Method for detecting diazepam and metabolite residues thereof in aquatic product tissue based on process capability evaluation

Abstract

The invention discloses a method for detecting the residual quantity of diazepam and metabolites thereof in aquatic product tissues based on process capability evaluation, which adopts a response surface method for design optimization, extracts diazepam and metabolites thereof in aquatic product tissues in an ultrasonic extraction mode, takes dispersive solid-phase extraction as a main purification means, and is used for detecting the residual quantity of diazepam and metabolites thereof in aquatic product tissues. The method for simultaneously detecting diazepam and metabolites in main tissues of aquatic products is established by optimizing extraction and purification methods and mass spectrum and chromatographic detection conditions in a pretreatment process, eliminating the influence of a matrix effect and improving the detection efficiency and the accuracy of a result by combining liquid chromatography-tandem mass spectrometry with an internal standard or external standard method, and is used for detecting diazepam and metabolites in the main tissues of the aquatic products. And a process capability evaluation method is adopted to evaluate the practicability of the method. The establishment of the detection method and the evaluation system can provide theoretical basis and technical support for relevant supervision departments of the aquaculture industry and provide data reference for improvement of relevant standards.

Description

Technical Field

[0001] The invention relates to a method for detecting the residual amount of diazepam and its metabolites in aquatic product tissue based on process capability evaluation, and belongs to the technical field of analysis and detection. Background Art

[0002] Diazepam (DZP) is a benzodiazepine sedative with sedative, anticonvulsant and hypnotic effects. It is also called Valium. It is an anti-stress drug illegally used in aquaculture, fishing and transportation of aquatic products in fishery production and breeding. Diazepam is mainly metabolized into oxazepam, temazepam, nitrazepam, nitrazepam, nordiazepam, 7-aminonitrazepam and 7-aminometrazepam in the body. It has a relatively long metabolic cycle. Long-term residual accumulation in the body can easily cause damage to the nervous system and pose a certain threat to human health and the ecological environment. Diazepam is listed as a banned veterinary drug, and it is stipulated that diazepam shall not be detected in animal food. The residue problem of diazepam and its metabolites has received widespread attention and needs to be studied and solved urgently.

[0003] At present, the main detection methods of diazepam are liquid chromatography, gas chromatography, liquid chromatography-tandem mass spectrometry, gas chromatography-tandem mass spectrometry, spectrophotometry, molecular imprinting, thin layer chromatography and immunochromatography, etc. At present, the main research reports on diazepam sedatives are that the pretreatment process is cumbersome, the detection time is long, and it is mainly the detection of diazepam original drug. There are few reports on the simultaneous detection of its metabolites, and there is insufficient attention to the detection of original drugs and their metabolites in the main tissues of aquatic products, which affects the pollution traceability and supervision of diazepam and metabolites in aquaculture. It is urgent to establish relevant reliable detection methods. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for detecting the residues of diazepam and its metabolites in aquatic product tissues based on process capability evaluation.

[0005] Technical solution: The method for detecting the residual amount of diazepam and its metabolites in aquatic product tissues based on process capability evaluation of the present invention comprises the following steps:

[0006] (1) Sample pretreatment: Ultrasonic extraction was used to extract diazepam and its metabolites from aquatic product tissues, and dispersed solid phase extraction was used to purify the extracted diazepam and its metabolites;

[0007] (2) Detection using liquid chromatography tandem mass spectrometry;

[0008] (3) Quantification using internal or external standard method.

[0009] Furthermore, the aquatic product tissue is one or more of the muscle, heart, liver, spleen, kidney, gall bladder, gonad, plasma, intestine or gill of aquatic products.

[0010] Furthermore, the diazepam metabolite is one or more of temazepam, nitrazepam, nordiazepam, 7-aminometazepam, 7-aminonitrazepam, oxazepam, and nimetazepam.

[0011] Furthermore, in step (1), the sample pretreatment includes the following steps:

[0012] (A1) adding the aquatic product tissue to ethyl acetate, vortexing, ultrasonic extraction, centrifugation, and collecting the supernatant;

[0013] (A2) adding the precipitate obtained after centrifugation in step (A1) to ethyl acetate again, repeating step (A1), collecting the supernatant, and combining the supernatant with the supernatant collected in step (A1);

[0014] (A3) blowing the supernatant to near dryness with nitrogen, re-dissolving with methanol solution, vortexing, ultrasonic treatment, adding purification materials for dispersive solid phase extraction, centrifuging, adding n-hexane to the supernatant, vortexing, freezing, centrifuging, and filtering the lower layer solution;

[0015] or,

[0016] (B1) adding the aquatic product tissue to the mixed standard internal standard solution, vortexing, standing, adding to ethyl acetate, vortexing, ultrasonic extraction, centrifuging, and collecting the supernatant;

[0017] (B2) adding the precipitate obtained after centrifugation in step (B1) to ethyl acetate, vortexing, ultrasonic extraction, centrifuging, collecting the supernatant, and combining the supernatant collected in step (B1);

[0018] (B3) The supernatant is blown to near dryness with nitrogen, reconstituted with methanol solution, vortexed, sonicated, added with purification materials for dispersed solid phase extraction, centrifuged, n-hexane is added to the supernatant, vortexed, frozen, centrifuged, and the lower layer of solution is removed and filtered.

[0019] Furthermore, the volume concentration of the methanol solution is 10%. If it is not this concentration, the retention time will drift during the analysis and detection, and the solvent effect will occur.

[0020] Furthermore, the purification material is a mixed purification material, wherein the mass ratio of NH2-based SPE bulk adsorbent to cellulose powder is 1-2:1-2.

[0021] Furthermore, the mass ratio of the aquatic product tissue to the purification material is 2000:5-30.

[0022] Furthermore, the ultrasonic extraction time is more than 10 minutes.

[0023] Furthermore, the freezing temperature is lower than -18°C and the freezing time is more than 20 minutes.

