A method for the efficient determination of 14 psychotropic drugs

Abstract

The present invention discloses a method for efficiently determining 14 kinds of psychotropic drugs, comprising the following steps: obtaining a sample to be tested; detecting the sample to be tested by high performance liquid chromatography tandem mass spectrometry, and performing quantitative analysis by an external standard method. The present invention simultaneously determines the concentrations of 14 common psychotropic drugs, which can not only avoid the problem of a single drug type, low throughput, affecting the efficiency of detection, and being unfavorable for carrying out more detection projects; but also avoids the phenomenon of detecting too many drug types, high costs, and waste of resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical testing, and in particular relates to a method for efficiently determining 14 kinds of psychotropic drugs. Background Art

[0002] Antipsychotic drugs, also known as strong tranquilizers or neuroleptics, are a group of medications used to treat schizophrenia and other psychotic disorders. While typical therapeutic doses do not affect a patient's intelligence or consciousness, they can effectively control symptoms such as psychomotor agitation, hallucinations, delusions, hostility, impaired thinking, and abnormal behavior. Patients often experience sleep disturbances and frequently take sedatives concurrently. Therefore, these two medications must be separated during testing to obtain stable antipsychotic blood concentrations and avoid interference from other concurrent medications.

[0003] Therapeutic drug monitoring (TDM) involves measuring the concentration of a specific drug in a patient's blood at specified intervals during clinical drug therapy. Currently, the main methods for clinical blood drug concentration monitoring in China include liquid chromatography (HPLC), radioimmunoassay (RIA), gas chromatography (GC), and high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Because patients often take multiple medications for underlying psychiatric conditions, different drug detection methods and equipment often differ. Conventional liquid chromatography lacks sensitivity for detecting certain drug concentrations in serum / plasma and typically only measures single drugs. Radioimmunoassay and gas chromatography often require sample derivatization, which is cumbersome and increases testing costs. High-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) offers high sensitivity, accuracy, specificity, and high throughput (capable of detecting multiple drugs within 3 minutes), making it suitable for analyzing clinical biological samples and the gold standard for detecting small organic molecules.

[0004] In recent years, liquid chromatography tandem mass spectrometry (LC-MS / MS) has been widely used in TDM. It has high specificity, short running time and can simultaneously measure and analyze multiple chemical structures. Among the existing methods, such as the "A method for detecting risperidone and 9-hydroxyrisperidone in plasma" disclosed in Chinese patent CN106918675A and the "Method for determining the concentration of clozapine in plasma by high performance liquid chromatography-mass spectrometry" disclosed in Chinese patent CN106168610B, the detection throughput is small and can only be used to simultaneously measure one or several drugs, rarely involving dozens of drugs, which affects the efficiency of the detection. For example, the "A method for determining 23 psychotropic drugs and metabolites in human serum" disclosed in Chinese patent CN113933423A uses the internal standard method for quantification, which greatly reduces the detection efficiency and greatly increases the detection cost. Therefore, it is necessary to develop a reasonable method based on clinical needs.

[0005] In general, the main defects in the clinical testing process of psychotropic drugs in the existing technology are:

[0006] First, the types of drugs tested are not clinically applicable: the types of drugs tested are too single or too many, and the throughput is low, which affects the efficiency of testing and is not conducive to carrying out more testing projects; too many types of drugs tested result in high costs and waste of resources, which are not easy to conduct large-scale clinical testing;

[0007] Second, the linear range is not suitable: the linear range of many drugs in the existing measurement methods is unreasonable and has not been verified with clinical samples, making it unsuitable for clinical testing;

[0008] Third, the cost is high. Currently, most LC-MS / MS methods use deuterated internal standards to correct for matrix effects. The high cost of such internal standards directly leads to higher costs for the entire detection process.

[0009] Fourth, the analysis time is long. In the existing measurement methods, the LC part of the analysis time accounts for the vast majority of the time, resulting in a long drug testing time, generally exceeding 5 minutes.

