A method for simultaneously detecting the contents of 7 psychotropic drugs and their metabolites

Abstract

The present invention provides a method for simultaneously detecting the content of 7 kinds of psychotropic drugs and their metabolites. The method comprises the following steps: preparing at least three standard solutions containing 7 kinds of psychotropic drugs, 3 kinds of psychotropic drug metabolites and their internal standards of different concentrations, detecting by liquid chromatography-mass spectrometry, and establishing a standard curve equation; mixing the sample to be tested and the internal standard, extracting with an extractant, and obtaining a sample to be tested after drying and redissolving; detecting the sample to be tested by liquid chromatography-mass spectrometry, and calculating the content of each target in the sample to be tested according to the test results and the standard curve equation. The method provided by the present invention can simultaneously detect 7 kinds of psychotropic drugs and 3 kinds of psychotropic drug metabolites, and has the advantages of high sensitivity, strong specificity, good precision and accuracy, and fast detection speed.

Description

Technical Field

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

[0002] Olanzapine, risperidone, chlorpromazine, haloperidol, perphenazine, aripiprazole, and chlorprothixene are all commonly used psychotropic drugs in clinical practice, with narrow therapeutic windows, long usage times, and easy side effects. Therefore, monitoring the concentrations of the prototype and active metabolites of psychotropic drugs and adjusting the dosage in a timely manner according to clinical manifestations and blood drug concentration monitoring results can not only improve clinical efficacy, but also reduce treatment costs and reduce the occurrence of adverse reactions, which is of great significance for patient recovery. In the "Establishment of an HPLC-MS / MS Method for Simultaneous Detection of the Concentrations of 11 Psychiatric Drugs in Human Plasma", the determination of haloperidol and others uses haloperidol-d4 as the internal standard, but the crosstalk peak of the haloperidol-d4 mass spectrometry channel of the reduced haloperidol metabolite in the human body is not separated, which may lead to inaccurate quantitative results. CN109655568B discloses a method and kit for simultaneously determining 35 psychotropic drugs by high performance liquid spectrometry-mass spectrometry. It determines haloperidol but does not detect reduced haloperidol. There is evidence that an increased ratio of reduced haloperidol to haloperidol (i.e., >5) indicates a poor clinical response, so the indicator of this project is incomplete; the upper limit of the quantitative determination of perphenazine is 10 ng / mL, which cannot take into account the serum reference range (5-30 ng / mL) given by MAYO; in addition, this method uses negative serum to prepare the standard solution, and there are risks in its preparation process and long-term storage. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention aims to provide a method for simultaneously detecting the contents of 7 psychotropic drugs and their metabolites. The detection method can simultaneously detect 7 psychotropic drugs and 3 psychotropic drug metabolites, and has the advantages of high sensitivity, strong specificity, good precision and accuracy, and fast detection speed.

[0004] To achieve this object, the present invention adopts the following technical solutions:

[0005] The present invention provides a method for simultaneously detecting the contents of seven psychotropic drugs and their metabolites, the method comprising the following steps:

[0006] Establishment of the standard curve equation: prepare at least three standard solutions containing seven psychotropic drugs, three psychotropic drug metabolites and their internal standards at different concentrations; detect the standard solutions using liquid chromatography-mass spectrometry, and establish a standard curve equation of each target substance / internal standard substance chromatographic peak area ratio-target substance / internal standard substance concentration ratio based on the test results;

[0007] Pretreatment of the sample to be tested: Mix the sample to be tested and the internal standard, extract with an extractant, dry the extracted load phase, and re-dissolve the residue with a re-solution to obtain the injection sample to be tested;

[0008] Detection of the sample to be tested: Detect the sample to be tested by liquid chromatography-mass spectrometry, and calculate the contents of 7 psychotropic drugs and 3 psychotropic drug metabolites in the sample to be tested according to the detection results and the standard curve equation;

[0009] Among them, the seven psychotropic drugs are olanzapine, risperidone, haloperidol, aripiprazole, chlorpromazine, chlorprothixene and fluphenazine; the three psychotropic drug metabolites are 9-hydroxyrisperidone, reduced haloperidol and dehydroaripiprazole.

[0010] Since the molecular properties of the above-mentioned 7 psychotropic drugs and 3 psychotropic drug metabolites are different, the clinical monitoring concentration range of perphenazine is large, and the blood quantification limit of other psychotropic drugs is low, it is difficult to detect the above-mentioned 10 targets at the same time.

[0011] The present invention adopts a liquid-liquid extraction pretreatment method, which is matched with appropriate chromatography-mass spectrometry conditions, and can fully reduce the blood matrix effect, so that the standard solution does not need to be pretreated, and can fully separate 10 target substances to avoid mutual interference and reduce the quantitative limit, so that the obtained standard curve has good linearity, high sensitivity, strong specificity, good precision and accuracy, fast detection speed and high throughput.

[0012] In the present invention, the structural formulas of the 7 psychotropic drugs and 3 psychotropic drug metabolites are as follows:

[0013]

[0014] Among them, 1. Olanzapine, OLZ; 2. Risperidone, RIP; 3. 9-hydroxyrisperidone, 9-OH-RIP; 4. Haloperidol, HLP; 5. Reduced haloperidol, RH; 6. Aripiprazole, ARPZ; 7. Dehydroaripiprazole, DPZ; 8. Chlorpromazine, CLP; 9. Chlorprothixene, CPX; 10. Perphenazine, PPZ.

