阅读说明：本技术 一种临床研究血浆样品中喹硫平浓度的分析方法 (Analysis method for clinically researching concentration of quetiapine in plasma sample ) 是由 陈云辉 钟勘 陈鑫 杨勇 钮小英 高妍 姜金方 周茂金 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种临床研究血浆样品中喹硫平浓度的分析方法,包括以下步骤：步骤1：血浆样品前处理；步骤2：色谱分离；步骤3：质谱检测。本发明的分析方法具有操作简单、通量高、灵敏度高、精密和准确。(The invention relates to an analysis method for clinically researching the concentration of quetiapine in a plasma sample, which comprises the following steps: step 1: plasma sample pretreatment; step 2: carrying out chromatographic separation; and step 3: and (4) detecting by mass spectrometry. The analysis method has the advantages of simple operation, high flux, high sensitivity, precision and accuracy.)

1. An analytical method for clinical study of quetiapine concentration in a plasma sample, comprising the steps of:

step 1: plasma sample pretreatment, adding the plasma sample, an internal standard solution and acetonitrile into a 96-well plate, carrying out vortex oscillation and centrifugation, taking out a supernatant, transferring the supernatant into the 96-well plate, and carrying out LC-MS/MS analysis;

step 2: carrying out chromatographic separation;

chromatographic conditions, chromatographic column: XDB-C18 column, 1.8 μm, 50X 4.6 mm; sample introduction amount: 1 mu L of the solution; column temperature: 40 ℃; the mobile phase A is 10mM ammonium acetate aqueous solution containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, and the volume ratio is 40: 60A: b, isocratic elution; the autosampler temperature was 4 ℃ and the flow rates of the mobile phases were: 0.5 mL/min;

and step 3: detecting mass spectrum;

mass spectrum conditions: an electrospray ionization source; the voltage is 5500V; the ion source temperature is 500 ℃; curtain Gas and CID pressures were 35 and 9psi, respectively; the scan mode is + MRM; the monitoring ion reaction is respectively as follows: quetiapine, m/z 384.1 → m/z 279.2; quetiapine-d 8, m/z 392.2 → m/z 286.1; CE is 38 eV; DP is 80V; the scan time is 250 ms.

2. The assay of claim 1, wherein the assay comprises: step 1, plasma sample pretreatment, 50.0 mu L of plasma sample is taken, 50.0 mu L of internal standard working solution and 500 mu L of methanol are respectively added, vortex flow is carried out for 10min, centrifugation is carried out for 10min at 4500rpm, 200 mu L of supernatant is taken out and transferred to a 96-well plate, and 1.00 mu L of supernatant is taken for LC-MS/MS analysis.

Technical Field

The invention relates to an analysis method for clinically researching the concentration of quetiapine in a plasma sample.

Background

The quetiapine fumarate sustained release tablet is an atypical antipsychotic drug and has interaction with various neurotransmitter receptors. Developed by the pharmaceutical company of Aslicon, 2007 is approved by the FDA to be marketed and becomes an FDA approved drug for treating schizophrenia. In order to support clinical research on quetiapine and the development of a simulated drug, a method for detecting the concentration of quetiapine in a human plasma sample is required. The related art currently used to detect the concentration of quetiapine in biological matrices is less disclosed. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for drug concentration determination in biological matrices due to its excellent selectivity, sensitivity, throughput and applicability. The invention develops a method for detecting the concentration of quetiapine in a clinical research plasma sample based on an LC-MS/MS technology.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an analysis method for clinically researching the concentration of quetiapine in a plasma sample.

