阅读说明：本技术 超高效液相色谱串联质谱技术检测血清中抗抑郁药物的试剂盒 (Kit for detecting antidepressant drug in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology ) 是由 成晓亮 李美娟 于 2020-06-01 设计创作，主要内容包括：本发明涉及超高效液相色谱串联质谱技术检测血清中抗抑郁药物的试剂盒,前处理过程简单,成本低,且灵敏度高、特异性强,4.5min之内完成抗抑郁药物的分离和检测,准确度与精密度基本满足要求,可用于临床上抗抑郁药物的定量分析,为临床上抗抑郁药物的治疗浓度监测提供一种可靠的检测方法。(The invention relates to a kit for detecting antidepressant drugs in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, which has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, completes the separation and detection of the antidepressant drugs within 4.5min, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical antidepressant drugs, and provides a reliable detection method for monitoring the treatment concentration of the antidepressant drugs in clinic.)

1. A kit for detecting an antidepressant medicament in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,

the antidepressant is BPP, AML, HBPP, NTP, NVLF, MAS, MTZ, VLF, ATP, DXP, NFXT, DLX, FXT, FVX, CTP, PXT, TZD, VXT;

the kit is characterized by comprising the following reagents:

(1) mobile phase: mobile phase A: 0.001 to 0.1 percent of formic acid aqueous solution; mobile phase B: 0.001 to 0.1 percent of formic acid methanol solution;

(2) a calibration sample:

calibrator samples at seven different concentration points:

s1, which comprises 1ng/mL each of AML/MAS/CTP/PXT, 10ng/mL each of BPP/HBPP/TZD, 2ng/mL each of NTP/MTZ/DXP/DLX, 5ng/mL each of VLF/ATP/NFXT/FXT, 20ng/mL of NVLF, FVX 4ng/mL and 0.5ng/mL of VXT;

s2, which comprises AML/MAS/CTP/PXT each 2.5ng/mL, BPP/HBPP/TZD each 25ng/mL, NTP/MTZ/DXP/DLX each 5ng/mL, VLF/ATP/NFXT/FXT each 12.5ng/mL, NVLF 50ng/mL, FVX 10ng/mL, VXT 1.25 ng/mL;

s3, which comprises AML/MAS/CTP/PXT each 5ng/mL, BPP/HBPP/TZD each 50ng/mL, NTP/MTZ/DXP/DLX each 10ng/mL, VLF/ATP/NFXT/FXT each 25ng/mL, NVLF 100ng/mL, FVX 20ng/mL, VXT 2.5 ng/mL;

s4, which comprises 25ng/mL each of AML/MAS/CTP/PXT, 250ng/mL each of BPP/HBPP/TZD, 50ng/mL each of NTP/MTZ/DXP/DLX, 125ng/mL each of VLF/ATP/NFXT/FXT, 500ng/mL each of NVLF, FVX 100ng/mL, and 12.5ng/mL each of VXT;

s5, which comprises AML/MAS/CTP/PXT each 50ng/mL, BPP/HBPP/TZD each 500ng/mL, NTP/MTZ/DXP/DLX each 100ng/mL, VLF/ATP/NFXT/FXT each 250ng/mL, NVLF 1000ng/mL, FVX 200ng/mL, VXT25 ng/mL;

s6, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

s7, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

(3) mixing internal standard working solution:

methanol solutions comprising 200ng/mL BPP-d9, 20ng/mL AML-d6, 200ng/mL HBPP-d6, 100ng/mL NVLF-d6, 50ng/mL NTP-d3, 20ng/mL MAS-d3, 50ng/mL MTZ-d3, 100ng/mL VLF-d6, 100ng/mL ATP-d3, 50ng/mL DXP-d3, 100ng/mL NFXT-d5, 50ng/mL DLX-d7, 100ng/mL FXT-d5, 100ng/mL FVXg-d 3, 20ng/mL CTP-d4, 20ng/mL T-d6, 200ng/mL TZD-d6, and 10ng/mL VXT-d 8.

(4) Protein precipitant:

any one of methanol and acetonitrile, or a mixed solution of methanol and acetonitrile;

(5) quality control product:

blank serum matrix containing antidepressant drugs is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H), wherein the QC (L) comprises the following components: 20ng/mL BPP, 2ng/mL AML, 20ng/mL HBPP, 4ng/mL NTP, 2ng/mLMAS, 4ng/mL MTZ, 10ng/mL VLF, 10ng/mL ATP, 4ng/mL DXP, 10ng/mL NFXT, 4ng/mL DLX, 10ng/mL FXT, 8ng/mL FVX, 2ng/mL CTP, 2ng/mL PXT, 20ng/mL TZD, 1ng/mL VXT and 40ng/mL LNVLF;

