CN111679002B

CN111679002B - Kit for anxiolytic and hypnotic drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology

Info

Publication number: CN111679002B
Application number: CN202010484196.1A
Authority: CN
Inventors: 成晓亮; 李美娟
Original assignee: Fuzhou Pinsheng Medical Technology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd; Wuhan Pinsheng Pharmaceutical Technology Co ltd; Nanjing Pinsheng Medical Technology Co ltd
Current assignee: Fuzhou Pinsheng Medical Technology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd; Nanjing Pinsheng Medical Technology Co ltd; Wuhan Pinsheng Pharmaceutical Technology Co ltd
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2023-05-09
Anticipated expiration: 2040-06-01
Also published as: CN111679002A

Abstract

The invention relates to a kit for detecting anxiolytic and hypnotic drugs in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, which can detect 11 anxiolytic and hypnotic drugs at one time, has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, can complete separation and detection of the anxiolytic and hypnotic drugs within 5 minutes, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of the anxiolytic and hypnotic drugs clinically, and provides a simple and rapid detection method for monitoring the treatment concentration of the anxiolytic and hypnotic drugs in the serum clinically.

Description

Kit for anxiolytic and hypnotic drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a kit for detecting anxiolytic and hypnotic drugs in serum by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology, which comprises the following specific drugs: nitrazepam (NZP), oxazepam (Oxazepam, OXP), estazolam (ESL), temazepam (Temazepam, TMP), alprazolam (Alprazolam, APL), bromazepam (Bromazepam, BZP), lorazepam (Lorazepam, LZP), midazolam (Midazolam, MDZ), zopiclone (Zopiclone, ZPC), diazepam (diappam, DZP), and Zolpidem (Zolpidem, ZPM).

Background

Anxiety insomnia is a common disorder, and is generally affected by three aspects of biology, psychology and society. The benzodiazepine medicine belongs to a psychotropic medicine, and has the functions of sedation, hypnosis, anxiolytic, anticonvulsant, muscle relaxation and the like. Benzodiazepines are drug-related benzodiazepine receptor agonists, have inhibitory effect on the Central Nervous System (CNS), and are suitable for treating sleep insufficiency, anxiety, increased muscle tone or epilepsy.

Benzodiazepines are diazepam (diazepam, DZP), nitrazepam (Nitrazepam, NZP), lorazepam (Lorazepam, LZP), oxazepam (Oxazepam, oxp), alprazolam (Alprazolam, APL), temazepam (Temazepam, TMP), bromozepam (Bromazepam, BZP), and the like.

Diazepam (7-chloro-1-methyl-5-phenyl-3H-1, 4-benzodiazepine-2 [ 1H)]-ketone) for the treatment of aniline diseases, long acting benzodiazepines. Oxazepam, temazepam (3-hydroxy diazepam) is a 1, 4-benzodiazepine derivative, and is also the major active metabolite of diazepam, mainly used as an anxiolytic and hypnotic drug. Lorazepam is also a 1, 4-benzodiazepine derivative which is clinically used for the treatment of anxiety, either associated or not with depressive symptoms, as a pre-operative drug, and also as an anticonvulsant for newborns. Nitroazepam has anti-epileptic hypnotic effects, which are stronger than diazepam, and lorazepam and oxazepam are substantially as effective as diazepam. The alprazolam is one of the most commonly used benzodiazepine medicines in China, is a safe anxiolytic, and has the advantages of rapid absorption and high bioavailability. Esmolol is a quick-absorption and half-life class Zhuo Anding hypnotic agent of benzodiazepine, which is benzodiazepine

The extraction of medication is often referred to as short-term insomnia prescription. Midazolam has typical benzodiazepine +.>

Pharmacological activities such as hypnotizing, tranquilizing, anticonvulsant, and relaxing musclesForgetfulness, and axiality. In clinical practice, systemic seizures, refractory status epilepticus and muscle spasms are treated by intravenous and intramuscular injection. Midazolam is also used for conscious sedation and induction of general anesthesia.

Besides benzodiazepines, zolpidem (An Bien) is a prescribed drug for short term treatment of insomnia (difficulty falling asleep or staying asleep) and some brain diseases. It is a short-acting non-benzodiazepine hypnotic, which has a hypnotic effect similar to that of benzodiazepine but is molecularly different from classical benzodiazepine molecules, belonging to the class of imidazopyridines, and zolpidem does not fully prove its effectiveness in maintaining sleep, but is effective in shortening the fall-asleep time and prolonging the sleep time. Zopiclone is a pyrrolidone sedative hypnotic and is useful in the treatment of insomnia, particularly in patients with temporary difficulty falling asleep and early awakening.

However, these anxiolytic and hypnotic drugs have similar adverse or toxic effects, mainly manifested by dizziness or prolonged sleep time, and reduced concentration. If taken in conjunction with other substances inhibiting the central nervous system, in particular alcohol, this may lead to serious and even life-threatening respiratory depression. In addition, these substances have a high potential to develop tolerance and dependence, which can lead to physiological and psychological withdrawal symptoms, leading to misuse and abuse.

Thus, for toxicology screening, identification and quantification in Clinical Toxicology (CT), a suitable analytical procedure is required. The detection of anxiolytic and hypnotic agents in serum or serum is of great clinical significance for compliance, monitoring and identifying abuse. Determining serum or serum concentrations helps to optimize chronic dosing, verify compliance, and determine pharmacokinetic changes.

So far, a general screening procedure for benzodiazepines, zolpidem and zopiclone in serum has not been published, unlike the method of quantification of this group of single substances or a combination of some of them (some also including metabolites). Serum analysis of different drugs has published a number of analytical methods. However, all of these methods involve only a single or a mixture of a few benzodiazepines.

There are many patents aiming at the detection of the concentration of anxiolytic and hypnotic drugs in vivo at present, for example, china patent application (publication No. CN 109085265A) discloses a kit for detecting anxiolytic/hypnotic drugs in serum and plasma by using a liquid chromatography tandem mass spectrometry, meanwhile, 8 substances are detected, the sample size is required to be 100 mu L, the pretreatment is required to be more than 35 minutes, china patent application (publication No. CN 107991421A) discloses a liquid chromatography analysis method for detecting the content of diazepam in blood, the sensitivity of the diazepam is low, the quantitative limit is 0.625 mu g/mL, the sample size is required to be 100 mu L, the pretreatment is complicated, and the extraction and nitrogen blowing concentration are required. In addition, there are also patents directed to detection of one or more of these drugs, which have relatively low efficiency, and have the disadvantages of long analysis time, low sensitivity, unsuitable linear range, complex pretreatment of the sample, interference of matrix effect, and the like for a single sample.