[0024] Furthermore, in step (2), the conditions for liquid chromatography analysis are as follows: the chromatographic column is a Kinetex SB-C18 (50 mm×2.1 mm, 1.7 μm) chromatographic column; the mobile phase A is a 5 mmol / L ammonium formate aqueous solution, and the mobile phase B is a methanol solution; the gradient elution program is: 0-1 min, 10% B, 1-2 min, 10%→50% B, 2-6 min, 50%→95% B, 8.0-8.1 min, 95%→10% B; 8.1-10 min, 10% B; the flow rate of the mobile phase is 0.3 mL / min; the column temperature is 40°C; and the injection volume is 3 μL.

[0025] Furthermore, in step (2), the conditions for mass spectrometry analysis are as follows: scanning mode: ESI+; monitoring mode: MRM; ion source temperature: 550°C; spray voltage: 5500V; collision gas: N2; collision gas pressure: 379kPa; curtain gas pressure: 241kPa; auxiliary gas pressure: 414kPa.

[0026] Further, the remaining mass spectrometry conditions are shown in the following table:

[0027]

[0028]

[0029] Note: *Quantitative ion.

[0030] Furthermore, in step (3), the internal standard used for quantification by the internal standard method is diazepam-D5, oxazepam-D5, temazepam-D5, nitrazepam-D5 and / or 7-aminonitrazepam-D5.

[0031] Furthermore, in step (3), the external standard used for quantification by the external standard method is diazepam, oxazepam, temazepam, nitrazepam, nordiazepam, nimetazepam, 7-aminonitrazepam and / or 7-aminometazepam.

[0032] Furthermore, in step (3), when the external standard method is used for quantification, the response surface methodology is used for optimization.

[0033] A method for evaluating the detection method of the present invention based on process capability, wherein the practicability of the detection method of claim 1 is evaluated using the process capability evaluation method.

[0034] Furthermore, the method process capability evaluation includes the following steps:

[0035] The recovery rates of the eight target compounds are used as the process capability evaluation objects, the accuracy requirements of 60% to 120% of the recovery rates of each target compound are used as the expected use requirements of the method, and 480% to 960% are used as the overall expected use requirements of the method; the recovery rate of 100% is used as the expected target of the method for each target compound, and 800% is used as the overall expected target of the method. The process capability index is used to evaluate the method grade, the target compounds of the evaluation method, and the overall detection capability level according to the formula to determine whether the detection method process can meet the expected detection use requirements.

[0036] Furthermore, the eight target compounds are diazepam, oxazepam, temazepam, nitrazepam, nordiazepam, nimetazepam, 7-aminonitrazepam and 7-aminometazepam.

[0037] The invention takes the detection method of diazepam and its metabolite residue as a special product based on process capability evaluation, adopts response surface method to design and optimize multivariate quality characteristic parameters, extracts diazepam and its metabolites in the heart, liver, spleen, kidney, gallbladder, gonad, edible muscle, plasma, intestine and gill tissues of aquatic products by ultrasonic extraction, uses dispersed solid phase extraction as the main purification means, eliminates the influence of matrix effect by optimizing the extraction and purification methods, mass spectrometry and chromatographic detection conditions in the pretreatment process, improves the detection efficiency and the accuracy of the results, establishes a method for simple, rapid, efficient and sensitive simultaneous detection of diazepam and its metabolites in the main tissues of aquatic products by liquid chromatography tandem mass spectrometry combined with external standard method or internal standard method, and adopts process capability evaluation method to evaluate the practicability of each target compound and the overall detection capability of the method, provides theoretical basis and technical support for relevant regulatory departments of aquaculture industry, and provides data reference for the improvement of relevant standards.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0039] The present invention uses 10 tissues of aquatic products, including heart, liver, spleen, kidney, gallbladder, gonad, edible muscle, plasma, intestine, and gill, as detection objects, systematically studies the matrix effects, extraction and purification methods of different tissues, and constructs a widely applicable detection method by integrating the characteristics of the matrix of different tissues. The wide expansion of its scope of application is based on my country's diversified eating habits, making up for the defect that the existing technology is only applicable to muscle tissue and / or liver, and provides necessary support for ensuring food safety and traceability law enforcement. Taking 8 drugs, including diazepam, oxazepam, temazepam, nitrazepam, nordiazepam, nimetazepam, 7-aminonitrazepam and 7-aminometrazepam, as detection target compounds, the adsorption effect of 12 common purifiers on different target compounds is systematically studied, and 2 purifiers are selected for detection based on purification capacity and economy using data statistical methods. The experimental results show that the solid-phase dispersed purifier reported in the prior art is not suitable for 8 target compounds. For example, PSA has a certain adsorption effect on temazepam and nitrazepam, C18 has a certain adsorption effect on oxazepam and temazepam, and GCB has a certain adsorption effect on nitrazepam and nimetazepam. The establishment of this method makes up for the deficiency that the prior art can only detect diazepam and / or nitrazepam, oxazepam, nordiazepam, and temazepam, and provides necessary technical support for pharmacokinetic research and traceability law enforcement.

[0040] Based on the process capability evaluation method, the practicality of 8 target compounds and the overall method detection capability were evaluated at the same time, and the evaluation results were used to screen out vulnerable factors, and targeted optimization measures were taken to cyclically improve the method detection performance and evaluate the method practical capability level. This makes up for the defect that the existing technical methods meet the requirements in methodological characteristics but lack practicality in production, making the method more suitable for practical application in grassroots inspection and testing institutions.

[0041] At the same time, internal standard method and external standard method were constructed. Both methods can meet daily testing needs and provide diversified selection options for testers. The internal standard method can be given priority based on method performance requirements, while the external standard method can be given priority based on economic and practical requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the interactive response diagram and contour diagram of quality characteristic parameters of diazepam recovery rate;

[0043] Figure 2 The interactive response diagram and contour diagram of the quality characteristic parameters of oxazepam recovery rate;

[0044] Figure 3 The interactive response diagram and contour diagram of the quality characteristic parameters of temazepam recovery rate;

[0045] Figure 4The quality characteristic parameter interaction response diagram and contour diagram of nitrazepam recovery rate;

[0046] Figure 5 Interaction response diagram and contour diagram of quality characteristic parameters of nordiazepam recovery rate

[0047] Figure 6 It is the interactive response diagram and contour diagram of the quality characteristic parameters of nitrazepam recovery rate;

[0048] Figure 7 It is the interactive response diagram and contour diagram of the quality characteristic parameters of 7-aminonitrazepam recovery rate;