[0015] Unless otherwise specified, plasma or serum is not used in the preparation of the standard solution of the present invention, and the liquid chromatography-mass spectrometry conditions for detecting the standard solution and the injected sample to be detected are the same.

[0016] It should be noted that in the standard solutions of different concentrations of the present invention, the concentration of the same target substance is different, while the concentration of the same internal standard substance is preferably the same; in the same standard solution, the concentrations of the 10 target substances and their internal standards are independent of each other, and may be the same or different, and the present invention does not make special requirements.

[0017] In the present invention, the "target substance / internal standard substance chromatographic peak area ratio" refers to the ratio of the chromatographic peak area of ​​the psychotropic drug or its metabolite to the chromatographic peak area of ​​the corresponding internal standard substance; the "target substance / internal standard substance concentration" refers to the ratio of the concentration of the psychotropic drug or its metabolite to the concentration of the corresponding internal standard substance.

[0018] In the present invention, the extraction loading phase refers to the extractant loaded with 7 kinds of psychotropic drugs and 3 kinds of psychotropic drug metabolites and their internal standards after extraction.

[0019] In some embodiments of the present invention, the internal standards of the seven psychotropic drugs and the three psychotropic drug metabolites are isotope internal standards.

[0020] In some embodiments of the present invention, the internal standards of the seven psychotropic drugs and the three psychotropic drug metabolites are:

[0021] Olanzapine: olanzapine-d8, risperidone: risperidone-d4, haloperidol and reduced haloperidol: haloperidol-d4, aripiprazole: aripiprazole-d8, chlorpromazine: chlorpromazine-d6, chlorprothixene: chlorprothixene-d6, perphenazine: perphenazine-d4, 9-hydroxyrisperidone: 9-hydroxyrisperidone-d4, dehydroaripiprazole: dehydroaripiprazole-d8.

[0022] Using isotope internal standards for calibration and optimizing the mass spectrometry parameters of the internal standards can avoid differences in matrix effects caused by differences in human blood.

[0023] In some embodiments of the present invention, the solvent of the standard solution is a mixture of methanol and water, preferably an aqueous solution containing 20-80 vol% methanol.

[0024] In some embodiments of the present invention, the concentration ranges of the seven psychotropic drugs and three psychotropic drug metabolites in the standard solutions of different concentrations are:

[0025] Olanzapine: 0.36-360ng / mL, Risperidone: 0.3-300ng / mL, Haloperidol: 0.1-100ng / mL, Aripiprazole: 2.56-2560ng / mL, Chlorpromazine: 1-1000ng / mL, Chlorprothixene: 1-1000ng / mL, Perphenazine: 0.1-100ng / mL, 9-hydroxyrisperidone: 0.3-300ng / mL, Reduced Haloperidol: 0.5-500ng / mL, Dehydroaripiprazole: 2.56-2560ng / mL.

[0026] In some embodiments of the present invention, the concentrations of the internal standards of the seven psychotropic drugs and three psychotropic drug metabolites in the standard solution are:

[0027] Olanzapine-d8: 10-30ng / mL, Risperidone-d4: 1-8ng / mL, Haloperidol-d4: 6-20ng / mL, Aripiprazole-d8: 20-60ng / mL, Chlorpromazine-d6: 10-30ng / mL, Chlorprothixene-d6: 20-60ng / mL, Perphenazine-d4: 20-70ng / mL, 9-hydroxyrisperidone-d4: 1-8ng / mL, Dehydroaripiprazole-d8: 10-30ng / mL.

[0028] In the present invention, the standard solution can be prepared according to the following method:

[0029] Target working solution: take the standard samples of the 7 psychotropic drugs and 3 psychotropic drug metabolites, dissolve them in aqueous solutions containing 0-100 vol% methanol, and prepare their respective target stock solutions; take the target stock solutions of the 7 psychotropic drugs and 3 psychotropic drug metabolites, dilute them with aqueous solutions containing 0-100 vol% methanol to prepare their respective target intermediate solutions; take the target intermediate solutions of the 7 psychotropic drugs and 3 psychotropic drug metabolites, mix them, and dilute them with aqueous solutions containing 0-100 vol% methanol to obtain a series of target working solutions containing 7 psychotropic drugs and 3 psychotropic drug metabolites of different concentrations;

[0030] Internal standard working solution: take the standard samples of the internal standards corresponding to the 7 psychotropic drugs and the 3 psychotropic drug metabolites, dissolve them in an aqueous solution containing 50-100 vol% methanol, and prepare their own internal standard stock solutions; take the internal standard stock solutions of each internal standard, dilute them with an aqueous solution containing 50-100 vol% methanol to prepare their own internal standard intermediate solutions; take the internal standard intermediate solutions of each internal standard, mix them, and dilute them with an aqueous solution containing 50-100 vol% methanol to obtain internal standard working solutions containing the internal standards of the 7 psychotropic drugs and the 3 psychotropic drug metabolites;

[0031] Take the target working solution (eg, 10 μL) and the internal standard working solution (eg, 10 μL), add an aqueous solution containing 20-80 vol% methanol (eg, 80 μL), and mix well to obtain a series of standard solutions with different concentrations.

[0032] In some embodiments of the present invention, the sample pretreatment comprises the following steps:

[0033] The sample to be tested and the internal standard are mixed, and an extractant is added for extraction. The mixture is centrifuged, and the extraction load phase is taken and blown dry with nitrogen. The residue is re-dissolved with a re-solution, and the mixture is centrifuged. The supernatant is taken to obtain the injection sample to be tested.

