Method for rapidly detecting sedative-hypnotic in human plasma

Abstract

The invention relates to a method for rapidly detecting sedative-hypnotics in human plasma, which comprises the following steps: uniformly mixing a test sample with an internal standard precipitator, oscillating and vortex to obtain a supernatant of the test sample, and carrying out qualitative detection and quantitative detection on the sedative-hypnotics in the supernatant of the test sample by using an ultra-high performance liquid chromatography-mass spectrometry technology, the conditions of the ultra-high performance liquid chromatography comprise: gradient elution; the mobile phase A is an acetonitrile solution of formic acid, and the volume fraction of the formic acid is 0.01%-1%; the mobile phase B is an aqueous solution containing formic acid and ammonium formate, the volume fraction of the formic acid is 0.01%-1%, and the concentration of the ammonium formate is 5-20 mM. The invention develops a method for rapidly detecting sedative-hypnotic drugs in human plasma by using an ultra-high performance liquid chromatography-mass spectrometry technology, provides a qualitative and quantitative scheme for 10 benzodiazepine drugs and 3 novel sedative-hypnotic drugs in human plasma, and provides a method support for rapid and accurate diagnosis and treatment of sedative-hypnotic drug poisoning patients.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection in plasma, and in particular relates to a method for rapidly detecting sedative hypnotic drugs in human plasma. Background Art

[0002] There are many types of sedatives and hypnotic drugs. In addition to the classic benzodiazepines (diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam and estazolam), new sedatives and hypnotic drugs such as zaleplon, zopiclone and zolpidem are also widely used. In clinical practice, poisoning cases caused by overdose of sedatives and hypnotic drugs are very common. Such poisoning patients can develop from mild sedation and hypnosis to coma or even death.

[0003] Patients poisoned by sedatives and hypnotic drugs are usually unconscious or comatose after admission, and it is difficult for clinicians to determine the type of poisoning. Therefore, it is necessary to use rapid and accurate qualitative detection methods to confirm the diagnosis of such patients and then carry out symptomatic treatment. During the later blood purification treatment of such poisoned patients, it is necessary to quantitatively detect the concentration of such drugs in human plasma, and evaluate the treatment effect by comparing the drug concentration before and after blood purification.

[0004] The reported detection methods for sedatives and hypnotic drugs have many disadvantages, which make them unusable in clinical practice: (1) The types of drugs that can be tested are incomplete, and new hypnotic drugs such as zaleplon, zopiclone and zolpidem are missing.

[0005] (2) The detection method is not suitable. The polarity of different drugs in the benzodiazepine class is very different. Liquid chromatography-ultraviolet method cannot separate the baseline of this class of drugs from the chromatogram, and the structural similarity of different drugs makes it difficult to distinguish them on the ultraviolet spectrum. In addition, the ultraviolet detector is not sensitive enough to detect low-concentration samples below 1μg / mL. Although gas chromatography-mass spectrometry can effectively improve the problem of poor separation, its detection limit is concentrated in 0.1-10μg / mL, which still cannot solve the sensitivity problem.

[0006] (3) The sample processing methods that have been reported mostly use liquid-liquid extraction with organic solvents followed by drying and re-dissolution. This not only requires a large amount of samples, but also has complex pre-processing and a reporting time limit of more than 3 hours, which cannot meet the urgent needs of clinical poisoning detection.

[0007] (4) Different sedatives and hypnotic drugs have different therapeutic concentration ranges and poisoning warning concentrations. The reported detection linear range does not fully cover this range, resulting in an inability to accurately detect the drug during use.

[0008] (5) When testing sedatives and hypnotic drugs for poisoning patients, it is first necessary to qualitatively identify the type of drug and then conduct quantitative testing. Currently, there is no reliable qualitative determination method in clinical practice.

[0009] Therefore, there is an urgent need for a method with simple pretreatment, appropriate linear range and sensitivity, low cost, and the ability to quickly and accurately qualitatively and quantitatively detect 13 sedative hypnotic drugs in human plasma samples. Summary of the invention

[0010] The present invention provides a method for rapidly detecting sedative hypnotic drugs in human plasma to solve the problems in the background technology.

[0011] The technical scheme adopted by the present invention is: a method for rapidly detecting sedative hypnotic drugs in human plasma, which comprises the steps of: mixing a test sample with an internal standard precipitant, vortexing to obtain a supernatant of the test sample, and performing qualitative and quantitative detection of the sedative hypnotic drugs in the supernatant of the test sample by ultra-high performance liquid chromatography coupled to mass spectrometry; The conditions of ultra-high performance liquid chromatography include: gradient elution; mobile phase A is an acetonitrile solution of formic acid, the volume fraction of formic acid is 0.01%~1%; mobile phase B is an aqueous solution containing formic acid and ammonium formate, the volume fraction of formic acid is 0.01%~1%, and the concentration of ammonium formate is 5~20mM; During gradient elution: the proportion of mobile phase B is ≥90% at the beginning, and the proportion of mobile phase B drops to 5%~10% after 4 minutes. Among them, the decrease rate of the proportion of mobile phase B is slow at first and then fast.

[0012] Furthermore, the conditions of ultra-high performance liquid chromatography include: the chromatographic column filler is octadecylsilane bonded silica gel; the flow rate is 0.3~0.5mL / min, the column temperature is 35~40℃, and the injection volume is 2~10μL.

[0013] Furthermore, the conditions for gradient elution are: 0.0min, mobile phase A, 10%; mobile phase B, 90%; 3.0min: mobile phase A, 40%; mobile phase B, 60%; 4.0min: mobile phase A, 90%; mobile phase B, 10%; 5.0min: mobile phase A, 10%; mobile phase B, 90%; or, 0.0min, mobile phase A, 10%; mobile phase B, 90%; 2.5 min: mobile phase A, 35%; mobile phase B, 65%; 4.0min: mobile phase A, 95%; mobile phase B, 5%; 5.0min: mobile phase A, 10%; mobile phase B, 90%.

