A purification extraction composition, a kit, and a method for determining drugs and fentanyl drugs in biological samples

Abstract

The present invention relates to the field of biological detection, and particularly to a purification extraction composition, a kit, and a method for determining drugs and fentanyl drugs in biological samples. The present invention provides a purification extraction composition, and the purification extraction composition includes C 18 adsorbent, EMR, aminopropyl extraction adsorbent, PSA, basic diatomaceous earth, and basic alumina; the C 18 mass ratio of the adsorbent, EMR, aminopropyl extraction adsorbent, N-propylethylenediamine solid-phase adsorbent, basic diatomaceous earth, and basic alumina is 32:52:100:100:100:100. The purification extraction composition of the present invention can be used for the purification of multi-components of 14 common drugs and 19 fentanyl drugs, and has better applicability and specificity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to a purification extraction composition, a kit, and a method for determining drugs and fentanyl drugs in biological samples. Background Art

[0002] Due to the diverse types of drugs and the synergistic effect of different drugs when used simultaneously, the probability of multiple drugs appearing simultaneously in actual samples increases, adding many difficulties to the actual detection of drugs. Currently, the main chemical methods for determining drugs include capillary electrophoresis, gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, etc. Compared with the combined use of chromatography and mass spectrometry, the separation efficiency and sensitivity of capillary electrophoresis and gas chromatography are relatively low. Gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry fully combine the advantages of high separation of chromatography and high sensitivity of mass spectrometry, and are currently the mainstream methods for determining drugs. Their application and research are relatively extensive. However, due to the complex biological sample matrix, purification treatment is required before mass spectrometer detection.

[0003] Currently, the pretreatment methods for common drugs and fentanyl drugs in biological samples include liquid-liquid extraction (SF / T 0066-2020, Liquid Chromatography-Tandem Mass Spectrometry Method for 31 New Psychoactive Substances of Fentanyl and Their Metabolites in Biological Samples), protein precipitation method (Wu Lina et al., Fudan University Journal (Medical Sciences), 2023, 50(3): 447-455), and direct purification method (Zhou Jian et al., A Method and Kit for Determining Fentanyl Drugs in Biological Samples, CN202011106966.5). The protein precipitation method and liquid-liquid extraction method are relatively simple, but the matrix effect is relatively serious during subsequent tandem mass spectrometry detection. Especially when multiple components are determined simultaneously, the matrix interference of some components is very serious. At the same time, due to the large number of types of fentanyl drugs and drugs, the names and chemical properties of the target compounds in SF / T 0066-2020 and the target compounds reported in other literatures are quite different, and the relevant literature methods lack universality. The invention patent of CN202011106966.5 only involves 7 types of fentanyl drugs. Therefore, it is still necessary to develop a more applicable and specific treatment method for biological matrices for the purification of biological samples and exclusive mass spectrometry detection. Summary of the Invention

[0004] The purpose of the present invention is to provide a purification extraction composition, a kit, and a method for determining drugs and fentanyl drugs in biological samples to solve the problems existing in the above-mentioned prior art. The purification extraction composition of the present invention can be used for the purification of multiple components of 14 common drugs and 19 fentanyl drugs, and has better applicability and specificity.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a purification extraction composition, and the purification extraction composition includes C 18 adsorbent, EMR, aminopropyl extraction adsorbent, PSA, basic diatomaceous earth, and basic alumina;

[0007] The C 18 mass ratio of the adsorbent, EMR, aminopropyl extraction adsorbent, N-propylethylenediamine solid phase adsorbent, basic diatomaceous earth, and basic alumina is 32:52:100:100:100:100.

[0008] Further preferably, the determination method of each component and its dosage in the purification extraction composition is determined by stoichiometry technology; the technology specifically includes response surface-central composite design.

[0009] The present invention provides a purification extraction tube containing the above purification extraction composition, and the purification extraction composition is loaded in the purification extraction tube.

[0010] Further preferably, the filter membrane is a 0.22 μm hydrophilic PTFE microporous filter membrane.

[0011] The present invention provides the application of the above purification extraction agent or the above purification extraction tube in the preparation of a kit, and the kit is a kit for simultaneously detecting drugs and fentanyl drugs.

[0012] Preferably, the fentanyl drugs include ofentanyl, acetylfentanyl, acryloylfentanyl, fentanyl, furanylfentanyl, parafluorofentanyl, α-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, parafluorobutyrylfentanyl, sufentanil, valeryl fentanyl, para-methylacetyl fentanyl, cyclopentyl fentanyl, methoxyacetyl fentanyl, ortho-fluorofentanyl, norfentanyl, and cyclopropionyl fentanyl;

[0013] The drugs include morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, nor-ketamine hydrochloride, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine, and methcathinone hydrochloride.

[0014] The present invention provides a kit for simultaneously detecting drugs and fentanyl drugs, and the kit includes the above purification extraction composition or the above purification extraction tube.

[0015] Preferably, the kit further includes an acetonitrile extraction package, a sodium chloride extraction package, a quality control product, and a supporting disposable consumable.

[0016] Preferably, the fentanyl drugs include norfentanyl, acetylfentanyl, acrylfentanyl, fentanyl, furanylfentanyl, parafluorofentanyl, alpha-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, parafluorobutyrylfentanyl, sufentanil, valerylfentanyl, para-methylacetylfentanyl, cyclopentafentanyl, methoxyacetylfentanyl, ortho-fluorofentanyl, norfentanyl and cyclopropionyl fentanyl;

[0017] The drugs include morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, nor-ketamine hydrochloride, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine and methcathinone hydrochloride.

