Method for detecting 58 psychotropic drugs and metabolites in plasma

Abstract

The invention discloses a method for detecting 58 psychotropic drugs and metabolites in plasma. The method comprises the following steps: 1) mixing a precipitant and plasma, and carrying out solid-liquid separation to obtain a to-be-detected sample solution; preparing a mixed standard working solution of 58 psychotropic drugs and metabolites; the precipitant is selected from methanol, acetonitrile and octanoic acid; and 2) detecting the mixed standard working solution and the to-be-detected sample solution by adopting a liquid chromatography-mass spectrometry method, and calculating the concentrations of psychotropic drugs and metabolites in the plasma according to the standard curve by adopting an external standard method. The method has the advantages that the plasma pretreatment is simple, the matrix effect of the protein in the plasma on the chromatographic column can be effectively reduced by adding acetonitrile, methanol and octanoic acid through a one-step precipitation method, and the detection efficiency is improved; in addition, the plasma sample treated by the deposition agent can be kept at normal temperature for 72 hours or more, and the problem that part of psychotropic drugs and metabolites are decomposed due to the fact that the plasma sample in remote areas is difficult to transport is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a method for detecting 58 kinds of psychotropic drugs and their metabolites in plasma. Background Art

[0002] Currently, poisoning by psychotropic drugs ranks first among clinical drug poisonings. Rapid detection and confirmation of toxic substances in suspected poisoned patients is crucial, especially in emergencies such as suicide attempts, accidental poisoning, or adverse drug reactions. Identifying the specific drug and its composition is crucial for accurate diagnosis and effective treatment of poisoned patients. In recent years, the use of LC-MS (particularly LC-MS / MS) technology has increased significantly in medical institutions. It is particularly useful for analyzing polar, non-volatile, and thermally unstable compounds that are difficult to analyze using gas chromatography (GC).

[0003] CN202111174772.3 discloses a method for determining 23 kinds of psychotropic drugs and metabolites in human serum, comprising: first performing sample pretreatment on the human serum sample to be tested, and then performing liquid chromatography tandem mass spectrometry detection on each sample after pretreatment, and simultaneously accurately and quantitatively analyzing 23 kinds of psychotropic drugs and metabolites in human serum, comprising the following detailed steps: step S1, preparing a series of concentrations of calibration solution, quality control solution and internal standard working solution; step S2, adding internal standard working solution to the sample to be tested and vortex mixing; step S3, taking each sample mixed in step S2 and centrifuging it, taking the supernatant after centrifugation and adding diluent to dilute it, and then performing LC-MS / MS analysis. It uses isotope internal standard to pretreat serum samples. CN201910058868.X discloses a method and kit for the simultaneous determination of 35 kinds of psychotropic drugs by high performance liquid chromatography-mass spectrometry, which adds methanol to the serum, obtains the supernatant after centrifugation, and uses external standard quantification method and high performance liquid chromatography-mass spectrometry to simultaneously determine 35 kinds of psychotropic drugs. The "Method for the Simultaneous Rapid Confirmation and Detection of 42 Psychotropic Drugs and Their Metabolites in Plasma and Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry" discloses the use of acetonitrile as a precipitant to pretreat plasma, with ammonium sulfate and methanol-acetonitrile as the mobile phase. Whether the aforementioned public documents and patents use an isotope internal standard method or an external standard method for quantification, the matrix components in the plasma sample (such as phospholipids, protein residues, etc.) may affect the ionization efficiency of the target analyte, resulting in deviations in the quantitative results. Although the isotope internal standard method can compensate for the matrix effect to a certain extent, completely eliminating the matrix effect in complex biological samples remains a challenge. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for 58 kinds of psychotropic drugs and metabolites in plasma. By screening the precipitants, it can effectively precipitate the proteins in plasma, reduce the matrix effect, improve the detection sensitivity, and realize the simultaneous detection of 58 kinds of psychotropic drugs and metabolites. In addition, the present invention also unexpectedly finds that the precipitant can improve the storage period of plasma samples, thus solving the problem that some psychotropic drugs and metabolites are decomposed due to difficult transportation in remote areas.