Disclosure of Invention

The invention aims to provide a kit for detecting anxiolytic and hypnotic drugs in serum by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology on the basis of the prior art.

The invention also aims to provide the application of the kit in detecting anxiolytic and hypnotic drugs in serum by utilizing an ultra-high performance liquid chromatography-tandem mass spectrometry technology.

The technical scheme of the invention is as follows:

a kit for detecting anxiolytic and hypnotic drugs in serum by using ultra-high performance liquid chromatography-tandem mass spectrometry technology,

the anxiolytic and hypnotic drugs are respectively: nitrazepam, oxazepam, esmolam, temazepam, alprazolam, bromazepam, lorazepam, midazolam, zopiclone, diazepam, and zolpidem;

the kit comprises the following reagents:

(1) Eluent:

eluent a:0.01 to 0.1 percent of acetic acid-0.05 to 0.5mM of ammonium acetate aqueous solution; eluent B: acetonitrile;

(2) Calibrator solution:

preparing a mixed standard stock solution containing nitrazepam 20000ng/mL, oxazepam 50000ng/mL, esmolam 20000ng/mL, temazepam 40000ng/mL, alprazolam 5000ng/mL, bromazepam 20000ng/mL, lorazepam 10000ng/mL, midazolam 5000ng/mL, zopiclone 20000ng/mL, diazepam 100000ng/mL and zolpidem 10000ng/mL into a calibrator solution with seven different concentration points in a blank serum matrix, wherein the seven concentration points of the calibrator solution are as follows:

The concentration of the alprazolam and the concentration of the midazolam are the same, and seven concentration points are as follows: 0.5ng/mL, 1.25ng/mL, 2.5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL;

the concentration of lorazepam and zolpidem are the same, and the seven concentration points are as follows: 1ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500ng/mL;

the concentration of nitrazepam, eszolam, bromazepam and zopiclone are the same, and seven concentration points are as follows: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL;

the seven concentration points of temazepam are in sequence: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL;

the seven concentration points of oxazepam are in sequence: 5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL;

the seven concentration points of diazepam are in turn: 10/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL;

(3) Mixing an internal standard solution:

a methanolic solution comprising nitrazepam-d 5.5 μg/mL, oxazepam-13 c6 μg/mL, esmolam-d 5.5 μg/mL, temazepam-d 5 1 μg/mL, alprazolam-d 5.5 μg/mL, bromazepam-d 4.5 μg/mL, lorazepam-d 4.2 μg/mL, midazolam-d 7.1 μg/mL, zopiclone-d 8.5 μg/mL, diazepam-d 5 2 μg/mL, and zolpidem-d 8 2 μg/mL;

(4) Protein precipitant:

a mixed solution of methanol and isopropanol;

(5) Quality control product:

the blank serum matrix containing anxiolytic and hypnotic drugs is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H), wherein,

QC (L) was diluted 5000-fold with blank serum matrix for the above mixed standard stock solution;

QC (M) was diluted 500-fold with blank serum matrix for the above mixed standard stock solution;

QC (H) was diluted 50-fold with blank serum matrix for the above mixed standard stock solution.

In a preferred embodiment, the eluent A is 0.01% to 0.05% acetic acid-0.05 to 0.1mM ammonium acetate aqueous solution, preferably 0.05% acetic acid-0.1 mM ammonium acetate aqueous solution.

In one scheme, the volume ratio of the methanol to the isopropanol in the protein precipitant is 1:1-5; preferably, the volume ratio of methanol to isopropanol in the protein precipitant is 1:4.

In one embodiment, the empty serum matrix is empty serum that does not contain the anxiolytic and hypnotic agent of interest.

The mixed standard stock solutions mentioned in the present invention were prepared as follows:

the anxiolytic and hypnotic drugs are prepared into standard mother liquor with the following concentration: 1mg/mL of nitrazepam, 1mg/mL of oxazepam, 1mg/mL of esmolam, 1mg/mL of temazepam, 0.1mg/mL of alprazolam, 1mg/mL of bromazepam, 1mg/mL of lorazepam, 1mg/mL of midazolam, 5mg/mL of zopiclone, 1mg/mL of diazepam and 0.1mg/mL of zolpidem;

And respectively transferring mother solutions of all standard substances: nitroazepam 20. Mu.L, oxazepam 50. Mu.L, esmolam 20. Mu.L, temazepam 40. Mu.L, alprazolam 50. Mu.L, bromazepam 20. Mu.L, lorazepam 10. Mu.L, idazopiclone 5. Mu.L, zopiclone 4. Mu.L, diazepam 100. Mu.L and zolpidem 100. Mu.L, and then added to 581. Mu.L of methanol to give 1mL of a mixed standard stock solution.

The mixed internal standard solution mentioned in the present invention is prepared as follows:

the following isotopic internal standard mother liquor is prepared by methanol: nitroazepam-d 5.1 mg/mL, oxazepam-13C 6 1mg/mL, esmolam-d 5.1 mg/mL, temazepam-d 5 1mg/mL, alprazolam-d 5.01 mg/mL, bromazepam-d 4.1 mg/mL, lorazepam-d 4.1 mg/mL, midazolam-d 7.1 mg/mL, zopiclone-d 8.1 mg/mL, diazepam-d 5.1 mg/mL, and zolpidem-d 81 mg/mL;

and respectively transferring the isotope internal standard mother solutions: nitroazepam-d 5 5. Mu.L, oxazepam-13C 6. Mu.L, esmolam-d 5 5. Mu.L, temazepam-d 5 1. Mu.L, alprazolam-d 5. Mu.L, bromazepam-d 4 5. Mu.L, lorazepam-d 4 2. Mu.L, midazolam-d 7 1. Mu.L, zopiclone-d 8 5. Mu.L, diazepam-d 5. Mu.L, and zolpidem-d 8 2. Mu.L were added to 903. Mu.L of methanol to give 1mL of a mixed internal standard solution.

The serum mentioned in the present invention is human or animal serum.