[0018] The present invention provides a method for determining drugs and fentanyl drugs in biological samples, comprising:

[0019] (1) Pretreatment of the sample to be tested: treating the sample to be tested with the above purification extraction composition or the above purification extraction tube to remove co-extracted impurities;

[0020] (2) LC-MS / MS detection: the mobile phase A of the liquid chromatography is 0.05% formic acid - 5 mM ammonium acetate - aqueous solution, the mobile phase B is 0.05% formic acid - acetonitrile, and gradient elution is performed; the mass spectrometry is detected by the positive ion multiple reaction monitoring mode of the electrospray ion source.

[0021] Preferably, the chromatographic column used in the liquid chromatography is an Acquity BEH C 18 chromatographic column, the flow rate is 300 μL / min, and the injection volume is 10 μL;

[0022] The condition parameters of the mass spectrometry are: adopting the positive ion multiple reaction monitoring mode based on the electrospray ion source; the quantitative detection mode is the multiple reaction monitoring mode; the curtain gas pressure is 20 psi, the collision gas pressure is 7 psi, the declustering voltage is 60 V, the ion source voltage is 4.5 kV, and the ion source temperature is 500 °C.

[0023] Preferably, the sample to be tested includes serum, urine or hair;

[0024] The fentanyl drugs include norfentanyl, acetylfentanyl, acrylfentanyl, fentanyl, furanylfentanyl, parafluorofentanyl, alpha-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, parafluorobutyrylfentanyl, sufentanil, valerylfentanyl, para-methylacetylfentanyl, cyclopentafentanyl, methoxyacetylfentanyl, ortho-fluorofentanyl, norfentanyl and cyclopropionyl fentanyl;

[0025] The drugs include morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, norketamine hydrochloride, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine, and methcathinone hydrochloride.

[0026] Further preferably, the centrifugation method in step (1) is oscillating centrifugation; the specific steps are as follows: Take the sample, add acetonitrile with a volume twice that of the sample, oscillate and extract for 5 min, and centrifuge at a centrifugal force of 15,000 g for 10 min.

[0027] The present invention discloses the following technical effects:

[0028] 1. The optimized purification and extraction composition of the present invention can effectively reduce the content of matrix co-extracted impurities in the sample, and control the matrix effect within an acceptable range; at the same time, the purification and extraction composition is uniform and has good stability, and the method precision, accuracy, and sensitivity are all satisfactory.

[0029] 2. The present invention designs and develops a kit suitable for rapid extraction and treatment of 14 common drugs and 19 fentanyl drugs in serum, urine, or hair. (1) It can take into account 14 common drugs and 19 new fentanyl drugs at the same time; (2) The designed kit takes the purification tube as the main body, can quickly complete the pretreatment and purification of complex biological matrices, and has few influencing factors and low requirements for the professional level of operators, which can avoid the problems of poor accuracy and precision of the measurement results caused by the lack of professional experience of operators; (3) The designed purification tube has low cost and good applicability, and can meet the detection applications in the field and laboratory. The content of the present invention can provide a new idea for the multi-component rapid detection of drugs and has important practical application value.

[0030] 3. The sample treatment of the present invention has high recovery rate and good stability. The present invention uses chemometric techniques to evaluate the adsorption effect and recovery rate of the purifying agent, and quickly screens out the types that have a significant impact on the test results; at the same time, it designs a central composite experimental matrix to efficiently predict and calculate the optimal ratio and dosage. Compared with the traditional control variable optimization method, it can evaluate the importance of each factor, and can greatly reduce the number of required experiments while ensuring the accuracy of the results and deriving the theoretical optimal value.

[0031] 4. All the reagents used in the intensive kit designed by the present invention are pre-filled and packaged in small bags. The sample only needs a small centrifuge to complete the entire pretreatment process, which is very suitable for on-site sampling and purification. Subsequently, it can be connected with equipment such as portable mass spectrometry or bench-top mass spectrometry, and can quickly qualitatively and quantitatively analyze 14 common drugs and 19 fentanyl drugs in the sample, which is also an advantage that traditional analysis methods currently lack. Description of the Drawings

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0033] Figure 1 It is a schematic diagram of a purification extraction tube;

[0034] Figure 2 It is the MRM spectrogram of standard solutions (5.0 ng / mL) of 14 common drugs and 19 fentanyl drugs;

[0035] Figure 3 It is the total ion current diagram for comparing the urine purification effect. Among them, the left picture is the total ion current diagram of urine without using the extraction purification tube, and the right picture is the total ion current diagram of purified urine after extraction;

[0036] Figure 4 It is the total ion current diagram for comparing the serum purification effect. Among them, the left picture is the total ion current diagram of serum without using the extraction purification tube, and the right picture is the total ion current diagram of purified serum after extraction. Detailed implementation manners

[0037] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0041] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0042] The reagents, materials and experimental instruments involved in the present invention are as follows:

[0043] A. Reagents and Materials

[0044] Olfentanil, acetylfentanyl, acryloylfentanyl, fentanyl, furanylfentanyl, para-fluorofentanyl, alpha-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, para-fluorobutyrylfentanyl, sufentanil, valeryl fentanyl, para-methylacetylfentanyl, cyclopentyl fentanyl, methoxyacetyl fentanyl, ortho-fluorofentanyl, norfentanyl, cyclopropionyl fentanyl, morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, norketamine hydrochloride, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine and methamphetamine hydrochloride standard solution (100 μg / mL, methanol solution), Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0045] Methanol (CH 3 OH, chromatographically pure), acetonitrile (CH 3 CN, chromatographically pure), Fisher Scientific (China) Co., Ltd.