[0005] The present invention provides a method for 58 kinds of psychotropic drugs and metabolites in plasma, comprising the following steps:

[0006] 1) Mix the precipitant and plasma, perform solid-liquid separation to obtain a sample solution to be detected; prepare a mixed standard working solution of 58 kinds of psychotropic drugs and metabolites; the precipitant is selected from methanol, acetonitrile and octanoic acid;

[0007] 2) Use liquid chromatography-mass spectrometry (LC-MS) to detect the mixed standard working solution and the sample solution to be detected, and calculate the concentrations of psychotropic drugs and metabolites in plasma by the external standard method according to the standard curve.

[0008] In liquid chromatography-mass spectrometry (LC-MS) analysis, the precipitant in the pretreatment of plasma samples is crucial for the accuracy of the analysis results. The selection of the precipitant not only affects the removal effect of proteins, but also may introduce matrix effects, which in turn affect the quantitative and qualitative analysis of target analytes. By screening the types of precipitants, the present invention unexpectedly finds that octanoic acid has a synergistic effect with organic solvents (methanol and acetonitrile), which helps to precipitate plasma proteins more thoroughly, thereby reducing the contamination of the chromatographic column and ion source by residual proteins and effectively reducing the matrix effect. In addition, the present invention also unexpectedly finds that adding octanoic acid can also extend the stability of plasma samples, eliminating the need for low-temperature dry ice transportation, and can maintain the stability of analytes at room temperature (25°C) for more than 72 hours, solving the problem that some psychotropic drugs and metabolites are decomposed due to difficult transportation in remote areas.

[0009] In some embodiments, the 58 psychotropic drugs and metabolites include: amisulpride, amitriptyline, aripiprazole, carbamazepine, chlorpromazine, citalopram, clomipramine, clozapine, dimenhydrinate, donepezil, doxepin, duloxetine, fluoxetine, fluphenazine, fluvoxamine, haloperidol, 9-hydroxyrisperidone, imipramine, lamotrigine, loxapine, memantine, mirtazapine, nordoxepin, norfluoxetine, nortriptyline, oxcarbazepine, paroxetine, penfluridol, perphenazine, primidone, quetiapine, risperidone, sertraline, sulpiride, tandospirone, trazodone, trihexyphenidyl, ziprasidone, N-desalkyl quetiapine, O-desmethylvenlafaxine, dehydroaripiprazole, phenytoin sodium, alprazolam, clonazepam, diazepam, estazolam, lidocaine, lorazepam, midazolam, oxazepam, tramadol, zolpidem, nitrazepam, triazolam, N-demethyl clozapine, N-desmethyl clomipramine, phenobarbital and bupropion. Among them, 9-hydroxyrisperidone is a metabolite of risperidone, N-desalkyl quetiapine is a metabolite of quetiapine, norfluoxetine is a metabolite of fluoxetine, nortriptyline is a metabolite of amitriptyline, N-demethyl clozapine is a metabolite of clozapine, and N-desmethyl clomipramine is a metabolite of clomipramine.

[0010] In some embodiments, the volume ratio of methanol to acetonitrile is (0.8 - 1.2):1. In this application, the precipitant was screened. First, 10% trichloroacetic acid solution, 5% perchloric acid solution, saturated ammonium sulfate solution, methanol, and acetonitrile were used for precipitation respectively, and the response value was very low when measured by liquid chromatography-mass spectrometry; then combinations of methanol and acetonitrile, and acetonitrile and saturated sodium chloride were used for measurement, and it was found that the response value was high when measured with methanol and acetonitrile; then by measuring the ratios of ethanol to acetonitrile in the combination of 1:1, 1:2, and 2:1, it was found that the ratio of methanol to acetonitrile could not vary too much, which also led to a very low response value. The optimal range was found to be (0.8 - 1.2):1.

