A kit and method for detecting phenothiazines antipsychotic drugs in plasma

Abstract

The present invention relates to a kit and method for detecting phenothiazine antipsychotic drugs in plasma, and belongs to the field of biological detection technology. The kit includes: a calibrator, a calibrator diluent, a high-level quality control product, a medium-level quality control product, a low-level quality control product, a protein precipitant (extract), a protective agent, and a mobile phase. The detection method includes: step one, treating the collected plasma sample with a protein precipitant containing a protective agent to obtain a supernatant; step two, after diluting the supernatant, obtaining a sample to be tested, and then using a high-performance liquid chromatography-tandem mass spectrometry method to detect the sample to be tested. By using a protein precipitant (extract) in combination with a protective agent, phenothiazine compounds are prevented from oxidizing and deteriorating, ensuring the stability of the test object during the extraction process and the on-machine detection process; and diluting the supernatant after protein precipitation, the matrix effect is small, and the results are accurate and reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a kit and a method for detecting phenothiazines antipsychotic drugs in plasma. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Schizophrenia is a complex, chronic mental illness with repeated and prolonged illness. Schizophrenia patients often need medication. At present, phenothiazine antipsychotics belong to the first generation of antipsychotics, mainly chlorpromazine (aliphatic), thioridazine (piperidine), perphenazine, fluphenazine, and perazine (piperazine). In actual medication, the individual's steady-state drug concentration is affected by individual metabolic enzyme differences, combined medication, pathological factors, life and eating habits, etc. The degree of drug metabolism in the body varies significantly, and the steady-state drug concentration of different individuals can differ by more than 20 times. Excessive drug concentration can easily cause poisoning or even death, and low drug concentration cannot achieve the therapeutic effect. Therefore, it is particularly important to monitor the free blood drug concentration during the patient's medication. The AGNP consensus guidelines (2017 edition) recommend that the monitoring level for chlorpromazine is level 2, and the monitoring level for thioridazine, perphenazine, fluphenazine, and perazine is level 1. Through drug concentration monitoring, it provides strong technical support for clinicians to determine the optimal dosage and time of medication for patients with arrhythmias, so that doctors can have a clear idea when using medication, greatly reducing the blindness of medication (including increasing or decreasing the dosage, changing medications, stopping medications, etc.), allowing the drugs to exert the best therapeutic effect in the patient's body, reducing adverse drug reactions, and saving treatment costs.

[0004] At present, the analytical techniques for determining drug concentrations in biological samples (blood drug concentrations, urine drug concentrations, other tissue fluid or homogenate drug concentrations) mainly include spectral analysis, immunological detection technology, liquid chromatography technology, liquid chromatography-mass spectrometry technology, etc. However, immunoassays have poor specificity and sensitivity, because drugs have similar chemical structures and similar antigenic surface regions, and can all undergo antigen-antibody reactions. It is difficult to completely distinguish them using immunoassays, resulting in deviations between the detected drug concentrations and the actual situation, making it impossible to better predict efficacy and evaluate adverse reactions.

[0005] Therefore, from the perspective of drug specificity and specificity, liquid chromatography and liquid chromatography-mass spectrometry are suitable. Liquid chromatography-tandem mass spectrometry determination technology: The sample uses an organic precipitant to precipitate protein, solid phase extraction or liquid-liquid microextraction to remove impurity interference and obtain the sample to be tested. The sample first enters the liquid phase system, and is separated by a chromatographic column and then introduced into the mass spectrometry system. After ionization in the ion source of the mass spectrometry system, it enters the triple quadrupole mass analyzer, separates according to the mass-to-charge ratio (m / z) of the ions, collects data in multiple reaction monitoring mode (MRM / SRM), enters the detector to form a mass spectrometry signal, and records the chromatogram and peak area of ​​each analyte and the corresponding internal standard in the test sample.

[0006] The current shortcomings of liquid chromatography-tandem mass spectrometry technology include: some methods only use external standard method for value determination, which reduces the cost, but is affected by the interference of endogenous impurities in the biological matrix, resulting in low accuracy; the extraction liquid in the determination method of psychotropic drugs is mostly only protein precipitants such as methanol, acetonitrile, and isopropanol. Phenothiazine compounds are easily oxidized and deteriorated due to the influence of phenothiazine parent nucleus, which will lead to reduced accuracy of the determination results. In addition, the detection process requires the use of multiple devices and the operation is complicated. Summary of the invention

[0007] In order to improve the deficiencies of the prior art, the purpose of the present invention is to provide a kit and method for detecting phenothiazines antipsychotic drugs in plasma, which improves the stability of the detection process and the accuracy of the detection results.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows.