[0034] In some embodiments of the present invention, the sample to be tested is plasma or serum.

[0035] In some embodiments of the present invention, in the pretreatment of the sample to be tested, the internal standard is added in the form of an internal standard working solution, and the internal standard working solution is a solution of internal standards containing the 7 psychotropic drugs and 3 psychotropic drug metabolites.

[0036] In some embodiments of the present invention, the volume ratio of the sample to be tested to the internal standard working solution is 1:0.5-3; for example, it can be 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8 or 1:3, etc.

[0037] In some embodiments of the present invention, the extractant is methyl tert-butyl ether.

[0038] In some embodiments of the present invention, the volume ratio of the extractant to the sample to be tested is 6-15:1, for example, it can be 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or 15:1, etc.

[0039] In some embodiments of the present invention, the complex solution is an aqueous solution containing 20-80 vol% (for example, 20 vol%, 25 vol%, 30 vol%, 35 vol%, 40 vol%, 45 vol%, 50 vol%, 55 vol%, 60 vol%, 65 vol%, 70 vol%, 75 vol% or 80 vol%, etc.) methanol.

[0040] In some embodiments of the present invention, the volume ratio of the reconstituted solution to the sample to be tested is 0.5-2 times the volume ratio of the extracted load phase to the extractant; for example, it can be 0.5 times, 0.8 times, 1 times, 1.2 times, 1.5 times, 1.8 times or 2 times, etc.

[0041] In some embodiments of the present invention, the conditions of the liquid chromatography include:

[0042] The chromatographic column is a C18 column;

[0043] and / or, the column temperature is 35-45° C. (e.g., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., etc.);

[0044] and / or, the mobile phase flow rate is 0.3-0.5 mL / min (e.g., 0.3 mL / min, 0.32 mL / min, 0.35 mL / min, 0.38 mL / min, 0.4 mL / min, 0.42 mL / min, 0.45 mL / min, 0.48 mL / min or 0.5 mL / min, etc.);

[0045] And / or, the mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.05-0.5 volt% formic acid, and the mobile phase B is a methanol solution containing 0.05-0.5 vol% formic acid and 1-10 mM ammonium formate; wherein the concentration of formic acid can be 0.05 vol%, 0.08 vol%, 0.1 vol%, 0.15 vol%, 0.2 vol%, 0.25 vol%, 0.3 vol%, 0.4 vol% or 0.5 vol%, etc.; the concentration of ammonium formate can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, etc.;

[0046] And / or, the elution method is gradient elution.

[0047] The mobile phase used in the present invention can reduce chromatographic peak tailing, strengthen the ionization of aripiprazole, dehydroaripiprazole and perphenazine at the ion source, improve the ion suppression of aripiprazole and dehydroaripiprazole in high-concentration samples due to ionization saturation, thereby increasing the upper limit of quantification, improving the response of perphenazine in low-concentration samples, thereby reducing the influence of baseline noise and lowering the lower limit of quantification.

[0048] In some embodiments of the present invention, the conditions of the gradient elution are:

[0049] 0-2.2min: 50% mobile phase A linearly decreased to 0%, 50% mobile phase B linearly increased to 100%, flow rate 0.3mL / min;

[0050] 2.2-3.2min: 100% mobile phase B, flow rate 0.4mL / min;

[0051] 3.2-4.7min: 50% mobile phase A, 50% mobile phase B, flow rate 0.4mL / min.

[0052] When the above-mentioned gradient elution conditions are used for liquid chromatography-mass spectrometry detection, it is preferred to adjust the flow rate back to 0.3 mL / min after 4.7 min to prepare for the next analysis.

[0053] It should be noted that in the gradient elution procedure of the present invention, the mobile phase composition changes around 3.2 min. The mobile phase composition at the 3.2 min time point can be 100% mobile phase B or 50% mobile phase A and 50% mobile phase B.

[0054] Haloperidol and reduced haloperidol contain chlorine atoms, and their natural isotope abundance is higher. Reduced haloperidol can produce crosstalk to the internal standard haloperidol-d4 channel, thereby affecting the quantification of the two. Since reduced haloperidol is the metabolite of haloperidol in the human body, it does not change with the change of the detection index. Therefore, when using haloperidol-d4 as an internal standard, whether or not to measure reduced haloperidol, its crosstalk to the haloperidol-d4 channel exists, but this problem has not been reported in the literature published in the past. The present invention separates haloperidol and reduced haloperidol completely by optimizing liquid phase conditions, and the retention time of haloperidol-d4 is consistent with that of haloperidol, so that the crosstalk peak of reduced haloperidol to haloperidol-d4 will not affect the chromatographic peak area of ​​haloperidol-d4, thereby ensuring the accurate quantification of the two substances.

[0055] In addition, using different flow rates in the above-mentioned gradient elution process, that is, using a high flow rate for elution when flushing the chromatographic column with a high proportion of organic phase and balancing the chromatographic column with the initial proportion of mobile phase, can achieve the purpose of flushing and balancing more quickly, thereby shortening the analysis time and improving detection efficiency.

