 to 30 Arb, and the purge gas is 1 to 5 Arb, and the sheath gas, auxiliary gas, and purge gas are nitrogen; the ion transfer tube temperature is 300 to 325° C., and the vaporization chamber temperature is 330 to 350° C. An internal standard is added to the test solution, and quantitative analysis is performed using ultra-performance liquid chromatography-tandem mass spectrometry; the internal standard is diazepam-D5 and nordiazepam-D5; The conditions of ultra-high performance liquid chromatography in the quantitative analysis include: the chromatographic column is C 18 Column; mobile phase A is a 0.1-0.3% volume fraction of formic acid aqueous solution, mobile phase B is acetonitrile; column temperature is 30-35°C, flow rate is 0.2-0.3 mL / min; gradient elution, the elution program of the gradient elution is: 0-2.00 min, 30% mobile phase B; 2.00-7.00 min, 30%-70% mobile phase B; 7.00-9.00 min, 70-100% mobile phase B; 9.00 min, 100% mobile phase B; 10.00-10.10 mobile phase min, 100%-30% mobile phase B, hold for 1.9 min; The mass spectrometry conditions in the quantitative analysis include: the ion source is an electrospray ion source; the capillary voltage is 0.5-1 kV, the ion source temperature is 100-150°C, the desolvation gas temperature is 300-500°C, the desolvation gas flow rate is 600-800 L / h, the cone hole backflush gas flow rate is 120-150 L / h, and the desolvation gas is nitrogen.

2. The detection method according to claim 1, wherein The ratio of the volume of the extractant to the mass of the fish sample is 3-10 mL:1-5 g.

3. The detection method according to claim 1 or 2, characterized in that The extraction comprises sequentially performing vortex extraction and ultrasonic extraction; The speed of the vortex is 1000-2000 rpm, and the frequency of the ultrasound is 30k-40kHz; the time of the vortex extraction and the ultrasound extraction is independently 10-30 minutes.

4. The detection method according to claim 1, wherein The extraction agent used in the extraction is sodium chloride; the mass ratio of the extraction agent to the fish sample is 1:1-3.

5. The detection method according to claim 1, wherein The filler of the solid phase extraction column in the purification process includes C 18 / PSA, EMR, C 18 / SAX、Silica / PSA、C 18 and one of SLC.

6. The detection method according to claim 1, characterized in that After the concentration, the method further includes re-dissolving and micro-filtering the obtained product; the reagent used for the re-dissolution is acetonitrile.

7. The detection method according to claim 6, characterized in that The pore size of the filter membrane used for microfiltration is 0.1-3 μm; the filter membrane is a polytetrafluoroethylene membrane.

8. The detection method according to claim 1, wherein The contents of diazepam, nordiazepam and nordiazepam isomer BP-271 were calculated using the internal standard method.

9. The detection method according to claim 8, characterized in that The concentration of the mixed standard solution of the internal standard is 100-400 ng / mL.

10. The detection method according to claim 1, characterized in that The injection volume of the chromatographic separation in the qualitative analysis is 1 to 10 μL; the injection volume of the ultra-high performance liquid chromatography in the quantitative analysis is 1 to 5 μL.

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