[0009] In a first aspect, a kit for detecting phenothiazine antipsychotic drugs in plasma, comprising: a calibrator, a calibrator diluent, a high-level quality control product, a mid-level quality control product, a low-level quality control product, a protein precipitant (extracting solution), a protective agent, and a mobile phase;

[0010] The calibrator refers to a standard solution prepared by adding a set volume of solvent of phenothiazine antipsychotic drugs chlorpromazine, thioridazine, perphenazine, fluphenazine and perazine to a calibrator diluent and mixing;

[0011] The calibrator diluent refers to blank plasma;

[0012] The high-level quality control product is prepared by adding a set volume of a calibration diluent to a standard solution;

[0013] The mid-level quality control product is prepared by adding a set volume of a calibration diluent to a high-level quality control product;

[0014] The low-level quality control product is prepared by adding a set volume of a calibration diluent to the mid-level quality control product;

[0015] The protective agent refers to an antioxidant that can delay or inhibit the occurrence of oxidation reactions and has no interference with the detection process.

[0016] The function of quality control products is to be used for internal quality control in the laboratory, to evaluate the measurement precision, measurement accuracy, or the analytical deviation that may occur in the detection system due to changes in reagents and analytical instruments.

[0017] Optionally, the protective agent comprises ascorbic acid.

[0018] In a second aspect, a method for detecting phenothiazines in plasma using the above kit comprises the following steps:

[0019] Step 1: treating the collected plasma sample with a protein precipitant containing a protective agent to obtain a supernatant;

[0020] Step 2: After the supernatant is diluted, a sample to be tested is obtained, and then the sample to be tested is detected by high performance liquid chromatography tandem mass spectrometry.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention uses a protein precipitant (extract) in combination with a protective agent to prevent phenothiazine compounds from oxidizing and deteriorating, thereby ensuring the stability of the analyte during the extraction process and the on-machine detection process; and dilutes the supernatant after protein precipitation, thereby reducing the matrix effect and providing accurate and reliable results.

[0023] 2. The present invention adopts three levels of quality control products, namely, high, medium and low, and can establish an accurate standard working curve. The detection method is accurate and effective, and distortion of the detection results is avoided.

[0024] 3. The present invention collects two pairs of parent ions / daughter ions for each compound for mass spectrometry detection. In addition to the quantitative ion pair used for quantification, there is also a pair of qualitative ions to assist in qualitative analysis, which can effectively avoid the occurrence of false positive results in the test results.

[0025] 4. The present invention adopts the internal standard method for quantitative detection, which effectively avoids the reduction of the accuracy of the detection result caused by the interference of impurities.

[0026] 5. The present invention obtains that all reagents in the kit have freeze-thaw stability and accelerated destruction stability, and can be used in situations requiring storage and carrying. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 It is a pre-processing flow chart in a specific implementation manner of the present invention.

[0029] Figure 2 It is a total ion current chromatogram (TIC) in a specific embodiment of the present invention.

[0030] Figure 3 This is the extracted ion chromatogram (EIC) of chlorpromazine in a specific embodiment of the present invention.

[0031] Figure 4 FIG. 5 is an extracted ion chromatogram (EIC) of thioridazine in a specific embodiment of the present invention.

[0032] Figure 5 FIG. 5 is an extracted ion chromatogram (EIC) of perphenazine in a specific embodiment of the present invention.

[0033] Figure 6 Extracted ion chromatogram (EIC) of fluphenazine in a specific embodiment of the present invention.

[0034] Figure 7 FIG. 5 is an extracted ion chromatogram (EIC) of perazine in a specific embodiment of the present invention.

[0035] Figure 8 It is the standard working curve of chlorpromazine in the specific embodiment of the present invention.

[0036] Fig. 9 This is the standard working curve of thioridazine in a specific embodiment of the present invention.

[0037] Fig.10 This is the standard working curve of perphenazine in a specific embodiment of the present invention.

[0038] Fig.11 This is the standard working curve of fluphenazine in a specific embodiment of the present invention.