QC (M) comprises: 200ng/mL BPP, 20ng/mL AML, 200ng/mL HBPP, 40ng/mL NTP, 20ng/mLMAS, 40ng/mL MTZ, 100ng/mL VLF, 100ng/mL ATP, 40ng/mL DXP, 100ng/mL NFXT, 40ng/mL LDLX, 100ng/mL FXT, 80ng/mL FVX, 20ng/mL CTP, 20ng/mL PXT, 200ng/mL TZD, 10ng/mL VXT and 400ng/mL NVLF;

QC (H) comprises 2000ng/mL BPP, 200ng/mL AML, 2000ng/mL HBPP, 400ng/mL NTP, 200ng/mL MAS, 400ng/mL MTZ, 1000ng/mL VLF, 1000ng/mL ATP, 400ng/mL DXP, 1000ng/mL NFXT, 400ng/mL DLX, 1000ng/mL FXT, 800ng/mL FVX, 200ng/mL CTP, 200ng/mL PXT, 2000ng/mL ZD, 100ng/mL LTT and 4000ng/mL NVLF.

2. A kit for detecting an antidepressant medicament in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,

the antidepressant is BPP, AML, HBPP, NTP, NVLF, MAS, MTZ, VLF, ATP, DXP, NFXT, DLX, FXT, FVX, CTP, PXT, TZD, VXT;

the kit is characterized by comprising the following reagents:

(1) mobile phase: mobile phase A: 0.001 to 0.1 percent of formic acid aqueous solution; mobile phase B: 0.001 to 0.1 percent of formic acid methanol solution;

(2) mixing the standard solution and the blank serum matrix;

the mixed standard solution comprises 100000ng/mL BPP, 10000ng/mL AML, 100000ng/mL HBPP, 20000ng/mL NTP, 10000ng/mL MAS, 20000ng/mL MTZ, 50000ng/mL VLF, 50000ng/mL ATP, 20000ng/mL DXP, 50000ng/mL NFXT, 20000ng/mL DLX, 50000ng/mL FXT, 40000ng/mL FVX, 10000ng/mL CTP, 10000ng/mL CTT, 100000ng/mL ZD, 5000ng/mL VXT and 200000ng/mL LNVLF;

before detection, the mixed standard solution is diluted by a blank serum substrate according to a formula recorded in an instruction manual to prepare calibration sample samples S1-S7 of seven different concentration points and quality control solution QC (L), QC (M) and QC (H) of low, medium and high three concentrations:

s1, which comprises 1ng/mL each of AML/MAS/CTP/PXT, 10ng/mL each of BPP/HBPP/TZD, 2ng/mL each of NTP/MTZ/DXP/DLX, 5ng/mL each of VLF/ATP/NFXT/FXT, 20ng/mL of NVLF, FVX 4ng/mL and 0.5ng/mL of VXT;

s2, which comprises AML/MAS/CTP/PXT each 2.5ng/mL, BPP/HBPP/TZD each 25ng/mL, NTP/MTZ/DXP/DLX each 5ng/mL, VLF/ATP/NFXT/FXT each 12.5ng/mL, NVLF 50ng/mL, FVX 10ng/mL, VXT 1.25 ng/mL;

s3, which comprises AML/MAS/CTP/PXT each 5ng/mL, BPP/HBPP/TZD each 50ng/mL, NTP/MTZ/DXP/DLX each 10ng/mL, VLF/ATP/NFXT/FXT each 25ng/mL, NVLF 100ng/mL, FVX 20ng/mL, VXT 2.5 ng/mL;

s4, which comprises 25ng/mL each of AML/MAS/CTP/PXT, 250ng/mL each of BPP/HBPP/TZD, 50ng/mL each of NTP/MTZ/DXP/DLX, 125ng/mL each of VLF/ATP/NFXT/FXT, 500ng/mL each of NVLF, FVX 100ng/mL, and 12.5ng/mL each of VXT;

s5, which comprises AML/MAS/CTP/PXT each 50ng/mL, BPP/HBPP/TZD each 500ng/mL, NTP/MTZ/DXP/DLX each 100ng/mL, VLF/ATP/NFXT/FXT each 250ng/mL, NVLF 1000ng/mL, FVX 200ng/mL, VXT25 ng/mL;

s6, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

s7, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

QC (L) includes: 20ng/mL BPP, 2ng/mL AML, 20ng/mL HBPP, 4ng/mL NTP, 2ng/mL MAS, 4ng/mL MTZ, 10ng/mL VLF, 10ng/mL ATP, 4ng/mL DXP, 10ng/mL NFXT, 4ng/mL DLX, 10ng/mL LFXT, 8ng/mL FVX, 2ng/mL CTP, 2ng/mL PXT, 20ng/mL TZD, 1ng/mL VXT, and 40ng/mL NVLF;

QC (M) comprises: 200ng/mL BPP, 20ng/mL AML, 200ng/mL HBPP, 40ng/mL NTP, 20ng/mLMAS, 40ng/mL MTZ, 100ng/mL VLF, 100ng/mL ATP, 40ng/mL DXP, 100ng/mL NFXT, 40ng/mL LDLX, 100ng/mL FXT, 80ng/mL FVX, 20ng/mL CTP, 20ng/mL PXT, 200ng/mL TZD, 10ng/mL VXT and 400ng/mL NVLF;