In a preferred embodiment, the kit for detecting anxiolytic and hypnotic agents in serum by ultra performance liquid chromatography tandem mass spectrometry comprises the following reagents:

(1) Eluent:

eluent a:0.05% acetic acid-0.1 mM ammonium acetate aqueous solution; eluent B: acetonitrile;

(2) Calibrator solution:

and respectively transferring mother solutions of all standard substances: nitroazepam 20. Mu.L, oxazepam 50. Mu.L, esmolam 20. Mu.L, temazepam 40. Mu.L, alprazolam 50. Mu.L, bromazepam 20. Mu.L, lorazepam 10. Mu.L, idazopiclone 5. Mu.L, zopiclone 4. Mu.L, diazepam 100. Mu.L and zolpidem 100. Mu.L were added to 581. Mu.L of methanol to give 1mL of a mixed standard stock solution, the concentrations of which are shown in Table 1 below.

Table 1 mixed standard stock solution

The mixed standard stock solution is prepared into seven calibration material solutions with different concentration points by a blank serum matrix (blank serum solution without anxiolytic and hypnotic target drugs), and the preparation process is as follows:

Adding 10 mu L of mixed standard stock solution into 190 mu L of blank serum matrix to serve as a first high-value concentration point; diluting the first high-value concentration point with an equal volume of blank serum matrix to obtain a second high-value concentration point; taking the first high-value concentration point and diluting the first high-value concentration point with 9 times of blank serum matrix to obtain a third high-value concentration point; taking the second high-value concentration point and diluting the second high-value concentration point with 9 times of blank serum matrix to obtain a fourth high-value concentration point; taking the third high-value concentration point and diluting the third high-value concentration point with 9 times of blank serum matrix to obtain a fifth high-value concentration point; taking a fourth high-value concentration point, and diluting the fourth high-value concentration point with a blank serum matrix with the volume of 9 times to obtain a sixth high-value concentration point; and taking the fifth high-value concentration point, and diluting the fifth high-value concentration point with 4 times of blank serum matrix to obtain a seventh high-value concentration point. Seven concentration points for the standard formulation are shown in table 2.

Table 2 standard curve preparation

(3) Mixing an internal standard solution:

the following isotopic internal standard mother liquor is prepared by methanol: nitroazepam-d 5.1 mg/mL, oxazepam-13C 6 1mg/mL, esmolam-d 5.1 mg/mL, temazepam-d 5 1mg/mL, alprazolam-d 5.01 mg/mL, bromazepam-d 4.1 mg/mL, lorazepam-d 4.1 mg/mL, midazolam-d 7.1 mg/mL, zopiclone-d 8.1 mg/mL, diazepam-d 5.1 mg/mL, and zolpidem-d 8 1mg/mL;

(4) Protein precipitant:

the volume ratio of the methanol to the isopropanol is 1:4;

(5) Quality control product:

the above mixed standard stock solution was prepared as a blank serum solution without anxiolytic and hypnotic target drug into three different concentrations QC (L), QC (M), QC (H), wherein the specific is shown in table 3.

TABLE 3 anxiolytic and hypnotic substance controls corresponding concentration (concentration Unit: ng/mL)

Numbering device	Component (A)	QC(L)	QC(M)	QC(H)
					1	NZP	4	40	400
2	OXP	10	100	1000
					3	ESL	4	40	400
4	TMP	8	80	800
					5	APL	1	10	100
6	BZP	4	40	400
					7	LZP	2	20	200
8	MDZ	1	10	100
					9	ZPC	4	40	400
10	DZP	20	200	2000
					11	ZPM	2	20	200

QC (L) contains: nitroazepam 4mg/mL, oxazepam 10mg/mL, esmolam 4mg/mL, temazepam 8mg/mL, alprazolam 1mg/mL, bromazepam 4mg/mL, lorazepam 2mg/mL, midazolam 1mg/mL, zopiclone 4mg/mL, diazepam 20mg/mL, and zolpidem 2mg/mL.

The QC (M) includes: nitroazepam 40mg/mL, oxazepam 100mg/mL, esmolam 40mg/mL, temazepam 80mg/mL, alprazolam 10mg/mL, bromazepam 40mg/mL, lorazepam 20mg/mL, midazolam 10mg/mL, zopiclone 40mg/mL, diazepam 200mg/mL, and zolpidem 20mg/mL.

The QC (H) includes: 400mg/mL of nitrazepam, 1000mg/mL of oxazepam, 400mg/mL of esmolam, 800mg/mL of temazepam, 100mg/mL of alprazolam, 400mg/mL of bromazepam, 200mg/mL of lorazepam, 100mg/mL of midazolam, 400mg/mL of zopiclone, 2000mg/mL of diazepam and 200mg/mL of zolpidem.

When the kit is used for detecting the anxiolytic and hypnotic drugs in serum, the protein precipitant containing the internal standard is prepared by mixing the mixed internal standard solution and the protein precipitant according to the volume ratio of 1:99.

The protein precipitant is a mixed solution of methanol and isopropanol, and in a preferred scheme, the volume ratio of the methanol to the isopropanol in the protein precipitant is 1:1-5; further preferably, the volume ratio of methanol to isopropanol in the protein precipitant is 1:4.

In a preferred embodiment, the internal standard-containing protein precipitant is prepared as follows:

respectively preparing isotope internal standard mother liquor of the anxiolytic and hypnotic drugs by methanol, adding the isotope internal standard mother liquor into 903 mu L of methanol, uniformly mixing to obtain 1mL of mixed internal standard solution, adding 200 mu L of the mixed internal standard solution into 19.8mL of mixed solution of methanol and acetonitrile (the volume ratio of the methanol to the isopropanol is 1:4), and obtaining the protein precipitant containing the internal standard, wherein the concentration is shown in the table 4 below.

Table 4 protein precipitant formulation containing internal standard

The application of the kit in detecting anxiolytic and hypnotic drugs in serum by utilizing the ultra-high performance liquid chromatography-tandem mass spectrometry technology is also within the protection scope of the invention.

The specific detection method comprises the following steps:

a method for detecting concentration of anxiolytic and hypnotic drugs in serum by using ultra-high performance liquid chromatography-tandem mass spectrometry technology,

the anxiolytic and hypnotic drugs are respectively: nitroazepam (NZP), oxazepam (OXP), esmolam (ESL), temazepam (TMP), alprazolam (Alprazolam, APL), bromazepam (BZP), lorazepam (LZP), midazolam (MDZ), zopiclone (ZPC), diazepam (DZP) and Zolpidem (ZPM).