[0049] Figure 8 The interactive response diagram and contour diagram of the quality characteristic parameters of 7-aminometrazepam recovery rate;

[0050] Fig. 9 This is the characteristic mass chromatogram of diazepam standard solution ions;

[0051] Fig.10 This is the characteristic mass chromatogram of ions of oxazepam standard solution;

[0052] Fig.11 This is the characteristic mass chromatogram of ions of temazepam standard solution;

[0053] Fig.12 This is the characteristic mass chromatogram of nitrazepam standard solution ions;

[0054] Fig.13 This is the characteristic mass chromatogram of the ions of the nordiazepam standard solution;

[0055] Fig.14 This is the characteristic mass chromatogram of ions of nimetazepam standard solution;

[0056] Fig.15 This is the characteristic mass chromatogram of ions of 7-aminonitrazepam standard solution;

[0057] Fig.16 This is the characteristic mass chromatogram of ions of 7-aminometrazepam standard solution;.

[0058] Fig.17 The total ion current chromatograms of standard solutions of 8 target compounds are shown in Figure 2. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0060] Example 1

[0061] 1. Materials and methods

[0062] 1.1 Instruments and reagents

[0063] AB QTRAP 5500 mass spectrometer, Sciex; LC-30A ultra-high performance liquid chromatograph, Shimadzu Corporation, Japan; H4-20K centrifuge, Hunan Kecheng Instrument Equipment Co., Ltd.; MS3 basic vortex mixer, IKA, Germany; UVF intelligent silent ultrasonic cleaning machine, Xiaomei Ultrasonic Instrument (Kunshan) Co., Ltd.; XOMFV-24 nitrogen blowdown instrument, Nanjing Xianou Instrument Manufacturing Co., Ltd.

[0064] Carp samples: commercially available and purchased from aquaculture bases;

[0065] Standard substances (concentration of 1000 μg / mL): diazepam standard solution, temazepam standard solution, nitrazepam standard solution, nordiazepam standard solution, 7-aminometazepam standard solution, 7-aminonitrazepam standard solution, oxazepam standard solution, nitrazepam standard solution, diazepam-D5 standard solution, oxazepam-D5 standard solution, nitrazepam-D5 standard solution, temazepam-D5 standard solution, 7-aminonitrazepam-D5 standard solution, purchased from Thermo Fisher, USA;

[0066] Chromatographic grade acetonitrile, acetic acid, formic acid, and methanol were purchased from Thermo Fisher, USA;

[0067] Purification materials:

[0068] Diatomaceous earth was purchased from Sigma-Aldrich, USA;

[0069] Multi-walled carbon nanotubes and amino-modified multi-walled carbon nanotubes were purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0070] Graphitized carbon black (GCB, 38~125μm), C 18 End-capped SPE bulk adsorbent (40 μm), ethylenediamine-N-propylsilane silica gel (PSA, 40 μm), and NH2-based SPE bulk adsorbent were purchased from Agilent, USA;

[0071] Neutral alumina was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0072] Ammonium formate, graphene oxide powder (>99%), cellulose powder (particle size: 25 μm), fullerene (>99.9%), and nano zirconium dioxide (>99.9%, 100 nm, monoclinic phase) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0073] 1.2. Carp tissue sample preparation

[0074] After dissecting the carp samples, the heart, liver, spleen, kidney, gall bladder, gonads, edible muscle, plasma, intestine, and gill tissues were cut into small pieces, minced into minced meat using a tissue grinder, mixed and packaged in sealed bags, and frozen at -18°C to obtain 10 types of carp tissue samples for later use.

[0075] 1.3 Experimental methods

[0076] 1.3.1 Preparation of standard solution

[0077] Mixed standard external standard intermediate solution: Accurately pipette a certain amount of diazepam standard solution, oxazepam standard solution, temazepam standard solution, nitrazepam standard solution, nordiazepam standard solution, nimetazepam standard solution, 7-aminonitrazepam solution, and 7-aminometrazepam standard solution into a 10mL volumetric flask, dilute to the scale with methanol to obtain a mixed standard external standard intermediate solution with a concentration of 1μg / mL, and store it in a freezer at -18℃.

[0078] Mixed standard internal standard intermediate solution: Accurately pipette a certain amount of diazepam-D5 standard solution, oxazepam-D5 standard solution, temazepam-D5 standard solution, nitrazepam-D5 standard solution, and 7-aminonitrazepam-D5 standard solution into a 10mL volumetric flask, dilute to the scale with methanol to obtain a mixed standard internal standard intermediate solution with a concentration of 1μg / mL, and store it in a freezer at -18°C.

[0079] Mixed standard working solution: Add a certain amount of mixed standard external standard intermediate solution to 50mL centrifuge tubes containing 2.00g blank carp 10 tissue matrix samples, and prepare a series of matrix mixed standard working solutions with concentrations of 0.1, 0.5, 1, 5, 10, 20, and 50ng / mL according to the pretreatment process.

[0080] 1.3.2 Sample pretreatment

[0081] 1.3.2.1. External Standard Method

[0082] Accurately weigh 2.00g of carp tissue sample in a 50mL centrifuge tube, add 10mL of ethyl acetate, vortex mix for 1min, ultrasonically extract for 14min, centrifuge at 10000r / min for 5min, transfer the supernatant to a 50mL centrifuge tube, add 10mL of ethyl acetate to the residue and repeat the extraction once, combine the two extraction solutions and vortex mix for 30s, blow all the solutions to near dryness with nitrogen at 40℃, re-dissolve with 1mL of 10% methanol solution, vortex for 30s, ultrasonicate for 5min, add to a 2mL centrifuge tube containing 21mg of mixed purification material (NH2-based SPE bulk adsorbent: cellulose powder mass ratio = 1:1), add 1mL of n-hexane and vortex mix for 1min, freeze at -18℃ for 20min, centrifuge at 10000r / min for 5min, take the lower layer of solution and pass it through a 0.22μm membrane for measurement.