QC (H) comprises 2000ng/mL BPP, 200ng/mL AML, 2000ng/mL HBPP, 400ng/mL NTP, 200ng/mL MAS, 400ng/mL MTZ, 1000ng/mL VLF, 1000ng/mL ATP, 400ng/mL DXP, 1000ng/mL NFXT, 400ng/mL DLX, 1000ng/mL FXT, 800ng/mL FVX, 200ng/mL CTP, 200ng/mL PXT, 2000ng/mL ZD, 100ng/mL VXT and 4000ng/mL NVLF;

(3) mixing internal standard working solution:

methanol solutions comprising 200ng/mL BPP-d9, 20ng/mL AML-d6, 200ng/mL HBPP-d6, 100ng/mL NVLF-d6, 50ng/mL NTP-d3, 20ng/mL MAS-d3, 50ng/mL MTZ-d3, 100ng/mL VLF-d6, 100ng/mL ATP-d3, 50ng/mL DXP-d3, 100ng/mL NFXT-d5, 50ng/mL DLX-d7, 100ng/mL FXT-d5, 100ng/mL FVXg-d 3, 20ng/mL CTP-d4, 20ng/mL T-d6, 200ng/mL TZD-d6, and 10ng/mL VXT-d 8.

(4) Protein precipitant:

any one of methanol and acetonitrile, or a mixed solution of methanol and acetonitrile;

(5) the use instruction comprises: comprises calibrator samples S1-S7 and quality control solution QC (L), QC (M) and QC (H) with low, medium and high concentrations.

3. The kit according to claim 1 or 2,

the mobile phase A is 0.001-0.1% aqueous formic acid solution, preferably 0.01% aqueous formic acid solution; the mobile phase B is 0.001 to 0.1 percent of formic acid methanol solution, and preferably 0.01 percent of formic acid methanol solution.

4. The kit according to claim 1 or 2,

the protein precipitator is a mixed solvent of methanol and acetonitrile, preferably a mixed solvent of methanol and acetonitrile in a volume ratio of 1: 1-1: 3, and more preferably a mixed solvent of methanol and acetonitrile in a volume ratio of 1: 2.

5. The kit according to claim 2,

the blank serum matrix is blank serum without antidepressant drugs.

6. The kit according to claim 2,

the mixed standard solution is prepared according to the following method: preparing standard mother liquor containing BPP 100000ng/mL, BPLAML 10000ng/mL, HBPP 100000ng/mL, NTP 50000ng/mL, DXP 20000/mL, NFXT 50000ng/mL, NTP 50000ng/mL, NFPP 50000ng/mL, NFXT 5005 mg/mL, MTZ 4mg/mL, MTZ 5mg/mL, VLF 2mg/mL ATP 2mg/mL DXP 5mg/mL NFXT 2mg/mL DLX 5mg/mL FXT 4mg/mL FVX 0.5mg/mL CTP 2mg/mL PXT 5mg/mL TZT 1mg/mL LVXT 1mg/mL NVLF with methanol solution, 20000ng/mL DLX, 50000ng/mLFXT, 40000ng/mL FVX, 10000ng/mL CTP, 10000ng/mL PXT, 100000ng/mL TZD, 5000ng/mLVXT and 200000ng/mL NVLF.

7. The kit according to claim 1 or 2,

mobile phase: the mobile phase A: 0.005-0.05% formic acid water solution; mobile phase B: 0.005 to 0.05 percent of methanoic acid solution;

the volume ratio of the protein precipitant methanol to the acetonitrile is 1: 1-1: 3.

8. The kit according to claim 7,

the mobile phase A: 0.01% aqueous formic acid; mobile phase B: 0.01% formic acid in methanol;

the protein precipitating agent:

the volume ratio of methanol to acetonitrile is 1: 2.

9. The kit according to claim 2, wherein the method for preparing the low, medium and high concentrations of the quality control solutions QC (L), QC (M), QC (H) described in the instructions for use is as follows:

QC (L) is medium concentration quality control diluted to 10 times with blank serum substrate;

QC (M) is the above mixed standard solution diluted 500 times with blank serum matrix;

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

10. The kit of any one of claims 1-2, for use in detecting antidepressant drugs in serum by using ultra high performance liquid chromatography tandem mass spectrometry.

Technical Field

The invention belongs to the technical field of serum detection, and particularly relates to a kit for detecting an antidepressant drug in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology.

Background

The current commonly used therapeutic drugs for depression are classified according to their mechanism of action: norepinephrine reuptake inhibitors, 5-hydroxytryptamine (5-HT) reuptake inhibitors (SSRIs), 5-HT and norepinephrine reuptake inhibitors (SNRIs), norepinephrine and specific 5-HT antidepressants (NaSSAs), 5-HT2 receptor antagonists and 5-HT reuptake inhibitors, norepinephrine and dopamine reuptake inhibitors, and melatonin receptor agonists.