The isotope internal standard corresponding to the anxiolytic and hypnotic drugs are respectively: nitroazepam-d 5 (NZP-d 5), oxazepam-13C 6 (OXP-13C 6), esmolam-d 5 (ESL-d 5), temazepam-d 5 (TMP-d 5), alprazolam-d 5 (APL-d 5), bromazepam-d 4 (BZP-d 4), lorazepam-d 4 (LZP-d 4), midazolam-d 7 (MDZ-d 7), zopiclone-d 8 (ZPC-d 8), diazepam-d 5 (DZP-d 5) and zolpidem-d 8 (ZPM-d 8).

The anxiolytic and hypnotic drugs in the pretreated serum are detected by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly, an ultra-high performance liquid chromatography is utilized to separate an object to be detected from an interference component in a serum matrix, then, a mass spectrometry isotope internal calibration method is utilized, the concentration ratio of a standard substance to an internal standard substance is taken as an X axis, the peak area ratio of the standard substance to the internal standard substance is taken as a Y axis, a calibration curve is established, and the content of the anxiolytic and hypnotic drugs in the serum is calculated, wherein the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase a:0.01 to 0.1 percent of acetic acid-0.05 to 0.5mM of ammonium acetate aqueous solution; mobile phase B: acetonitrile;

chromatographic column model: ACQUITY UPLC CSH C18 (2.1X105 mm,1.7 μm);

gradient elution is carried out by adopting a mobile phase A and a mobile phase B as mixed mobile phases, wherein the initial proportion of the mobile phase A to the mobile phase B is 80-100:20-0; the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial proportion to 35:65 at a constant speed within 0.0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 35:65 to 2:98 at a constant speed within 1.0-3.0 minutes; in 3.0-5.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to the initial ratio at a constant speed; each sample was collected for 5.0 minutes.

(2) Mass spectrometry conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 2.5kV (ESI+); the source temperature was 120 ℃; the desolventizing gas temperature is 500 ℃, the desolventizing gas flow rate is 800L/h, and the taper hole gas flow rate is 150L/h; and simultaneously monitoring each target object and the corresponding isotope internal standard.

In order to improve the chromatographic selectivity, it may be considered to adjust the polarity of the mobile phase. According to the invention, acetic acid and ammonium acetate are added into the mobile phase A, so that the ionization efficiency of certain target compounds can be effectively improved, and under the cooperation of other conditions, compared with the detection of anxiolytic and hypnotic drugs in serum by adopting an LC-MS/MS method in the prior art, the separation and detection of the anxiolytic and hypnotic drugs are completed within 5 minutes, and the preparation method has the advantages of simple pretreatment process, low cost and high sensitivity. In a preferred embodiment, mobile phase A is 0.01% to 0.05% acetic acid-0.05 to 0.1mM ammonium acetate in water without affecting the effectiveness of the invention. In a more preferred embodiment, mobile phase A is 0.05% acetic acid-0.1 mM ammonium acetate in water.

In chromatography, the choice of the chromatographic column is important, and the requirements for the chromatographic column are: high column efficiency, good selectivity, high analysis speed, etc. The invention adopts 0.01 to 0.1 percent of acetic acid-0.05 to 0.5mM of ammonium acetate aqueous solution and acetonitrile as mobile phases, and the chromatographic column is of the type: ACQUITY UPLC CSH C18 (2.1X105 mm,1.7 μm), under the cooperation of other conditions, endogenous substances do not interfere with the measurement of the sample, and the method has the advantages of high sensitivity, strong specificity, low cost, simple pretreatment process, capability of completing separation and detection within 5.0 minutes, and precision and accuracy meeting the requirements.

The selection of the internal standard is a very important task when using the internal standard method. The ideal internal standard should be capable of being added to the sample in accurate, known amounts and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior and response characteristics as the sample being analyzed; the internal standard must be sufficiently separated from the components of the sample under chromatographic conditions. According to the invention, nitrazepam-d 5 (NZP-d 5), oxazepam-13C 6 (OXP-13C 6), esL-d5, temazepam-d 5 (TMP-d 5), alprazolam-d 5 (APL-d 5), bromazepam-d 4 (BZP-d 4), lorazepam-d 4 (LZP-d 4), imidazopran-d 7 (MDZ-d 7), zopiclone-d 8 (ZPC-d 8), diazepam-d 5 (DZP-d 5) and zolpidem-d 8 (ZPM-d 8) are respectively adopted as internal standards, and the deuterated internal standards and the substances to be measured have the same retention time, chemical property and matrix effect, so that the reproducibility and the accuracy in the measurement of anxiolytic and hypnotic drugs in serum are good.

In a preferred embodiment, the initial ratio of mobile phase A to mobile phase B is 85-95:15-5. Further preferably, the initial ratio of mobile phase a to mobile phase B is 90:10.

In one embodiment, the flow rate is 0.2 to 0.5mL/min, preferably 0.3mL/min.

Further, the column temperature is 35 to 45 ℃, preferably 45 ℃.

Further, the sample volume is 0.2 to 5. Mu.L, preferably 1. Mu.L.

In a preferred embodiment, when the anti-anxiety and hypnotic drugs in serum are detected by adopting the ultra-high performance liquid chromatography tandem mass spectrometry technology, specific chromatographic conditions are as follows:

(1) High performance liquid chromatography conditions:

mobile phase a:0.05% acetic acid-0.1 mM ammonium acetate aqueous solution;

mobile phase B: acetonitrile;

chromatographic column model: ACQUITY UPLC CSH C18 (2.1X105 mm,1.7 μm);

the initial ratio of mobile phase A to mobile phase B was 90:10; the gradient elution mode is adopted, and the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 99:10 to 35:65 at a constant speed within 0.0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 35:65 to 2:98 at a constant speed within 1.0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 90:10 at a constant speed within 3.0-5.0 minutes; each sample was collected for 5.0 minutes. The gradient elution mode is specifically shown in Table 5; the flow rate is 0.3mL/min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;

TABLE 5 gradient elution parameters for mobile phases

Time (min)	Flow rate (mL/min)	％A	％B
				0.0	0.3	90	10
1.0	0.3	35	65
				3.0	0.3	2	98
5.0	0.3	90	10

(2) Mass spectrometry conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 2.5kV (ESI+); the source temperature was 120 ℃; the desolventizing gas temperature is 500 ℃, the desolventizing gas flow rate is 800L/h, the taper hole gas flow rate is 150L/h, and the mass spectrum source parameters are shown in Table 6; simultaneously monitoring each target and the corresponding isotope internal standard thereof; the mass spectrum acquisition parameters of each target object to be detected are shown in table 7.