[0056] In some embodiments of the present invention, the mass spectrometry conditions include:

[0057] The mass spectrometer detector used an electrospray ion source, positive ion mode, drying gas temperature 300-400°C, drying gas flow rate 8-15L / min, nebulizer pressure 20-40psi, capillary voltage 2500-3500V.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The present invention adopts a suitable pretreatment method in combination with chromatographic conditions, so that the provided detection method can simultaneously detect seven psychotropic drugs, namely olanzapine, risperidone, haloperidol, aripiprazole, chlorpromazine, chlorprothixene and perphenazine, and three psychotropic drug metabolites, namely 9-hydroxyrisperidone, reduced haloperidol and dehydroaripiprazole, and the method has the advantages of reasonable linear range, high sensitivity, strong specificity, good precision and accuracy, fast detection speed and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is the ion channel chromatogram of 10 target substances in the blank serum sample in the specificity experiment;

[0061] Figure 2 This is the ion channel chromatogram of 10 target substances in the quality control serum sample in the specific experiment;

[0062] Figure 3 This is the chromatogram of ion channels of 10 target substances in the standard solution in the specificity experiment;

[0063] Figure 4 ion channel chromatograms of haloperidol, reduced haloperidol and haloperidol-d4 in the standard solution prepared using the target working solution L7 in Example 1;

[0064] Figure 5 This is the ion channel chromatogram of olanzapine in Comparative Example 2. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. It should be understood by those skilled in the art that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0066] Example 1

[0067] A method for simultaneously detecting the contents of 7 psychotropic drugs and their metabolites, the specific steps are as follows:

[0068] 1. Preparation of standard solution

[0069] 1.1 Preparation of target working solution

[0070] Accurately weigh the standard samples of 7 psychotropic drugs and 3 psychotropic drug metabolites, and dissolve them in aqueous solution containing 70 vol% methanol to obtain 10 target stock solutions (with a concentration of not less than 500 μg / mL);

[0071] The 10 target stock solutions were diluted with an aqueous solution containing 70 vol% methanol to obtain 10 target intermediate solutions;

[0072] The above 10 target intermediate solutions were mixed respectively and diluted with an aqueous solution containing 70 vol% methanol to obtain 7 target working solutions containing 7 psychotropic drugs and 3 psychotropic drug metabolites at 7 different concentrations.

[0073] The concentrations of the seven target working solutions are shown in Table 1 below:

[0074] Table 1

[0075]

[0076] 1.2 Preparation of internal standard working solution

[0077] Accurately weigh the internal standard substances corresponding to 7 psychotropic drugs and 3 psychotropic drug metabolites, add methanol to dissolve them respectively, and obtain 9 internal standard stock solutions;

[0078] The above 9 internal standard stock solutions were diluted with an aqueous solution containing 70 vol% methanol to obtain 9 internal standard intermediate solutions with a concentration of 10 μg / mL;

[0079] The above 9 internal standard intermediate solutions were respectively mixed and diluted with an aqueous solution containing 70 vol% methanol to obtain an internal standard working solution.

[0080] The types of internal standards and the concentrations of internal standard working solutions are shown in Table 2 below:

[0081] Table 2

[0082]

[0083]

[0084] 1.3 Preparation of standard solution

[0085] Take 10 μL of the target working solution in a 1.5 mL centrifuge tube, then add 10 μL of the internal standard working solution, add 80 μL of the reconstitution solution (methanol: water = 1:1, v / v), vortex and mix at 2500 rpm for 1 min to obtain 7 standard solutions of different concentrations, and transfer them to the injection bottle for analysis.

[0086] 2. Pretreatment of samples to be tested

[0087] Use a pipette to transfer 100 μL of the serum sample to be tested into a 1.5 mL centrifuge tube, then add 10 μL of the internal standard working solution, add 1 mL of methyl tert-butyl ether, vortex and mix at 2500 rpm for 5 min, and then centrifuge at 12000 rpm for 5 min. Transfer 950 μL of the supernatant to a 1.5 mL centrifuge tube and blow dry under nitrogen. The residue is re-dissolved with 100 μL of re-solution (methanol: water = 1:1, v / v), vortex and mix at 2500 rpm for 1 min, and then centrifuge at 12000 rpm for 5 min. Transfer 90 μL of the supernatant obtained by high-speed centrifugation, which is the injection sample to be tested.

[0088] 3. High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS / MS) Detection

[0089] Take the above standard solution, use high performance liquid chromatography-mass spectrometry to detect, obtain a series of chromatograms of standard solutions with different concentrations, use the ratio of the chromatographic peak area of ​​the target substance to the chromatographic peak area of ​​the corresponding internal standard substance as the ordinate (y), and the ratio of the concentration of the target substance to the concentration of the corresponding internal standard substance as the abscissa (x), perform linear regression, and fit to obtain the standard curve equation of each target substance (the standard curve equation needs to be re-determined before each detection);

[0090] The above-mentioned samples to be tested were taken and detected by high performance liquid chromatography-mass spectrometry to obtain a chromatogram. The concentrations of the seven psychotropic drugs and three psychotropic drug metabolites in the serum samples to be tested were calculated based on the chromatographic peak area of ​​each target, the chromatographic peak area of ​​the corresponding internal standard and the corresponding standard curve equation.

[0091] The HPLC conditions are as follows:

[0092] Chromatographic column: Waters XBridge C18 column;

[0093] Column temperature: 40°C;

[0094] Mobile phase: It consists of mobile phase A and mobile phase B. Mobile phase A is an aqueous solution containing 0.1 vol% formic acid, and mobile phase B is a methanol solution containing 0.1 vol% formic acid and 10 mM ammonium formate.