[0039] Fig.12 This is the standard working curve of perazine in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0041] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0042] In a specific embodiment, the method for detecting phenothiazine antipsychotic drugs in plasma is described from the aspects of linearity, carryover contamination, accuracy, precision, matrix effect, etc., and the process formula of the kit is described, and the performance of the kit is verified from the aspects of freeze-thaw stability, accelerated destruction stability, and protective agent stability.

[0043] The Chinese and English names, chemical formulas, and CAS numbers of the five phenothiazine antipsychotic drugs involved are shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] The instruments involved include: YS EXACT 9900MD liquid spectrometer-mass spectrometer (Shandong Yingsheng Biotechnology Co., Ltd.); electronic balance (RADWAG, Germany); 24-position vortex mixer (eppendorf, Germany); centrifuge (eppendorf, Germany); ultrapure water machine (millipore, USA).

[0048] The reagents and standards involved include:

[0049] Chlorpromazine (1 mg / mL), perphenazine (1 mg / mL), fluphenazine (1 mg / mL), perazine (1 mg / mL) standard solutions, thioridazine (10 mg) (all from Tianjin Alta Technology Co., Ltd.); methanol of chromatographic grade (Sigma-Aldrich); ammonium acetate (LC-MS grade, Fisher); Watsons distilled water (purified by Milli-Q ultrapure water device); chromatographic column (Shandong Yingsheng Biotechnology Co., Ltd.).

[0050] 1. Drug preparation

[0051] 1.1 Preparation of standard stock solution

[0052] Accurately weigh 10 mg ± 0.01 mg of thioridazine standard into a 5 mL brown volumetric flask, dissolve it in methanol and dilute to volume to obtain a 2 mg / mL standard stock solution. Other standards are provided in liquid form.

[0053] 1.2 Preparation of standard mixed stock solution

[0054] Transfer 160 μL, 200 μL, 8 μL, 8 μL, and 400 μL of the standard stock solutions of chlorpromazine, thioridazine, perphenazine, fluphenazine, and perazine into 10 mL brown volumetric flasks, and dilute to the mark with methanol to obtain a mixed stock solution.

[0055] 1.3 Preparation of high concentration calibrant solution and standard working solution (plasma matrix)

[0056] The high concentration calibrator is prepared by adding a certain volume of a solvent mixed standard solution of phenothiazine antipsychotic drugs chlorpromazine, thioridazine, perphenazine, fluphenazine and perazine to the calibrator diluent (blank plasma).

[0057] Specifically, 40 μL of the standard mixed stock solution (1.2) was transferred, 1 mL of the calibration diluent (plasma) was added, and the mixture was vortexed to obtain a high-concentration calibration solution S1.

[0058] The standard working solution has 6 concentration points from S1 to S6, which are prepared by stepwise dilution of the high-concentration calibrator with the calibrator diluent (blank plasma). The dilution ratio (volume ratio, the same below) and concentration points are shown in Table 2.

[0059] Table 2

[0060] Calibrator working solution S1 S2 S3 S4 S5 S6 Dilution ratio 1 1 / 2 1 / 4 1 / 8 1 / 16 1 / 64 Chlorpromazine (ng / mL) 640 320 160 80 40 10 Perphenazine (ng / mL) 32 16 8 4 2 0.5 Fluphenazine (ng / mL) 32 16 8 4 2 0.5 Thioridazine (ng / mL) 1600 800 400 200 100 25 Perazine (ng / mL) 1600 800 400 200 100 25

[0061] 1.4 Preparation of high / medium / low level quality control products (plasma matrix)

[0062] Quality control product III is prepared by adding a certain volume of calibration diluent (blank plasma) to the calibration solution (1.2), quality control product II is prepared by adding a certain volume of calibration diluent (blank plasma) to quality control product III, and quality control product I is prepared by adding a certain volume of calibration diluent (blank plasma) to quality control product II.

[0063] Specifically, the calibration solution (1.2) and the calibration diluent (blank plasma) are mixed in a ratio of 1:1 (volume ratio, the same below) to obtain a control product III solution (i.e., a high-level control product); the control product III solution and the calibration diluent (blank plasma) are mixed in a ratio of 1:1 to obtain a control product II solution (i.e., a medium-level control product); the control product II solution and the calibration diluent (blank plasma) are mixed in a ratio of 1:3 to obtain a control product I solution (i.e., a low-level control product). The concentrations of different control products are shown in Table 3.