[0083] 1.3.2.2. Internal standard method

[0084] Accurately weigh 2.00g of carp tissue sample in a 50mL centrifuge tube, add 50μL of mixed standard internal standard intermediate solution, vortex mix for 1min, and let stand for 10min. Add 10mL of ethyl acetate, vortex mix for 1min, ultrasonic extract for 14min, centrifuge at 10000r / min for 5min, transfer the supernatant to a 50mL centrifuge tube, add 10mL of ethyl acetate solution to the residue and repeat the extraction once, combine the two extraction solutions and vortex mix for 30s. Transfer 10mL of the extraction solution to 40℃ nitrogen blow until nearly dry, re-dissolve with 1mL of 10% methanol solution, vortex 30s, ultrasonic 5min, add to a 2mL centrifuge tube containing 21mg of mixed purification material (NH2-based SPE bulk adsorbent: cellulose powder mass ratio = 1:1), add 1mL of n-hexane and vortex mix for 1min, freeze at -18℃ for 20min, centrifuge at 10000r / min for 5min, take the lower layer of solution and pass it through a 0.22μm membrane, and wait for the machine to measure.

[0085] 1.3.3 Chromatography-mass spectrometry conditions

[0086] Chromatographic column: Kinetex SB-C18 (50mm×2.1mm, 1.7μm); mobile phase A was 5mmol / L ammonium formate aqueous solution, and mobile phase B was methanol solution; gradient elution program was 0-1min, 10% B, 1-2min, 10%→50% B, 2-6min, 50%→95% B, 8.0-8.1min, 95%→10% B; 8.1-10min, 10% B; flow rate was 0.3mL / min; column temperature was 40℃; injection volume was 3μL.

[0087] Mass spectrometry scanning mode: ESI +; Monitoring mode: MRM; Ion source temperature: 550°C; Spray voltage: 5500V; Collision gas: N2; Collision gas pressure: 379 kPa; Curtain gas pressure: 241 kPa; Auxiliary gas pressure: 414 kPa. The other mass spectrometry conditions are shown in Table 1.

[0088] 2. Results and Discussion

[0089] 2.1 Optimization of chromatographic and mass spectrometric conditions

[0090] 2.1.1 Optimization of chromatographic conditions

[0091] The response value and separation effect of diazepam and its metabolites were investigated when the mobile phase A was water, formic acid aqueous solution (0.1%), 5mmol / L ammonium formate aqueous solution, 5mmol / L ammonium acetate aqueous solution, 5mmol / L ammonium formate-formic acid (0.1%) solution, and 5mmol / L ammonium acetate-formic acid (0.1%) solution, and the mobile phase B was methanol and acetonitrile. The results showed that when the mobile phase A was 5mmol / L ammonium formate water, the response value and separation degree of diazepam and its metabolites were the best, and when the mobile phase B was methanol, the peak shape and separation degree were better than acetonitrile. Therefore, the mobile phase A was determined to be 5mmol / L ammonium formate water, and the mobile phase B was methanol. The extracted ion chromatograms of diazepam and its metabolites obtained under this condition are shown in the attached figure. Figure 1-9 .

[0092] 2.1.2 Optimization of mass spectrometry conditions

[0093] The external and internal standard target compounds of diazepam and its metabolites were prepared into standard solutions with a concentration of 50 ng / mL, and the positive ion mode was selected to scan the parent ion first. After the parent ion was determined, the daughter ion was scanned. Two ions with strong and stable response values ​​were selected as the quantitative examples and qualitative ions for the external standard, and an ion with a strong response value was selected as the qualitative ion for the internal standard. The optimal declustering voltage (DP) and the optimal collision voltage (CE) were optimized for the determined parent ion and daughter ion MRM, and finally the compound with the best synchronization correspondence with the external standard solution was determined as its internal standard solution. The specific mass spectrometry parameters of diazepam and its metabolites are shown in Table 1, and the ion characteristic mass chromatogram is shown in detail. Figure 9-17 .

[0094] Table 1 Mass spectrometry detection conditions for diazepam and its metabolites

[0095]

[0096]

[0097] Note: *Quantitative ion.

[0098] 2.2 Establishment of sample pretreatment method

[0099] 2.2.1 Investigation of matrix effects

[0100] The matrix of the sample has a certain interference on the accuracy of the detection results of the target compound. This method investigates the matrix effects of 10 matrices by adding 10 ng / mL of the target compound to 10 blank matrices of aquatic product heart, liver, spleen, kidney, gall bladder, gonad, edible muscle, plasma, intestine, and gill tissues, and comparing the response values ​​of the target compound at the same concentration in the solvent. If the matrix effect is greater than 1, it indicates a matrix enhancement effect; if the matrix effect is less than 1, it indicates a matrix inhibition effect; if the matrix effect = 1, it indicates that there is no matrix effect; when the matrix effect is between 0.8 and 1.2, the matrix effect is not obvious, otherwise the matrix effect is obvious.

[0101] The experiment found that when acetonitrile, 1% acetic acid acetonitrile, and ethyl acetate were used as extraction solvents, diazepam and its metabolites in 10 tissue matrices showed matrix inhibition effects, and the matrix effect was the smallest when ethyl acetate was used as the solvent. Figure 1 The results show that diazepam, oxazepam, temazepam, nimetazepam, 7-aminonitrazepam, and 7-aminometrazepam show weak matrix inhibition, while nitrazepam and nordiazepam show moderate to strong matrix inhibition; gallbladder, gonad, and intestinal tissues have strong matrix inhibition effects on most target compounds, so effective purification measures, matrix standard solutions, or internal standard methods are required for correction. The matrix effect results are shown in Table 2.