[0095] Elution mode: gradient elution;

[0096] Injection volume: 5 μL;

[0097] The needle washing mode is to wash the needle before and after injection;

[0098] The gradient elution conditions are shown in Table 3 below:

[0099] Table 3

[0100]

[0101] This mobile phase gradient can make the compounds that cause mutual interference in mass spectrometry behavior be retained on the chromatographic column and completely separated at the same time. Among them, 0-2.2min, as the concentration of the organic phase gradually increases, the compounds can be separated one by one according to the different polarities of the compounds; 2.2-3.2min is a high-concentration organic phase, which can elute other analytes in the sample and impurities remaining in the chromatographic column, and the increase in flow rate can speed up the elution speed; 3.2-4.7min is the equilibrium time, which allows the mobile phase to quickly return to its initial state, that is, the proportion of mobile phase B is restored to 50%; 4.71min, the flow rate is adjusted back to 0.3mL / min to prepare for the next analysis.

[0102] The mass spectrometry conditions were as follows:

[0103] The mass spectrometer detector (Agilent LC1260 / MSG6410B) used an electrospray ion source (ESI) in positive ion mode; the drying gas temperature was 350°C, the drying gas flow rate was 10 L / min, the nebulizer pressure was 30 psi, the capillary voltage was 3000 V, and the ion pair parameter values ​​were shown in Table 4 below:

[0104] Table 4

[0105] Substance name Precursor ion Product ions Capillary outlet voltage (V) Impact voltage(V) ARPZ-d8 456 293.1 120 28 DPZ-d8 454 293.1 150 20 ARPZ 448.1 285 120 28 DPZ 446.1 285 150 20 9-OH-RIP-d4 431.1 211.1 160 30 9-OH-RIP 427.1 207.1 160 30 RIP-d4 415.1 195.1 160 30 RIP 411.1 191.1 160 30 PPZ-d4 408.1 171.1 100 24 PPZ 404.1 171 100 22 HLP-d4 380.2 127.1 175 40 RH 378.2 123.1 135 36 HLP 376.1 123.1 140 40 CLP-d6 325 92 135 20 CPX-d6 322.1 270.9 150 22 OLZ-d8 321.2 260.1 120 22 CLP 319 86 135 18 CPX 316.1 271 150 20 OLZ 313.2 256.1 120 22

[0106] The linear range and linear correlation coefficient of each target in this embodiment are shown in Table 5 below:

[0107] Table 5

[0108]

[0109]

[0110] Methodological validation

[0111] According to the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples (Part IV of the Chinese Pharmacopoeia 2020 Edition)" and "Recommendations for the Clinical Application of Liquid Chromatography-Mass Spectrometry (Guidelines and Consensus)" (Chinese Journal of Laboratory Medicine, 2017, 40(10): 770-779), the detection method of the present invention was methodologically validated, mainly including linearity, specificity, sensitivity, accuracy and precision.

[0112] Preparation of quality control serum samples:

[0113] Standard samples of 7 psychotropic drugs and 3 psychotropic drug metabolites were dissolved in a diluent (methanol: water = 1:1, v / v) to prepare quality control intermediate solutions of low, medium and high concentrations, each of which contained 10 target substances at the same time; the quality control intermediate solution was mixed with blank serum in a volume ratio of 10:90 to obtain quality control serum samples of low, medium and high concentrations.

[0114] The concentrations of each target in the quality control serum samples are shown in Table 6 below:

[0115] Table 6

[0116]

[0117]

[0118] 1. Linear

[0119] The results of the standard curve determination are shown in Table 5. It can be seen from Table 5 that the linear range of the standard curve can cover the clinical treatment reference range and warning value of drugs and metabolites in the official website and documents. The standard curves of each drug have good linear relationships within the concentration range (R 2 >0.9900), which can not only meet clinical needs and guide rational medication adjustments, but also avoid the confusion caused by excessive monitoring without a reference range.

[0120] 2. Specificity

[0121] Take 100 μL of blank serum without adding drugs and internal standards, and treat according to the conditions of "II. Pretreatment of test samples" in Example 1, and detect according to the conditions of "III. HPLC-MS / MS detection" in Example 1, record the chromatogram and mass spectrum, and extract the characteristic ion pair channels of each target in the chromatogram. The results are as follows: Figure 1 shown.

[0122] Take 100 μL of the medium concentration quality control serum sample shown in Table 6, treat it according to the item "II. Pretreatment of the test sample" in Example 1, and detect it according to the conditions of "III. HPLC-MS / MS detection" in Example 1, record the chromatogram and mass spectrum, and extract the characteristic ion pair channel of each target in the chromatogram. The results are as follows: Figure 2 shown. Figure 2 The retention times of the target substances are as follows: 9-hydroxyrisperidone (9-OH-RIP): 1.23min; aripiprazole (ARPZ): 2.78min; chlorpromazine (CLP): 3.08min; chlorprothixene (CPX): 3.11min; dehydroaripiprazole (DPZ): 2.72min; haloperidol (HLP): 2.37min; reduced haloperidol (RH): 2.04min; olanzapine (OLZ): 0.82min; perphenazine (PPZ): 3.20min; risperidone (RIP): 1.33min.

[0123] Take 100 μL of the standard solution prepared in Example 1 (prepared from the target working solution L3 in Table 1), and perform detection according to the conditions of "III. HPLC-MS / MS detection" in Example 1, record the chromatogram and mass spectrum, and extract the characteristic ion pair channels of each target in the chromatogram. The results are as follows: Figure 3 shown.

