CN112697935A

CN112697935A - Method for simultaneous determination of perindopril and perindopril A concentration in human plasma

Info

Publication number: CN112697935A
Application number: CN202011436093.4A
Authority: CN
Inventors: 周艺; 戴智; 尹小丽
Original assignee: Hunan Taixin Medicine Technology Co ltd
Current assignee: Hunan Taixin Medicine Technology Co ltd
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-04-23
Anticipated expiration: 2040-12-10
Also published as: CN112697935B

Abstract

The invention belongs to the technical field of medical detection, and discloses a method for simultaneously measuring the concentrations of perindopril and perindopril A in human plasma, wherein perindopril tert-butylammonium salt and perindopril A are respectively weighed and dissolved by methanol and DMSO to prepare standard reference substance stock solutions; diluting with acetonitrile aqueous solution to prepare perindopril and perindopril A working solution; mixing perindopril and perindopril A working solution to prepare a mixed standard curve working solution; diluting the mixed standard curve working solution into blank plasma to prepare a standard curve plasma sample; and sequentially adding a precipitator, a mixed internal standard working solution and a standard curve plasma sample into the sample hole, carrying out vortex centrifugation, and carrying out sample injection on an LC-MS/MS system to obtain a chromatogram. The invention solves the problem of much interference of plasma matrix, has high sensitivity, can ensure that plasma samples from different matrix sources are all at the same response level, has accurate and reliable detection result without being influenced by the matrix.

Description

Method for simultaneous determination of perindopril and perindopril A concentration in human plasma

Technical Field

The invention relates to the technical field of medicine detection, in particular to a method for simultaneously measuring the concentrations of perindopril and perindopril A in human plasma.

Background

Perindopril (Perindopril) is mainly used for treating essential hypertension and heart failure, is an Angiotensin Converting Enzyme (ACE) inhibitor which is developed by Schweiya, France, in the eighties of the last century, does not contain a sulfhydryl group, is a chiral medicine with good clinical curative effect, high safety and small side effect in the current Angiotensin Converting Enzyme Inhibitor (ACEIs) antihypertensive medicines in the world, and has long action time, good tolerance and good market prospect. Perindopril is a metabolite of perindopril that differs from perindopril in chemical and physical properties, and in order to ensure that both are present in plasma at concentrations, the assay procedure needs to be reasonably adjusted and matched during the assay.

In the existing detection method, plasma is pretreated mainly by a protein precipitation method, so that the condition that phospholipid in plasma impurities cannot be effectively removed exists, matrix effect is difficult to avoid in the detection process, and the quantification of analytes is influenced. The existing method for analyzing perindopril related substances, for example, in the literature (chinese new drug journal, vol.17, No. 21, No. 1860-1864, gecko, etc., liquid chromatography-tandem mass spectrometry is used for measuring perindopril and its active metabolite perindopril A in human plasma), the concentrations of perindopril and perindopril A in plasma are measured by adopting a protein precipitation method, and detection is carried out by adopting a Phenomenex Cu-tandem mass spectrometry in a manner of detecting positive ions by selective reaction, but the method has obvious matrix effect and inconsistent matrix effect levels of different concentrations, and has limitation in the practical application process.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a method for simultaneously measuring the concentrations of perindopril and perindopril A in human plasma, the concentrations of perindopril and perindopril A in human plasma are simultaneously measured by adopting an LC-MS/MS method, effective separation of perindopril and perindopril A is realized by optimizing liquid phase conditions, the problem of plasma matrix effect is solved, plasma samples from different matrix sources can be ensured to be in the same response level, the detection sensitivity is high, and the result is accurate and reliable.

In order to solve the technical problems, the invention adopts the following technical scheme:

in one aspect, the present invention provides a method for the simultaneous determination of perindopril and perindopril A concentration in human plasma comprising the steps of:

1) preparation of standard series working solution: weighing perindopril tert-butyl ammonium salt and perindopril A, respectively placing the perindopril T-butyl ammonium salt and the perindopril A in volumetric flasks, and respectively dissolving the perindopril T-butyl ammonium salt and the perindopril A in methanol and DMSO to prepare standard reference substance stock solutions; diluting the standard reference substance stock solution with 50% acetonitrile water solution to respectively prepare a standard series of perindopril working solution and perindopril base working solution; mixing a perindopril working solution and a perindopril base working solution of a standard series with equal volume to prepare a mixed standard curve working solution;

2) preparation of standard curve plasma samples: taking a standard series of mixed standard curve working solutions, diluting the standard series of mixed standard curve working solutions into blank plasma, and preparing a series of concentration standard curve plasma samples;

3) and (3) drawing a standard curve regression equation: adding a precipitator into the sample hole, adding a mixed internal standard working solution and a standard curve plasma sample, carrying out vortex and centrifugation, and then carrying out sample injection on an LC-MS/MS system to obtain a chromatogram; calculating the concentration of perindopril in blood plasma according to the ratio of the chromatographic peak area of perindopril to the peak area of the internal standard in the obtained chromatogram, and making a regression equation of a standard curve of perindopril; and calculating the concentration of perindopril A in blood plasma according to the ratio of the chromatographic peak area of perindopril A to the internal standard peak area in the obtained chromatogram, and establishing a standard curve regression equation of perindopril A.