[0046] QuEChERS purification agents (including C 18 adsorbents, PSA (aminopropyl extraction adsorbent), EMR (Bond Elut EMR-Lipid enhanced lipid removal agent), basic diatomaceous earth, neutral diatomaceous earth, Florisil magnesium silicate adsorbent, acidic alumina, neutral alumina, basic alumina, NH 2 (N-propylethylenediamine solid phase adsorbent), GCB (graphitized carbon black adsorbent), Phenomenex & Agela Technologies Co., Ltd., Agilent Technologies (China) Co., Ltd.

[0047] The remaining consumables in the kit (including centrifuge tubes, filter membranes, sieve plates, gloves, pipettes, etc.) are purchased from Hamai Technology Co., Ltd.

[0048] In addition, blank serum, saliva, and urine are also provided for preparing matrix matching curves (which need to be stored under the conditions of -20°C to -80°C and are suitable for accurate quantitative analysis scenarios in the laboratory).

[0049] B. Experimental Instruments

[0050] The UHPLC-MS / MS system consists of an ExionLC UHPLC and a Sciex Q-Trap 6500plus mass spectrometer (ABSciex Company, USA). It is mainly used for quantitative analysis and can also qualitatively analyze some suspicious results with low contents based on retention time and fragment ion ratio. The UHPLC-MS / MS data acquisition is based on the multiple reaction monitoring (MRM) mode. First, various single-standard solutions of target drugs are directly input into the mass spectrometer through a syringe pump for optimization. Table 1 shows the hardware conditions and operation parameters of the instrument method, and Table 2 shows the gradient elution conditions of the detection mode. The specific optimized MRM parameters are shown in Table 3. Instrument control, data acquisition, and processing are carried out through the software Analyst 1.7.1 (Sciex). Experimental design and statistical analysis are respectively carried out using Design-expert 8.0.6.0 (Stat-Ease Inc., Minneapolis, USA), Minitab 17.1.0 (Minitab Inc., USA), and IBM SPSS Statistics 24 (SPSS Inc., Chicago, USA). Retsch CryoMill fully automatic cryogenic grinder (Retsch, Germany).

[0051] Table 1 UHPLC-MS / MS Operating Conditions

[0052]

[0053]

[0054] Table 2 Gradient Elution Program Used for Detection

[0055]

[0056] Table 3 Mass Spectrometry Multiple Reaction Monitoring Determination Parameters for Common Drugs and Fentanyl-Type Drugs

[0057]

[0058]

[0059]

[0060] Example 1 Optimization Design of Purification and Extraction Composition

[0061] Select common QuEChERS purification agents, including C18 Adsorbents, N-propylethylenediamine solid-phase adsorbent (PSA), Bond Elut EMR-Lipid enhanced lipid removal agent (EMR), basic diatomaceous earth, neutral diatomaceous earth, Florisil magnesium silicate adsorbent, acidic alumina, neutral alumina, basic alumina, aminopropyl extraction adsorbent (NH 2 ) and graphitized carbon black adsorbent (GCB) were used to conduct the recovery test of target compounds to determine the type of adsorbent for purification.

[0062] Experimental conditions: 1.0 mL of pure water spiked sample (5.0 ng / mL) was used instead of the experimental sample, mixed with 2.0 mL of acetonitrile, and 300 mg of the filler to be investigated was added to each. After mixing for 10 min, the supernatant of 200 μL was taken by centrifugation and placed in an injection vial, and 800 μL of pure water was added for mixing before injection analysis. The results showed that Florisil magnesium silicate adsorbent and alumina-based purifying agents had a very strong adsorption effect on fentanyl drugs, resulting in low recovery rates. On the other hand, GCB filler had obvious adsorption on almost all drugs (except cotinine) (the recovery rate was close to 0%), so the above three fillers were not suitable for the current situation analysis. NH 2 , PSA, basic diatomaceous earth, and basic alumina did not show a significant adsorption effect on the target compounds (the average recovery rates were all greater than 80%). At the same time, C 18 The recovery rates of the adsorbent and EMR for individual drugs were below 80%, and their dosages needed to be further optimized.

[0063] Regarding the dosages of C 18 adsorbent and EMR as key factors, response surface-central composite design optimization was carried out. At the same time, a dummy variable was set to investigate the model quality. Five different levels of experiments were conducted for each factor to reduce the influence of accidental errors in the operation. The experimental matrix design and results are shown in Table 4.

[0064] Table 4 Experimental matrix for three-factor response surface-central composite design optimization

[0065]

[0066]

[0067] After conducting the experiments according to the experimental matrix, the response values were input back, and the software was used to conduct the model fitting degree analysis in combination with the experimental results. In this experiment, the preferred model was the quadratic polynomial model. Subsequently, the obtained data was subjected to quadratic multiple regression fitting, and the regression equation for evaluating the first-order terms, interaction terms, and quadratic terms of the experimental factors was sorted out as follows:

[0068]

[0069] Wherein, Y is the predicted response value, X i and X j represent independent variables, δ 0 is the constant term, δ i is the linear coefficient, δ ii is the quadratic term coefficient, δ ij is the interaction term coefficient, ε is the random error compensation term. After obtaining the regression equation, ANOVA variance analysis is performed on the results. The generated polynomial model equation is evaluated by the fitting equation model term (Model), lack of fit term (Lack of Fit), coefficient of determination (R 2 ), and adjusted coefficient of determination (Adj-R 2 ). The results are shown in Table 5 below.