[0124] from Figure 1 It can be seen that no characteristic ion pair chromatographic peaks were detected in the blank serum without drug addition; Figure 2 It can be seen that obvious characteristic ion pair chromatographic peaks can be detected in the quality control serum samples with added drugs, and the peak shapes of each target are good without interference from other peaks. Figure 3 The target chromatographic peaks in each ion channel are respectively Figure 3 The retention time of the target chromatographic peaks in each ion channel in the standard solution is consistent, which shows that the endogenous substances in the serum sample do not interfere with the determination of each drug. The chromatographic peaks in the quality control serum sample and the chromatographic peaks in the standard solution have almost the same retention time and peak shape, proving that the detection method provided by the present invention has strong specificity for the detection of the above 10 targets in serum.

[0125] 3. Sensitivity

[0126] The low-concentration quality control serum samples shown in Table 6 were taken and diluted with blank serum at different times. The diluted drug-containing serum samples were treated according to the item "II. Pretreatment of the test sample" in Example 1, and tested according to the conditions of "III. HPLC-MS / MS detection" in Example 1 to obtain the chromatogram of the low-concentration drug-containing serum samples. The lowest concentration of the substance with a signal-to-noise ratio of not less than 3:1 was taken as the detection limit of each target substance, and the lowest drug concentration with a signal-to-noise ratio of not less than 10:1 was taken as the quantitative limit of each target substance. The test results are shown in Table 7 below:

[0127] Table 7

[0128]

[0129]

[0130] 4. Accuracy

[0131] The accuracy of the method was verified by recovery experiments. The standard samples of 7 psychotropic drugs and 3 psychotropic drug metabolites were dissolved in a diluent (methanol: water = 1:1, v / v) to prepare solutions of low, medium and high concentrations, each of which contained 10 targets at the same time; the solution was mixed with a blank serum sample at a volume ratio of 10:90, and 5 drug-added serum samples were prepared in parallel for each concentration, and processed according to the item "II. Pretreatment of the sample to be tested" in Example 1, and tested according to the conditions of "III. HPLC-MS / MS detection" in Example 1, and the concentration of each target in the drug-added serum sample was determined. The degree of dispersion of the concentration of the 5 parallel samples was expressed by the relative standard deviation (RSD). Since each target in the drug-added serum sample is an exogenous substance, the ratio of the measured value to the theoretical value is the spike recovery rate, which is used to evaluate the accuracy. The measured value is within ±15% of the theoretical value, indicating that the accuracy is qualified. The specific data are shown in Table 8 below:

[0132] Table 8

[0133]

[0134]

[0135] It can be seen from the experimental results in Table 8 that the accuracy of the test concentration of each target in the drug-added serum samples is in the range of 94.1-104.7%, all within the qualified range of 85-115%, proving that the method provided by the present invention has good accuracy in detecting the target in serum.

[0136] 5. Precision

[0137] Prepare the quality control serum samples of low, medium and high concentrations shown in Table 6 (prepare 6 copies of each concentration, prepare a batch of samples every day, for 5 consecutive days), each quality control serum sample is treated according to the item "II. Pretreatment of the sample to be tested" in Example 1, and tested according to the conditions of "III. HPLC-MS / MS detection" in Example 1, substitute the obtained data into the standard curve obtained on the same day to calculate the concentration, calculate the coefficient of variation CV (expressed as RSD) of the intra-batch and inter-batch measurement values, and the absolute value of the value is less than 15% to be qualified. The specific results are shown in Table 9 below:

[0138] Table 9

[0139]

[0140]

[0141] It can be seen from the experimental results in Table 9 that the intra-batch precision of the test concentration of each target in serum is 0.9-4.3%, and the inter-batch precision is 2.6-8.1%, both of which are less than 15%, proving that the method provided by the present invention has good detection precision for the target in serum.

[0142] 6. Inter-laboratory quality assessment comparison test

[0143] The samples of the British LGC international inter-laboratory comparison olanzapine and aripiprazole project were processed according to the item "II. Pretreatment of the test samples" in Example 1, and tested according to the conditions of "III. HPLC-MS / MS detection" in Example 1, and the obtained data were substituted into the standard curve to calculate the concentration. The test results are shown in Table 10 below:

[0144] Table 10

[0145]

[0146] The above comparison results show that the deviation between the numerical values ​​of aripiprazole and olanzapine measured by the method provided by the present invention and the target values ​​is small, which further proves that the method has high accuracy and can enable doctors to obtain more accurate test results, and has important clinical significance and application value.

[0147] 7. To further illustrate the importance of separation of haloperidol (HLP), haloperidol-d4 (HLP-d4), and reduced haloperidol (RH), the peak area data of HLP, HLP-d4, RH, and crosstalk peaks in the standard solution of this embodiment are given below, and the deviation that will be caused if the separation of haloperidol-d4 and crosstalk peaks cannot be achieved is calculated. The data are shown in Table 11 below:

[0148] Table 11

[0149]

[0150] Among them, AR is the chromatographic peak area ratio of haloperidol to haloperidol-d4, which is used to calculate the quantitative results.

[0151] The ion channel chromatograms of haloperidol, reduced haloperidol, and haloperidol-d4 in the standard solution prepared using the target working solution L7 are shown in the figure. Figure 4 As shown, the chromatographic peaks marked with # are crosstalk peaks caused by reduced haloperidol on the haloperidol-d4 ion channel.