The methods for measuring the antidepressant in human serum or plasma in domestic and foreign literature reports mainly comprise an HPLC-UV method, a GC-MS method and an LC-MS/MS method, for example, the literature of ' Quantification of 33 antidepressants by LC-MS/MS-compatible evaluation in white blood, and the literature of ' plasma and serum ' reports a method for detecting 33 antidepressants by liquid-liquid extraction, but the pretreatment requires complex procedures such as liquid-liquid extraction and nitrogen blowing concentration, and each sample collection requires 30 minutes. For example, Chinese patent (CN 108982714A) discloses "a simple and efficient method for detecting venlafaxine and its active metabolite o-desmethylvenlafaxine concentration in human plasma", the invention is to add 500 μ L of plasma sample into ether to perform liquid-liquid extraction, then re-dissolve and sample injection after nitrogen blowing, and then detect the concentration of the substance to be detected by high-efficiency liquid phase fluorescence method; the pretreatment of the method is time-consuming and labor-consuming, the sample consumption is large, and the analysis time of a single sample is longer. In addition, Chinese patent (CN 109655568A) discloses a method and a kit for simultaneously measuring 35 psychotropic drugs by high-efficiency liquid chromatography-mass spectrometry, and the method and the kit have the advantages of simple pretreatment, but poor accuracy due to the adoption of an external standard method for quantification. For another example, the journal of northwest pharmacy 2019, volume 34, No. 6, entitled "LC-MS/MS method for simultaneously determining mass concentrations of fluoxetine, norfluoxetine, paroxetine, and fluvoxamine in human plasma" used 200 μ L of plasma for protein precipitation, which is a large sample dosage; the roxithromycin is used as an internal standard of all objects to be detected, so that the universal applicability and accuracy are lacked, and matrix effect interference may exist; the linear range of 4 drugs was the same, and there was no clinical validation and lack of rationality. In addition, because clinical samples are extremely precious, the amount of pretreatment samples is as small as possible, and the large amount of samples can seriously pollute the instrument and increase the maintenance cost of the instrument. The single method for evaluating different antidepressants is greatly simplified and convenient for laboratory monitoring, and meets the clinical requirements.

Disclosure of Invention

The invention aims to provide a kit for detecting antidepressant 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 the antidepressant drugs in the serum by using the ultra-performance liquid chromatography tandem mass spectrometry technology.

The technical scheme of the invention is as follows:

a kit for detecting an antidepressant medicament in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,

the antidepressant drugs are respectively: bupropion (BPP), Agomelatine (AML), Hydroxyamphetanone (HBPP), Nortriptyline (NTP), o-desmethylvenlafaxine (NVLF), Mianserin (MAS), Mirtazapine (MTZ), Venlafaxine (VLF), Amitriptyline (ATP), Doxepin (DXP), Norfluoxetine (NFXT), Duloxetine (DLX), Fluoxetine (FXT), Fluvoxamine (FVX), Citalopram (CTP), Paroxetine (PXT), trazodone (TtS), Vortioxetine (VXT);

the isotope internal standard substances corresponding to the antidepressant drugs are respectively as follows: bupropion-d 9(BPP-d9), agomelatine-d 9 (AML-d 9), hydroxybupropion-d 9 (HBPP-d 9), o-desmethylvenlafaxine-d 9 (NVLF-d 9), nortriptyline-d 9 (NTP-d 9), mianserin-d 9 (MAS-d 9), mirtazapine-d 9 (MTZ-d 9), venlafaxine-d 9 (VLF-d 9), amitriptyline-d 9 (ATP-d 9), doxepin-d 9 (DXP-d 9), norfluoxetine-d 9 (NFXT-d 9), duloxetine-d 9 (DLX-d 9), fluoxetine-d 9 (FXT-d 9), fluvoxamine-d 9 (9-d 9), citalopram-d 9 (PXX-d 9), and CTP-d 9 (PXT-d 9), Trazodone-d 6(TZD-d6) and Volvoxetine-d 8(VXT-d 8).

The kit comprises the following reagents:

(1) mobile phase:

mobile phase A: water (containing 0.001-0.1% formic acid); mobile phase B: methanol (containing 0.001 to 0.1 percent of formic acid);