[0064] Table 3

[0065]

[0066] 2. Detection Method

[0067] 2.1 Pre-treatment method

[0068] Accurately pipette 50 μL of standard solution (plasma matrix), quality control sample, or human plasma into a clean centrifuge tube; add 150 μL of methanol solution containing internal standard and protective agent (ascorbic acid), vortex and shake at 3000 rpm for 5 minutes, and centrifuge at 12000 rpm for 5 minutes at room temperature; transfer 100 μL of supernatant to a 1 mL 96-well plate, add 100 μL of ultrapure water to dilute, mix thoroughly, and then inject. For the specific pretreatment process, see Figure 1 .

[0069] 2.2 Test conditions

[0070] 2.2.1 Chromatographic conditions

[0071] Chromatographic column model: ACE EXCEL 3C18 (2.1*100mm, 3μm);

[0072] Mobile phase A: 5mmol / L ammonium acetate aqueous solution;

[0073] Mobile phase B: methanol;

[0074] Flow rate: 0.35 mL / min;

[0075] Column temperature: 45°C;

[0076] Injection volume: 5 μL; gradient elution program see Table 4.

[0077] Table 4

[0078] Time (min) Flow rate (mL / min) ％A ％B 0.0 0.35 60 40 1.0 0.35 2 98 3.0 0.35 2 98 3.1 0.35 60 40 4 0.35 60 40

[0079] 2.2.2 Mass spectrometry conditions

[0080] Ion source: electrospray ion source (ESI), positive ion mode; ion spray voltage: 3000 V;

[0081] Sheath gas: 5.08L / min;

[0082] Auxiliary gas: 6.97L / min;

[0083] Ion transfer tube temperature: 350°C;

[0084] Atomization temperature: 350℃;

[0085] Scan mode: The mass spectrometry parameters of each target compound in multiple reaction monitoring are shown in Table 5.

[0086] Table 5

[0087]

[0088] 3. Test results

[0089] 3.1 Optimization of precipitant type and dosage

[0090] In the specific implementation, the effects of the addition amount of three precipitants, methanol, acetonitrile and methanol mixed with acetonitrile (mixing ratio (v / v) of 1:1) on the response of the compound were investigated when the sample volume was 50 μL, and the volume ratios investigated were sample: precipitant = 1:2, sample: precipitant = 1:3 and sample: precipitant = 1:4, respectively. The peak areas of the five compounds under different precipitant types and precipitation ratios are shown in Table 6. In the table, ratio refers to the volume ratio, and the value corresponding to Area is the peak area of ​​the drug.

[0091] Table 6

[0092]

[0093] The results showed that when methanol and acetonitrile were used as precipitants, the responses of the five compounds did not change significantly under different extraction ratios; when methanol:acetonitrile = 1:1 (v / v) was used as a precipitant, the responses of the five compounds were lower than those of the other two precipitants. Considering the cost saving, reducing the toxicity of organic solvents and the dosage, methanol was finally selected as the precipitant, and the precipitation ratio was sample:precipitant = 1:3.

[0094] 3.2 Optimization of protective agent dosage

[0095] In the specific implementation, ascorbic acid, which has good antioxidant effect, is non-toxic and inexpensive, is selected as a protective agent. When different concentrations of the protective agent are added to the extract, the in-machine stabilities of five phenothiazine compounds at an automatic sampler temperature of 5°C for 0h, 4h, 8h, 12h and 24h are investigated; the S6 concentration point of the calibration curve is used as the sample to be tested.

[0096] Pretreatment was performed according to the pretreatment method in 2.1, and the detection values ​​of the compound at different time periods were compared with the detection values ​​at 0 h, and the deviation (%) was calculated. The results of the investigation are shown in Table 7.

[0097] Table 7

[0098]

[0099] The results showed that when ascorbic acid was not added to the extract, the response of the five compounds gradually decreased with the increase of injection time compared with 0h, and the deviation became larger and larger. The 24h deviation of perphenazine and perazine even exceeded 30%. When ascorbic acid was added to the extract, the stability of the compound was improved. When the concentration of ascorbic acid was 250mg / L, the deviation of the results of 4h, 8h, 12h, and 24h of the compound and 0h was controlled within ±5%, and the result was the best. Therefore, the amount of ascorbic acid added to the extract was selected to be 250mg / L.

[0100] 3.3 Total ion current chromatogram (TIC) and extracted ion chromatogram (EIC)

[0101] The MRM mode was used to monitor the ion pairs of five phenothiazine antipsychotic drugs. The quantitative ion pair was used for quantitative results, and a pair of qualitative ion pairs was used to assist in qualitative confirmation.