[0014] Furthermore, the particle size of the chromatographic column filler is 1.7 μm.

[0015] Furthermore, the model of the chromatographic column in the ultra-high performance liquid chromatography is Waters ACQUITY BEH C18, and the column length is 50 mm and the inner diameter is 2.1 mm.

[0016] Furthermore, the mass spectrometry conditions are: Positive ion electrospray ion source, multiple reaction monitoring MRM, capillary voltage 0.5~1.0 kV, desolvation gas temperature 400~500℃, desolvation gas flow rate 600~800 L / Hr, cone gas flow rate 40~60 L / Hr.

[0017] Furthermore, the 13 sedative hypnotic drugs that can be detected by the method include: diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam, estazolam, zopiclone, zolpidem, and zaleplon; when the 13 sedative hypnotic drugs are detected by the method, the internal standard precipitant contains an internal standard and a second solvent, the internal standard includes diazepam-D5 and / or zopiclone-D4, the second solvent contains acetonitrile and formic acid, and the volume fraction of the formic acid is 0.01%~1%.

[0018] Further, the step of performing qualitative detection on the supernatant of the test sample includes: Prepare quality control samples, which contain sedative hypnotic standards and blank plasma; The quality control sample and the internal standard precipitant were mixed and vortexed to obtain the quality control sample supernatant. The 13 sedative hypnotic drugs in the quality control sample supernatant were separated and qualitatively detected by ultra-high performance liquid chromatography-mass spectrometry. The detection conditions of the quality control sample supernatant and the test sample supernatant were the same. By comparing the test results of the supernatant of the quality control samples and the supernatant of the test samples, the types of sedative hypnotic drugs contained in the supernatant of the test samples were gradually determined based on the retention time, dual-channel MRM chromatogram, and product ion abundance ratio.

[0019] Furthermore, the step of quantitatively detecting the supernatant of the test sample includes calibrating the standard sample, and the operation is as follows: A standard sample is prepared, wherein the standard sample contains a sedative hypnotic standard and blank plasma; the standard sample is mixed with an internal standard precipitant, and vortexed to obtain a standard sample supernatant; 13 sedative hypnotic drugs in the standard sample supernatant are separated and quantitatively detected by ultra-high performance liquid chromatography-mass spectrometry, and the detection conditions of the standard sample supernatant and the test sample supernatant are the same; The mass concentration of each sedative hypnotic is used as the horizontal axis.X , the chromatographic peak area ratio of each sedative hypnotic to the internal standard is the ordinate - Y , weighted least squares method is used for linear regression, and the weighting factor is 1 / X 2 , and the standard curve equations of 13 sedative-hypnotic drugs were obtained respectively.

[0020] Furthermore, the quantitative lower limits of the method for detecting 13 sedative hypnotic drugs are: Diazepam: 62.5 ng / mL; Alprazolam: 3.125 ng / mL; Estazolam: 31.25 ng / mL; Lorazepam: 12.5 ng / mL; Triazolam: 3.125 ng / mL; Midazolam: 62.5 ng / mL; Nitrazepam: 6.25 ng / mL; Flunitrazepam: 6.25 ng / mL; Clonazepam: 3.125 ng / mL; Oxazepam: 31.25 ng / mL; Zolpidem: 12.5 ng / mL; Zopiclone: ​​12.5 ng / mL; Zalaplon: 3.125 ng / mL.

[0021] The advantages and positive effects of the present invention include at least: (1) A second solvent is provided that can stably and fully dissolve sedative hypnotic standards and isotope internal standards, and can precipitate proteins in plasma. The second solvent can be used to complete the two steps of internal standard addition and protein precipitation at one time, greatly reducing the sample pretreatment time and facilitating the rapid diagnosis of sedative hypnotic poisoning patients in clinical practice.

[0022] (2) A method for rapid detection of sedatives and hypnotics in human plasma using ultra-high performance liquid chromatography-mass spectrometry was provided. A qualitative and quantitative method for the detection of 10 benzodiazepines (diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam and estazolam) and three new sedatives and hypnotics (zopiclone, zolpidem and zaleplon) in human plasma was developed. Different detection concentration linear ranges were set for the therapeutic concentration ranges and poisoning warning concentrations of different sedatives and hypnotics, and methodological verification was carried out. The method is accurate in analysis, has a low limit of quantification, requires a small amount of sample (only 200 μL), is simple and time-consuming for sample pretreatment, and has a short detection time (the sample loading and analysis time is only 5 minutes, and the test report can be issued within 1 hour). It provides methodological support for the rapid and accurate diagnosis and treatment of patients poisoned by sedatives and hypnotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A chromatogram obtained by using the method provided in Example 1 of the present invention to detect the methodological validation sample 1; Figure 2 The chromatogram obtained by detecting a blank sample using the method provided in Example 1 of the present invention; Figure 3 The chromatogram obtained by detecting the standard sample using the method provided in Example 2 of the present invention; Figure 4 The chromatogram is obtained by detecting a blank sample using the method provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with embodiments and drawings: Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and comparative examples, and are not intended to limit the scope of protection of the present invention. It should be particularly noted that there may be multiple names for the same organic structure, as long as the structure is within the scope of this patent, it belongs to the protection object of this patent.

[0025] Unless otherwise defined, the raw materials, reagents, etc. in the following examples can be purchased from the market or prepared according to reported methods.

[0026]

Drugs and reagents

[0027] Flunitrazepam methanol solution (1.0 mg / mL, batch number: FE08051602), oxazepam methanol solution (1.0 mg / mL, batch number: FE10241801), estazolam methanol solution (1.0 mg / mL, batch number: FE05231705), and zolpidem methanol solution (1.0 mg / mL, batch number: FE02261904) were purchased from Cerilliant (USA).

[0028] Zopiclone (purity: 99.3%, batch number: 145712) was purchased from LGC (UK).

[0029] Zaleplon (purity: 99.92%, batch number: SM190622-14) was purchased from Stanford (USA).