(2) a calibration sample:

calibrator samples at seven different concentration points:

s1, which comprises 1ng/mL each of AML/MAS/CTP/PXT, 10ng/mL each of BPP/HBPP/TZD, 2ng/mL each of NTP/MTZ/DXP/DLX, 5ng/mL each of VLF/ATP/NFXT/FXT, 20ng/mL of NVLF, FVX 4ng/mL and 0.5ng/mL of VXT;

s2, which comprises AML/MAS/CTP/PXT each 2.5ng/mL, BPP/HBPP/TZD each 25ng/mL, NTP/MTZ/DXP/DLX each 5ng/mL, VLF/ATP/NFXT/FXT each 12.5ng/mL, NVLF 50ng/mL, FVX 10ng/mL, VXT 1.25 ng/mL;

s3, which comprises AML/MAS/CTP/PXT each 5ng/mL, BPP/HBPP/TZD each 50ng/mL, NTP/MTZ/DXP/DLX each 10ng/mL, VLF/ATP/NFXT/FXT each 25ng/mL, NVLF 100ng/mL, FVX 20ng/mL, VXT 2.5 ng/mL;

s4, which comprises 25ng/mL each of AML/MAS/CTP/PXT, 250ng/mL each of BPP/HBPP/TZD, 50ng/mL each of NTP/MTZ/DXP/DLX, 125ng/mL each of VLF/ATP/NFXT/FXT, 500ng/mL each of NVLF, FVX 100ng/mL, VXT12.5 ng/mL;

s5, which comprises AML/MAS/CTP/PXT each 50ng/mL, BPP/HBPP/TZD each 500ng/mL, NTP/MTZ/DXP/DLX each 100ng/mL, VLF/ATP/NFXT/FXT each 250ng/mL, NVLF 1000ng/mL, FVX 200ng/mL, VXT25 ng/mL;

s6, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

s7, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

(3) mixing internal standard working solution:

methanol solutions comprising 200ng/mL BPP-d9, 20ng/mL AML-d6, 200ng/mL HBPP-d6, 100ng/mL NVLF-d6, 50ng/mL NTP-d3, 20ng/mL MAS-d3, 50ng/mL MTZ-d3, 100ng/mL VLF-d6, 100ng/mL ATP-d3, 50ng/mL DXP-d3, 100ng/mL NFXT-d5, 50ng/mL DLX-d7, 100ng/mL FXT-d5, 100ng/mL LFVX-d3, 20ng/mL CTP-d4, 20ng/mL T-d6, 200ng/mL TZD-d6, and 10ng/mL VXT-d 8.

(4) Protein precipitant:

any one of methanol and acetonitrile, or a mixed solution of methanol and acetonitrile;

(5) quality control product:

blank serum matrix containing antidepressant drugs is divided into low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,

QC (L) includes: 20ng/mL BPP, 2ng/mL AML, 20ng/mL HBPP, 4ng/mL NTP, 2ng/mLMAS, 4ng/mL MTZ, 10ng/mL VLF, 10ng/mL ATP, 4ng/mL DXP, 10ng/mL NFXT, 4ng/mL DLX, 10ng/mL FXT, 8ng/mL FVX, 2ng/mL CTP, 2ng/mL PXT, 20ng/mL TZD, 1ng/mL VXT and 40ng/mL LNVLF;

QC (M) comprises: 200ng/mL BPP, 20ng/mL AML, 200ng/mL HBPP, 40ng/mL NTP, 20ng/mL MAS, 40ng/mL MTZ, 100ng/mL VLF, 100ng/mL ATP, 40ng/mL DXP, 100ng/mL NFXT, 40ng/mL DLX, 100ng/mL FXT, 80ng/mL FVX, 20ng/mL CTP, 20ng/mL PXT, 200ng/mL TZD, 10ng/mL LVXT, and 400ng/mL NVLF;

QC (H) comprises 2000ng/mL BPP, 200ng/mL AML, 2000ng/mL HBPP, 400ng/mL NTP, 200ng/mL MAS, 400ng/mL MTZ, 1000ng/mL VLF, 1000ng/mL ATP, 400ng/mL DXP, 1000ng/mL LNFXT, 400ng/mL DLX, 1000ng/mL FXT, 800ng/mL FVX, 200ng/mL CTP, 200ng/mL PXT, 2000ng/mL TZD, 100ng/mL VXT and 4000ng/mL NVLF.

A kit for detecting an antidepressant medicament in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,

the antidepressant is BPP, AML, HBPP, NTP, NVLF, MAS, MTZ, VLF, ATP, DXP, NFXT, DLX, FXT, FVX, CTP, PXT, TZD, VXT;

the kit comprises the following reagents:

(1) mobile phase:

mobile phase A: 0.001 to 0.1 percent of formic acid aqueous solution; mobile phase B: 0.001 to 0.1 percent of formic acid methanol solution;

(2) mixing the standard solution and the blank serum matrix;

the mixed standard solution comprises 100000ng/mL BPP, 10000ng/mL AML, 100000ng/mL HBPP, 20000ng/mL NTP, 10000ng/mL MAS, 20000ng/mL MTZ, 50000ng/mL VLF, 50000ng/mL ATP, 20000ng/mL DXP, 50000ng/mL NFXT, 20000ng/mL DLX, 50000ng/mL FXT, 40000ng/mL FVX, 10000ng/mL CTP, 10000ng/mL CTT, 100000ng/mL ZD, 5000ng/mL VXT and 200000ng/mL LNVLF;

before detection, the mixed standard solution is diluted by a blank serum substrate according to a formula recorded in an instruction manual to prepare calibration sample samples S1-S7 of seven different concentration points and quality control solution QC (L), QC (M) and QC (H) of low, medium and high three concentrations:

s1, which comprises 1ng/mL each of AML/MAS/CTP/PXT, 10ng/mL each of BPP/HBPP/TZD, 2ng/mL each of NTP/MTZ/DXP/DLX, 5ng/mL each of VLF/ATP/NFXT/FXT, 20ng/mL of NVLF, FVX 4ng/mL and 0.5ng/mL of VXT;

s2, which comprises AML/MAS/CTP/PXT each 2.5ng/mL, BPP/HBPP/TZD each 25ng/mL, NTP/MTZ/DXP/DLX each 5ng/mL, VLF/ATP/NFXT/FXT each 12.5ng/mL, NVLF 50ng/mL, FVX 10ng/mL, VXT 1.25 ng/mL;

s3, which comprises AML/MAS/CTP/PXT each 5ng/mL, BPP/HBPP/TZD each 50ng/mL, NTP/MTZ/DXP/DLX each 10ng/mL, VLF/ATP/NFXT/FXT each 25ng/mL, NVLF 100ng/mL, FVX 20ng/mL, VXT 2.5 ng/mL;

s4, which comprises 25ng/mL each of AML/MAS/CTP/PXT, 250ng/mL each of BPP/HBPP/TZD, 50ng/mL each of NTP/MTZ/DXP/DLX, 125ng/mL each of VLF/ATP/NFXT/FXT, 500ng/mL each of NVLF, FVX 100ng/mL, VXT12.5 ng/mL;

s5, which comprises AML/MAS/CTP/PXT each 50ng/mL, BPP/HBPP/TZD each 500ng/mL, NTP/MTZ/DXP/DLX each 100ng/mL, VLF/ATP/NFXT/FXT each 250ng/mL, NVLF 1000ng/mL, FVX 200ng/mL, VXT25 ng/mL;

s6, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

s7, including AML/MAS/CTP/PXT each 250ng/mL, BPP/HBPP/TZD each 2500ng/mL, NTP/MTZ/DXP/DLX each 500ng/mL, VLF/ATP/NFXT/FXT each 1250ng/mL, NVLF 5000ng/mL, FVX 1000ng/mL, VXT125 ng/mL;

QC (L) includes: 20ng/mL BPP, 2ng/mL AML, 20ng/mL HBPP, 4ng/mL NTP, 2ng/mLMAS, 4ng/mL MTZ, 10ng/mL VLF, 10ng/mL ATP, 4ng/mL DXP, 10ng/mL NFXT, 4ng/mL DLX, 10ng/mL FXT, 8ng/mL FVX, 2ng/mL CTP, 2ng/mL PXT, 20ng/mL TZD, 1ng/mL VXT and 40ng/mL LNVLF;

QC (M) comprises: 200ng/mL BPP, 20ng/mL AML, 200ng/mL HBPP, 40ng/mL NTP, 20ng/mL MAS, 40ng/mL MTZ, 100ng/mL VLF, 100ng/mL ATP, 40ng/mL DXP, 100ng/mL NFXT, 40ng/mL DLX, 100ng/mL FXT, 80ng/mL FVX, 20ng/mL CTP, 20ng/mL PXT, 200ng/mL TZD, 10ng/mL LVXT, and 400ng/mL NVLF;

QC (H) comprises 2000ng/mL BPP, 200ng/mL AML, 2000ng/mL HBPP, 400ng/mL NTP, 200ng/mL MAS, 400ng/mL MTZ, 1000ng/mL VLF, 1000ng/mL ATP, 400ng/mL DXP, 1000ng/mL LNFXT, 400ng/mL DLX, 1000ng/mL FXT, 800ng/mL FVX, 200ng/mL CTP, 200ng/mL PXT, 2000ng/mL TZD, 100ng/mL VXT and 4000ng/mL NVLF;

(3) mixing internal standard working solution:

methanol solutions comprising 200ng/mL BPP-d9, 20ng/mL AML-d6, 200ng/mL HBPP-d6, 100ng/mL NVLF-d6, 50ng/mL NTP-d3, 20ng/mL MAS-d3, 50ng/mL MTZ-d3, 100ng/mL VLF-d6, 100ng/mL ATP-d3, 50ng/mL DXP-d3, 100ng/mL NFXT-d5, 50ng/mL DLX-d7, 100ng/mL FXT-d5, 100ng/mL LFVX-d3, 20ng/mL CTP-d4, 20ng/mL T-d6, 200ng/mL TZD-d6, and 10ng/mL VXT-d 8.

(4) Protein precipitant:

any one of methanol and acetonitrile, or a mixed solution of methanol and acetonitrile;

(5) the use instruction comprises: comprises calibrator samples S1-S7 and quality control solution QC (L), QC (M) and QC (H) with low, medium and high concentrations.

In a preferable embodiment, the mobile phase a is 0.005% to 0.05% aqueous formic acid, preferably 0.01% aqueous formic acid.