[0102] The total ion current chromatogram (TIC) obtained is as follows Figure 2 As shown in the figure, the extracted ion chromatogram (EIC) of chlorpromazine is as follows Figure 3 As shown in Figure 2, the extracted ion chromatogram (EIC) of thioridazine is as follows: Figure 4 As shown in Figure 2, the extracted ion chromatogram (EIC) of perphenazine is as follows Figure 5 As shown in Figure 2, the extracted ion chromatogram (EIC) of fluphenazine is as follows: Figure 6 The extracted ion chromatogram (EIC) of perazine is shown in Figure 7 shown.

[0103] The results showed that the peaks of the five compounds were sharp and symmetrical, and there was no mutual interference between the quantitative and qualitative ion pairs of different compounds.

[0104] 3.4 Internal standard method for quantification

[0105] The internal standard method was used for quantification. The six concentrations of the standard working solution (plasma matrix) S1-S6 prepared in 1.3 were used as the X-axis, and the peak area ratio of the standard working solution and its internal standard was used as the Y-axis to establish an internal standard standard curve. Thioridazine, perphenazine and fluphenazine could be linearly fitted within their respective concentration ranges, while chlorpromazine and perazine required quadratic fitting. The correlation coefficients of the five compounds were all above 0.99. The specific regression equations and correlation coefficients are shown in Table 8.

[0106] Table 8

[0107]

[0108] The standard working curve obtained is as follows Figure 8-Figure 12 As shown, Figure 8 is the standard working curve of chlorpromazine, Fig. 9 is the standard working curve of thioridazine, Fig.10This is the standard working curve of perphenazine. Fig.11 is the standard working curve of fluphenazine, Fig.12 This is the standard working curve of perazine.

[0109] 3.5 Carryover Contamination Verification

[0110] In the process of detecting target analytes in samples using high performance liquid chromatography tandem mass spectrometry, the problem of carryover contamination is often encountered, which affects the accuracy and reliability of the detection results. The present invention verifies the carryover contamination in two ways.

[0111] One verification method is: repeatedly inject the standard curve low concentration sample 5 times, then alternately inject the standard curve high concentration sample 1 time and the low concentration sample 2 times in a row, repeat 5 times, and compare the deviation of the high-low concentration value conversion sample mean (HL) and the low-low concentration value conversion sample mean (LL); the second verification method is to alternately inject the standard curve high concentration sample and the blank sample 5 times, and compare the average value of the analyte peak area of ​​the blank sample for 5 injections and the average value of the peak area of ​​the standard curve low concentration sample repeated 5 times in the first verification method.

[0112] The results of the first verification method showed that the deviation between the mean test results of the standard curve low-concentration sample injected 5 times and the mean test results of the standard curve low-concentration sample injected alternately with HL concentrations was less than 20%; and the difference between the mean of the HL concentration value converted sample and the mean of the LL concentration value converted sample was less than 3SD of the LL concentration value converted sample; the results of the second verification method showed that the mean peak area of ​​the analyte in the blank sample after the high-concentration sample was lower than 20% of the mean peak area of ​​the analyte in the standard curve low-concentration sample, see Table 9 for details.

[0113] Table 9

[0114]

[0115]

[0116] It was found that the problem of carryover contamination was not significant in this scheme.

[0117] 3.6 Accuracy Verification

[0118] The accuracy of the method was evaluated by the spiked recovery test: according to the preparation method of 1.4 high / medium / low level quality control product preparation (plasma matrix), three different concentrations of high / medium / low were prepared. Each concentration sample was processed in parallel for 5 times, which are indicated in the table below as "Parallel-1", "Parallel-2", "Parallel-3", "Parallel-4" and "Parallel-5". The test results are shown in Table 10.

[0119] Table 10

[0120]

[0121] The results showed that the average recoveries of the five phenothiazines were between 96.49% and 113.35%, meeting the requirement of 85% to 115%, and the CVs of five repeated experiments were between 0.92% and 4.12%.

[0122] 3.7 Precision Verification

[0123] According to 1.4 high / medium / low level quality control product preparation (plasma matrix), three different concentrations of high / medium / low were prepared for testing. Five samples of each concentration were processed in parallel within one day for three consecutive days. The results are shown in Table 11.