[0070] Table 5 ANOVA Results of Quadratic Polynomial Model

[0071]

[0072]

[0073] As can be seen from Table 5, by analyzing all coefficient terms, it is found that the P-values of all established quadratic polynomial model terms are far less than 0.05, indicating that the model shows a significant correlation with the data and is very suitable for the analysis and prediction of the current situation. The fitting degree and model quality of the polynomial model equation are represented by the coefficient of determination (R 2 ). Generally, for a good model fit, it is required that R 2 is at least greater than 0.8. The results show that for all target compounds, R 2 are all greater than 0.9463, and Adj R 2 are all greater than 0.8980, indicating that the generated equation has a 94.63% compliance with the experimental data, can explain 89.80% of the variation effect, has excellent predictive ability for the response value, and high credibility. On the other hand, the dosage of EMR has a significant impact on 24 compounds, and the dosage of C 18 adsorbent has a significant impact on 29 compounds, while the dummy variable has no significant correlation. Finally, after analyzing the obtained quadratic regression equation and setting the target term range (A, B, and C are all within the set range, and the factor terms are all at the maximum value), the theoretical optimal ratio is calculated using the Numerical Solution function of the software: the dosage of EMR is 52 mg, and the dosage of C 18 adsorbent is 32 mg. At the same time, considering that purifying agents such as NH 2 , PSA, basic diatomaceous earth, and basic alumina have an adsorption effect on organic acid components, some pigments, metal ions, and phenolic impurities in body fluid samples. Therefore, in summary, the final ratio of the purification extraction composition is set as: the dosage of EMR is 52 mg, the dosage of C 18 is 32 mg, and the dosage of NH2 100 mg each of PSA, basic diatomaceous earth, and basic alumina.

[0074] Methodology verification:

[0075] For the purification and extraction composition established above, the method selectivity, spiked recovery rate, matrix effect, sensitivity, linear range, and correlation coefficient were investigated in artificial serum, urine, and hair. First, the MRM spectra ( Figure 2 ) of the standard solution, blank sample solution, and blank matrix spiked solution (5.0 ng / mL) were compared. The results showed that no interference signals were detected near the retention time of all target compounds, indicating that the established purification and extraction composition and instrumental method were capable of distinguishing multiple common drugs, fentanyl drugs, and matrix interference components, and the method selectivity was good. A matrix-matched curve was prepared in the blank matrix, and then a standard working curve with the same concentration was prepared with the initial mobile phase (5 mM ammonium acetate + 0.05% formic acid aqueous solution (A), 0.05% formic acid / methanol (B), A / B = 70:30 (V / V)). The ratio of the slopes of the two was taken as the matrix effect (see Table 6-8). The results showed that there was a relatively obvious matrix suppression effect on some highly polar target compounds (retention time 2-4 min) such as cotinine, codeine, morphine, and methcathinone in the two matrices of urine and serum, which might be related to the relatively large amount of highly polar impurities such as inorganic salts in the two matrices. At the same time, the actual purification effect of the purification tube on two complex matrices such as urine and serum was evaluated. After purification by the purification tube, it was found that the pigment content in the urine and serum sample re-solutions was significantly reduced. On the other hand, by comparing the total ion chromatograms ( Figure 3 and Figure 4 ), it was found that the signal intensity of the impurity peaks with a retention time of 3-4 min in the two matrices decreased significantly after purification, indicating that the purification tube had an obvious effect on controlling matrix impurity interference and reducing the matrix effect.

[0076] Table 6 Methodology parameters in serum matrix

[0077]

[0078] Table 7 Methodology parameters in urine matrix

[0079]

[0080]

[0081] Table 8 Methodology parameters in hair matrix

[0082]

[0083]

[0084] It can be found from the data in Tables 6 - 8 that among the three matrices, the matrix effect ranges from 55.79% to 106.98% (the average matrix effect of serum is 91.35%, the average matrix effect of urine is 87.29%, and the average matrix effect of hair is 93.24%). In most cases, the matrix inhibition effect can be controlled within 30%, indicating that the purification effect of this purification extractant is acceptable, and the matrix effect is not sufficient to have a significant impact on the quantitative results of trace concentrations after correction. On the other hand, the recoveries of all target compounds in serum are between 75.2% and 102.6%, and the RSDs are all less than 15.7%. The spiked recoveries in urine are between 69.1% and 102.7%, and the RSDs are all less than 14.8%. The recoveries in hair are between 77.5% and 102.9%, and the RSDs are all less than 14.6%. The results of method accuracy and precision are satisfactory. The method sensitivity is determined by spiking tests at different low concentration levels, with a signal-to-noise ratio of 10:1 as the limit of quantification (summarized in Tables 6 - 8), and the detection sensitivity of actual samples can fully meet the detection requirements for ultra-low concentrations.

[0085] Comparison of purification effects

[0086] After completing the method validation, the actual purification effect of the purification tube was investigated. Since there are many target compounds involved, the results were evaluated by the matrix effect of the blank matrix and the matrix after matrix purification. If the matrix (inhibition / enhancement) effect is significant, it indicates that the concentration of matrix components is high and has an impact on the direct quantification of target compounds. If the matrix effect is low, it can be judged that there are few interfering matrix components and the purification effect of the pretreatment method is good. The specific results are shown in Table 9.