[0102] Table 2 Matrix effects of diazepam and its metabolites

[0103]

[0104]

[0105] 2.2.2 Investigation of purification materials

[0106] The adsorption effects of 12 purification materials, including PSA, C18-capped SPE bulk adsorbent, NH2-based SPE bulk adsorbent, GCB, neutral alumina, diatomaceous earth, multi-walled carbon nanotubes, amino-modified multi-walled carbon nanotubes, cellulose powder, fullerene, graphene oxide, and zirconium dioxide, on diazepam and its metabolites were investigated. The recovery rate was characterized by the quantitative ion pair response value of each target compound in methanol solution with a concentration of 20 ng / mL. After adding 10 mg of each of the 12 purification materials, vortex mixing was performed for 1 min, centrifuged at 10000 r / min for 5 min, and then passed through the membrane and measured on the machine. The results showed that multi-walled carbon nanotubes, amino-modified multi-walled carbon nanotubes, and graphene oxide had strong adsorption effects on most target compounds, and neutral alumina and zirconium dioxide had strong adsorption effects on oxazepam and temazepam, so these purification materials were not suitable for this method. Seven purification materials, PSA, C18, NH2, GCB, diatomaceous earth, cellulose, and fullerene, were selected for further analysis of adsorption by F test, and the significant results were compared at the confidence level of α = 0.05, and the results are shown in Table 3. The results show that PSA has a certain adsorption effect on temazepam and nimetazepam, C18 has a certain adsorption effect on oxazepam and temazepam, and GCB has a certain adsorption effect on nitrazepam and nimetazepam. Considering that diatomaceous earth is used in large quantities, fullerene has a high cost, and the purification effect is not significantly different from NH2 and cellulose, it is not considered. NH2-based SPE bulk adsorbent and cellulose powder are selected as mixed purification materials with a mass ratio of 1:1.

[0107] Table 3 Evaluation of the adsorption of diazepam and its metabolites by seven purifiers

[0108]

[0109]

[0110] 2.2.3. Investigation of extraction solvent and concentration method

[0111] The present invention investigates the effects of three extraction solvents, acetonitrile, 1% acetic acid acetonitrile, and ethyl acetate, and two concentration methods, nitrogen blowing and rotary evaporation, on the extraction effects of diazepam and its metabolites in 10 tissue matrices of aquatic products, and the results are shown in Table 4. The results show that the significance of the Levene's equivalence test for the concentration method of diazepam and its metabolites is greater than 0.05, indicating that there is no significant difference, and the significance of the F test for the heteroscedasticity of the extraction solvents of diazepam and its metabolites is greater than 0.05, indicating that there are differences within the group, that is, different extraction solvents are different, but there is no difference in the concentration method. Therefore, considering the cost and efficiency issues, nitrogen blowing is selected as the concentration method of this method, and the extraction solvent is further investigated.

[0112] Table 4 Evaluation of the effects of extraction solvents and concentration methods for diazepam and its metabolites

[0113]

[0114] 2.2.4. Initial evaluation of method and process capability

[0115] Using process capability index C P (Complex process Capability) evaluates the detection capability level of the method to determine whether the detection method process can meet the expected detection purpose requirements. The recovery rates of 8 target compounds (diazepam, oxazepam, temazepam, nitrazepam, nordiazepam, nimetazepam, 7-aminonitrazepam and 7-aminometrazepam) are used as process capability evaluation objects, the accuracy requirements of 60% to 120% of the recovery rate of each target compound are used as the expected use requirements of the method, and 480% to 960% are used as the overall expected use requirements of the method; the recovery rate of 100% is used as the expected target of each target compound method, and 800% is used as the overall expected target of the method. According to formulas 1 to 10, the process capability index is used to evaluate the method level, the target compounds of the evaluation method and the overall detection capability level to determine whether the detection method process can meet the expected detection purpose requirements. The t-test method was used to evaluate the accuracy bias. According to the significance level α=0.005, degrees of freedom ν=n-1=6, t(1-α / 2, ν)=2.447, the specific evaluation criteria are shown in Table 5.

[0116] T=USL-LSL (1)

[0117]

[0118] or

[0119]

[0120] C Pk =C P ×(1-C a ) (6)

[0121]

[0122] Where, T is the required tolerance; USL is the upper limit of recovery; LSL is the lower limit of recovery; M is the standard center, σ is the standard deviation; S is the overall standard deviation; C4 is a constant related to the sample size n, which can be obtained by looking up the table; R is the range, d2 is a constant related to the sample size, which can be obtained by looking up the table; C p is the evaluation indicator capability index; is the average value of the evaluation index; U is the expected value requirement, the center of the controlled process; C pk The ability index to meet the expected goals; C pL C is the lower limit of the process capability index of the evaluation index recovery rate; pUThe upper limit of the process capability index for the evaluation index; The actual process capability.

[0123] In Six Sigma management activities, Sigma level Z is more often used to evaluate process capability. The advantage is that it corresponds one-to-one with the defective rate or DPMO of the process. When the two-sided specification limit is:

[0124] Z USL =(USL-U) / σ, Z lSL =(U-LSL) / σ (10)

[0125] Where Z is the process capability index; USL is the upper limit of recovery rate; LSL is the lower limit of recovery rate.

[0126] Table 5C P Process capability index evaluation criteria

[0127]

[0128] 2.2.4.1 External standard method

[0129] The process capability evaluation results of diazepam and its metabolites before and after optimization of 10 main tissue matrices of aquatic products by external standard method showed that before method optimization, the C pk The level is between -116.26 and 2.84, and most of them are less than 0.67. The short-term process capability Z st The levels were between -21.21 and 5.33, all lower than 6. The detection capacity could not meet the general recovery rate requirement of 60-120%, and the C pm The process capability was significantly different from the expected recovery rate of 100%, and measures should be taken to optimize and improve it. Comparison of the process capability levels of the 10 matrices showed that the process capability levels of the 8 target compounds in the gallbladder matrix were relatively low, which was a vulnerable factor in the entire detection system and was listed as the target matrix for method optimization.

[0130] 2.2.4.2. Internal standard method

[0131] The evaluation results of diazepam and its metabolites in 10 major tissue matrices of aquatic products before optimization by internal standard method showed that C pk The level is between 1.65 and 21.11, all > 0.67, and the short-term process capability Z st The levels were between 2.53 and 21.18, and most of them were >6. The detection capacity basically met the general recovery rate requirement of 60-120%, and the C pm All of them are > 0.67, and the process capability is close to the expected target of 100% recovery rate, and the expected requirements can be met without optimization.