[0124] Table 11

[0125]

[0126]

[0127] Results show that the intra-batch precision of the five phenothiazines antipsychotics, expressed as coefficient of variation (CV), was 0.92%-5.31%, and the inter-batch precision (CV) was between 1.39%-5.19%.

[0128] 3.8 Matrix effect detection

[0129] The target analyte (the analyte concentration is the midpoint concentration of the calibration curve) was added to the calibrator dilution solution as the matrix sample, the biological matrix sample and a 1:1 mixture of the two. They were pretreated separately and then tested on the machine. The average values ​​of the ratios of the peak area of ​​the matrix sample with the target analyte to the peak area of ​​its internal standard and the ratios of the peak area of ​​the biological matrix sample to the peak area of ​​its internal standard were compared, and the deviations from the ratios of the peak area of ​​the 1:1 mixture to the peak area of ​​its internal standard were compared. The results are shown in Table 12.

[0130] Table 12

[0131]

[0132]

[0133] Result explanation: The deviation is between -5.24% and 13.50%.

[0134] It can be seen from the above test results that the detection method of this embodiment has good reproducibility, stability and accuracy for detecting 5 phenothiazine antipsychotic drugs.

[0135] Preferably, methanol is used as the precipitant, and the precipitation ratio is sample:precipitant=1:3; when the protective agent is ascorbic acid, the added amount is 250 mg / L.

[0136] 4. Kit Composition

[0137] The composition, formula, specifications and loading volume of the kit are shown in Table 13.

[0138] Table 13

[0139]

[0140] The function of quality control products is to be used for internal quality control in the laboratory, to evaluate the measurement precision, measurement accuracy, or the analytical deviations that may be caused by changes in reagents and analytical instruments in the detection system.

[0141] Internal standard refers to: a reference substance used for quantification and correction of analytical results; it has similar properties to the analyte or target analyte, and is generally labeled with a stable isotope (deuterated internal standard or 13C isotope internal standard is used as internal standard and analyzed together with the analyte during sample processing, extraction, separation and detection);

[0142] Its role in the test kit is to correct analytical errors caused by factors such as sample processing, instrument response changes, sample loss and chromatographic peak shape changes, and to improve the stability, accuracy and reliability of the analysis results.

[0143] 4.1 Preparation of lyophilized calibrators

[0144] Prepare the standard mixed stock solution according to the preparation method of 1.2 standard mixed stock solution, and then dispense it into 3 mL brown lyophilized bottles at 200 μL / bottle. Place the dispensed calibration solution in a freeze dryer to freeze into dry powder and store it at -18°C or below. Take it out when using and add 200 μL of water to reconstitute it.

[0145] 4.2 Preparation of freeze-dried quality control products

[0146] Prepare three types of quality control products according to the preparation method of 1.4 high / medium / low level quality control products (plasma matrix). Dispense them into 3 mL brown lyophilized bottles at 200 μL / bottle. Freeze the dispensed solutions into dry powder in a freeze dryer and store at -18°C or below. Take out the products when using and add 200 μL of water to reconstitute them.

[0147] 4.3 Preparation of lyophilized calibrator diluent

[0148] Take blank plasma and dispense it into 3 mL brown freeze-dried bottles at 800 μL / bottle, freeze it into dry powder in a freeze dryer, and store it at -18°C or below. Take it out when using and add 800 μL of water to reconstitute it.

[0149] 4.4 Preparation of freeze-dried protective agent

[0150] Accurately weigh 100 mg ± 0.01 mg of ascorbic acid standard into a 10 mL brown volumetric flask, dissolve it with ultrapure water and dilute to the mark to obtain a 10 g / L ascorbic acid protective agent solution.

[0151] Dispense the protective agent solution into 3 mL brown freeze-dried bottles at 200 μL / bottle, freeze the calibrant solution into dry powder in a freeze dryer, inspect the quality after freeze-drying, cap and label after passing the quality test, and store at -18°C or below. Take out and add 200 μL water to reconstitute when using.

[0152] 4.5 Preparation of mobile phase A

[0153] Accurately weigh 38.541 g of LC-MS grade ammonium acetate using an electronic balance, add ultrapure water to dissolve by ultrasonication and make up to 100 mL, mix thoroughly to obtain a 5 mol / L aqueous solution of ammonium acetate, and place in a 4°C refrigerator for later use.

[0154] Take a certain amount of ultrapure water with a measuring cylinder to prepare 2L, add 2mL of 5mol / L ammonium acetate aqueous solution, make up to 2L with ultrapure water, mix thoroughly with ultrasound, and package according to the 400mL filling volume in Table 13 after passing the quality inspection, label, and store at room temperature.