Table 6 mass spectrometry source parameters

Table 7 anxiolytic and hypnotic drug detection mass spectral parameters

The serum mentioned in the present invention is human or animal serum.

The pretreated serum was prepared as follows: adding a protein precipitant containing an internal standard into serum, oscillating and centrifuging, and taking supernatant; the protein precipitant is a mixed solution of methanol and isopropanol; preferably, the volume ratio of the methanol to the isopropanol in the mixed solution of the methanol and the isopropanol is 1:1-5. For example, the volume ratio of methanol to isopropanol in the mixed solution of methanol and isopropanol is 1:4 without affecting the effect of the invention.

In a preferred embodiment, the pretreated serum is prepared as follows: taking 50 mu L of serum in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to isopropanol is 1:4) into the centrifuge tube, oscillating for 3-5 min, centrifuging for 4-10 min at the temperature of 12000-15000 r/min and the temperature of 1-5 ℃, and transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner liner tube for sample injection.

In a more preferred embodiment, the pretreated serum is prepared as follows: taking 50 mu L of serum in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to isopropanol is 1:4) into the centrifuge tube, oscillating for 5min, centrifuging for 5min at 14000r/min at 4 ℃, transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner liner tube, and carrying out sample injection with the sample injection amount of 1 mu L.

In one embodiment, the internal standard-containing protein precipitant is prepared according to the following method:

the following isotopic internal standard mother liquor is prepared by methanol: nitroazepam-d 5 (NZP-d 5) 0.1mg/mL, oxazepam-13C 6 (OXP-13C 6) 1mg/mL, esmolam-d 5 (ESL-d 5) 0.1mg/mL, temazepam-d 5 (TMP-d 5) 1mg/mL, alprazolam-d 5 (APL-d 5) 0.01mg/mL, bromazepam-d 4 (BZP-d 4) 0.1mg/mL, lorazepam-d 4 (LZP-d 4) 0.1mg/mL, idazopran-d 7 (MDZ-d 7) 0.1mg/mL, zopiclone-d 8 (ZPC-d 8) 0.1mg/mL, diazepam-d 5 (DZP-d 5) 0.1mg/mL, and zolpidem-d 8 (ZPM-d 8) 1mg/mL.

And respectively transferring the isotope internal standard mother solutions: nitroazepam-d 5 (NZP-d 5) 5. Mu.L, oxazepam-13C 6 (OXP-13C 6) 1. Mu.L, esL-d5 (ESL-d 5) 5. Mu.L, temazepam-d 5 (TMP-d 5) 1. Mu.L, alprazolam-d 5 (APL-d 5) 50. Mu.L, bromozepam-d 4 (BZP-d 4) 5. Mu.L, lorazepam-d 4 (LZP-d 4) 2. Mu.L, midazolam-d 7 (MDZ-d 7) 1. Mu.L, zopiclone-d 8 (ZPC-d 8) 5. Mu.L, diazepam-d 5 (DZP-d 5) 20. Mu.L and zolpidem-d 8 (ZPM-d 8) 2. Mu.L, and then added to 903. Mu.L of methanol to give 1mL of the mixed internal standard solution.

200uL of the mixed internal standard solution is added into 19.8mL of protein precipitant (the volume ratio of methanol to isopropanol is 1:4), and the protein precipitant containing the internal standard is obtained.

In one embodiment, the standard solution is prepared as follows:

the anxiolytic and hypnotic drugs are prepared into standard mother liquor with the following concentration: nitroazepam (NZP) 1mg/mL, oxazepam (OXP) 1mg/mL, esmolam (ESL) 1mg/mL, temazepam (TMP) 1mg/mL, alprazolam (APL) 0.1mg/mL, bromazepam (BZP) 1mg/mL, lorazepam (LZP) 1mg/mL, midazolam (MDZ) 1mg/mL, zopiclone (ZPC) 5mg/mL, diazepam (DZP) 1mg/mL, and Zolpidem (ZPM) 0.1mg/mL.

And respectively transferring mother solutions of all standard substances: nitroazepam (NZP) 20. Mu.L, oxazepam (OXP) 50. Mu.L, esmolam (ESL) 20. Mu.L, temazepam (TMP) 40. Mu.L, alprazolam (APL) 50. Mu.L, bromazepam (BZP) 20. Mu.L, lorazepam (LZP) 10. Mu.L, midazolam (MDZ) 5. Mu.L, zopiclone (ZPC) 4. Mu.L, diazepam (DZP) 100. Mu.L and Zolpidem (ZPM) 100. Mu.L, and then added to 581. Mu.L of methanol to give 1mL of a mixed standard stock solution.

The invention prepares the mixed standard stock solution into seven calibrator solutions with different concentration points by a blank serum matrix (blank serum without anxiolytic and hypnotic target drugs), and the preparation process is as follows:

Adding 10 mu L of mixed standard stock solution into 190 mu L of blank serum matrix to serve as a first high-value concentration point; diluting the first high-value concentration point with an equal volume of blank serum matrix to obtain a second high-value concentration point; taking the first high-value concentration point and diluting the first high-value concentration point with 9 times of blank serum matrix to obtain a third high-value concentration point; taking the second high-value concentration point and diluting the second high-value concentration point with 9 times of blank serum matrix to obtain a fourth high-value concentration point; taking the third high-value concentration point and diluting the third high-value concentration point with 9 times of blank serum matrix to obtain a fifth high-value concentration point; taking a fourth high-value concentration point, and diluting the fourth high-value concentration point with a blank serum matrix with the volume of 9 times to obtain a sixth high-value concentration point; and taking the fifth high-value concentration point, and diluting the fifth high-value concentration point with 4 times of blank serum matrix to obtain a seventh high-value concentration point.

Seven concentration points of the calibrator solution are:

the concentration of Alprazolam (APL) and Midazolam (MDZ) are the same, and the seven concentration points are as follows: 0.5ng/mL, 1.25ng/mL, 2.5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL.

The concentration of Lorazepam (LZP) and Zolpidem (ZPM) are the same, and the seven concentration points are as follows: 1ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500ng/mL.

The concentrations of Nitrazepam (NZP), esmolam (ESL), bromoazepam (BZP) and Zopiclone (ZPC) are the same, and the seven concentration points are as follows: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL.