In order to achieve the purpose, the invention adopts the following technical scheme:

an analysis method for clinically researching the concentration of quetiapine in a plasma sample comprises the following steps:

step 1: plasma sample pretreatment, adding the plasma sample, an internal standard solution and acetonitrile into a 96-well plate, carrying out vortex oscillation and centrifugation, taking out a supernatant, transferring the supernatant into the 96-well plate, and carrying out LC-MS/MS analysis;

step 2: carrying out chromatographic separation;

chromatographic conditions, chromatographic column: XDB-C18 column, 1.8 μm, 50X 4.6 mm; sample introduction amount: 1 mu L of the solution; column temperature: 40 ℃; the mobile phase A is 10mM ammonium acetate aqueous solution containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, and the volume ratio is 40: 60A: b, isocratic elution; the autosampler temperature was 4 ℃ and the flow rates of the mobile phases were: 0.5 mL/min;

and step 3: detecting mass spectrum;

mass spectrum conditions: an electrospray ionization source; the voltage is 5500V; the ion source temperature is 500 ℃; curtain Gas and CID pressures were 35 and 9psi, respectively; the scan mode is + MRM; the monitoring ion reaction is respectively as follows: quetiapine, m/z 384.1 → m/z 279.2; quetiapine-d 8, m/z 392.2 → m/z 286.1; CE is 38 eV; DP is 80V; the scan time is 250 ms.

Preferably, the plasma sample is pretreated in the step 1, 50.0 μ L of plasma sample is taken, 50.0 μ L of internal standard working solution and 500 μ L of methanol are respectively added, vortex flow is carried out for 10min, centrifugation is carried out for 10min at 4500rpm, 200 μ L of supernatant is taken out and transferred to a 96-well plate, and 1.00 μ L of supernatant is taken for LC-MS/MS analysis.

By the scheme, the invention at least has the following advantages:

1. the invention adopts a simple protein precipitation method to treat the plasma sample, does not have a complex concentration process and has higher pretreatment flux.

2. The detection method has high sensitivity, the signal-to-noise ratio of the quantitative lower limit sample with the concentration of 2.00ng/mL is about 69, the detection limit is about 0.100ng/mL through calculation, and the chromatographic sample injection amount is only 1.00 mu L.

3. The instrument for detecting the quetiapine plasma concentration has high analysis speed, and the chromatographic running time is only 2 min.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a full scan mass spectrum of the product ion of quetiapine of the present invention;

FIG. 2 is a product ion full scan mass spectrum of internal standard quetiapine-d 8 of the present invention;

FIG. 3 is an MRM chromatogram of a human plasma blank human plasma sample of quetiapine (left) and quetiapine-d 8 (right) of the present invention;

FIG. 4 is an MRM chromatogram of quetiapine (left) and quetiapine-d 8 (right) of the present invention in a lower limit sample of quantitation;

FIG. 5 is a graph of drug concentration versus time for 1 subject of the present invention after oral administration of quetiapine fumarate sustained release tablets.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

The development of a method for detecting the concentration of a drug in blood plasma by liquid chromatography-tandem mass spectrometry can be generally divided into three parts, namely an extraction method (namely a pretreatment method), a liquid chromatography method and a mass spectrometry method. The invention aims at the defects of the prior art and sets up an analysis method from the three aspects.

Examples

As shown in fig. 1 and 2, an analysis method for clinical research of quetiapine concentration in plasma samples comprises the following steps:

step 1: plasma sample pretreatment, adding the plasma sample, an internal standard solution and acetonitrile into a 96-well plate, carrying out vortex oscillation and centrifugation, taking out a supernatant, transferring the supernatant into the 96-well plate, and carrying out LC-MS/MS analysis;

step 2: carrying out chromatographic separation;

chromatographic conditions, chromatographic column: XDB-C18 column, 1.8 μm, 50X 4.6 mm; sample introduction amount: 1 mu L of the solution; column temperature: 40 ℃; the mobile phase A is 10mM ammonium acetate aqueous solution containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, and the volume ratio is 40: 60A: b, isocratic elution; the autosampler temperature was 4 ℃ and the flow rates of the mobile phases were: 0.5 mL/min;

and step 3: detecting mass spectrum;

mass spectrum conditions: an electrospray ionization source; the voltage is 5500V; the ion source temperature is 500 ℃; curtain Gas and CID pressures were 35 and 9psi, respectively; the scan mode is + MRM; the monitoring ion reaction is respectively as follows: quetiapine, m/z 384.1 → m/z 279.2; quetiapine-d 8, m/z 392.2 → m/z 286.1; CE is 38 eV; DP is 80V; the scan time is 250 ms.