[0155] 4.6 Verification of moisture content and status of calibrators, calibrator diluents, and freeze-dried quality control products

[0156] Three lyophilized samples of calibration material, calibration material diluent and three-level quality control materials from the same batch were randomly selected and stored at room temperature away from light. The water content of the lyophilized samples was determined by volumetric titration using a Karl Fischer titrator. The appearance of the lyophilized samples and the state after reconstitution were also investigated. The specific results are shown in Table 14.

[0157] Table 14

[0158]

[0159] From the results, we can see that the moisture content of the calibrator, calibrator diluent and quality control freeze-dried products does not exceed 3%, and the freeze-dried products are all light yellow dry powder, and after reconstitution, they are light yellow clear liquid. Therefore, the moisture content and appearance of the calibrator, calibrator diluent and low, medium and high level quality control freeze-dried products in the kit are good.

[0160] 4.7 Freeze-thaw stability test of lyophilized products

[0161] The stability of two levels of quality control products was investigated after being placed below -70°C and frozen and thawed four times. Prepare 8 portions of freeze-dried powder of two levels of quality control products, cover them after reconstitution, and divide each 2 portions into a group of parallel, and divide them into 4 groups in total. The first group was taken out from the condition below -70°C on the 1st day, 3rd day, 5th day and 7th day, and then thawed and stored below -70°C (4 freeze-thaw times). The second group was taken out from the condition below -70°C on the 3rd day, 5th day and 7th day, and then thawed and stored below -70°C (3 freeze-thaw times). The third group was taken out from the condition below -70°C on the 5th day and 7th day, and then thawed and stored below -70°C (2 freeze-thaw times). The fourth group was taken out and thawed at -70°C on the 7th day (1 freeze-thaw). The quality control products with 1 freeze-thaw, 2 freeze-thaw, 3 freeze-thaw and 4 freeze-thaw were tested on the machine according to the detection method, and compared with the measurement results of the products opened for the first time, and the relative deviation was calculated. The results are shown in Table 15.

[0162] Table 15

[0163]

[0164]

[0165] The results show that after the freeze-dried product of the quality control product is reconstituted, it is stored below -70°C and frozen and thawed four times, and the stability of the compound can be guaranteed.

[0166] 4.8 On-machine stability verification

[0167] The lyophilized products of quality control products I, II and III in the kit were reconstituted by adding 200 μL of water according to 4.2, and then treated with the pretreatment method in 2.1. Under the condition of automatic sampler temperature of 5°C, the products were measured at 0h, 4h, 8h and 24h, respectively. Each time point was measured 3 times, and a calibration curve was used for each time point. The relative deviation from the 0h result was calculated. The results are shown in Table 16.

[0168] Table 16

[0169]

[0170] The results showed that after the freeze-dried products of quality control products I, II and III were reconstituted and subjected to sample pretreatment, the deviations of the on-machine test results at 4h, 8h and 24h were all controlled within 10% compared with those at 0h. Therefore, the on-machine detection stability within 24h met the requirements.

[0171] 4.9 Accelerated Destruction Stability Verification

[0172] The freeze-dried products of the calibration substance and the quality control substance II were stored at room temperature for 0d, 3d, 7d, and 14d respectively from day 0. After each time, they were quickly transferred from room temperature to a storage temperature below -70°C. After the 14th day, they were taken out and re-dissolved, and tested on the machine according to the detection method. Two parallels were used for each time point. The measurement results at each time point were compared with the measurement results at 0d, and the relative deviation was calculated to examine the stability. The results are shown in Table 17.

[0173] Table 17

[0174]

[0175] The results show that the five phenothiazine compounds are stable during the storage of calibrator freeze-dried products and quality control freeze-dried products at room temperature for 14 days, and there is no need to add protective agents when preparing calibrator freeze-dried products and quality control freeze-dried products.

[0176] The test was conducted using a third-party quality control product from a different manufacturer than the above-mentioned manufacturer, and the test results were compared with the target values ​​marked on the third-party quality control product. The test results were consistent with the above-mentioned test. This shows that the kit and the detection method of the present technical solution use drugs from a third-party source, and they are also accurate and reliable, thus illustrating the accuracy and reliability of the kit and the detection method of the present technical solution.