[0152] From Table 11 and Figure 4 It can be seen that if the chromatographic peak of haloperidol-d4 is not separated from the crosstalk peak of its ion channel, when the concentration of reduced haloperidol in the blood of clinical patients is high and the concentration of haloperidol-d4 is low, it will affect the peak area of ​​haloperidol-d4 and lead to serious deviation in the fixed value.

[0153] Comparative Example 1

[0154] This comparative example provides a method for simultaneously detecting the contents of 7 psychotropic drugs and their metabolites, which is different from Example 1 in that:

[0155] The preparation method of the standard solution is as follows: take 10 μL of the target working solution into a 1.5 mL centrifuge tube, then add 10 μL of the internal standard working solution, add 390 μL of protein precipitant methanol, and vortex and mix at 2500 rpm for 1 min to obtain the standard solution.

[0156] The pretreatment method of the sample to be tested is as follows: take 100 μL of the serum sample to be tested into a 1.5 mL centrifuge tube, then add 10 μL of the internal standard working solution, add 300 μL of protein precipitant methanol, vortex and mix at a speed of 2500 rpm for 5 minutes, and then centrifuge at a speed of 14000 rpm for 10 minutes, and take the supernatant, which is the sample to be tested.

[0157] In this comparative example, 10 μL of the target working solution L1 was mixed with 90 μL of blank serum to prepare 100 μL spiked serum, which was treated according to the pretreatment method of the test sample described above, and then injected for testing. The injection was repeated three times, and the test results of perphenazine are shown in Table 12 below:

[0158] Table 12

[0159]

[0160] The AR (Area Ratio) value is the ratio of the chromatographic peak areas of perphenazine and perphenazine-d4.

[0161] It can be seen from Table 12 that when the protein precipitation method is used for sample pretreatment, the detection precision of perphenazine is poor in samples with low perphenazine concentration.

[0162] In addition, due to the early elution time of olanzapine and the high proportion of water in the mobile phase, the mass spectrometric response of olanzapine was further reduced when the protein precipitation method was used for sample pretreatment, resulting in poor linearity of the standard curve and R 2 <0.9900. In order to achieve accurate quantification of olanzapine in the range of 0.36-360 ng / mL, this application uses liquid-liquid extraction for pretreatment.

[0163] Comparative Example 2

[0164] This comparative example provides a method for simultaneously detecting the contents of 7 psychotropic drugs and their metabolites. The difference from Example 1 is that the liquid chromatography gradient elution procedure is as follows:

[0165]

[0166] In this comparative example, after instrumental analysis, the peak area of ​​dehydrogenated aripiprazole in the standard solution prepared with the target working solution L1 was 1114, and the peak area of ​​dehydrogenated aripiprazole in the standard solution prepared with the target working solution L7 was 673166. After the standard solution prepared with the target working solution L7 was tested, a blank sample was injected, and the residual peak area at the retention time of dehydrogenated aripiprazole was measured to be 515, that is, the residual exceeded 20% of the peak area of ​​dehydrogenated aripiprazole in the standard solution prepared with the target working solution L1, which did not meet the requirements of the methodological investigation. In actual testing, high-concentration samples will affect the fixed value results of the subsequent test samples. However, this application does not have this residual problem by optimizing the gradient elution conditions.

[0167] In addition, when eluted using the gradient elution program of this comparative example, the ion channel chromatogram of olanzapine is as follows: Figure 5 As shown, the chromatographic peaks are obviously tailed. However, after optimizing the gradient elution procedure, the present application effectively reduces the tailing degree of the olanzapine chromatographic peaks (e.g. Figure 2 and Figure 3 shown).

[0168] Comparative Example 3

[0169] This comparative example provides a method for simultaneously detecting the contents of 7 psychotropic drugs and their metabolites. The difference from comparative example 2 is that the mobile phase B in the liquid chromatography is a methanol solution containing 0.1 vol% formic acid.

[0170] The linear data of the standard curves obtained in Comparative Examples 2 and 3 are shown in Table 13 below:

[0171] Table 13

[0172]

[0173]

[0174] It can be seen from Table 13 that compared with Comparative Example 2, the linear correlation coefficient R of the standard curve equation of the target compound excluding hydrogen aripiprazole in Comparative Example 3 is 2 Among them, the R 2 The R of perphenazine is only 0.9925. 2 is 0.9870, R 2 The linearity is not up to standard, which shows the importance of adding an appropriate amount of ammonium formate to the mobile phase.

[0175] In summary, the method provided by the present invention has the advantages of reasonable linear range, high sensitivity, strong specificity, good precision and accuracy, fast detection speed and high throughput. In the inter-laboratory quality assessment comparison test, the results of the concentration determination of olanzapine, aripiprazole and dehydro-aripiprazole in blood samples further proved that the method of the present invention is suitable for the detection of drug concentration in clinical blood. When the current processing method or chromatographic conditions exceed the scope of the present invention, it is easy to cause the detection effect to deteriorate.