[0010] Fifth, low sensitivity: When testing multiple drugs simultaneously, there is a problem of relatively low sensitivity of multiple targets, which affects the accuracy of detection;

[0011] Sixth, some methods cannot detect drugs and active metabolites at the same time: Some drugs not only have pharmacological activity themselves, but their metabolites also have the same pharmacological effects as the original drugs. Therefore, including the active metabolites of drugs in the monitoring scope can better explain the correlation between patients' blood drug concentrations and treatment effects. Summary of the Invention

[0012] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a method for efficiently determining 14 kinds of psychotropic drugs.

[0013] In order to achieve the above object, the present invention adopts the following technical means:

[0014] A method for efficiently determining 14 psychotropic drugs comprises the following steps:

[0015] Obtaining a sample to be tested; detecting the sample to be tested by high performance liquid chromatography tandem mass spectrometry;

[0016] The 14 psychotropic drugs are: estazolam, amitriptyline, nortriptyline, memantine, quetiapine, norquetiapine, mianserin, vortioxetine, doxepin, nordoxepin, agomelatine, perampanel, lacosamide, and zonisamide; their English names and structural formulas are shown in the table below.

[0017]

[0018]

[0019]

[0020] Among them, the mass spectrometry conditions were as follows: ion source: electrospray ionization source (ESI), positive ion mode;

[0021] Spray capillary voltage (Capliary): 5500V;

[0022] Ion source temperature: 350°C;

[0023] Ion source nebulizer gas (Gas1): 40 psi;

[0024] Ion source heating auxiliary gas (Gas2): 50psi;

[0025] Curtain air (CUR): 20psi;

[0026] Collision gas (CAD): 4 psi;

[0027] Scan mode: Multiple reaction monitoring

[0028] The MRM parameters of the compounds are shown in the following table.

[0029]

[0030]

[0031] Wherein, the HPLC conditions are as follows:

[0032] Mobile phase A: 0.01-0.5% formic acid aqueous solution; specifically, the formic acid concentration can be any value selected from 0.01%, 0.04%, 0.12%, 0.18%, 0.24%, 0.30%, 0.36%, 0.42% and 0.5%;

[0033] Mobile phase B: 0.01-0.5% formic acid methanol solution; the formic acid concentration can be selected from 0.01%, 0.04%, 0.12%, 0.18%, 0.24%, 0.30%, 0.36%, 0.42% and 0.5%;

[0034] The chromatographic column is selected from one of C18, T3 and PFP columns; the column specifications are 2.1 mm × 100 mm, 2.6 μm or 2.1 mm × 150 mm, 3 μm;

[0035] Gradient elution was used, and the elution gradient was as follows: from 0 to 2.5 min, the volume fraction of mobile phase B was increased to 90-100%, from 2.5 to 2.51 min, the volume fraction of mobile phase B was reduced to 70-50%, and from 2.51 to 3 min, the volume fraction of mobile phase B was maintained unchanged;

[0036] The flow rate is 0.1-0.5 mL / min; the column temperature is 25-40°C, and the injection volume is 1-10 μL;

[0037] That is, the flow rate can be selected from any value among 0.1, 0.12, 0.25, 0.3, 0.4 and 0.5;

[0038] The column temperature can be selected from any value among 25, 28, 30, 32, 36 and 40;

[0039] For injection, select any value from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0040] Preferably, the method further comprises the step of obtaining a sample to be tested: first, accurately measuring 5-500 μL of a biological sample and placing it in a clean centrifuge tube; then adding 100-200 μL of methanol, vortexing and mixing for 2 minutes to precipitate the protein, and centrifuging at 4°C and 13,000 rpm for 5 minutes to obtain a supernatant; 100 μL of the supernatant is respectively aspirated and transferred to a 96-well plate to obtain a sample to be tested; wherein the biological sample is human serum or plasma.