[0132] 2.2.5. Optimize the experimental design

[0133] Using Design-Expert 13 software and surface response method, the ultrasonic time, purification agent dosage, and extraction solvent that have the main influence on the test results are used as design variables. A 4-factor 3-level test is conducted to investigate the mean and variance indicators of the multivariate quality characteristic recovery rate of 8 target compounds, among which the recovery rate is the target characteristic. The design variable range of each parameter is: the ultrasonic time is investigated as 10min≤A≤30min; the mixed purification material NH2-based SPE bulk adsorbent and cellulose powder (mass ratio is 1:1), and the dosage is investigated as 25mg≤B≤100mg; the extraction solvent is acetonitrile, 1% acetic acid acidified acetonitrile, and ethyl acetate, and the digital assignment is -1, 0, 1, that is, -1≤C≤1.

[0134] 2.2.6 Response surface methodology optimization

[0135] In view of the insufficient process capability of the external standard method, the gallbladder matrix was taken as the target, and the recovery rate of the eight target compounds was taken as the multivariate quality characteristic target. The experimental conditions were further optimized to meet the detection requirements. The response surface method was used for optimization, and the optimal design variable combination was obtained using Design Expert 13 software. The three-dimensional surface diagram is attached. Figures 1 to 8 The approximate models of the standard deviation and mean of each quality characteristic of diazepam and its metabolites are shown in formulas 11 to 18. The results show that the ultrasonic time, the amount of purifier, and the extraction solvent have different effects on the recovery rate. The results show that the REC model is significant, with a P value of <0.0001, indicating that the noise influence of instrument equipment status, environmental conditions, etc. has only a 0.01% probability. The important parameters of the model are A, B, C, AB, AC, BC, A 2 , B 2 , C 2 , correlation coefficient r 2 The range is 0.982~0.999.

[0136] Diazepam: REC = 24.62367-0.11713×A+0.0803×B+21.14474×C-0.00056×A×B-0.30925×A×C-0.08928×B×C+0.002027×A 2 -0.00056×B 2 +

[0137] 41.5252×C 2 (11)

[0138] Oxazepam: REC = 30.69518 + 1.39236 × A - 0.133011 × B + 14.36947 × C +

[0139] 0.003211×A×B+0.086×A×C-0.135968×B×C-0.04437×A 2 +0.000048×B 2 +38.50575×C 2 (12)

[0140] Temazepam: REC = 24.59007 + 1.49938 × A - 0.075904 × B + 16.09842 × C - 0.00494 × A × B - 0.35475 × A × C - 0.06791 × B × C - 0.03066 × A 2 +0.001183×B 2 +34.857×C 2 (13)

[0141] Nitrazepam: REC = 34.36494 + 1.18619 × A - 0.464286 × B + 3.09079 × C +

[0142] 0.000772×A×B-0.12675×A×C-0.03035×B×C-0.03168×A 2 +0.003095×B 2 +41.92362×C 2 (14)

[0143] Nimetazepam: REC = 51.46618 + 0.412916 × A - 0.470371 × B + 8.60579 × C - 0.002679 × A × B - 0.2365 × A × C - 0.009547 × B × C - 0.010515 × A 2 +

[0144] 0.003451×B 2 +31.9885×C 2 (15)

[0145] Nordiazepam: REC = 32.06201 + 0.535777 × A - 0.343721 × B + 11.52316 × C - 0.00038 × A × B - 0.30425 × A × C - 0.026589 × B × C - 0.014186 × A 2 +0.00222×B 2 +34.8999×C 2 (16)

[0146] 7-aminometrazepam: REC = 21.86112 + 0.483632 × A - 0.070767 × B + 17.54447 ×

[0147] C-0.0001082×A×B-0.19775×A×C-0.067368×B×C-0.01146×A 2 +

[0148] 0.000821×B 2 +40.6615×C 2 (17)

[0149] 7-aminonitrazepam: REC = 21.90467 + 0.576979 × A - 0.08738 × B + 15.76842 × C - 0.001415 × A × B - 0.05425 × A × C - 0.086705 × B × C - 0.01416 × A 2 +

[0150] 0.000886×B 2 +40.3615×C 2 (18)

[0151] The optimal quality characteristic of multivariate quality characteristic recovery rate was set at 100%. The results showed that the desirability of the optimal parameter design variable combination was 1.0, and the ultrasonic time of 14min, the amount of mixed purification material of 21mg, and the extraction solvent of ethyl acetate were the optimal parameter design variable combination. Under the 95% confidence level, the bilateral test predicted the recovery rate of each target compound in gallbladder tissue to be in the range of 70.2% to 88.7%. The actual verification of the process capability index evaluation range of the results in gallbladder tissue was 1.37 to 5.31, and the mean recovery rate of each target compound ranged from 73.5% to 89.8%, which basically achieved the expected results, and the recovery rate of diazepam and its metabolites was significantly improved.

[0152] 2.2.7 Post-evaluation of method and process capability

[0153] The test was carried out according to the optimized detection method conditions, and the process capability of the optimized method was evaluated using the process capability method. The process capability evaluation results of diazepam and its metabolites in 10 main tissue matrices of aquatic products before and after optimization by the external standard method showed that after the method was optimized, the short-term process capability Z of 8 target compounds in 10 matrices was st The level is between 5.95 and 21.18, and the detection capacity can basically meet the general recovery rate requirement of 60% to 120%. pm Between 0.38 and 6.49, 71% of C pmThe value is greater than 0.67, and the process capability is close to the expected recovery rate of 100%. The results show that through the optimization of the pretreatment method, the detection capability of the external standard method has been improved, and its practicality has been enhanced. It can meet the needs of daily detection and can replace the internal standard method, greatly reducing the cost of detection. After the optimization of the internal standard method, the detection limit of the method is reduced, and the process capability is still very good and practical, and it can be fully used for daily detection activities.

[0154] 2.3 Methodological Investigation

[0155] 2.3.1 External Standard Method

[0156] 2.3.1.1. Linearity, detection limit, and quantification limit of the method

[0157] Since the matrix effect of 10 tissue matrices of aquatic products is relatively obvious, in order to reduce the influence of matrix effect on the detection results of diazepam and its metabolites, 10 matrix standard solution working curves were prepared according to Section 1.3.2.1. The detection limit (LOD) and quantification limit (LOQ) of the method were evaluated by the signal-to-noise ratio method. The detection limit (LOD) of this method was calculated as 3 times of (S / N), and the quantification limit (LOQ) of this method was calculated as 10 times of the signal-to-noise ratio (S / N). The detection limit and quantification limit of diazepam and its metabolites in 10 matrices are shown in Table 6. The results show that diazepam and its metabolites have a good linear relationship in the linear range of mass concentration 0.1-50 ng / mL, and the correlation coefficients are all greater than 0.999, the detection limit is 0.13-1.55 μg / kg, and the quantification limit is 0.43-5.17 μg / kg.