[0011] In some specific embodiments, the volume ratio of methanol to acetonitrile is 1:1.

[0012] In some embodiments, based on the total volume of the precipitant, the concentration of caprylic acid is 0.1 - 0.4 v / v%. In this application, caprylic acid was accidentally added on the basis of methanol and acetonitrile, and it was found that adding caprylic acid could improve the storage stability of plasma samples.

[0013] In some specific embodiments, the concentration of caprylic acid is 0.1 v / v%.

[0014] In some embodiments, the volume ratio of plasma to precipitant is 100:(300 - 500).

[0015] In certain specific embodiments, the volume ratio of the plasma to the precipitant is 100:400.

[0016] In certain embodiments, the solid-liquid separation is centrifugation, and the rotation speed of the centrifugation is 10,000 - 14,000 r / min.

[0017] In certain specific embodiments, the rotation speed of the centrifugation is 12,000 r / min.

[0018] In certain embodiments, the preparation method of the mixed standard working solution is as follows: The preparation method of the mixed standard working solution is to dissolve the standards of 58 psychotropic drugs and metabolites in methanol, then serially dilute with acetonitrile, and then mix with the negative blank plasma extract, where the negative blank plasma extract is obtained by mixing negative blank plasma and a precipitant.

[0019] In certain specific embodiments, the concentration of the mixed standard working solution is 0.5 - 20,000.0 μg / L.

[0020] In certain embodiments, the standard working solution uses the concentration of the standards in the mixed standard working solution as the X-axis and the corresponding peak area for each concentration as the Y-axis for linear regression analysis to obtain.

[0021] In certain embodiments, in the liquid chromatography - mass spectrometry (LC - MS) method,

[0022] The conditions for liquid chromatography are as follows:

[0023] Reverse-phase C18 high-performance liquid chromatography column;

[0024] Column temperature: 40 - 45 °C;

[0025] Injection volume: 3 - 7 μL;

[0026] Mobile phase A is: aqueous ammonium acetate solution,

[0027] Mobile phase B is: ammonium acetate solution in methanol;

[0028] Flow rate: 0.4 - 0.8 μL / min;

[0029] Gradient elution is adopted, and the elution gradient of the mobile phase is: 0 min, 95% A + 5% B; 2 min, 95% A + 5% B; 3.0 min, 70% A + 30% B; 10.0 min, 5% A + 95% B; 12.5 min, 5% A + 95% B; 13 min, 95% A + 15% B.

[0030] In certain embodiments, in the liquid chromatography - mass spectrometry (LC - MS) method,

[0031] The conditions for mass spectrometry are as follows:

[0032] Curtain gas: 28 - 32 psi;

[0033] Electrospray voltage: 5300 - 5700 V, -4300 - -4700 V;

[0034] Temperature: 450 - 550 °C;

[0035] Nebulizing gas: 45 - 55 psi;

[0036] Auxiliary gas: 45 - 55 psi;

[0037] Data acquisition mode: Multiple reaction monitoring.

[0038] The present invention has the following beneficial effects:

[0039] 1) The pretreatment of plasma in the present invention is simple. By adding acetonitrile, methanol and octanoic acid in a one-step precipitation method, the matrix effect of proteins in plasma on the chromatographic column can be effectively reduced, and the detection efficiency can be improved. In addition, the pretreated plasma samples can maintain the stability of analytes at room temperature (25 °C) for more than 72 hours, solving the problem of sample transportation in remote areas.

[0040] 2) For the detection method established in the present invention, 58 kinds of psychotropic drugs and metabolites have good linear relationships within a certain mass concentration range, and their correlation coefficients are all ≥ 0.996. The detection limit range of the method is 0.03 - 3 μg / L (except phenobarbital), the lower limit of quantification range is 0.1 - 10 μg / L (except phenobarbital), and the recovery rate is in the range of 90.93 - 121.72%, which can meet the requirements of accurate quantitative detection.