The seven concentration points of Temazepam (TMP) are in order: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL.

The seven concentration points of Oxazepam (OXP) are in order: 5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL.

The seven concentration points of Diazepam (DZP) are in order: 10/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL.

And taking 50 mu L of each concentration point sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to isopropanol is 1:4) into the centrifuge tube, oscillating for 5min, centrifuging for 5min at 14000r/min at 4 ℃, transferring 60 mu L of supernatant in the centrifuge tube into a plastic lining tube, and carrying out sample injection with the sample injection amount of 1 mu L.

The invention also comprises the preparation of quality control products, wherein the quality control products are prepared according to the following method: the mixed standard stock solution is prepared into three different concentrations of QC (L), QC (M) and QC (H) by using blank serum without anxiolytic and hypnotic target drugs, wherein,

QC (L) was diluted 5000-fold with the above mixed standard stock solution as a blank serum matrix.

QC (M) was diluted 500-fold with the above mixed standard stock solution as a blank serum matrix.

When the kit is used for detecting the anxiolytic and hypnotic drugs in serum, the sample size is only 50uL, the pretreatment can be completed within about 10 minutes, 11 substances can be detected at the same time, and the flux is higher.

By adopting the technical scheme of the invention, the advantages are as follows:

When the kit provided by the invention is used for detecting the anxiolytic and hypnotic drugs in serum, 11 anxiolytic and hypnotic drugs can be detected at one time, the target drugs and the metabolites are monitored simultaneously, the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and detection of the anxiolytic and hypnotic drugs are completed within 5 minutes, the accuracy and precision basically meet the requirements, the kit can be used for quantitative analysis of the clinical anxiolytic and hypnotic drugs, and a simple and quick detection method is provided for monitoring the treatment concentration of the anxiolytic and hypnotic drugs in the clinical serum.

Drawings

FIG. 1 is a selected ion flow chromatogram of an anxiolytic and hypnotic drug standard;

FIG. 2 is a graph of a selected ion flow chromatogram of anxiolytic and hypnotic drugs in a serum sample.

Detailed Description

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.

Example 1:

1. experimental materials and instruments

1. Material

The samples were from serum samples collected from the 11 month clinic in 2019 of the wuhan asian heart disease hospital.

(1) Instrument: a Xex TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra-high performance liquid chromatography system (auto sampler, waters Corporation); SCILOGEX D2012 high speed table centrifuge (usa); ultrapure water meter (ELGA LabWater, uk); multitube Vortex mixer (Vortex genie2, usa); an adjustable pipette (Eppendorf 0.5-10. Mu.L, 10-100. Mu.L, 100-1000. Mu.L); glassware, measuring cylinders, etc. .

(2) Reagent consumable: MS grade methanol (Fisher, USA); MS grade acetonitrile (Fisher, USA); HPLC grade acetonitrile (Honeywell, usa); MS grade acetic acid (Fisher, USA); MS grade ammonium acetate (Fisher, USA); HPLC grade methanol (Honeywell, usa); column ACQUITY UPLC CSH C (2.1X105 mm,1.7 μm).

(3) Standard substance: standards and their corresponding internal standards are shown in Table 8

Table 8 standards and internal standards

Sequence number	Chinese name	Manufacturer' s
			1	Nitrazepam	Sigma
2	Nitrazepam-d 5	Screening quasi-living things
			3	Diazepam	Sigma
4	Diazepam-d 5	Sigma
			5	Oxazepam	Sigma
6	oxazepam-13C 6	Screening quasi-living things
			7	Esmolam	Sigma
8	Eszolam-d 5	Sigma
			9	Temazepam	Screening quasi-living things
10	Temazepam-d 5	Screening quasi-living things
			11	Zolpidem	National standard substance net
12	Zolpidem-d 8	Sigma
			13	Alprazolam	Middle check station
14	Alprazolam-d 5	Sigma
			15	Bromozepam	Sigma
16	Bromozepam-d 4	Screening quasi-living things
			17	Lorazepam	Cerilliant
18	Lalazepam-d 4	Sigma
			19	Midazolam	Cerilliant
20	Midazolam-d 7	Screening quasi-living things
			21	Zopiclone	TRC
22	Zopiclone-d 8	Screening quasi-living things

(4) Quality control product: the blank serum matrix containing anxiolytic and hypnotic substances was divided into low, medium and high concentrations of QC (L), QC (M) and QC (H), respectively, as shown in table 3.

2. Liquid condition

(1) Chromatographic conditions: chromatographic conditions: mobile phase a:0.05% acetic acid-0.1 mM ammonium acetate-water solution; mobile phase B: acetonitrile. Chromatographic column model: ACQUITY UPLC CSH C18 (2.1X105 mm,1.7 μm) by gradient elution, see Table 5 for details. The flow rate was 0.3mL/min, the column temperature was 45℃and the sample volume was 1. Mu.L.

(2) Mass spectrometry conditions: in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 2.5kV (ESI+); the source temperature was 120 ℃; the desolventizing gas temperature is 500 ℃, the desolventizing gas flow rate is 800L/h, the taper hole gas flow rate is 150L/h, and the mass spectrum source parameters are shown in Table 6; simultaneously monitoring each target and the corresponding isotope internal standard thereof; the mass spectrum acquisition parameters of each target object to be detected are shown in table 7.

2. Experimental procedure

(1) Preparing a standard substance:

The anxiolytic and hypnotic drugs are prepared into standard mother liquor with the following concentration: nitroazepam (NZP) 1mg/mL, oxazepam (OXP) 1mg/mL, esmolam (ESL) 1mg/mL, temazepam (TMP) 1mg/mL, alprazolam (APL) 0.1mg/mL, bromazepam (BZP) 1mg/mL, lorazepam (LZP) 1mg/mL, midazolam (MDZ) 1mg/mL, zopiclone (ZPC) 5mg/mL, diazepam (DZP) 1mg/mL, and Zolpidem (ZPM) 0.1mg/mL;

and respectively transferring mother solutions of all standard substances: nitroazepam (NZP) 20. Mu.L, oxazepam (OXP) 50. Mu.L, esmolam (ESL) 20. Mu.L, temazepam (TMP) 40. Mu.L, alprazolam (APL) 50. Mu.L, bromazepam (BZP) 20. Mu.L, lorazepam (LZP) 10. Mu.L, midazolam (MDZ) 5. Mu.L, zopiclone (ZPC) 4. Mu.L, diazepam (DZP) 100. Mu.L and Zolpidem (ZPM) 100. Mu.L, and then added to 581. Mu.L of methanol to give 1mL of a mixed standard stock solution. Details are shown in Table 1.