[0176] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously detecting the contents of 7 psychotropic drugs and their metabolites, characterized in that: The method comprises the following steps: Establishment of the standard curve equation: prepare at least three standard solutions of different concentrations containing seven psychotropic drugs, three psychotropic drug metabolites and their internal standards; the solvent of the standard solution is a mixture of methanol and water; use liquid chromatography-mass spectrometry to detect the standard solution, and establish a standard curve equation of each target / internal standard chromatographic peak area ratio-target / internal standard concentration ratio based on the detection results; Pretreatment of the sample to be tested: mixing the sample to be tested and the internal standard, wherein the sample to be tested is plasma or serum, extracting with an extractant, drying the extracted load phase, and re-dissolving the residue with a re-solution to obtain an injection sample to be tested; Detection of the sample to be tested: Detect the sample to be tested by liquid chromatography-mass spectrometry, and calculate the contents of 7 psychotropic drugs and 3 psychotropic drug metabolites in the sample to be tested according to the detection results and the standard curve equation; Among them, the seven psychotropic drugs are olanzapine, risperidone, haloperidol, aripiprazole, chlorpromazine, chlorprothixene and perphenazine; the metabolites of the three psychotropic drugs are 9-hydroxyrisperidone, reduced haloperidol and dehydroaripiprazole; In the pretreatment of the sample to be tested, the extractant is methyl tert-butyl ether, and the volume ratio of the extractant to the sample to be tested is 6-15:1; The liquid chromatography conditions include: the mobile phase is composed of mobile phase A and mobile phase B, the mobile phase A is an aqueous solution containing 0.05-0.5 vol% formic acid, and the mobile phase B is a methanol solution containing 0.05-0.5 vol% formic acid and 1-10 mM ammonium formate; the elution method is gradient elution; the chromatographic column is a Waters XBridge C18 chromatographic column; The conditions of the gradient elution are: 0-2.2 min: 50% mobile phase A linearly decreased to 0%, 50% mobile phase B linearly increased to 100%, flow rate 0.3 mL / min; 2.2-3.2 min: 100% mobile phase B, flow rate 0.4 mL / min; 3.2-4.7 min: 50% mobile phase A, 50% mobile phase B, flow rate 0.4 mL / min.

2. The method according to claim 1, characterized in that: The internal standards of the 7 psychotropic drugs and 3 psychotropic drug metabolites are isotope internal standards.

3. The method according to claim 2, characterized in that The internal standards of the 7 psychotropic drugs and 3 psychotropic drug metabolites are: Olanzapine: olanzapine-d8, risperidone: risperidone-d4, haloperidol and reduced haloperidol: haloperidol-d4, aripiprazole: aripiprazole-d8, chlorpromazine: chlorpromazine-d6, chlorprothixene: chlorprothixene-d6, perphenazine: perphenazine-d4, 9-hydroxyrisperidone: 9-hydroxyrisperidone-d4, dehydroaripiprazole: dehydroaripiprazole-d8.

4. The method according to claim 1, characterized in that: The solvent of the standard solution is an aqueous solution containing 20-80 vol% methanol.

5. The method according to claim 1, characterized in that The concentration ranges of the 7 psychotropic drugs and 3 psychotropic drug metabolites in the standard solutions of different concentrations are: Olanzapine: 0.36-360 ng / mL, Risperidone: 0.3-300 ng / mL, Haloperidol: 0.1-100 ng / mL, Aripiprazole: 2.56-2560 ng / mL, Chlorpromazine: 1-1000 ng / mL, Chlorprothixene: 1-1000 ng / mL, Perphenazine: 0.1-100 ng / mL, 9-hydroxyrisperidone: 0.3-300 ng / mL, Reduced Haloperidol: 0.5-500 ng / mL, Dehydroaripiprazole: 2.56-2560 ng / mL.

6. The method according to claim 1, characterized in that The concentrations of the internal standards of the seven psychotropic drugs and three psychotropic drug metabolites in the standard solution are: Olanzapine-d8: 10-30 ng / mL, risperidone-d4: 1-8 ng / mL, haloperidol-d4: 6-20 ng / mL, aripiprazole-d8: 20-60 ng / mL, chlorpromazine-d6: 10-30 ng / mL, chlorprothixene-d6: 20-60 ng / mL, perphenazine-d4: 20-70 ng / mL, 9-hydroxyrisperidone-d4: 1-8 ng / mL, dehydroaripiprazole-d8: 10-30 ng / mL.

7. The method according to claim 1 or 6, characterized in that: The concentration of the same internal standard in standard solutions of different concentrations is the same.

8. The method according to claim 1, characterized in that The sample pretreatment comprises the following steps: The sample to be tested and the internal standard are mixed, and an extractant is added for extraction. The mixture is centrifuged, and the extraction load phase is taken and blown dry with nitrogen. The residue is re-dissolved with a re-solution, and the mixture is centrifuged. The supernatant is taken to obtain the injection sample to be tested.

9. The method according to claim 1 or 8, characterized in that: In the pretreatment of the sample to be tested, the internal standard is added in the form of an internal standard working solution, and the internal standard working solution is a solution containing the internal standards of the 7 psychotropic drugs and 3 psychotropic drug metabolites.

10. The method according to claim 9, characterized in that The solvent of the internal standard working solution is a mixture of methanol and water.

11. The method according to claim 9, characterized in that The volume ratio of the sample to be tested to the internal standard working solution is 1:0.5-3.

12. The method according to claim 1, characterized in that The reconstitution solution is an aqueous solution containing 20-80 vol% methanol.

13. The method according to claim 1 or 12, characterized in that: The volume ratio of the reconstituted solution to the sample to be tested is 0.5-2 times the volume ratio of the extracted load phase to the extractant.

14. The method according to claim 1, characterized in that The conditions of the liquid chromatography also include: The column temperature was 35-45°C.

15. The method according to claim 1, characterized in that The mass spectrometry conditions include: The mass spectrometer detector used an electrospray ion source, positive ion mode, drying gas temperature 300-400°C, drying gas flow rate 8-15L / min, nebulizer pressure 20-40psi, capillary voltage 2500-3500V.

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