In one scheme, the protein precipitator is a mixed solvent of methanol and acetonitrile in a volume ratio of 1: 1-3, and preferably a mixed solvent of methanol and acetonitrile in a volume ratio of 1: 2.

The mixed standard solution mentioned in the present invention is prepared as follows: 5mg/mL of mother solution of an bupropion standard product, 2mg/mL of mother solution of an agomelatine standard product, 5mg/mL of mother solution of a hydroxybupropion standard product, 2mg/mL of mother solution of a nortriptyline standard product, 0.2mg/mL of mother solution of a mianserin standard product, 4mg/mL of mother solution of a mirtazapine standard product, 5mg/mL of mother solution of a venlafaxine standard product, 2mg/mL of mother solution of an amitriptyline standard product, 2mg/mL of mother solution of a doxepin standard product, 5mg/mL of mother solution of a norfluoxetine standard product, 2mg/mL of mother solution of a duloxetine standard product, 5mg/mL of mother solution of a fluoxetine standard product, 4mg/mL of mother solution of a fluvoxamine standard product, 0.5mg/mL of mother solution of a citalopram standard product, 2mg/mL of mother solution of a paroxetine standard product, 5mg/mL of a mother solution of a standard product of a trazodone, 1mg/mL of a valacitin standard product and 1mg/mL of desvenlaf The mother liquor of the standard substance is prepared into a mixed standard solution containing 100000ng/mL of bupropion, 10000ng/mL of agomelatine, 100000ng/mL of hydroxyamphetanone, 20000ng/mL of nortriptyline, 10000ng/mL of mianserin, 20000ng/mL of mirtazapine, 50000ng/mL of venlafaxine, 50000ng/mL of amitriptyline, 20000ng/mL of doxepin, 50000ng/mL of norfluoxetine, 20000ng/mL of duloxetine, 50000ng/mL of fluoxetine, 40000ng/mL of fluvoxamine, 10000ng/mL of citalopram, 10000ng/mL of paroxetine, 100000ng/mL of trazodone, 5000ng/mL of vortioxetine and 200000ng/mL of O-desmethylvenlafaxine by using a methanol solution.

The mixed internal standard working solution provided by the invention is prepared according to the following method: 1mg/mL BPP-d9, 0.1mg/mL TML-d 6, 0.1mg/mL HBPP-d6, 0.1mg/mL NVLF-d6, 0.1mg/mL NTP-d3, 0.1mg/mL MAS-d3, 0.1mg/mL MTZ-d3, 0.1mg/mL VLF-d6, 0.1mg/mL ATP-d3, 0.1mg/mL DXP-d3, 0.1mg/mL DXT-d 5, 0.1mg/mL DLX-d7, 0.1mg/mL T-d5, 0.1mg/mL FVX-d3, 0.1mg/mL CTP-d4, 0.1mg/mL PXT-d 8, 0.1mg/mL Tzd 6, and 0.01mg/mL VXT-d 9, 200ng/mL AML-d6, 2000ng/mL HBPP-d6, 1000ng/mL NVLF-d6, 500ng/mL NTP-d3, 200ng/mL MAS-d3, 500ng/mL MTZ-d3, 1000ng/mL VLF-d6, 1000ng/mL LATP-d3, 500ng/mL DXP-d3, 1000ng/mL NFXT-d5, 500ng/mL DLX-d7, 1000ng/mL FXT-d5, 1000ng/mL FVX-d3, 200ng/mL CTP-d4, 200ng/mL PXT-d6, 2000ng/mL Tml Td 6, and 100ng/mL ZD-d 8; and adding 900 mu L of methanol solution into 100 mu L of the mixed internal standard solution, and uniformly mixing to obtain the mixed internal standard working solution.

The substrate mentioned in the invention is serum or animal serum.

The blank serum matrix mentioned in the invention is blank serum without antidepressant drugs.

The application of the kit in detecting the antidepressant drug in the serum by using the ultra-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 antidepressant drugs in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology,

detecting the antidepressant in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly separating a target object to be detected from an interference component in a serum matrix by utilizing the ultra-high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method and 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 content of the antidepressant in the serum, wherein the specific chromatographic conditions are as follows:

(1) ultra-high performance liquid chromatography conditions:

mobile phase A: water (containing 0.001-0.1% formic acid);

mobile phase B: methanol;

a chromatographic column: phenomenex Kintex C18 (2.1X 100mm, 2.6 μm);

and (3) performing gradient elution by adopting the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 85:15 to 25:75 at a constant speed within 0-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 25:75 to 3:97 at a constant speed within 2.0-2.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 3:97 to 85:15 at a constant speed within 2.5-4.5 minutes. The flow rate is 0.2-0.5 mL/min, the collection time of each sample is 4.5min, the column temperature is 30-50 ℃, and the sample injection volume is 0.2-10 mu L;

(2) mass spectrum conditions:

in an electrospray ionization detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spray voltage was 3.0kV (ESI +); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; each target was monitored simultaneously with the isotope internal standard.