The above mixed standard stock solution was formulated as a calibrator solution with seven different concentration points in a blank serum matrix (blank serum without anxiolytic and hypnotic target drug), see table 2 for details, the seven concentration points of the calibrator solution being:

(2) Preparation of mixed internal standard liquid

The following isotopic internal standard mother liquor is prepared by methanol: nitroazepam-d 5 (NZP-d 5) 0.1mg/mL, oxazepam-13C 6 (OXP-13C 6) 1mg/mL, esmolam-d 5 (ESL-d 5) 0.1mg/mL, temazepam-d 5 (TMP-d 5) 1mg/mL, alprazolam-d 5 (APL-d 5) 0.01mg/mL, bromazepam-d 4 (BZP-d 4) 0.1mg/mL, lorazepam-d 4 (LZP-d 4) 0.1mg/mL, idazopran-d 7 (MDZ-d 7) 0.1mg/mL, zopiclone-d 8 (ZPC-d 8) 0.1mg/mL, diazepam-d 5 (DZP-d 5) 0.1mg/mL, and zolpidem-d 8 (ZPM-d 8) 1mg/mL;

(3) Preparing a quality control product:

the above mixed standard stock solution was prepared as three different concentrations of QC (L), QC (M), QC (H) in blank serum without anxiolytic and hypnotic target drug, as detailed in table 3.

QC (L) contains: nitroazepam 4mg/mL, oxazepam 10mg/mL, esmolam 4mg/mL, temazepam 8mg/mL, alprazolam 1mg/mL, bromazepam 4mg/mL, lorazepam 2mg/mL, midazolam 1mg/mL, zopiclone 4mg/mL, diazepam 20mg/mL, and zolpidem 2mg/mL;

the QC (M) includes: nitroazepam 40mg/mL, oxazepam 100mg/mL, esmolam 40mg/mL, temazepam 80mg/mL, alprazolam 10mg/mL, bromazepam 40mg/mL, lorazepam 20mg/mL, midazolam 10mg/mL, zopiclone 40mg/mL, diazepam 200mg/mL, and zolpidem 20mg/mL;

(4) Sample processing

200 mu L of the mixed internal standard solution is added into 19.8mL of protein precipitant (the volume ratio of methanol to isopropanol is 1:4), and the protein precipitant containing the internal standard is obtained.

1) Pretreatment of standard products: and taking 50 mu L of each concentration point sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to isopropanol is 1:4) into the centrifuge tube, oscillating for 5min, centrifuging for 5min at 14000r/min at 4 ℃, transferring 60 mu L of supernatant in the centrifuge tube into a plastic lining tube, and carrying out sample injection with the sample injection amount of 1 mu L.

2) Pretreatment of serum samples: taking 50 mu L of serum in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to isopropanol is 1:4) into the centrifuge tube, oscillating for 5min, centrifuging for 5min at 14000r/min at 4 ℃, transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner liner tube, and carrying out sample injection with the sample injection amount of 1 mu L.

3) Pretreatment of quality control products: respectively taking 50 mu L of quality control product solution QC (L), QC (M) and QC (H) in a 1.5mL centrifuge tube, and then consistent with pretreatment of serum samples, which are not described herein.

The components of the assay kit are shown in Table 9.

Table 9 preparation of components of anxiolytic and hypnotic concentration analysis kit

4. Method verification

1. Extracting an ion flow chromatogram: the peak shapes of the standard substance of the anxiolytic and the hypnotic drug and the serum sample are symmetrical, and the peak shape is not interfered by miscellaneous peaks, which indicates that the standard substance of the anxiolytic and the hypnotic drug can be well detected under the condition, and the figure 1 is a selective ion flow chromatogram of the standard substance of the anxiolytic and the hypnotic drug; FIG. 2 is a graph of a selected ion flow chromatogram of anxiolytic and hypnotic drugs in a serum sample.

2. Calibration curve: and (3) using an isotope internal calibration method, using TargetLynx software to establish a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the concentration of an object to be detected in serum. The linear fitting equation of the anxiolytic and hypnotic drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, as shown in table 10.

TABLE 10 Linear regression equation and Linear correlation coefficient for anxiolytic and hypnotic drugs

3. Accuracy investigation: and (5) evaluating the accuracy of the method by adopting a standard adding recovery rate test. A mixed blank serum sample was prepared, mixed standard substances of low, medium and high 3 concentrations were added respectively, and the treatment and measurement were repeated 5 times in the same procedure, and the results showed that the labeled recovery rate of the anxiolytic and hypnotic drugs was 88.63% -111.62%, and the RSD of the 5 repeated tests was 1.34% -7.32%, and the statistical results are shown in Table 11.

TABLE 11 results of recovery of anxiolytic and hypnotic drug addition

4. Precision test: taking a non-interference blank serum sample, adding anti-anxiety and hypnotic drug standard substances with different concentrations to obtain low, medium and high concentration serum samples, repeatedly treating for 6 batches in one day, continuously treating for three days, quantitatively measuring the concentration of the anti-anxiety and hypnotic drug by an isotope internal standard method, treating for 3 batches in three days, and calculating the precision between batches to be 2.64-11.53%, wherein the result is shown in Table 12.

TABLE 12 results of batch-to-batch precision test (concentration units: ng/mL)

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5. Discussion of the invention

The invention adopts an ID-UPLC-MS/MS method to measure the concentration of the anxiolytic and hypnotic drugs in human serum. Meanwhile, the detection is carried out on the peak-out time and the ion pair of the target object, the sensitivity is high, meanwhile, the substrate interference can be greatly eliminated by adopting the isotope internal standard method for quantification, and the accurate quantification can be achieved without being influenced by the pretreatment process, the sample loading volume, the flow and other conditions.

The accuracy of the method is evaluated by a labeling recovery rate test, and the result shows that the labeling recovery rate of the anxiolytic and hypnotic drugs is 88.63-111.62%, the RSD of 5 repeated tests is 1.34-7.32%, and the accuracy is good.

The reproducibility of the method shows that the precision of the anxiolytic and hypnotic drug in the batch is 1.87-8.00% and the precision of the anxiolytic and hypnotic drug in the batch is 2.64-11.53%. And the pretreatment process of the established serum sample is very simple, the protein precipitation is carried out in one step, and the serum dosage is only 50 mu L.