[0030] Diazepam-D5 methanol solution (100 μg / mL, chemical purity: 99.9%, isotopic purity: 98.7%, batch number: S126697) was purchased from Tianjin Alta Technology (China) Co., Ltd.

[0031] Acetonitrile (mass spectrometry grade, batch number: 217207), methanol (mass spectrometry grade, batch number: 224617), and formic acid (mass spectrometry grade, batch number: 220614) were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0032] The water used in the experiment was prepared by an ultrapure water system.

[0033] Blank plasma can be obtained from a variety of channels, including waste plasma from blood banks and reagent companies.

[0034]

Equipment and consumables

[0035] Low-temperature high-speed centrifuge (model: SCIENTIFIC Legend Micro 21R), low-speed centrifuge (model: SL8), and vortex mixer (model: LP Vortex Mixer) were purchased from Thermo Fisher, USA.

[0036] The 1 / 100,000 balance (model: MS105) was purchased from Mettler, USA.

[0037] The ultrapure water system was purchased from Millipore Corporation, USA.

[0038] Pipettes (specifications: 10, 20, 100, 200, 1000 μL) were purchased from Eppendorf, Germany.

[0039] Volumetric flasks (specifications: 10 mL, 50 mL, 100 mL) were purchased from Beijing Xinweier Glass Instrument Co., Ltd.

[0040] Centrifuge tubes (specifications: 0.2 mL, 1.5 mL, 10 mL) were purchased from Aisheng Biotechnology (Hangzhou) Co., Ltd.

[0041] The embodiment of the present invention provides a method for rapidly detecting sedative hypnotic drugs in human plasma. The method can detect 10 benzodiazepine sedative hypnotic drugs and 3 new sedative hypnotic drugs. The 10 benzodiazepine drugs are diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam and estazolam, and the 3 new sedative hypnotic drugs are zopiclone, zolpidem and zaleplon.

[0042] The separation and detection technology used in the method is ultra-high performance liquid chromatography coupled to mass spectrometry, the qualitative method used is preferably retention time, dual-channel MRM chromatogram, and step-by-step determination of the product ion abundance ratio, and the quantitative method used can be an external standard method or an isotope internal standard-standard curve method. In order to briefly explain the technical solution of the present invention, the isotope internal standard-standard curve method is specifically introduced below.

[0043] The method provided in the embodiment of the present invention comprises the following steps: (1) Testing blank samples The blank sample is blank plasma. The blank sample is mixed with the second solvent in the internal standard precipitant, and vortexed to obtain the blank sample supernatant. The blank sample supernatant is detected using ultra-high performance liquid chromatography-mass spectrometry.

[0044] (2) Calibration standard samples The standard sample and the internal standard precipitant were mixed and vortexed to obtain the standard sample supernatant, and 13 sedative hypnotic drugs in the standard sample supernatant were separated and detected by ultra-high performance liquid chromatography-mass spectrometry. The mass concentration of each sedative hypnotic is taken as the horizontal axis ( X ), the chromatographic peak area ratio of each sedative hypnotic to the internal standard is the vertical axis ( Y ), weighted least squares method was used for linear regression, and the weighting factor was 1 / X 2 , and the standard curve equations of 13 sedative-hypnotic drugs were obtained respectively.

[0045] (3) Testing quality control samples The quality control sample and the internal standard precipitant were mixed and vortexed to obtain the quality control sample supernatant. The 13 sedative hypnotic drugs in the quality control sample supernatant were separated and detected by ultra performance liquid chromatography-mass spectrometry.

[0046] (4) Testing of test samples The test sample and the internal standard precipitant are mixed, vortexed to obtain the test sample supernatant, and the sedative hypnotic drugs in the test sample supernatant are separated and detected by ultra-high performance liquid chromatography-mass spectrometry.

[0047] In this method, the separation and detection conditions for the blank sample supernatant, standard sample supernatant, quality control sample supernatant and test sample supernatant are the same, which are: ①Ultra-high performance liquid chromatography conditions: Chromatographic column: Model: Waters ACQUITY BEH C18, column length: 50 mm, column inner diameter: 2.1 mm, filler: octadecylsilane bonded silica gel, filler particle size: 1.7~5 μm, preferably 1.7 μm, with higher column efficiency and faster analysis speed; Mobile phase A: formic acid in acetonitrile solution, the volume fraction of formic acid is 0.01%~1%, preferably 0.1%; Mobile phase B: an aqueous solution containing formic acid and ammonium formate, wherein the volume fraction of formic acid is 0.01% to 1%, preferably 0.1%, and the concentration of ammonium formate is 5 to 20 mM, preferably 5 mM; Flow rate: 0.3~0.5mL / min, preferably 0.4mL / min; Column temperature: 35~40℃, preferably 40℃; Injection volume: 2~10μL, preferably 2μL.

[0048] During gradient elution, due to the strong polarity of zopiclone and zolpidem, the initial mobile phase needs to be set to a higher proportion of 90% water phase to delay the elution time of zopiclone and zolpidem. In the first 3 minutes, the proportion of the water phase is slowly reduced to 60%~65%, which can effectively remove the residual biological matrix. Then the proportion of the water phase is quickly reduced to 5%~10% at the 4th minute, and the benzodiazepine sedative hypnotic can be eluted. There is no need to maintain the 5%~10% water phase. Adjust the mobile phase to the initial mobile phase within 1 minute to balance the chromatographic system, and then prepare to inject the next sample.

[0049] Gradient elution conditions: 0.0 min, mobile phase A, 10%; mobile phase B, 90%; 3.0 min: mobile phase A, 40%; mobile phase B, 60%; 4.0 min: mobile phase A, 90%; mobile phase B, 10%; 5.0 min: mobile phase A, 10%; mobile phase B, 90%; or, 0.0 min, mobile phase A, 10%; mobile phase B, 90%; 2.5 min: mobile phase A, 35%; mobile phase B, 65%; 4.0 min: mobile phase A, 95%; mobile phase B, 5%; 5.0 min: mobile phase A, 10%; mobile phase B, 90%.