[0041] Preferably, the method further comprises the step of preparing a standard solution: weighing the corresponding 14 psychotropic drug standards, preparing the standard stock solution with methanol, diluting the standard stock solution 100 times with methanol to obtain a standard stock solution W1, and diluting the standard stock solution W2-W9 stepwise to obtain standard stock solutions of another 8 concentration points; taking the standard stock solutions W1-W9, respectively, adding negative serum at a ratio of standard stock solution: negative serum = 1:50, and shaking and mixing to obtain standard solutions of 14 psychotropic drugs of different concentrations; wherein the reference concentration ranges of the standard solutions of the 14 psychotropic drugs are as follows: 50-2500 ng / ml of estazolam, 50-2500 ng / ml of estazolam, and 2500 ng / ml of negative serum. mL, amitriptyline 10-500 ng / mL, nortriptyline 10-500 ng / mL, memantine 10-500 ng / mL, quetiapine 100-500 ng / mL, norquetiapine 24-1200 ng / mL, mianserin 5-250 ng / mL, vortioxetine 5-250 ng / mL, doxepin 10-500 ng / mL, nordoxepin 10-500 ng / mL, agomelatine 2-800 ng / mL, perampanel 30-1500 ng / mL, lacosamide 500-25000 ng / mL, and zonisamide 2000-100000 ng / mL.

[0042] Preferably, the method further comprises a quality control sample preparation step: preparing quality control stock solutions of 14 psychotropic drugs using methanol respectively, taking 10 μL of each quality control stock solution and adding 500 μL of negative human serum respectively, mixing, to obtain quality control samples of 14 psychotropic drugs.

[0043] Preferably, the method further comprises the step of quantitative analysis: S1, separating and detecting the standard solution, and drawing a standard curve: using the concentration of the standard solution of 14 psychotropic drugs as the X-axis and the peak area corresponding to the concentration of the standard solution as the Y-axis, performing linear regression analysis to obtain the corresponding external standard curves of the 14 psychotropic drugs; S2, substituting the peak area of ​​the biological sample obtained by high performance liquid chromatography tandem mass spectrometry into the drawn standard curve, and calculating the concentrations of the 14 psychotropic drugs respectively.

[0044] The present invention also provides a kit for the method of efficiently determining 14 psychotropic drugs, the kit comprising 14 standard substances, mobile phase A liquid and mobile phase B liquid;

[0045] The 14 standards are as follows: estazolam, amitriptyline, nortriptyline, memantine, quetiapine, norquetiapine, mianserin, vortioxetine, doxepin, nordoxepin, agomelatine, perampanel, lacosamide, and zonisamide;

[0046] Mobile phase A is 0.01-0.5% formic acid in water; mobile phase B is 0.01-0.5% formic acid in methanol.

[0047] Beneficial effects of the present invention

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The present invention can not only detect psychotropic drugs, but also include the active metabolites of drugs in the monitoring range, which can better explain the correlation between the patient's blood drug concentration and the treatment effect.

[0050] (2) The present invention can simultaneously measure the concentrations of 14 common psychotropic drugs, which can not only avoid the problem of a single drug type, low throughput, affecting the efficiency of detection, and being unfavorable for carrying out more detection projects; but also avoid the phenomenon of detecting too many drug types, high costs, and waste of resources.

[0051] (3) The reference ranges of the present invention are reasonable, have been verified by clinical samples, and are suitable for clinical testing.

[0052] (4) The present invention avoids the use of expensive internal standards and adopts a simple external standard method to quantitatively detect the concentration of psychotropic drugs, which greatly reduces the detection cost; in addition, the standard curve can be reused, which improves the efficiency of detection.

[0053] (5) The present invention greatly optimizes the entire analysis time by optimizing the liquid phase conditions, shortening the analysis time to 3 minutes, and can quickly and efficiently obtain the concentrations of multiple psychotropic drugs, thereby improving detection efficiency.