Further, the concentration of the standard reference substance stock solution is 1mg/mL^-1(ii) a The precipitant is 3% HClO₄And (3) solution.

Further, the concentration range of the standard series of perindopril working solutions is 40-4000 ng/mL; the concentration range of the perindopril A working solution of the standard series is 16-800 ng/mL; the concentration range of the perindopril working solution in the mixed standard curve working solution is 20-2000 ng/mL, and the concentration range of the perindopril working solution is 8-400 ng/mL; the concentration range of the perindopril working solution in the mixed standard curve working solution is preferably 2000, 1700, 1200, 600, 300, 160, 40 and 20ng/mL, and the concentration range of the perindopril base working solution is preferably 400, 340, 240, 120, 60, 30, 16 and 8 ng/mL.

Further, in the plasma sample of the standard curve, the concentration range of perindopril is 1-100 ng/mL, preferably 1, 2, 8, 15, 30, 60, 85, 100ng/mL, and the concentration range of perindopril is 0.4-20 ng/mL, preferably 0.4, 0.8, 1.5, 3, 6, 12, 17, 20 ng/mL.

Further, the standard curve regression equation is obtained as follows: adding 200 μ L of precipitant into the sample well, adding 50 μ L of mixed internal standard working solution, 50 μ L of standard curve plasma sample, sealing the plate, vortexing for 5min, centrifuging, 2490g, and 10 min; 15 mu L of sample injection LC-MS/MS system to obtain a chromatogram; calculating the concentration of perindopril in blood plasma according to the ratio of the chromatographic peak area of perindopril to the peak area of the internal standard in the obtained chromatogram, and making a regression equation of a standard curve of perindopril; and calculating the concentration of perindopril A in blood plasma according to the ratio of the chromatographic peak area of perindopril A to the internal standard peak area in the obtained chromatogram, and establishing a standard curve regression equation of perindopril A.

Further, the preparation process of the mixed internal standard working solution is as follows: weighing perindopril tert-butylamine salt-d₄And perindopril-d₄Respectively dissolving in DMSO in volumetric flasks to obtain 10mg/mL solutions^-1Perindopril-d of₄Internal standard reference stock solution and perindopril-d₄An internal standard control stock solution;

perindopril-d₄Diluting the stock solution of the internal standard reference substance with DMSO to obtain the concentration of 1mg/mL^-1Perindopril-d of₄Internal standard working solution;

perindoprilat-d₄Diluting the stock solution of the internal standard reference substance with DMSO to obtain the concentration of 1mg/mL^-1Perindoprilat-d of₄Internal standard working solution;

respectively taking perindopril-d₄Internal standard working solution 50 mu L, perindopril A-d₄Placing 5 mu L of internal standard working solution into a 1ml brown volumetric flask, adding 50% acetonitrile aqueous solution to constant volume to a scale mark, and shaking up; further diluting 50 μ L of the mixed solution with 50% acetonitrile water solution to 50mL to obtain perindopril-d₄Perindoprilat-d₄＝50/5ng·mL^-1Mixed internal standard working solution of (1).

Further wherein the conditions of the LC-MS/MS system are as follows:

chromatographic conditions

A chromatographic column: agilent ZORBAX SB-Aq (4.6X 150mm, 5 μm)

Protection of the column: ACE 5C18 ANALYTICL GRD CART (X5)

Mobile phase A: 0.5% aqueous formic acid; mobile phase B: methanol

Flow rate: 1 mL/min; stopping time: 5min

Washing the needle washing liquid: 5 s; sample introduction amount: 15 mu L of the solution; column temperature: 40 ℃;

the mobile phase ratio was set as follows:

	Time	A	B	Flow	Max Pressure
						1	0.00min	95.0％	5.0％	1mL/min	1300bar
2	1.80min	20.0％	80.0％	1mL/min	1300bar
						3	3.80min	20.0％	80.0％	1mL/min	1300bar
4	3.81min	95.0％	5.0％	1mL/min	1300bar
						5	5.00min	95.0％	5.0％	1mL/min	1300bar

conditions of Mass Spectrometry

ESI source is adopted, ionization mode: electrospray ionization positive ion mode (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM);

the mass spectrum switching valve is set as follows:

	Start Time(min)	Div Valve
			1	0	To Waste
2	2	To MS

the ion source parameters were set as follows:

Parameter	Value(+)
		Gas Temp(℃)	350
Gas Flow(L/min)	10
		Nebulizer(psi)	45
SheathGasHeater	350
		SheathGasFlow	11
Capillary(V)	4000
		VCharging	1000

。

furthermore, after the detection of the LC-MS/MS system, perindopril and perindopril A respectively generate peaks at about 3.38min and 3.34min, the peak shapes are good, and no impurity peak in human plasma interferes with the determination of the compounds, which shows that the method has strong specificity.