[0087] Table 9 Comparison of matrix effects of purified and unpurified matrices

[0088]

[0089] It can be found from the experimental data recorded in Table 9 that in the unpurified serum matrix, the average matrix effect of fentanyl compounds is 85.97%, and the matrix effect after purification is 93.14%. The difference is not significant. However, for morphine (unpurified matrix effect: 29.38%, purified matrix effect: 70.29%), MDMA (unpurified matrix effect: 59.27%, purified matrix effect: 86.41%), MDA (unpurified matrix effect: 45.86%, purified matrix effect: 88.47%), amphetamine (unpurified matrix effect: 66.44%, purified matrix effect: 106.86%), codeine (unpurified matrix effect: 34.29%, purified matrix effect: 72.45%), methadone (unpurified matrix effect: 55.97%, purified matrix effect: 83.73%), cotinine (unpurified matrix effect: 31.02%, purified matrix effect: 70.36%), and methcathinone hydrochloride (unpurified matrix effect: 68.44%, purified matrix effect: 101.20%), the purification step has a very significant control over the matrix inhibition effect. The average matrix effect for 14 typical drugs improved from 63.70% to 88.93%. For urine matrix, similar results exist. The average matrix effect of fentanyl compounds improved from 82.24% to 91.28%, and the difference is not significant. The average matrix effect of typical drugs improved from 61.35% to 81.88%. For morphine (unpurified matrix effect: 31.61%, purified matrix effect: 82.75%), amphetamine (unpurified matrix effect: 61.33%, purified matrix effect: 88.52%), MDA (unpurified matrix effect: 54.66%, purified matrix effect: 102.80%), cocaine (unpurified matrix effect: 28.46%, purified matrix effect: 55.79%), benzoylecgonine (unpurified matrix effect: 49.12%, purified matrix effect: 79.19%), and cotinine (unpurified matrix effect: 24.52%, purified matrix effect: 72.76%), there are also significant matrix inhibition effects. The above phenomena can be explained by the removal effects of acetonitrile layered extraction and the purifying agent on strongly polar interfering substances (inorganic salts, urea, polar small molecules, etc.) and weakly polar lipid substances (various proteins, phospholipids, fatty acids, etc.). Due to the presence of these two major types of substances in the unpurified samples, competitive ionization with the target compounds occurs, resulting in an obvious matrix inhibition effect. After purification, it can be significantly improved to meet the drug detection at ultra-low concentration levels. Finally, for hair matrix, because its composition is relatively fixed and simple, and the grinding process will not cause a large amount of endogenous precipitation, the matrix effect comparison is not significant (the average matrix effect before purification is 89.70%, and the average matrix effect after purification is 93.25%). In summary, the purification and extraction composition provided by the present invention has a good purification effect on treating serum and urine samples, especially improving the sensitivity for strongly polar and fat-soluble target compounds.

[0090] Preparation of the Kit in Example 2

[0091] 1. Preparation of the mixed standard working solution:

[0092] Preparation of the stock solutions of 14 common drugs (10 μg / mL): Accurately pipette 100 μL each of the liquid standard solutions of morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, nor-ketamine hydrochloride, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine, and methcathinone hydrochloride (1.0 mg / mL) into a 10-mL volumetric flask, make up to the scale line with methanol, mix well, seal, and store at -20 °C (frozen) in a refrigerator. The validity period is 6 months;

[0093] Preparation of the stock solutions of 19 fentanyl drugs (10 μg / mL): Accurately pipette 1000 μL each of the liquid standard solutions of ofentanil, acetylfentanyl, acrylfentanyl, fentanyl, furanylfentanyl, para-fluorofentanyl, α-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, para-fluorobutyrylfentanyl, sufentanil, valerylfentanyl, para-methylacetylfentanyl, cyclopentylfentanyl, methoxyacetylfentanyl, ortho-fluorofentanyl, norfentanyl, and cyclopropionylfentanyl (0.1 mg / mL) into a clean glass tube, concentrate by nitrogen blowing to about 5 mL, then transfer to a 10-mL volumetric flask, make up to the scale line with methanol, mix well, seal, and store at -20 °C (frozen) in a refrigerator. The validity period is 6 months;

[0094] Mixed standard working solution of 14 common drugs and 19 fentanyl substances (1.0 μg / mL, with the concentration of fentanyl drugs being 0.5 μg / mL): Pipette 1.0 mL of the stock solution of 14 common drugs and 0.5 mL of the stock solution of 19 fentanyl drugs into a 10-mL volumetric flask, make up to the scale with acetonitrile, seal and store at -20 °C. The validity period is 1 month.

[0095] 2. Contents of the kit:

[0096] The kit mainly includes 4 small packages. Package A: 20 purification extraction tubes and 20 outer shells of 50 mL high-recovery centrifuge tubes; Package B: 20 5 mL centrifuge tubes, 20 droppers, 20 syringes, and 5 pairs of disposable gloves; Package C: 20 packages of acetonitrile extraction packages (each containing 2 mL of acetonitrile) and 20 packages of sodium chloride extraction packages (each containing 0.5 g of sodium chloride); Package D: 1 bottle (2 mL) of 14 common drugs (10.0 μg / mL) and 1 bottle (2 mL) of 19 kinds of fentanyl mixed standard solutions (10.0 μg / mL), 3 bottles of serum matrix and urine matrix quality control products, covering 5 common drugs (including morphine, methamphetamine, codeine, cotinine, methcathinone) and 2 kinds of fentanyl drugs (fentanyl, valeryl fentanyl). It is stored at -20 °C, and the concentration range is divided into three levels for selection according to the actual detection situation. The low-level concentration is 10 - 20 ng / mL, the medium-level concentration is 20 - 50 ng / mL, and the high-level concentration is 50 - 100 ng / mL.