In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid into the mobile phase A, can effectively improve the ionization efficiency of certain target compounds, has higher sensitivity than the method for detecting the antidepressant drug in the serum by adopting an LC-MS/MS method in the prior art under the coordination of other conditions, has simple pretreatment process, low cost, high sensitivity and strong specificity, and completes the separation and detection of the antidepressant drug within 4.5 min. In a preferable embodiment, the mobile phase A is 0.005-0.05% formic acid solution in water, and the mobile phase B is 0.005-0.05% formic acid solution in methanol without affecting the effect of the invention. In a more preferred embodiment, mobile phase A is 0.01% formic acid in water and mobile phase B is 0.01% formic acid in methanol.

In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts 0.001-0.1% formic acid water solution and 0.001-0.1% formic acid methanol solution as mobile phase, the type of chromatographic column is as follows: phenomenex Kintex C18(2.1 × 100mm, 2.6 μm), under the cooperation of other conditions, endogenous substances do not interfere with the determination of the sample, the sensitivity is high, the specificity is strong, the cost is low, the pretreatment process is simple, the separation and the detection can be completed within 4.5min, and the precision and the accuracy meet the requirements.

When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The invention respectively adopts bupropion-d 9(BPP-d9), agomelatine-d 9 (AML-d 9), hydroxybupropion-d 9 (HBPP-d 9), o-norvenlafaxine-d 9 (NVLF-d 9), nortriptyline-d 9 (NTP-d 9), mianserin-d 9 (MAS-d 9), mirtazapine-d 9 (MTZ-d 9), venlafaxine-d 9 (VLF-d 9), amitriptyline-d 9 (ATP-d 9), doxepin-d 9 (DXP-d 9), norfluoxetine-d 9 (NFXT-d 9), duloxetine-d 9 (DLX-d 9), fluoxetine-d 9 (FXT-d 9), fluoxetine-d 9 (NFXT-d 9), and citalopram-d 9 (CTP-d 9), CTP-d 9 (CTP-d 9), duloxetine-d 9 (DLX-d 9), CTP-d 9, Trazodone-d 6(TZD-d6) and Volumin-d 8(VXT-d8) are used as internal standards, have the same retention time, chemical properties and matrix effect with the object to be tested, and have better reproducibility and accuracy when used for determining the antidepressant drug in serum.

In one embodiment, the flow rate is 0.2-0.5 mL/min, preferably 0.4 mL/min.

Further, the column temperature is 30-50 ℃, preferably 45 ℃.

Furthermore, the injection volume is 0.2-10 μ L, preferably 1 μ L.

In a preferred scheme, when the ultra-high performance liquid chromatography tandem mass spectrometry technology is adopted to detect the antidepressant drug in the serum, the specific chromatographic conditions are as follows:

(1) high performance liquid chromatography conditions:

mobile phase A: water (0.01% formic acid);

mobile phase B: methanol (containing 0.01% formic acid);

a chromatographic column: phenomenex Kintex C18 (2.1X 100mm, 2.6 μm);

gradient elution is carried out by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, and the gradient elution is shown in a table 1; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 85:15 to 25:75 at a constant speed within 0-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 25:75 to 3:97 at a constant speed within 2.0-2.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 3:97 to 85:15 at a constant speed within 2.5-4.5 minutes; the flow rate was 0.4mL/min, the time for each sample collection was 4.5min, the column temperature was 45 ℃ and the sample injection volume was 1. mu.L.

TABLE 1 mobile phase gradient elution parameters

(2) Mass spectrum conditions:

in an electrospray ionization detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spray voltage was 3.0kV (ESI +); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; simultaneously monitoring each target and the isotope internal standard; the declustering voltage and collision voltage of each target are shown in table 2:

TABLE 2 antidepressant drug detection Mass Spectrometry parameters

In one protocol, pre-treated serum was prepared as follows: adding the mixed internal standard working solution into the serum, adding a protein precipitator after vortex, and taking supernatant after vortex centrifugation; wherein the protein precipitant is any one of methanol and acetonitrile, or a mixed solution of methanol and acetonitrile.

Preferably, the protein precipitant is a mixed solution of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 1: 1-3, and the volume ratio of methanol to acetonitrile in the protein precipitant is 1:2, for example, without affecting the effect of the invention.

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

when the kit is used for detecting the antidepressant drugs in the serum, the pretreatment process is simple, the sensitivity is high, the sample dosage is small, the analysis time of a single sample is short, the flux is high, the separation and detection of the 18 antidepressant drugs in the serum are completed within 4.5min, the isotope internal standard quantitative method is high in accuracy, and the accuracy and the precision basically meet the requirements, so that the kit can be used for pharmacokinetic and clinical research of clinical combined medication, and provides a reliable detection method for monitoring the concentration of the antidepressant drugs in clinical.

Drawings

FIG. 1 is an extracted ion flow chromatogram of an antidepressant drug standard.

FIG. 2 is an extracted ion flow chromatogram of an antidepressant drug in serum.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.