[0050] If only benzodiazepine sedatives and hypnotics with lower polarity are detected, the gradient elution time can be adjusted to 3~3.5min.

[0051] ②Mass spectrometry conditions: Positive ion electrospray ion source; The capillary voltage is 0.5-1.0 kV, preferably 1.0 kV; The desolvation temperature is 400-500°C, preferably 450°C; The desolvation gas flow rate is 600-800 L / Hr, preferably 800 L / Hr; The cone hole gas flow rate is 40~60L / Hr, preferably 60L / Hr; The multiple reaction monitoring (MRM) mode was used, and the ion information, collision energy, and cone voltage are shown in Table 1.

[0052] Table 1 Ion information, collision energy and cone voltage

[0053] In this method, the qualitative detection method for the supernatant of the test sample is: compare the detection results of the supernatant of the quality control sample and the supernatant of the test sample, and gradually determine the type of sedative hypnotic contained in the supernatant of the test sample according to the retention time, dual-channel MRM chromatogram, and product ion abundance ratio. Specifically: if the supernatant of the quality control sample has chromatographic peaks in all the total ion flow chromatogram channels of sedatives and hypnotic drugs, and the supernatant of the blank sample has no peaks, the qualitative test of this batch of test samples can be carried out, otherwise it proves that this batch of experiments has failed; in the qualitative test, if the supernatant of the test sample does not have any chromatographic peaks, the test sample is judged as a negative result, that is, it does not contain sedatives and hypnotic drugs. If a chromatographic peak appears in the total ion flow channel of a certain sedative and hypnotic drug, the judgment is continued downward. If chromatographic peaks with the same retention time as the supernatant of the quality control sample appear in the two daughter ion pair channels of the sedative and hypnotic drug, the judgment is continued downward. If in the average mass spectrum of the chromatographic peak of the sedative and hypnotic drug, the relative error of the abundance ratio of the two qualitative daughter ions is less than the specified value, it can be finally judged that the sedative and hypnotic drug in the test sample is positive.

[0054] In this method, the quantitative detection method for the supernatant of the test sample is as follows: the chromatographic peak area ratio of the sedative hypnotic that tests positive in the supernatant of the test sample to the internal standard is substituted into the standard curve equation to calculate the concentration of the sedative hypnotic that tests positive in the test sample; the chromatographic peak area ratio of the sedative hypnotic that tests positive in the supernatant of the quality control sample to the internal standard is substituted into the standard curve equation to calculate the measured concentration of the sedative hypnotic in the quality control sample, and the degree of deviation from the theoretical concentration of the sedative hypnotic in the quality control sample is compared to determine the quality of this quantitative detection.

[0055] This method can be used to analyze and detect the above 13 sedative hypnotic drugs at the same time, and can also be used to analyze and detect one or a mixture of two or more of the above 13 sedative hypnotic drugs according to actual testing needs.

[0056] The internal standard used in this method is an isotope internal standard. According to the actual conditions and the difference of the above-mentioned test drugs, one or both of diazepam-D5 and zopiclone-D4 can be selected, preferably diazepam-D5. If the drug to be tested is only one or more of zopiclone, zolpidem, zaleplon and midazolam, the internal standard is preferably zopiclone-D4.

[0057] The preparation method of standard samples and quality control samples in this method includes: ① Prepare 13 stock solutions of standard substances: dissolve the standard substances of diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam, estazolam, zopiclone, zolpidem and zaleplon in the first solvent and make up to volume respectively; ②Preparing mixed stock solutions of standard substances: according to actual testing requirements, several of the above 13 stock solutions of standard substances are mixed in proportion, diluted with the first solvent and fixed to volume, to obtain mixed stock solutions of standard substances respectively; ③ Prepare standard working solution and quality control working solution: Use the multiple dilution method to dilute the standard mixed stock solution downward with the first solvent to obtain a standard working solution with at least 5 concentration points, and use the first solvent to dilute the standard mixed stock solution downward to obtain a quality control working solution with at least 2 concentration points.

[0058] The first solvent includes one or more of methanol, acetonitrile and water, preferably a 75% methanol aqueous solution, because it has good solubility, is liquid at -80°C, the solution is a homogeneous system, and is not easily volatile.

[0059] When preparing the standard working solution and the quality control working solution, a 2 to 4-fold dilution ratio can be used. The dilution ratio of the standard working solution is preferably 2 times, and the dilution ratio of the quality control working solution is preferably 3 times.

[0060] ④Prepare standard samples and quality control samples: Mix the standard working solution and blank plasma according to the ratio to obtain a standard sample; The quality control working solution and blank plasma were mixed in a certain ratio to obtain the quality control sample.

[0061] When preparing standard samples and quality control samples, the volume ratio of blank plasma to standard working solution and the volume ratio of blank plasma to quality control working solution are (5-10):1, preferably 9:1.

[0062] The preparation method of the internal standard precipitant in this method includes: ① Prepare internal standard stock solution: dissolve the internal standard substance in the first solvent and then make up the volume to obtain the internal standard stock solution.

[0063] ②Preparing an internal standard precipitant: dissolving the internal standard stock solution with a second solvent and then fixing the volume to obtain an internal standard precipitant; wherein the second solvent includes one or more of acetonitrile, methanol and formic acid, preferably a mixed solution of acetonitrile and formic acid, wherein the volume fraction of formic acid is 0.01% to 1%, preferably 0.1%. The component system of the second solvent can not only stably dissolve the internal standard substances selected in the embodiment of the present invention, namely diazepam-D5 and zopiclone-D4, but also precipitate proteins in plasma. After the internal standard precipitant prepared in this way is mixed with the sample, the two processes of adding the internal standard substance and precipitating the protein are completed at one time, which greatly reduces the pre-treatment time of the sample.

[0064] When preparing the standard sample supernatant and the quality control sample supernatant, the volume ratio of the standard sample to the internal standard precipitant and the volume ratio of the quality control sample to the internal standard precipitant are 1:(1.5-4), respectively, preferably 1:2.