[0054] (6) The present invention has a simple pre-processing for the test samples, and achieves the effect of high-efficiency and high-throughput simultaneous detection by optimizing the selection of instruments and test conditions, simplifying the operation steps and improving the detection efficiency; by continuously optimizing the pre-processing and instrument methods, each drug can achieve high sensitivity and accuracy to meet the needs of clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 shows MRM extracted ion chromatograms of 14 psychotropic drug standards according to an embodiment of the present invention;

[0056] Figure 2 shows the MRM extracted ion chromatogram of the test sample 1 according to one embodiment of the present invention;

[0057] Figure 3 shows the MRM extracted ion chromatogram of the test sample 2 according to one embodiment of the present invention;

[0058] Figure 4 shows the MRM extracted ion chromatogram of the test sample 3 according to one embodiment of the present invention;

[0059] Figure 5 shows the MRM extracted ion chromatogram of the test sample 4 according to one embodiment of the present invention;

[0060] Figure 6 The MRM extracted ion chromatogram of the sample 5 to be tested according to one embodiment of the present invention is shown.

[0061] Figure 7 A standard curve graph of vortioxetine is shown;

[0062] Figure 8 A standard curve graph of doxepin is shown;

[0063] Figure 9 A standard curve graph of nordoxepin is shown;

[0064] Figure 10 A standard curve graph of mianserin is shown;

[0065] Figure 11 A standard curve graph of norquetiapine is shown;

[0066] Figure 12 A standard curve graph for amitriptyline is shown;

[0067] Figure 13 A standard curve graph of nortriptyline is shown;

[0068] Figure 14 A standard curve graph of memantine is shown;

[0069] Figure 15 A standard curve graph of estazolam is shown;

[0070] Figure 16 A standard curve graph of perampanel is shown;

[0071] Figure 17 A standard curve graph of agomelatine is shown;

[0072] Figure 18 A standard curve graph of lacosamide is shown;

[0073] Figure 19 A standard curve graph of zonisamide is shown;

[0074] Figure 20 A standard curve graph showing quetiapine

[0075] Figure 21 The MRM extracted ion chromatogram of Comparative Example 1 is shown. DETAILED DESCRIPTION

[0076] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, especially with respect to definitions of synthetic techniques, product and processing designs, polymers, comonomers, initiators, or catalysts disclosed in these documents in the art. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0077] Numerical ranges in this application are approximate values, so unless otherwise stated, they may include numerical values ​​outside the scope. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if the description component, physical or other properties (such as molecular weight, melt index, etc.) is 100 to 1000, it means that all individual numerical values ​​are clearly enumerated, such as 100, 101, 102, etc., and all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For the scope comprising a numerical value less than 1 or comprising a fraction greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately considered as 0.0001, 0.001, 0.01 or 0.1. For the scope comprising a single digit less than 10 (such as 1 to 5), 1 unit is usually considered as 0.1. These are only specific examples of what is intended, and all possible combinations of numerical values ​​between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0078] When used with respect to chemical compounds, unless expressly stated otherwise, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). In addition, nouns using "a," "an," or "the" also include their plural forms unless expressly stated otherwise.

[0079] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination.

[0080] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments.

[0081] Example

[0082] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0083] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials they cite are hereby incorporated by reference.

[0084] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0085] The samples of the present invention were obtained from Hangzhou Duan Medical Testing Laboratory;

[0086] The instruments used in the present invention are as follows: a Waters Xevo TQD triple quadrupole mass spectrometer (USA, Waters), including an ultra-high performance liquid chromatography Accurity UPLC system; a KQ-500E ultrasonic cleaner; an H1650R desktop high-speed refrigerated centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., China); a G560E vortex mixer (Scientific Industries, USA); Watsons distilled water, purchased from Guangzhou Watsons Food and Beverage Co., Ltd. (China); a BT125D electronic balance (Sartorius AG, Germany); and a pipette (EPPENDORF, Germany).

[0087] The reagents and consumables used in the present invention are as follows: acetonitrile, methanol (Merck, USA); high-purity water (Wahaha); formic acid (SIGMA Aldrich, USA); chromatographic column (ACE, UK);

[0088] Standards: Estazolam Etzl liquid (1 mg / mL) Sigma; Amitriptyline AM solid Shanghai Zhenzhun; Nortriptyline N-AM solid Shanghai Zhenzhun; Memantine MJG solid Shanghai Zhenzhun; Quetiapine QTP solid McLean; Norquetiapine N-QTP solid McLean; Mianserin MSN solid McLean; Vortioxetine VTE solid McLean; Doxepin DXP solid McLean; Nordoxepin N-DXP liquid Sigma; Agomelatine AME solid McLean; Perampanel PM solid McLean; Lacosamide Lasamd solid McLean; Zonisamide Zosamd solid McLean.