[0097] The purification extraction tube contains a purification extraction composition, and the purification extraction composition consists of C 18 adsorbent, EMR, aminopropyl extraction adsorbent, PSA, basic diatomaceous earth, and basic alumina; C 18 The masses of the adsorbent, EMR, aminopropyl extraction adsorbent, N-propylethylenediamine solid-phase adsorbent, basic diatomaceous earth, and basic alumina are 32 mg, 52 mg, 100 mg, 100 mg, 100 mg, and 100 mg respectively;

[0098] The purification extraction tube is composed of four parts: a purification tube, an outer shell of a centrifuge tube, a polypropylene lid (centrifuge tube lid), and a sieve plate. The mixed purification filler (purification extraction composition) is filled in the purification tube, fixed with a 10 μm sieve plate at the upper end and a 1 μm filter sieve plate at the lower end. The purification tube is deoxidized and sealed for packaging, and should be used as soon as possible after opening. The structure of the purification extraction tube is as Figure 1 shown.

[0099] 3. Structure design and principle of the purification extraction tube

[0100] Transfer the obtained purification extraction composition to the center of the purification extraction tube. Because the dosage of the main purifying agent in the purification extraction composition (EMR 52 mg, C 1832 mg) is less than that of traditional solid-phase extraction technology, which will result in less contact time and area of the extraction solution. At the same time, in order to balance the processing speed and efficiency, the purification extraction tube storage and purification extraction composition part is designed to be slender and tubular, so as to increase the contact efficiency between the extraction solution and the purification extraction composition. Both the purification extraction tube and the centrifuge tube housing are made of high-quality polypropylene material, with high strength and good transparency, can withstand a high-speed centrifugal force of 5000 g, have few organic eluents, and there are no interfering substances. At the same time, the purification tube part adopts a universal design and can be matched with the conventional 50 mL centrifuge tube in the laboratory. The end of the purification extraction tube is fixed with a 1μm protein filter sieve plate, and the packing is compressed and fastened with the actual centrifugal force (3500 g) during the filling process. Finally, it is fixed with a 10μm sieve plate at the upper end. The volume of the liquid residue part in the purification extraction tube is measured by the difference weight method. Usually, the volume of the retained liquid in the purification extraction tube does not exceed 100 μL. At the same time, the bottom of the centrifuge tube adopts a high-recovery design, and almost all the organic solvents can be easily recovered, which is very suitable for the purification and disposal of precious and small samples.

[0101] Determination of the Contents of 14 Common Poisons and 19 Fentanyl Drugs in Biological Samples in Example 3

[0102] The sample is pretreated with the kit in Example 2, specifically as follows:

[0103] Serum sample: Take 0.1 - 2.0 mL of serum sample (specifically depending on the instrument sensitivity and the concentration of the target substance in the serum) and transfer it to a blank 5 mL centrifuge tube in Package B. Add pure water to make up to the 2 mL scale line, then add the acetonitrile extraction package in Package C (containing 2 mL of acetonitrile). After manual shaking and mixing, add the sodium chloride extraction package in Package C (containing 0.5 g of sodium chloride). After shaking and mixing for 2 min, centrifuge at 3000 - 5000 rpm for 2 min, and transfer the upper acetonitrile layer for purification.

[0104] Urine sample: Take 2.0 mL of urine sample and transfer it to a blank 5 mL centrifuge tube in Package B. Add the acetonitrile extraction package in Package C (containing 2 mL of acetonitrile). After manual shaking and mixing, add the sodium chloride extraction package in Package C (containing 0.5 g of sodium chloride). After shaking and mixing for 2 min, centrifuge at 3000 - 5000 rpm for 2 min, and transfer the upper acetonitrile layer for purification.

[0105] Hair sample: Weigh 20 - 200 mg of hair sample (specifically depending on the instrument sensitivity and the hair concentration) and mix it with several stainless steel beads (2 mm in diameter) and place it in a 2 mL extraction tank. Grind it by impact at a frequency of 50 Hz, then add the acetonitrile extraction package in Package C (containing 2 mL of acetonitrile), and fully shake and extract. After skimming off the stainless steel beads from the mixed solution, it is ready for purification (the presence of hair does not affect the subsequent purification).

[0106] Purification and determination: The acetonitrile extract was transferred to the purification extraction tube in package A and purified by centrifugation at 3000 rpm. The purified solution was collected and blown to dryness under nitrogen at 40°C. After being reconstituted with the initial mobile phase, it was filtered through a 0.22 μm PTFE filter membrane and analyzed by UPLC-MS / MS.

[0107] Example 4

[0108] The kit prepared in Example 2 was used to test three hair quality control assessment samples in 2021. 20 mg of hair sample was accurately weighed and mixed with several stainless steel beads (2 mm diameter) in a 2 mL grinding jar, and ground at 50 Hz on a Retsch CryoMill fully automatic cryo-grinder, followed by adding the acetonitrile extraction package in package C (containing 2 mL acetonitrile), fully shaken and extracted, the mixed solution was skimmed off the stainless steel beads and transferred to the mixed filler purification tube in package A, and purified by centrifugation at 3000 rpm, the purified liquid was collected and blown to dryness with nitrogen at 40 ° C, re-dissolved with the initial mobile phase and passed through a 0.22 μm PTFE filter membrane, 5 μL of the supernatant was aspirated, and UPLC-MS / MS injection analysis was performed.

[0109] At the same time, sample processing and LC-MS / MS analysis were performed in accordance with the technical specification for forensic identification "Liquid Chromatography-Tandem Mass Spectrometry Test Method for 15 Drugs and Metabolites in Hair" (SF / Z JD0107025-2018). 20 mg of hair sample was accurately weighed and mixed with several stainless steel beads (2 mm diameter) in a 2 mL grinding jar, and then ground at a frequency of 50 Hz on a Retsch CryoMill fully automatic cryo-grinder, followed by adding 1.0 mL of internal standard methoxyphenamine standard working solution (methoxyphenamine 1 ng / mL), ultrasonicating in an ice bath for 30 min, centrifuging, removing the supernatant, and drying in a 60 ° C water bath under air flow. The residue was re-dissolved with 100 μL of methanol, and 5 μL of the supernatant was drawn for UPLC-MS / MS injection analysis.