[0065] When preparing the supernatant of the standard sample and the supernatant of the quality control sample, it is preferred to perform vortexing twice or more, and each time the supernatant obtained from the previous vortexing needs to be diluted with a diluent. The diluent is preferably ultrapure water, and the volume ratio of the supernatant to the diluent is 1: (0.5~2), preferably 1:1.

[0066] The quantitative lower limits of 13 sedatives and hypnotics detected by the above method are as follows: diazepam: 62.5 ng / mL; alprazolam: 3.125 ng / mL; estazolam: 31.25 ng / mL; lorazepam: 12.5 ng / mL; triazolam: 3.125 ng / mL; midazolam: 62.5 ng / mL; nitrazepam: 6.25 ng / mL; flunitrazepam: 6.25 ng / mL; clonazepam: 3.125 ng / mL; oxazepam: 31.25 ng / mL; zolpidem: 12.5 ng / mL; zopiclone: ​​12.5 ng / mL; zalapron: 3.125 ng / mL.

[0067] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can also be made, which all belong to the protection scope of the present invention.

[0068] Example 1: Rapid detection of the types and contents of sedative hypnotic drugs in human plasma

[0069] S1. Prepare solution ①Prepare 13 kinds of standard stock solutions and internal standard stock solutions Accurately weigh 10 mg each of 9 solid standards (diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, zopiclone, and zaleplon), dissolve them in 75% methanol aqueous solution (i.e., the first solvent), transfer them to 100 mL volumetric flasks, make up to volume with 75% methanol aqueous solution, and shake well to obtain 9 standard stock solutions with a concentration of 100,000 ng / mL.

[0070] Accurately measure 1 mL each of the four liquid standards (flunitrazepam methanol solution, oxazepam methanol solution, estazolam methanol solution, and zolpidem methanol solution), dissolve them in 75% methanol aqueous solution (i.e., the first solvent), transfer them to 10 mL volumetric flasks, make up to volume with 75% methanol aqueous solution, and shake well to obtain four standard stock solutions with a concentration of 100,000 ng / mL.

[0071] Accurately measure 1 mL of diazepam-D5 methanol solution (i.e., internal standard), place it in a 10 mL volumetric flask, make up to volume with 75% methanol aqueous solution (i.e., the first solvent), and shake well to obtain an internal standard stock solution of diazepam-D5 with a concentration of 10,000 ng / mL.

[0072] All the above stock solutions can be stored at -80℃.

[0073] ②Prepare standard mixed stock solution, standard working solution and quality control working solution Accurately measure 13 kinds of standard stock solutions, place them in the same volumetric flask, and make up to volume with 75% methanol aqueous solution (i.e., the first solvent) to obtain a mixed standard stock solution.

[0074] The mixed stock solution of the standard was diluted with 75% methanol aqueous solution (first solvent) by 2-fold serial dilution to obtain standard working solutions with 6 concentration points.

[0075] The standard mixed stock solution was diluted with 75% methanol aqueous solution by 3-fold stepwise dilution to obtain quality control working solutions at 3 concentration points.

[0076] Both the standard working solution and the quality control working solution can be stored at -20°C. Their concentrations are shown in Table 2.

[0077] Table 2 Concentrations of standard working solutions and quality control working solutions

[0078] ③Prepare standard samples and quality control samples Accurately add 180 μL of blank plasma and 20 μL of standard working solution into a 1.5 mL centrifuge tube to obtain a standard sample; Accurately add 180 μL of blank plasma and 20 μL of quality control working solution to a 1.5 mL centrifuge tube to obtain a quality control sample.

[0079] ④Prepare internal standard precipitant Accurately measure 100 μL of the internal standard stock solution, place it in a 15 mL volumetric flask, make up to volume with acetonitrile solution containing 0.1% formic acid (i.e., the second solvent), and shake well to obtain an internal standard precipitant with a diazepam-D5 concentration of 67 ng / mL.

[0080] S2. Sample processing and sample injection testing The conditions of ultra-high performance liquid chromatography and mass spectrometry are as follows: ①Ultra-high performance liquid chromatography conditions: Chromatographic column: Model: Waters ACQUITY BEH C18, column length: 50 mm, column inner diameter: 2.1 mm, filler: octadecylsilane bonded silica gel, filler particle size: 1.7~5 μm, preferably 1.7 μm, with higher column efficiency and faster analysis speed; Mobile phase A: formic acid in acetonitrile solution, the volume fraction of formic acid is 0.01%~1%, preferably 0.1%; Mobile phase B: an aqueous solution containing formic acid and ammonium formate, wherein the volume fraction of formic acid is 0.01% to 1%, preferably 0.1%, and the concentration of ammonium formate is 5 to 20 mM, preferably 5 mM; Flow rate: 0.3~0.5mL / min, preferably 0.4mL / min; Column temperature: 35~40℃, preferably 40℃; Injection volume: 2~10μL, preferably 2μL.

[0081] Gradient elution conditions: 0.0 min, mobile phase A, 10%; mobile phase B, 90%; 3.0 min: mobile phase A, 40%; mobile phase B, 60%; 4.0 min: mobile phase A, 90%; mobile phase B, 10%; 5.0 min: mobile phase A, 10%; mobile phase B, 90%.

[0082] If only benzodiazepine sedatives and hypnotics with lower polarity are detected, the gradient elution time can be adjusted to 3~3.5min.

[0083] ②Mass spectrometry conditions: Positive ion electrospray ion source; The capillary voltage is 0.5-1.0 kV, preferably 1.0 kV; The desolvation temperature is 400-500°C, preferably 450°C; The desolvation gas flow rate is 600-800 L / Hr, preferably 800 L / Hr; The cone hole gas flow rate is 40~60L / Hr, preferably 60L / Hr.