[0177] 4.10 Stability verification of freeze-dried products with protective agents

[0178] The protective agent ascorbic acid itself is easily oxidized. If the protective agent itself is not stored properly and degraded, it may affect the analyte during the pretreatment process and the machine process, thereby affecting the accuracy of the test results. Therefore, the stability of the protective agent in the kit was investigated: one part was stored in liquid form below -20°C, and was placed for 0d, 1d, 3d, 6d, 9d, 12d, and 14d from 0 day, and was taken out and placed below -70°C after the time was up; one part was freeze-dried and stored at room temperature, and was placed for 0d, 3d, 6d, 9d, 12d, and 14d from 0 day, and was taken out and placed below -70°C after the time was up, and finally taken out together, diluted to a machine concentration of 100 mg / L, and a fresh ascorbic acid standard curve was prepared, and tested together on the machine; 2 parallels were performed at each time point, and the measurement results at each time point were compared with the measurement results stored in liquid form below -20°C for 0d, and the relative deviation was calculated to investigate the stability. The results are shown in Table 19.

[0179] Table 19

[0180]

[0181]

[0182] According to the test results, the concentration deviation of the freeze-dried product of the protective agent is less than 5% when stored at room temperature for 14 days, and the concentration deviation of the liquid protective agent is also less than 5% when stored below -20°C for 14 days. This shows that the freeze-dried product of the protective agent is stable and can be stored in liquid form below -20°C for at least 14 days after reconstitution.

[0183] Therefore, it can be seen that the calibrator, calibrator diluent, high-level quality control, mid-level quality control, low-level quality control and protective agent in the kit can be stored in the form of lyophilized products and reconstituted into solutions when used.

[0184] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting phenothiazines antipsychotic drugs in plasma, The method is characterized in that it comprises the steps of: Step 1: treating the collected plasma sample with a protein precipitant containing a protective agent to obtain a supernatant; Step 2: After the supernatant is diluted, a sample to be tested is obtained, and then the sample to be tested is detected by high performance liquid chromatography tandem mass spectrometry; wherein, In the positive ion mode, two pairs of parent ions / daughter ions were collected for each compound for mass spectrometry detection; the amount of protective agent added was 250 mg / L; Wherein, the protective agent is ascorbic acid; the protein precipitant is methanol; the addition ratio of the precipitant is sample:precipitant=1:3; The HPLC detection conditions are: Chromatographic column model: ACE EXCEL 3C18, 2.1*100mm, 3μm; mobile phase A: 5mmol / L ammonium acetate aqueous solution; mobile phase B: methanol; flow rate: 0.1-0.5mL / min; column temperature: 25-50℃; injection volume: 1-20μL; gradient elution program: 0min: 40%B, 1min: 98%B, 3min: 98%B, 3.1min: 40%B, 4min: 40%B; The mass spectrometry detection conditions were: Ion source: electrospray ionization source ESI, positive ion mode; ion spray voltage: 3000V; Sheath gas: 5.08 L / min; Auxiliary gas: 6.97 L / min; Ion transfer tube temperature: 350 °C; Nebulizer temperature: 350 °C; Scan mode: Multiple reaction monitoring; The mass spectrometry parameters of each target are as follows: 。 2. A kit for detecting phenothiazine antipsychotic drugs in plasma, which is used for detection by the method for detecting phenothiazine antipsychotic drugs in plasma according to claim 1, characterized in that: include: Calibrators, calibrator diluents, high-level quality control products, mid-level quality control products, low-level quality control products, protein precipitants, protective agents, mobile phases; The calibrator refers to a standard solution prepared by adding a set volume of solvent of phenothiazine antipsychotic drugs chlorpromazine, thioridazine, perphenazine, fluphenazine and perazine to a calibrator diluent and mixing; The calibrator diluent refers to blank plasma; The high-level quality control product is prepared by adding a set volume of a calibration diluent to a standard solution; The mid-level quality control product is prepared by adding a set volume of a calibration diluent to a high-level quality control product; The low-level quality control product is prepared by adding a set volume of a calibration diluent to the mid-level quality control product; The protective agent refers to an antioxidant that can delay or inhibit the occurrence of oxidation reactions and has no interference with the detection process.

3. The kit for detecting phenothiazines antipsychotic drugs in plasma according to claim 2, characterized in that: The calibrator, calibrator diluent, high-level quality control product, mid-level quality control product, low-level quality control product and protective agent are stored in the form of freeze-dried products and are reconstituted into solutions when used.

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