[0041] 3) By setting the liquid chromatography and mass spectrometry conditions, the present invention can achieve the effect of simultaneously detecting 58 kinds of psychotropic drugs and metabolites, greatly improving the throughput of the whole determination process.

[0042] 4) The method of the present invention greatly shortens the analysis time: by optimizing the liquid phase conditions, the whole analysis time is greatly optimized, and the analysis time is shortened to 14 min. The concentrations of 58 kinds of psychotropic drugs and metabolites can be obtained quickly and efficiently, improving the detection efficiency.

[0043] 5) The present invention has high detection sensitivity: by continuously optimizing the pretreatment and instrument methods, high sensitivity can be achieved for each drug, meeting the requirements of clinical detection.

[0044] 6) The detection method provided by the present invention has the characteristics of simple and convenient sample pretreatment, small required sample volume, high sensitivity, high accuracy, and high throughput. Detailed implementation mode

[0045] To describe the present invention more specifically, the technical solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. These descriptions merely illustrate how the present invention is implemented and do not limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0046] The following standard products of this application are all obtained through commercial channels. Preferably, the standard products of Tianjin Aladdin Biochemical Technology Co., Ltd., SIGMA, TM Standard and the National Institutes for Food and Drug Control have a purity of ≥98%; methanol and acetonitrile are both of chromatographic grade.

[0047] Liquid chromatography - mass spectrometry: SCIEX TRIPLE QUAD 4500 equipped with an electrospray ionization source

[0048] The liquid chromatography analysis conditions are as follows:

[0049] The chromatographic column is: 00D - 4633 - AN( 5μm EVO C18 LC Column 100×2.1mm)

[0050] Mobile phase A is: water (5mmol / L ammonium acetate)

[0051] Mobile phase B is: methanol (5mmol / L ammonium acetate)

[0052] The initial ratio of the mobile phase is: 2% concentration of pump B

[0053] The mobile phase gradient elution program (see Table 1):

[0054] Table 1

[0055]

[0056]

[0057] The flow rate is: 0.6mL / min;

[0058] The column temperature is: 40°C;

[0059] The injection volume is: 5μL.

[0060] The mass spectrometry conditions are as follows:

[0061] Positive ion mode (ESI+) electrospray voltage: 5500V;

[0062] Negative ion mode (ESI-) electrospray voltage: -4500V;

[0063] Curtain gas: 30psi;

[0064] Nebulizing gas (gas 1): 50 psi;

[0065] Auxiliary gas (gas 2): 50 psi;

[0066] Ion source temperature (TEM): 500 °C;

[0067] Electrospray ionization positive and negative ion switching mode, segmented multiple reaction monitoring (MRM) acquisition mode. The mass spectrometry ion pair parameters of each target analyte are shown in Table 2.

[0068] Table 2 MRM parameters of 58 drugs

[0069]

[0070]

[0071]

[0072]

[0073] Take 160 μL of 42 kinds of mixed standard solutions, 160 μL of each of the 14 kinds of mixed standard solutions, 400 μL of 10 mg / L bupropion standard solution, and 400 μL of 1000 mg / L phenobarbital standard solution, add 880 μL of methanol, mix well to obtain 58 kinds of mixed standard solutions (volume is 2.0 mL), and label them as No. 1 mixed standard solution. Among them, the 42 kinds of mixed standard solutions are obtained by mixing 42 kinds of standard products (amisulpride, amitriptyline, aripiprazole, carbamazepine, chlorpromazine, citalopram, clomipramine, clozapine, dimenhydrinate, donepezil, doxepin, duloxetine, fluoxetine, fluphenazine, fluvoxamine, haloperidol, 9-hydroxyrisperidone, imipramine, lamotrigine, loxapine, memantine, mirtazapine, nordoxepin, norfluoxetine, nortriptyline, oxcarbazepine, paroxetine, penfluridol, perphenazine, primidone, quetiapine, risperidone, sertraline, sulpiride, tandospirone, trazodone, trihexyphenidyl, ziprasidone, N-desalkyl quetiapine, O-desmethyl venlafaxine, dehydroaripiprazole and phenytoin) and dissolving them in methanol. The concentration of each standard product is shown in Table 8; the 14 kinds of mixed standard solutions are obtained by mixing 14 kinds of standard products (alprazolam, clonazepam, diazepam, estazolam, lidocaine, lorazepam, midazolam, oxazepam, tramadol, zolpidem, nitrazepam, triazolam, N-demethyl clozapine, N-desmethyl clomipramine) and dissolving them in methanol. The concentration of each standard product is shown in Table 8; the 10 mg / L bupropion standard solution is obtained by dissolving bupropion in methanol, and the concentration of bupropion is 10 mg / L; the 1000 mg / L phenobarbital standard solution is obtained by dissolving phenobarbital in methanol, and the concentration of phenobarbital is 1000 mg / L.