[0110] The test results obtained by the method of the present invention and the SF / Z JD0107025-2018 method were compared and compared with the standard values. The comparison results are shown in Table 10.

[0111] Table 10 Comparison of hair quality control sample test results in 2021 (n=3)

[0112]

[0113]

[0114] The results recorded in Table 5 were compared with the standard values. The results showed that for hair samples, the sensitivity of the method of the present invention was better than that of the existing standard method (SF / Z JD0107025-2018), and the detection results were almost consistent with the existing standard method, and the maximum deviation from the standard value was less than 6%. The matrix effect of the method of the present invention was lower, avoiding the use of internal standards, and 14 common drugs and 19 fentanyl drugs could be measured simultaneously, which expanded the applicability of the method, had better practicality, and was convenient for the determination of batch samples.

[0115] Example 5

[0116] The kit prepared in Example 2 was used to test three hair quality control samples in 2023. 20 mg of hair sample was accurately weighed and mixed with several stainless steel beads (2 mm diameter) in a 2 mL grinding jar, and ground at a frequency of 50 Hz on a Retsch CryoMill fully automatic cryo-grinder, followed by adding the acetonitrile extraction package in package C (containing 2 mL of acetonitrile), fully shaken and extracted, the mixed solution was skimmed off the stainless steel beads and transferred to the mixed filler purification tube in package A, centrifuged at 3000 rpm to complete purification, the purified liquid was collected and blown to dryness with nitrogen at 40 ° C, re-dissolved with the initial mobile phase and passed through a 0.22 μm PTFE filter membrane, 5 μL of the supernatant was aspirated, and UPLC-MS / MS injection analysis was performed.

[0117] At the same time, sample processing and LC-MS / MS analysis were performed in accordance with the technical specification for forensic identification "Liquid Chromatography-Tandem Mass Spectrometry Test Method for 15 Drugs and Metabolites in Hair" (SF / Z JD0107025-2018). 20 mg of hair sample was accurately weighed and mixed with several stainless steel beads (2 mm diameter) in a 2 mL grinding jar, and then ground at a frequency of 50 Hz on a Retsch CryoMill fully automatic cryo-grinder, followed by adding 1.0 mL of internal standard methoxyphenamine standard working solution (methoxyphenamine 1 ng / mL), ultrasonicating in an ice bath for 30 min, centrifuging, removing the supernatant, and drying in a 60 ° C water bath under air flow. The residue was re-dissolved with 100 μL of methanol, and 5 μL of the supernatant was drawn for UPLC-MS / MS injection analysis.

[0118] The test results obtained by the method of the present invention and the SF / Z JD0107025-2018 method were compared and compared with the standard values. The comparison results are shown in Table 11.

[0119] Table 11 Comparison of hair quality control sample test results in 2023 (n=3)

[0120]

[0121] The results recorded in Table 11 show that for hair samples, the sensitivity of the method of the present invention is superior to the existing standard method (SF / Z JD0107025-2018), and the detection results are almost the same as those of the existing standard method. The maximum deviation from the standard value is less than 6%. The method of the present invention significantly reduces the matrix effect, avoids the use of internal standards, and can simultaneously measure 14 common drugs and 19 fentanyl drugs, expanding the applicability of the method, having better practicability, and facilitating the determination of batch samples.

[0122] Example 6

[0123] The kit prepared in Example 2 was used to detect 5 serum quality control samples in 2023. Accurately pipette 0.50 mL of the serum sample and transfer it to a blank 5 mL centrifuge tube in Package B. Add pure water to make up to the 2 mL scale line. Then add the acetonitrile extraction package in Package C (containing 2 mL of acetonitrile). After manual shaking and mixing, add the sodium chloride extraction package in Package C (containing 0.5 g of sodium chloride). Shake and mix for 2 min and then centrifuge at 5000 rpm for 2 min. Transfer the upper acetonitrile layer to the mixed packing purification tube in Package A and centrifuge at 3000 rpm to complete the purification. Collect the purified liquid and blow it to dry under nitrogen at 40 °C. Re-dissolve it with the initial mobile phase and filter it through a 0.22 μm PTFE filter membrane. Pipette 5 μL of the supernatant for injection analysis by UPLC-MS / MS, and perform parallel determination 6 times. The results show that the spiked recoveries of the 5 serum samples are between 85.2% and 96.5%, and the concentrations are between 0.23 ng / mL and 6.78 ng / mL.

[0124] Example 7

[0125] The kit prepared in Example 2 was used to detect 6 urine quality control samples. Accurately pipette 2.0 mL of the urine sample and transfer it to a blank 5 mL centrifuge tube in Package B. Add the acetonitrile extraction package in Package C (containing 2 mL of acetonitrile). After manual shaking and mixing, add the sodium chloride extraction package in Package C (containing 0.5 g of sodium chloride). Shake and mix for 2 min and then centrifuge at 4000 rpm for 2 min. Transfer the upper acetonitrile layer to the mixed packing purification tube in Package A and centrifuge at 3000 rpm to complete the purification. Collect the purified liquid and blow it to dry under nitrogen at 40 °C. Re-dissolve it with the initial mobile phase and filter it through a 0.22 μm PTFE filter membrane. Pipette 5 μL of the supernatant for injection analysis by UPLC-MS / MS, and perform parallel determination 6 times. The results show that the spiked recoveries of the 6 urine quality control samples are between 88.6% and 95.7%, and the concentrations are between 0.67 ng / mL and 9.45 ng / mL.