[0089] Example 1

[0090] A method for efficiently determining 14 psychotropic drugs comprises the following steps:

[0091] (1) Preparation of standard solution (taking amitriptyline as an example):

[0092] (2) a. Preparation of standard stock solution: Weigh 11.54 mg of amitriptyline standard (purity 98%) and dilute to 10 mL with methanol to obtain a standard stock solution (1 mg / mL);

[0093] b. Preparation of standard stock solutions: 20 μL of amitriptyline mother solution was diluted to 2 mL with methanol to obtain amitriptyline standard stock solution W1 (10,000 ng / mL); W2 was prepared by mixing 700 μL of W1 with 300 μL of methanol; W3 was prepared by mixing 400 μL of W1 with 600 μL of methanol; W4 was prepared by mixing 200 μL of W1 with 800 μL of methanol; W5 was prepared by mixing 100 μL of W1 with 900 μL of methanol; W6 was prepared by mixing 160 μL of W1 with 1,840 μL of methanol; W7 was prepared by mixing 500 μL of W6 with 500 μL of methanol; W8 was prepared by mixing 250 μL of W6 with 750 μL of methanol; W9 was prepared by mixing 150 μL of W6 with 1,050 μL of methanol;

[0094] c. Select 10 μL of each concentration of amitriptyline standard stock solution, add 500 μL of negative serum, and vortex to mix to obtain amitriptyline standard solutions of the corresponding concentrations. Other standard solutions were prepared as above. The concentrations of the standard stock solutions and standard solutions are shown in Table 1.

[0095] Table 1. Concentrations of standard stock solutions and standard solutions

[0096]

[0097]

[0098] (3) Preparation of quality control products

[0099] Prepare a certain concentration of quality control product stock solution (methanol matrix), take 10 μL of quality control product stock solution (methanol matrix), add 500 μL of negative human serum, mix well, and obtain the quality control product.

[0100] (4) Sample processing

[0101] Take 20uL of biological sample and place it in a clean centrifuge tube; add 1000μL of methanol, vortex and shake for 4 minutes to precipitate the protein, and centrifuge at 13000rpm for 5 minutes at 4℃; aspirate 100μL of supernatant to a 96-well plate to obtain the sample to be tested;

[0102] Treatment of quality control products: the same as that of samples;

[0103] Treatment of standard solution: the same as that of sample.

[0104] (5) Instrument testing

[0105] A triple quadrupole mass spectrometer is used to analyze and detect the samples to be tested;

[0106] The chromatographic and mass spectrometric conditions, as described above, enable simultaneous detection of 14 psychotropic drugs through control of the chromatographic and mass spectrometric conditions. Furthermore, by optimizing the chromatographic and mass spectrometric conditions, sample detection sensitivity is enhanced, and simple pretreatment is required, thereby improving detection efficiency and quality.

[0107] In this embodiment, the chromatographic conditions and mass spectrometry conditions are specifically selected as follows, but it is worth noting that the other parameter selections mentioned in the above chromatographic and mass spectrometry conditions are also within the operable range:

[0108] The chromatographic conditions are as follows:

[0109] Column: ACE Excel-2 C18-PFP, 2.1 mm × 100 mm, 2.6 μm;

[0110] Mobile phase A: 0.2% formic acid in water;

[0111] Mobile phase B: 0.2% formic acid in methanol;

[0112] Flow rate: 0.3 mL / min, column temperature 40 °C, injection 5 μL;

[0113] The elution gradient is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%, from 0 to 2.5 min, the volume fraction of mobile phase B increases from 0 to 95%, from 2.5 to 2.51 min, the volume fraction of mobile phase B decreases from 95% to 65%, and from 2.51 to 3 min, the volume fraction of mobile phase B is maintained at 65%.