[0074] According to Table 3, the 1# mixed standard solution was diluted step by step with acetonitrile to prepare the 2# to 7# mixed standard solutions; then, 7# mixed standard solution was taken and diluted with acetonitrile to prepare the 8# and 9# mixed standard solutions.

[0075] Table 3 Preparation of Standard Solutions

[0076]

[0077]

[0078] Preparation of mixed standard working solution: Take 10 μL of each of the 1#, 3#, 5#, 6#, 8#, and 9# mixed standard solutions, and add 490 μL of negative blank extract respectively, and mix well to obtain the standard working solution. Among them, the negative blank extract refers to: take 100 μL of mixed plasma sample from healthy people (without taking various psychotropic drugs and metabolites), add 400 μL of precipitant, vortex for 1 min, centrifuge at 12000 r / min for 10 min, and take the supernatant as the blank extract; the precipitant is 0.1% octanoic acid + methanol + acetonitrile, that is, after methanol and acetonitrile are mixed in a volume ratio of 1:1, octanoic acid is added, and the concentration of octanoic acid is 0.1 v / v%.

[0079] Drawing of working curve: Using the concentration of 58 standard substances in the mixed standard working solution as the X-axis and the peak area corresponding to each concentration as the Y-axis, perform linear regression analysis to obtain the standard working curve of 6 standard points. See Table 4.

[0080] Table 4 Working Curve Concentrations

[0081]

[0082]

[0083] * The concentration unit is mg / L

[0084] Example 1 Screening of Precipitant

[0085] In this Example 1, the effects of different precipitants on the detection method were investigated, including the effects on proteins in plasma samples and the stability of plasma samples, in order to screen suitable precipitants. The details are as follows:

[0086] 1.1. Effects on Proteins in Plasma Samples

[0087] Take 10 μL of the 4# mixed standard solution, add 490 μL of precipitant, mix well as the control solution, and then immediately detect it by liquid chromatography-mass spectrometry. Among them, the precipitant is obtained by mixing methanol and acetonitrile in a volume ratio of 1:1.

[0088] Another 4 portions of healthy human mixed plasma samples (100 μL each) were taken. 10 μL of the 4# mixed standard solution and 390 μL of the precipitant in Table 5 were added to each portion to form 4 experimental group solutions. Each group of solutions was vortexed for 1 min and centrifuged at 12,000 r / min for 10 min. The supernatant was taken as the sample solution to be measured, and then immediately detected by liquid chromatography-mass spectrometry. By detecting the response values of 7 psychotropic drugs and metabolites, namely alprazolam, lorazepam, mirtazapine, norfluoxetine, sulpiride, tramadol, and zolpidem in plasma, a suitable precipitant combination was screened.

[0089] Methanol + acetonitrile means methanol and acetonitrile obtained according to a volume ratio of 1:1; 0.1% octanoic acid + methanol means that after mixing octanoic acid and methanol, the concentration of octanoic acid is 0.1 v / v%; 0.1% octanoic acid + acetonitrile means that after mixing octanoic acid and acetonitrile, the concentration of octanoic acid is 0.1 v / v%; 0.1% octanoic acid + methanol + acetonitrile means that after mixing methanol and acetonitrile according to a volume ratio of 1:1, octanoic acid is added, and the concentration of octanoic acid is 0.1 v / v%.