In a word, the method has high sensitivity, strong specificity, accuracy and simpler pretreatment process, can finish separation and detection of the compound within 5.0 minutes, has accuracy and precision meeting the requirements, can be used for quantitative analysis of the concentration of the serum anxiolytic and hypnotic drugs clinically, and provides a reliable detection method for the treatment and monitoring of the concentration of the anxiolytic and hypnotic drugs clinically.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An application of a kit in detecting anxiolytic and hypnotic drugs in serum by utilizing an ultra-high performance liquid chromatography-tandem mass spectrometry technology,

the kit comprises the following reagents:

(1) Eluent:

(2) Calibrator solution:

and respectively transferring mother solutions of all standard substances: nitroazepam 20. Mu.L, oxazepam 50. Mu.L, esmolam 20. Mu.L, temazepam 40. Mu.L, alprazolam 50. Mu.L, bromazepam 20. Mu.L, lorazepam 10. Mu.L, idazopiclone 5. Mu.L, zopiclone 4. Mu.L, diazepam 100. Mu.L and zolpidem 100. Mu.L, and then added to 581. Mu.L of methanol to obtain 1mL of a mixed standard stock solution; preparing the mixed standard stock solution into seven calibration material solutions with different concentration points by using blank serum without anxiolytic and hypnotic target drugs; seven concentration points of the calibrator solution are:

the seven concentration points of temazepam are in sequence: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000 ng/mL;

the seven concentration points of oxazepam are in sequence: 5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL, 1250 ng/mL, 2500ng/mL;

the seven concentration points of diazepam are in turn: 10ng/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500ng/mL, 2500ng/mL, 5000 ng/mL;

(3) Mixing an internal standard solution:

And respectively transferring the isotope internal standard mother solutions: nitroazepam-d 5 5. Mu.L, oxazepam-13C 6. Mu.L, esmolam-d 5 5. Mu.L, temazepam-d 5 1. Mu.L, alprazolam-d 5. Mu.L, bromazepam-d 4 5. Mu.L, lorazepam-d 4 2. Mu.L, midazolam-d 7 1. Mu.L, zopiclone-d 8 5. Mu.L, diazepam-d 5. Mu.L, and zolpidem-d 8 2. Mu.L, and then added to 903. Mu.L of methanol to give 1mL of a mixed internal standard solution;

(4) Protein precipitant:

the volume ratio of the methanol to the isopropanol is 1:4;

(5) Quality control product:

the mixed standard stock solution is prepared into three different concentrations of L-QC, M-QC and H-QC by using blank serum without anxiolytic and hypnotic target drugs, wherein,

the L-QC comprises: nitroazepam 4mg/mL, oxazepam 10mg/mL, esmolam 4mg/mL, temazepam 8mg/mL, alprazolam 1mg/mL, bromazepam 4mg/mL, lorazepam 2mg/mL, midazolam 1mg/mL, zopiclone 4mg/mL, diazepam 20mg/mL, and zolpidem 2mg/mL;

the M-QC comprises: nitroazepam 40mg/mL, oxazepam 100mg/mL, esmolam 40mg/mL, temazepam 80mg/mL, alprazolam 10mg/mL, bromazepam 40mg/mL, lorazepam 20mg/mL, midazolam 10mg/mL, zopiclone 40mg/mL, diazepam 200mg/mL, and zolpidem 20mg/mL;

The H-QC comprises: 400mg/mL of nitrazepam, 1000mg/mL of oxazepam, 400mg/mL of esmolam, 800mg/mL of temazepam, 100mg/mL of alprazolam, 400mg/mL of bromazepam, 200mg/mL of lorazepam, 100mg/mL of midazolam, 400mg/mL of zopiclone, 2000mg/mL of diazepam and 200mg/mL of zolpidem.

The application comprises the following steps: the anxiolytic and hypnotic drugs in the pretreated serum are detected by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly, an ultra-high performance liquid chromatography is utilized to separate an object to be detected from an interference component in a serum matrix, then, a mass spectrometry isotope internal calibration method is utilized, the concentration ratio of a standard substance to an internal standard substance is taken as an X axis, the peak area ratio of the standard substance to the internal standard substance is taken as a Y axis, a calibration curve is established, and the content of the anxiolytic and hypnotic drugs in the serum is calculated, wherein the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

chromatographic column model: ACQUITY UPLC CSH C18 2.1X150 mm,1.7μm;

gradient elution is carried out by adopting a mobile phase A and a mobile phase B as mixed mobile phases, wherein the initial proportion of the mobile phase A to the mobile phase B is 90:10; the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B gradually changes from 90:10 to 35:65 at a constant speed within 0.0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 35:65 to 2:98 at a constant speed within 1.0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 90:10 at a constant speed within 3.0-5.0 minutes; each sample collection time was 5.0 minutes;

(2) Mass spectrometry conditions:

in an electrospray ionization positive ion detection mode, a multi-reaction monitoring mass spectrum scanning mode is adopted, and the capillary voltage is 2.5kVESI+; the source temperature was 120 ℃; the desolventizing gas temperature is 500 ℃, the desolventizing gas flow rate is 800L/h, and the taper hole gas flow rate is 150L/h; simultaneously monitoring each target and the corresponding isotope internal standard thereof;

the pretreated serum was prepared as follows: taking 50 mu L of serum in a 1.5 mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard into the centrifuge tube, oscillating for 5min, centrifuging at 14000 r/min and 4 ℃ for 5min, transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner liner tube, and carrying out sample injection with a sample injection amount of 1 mu L;

pretreatment of the calibrator was prepared as follows: taking 50 mu L of each concentration point sample in a 1.5 mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard into the centrifuge tube, oscillating for 5min, centrifuging at 14000 r/min and 4 ℃ for 5min, transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner liner tube, and carrying out sample injection with a sample injection amount of 1 mu L;

the pretreatment of the quality control product is prepared according to the following method: respectively taking quality control product solutions L-QC, M-QC and H-QC, respectively 50 mu L of each solution in a 1.5 mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard into each solution, oscillating for 5min, centrifuging for 5min at 14000 r/min at 4 ℃, and transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner liner tube, wherein the sample injection amount is 1 mu L;

Wherein: the protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the volume ratio of the mixed internal standard solution to the protein precipitant is 1:99.