[0114] Mass spectrometry conditions are as follows:

[0115] Ion source: electrospray ionization (ESI), positive ion mode;

[0116] Spray capillary voltage (Capliary): 5500V;

[0117] Ion source temperature: 350°C; ion source atomizing gas (Gas1): 40 psi;

[0118] Ion source heating auxiliary gas (Gas2): 50psi;

[0119] Curtain gas (CUR): 20 psi; Collision gas (CAD): 4 psi;

[0120] Scan mode: multiple reaction monitoring. The MRM parameters of the compounds are shown in Table 2.

[0121] like Figure 1As shown in the figure, the standard solutions of 14 psychotropic drugs have basically no interference from other peaks within ±1 min of the target peak elution time, indicating that good detection can be achieved under this condition.

[0122] The chromatogram obtained by detecting the sample 1 is as follows: Figure 2 , it can be seen that the measured data are as follows: the concentration of estazolam is 102.11 ng / mL, the concentration of amitriptyline is 172.45 ng / mL, and the concentration of nortriptyline is 89.52 ng / mL, all of which are within the linear range.

[0123] The chromatogram obtained by detecting the sample 2 is as follows: Figure 3 As shown, the measured data are as follows: quetiapine concentration is 106.23 ng / mL, norquetiapine concentration is 85.41 ng / mL, and memantine concentration is 81.13 ng / mL, all within the linear range.

[0124] The chromatogram obtained by detecting the sample 3 is as follows: Figure 4 As shown, the measured data are as follows: vortioxetine concentration is 68.32 ng / mL, doxepin concentration is 210.21 ng / mL, nordoxepin concentration is 53.24 ng / mL, and mianserin concentration is 123.21 ng / mL, all within the linear range.

[0125] The chromatogram obtained by detecting the sample 4 is as follows: Figure 5 As shown, the measured data are as follows: the concentration of agomelatine is 265.3 ng / mL, and the concentration of perampanel is 310.36 ng / mL, both of which are within the linear range.

[0126] The chromatogram obtained by detecting the sample 5 is as follows: Figure 6 As shown, the measured data are as follows: the concentration of lacosamide is 5.16ug / mL, and the concentration of zonisamide is 56.34ug / mL, both of which are within the linear range.

[0127] (6) Data processing

[0128] The experiment used the MRM mode to monitor the ion pairs (Q1 / Q3) of 14 psychotropic drugs, and selected DP / CE / CXP of different ion pairs for optimization. The best result corresponding to the highest ion signal intensity can be seen from the optimized chromatogram. Other parameters can select appropriate values ​​according to the reference value range provided by the instrument to ensure a strong signal intensity.

[0129] The external standard quantitative method was used, with the concentration of the standard solution as the X-axis and the peak area of ​​the standard solution as the Y-axis, to establish an external standard standard curve. The linear fitting equations of 14 psychotropic drugs in their respective concentration ranges showed good linearity, with correlation coefficients all above 0.99. The curves are shown in Figure 2. Figures 7 to 20The specific parameters are shown in Table 2;

[0130] Table 2. Linear regression equations and linear correlation coefficients of 14 psychotropic drugs

[0131] Analytes Curve concentration (ng / mL) Linear equations Linear coefficient Estazolam 50-2500 y=134.973*x+60.2242 0.998 Amitriptyline 10-500 y=152.799*x+-61.5513 0.998 Nortriptyline 10-500 y=140.132*x+6.77616 0.999 Memantine 10-500 y=21082.3*x+70591.6 0.999 Quetiapine 10-500 y=45.3052*x+-182.238 0.999 norquetiapine 24-1200 y=252.306*x+-842.109 0.999 Mianserin 5-250 y=203.51*x+24.0578 0.999 Vortioxetine 5-250 y=365.411*x+54.599 0.999 Doxepin 10-500 y=220.888*x+-179.935 0.999 nordoxepin 10-500 y=162.758*x+-132.571 0.999 Agomelatine 2-800 y=766.057*x+295.563 0.999 Perampanel 30-1500 y=540.64*x+-1481.41 0.999 Lacosamide 500-25000 y=112247*x+9678.13 0.999 Zonisamide 2000-100000 y=31689*x+19636.2 0.999