Further, the regression equation of the standard curve is as follows:

the regression equation of the standard curve of perindopril in human plasma was determined as: y 0.017626x +7.093819E-004, R²0.99797166; the linear range of perindopril is 1-100 ng/mL, and the lowest quantitative lower limit of blood concentration of perindopril is 1 ng/mL;

the regression equation of the standard curve of perindopril measured in human plasma is: 0.246053x-0.003765, R²0.99374085; the linear range of perindopril A is 0.4-20 ng/mL, and the lowest quantitative lower limit of blood concentration of perindopril A is 0.4 ng/mL.

The invention selects a chromatographic column: agilent ZORBAX SB-Aq (4.6X 150mm, 5 μm); the response value to perindopril and perindopril in blood plasma is highest, the peak shape is good, the response value is symmetrical and does not trail, and the peak-off time is not too early, nor too late, and is just right.

Considering the influence of the flow rate on the drug peak-out time and the influence of the peak-out effect caused by overlarge sample injection amount, the flow rate is selected to be 1mL/min, and the sample injection amount is 15 mu L, so that the drug peak-out effect and the response value can be ensured, the drug can be completely eluted, the pollution to a chromatographic column is avoided, and the recovery rate and the sensitivity of mass spectrometry detection are improved.

According to the invention, perindopril and perindopril in blood plasma are detected by adopting a gradient elution mode, the elution proportion of a mobile phase is changed at different time, and the problems of blood plasma matrix effect and the like are comprehensively considered, so that the peak shape of a drug is symmetrical and perfect, the drug can be sufficiently eluted, the recovery rate is high, and the interference is small.

The method can obtain high recovery rate, effectively reduce the interference of impurity peaks and avoid the influence caused by plasma matrix effect in the traditional method.

The method has systematic applicability, has no selectivity on blank blood plasma from different sources, can specifically measure perindopril and perindopril A, and has good separation degree.

The method has small dosage, only 50 mu L of blood plasma sample is needed for each test, only 15 mu L of sample is needed for sample injection, and the compound can be accurately determined.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a simple method for measuring perindopril and perindopril A concentration in human plasma by a pretreatment method, adopts a protein precipitation method, and is suitable for conventional measurement; meanwhile, under the chromatographic conditions adopted in the experiment, the retention time of perindopril is about 3.38min, the retention time of perindopril is about 3.34min, the peak shape is good, the measurement is free from the interference of miscellaneous peaks, and the base line is stable; the method has higher specificity, can accurately measure the concentrations of perindopril and perindopril A in blood plasma, has higher sensitivity, and has the lowest quantitative limit of the blood plasma of perindopril: 1ng/mL, perindopril A: 0.4 ng/mL; the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of perindopril and perindopril A. According to the method, the linear ranges of the standard plasma curves of perindopril and perindopril A are respectively perindopril: 1-100 ng/mL, perindopril A: 0.4-20 ng/mL, and the precision RSD in and among batches is less than +/-15%. The method has good reproducibility and high accuracy, is not interfered by a substrate, a hyperlipemia substrate and a hemolysis substrate, and has no interference phenomenon of the blood plasma of different crowds to the method.

The method can meet the pharmacokinetic requirement of human bodies and can be applied to clinical pharmacokinetic research.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 standard graph of perindopril in human plasma measured by LC-MS/MS method;

FIG. 2 standard graph of perindopril A in human plasma measured by LC-MS/MS method;

FIG. 3 human plasma perindopril and perindopril-d measured by LC-MS/MS method₄Liquid mass diagram of (1);

FIG. 4 Perindoprilat and Perindoprilat-d in human plasma measured by LC-MS/MS method₄Liquid mass diagram of (1);

FIG. 5 Perindopril positive ion multi-reaction detection scanning mass spectrogram;

FIG. 6 Perindopril-d₄Scanning mass spectrogram by positive ion multi-reaction detection;

FIG. 7 Perindoprilat positive ion multi-reaction detection scanning mass spectrogram;

FIG. 8 Perindoprilat-d₄And (3) detecting and scanning a mass spectrogram by positive ion multi-reaction.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

1. Main instrument

An Agilent G6470 model triple quadrupole LC MS equipped with a liquid chromatograph (including an autosampler), electrospray ionization ion source (ESI), and Agilent Mass Hunter Workstation Software data processing System (Agilent, USA); the METTLER TOLEDO AB 135-model S one hundred thousandth of a balance (METTLER, switzerland).