[0084] Perform the following operations on the blank sample: accurately add 200 μL of blank sample (i.e., blank plasma) and 400 μL of acetonitrile solution containing 0.1% formic acid (i.e., the second solvent) into a 1.5 mL centrifuge tube, vortex and shake, centrifuge at 14000 rpm / min for 5 min at 10°C, accurately measure 200 μL of supernatant, place it in a new 1.5 mL centrifuge tube, accurately add 200 μL of ultrapure water (i.e., diluent), vortex and shake again, centrifuge at 14000 rpm / min for 10 min at 10°C, accurately measure the supernatant, and inject it for detection.

[0085] Perform the following operations on the standard sample, quality control sample and test sample respectively: accurately add 200 μL of sample and 400 μL of internal standard precipitant to a 1.5 mL centrifuge tube, vortex and shake, centrifuge at 14000 rpm / min at 10°C for 5 min, accurately measure 200 μL of supernatant, place it in a new 1.5 mL centrifuge tube, accurately add 200 μL of ultrapure water (i.e., diluent), vortex again, centrifuge at 14000 rpm / min at 10°C for 10 min, accurately measure the supernatant, and inject for detection.

[0086] S3. Obtain the standard curve equation The mass concentration of each sedative hypnotic in the standard sample is taken as the horizontal axis ( X ), the chromatographic peak area ratio of each sedative hypnotic to the internal standard is the vertical axis ( Y ), weighted least squares method was used for linear regression, and the weighting factor was 1 / X 2 , and the standard curve equations of 13 sedative-hypnotic drugs were obtained respectively.

[0087] S4. Qualitative determination of the types of sedative hypnotic drugs contained in the test samples If the supernatant of the quality control sample has chromatographic peaks in the total ion flow chromatogram channels of all sedatives and hypnotic drugs, and the supernatant of the blank sample has no peaks, the qualitative test of this batch of test samples can be carried out. Other cases prove that this batch of experiments has failed. In the qualitative test, if no chromatographic peaks appear in the supernatant of the test sample, the test sample is judged as a negative result, that is, it does not contain sedatives and hypnotic drugs. If a chromatographic peak appears in the total ion flow channel of a certain sedative and hypnotic drug, the judgment is continued downward. If chromatographic peaks with the same retention time as the supernatant of the quality control sample appear in the two daughter ion pair channels of the sedative and hypnotic drug, the judgment is continued downward. If in the average mass spectrum of the chromatographic peak of the sedative and hypnotic drug, the relative error of the abundance ratio of the two qualitative daughter ions is less than the specified value, it can be finally judged that the sedative and hypnotic drug in the test sample is positive.

[0088] S5. Quantitative calculation of sedative hypnotic drugs contained in the test samples Substitute the chromatographic peak area ratio of the sedative-hypnotic drugs that test positive in the supernatant of the test sample to the internal standard into the standard curve equation to calculate the concentration of the sedative-hypnotic drugs that test positive in the test sample; substitute the chromatographic peak area ratio of the sedative-hypnotic drugs in the supernatant of the quality control sample to the internal standard into the standard curve equation to calculate the measured concentration of the sedative-hypnotic drugs in the quality control sample, compare the degree of deviation from the theoretical concentration of the sedative-hypnotic drugs in the quality control sample, and determine the quality of this quantitative test.

[0089] The chromatograms obtained by separating and detecting 13 sedative hypnotic drugs using the method provided in this embodiment can be referred to in the attached Figure 1 and attached Figure 2 Among them, Figure 1 The chromatogram obtained by using the method provided in this embodiment to detect the methodological verification sample 1 is attached. Figure 2 The chromatogram obtained by detecting a blank sample using the method provided in this embodiment is specifically shown in the detection and analysis of Example 4.

[0090] Example 2: Rapid detection of the types and contents of sedative hypnotic drugs in human plasma The difference from Example 1 is that the gradient elution conditions in the ultra-high performance liquid chromatography in Example 2 are: 0.0 min, mobile phase A, 10%; mobile phase B, 90%; 2.5 min: mobile phase A, 35%; mobile phase B, 65%; 4.0 min: mobile phase A, 95%; mobile phase B, 5%; 5.0 min: mobile phase A, 10%; mobile phase B, 90%.

[0091] The remaining operations and reagents are the same as those in Example 1. The chromatograms obtained by separating and detecting 13 sedative hypnotic drugs using the method provided in this example can be referred to in the attached Figure 3 and attached Figure 4 Among them, Figure 3 The chromatogram obtained by detecting the standard sample using the method provided in this embodiment is as follows: Figure 4 This is a chromatogram obtained by detecting a blank sample using the method provided in this embodiment.

[0092] By the attached Figure 3 It can be seen that the retention times of diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam, estazolam, zopiclone, zolpidem and zaleplon are 3.48min, 3.19min, 3.17min, 3.24min, 2.51min, 2.95min, 3.14min, 3.27min, 3.08min, 3.09min, 1.46min, 1.90min and 2.78min respectively, and the retention time of the internal standard (diazepam-D5) is 3.46min. The above 14 detection channels do not interfere with each other. Figure 4 It can be seen that the endogenous substances in the blank sample (i.e., blank plasma) do not interfere with the detection of the target substances (i.e., 13 sedative hypnotic drugs) and internal standards.

[0093] Examples 3-8 carried out methodological validation on the method of Example 1 in accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0094] Example 3: Linearity and Range

[0095] According to the detection method of the standard sample in "Sample Processing and Sample Injection Detection" in Example 1, the operation was continued and the measurement was performed for 3 days to obtain the standard curve equations and correlation coefficients of 13 sedative hypnotic drugs ( r ), linear range and lower limit of quantification (LLOQ), see Table 3.

[0096] The method for determining the quantitative lower limit is the concentration of the analyte in the sample when the signal-to-noise ratio is 10: 1. The quantitative lower limit test sample detected in this embodiment is obtained by diluting the standard working solution at the S1 concentration point by 2 times with blank plasma.

[0097] Table 3 Linearity and range

[0098] From Table 3, we can see that the correlation coefficient ( r ) were all greater than 0.99. When this method was used to detect 13 sedative hypnotic drugs in human plasma, a good linear relationship was found within their respective concentration detection ranges.