[0158] Table 6 Regression equations and method detection limits and quantification limits of diazepam and its metabolites in different matrices

[0159]

[0160]

[0161]

[0162] 2.3.1.2 Recovery and precision of the method

[0163] The mixed standard intermediate solution of diazepam and its metabolites at three concentration levels of limit of quantitation, 2 times the limit of quantitation and 5 times the limit of quantitation were added to the blank matrices of 10 tissues of aquatic products. The determination was repeated 7 times at each addition level according to this method. The recoveries of diazepam and its metabolites in the 10 tissue matrices of aquatic products are shown in Table 7. The results showed that the average recovery rate of diazepam and its metabolites in the heart tissue matrix of aquatic products by the external standard method was between 86.5% and 98.6%, and the relative standard deviation was between 1.34% and 7.34%; the average recovery rate of diazepam and its metabolites in the liver tissue matrix was between 77.1% and 96.5%, and the relative standard deviation was between 1.34% and 8.57%; the average recovery rate of diazepam and its metabolites in the spleen tissue matrix was between 72.1% and 96.7%, and the relative standard deviation was between 1.26% and 5.69%; the average recovery rate of diazepam and its metabolites in the kidney tissue matrix was between 73.2% and 90.2%, and the relative standard deviation was between 1.01% and 6.83%; the average recovery rate of diazepam and its metabolites in the gallbladder tissue matrix was between 73.5% and 89.8%, and the relative standard deviation was between 1.10% and 5.79%; The average recovery rates of diazepam and its metabolites in fish were between 76.3% and 100%, and the relative standard deviations were between 1.35% and 4.98%; the average recovery rates of diazepam and its metabolites in muscle tissue matrix were between 79.4% and 103%, and the relative standard deviations were between 1.34% and 6.39%; the average recovery rates of diazepam and its metabolites in plasma matrix were between 73.7% and 102%, and the relative standard deviations were between 1.15% and 5.96%; the average recovery rates of diazepam and its metabolites in intestinal tissue matrix were between 81.2% and 102%, and the relative standard deviations were between 1.05% and 5.90%; the average recovery rates of diazepam and its metabolites in gill tissue matrix were between 78.5% and 104%, and the relative standard deviations were between 1.03% and 5.72%, indicating that this method has certain reliability for the detection of diazepam and its metabolites in aquatic product tissues.

[0164] Table 7 Average recovery and relative standard deviation (RSD) of diazepam and its metabolites in different matrices (n=7)

[0165]

[0166]

[0167]

[0168]

[0169] 2.3.1.3 Method robustness

[0170] The samples with the same added concentration level were measured three times in parallel by different testers at intervals of 0, 7, and 30 days. The RSD results of the external standard method for detecting diazepam and its metabolites in 10 tissue matrices of aquatic products were 1.20% to 6.52%, all <15%, as shown in Table 8, indicating that this method has good feasibility and stability.

[0171] Table 8 Method robustness of diazepam and its metabolites in different matrices

[0172]

[0173] 2.3.2. Internal standard method

[0174] 2.3.2.1. Linearity, detection limit, and quantification limit of the method

[0175] The detection limits and quantification limits of diazepam and its metabolites in 10 matrices are shown in Table 9. The results showed that diazepam and its metabolites had a good linear relationship in the linear range of mass concentration of 0.1-50 ng / mL, with correlation coefficients greater than 0.999, detection limits of 0.04-1.43 μg / kg, and quantification limits of 0.13-4.77 μg / kg.

[0176] Table 9 Regression equations and method detection limits and quantification limits of diazepam and its metabolites in different matrices

[0177]

[0178]

[0179]

[0180] 2.3.2.2 Recovery and precision of the method

[0181] The mixed standard intermediate solution of diazepam and its metabolites at three concentration levels of limit of quantitation, 2 times the limit of quantitation and 5 times the limit of quantitation were added to the blank matrices of 10 tissues of aquatic products. The determination was repeated 7 times at each addition level according to this method. The recoveries of diazepam and its metabolites in the 10 tissue matrices of aquatic products are shown in Table 10. The results showed that the average recovery rate of diazepam and its metabolites in the matrix of aquatic product heart tissue by the internal standard method was between 89.0% and 107%, and the relative standard deviation was between 1.33% and 6.69%; the average recovery rate of diazepam and its metabolites in the matrix of liver tissue was between 84.5% and 110%, and the relative standard deviation was between 1.09% and 7.54%; the average recovery rate of diazepam and its metabolites in the matrix of spleen tissue was between 82.2% and 108%, and the relative standard deviation was between 1.50% and 7.33%; the average recovery rate of diazepam and its metabolites in the matrix of kidney tissue was between 83.9% and 105%, and the relative standard deviation was between 1.70% and 7.67%; the average recovery rate of diazepam and its metabolites in the matrix of gallbladder tissue was between 88.0% and 99.3%, and the relative standard deviation was between 1.88% and 5.88%; the average recovery rate of diazepam and its metabolites in the matrix of gonad tissue was between 1.38% and 1.70%; the average recovery rate of diazepam and its metabolites in the matrix of gallbladder tissue was between 1.86% and 1.91%; the average recovery rate of diazepam and its metabolites in the matrix of gonad tissue was between 1.10% and 1.31%. The average recovery of diazepam and its metabolites was between 90.8% and 107%, and the relative standard deviation was between 1.52% and 6.27%; the average recovery of diazepam and its metabolites in muscle tissue matrix was between 83.5% and 106%, and the relative standard deviation was between 1.10% and 7.14%; the average recovery of diazepam and its metabolites in plasma matrix was between 89.5% and 108%, and the relative standard deviation was between 1.26% and 6.57%; the average recovery of diazepam and its metabolites in intestinal tissue matrix was between 83.7% and 106%, and the relative standard deviation was between 1.14% and 6.84%; the average recovery of diazepam and its metabolites in gill tissue matrix was between 90.9% and 108%, and the relative standard deviation was between 1.47% and 5.90%, indicating that this method has good reliability for the detection of diazepam and its metabolites in aquatic product tissues.