[0090] Table 5

[0091]

[0092] As can be seen from Table 5, when using the combination of methanol, acetonitrile, and octanoic acid as the precipitant for plasma pretreatment, higher response values were shown in liquid chromatography-mass spectrometry analysis, indicating that this precipitant combination can effectively reduce the matrix effect and improve the sensitivity and accuracy of detection when treating plasma samples.

[0093] 1.2. Influence on the stability of plasma samples

[0094] In remote areas, when the sample transportation time may be long and the transportation conditions may not be ideal, it may lead to the degradation or denaturation of analytes, thus affecting the accuracy and reliability of the detection results. Therefore, the influence of different precipitants on the storage stability of plasma samples was further investigated.

[0095] The 4 experimental group solutions obtained in Step 1.1 were placed at room temperature for 0 d (labeled as D0), 1 d (labeled as D1), 2 d (labeled as D2), and 3 d (labeled as D3) respectively. The concentrations of alprazolam, lorazepam, mirtazapine, norfluoxetine, sulpiride, tramadol, and zolpidem were determined by liquid chromatography-mass spectrometry, and the change rates of the concentrations of the above 7 substances at 1 d, 2 d, and 3 d were calculated to evaluate the stability of the blood samples. The results are shown in Table 6.

[0096] Table 6

[0097]

[0098]

[0099] As can be seen from Table 6, after treating plasma samples with a precipitant formed by methanol, acetonitrile and octanoic acid for 3 days, the change rates of these 7 psychotropic drugs all exceeded 5% with the extension of storage time, while the change rates of the composition formed by methanol, acetonitrile and octanoic acid were all lower than 5%, indicating that this precipitant combination can extend the stability of plasma samples and solve the problem of sample transportation in remote areas.

[0100] Methodology verification of the detection method in Example 2

[0101] In this example, methodology verification was carried out on the liquid chromatography - mass spectrometry method, including matrix effect, detection limit, lower limit of quantification, precision and accuracy. Specifically as follows:

[0102] 2.1. Matrix effect evaluation

[0103] According to the CLSI guidelines, the matrix effect was calculated using a linear standard solution. Specifically as follows:

[0104] Group A: Take 10 μL of each of the mixed standard solutions of 1#, 3#, 5#, 6#, 8#, and 9# in Table 3, add 490 μL of the precipitant, and draw a 6 - point standard curve. The precipitant is 0.1% octanoic acid + methanol + acetonitrile, that is, after methanol and acetonitrile are mixed in a volume ratio of 1:1, octanoic acid is added, and the concentration of octanoic acid is 0.1 v / v%.

[0105] Group B: Take 10 μL of each of the mixed standard solutions of 1#, 3#, 5#, 6#, 8#, and 9# in Table 3, add 490 μL of negative blank plasma extract, and draw a 6 - point working curve. The negative blank plasma extract is obtained by adding 400 μL of the precipitant to 100 μL of a mixed plasma sample of healthy people, vortexing for 1 min, and centrifuging at 12000 r / min for 10 min; the precipitant is the same as that in Group A.

[0106] Group C: Take 10 μL of each of the mixed standard solutions of 1#, 3#, 5#, 6#, 8#, and 9# in Table 3, 100 μL of a mixed plasma sample of healthy people, add 390 μL of the precipitant, vortex for 1 min, and centrifuge at 12000 r / min for 10 min to obtain; draw a 6 - point spiked curve. The precipitant is the same as that in Group A.

[0107] Calculate the linear slope of the analytes in Group C, and calculate the ratio of its linear slope to that of the analytes in Group A to investigate the matrix effect. Calculate the linear slope of the analytes in Group C, and calculate the ratio of its linear slope to that of the analytes in Group B to investigate the extraction recovery rate. The results are shown in Table 7.