In the mass spectrum condition, the + MRM scanning mode is selected, and the positive ion scanning mode is selected finally because the quetiapine positive ion response is good, the interference peak is less in the positive ion mode, and the signal to noise ratio is high.

Example one

Description of abbreviations

1 Material

1.1 instruments

LC-30AD ultra-high performance liquid chromatography system and AB Sciex Triple Quad manufactured by Shimadzu corporation^TM6500⁺A type triple quadrupole tandem mass spectrometer equipped with an ESI source.

The data processing adopts Analyst 1.6.3 software.

Model CP225D analytical balance, beijing sydolis instruments ltd.

Japanese greeting-silent Z326K type desk type centrifugal machine

Eppendorf company 5424R, Germany, small high-speed refrigerated centrifuge.

1.2 standards and reagents

Quetiapine fumarate (content 99.8%); quetiapine fumarate-d 8 (99.0% content, 98.8% isotopic purity); methanol was purchased from Sigma, usa; formic acid was purchased from TCI corporation, japan; ammonium acetate was purchased from ROE corporation, usa; deionized water was prepared by a Millipore water purifier. The reagents are all chromatographically pure.

2 method

2.1 preparation of the solution

Standard Curve sample preparation

Quetiapine fumarate control was weighed, dissolved in methanol and brought to volume at a concentration of about 1.00 mg/mL. Sucking appropriate amount of the quetiapine stock solution, and gradually diluting with methanol-water (50/50, v/v) to obtain series concentration standard solutions, wherein the quetiapine concentration is 40.0, 80.0, 200, 600, 2000, 4000, 8000, and 10000ng/mL respectively. The working solutions were diluted with blank plasma to obtain standard curve samples with quetiapine concentrations of 2.00, 4.00, 10.0, 30.0, 100, 200, 400, and 500ng/mL, respectively.

Internal standard working fluid

Quetiapine fumarate-d 8 control was weighed, dissolved in methanol and metered. And precisely sucking a proper amount of internal standard stock solution, and diluting the internal standard stock solution by methanol-water (50/50, v/v) step by step to obtain an internal standard working solution with the concentration of 20.0 ng/mL.

2.2 plasma sample treatment

50.0 mu L of plasma sample is taken, 50.0 mu L of internal standard working solution and 500 mu L of methanol are respectively added, vortex flow is carried out for 10min, centrifugation is carried out for 10min at the rotating speed of 4500rpm, 200 mu L of supernatant is taken out and transferred to a 96-well plate, and 1.00 mu L of supernatant is taken for LC-MS/MS analysis.

2.3 chromatographic and Mass Spectrometry conditions

Chromatographic conditions

Conditions of Mass Spectrometry

2.4 methodological validation

Selectivity is

And respectively taking blank human plasma, a LLOQ sample and a plasma sample after a subject takes 200mg quetiapine fumarate sustained release tablets, processing and analyzing to obtain corresponding chromatograms (figure 3 and figure 4). The retention time of both quetiapine and internal standard quetiapine-d 8 is 1.09min, and no interference peak exists at the retention time in a blank plasma spectrogram.

Standard curve

Linear regression calculation was performed with the theoretical concentration of quetiapine as the abscissa (x) and the peak area ratio of quetiapine to quetiapine-d 8 as the ordinate (y) (weight factor W is 1/x)²) The regression equation of quetiapine is typically 0.0345x +0.00187, 0.9985, and quetiapine is obviously linear in the range of 2.00-500 ng/mL.