[0126] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a purification extraction composition or a purification extraction tube in the preparation of a kit, characterized in that, The kit is a kit for simultaneously detecting drugs and fentanyl drugs; the purification and extraction composition is C 18 adsorbent, Bond Elut EMR-Lipid enhanced lipid removal agent, aminopropyl extraction adsorbent, N-propylethylenediamine solid phase adsorbent, basic diatomaceous earth and basic alumina; the C 18 mass ratio of the adsorbent, Bond Elut EMR-Lipid enhanced lipid removal agent, aminopropyl extraction adsorbent, N-propylethylenediamine solid phase adsorbent, basic diatomaceous earth and basic alumina is 32:52:100:100:100:100; the purification extraction tube is loaded with the purification extraction composition; the purification extraction tube is composed of four parts: a purification tube, a centrifuge tube housing, a polypropylene lid, and a sieve plate; the fentanyl drugs are ofentanyl, acetylfentanyl, acrylfentanyl, fentanyl, furanylfentanyl, parafluorofentanyl, α-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, parafluorobutyrylfentanyl, sufentanil, valeryl fentanyl, para-methylacetyl fentanyl, cyclopentyl fentanyl, methoxyacetyl fentanyl, ortho-fluorofentanyl, norfentanyl, and cyclopropionyl fentanyl; the drugs are morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, norketamine hydrochloride, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine, and methcathinone hydrochloride.

2. A kit for simultaneously detecting drugs and fentanyl drugs, characterized in that, the kit includes a purification extraction composition or a purification extraction tube; The purification and extraction composition is C 18 adsorbent, Bond Elut EMR-Lipid enhanced lipid removal agent, aminopropyl extraction adsorbent, N-propylethylenediamine solid phase adsorbent, basic diatomaceous earth and basic alumina; the C 18 mass ratio of the adsorbent, Bond Elut EMR-Lipid enhanced lipid removal agent, aminopropyl extraction adsorbent, N-propylethylenediamine solid phase adsorbent, basic diatomaceous earth and basic alumina is 32:52:100:100:100:100; the purification extraction tube is loaded with the purification extraction composition; the purification extraction tube is composed of four parts: a purification tube, a centrifuge tube housing, a polypropylene lid, and a sieve plate; the fentanyl drugs are ofentanyl, acetylfentanyl, acrylfentanyl, fentanyl, furanylfentanyl, parafluorofentanyl, α-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, parafluorobutyrylfentanyl, sufentanil, valeryl fentanyl, para-methylacetyl fentanyl, cyclopentyl fentanyl, methoxyacetyl fentanyl, ortho-fluorofentanyl, norfentanyl, and cyclopropionyl fentanyl; the drugs are morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, norketamine hydrochloride, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine, and methcathinone hydrochloride.

3. The kit according to claim 2, characterized in that, the kit further includes an acetonitrile extraction package, a sodium chloride extraction package, a quality control product, and a supporting disposable consumable.

4. A method for determining drugs and fentanyl drugs in a biological sample, characterized in that, comprising: (1)Pretreatment of the sample to be tested: The sample to be tested is treated with a purification extraction composition or a purification extraction tube to remove co-extracted impurities; the purification extraction composition is C 18 adsorbent, Bond Elut EMR-Lipid enhanced lipid removal agent, aminopropyl extraction adsorbent, N-propylethylenediamine solid phase adsorbent, basic diatomaceous earth and basic alumina; the C 18 mass ratio of the adsorbent, Bond Elut EMR-Lipid enhanced lipid removal agent, aminopropyl extraction adsorbent, N-propylethylenediamine solid phase adsorbent, basic diatomaceous earth and basic alumina is 32:52:100:100:100:100; the purification extraction tube is loaded with the purification extraction composition; the purification extraction tube is composed of four parts: a purification tube, a centrifuge tube housing, a polypropylene lid, and a sieve plate; (2) LC-MS / MS detection: the mobile phase A of the liquid chromatography is 0.05% formic acid - 5 mM ammonium acetate - aqueous solution, the mobile phase B is 0.05% formic acid - acetonitrile, and gradient elution is performed; the mass spectrometry is detected by the positive ion multiple reaction monitoring mode of the electrospray ion source; the sample to be tested includes serum, urine, or hair; The fentanyl drugs are norfentanyl, acetylfentanyl, acrylfentanyl, fentanyl, furanylfentanyl, parafluorofentanyl, alpha-methylfentanyl, carfentanil, alfentanil, isobutyrylfentanyl, parafluorobutyrylfentanyl, sufentanil, valerylfentanyl, para-methylacetylfentanyl, cyclopentafentanyl, methoxyacetylfentanyl, orthofluorofentanyl, norfentanyl and cyclopropionyl fentanyl; The drugs are morphine, 6-acetylmorphine, methamphetamine, amphetamine, ketamine hydrochloride, norketamine hydrochloride, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine hydrochloride, benzoylecgonine, codeine, methadone, cotinine and methcathinone hydrochloride.

5. The method according to claim 4, characterized in that, The chromatographic column used in the liquid chromatography is AcquityBEH C 18 chromatographic column, the flow rate is 300 μL / min, and the injection volume is 10 μL; the condition parameters of the mass spectrometry are: adopting the positive ion multi-reaction monitoring mode based on the electrospray ion source; the quantitative detection mode is the multi-reaction monitoring mode; the curtain gas pressure is 20 psi, the collision gas pressure is 7 psi, the declustering voltage is 60 V, the ion source voltage is 4.5 kV, and the ion source temperature is 500 °C.

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