[0108] Table 7

[0109]

[0110] [[ID=�5]]

[0111] As can be seen from Table 7, the C / A slope ratio (%) is between 90 - 100%, indicating no matrix effect. To make the test matrix as consistent as possible, a working curve was prepared with a blank plasma extract for quantitative analysis. The C / B slope ratio (%) is between 90 - 100%, indicating a relatively high extraction recovery rate that meets the detection requirements.

[0112] 2.2, Detection Limit and Lower Limit of Quantification

[0113] Take 10 μL of each of the mixed standard solutions of 1#, 3#, 5#, 6#, 8#, and 9# in Table 3, and add them to 490 μL of negative blank plasma extract respectively, and mix well to obtain 6 standard working solutions. Among them, the negative blank plasma extract refers to: take 100 μL of mixed plasma from healthy people (without taking various psychotropic drugs and metabolites), add 400 μL of precipitant, vortex for 1 min, centrifuge at 12000 r / min for 10 min, and take the supernatant as the blank extract; the precipitant is 0.1% octanoic acid + methanol + acetonitrile, that is, after methanol and acetonitrile are mixed in a volume ratio of 1:1, octanoic acid is added, and the concentration of octanoic acid is 0.1 v / v%.

[0114] Linear Range: A working curve was plotted with the peak area of each analyte against the mass concentration to obtain the linear regression equation and correlation coefficient. The lower limit of quantification (LOQ) was calculated as 10 times the noise response value of the 6 standard working solutions in Step 2.2 compared with the blank solution (the control sample refers to: take 10 μL of each of the mixed standard solutions of 1#, 3#, 5#, 6#, 8#, and 9# in Table 3, add 490 μL of precipitant and mix well; the precipitant is obtained by mixing methanol and acetonitrile in a volume ratio of 1:1). The detection limit (LOD) was calculated as 3 times the value of the response compared with the noise response value of the blank sample. The results are shown in Table 8.

[0115] Table 8 Linear Range, Detection Limit and Lower Limit of Quantification

[0116]

[0117]

[0118] * The concentration unit is mg / L

[0119] As can be seen from Table 8, for the detection method established in the present invention, 58 psychotropic drugs and metabolites have good linear relationships within a certain mass concentration range, and their correlation coefficients are all ≥ 0.996. The detection limit range of the method is 0.03 - 3 μg / L (except for phenobarbital), and the lower limit of quantification range is 0.1 - 10 μg / L (except for phenobarbital), indicating that the accurate quantitative detection requirements can be met.

[0120] 2.3, Precision and Accuracy

[0121] Three groups were set up. 100 μL of pooled plasma from healthy individuals was taken for each group. Then, 10 μL of the mixed standard solutions of 2#, 4#, and 7# were added to each group, and then 390 μL of the precipitant was added. After vortexing for 1 min and centrifuging at 12,000 r / min for 10 min, the supernatant was taken for determination. Among them, the precipitant was 0.1% octanoic acid + methanol + acetonitrile, that is, after methanol and acetonitrile were mixed at a volume ratio of 1:1, octanoic acid was added, and the concentration of octanoic acid was 0.1 v / v%.

[0122] Six parallel samples were taken for each concentration, and the spiked recovery rate (%) at each concentration was calculated. The above operations were repeated on the 1st, 2nd, and 3rd days respectively, and the intra-day precision and inter-day precision were calculated. The results are shown in Table 9.

[0123] Table 9

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] * The concentration unit is mg / L

[0130] As can be seen from Table 9, the RSDs of the intra-day precision and inter-day precision are between 1.0% and 12.8%, both < 15%, and the recovery rates are between 91.4% and 108.0%. This shows that the detection method provided by the present invention has good precision and high accuracy, and can meet the needs of analytical detection.