[0182] Table 10 Average recovery and relative standard deviation (RSD) of diazepam and its metabolites in different matrices (n=7)

[0183]

[0184]

[0185]

[0186] 2.3.2.3. Method robustness

[0187] The samples with the same added concentration level were measured three times in parallel by different testers at intervals of 0, 7, and 30 days. The RSD results of the internal standard method for detecting diazepam and its metabolites in 10 tissue matrices of aquatic products were 4.37% to 7.36%, all <15%, as shown in Table 11, indicating that this method has good feasibility and stability.

[0188] Table 11 Method robustness of diazepam and its metabolites in different matrices

[0189]

[0190] The present invention optimizes the design of multivariate quality characteristic parameters of pretreatment conditions based on process capability evaluation, combines dispersed solid phase extraction purification method, and respectively uses external standard method and internal standard method to establish liquid chromatography tandem mass spectrometry analysis methods for diazepam and metabolites in the center, liver, spleen, kidney, gallbladder, seed, muscle, blood, intestine, and gill tissues of aquatic products. It has been verified that both the external standard method and the internal standard method can achieve the expected process capability goals, the internal standard method has excellent process capability, and the external standard method can greatly reduce the detection cost. The two methods have little matrix effect interference, and the detection limit is lower than the relevant standard. They are simple, reliable, low-cost, and highly practical, and are suitable for batch detection purposes. It provides a diversified selection scheme for actual detection work and provides technical support for the daily detection of diazepam and metabolites in 10 tissues of aquatic products and related source tracking.

Claims

1. A method for detecting the residual amount of diazepam and its metabolites in aquatic product tissues based on process capability evaluation, characterized in that: The following steps are involved: (1) Sample pretreatment: Ultrasonic extraction was used to extract diazepam and its metabolites from aquatic product tissues, and dispersed solid phase extraction was used to purify the extracted diazepam and its metabolites; (2) Detection using liquid chromatography tandem mass spectrometry; (3) Quantification using internal or external standard method.

2. The detection method according to claim 1, characterized in that: Aquatic product tissues include one or more of the muscles, heart, liver, spleen, kidney, gall bladder, gonads, plasma, intestines or gills of aquatic products.

3. The detection method according to claim 1, characterized in that: The metabolites of diazepam are one or more of temazepam, nitrazepam, nordiazepam, 7-aminometazepam, 7-aminonitrazepam, oxazepam, and nimetazepam.

4. The detection method according to claim 1, characterized in that: In step (1), the sample pretreatment includes the following steps: (A1) adding the aquatic product tissue to ethyl acetate, vortexing, ultrasonic extraction, centrifugation, and collecting the supernatant; (A2) adding the precipitate obtained after centrifugation in step (A1) to ethyl acetate again, repeating step (A1), collecting the supernatant, and combining the supernatant with the supernatant collected in step (A1); (A3) blowing the supernatant to near dryness with nitrogen, re-dissolving with methanol solution, vortexing, ultrasonic treatment, adding purification materials for dispersive solid phase extraction, centrifuging, adding n-hexane to the supernatant, vortexing, freezing, centrifuging, and filtering the lower layer solution; or, (B1) adding the aquatic product tissue to the mixed standard internal standard solution, vortexing, standing, adding to ethyl acetate, vortexing, ultrasonic extraction, centrifuging, and collecting the supernatant; (B2) adding the precipitate obtained after centrifugation in step (B1) to ethyl acetate, vortexing, ultrasonic extraction, centrifuging, collecting the supernatant, and combining the supernatant collected in step (B1); (B3) The supernatant is blown to near dryness with nitrogen, reconstituted with methanol solution, vortexed, sonicated, added with purification materials for dispersed solid phase extraction, centrifuged, n-hexane is added to the supernatant, vortexed, frozen, centrifuged, and the lower layer of solution is removed and filtered.

5. The detection method according to claim 4, characterized in that: The purification material is a mixed purification material, wherein the mass ratio of NH2-based SPE bulk adsorbent: cellulose powder is 1-2:1-2, the mass ratio of aquatic product tissue to purification material is 2000:5-30, the ultrasonic extraction time is more than 10 minutes, the freezing temperature is lower than -18°C, and the freezing time is more than 20 minutes.

6. The detection method according to claim 1, characterized in that: In step (2), the conditions of liquid chromatography analysis are as follows: the chromatographic column is a Kinetex SB-C18 (50mm×2.1mm, 1.7μm) chromatographic column; the mobile phase A is a 5mmol / L ammonium formate aqueous solution, and the mobile phase B is a methanol solution; the gradient elution program is: 0-1min, 10% B, 1-2min, 10%→50% B, 2-6min, 50%→95% B, 8.0-8.1min, 95%→10% B; 8.1-10min, 10% B; the flow rate of the mobile phase is 0.3mL / min; the column temperature is 40°C; and the injection volume is 3μL.

7. The detection method according to claim 1, characterized in that: In step (2), the conditions for mass spectrometry analysis are as follows: scanning mode: ESI+; monitoring mode: MRM; ion source temperature: 550°C; spray voltage: 5500V; collision gas: N2; collision gas pressure: 379kPa; curtain gas pressure: 241kPa; auxiliary gas pressure: 414kPa.

8. The detection method according to claim 1, characterized in that: In step (3), the internal standard used for quantification by the internal standard method is diazepam-D5, oxazepam-D5, temazepam-D5, nitrazepam-D5 and / or 7-aminonitrazepam-D5, and the external standard used for quantification by the external standard method is diazepam, oxazepam, temazepam, nitrazepam, nordiazepam, nimetazepam, 7-aminonitrazepam and / or 7-aminometazepam.

9. The detection method according to claim 1, characterized in that: In step (3), when the external standard method is used for quantification, the response surface methodology is used for optimization.

10. A method for evaluating the detection method of claim 1 based on process capability, characterized in that: The practicability of the detection method described in claim 1 is evaluated using a process capability evaluation method.

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