[0099] Example 4: Specificity

[0100] The standard substance was mixed with the stock solution, and the methodological verification sample 1 was accurately prepared according to the concentration value of the "lower limit of quantitation" in Example 2 and the preparation method of the "standard sample" in Example 1.

[0101] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the method validation sample 1 and the blank sample were detected respectively, and the chromatograms of the method validation sample 1 were obtained respectively (see Appendix Figure 1 ) and blank samples (see Appendix Figure 2 ), by the attached Figure 1 It can be seen that the retention times of diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam, estazolam, zopiclone, zolpidem and zaleplon are 3.72 min, 3.27 min, 3.23 min, 3.36 min, 2.51 min, 2.94 min, 3.19 min, 3.41 min, 3.10 min, 3.09 min, 1.46 min, 1.89 min and 2.79 min, respectively, and the retention time of the internal standard (diazepam-D5) is 3.70 min. The above 14 detection channels do not interfere with each other. Figure 2 It can be seen that the endogenous substances in the blank sample (i.e., blank plasma) do not interfere with the detection of the target substances (i.e., 13 sedative hypnotic drugs) and internal standards.

[0102] Example 5: Accuracy and Precision

[0103] The standard substance was mixed with the stock solution, and 5 portions of the method validation sample 2 were accurately prepared in parallel according to the concentration value of the "lower limit of quantitation" in Example 2 and the preparation method of the "standard sample" in Example 1.

[0104] The standard substance was mixed with the stock solution, and five quality control samples of three concentration points (L, M and H) were accurately prepared in parallel according to the preparation method of the "quality control sample" in Example 1, as the third method verification sample.

[0105] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the method validation sample 2 and the method validation sample 3 were detected respectively for 3 consecutive days, and the concentration was calculated every day using the newly determined standard curve equation.

[0106] Relative error (RE%) and relative standard deviation (RSD%) were used to evaluate accuracy and precision. The inter-day and intra-day RE% and RSD% of the 13 sedative-hypnotic drugs were all within 15% (the LLOQ limit was 20%). The results are shown in Table 4.

[0107] Table 4 Accuracy and precision

[0108]

[0109] Example 6: Residual Effect ① Use the standard mixed stock solution to accurately prepare 5 methodological verification samples 4 in parallel according to the concentration value of the "lower limit of quantitation" in Example 2 and the preparation method of the "standard sample" in Example 1.

[0110] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the methodological verification sample 4 was detected respectively, and the average chromatographic peak areas of the target sedative hypnotic and the internal standard were recorded as a1 and a2, respectively.

[0111] ② Use the standard mixed stock solution, and according to the preparation method of the "standard sample" in Example 1, accurately prepare 5 standard samples with a concentration point of S6 in parallel as the method verification sample 5.

[0112] According to the detection method of "Sample Processing and Injection Detection" in Example 1, the method validation sample 5 and the blank sample were detected respectively, and the injection sequence of "method validation sample 5-blank sample" was repeated 5 times, and the average chromatographic peak areas of the target sedative hypnotic and the internal standard in the blank sample were recorded as b1 and b2 respectively.

[0113] The calculated residual ratio of the target sedative-hypnotic drug is b1 / a1, which is less than 20%, and the residual ratio of the internal standard is b2 / a2, which is less than 5%.

[0114] Example 7: Extraction recovery and matrix effect

[0115] ① Use the standard mixed stock solution, and according to the preparation method of the "quality control sample" in Example 1, accurately prepare 6 copies of quality control samples at 3 concentration points (L, M and H) in parallel as the method verification sample 6.

[0116] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the methodological verification sample 6 was detected respectively, and the peak areas of the target sedative hypnotic and the internal standard were recorded as A1 and A2 respectively.

[0117] ② Six portions of blank plasma from different sources were treated according to the method of “Sample Processing and Injection Testing” in Example 1 to obtain blank sample supernatants, and corresponding volumes of quality control working solution and internal standard precipitant were added to the blank sample supernatants to obtain methodology verification sample seven. The injection concentration of methodology verification sample seven was the same as the theoretical concentration of sedative hypnotic drugs and the theoretical concentration of the internal standard in methodology verification sample six, respectively. Methodology verification sample seven was injected and tested, and the peak areas of the target sedative hypnotic drugs and the internal standard were recorded as B1 and B2, respectively.

[0118] ③ Treat ultrapure water according to the method for treating blank plasma in "Sample Processing and Injection Testing" in Example 1 to obtain an ultrapure water supernatant, add corresponding volumes of quality control working solution and internal standard precipitant to the ultrapure water supernatant to obtain methodology verification sample eight, the injection concentration of methodology verification sample eight is the same as the theoretical concentration of sedative hypnotic and the theoretical concentration of internal standard in methodology verification sample six, respectively, inject and test methodology verification sample eight, and record the peak areas of target sedative hypnotic and internal standard as C1 and C2, respectively.

[0119] According to the calculation of extraction recovery rate (%) = A / B × 100%, matrix effect (%) = B / C × 100%, and internal standard normalized matrix factor = target sedative-hypnotic drug matrix effect / internal standard matrix effect, the extraction recovery rate was between 98.32% and 127.03%, and the internal standard normalized matrix factor was between 0.86 and 1.60. The results are shown in Table 5.

[0120] Table 5 Extraction recovery and matrix effect (n=6)

[0121]

[0122] Example 8: Stability The stock solution was mixed with the standard, and quality control samples of three concentration points (L, M and H) were accurately prepared according to the preparation method of the "quality control sample" in Example 1, as method validation sample nine, and the stability of method validation sample nine under the following five conditions was investigated: after treatment, it was placed in an automatic sampler (15°C) for 12 hours, placed at room temperature (22°C) for 24 hours, repeatedly frozen and thawed in a -20°C refrigerator for 3 times (once a day), refrigerated in a 4°C refrigerator for 48 hours, and frozen in a -20°C refrigerator for 30 days. Six samples were investigated in parallel at each concentration point under each condition. The stability was good under various conditions. The results are shown in Table 6.