[0131] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. Method for detecting 58 kinds of psychotropic drugs and metabolites in plasma, characterized in that, It includes the following steps: 1) Mix the precipitant and plasma, perform solid-liquid separation to obtain the sample solution to be tested; prepare a mixed standard working solution of 58 kinds of psychotropic drugs and metabolites; the precipitant is selected from methanol, acetonitrile, and octanoic acid; 2) Use liquid chromatography-mass spectrometry to detect the mixed standard working solution and the sample solution to be tested, and calculate the concentrations of psychotropic drugs and metabolites in plasma by the external standard method according to the standard curve.

2. The method according to claim 1, wherein The 58 kinds of psychotropic drugs and metabolites include amisulpride, amitriptyline, aripiprazole, carbamazepine, chlorpromazine, citalopram, clomipramine, clozapine, diphenidol, donepezil, doxepin, duloxetine, fluoxetine, fluphenazine, fluvoxamine, haloperidol, 9-hydroxyrisperidone, imipramine, lamotrigine, loxapine, memantine, mirtazapine, nortriptyline, norfluoxetine, nortriptyline, oxcarbazepine, paroxetine, penfluridol, perphenazine, primidone, quetiapine, risperidone, sertraline, sulpiride, tandospirone, trazodone, trihexyphenidyl, ziprasidone, N-desalkyl quetiapine, O-desmethylvenlafaxine, dehydroaripiprazole, phenytoin sodium, alprazolam, clonazepam, diazepam, estazolam, lidocaine, lorazepam, midazolam, oxazepam, tramadol, zolpidem, nitrazepam, triazolam, N-demethylclozapine, N-desmethylclomipramine, phenobarbital, and bupropion.

3. The method according to claim 1, wherein The volume ratio of methanol to acetonitrile is (0.8 - 1.2):1; and / or, based on the total volume of the precipitant, the concentration of octanoic acid is 0.1 - 0.4 v / v%; and / or, the volume ratio of plasma to precipitant is 100:(300 - 500).

4. The method according to claim 1, characterized in that The solid-liquid separation is centrifugation, and the rotation speed of centrifugation is 10000 - 14000 r / min.

5. The method according to claim 1, wherein The preparation method of the mixed standard working solution is: dissolve the standards of 58 kinds of psychotropic drugs and metabolites in methanol, then gradually dilute with acetonitrile, and then mix with the negative blank plasma extract, and the negative blank plasma extract is obtained by mixing negative blank plasma and precipitant.

6. The method according to claim 5, wherein The concentration of the mixed standard working solution is 0.5 - 20000.0 μg / L.

7. The method according to claim 1, characterized in that The standard curve is obtained by performing linear regression analysis with the concentration of the standard in the mixed standard working solution as the X-axis and the peak area corresponding to each concentration as the Y-axis.

8. The method according to claim 1, wherein In the liquid chromatography-mass spectrometry method, the conditions of liquid chromatography are: Reverse-phase C18 high-performance liquid chromatography column; Column temperature: 40 - 45 °C; Injection volume: 3 - 7 μL; Mobile phase A is: ammonium acetate aqueous solution, Mobile phase B is: ammonium acetate solution of methanol; Flow rate: 0.4 - 0.8 μL / min; Gradient elution mode is adopted, and the elution gradient of the mobile phase is: 0 min, 95% A + 5% B; 2 min, 95% A + 5% B; 3.0 min, 70% A + 30% B; 10.0 min, 5% A + 95% B; 12.5 min, 5% A + 95% B; 13 min, 95% A + 15% B.

9. The method according to claim 1, wherein In the liquid chromatography-mass spectrometry method, the conditions of mass spectrometry are: Curtain gas: 28 - 32 psi; Electrospray voltage: 5300 - 5700 V, -4300 - -4700 V; Temperature: 450 - 550 °C; Nebulizing gas: 45 - 55 psi; Auxiliary gas: 45 - 55 psi; Data acquisition mode: Multiple reaction monitoring.

10. Use of a precipitating agent in enhancing the detection sensitivity of 58 psychotropic drugs and metabolites in plasma and / or enhancing the stability of 58 psychotropic drugs and metabolites in plasma, wherein the precipitating agent is selected from methanol, acetonitrile, and octanoic acid.

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