[0132] Get the ion chromatogram corresponding to the quality control product:

[0133] Quality control samples have known concentrations and are used to verify the accuracy of the method's results. The concentration of the quality control sample is calibrated using the standard curve for the day. The deviation between the calibrated concentration and the known concentration is recorded. Once a sufficient number of samples have been accumulated, the mean and CV values ​​are calculated. A quality control chart is then created, and reference is made to determine if the method is under control.

[0134] (7) Accuracy verification

[0135] Accuracy experiment: Inter-group accuracy was determined by analyzing two samples at different concentrations on at least three different days (with six replicates at each concentration point). The acceptance criteria required that the average measured concentration at each level be within 15% of the theoretical value, indicating an accuracy range of 85%-115%. Quality controls included: Low-concentration quality control (LQC); Medium-concentration quality control (MQC); and High-concentration quality control (HQC). Results are shown in Table 3.

[0136] Table 3. Accuracy data of quality control samples tested over three days

[0137]

[0138]

[0139] (8) Precision verification

[0140] Precision measurement: Three different concentrations of QC were measured at a time. The number of samples for each concentration was required to be no less than 5. The acceptance standard required that the CV of the QC samples at each concentration level be ≤15%. The results are shown in Table 4.

[0141] Table 4 Precision verification results

[0142] Sample type Precision LQC (%) Precision MQC (%) Precision HQC (%) Etzl 3.2 2.8 3.0 AM 2.8 1.8 4.2 N-AM 3.6 4.1 5.2 MJG 1.2 2.9 4.2 QTP 3.1 2.4 3.2 N-QTP 3.1 4.6 5.1 MSN 3.2 4.8 3.9 VTE 3.9 4.1 3.8 DXP 3.1 2.6 3.9 N-DXP 2.9 3.2 3.4 AME 5.8 2.8 4.9 PM 4.2 5.1 3.5 Lasamd 1.4 2.6 3.8 Zosamd 3.1 4.6 2.6

[0143] Note: LQC is low concentration quality control; MQC is medium concentration quality control; HQC is high concentration quality control

[0144] Comparative Example 1

[0145] The difference between Comparative Example 1 and Example 1 is that the chromatographic conditions of Comparative Example 1 adopt the following elution gradient: from 0 to 3 minutes, the volume fraction of mobile phase A is maintained at 20%, and the volume fraction of mobile phase B is maintained at 80%.

[0146] Comparative Example 1 The results of testing 14 psychotropic drugs and their active metabolites are as follows Figure 21 As shown by Figure 21 It can be seen that when the elution gradient is changed, the sample cannot be completely separated.

[0147] The present invention uses the HPLC-MS method to simultaneously determine the presence and content of 14 psychotropic drugs and their active metabolites in human serum / plasma. And the drug types detected by this scheme are more suitable for clinical use, and there is neither too low a detection flux nor too many detection types that cause waste of resources. The linear range has been verified by clinical samples and is suitable for clinical testing, safe and reliable. And in view of the fact that psychotropic drugs are externally taken drugs and the content in serum / plasma is relatively large, this study uses the external standard method for quantitative measurement, that is, no expensive internal standard is required to reduce the detection cost. And this study optimizes the chromatographic and mass spectrometry conditions, so that 14 psychotropic drugs and their active metabolites can be detected simultaneously with high throughput and high sensitivity within 3 minutes, and the sample pre-treatment steps are simple. In view of the accuracy and repeatability of the results of this experiment, and by comparing the precision and accuracy data, it can be seen that the accuracy and repeatability of this method are very high. In summary, this method has high sensitivity, strong specificity, accuracy and simple pretreatment steps. It can simultaneously complete the separation and detection of 14 psychotropic drugs and their active metabolites within 3 minutes, and simply achieve the quantification of psychotropic drugs by using the external standard method. It has high repeatability and low cost. The precision and repeatability meet the basic requirements. It can be used for the quantitative analysis of psychotropic drugs in clinical practice, providing a reliable detection method for clinical medicine.