[0123] Table 6 Stability under five conditions

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for rapid detection of sedative hypnotic drugs in human plasma, characterized in that: The method comprises the following steps: mixing the test sample and the internal standard precipitant, vortexing to obtain the supernatant of the test sample, and performing qualitative and quantitative detection of the sedative hypnotic drug in the supernatant of the test sample by ultra-high performance liquid chromatography coupled with mass spectrometry; The conditions of ultra-high performance liquid chromatography include: gradient elution; mobile phase A is an acetonitrile solution of formic acid, the volume fraction of formic acid is 0.01%~1%; mobile phase B is an aqueous solution containing formic acid and ammonium formate, the volume fraction of formic acid is 0.01%~1%, and the concentration of ammonium formate is 5~20mM; During gradient elution: the proportion of mobile phase B is ≥90% at the beginning, and the proportion of mobile phase B drops to 5%~10% after 4 minutes. Among them, the rate of decrease of the proportion of mobile phase B is slow at first and then fast.

2. The method for rapid detection of sedative hypnotic drugs in human plasma according to claim 1, characterized in that: The conditions of ultra-high performance liquid chromatography include: the chromatographic column filler is octadecylsilane bonded silica gel; the flow rate is 0.3~0.5mL / min, the column temperature is 35~40℃, and the injection volume is 2~10μL.

3. The method for rapid detection of sedative hypnotic drugs in human plasma according to claim 2, characterized in that: The conditions for gradient elution were: 0.0min, mobile phase A, 10%; mobile phase B, 90%; 3.0min: mobile phase A, 40%; mobile phase B, 60%; 4.0min: mobile phase A, 90%; mobile phase B, 10%; 5.0min: mobile phase A, 10%; mobile phase B, 90%; or, 0.0min, mobile phase A, 10%; mobile phase B, 90%; 2.5 min: mobile phase A, 35%; mobile phase B, 65%; 4.0min: mobile phase A, 95%; mobile phase B, 5%; 5.0min: mobile phase A, 10%; mobile phase B, 90%.

4. The method for rapid detection of sedative hypnotic drugs in human plasma according to claim 2 or 3, characterized in that: The particle size of the column packing is 1.7 μm.

5. The method for rapid detection of sedative hypnotic drugs in human plasma according to claim 4, characterized in that: The model of the chromatographic column in the ultra-high performance liquid chromatography is Waters ACQUITY BEH C18, and the column length is 50 mm and the inner diameter is 2.1 mm.

6. The method for rapid detection of sedative hypnotic drugs in human plasma according to any one of claims 1 to 3 and 5, characterized in that: The mass spectrometry conditions were: Positive ion electrospray ion source, multiple reaction monitoring MRM, capillary voltage 0.5~1.0 kV, desolvation gas temperature 400~500℃, desolvation gas flow rate 600~800 L / Hr, cone gas flow rate 40~60 L / Hr.

7. The method for rapid detection of sedative hypnotic drugs in human plasma according to claim 6, characterized in that: The 13 sedative hypnotic drugs that can be detected by the method include: diazepam, alprazolam, lorazepam, triazolam, midazolam, nitrazepam, clonazepam, flunitrazepam, oxazepam, estazolam, zopiclone, zolpidem, and zaleplon; when the method is used to detect the 13 sedative hypnotic drugs, the internal standard precipitant contains an internal standard substance and a second solvent, the internal standard substance includes diazepam-D5 and / or zopiclone-D4, the second solvent contains acetonitrile and formic acid, and the volume fraction of the formic acid is 0.01%~1%.

8. The method for rapid detection of sedative hypnotic drugs in human plasma according to claim 7, characterized in that: The steps for qualitative detection of the supernatant of the test sample include: Prepare quality control samples, which contain sedative hypnotic standards and blank plasma; The quality control sample and the internal standard precipitant were mixed and vortexed to obtain the quality control sample supernatant. The 13 sedative hypnotic drugs in the quality control sample supernatant were separated and qualitatively detected by ultra-high performance liquid chromatography-mass spectrometry. The detection conditions of the quality control sample supernatant and the test sample supernatant were the same. By comparing the test results of the supernatant of the quality control samples and the supernatant of the test samples, the types of sedative hypnotic drugs contained in the supernatant of the test samples were gradually determined based on the retention time, dual-channel MRM chromatogram, and product ion abundance ratio.

9. The method for rapid detection of sedative hypnotic drugs in human plasma according to claim 8, characterized in that: The steps of quantitatively detecting the supernatant of the test sample include calibrating the standard sample, and the operation is as follows: A standard sample is prepared, wherein the standard sample contains a sedative hypnotic standard and blank plasma; the standard sample is mixed with an internal standard precipitant, and vortexed to obtain a standard sample supernatant; 13 sedative hypnotic drugs in the standard sample supernatant are separated and quantitatively detected by ultra-high performance liquid chromatography-mass spectrometry, and the detection conditions of the standard sample supernatant and the test sample supernatant are the same; The mass concentration of each sedative hypnotic is used as the horizontal axis. X , the chromatographic peak area ratio of each sedative hypnotic to the internal standard is the ordinate - Y , weighted least squares method is used for linear regression, and the weighting factor is 1 / X 2 , and the standard curve equations of 13 sedative-hypnotic drugs were obtained respectively.

10. The method for rapid detection of sedative hypnotic drugs in human plasma according to any one of claims 7 to 9, characterized in that: The quantitative lower limits of the method for detecting 13 sedative hypnotic drugs are: Diazepam: 62.5 ng / mL; Alprazolam: 3.125 ng / mL; Estazolam: 31.25 ng / mL; Lorazepam: 12.5 ng / mL; Triazolam: 3.125 ng / mL; Midazolam: 62.5 ng / mL; Nitrazepam: 6.25 ng / mL; Flunitrazepam: 6.25 ng / mL; Clonazepam: 3.125 ng / mL; Oxazepam: 31.25 ng / mL; Zolpidem: 12.5 ng / mL; Zopiclone: ​​12.5 ng / mL; Zaleplon: 3.125 ng / mL.

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