Accuracy, precision and stability

And diluting quetiapine stock solution by using human blank plasma to prepare LQC, MQC, HQC (quetiapine concentration is 6.00, 30.0 and 375ng/mL) and LLOQ (quetiapine concentration is 2.00ng/mL), and verifying six samples of quality control samples with four concentrations determined in each analysis batch by using the method. The precision within and during LLOQ day is acceptable as the Relative Standard Deviation (RSD) < 20%, and the precision (RE) is acceptable between-20% and 20%. The QC samples of the other concentration levels have within-day and daytime precision (RSD) of less than 15 percent and have accuracy (RE) of between-15 and 15 percent.

The results shown in Table 1 show that the precision and accuracy of the quetiapine determined by the method are both acceptable, and the LLOQ of the quetiapine is 2.00 ng/mL.

TABLE 1

And taking LQC and HQC samples, and observing the stability of the LQC and HQC samples after the LQC and the HQC samples are placed for 20 hours at room temperature, repeatedly frozen and thawed for 5 times and placed for 41 days at the temperature of minus 70 ℃.

The results, shown in Table 2, indicate that quetiapine is stable under the above conditions, and RE is between-6.4% and-2.7%.

TABLE 2

Recovery and matrix effects

Six samples of QC samples were analyzed at low, medium and high concentrations. Meanwhile, taking 50.0 mu L of blank plasma, processing according to plasma samples, adding a quetiapine control solution and an internal standard working solution into the supernatant, mixing by vortex, and carrying out sample injection measurement, wherein the average peak area ratio of quetiapine in 2 samples is the recovery rate. The results show that the extraction recovery rates of the three concentration levels are respectively 99.9%, 100.9% and 101.8%, and the CV% of the different concentration levels are respectively 1.3%, 0.7% and 1.7%. The recovery of the internal standard was 96.2%.

Taking blank plasma (n is 6) from different sources, processing according to plasma samples (without adding internal standard), adding a control solution and a mixed internal standard working solution into supernate, and carrying out sample injection and determination. Another 50.0 μ L deionized water was treated as above and subjected to sample injection for measurement. And calculating the matrix factor according to the peak area ratio of the two samples of quetiapine. The matrix factors of quetiapine at LQC and HQC concentrations were 97.3% and 94.5%, respectively, with RSDs less than 2.0%, indicating that the effect of matrix effects on quetiapine assay was negligible under the conditions of this experiment.

Summary of method verification

The method is verified by methodology, the method is extremely high in sensitivity, good in selectivity, accuracy, precision and stability and good in linearity, and all indexes meet the requirements of 2015 national drug code appendix 9012 biological sample quantitative analysis method verification guiding principle

Pharmacokinetics study

The kit is used for determining the concentration of quetiapine in blood plasma and is used for human pharmacokinetics research of quetiapine tablets. The clinical study was approved by the hospital ethics committee, and subjects were informed of trial risk prior to the trial and voluntarily signed an informed consent. Healthy subjects were given 200mg quetiapine fumarate sustained release tablets. At different time points before (0h) and after administration, blood is taken from veins, placed in an anticoagulation centrifuge tube, centrifuged, and plasma is separated for determination. As shown in fig. 5, one subject showed a drug concentration-time curve. The invention can be used for human pharmacokinetics research and bioequivalence research of quetiapine tablets.

The invention has at least the following advantages:

1. the invention adopts a simple protein precipitation method to treat the plasma sample, does not have a complex concentration process and has higher pretreatment flux.

2. The detection method has high sensitivity, the signal-to-noise ratio of the quantitative lower limit sample with the concentration of 2.00ng/mL is about 69, the detection limit is about 0.100ng/mL through calculation, and the chromatographic sample injection amount is only 1.00 mu L.

3. The instrument for detecting the quetiapine plasma concentration has high analysis speed, and the chromatographic running time is only 2 min.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.