[0148] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for efficiently determining 14 psychotropic drugs, characterized in that: The following steps are involved: Obtaining a sample to be tested, and detecting the sample to be tested by high performance liquid chromatography tandem mass spectrometry; The 14 psychotropic drugs were: estazolam, amitriptyline, nortriptyline, memantine, quetiapine, norquetiapine, mianserin, vortioxetine, doxepin, nordoxepin, agomelatine, perampanel, lacosamide, and zonisamide; The chromatographic conditions for the test are as follows: Mobile phase A: 0.01-0.5% formic acid aqueous solution; Mobile phase B: 0.01-0.5% formic acid methanol solution; The chromatographic column was ACEExcel-2C18-PFP, with column specifications of 2.1 mm × 100 mm, 2.6 μm; Gradient elution was used, and the elution gradient was as follows: the volume fraction of mobile phase A liquid + the volume fraction of mobile phase B liquid = 100%, at 0-2.5min, the volume fraction of mobile phase B liquid increased from 0 to 95%, at 2.5-2.51min, the volume fraction of mobile phase B liquid decreased from 95% to 65%, at 2.51-3min, the volume fraction of mobile phase B liquid was maintained at 65%; The flow rate is 0.1-0.5mL / min; the column temperature is 25-40℃, and the injection volume is 1-10μL; Steps for obtaining the sample to be tested: First, accurately measure the biological sample and place it in a clean centrifuge tube; Then, methanol was added, vortexed and mixed, protein was precipitated, and centrifuged to obtain supernatant; the supernatant was transferred to the well plate to obtain the sample to be tested; The biological sample is a serum sample.

2. The method for efficiently determining 14 kinds of psychotropic drugs according to claim 1, characterized in that: The mass spectrometry conditions for the detection are as follows: ion source: electrospray ion source, positive ion mode; Spray capillary voltage: 5500V; Ion source temperature: 350°C; Ion source nebulizer gas: 40psi; Ion source heating auxiliary gas: 50psi; Air curtain gas: 20psi; Collision gas: 4psi; Scan mode: multiple reaction monitoring; The MRM parameters of the compounds are as follows: 。 3. The method for efficiently determining 14 kinds of psychotropic drugs according to claim 1, characterized in that: The method also includes the steps of preparing standard solutions: weighing standard substances corresponding to 14 psychotropic drugs respectively, preparing standard stock solutions with methanol, diluting the standard stock solutions with methanol to obtain standard stock solution W1, and diluting the standard stock solutions step by step to obtain standard stock solutions W2-W9 of another 8 concentration points; taking standard stock solutions W1-W9 respectively, adding negative serum at a ratio of standard stock solution: negative serum = 1:50, oscillating and mixing, and obtaining standard solutions of 14 psychotropic drugs of different concentrations respectively.

4. The method for efficiently determining 14 kinds of psychotropic drugs according to claim 3, characterized in that: The method also includes the steps of preparing quality control samples: preparing quality control stock solutions of 14 psychotropic drugs with methanol respectively, taking 10 μL of the quality control stock solutions and adding 500 μL of negative human serum respectively, mixing well, and obtaining quality control samples of 14 psychotropic drugs.

5. The method for efficiently determining 14 kinds of psychotropic drugs according to claim 1, characterized in that: The method also includes the steps of quantitative analysis: S1, separating and detecting the standard solution, and drawing a standard curve: taking the concentration of the standard solution of 14 psychotropic drugs as the X-axis and the peak area corresponding to the concentration of the standard solution as the Y-axis, a linear regression analysis is performed to obtain the corresponding external standard curves of the 14 psychotropic drugs; S2, substituting the peak area of ​​the biological sample obtained by high performance liquid chromatography tandem mass spectrometry into the drawn standard curve, and calculating the concentrations of the 14 psychotropic drugs respectively.

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