2. Chromatographic conditions

A chromatographic column: agilent ZORBAX SB-Aq (4.6X 150mm, 5 μm)

Protection of the column: ACE 5C18 ANALYTICL GRD CART (X5)

Mobile phase A: 0.5% aqueous formic acid; mobile phase B: methanol

Flow rate: 1 mL/min; stopping time: 5min

the mobile phase ratio was set as follows:

Time

A

B

Flow

Max Pressure

1

0.00min

95.0％

5.0％

1mL/min

1300bar

2

1.80min

20.0％

80.0％

1mL/min

1300bar

3

3.80min

20.0％

80.0％

1mL/min

1300bar

4

3.81min

95.0％

5.0％

1mL/min

1300bar

5

5.00min

95.0％

5.0％

1mL/min

1300bar

3. conditions of Mass Spectrometry

the mass spectrum switching valve is set as follows:

	Start Time(min)	Div Valve
			1	0	To Waste
2	2	To MS

the ion source parameters were set as follows:

4. anticoagulant: heparin sodium

5. Internal standard: perindopril-d₄Perindoprilat-d₄

6. Data processing

The retention time and peak area of the chromatogram were collected and processed by Agilent Mass Hunter Workstation Software and analyzed quantitatively, using Agilent ECM network system. And (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation.

7. The pretreatment method before the detection of the sample comprises the following steps:

taking whole blood, adding anticoagulant heparin sodium, centrifuging to obtain blank plasma, refrigerating for sample dilution or preparation, and thawing before use.

Adding 3% HClO₄Adding 50 mu L of mixed internal standard working solution into the solution, finally adding 50 mu L of standard curve plasma sample, whirling for 5min, centrifuging, 2490g, and carrying out sample injection at 4 ℃ for 10 min.

Adding 3% HClO₄Adding 50 mu L of mixed internal standard working solution into the solution, finally adding 50 mu L of SST, vortexing for 5min, centrifuging, 2490g, and carrying out sample injection at 4 ℃ for 10 min.

Adding 3% HClO₄Adding 50 mu L of mixed internal standard working solution into the solution, finally adding 50 mu L of quality control plasma sample, whirling for 5min, centrifuging, 2490g, and carrying out sample injection at 4 ℃ for 10 min.

Adding 3% HClO₄Solution, 50 μ L of 50% acetonitrile in water, final addition of 50 μ L of blank plasma sample, vortexed for 5min, centrifuged, 2490g, 4 ℃, 10min, ready for injection, recorded as: double blank or Carryover samples.

Adding 3% HClO₄Adding 50 mu L of mixed internal standard working solution into the solution, finally adding 50 mu L of blank plasma sample, vortexing for 5min, centrifuging, 2490g, 4 ℃, and 10min, and recording as: blank sample.

Adding 3% HClO₄Adding 50 mu L of 50% acetonitrile water solution into the solution, finally adding 50 mu L of ultrapure water, whirling for 5min, centrifuging, 2490g, 4 ℃, 10min, and recording the sample as: reagent and Materials blank samples.

Remarking: double blank for Double blank, carryover for residual investigation, blank for single blank with internal standard only, Reagent and Materials blank for Reagent consumables acceptance.

Example 1

1. Solution preparation

Mobile phase A: 1L of ultrapure water (0.5% formic acid solution) was weighed, and 5mL of formic acid was added and mixed.

Mobile phase B: (methanol) 1L of methanol was taken into a suitable solvent bottle.

Diluted solution (acetonitrile: water, 50:50, v/v): 50mL of acetonitrile and 50mL of ultrapure water were transferred to an appropriate solvent bottle and mixed.

Preparing a needle washing solution (methanol: water, 50:50, v/v): 500mL of methanol and 500mL of purified water were taken in an appropriate solvent bottle and mixed well.

Precipitant (3% HClO)₄Solution, v/v): 97mL of purified water and 3mLHClO are measured₄Transferring to a suitable solvent bottle and mixing to obtain 3% HClO₄And (5) mixing the solution and the mixture.

2. Preparing a standard solution:

preparation of standard reference substance stock solution

Preparation of Perindopril Standard control Stock solution (PDPL STD Stock,1mg/mL)

Weighing perindopril tert-butylammonium salt reference substance about 10mg, placing into self-made cup-shaped aluminum foil paper, and accurately recording the weight. Placing the aluminum foil paper in a 10mL brown wide-mouth glass bottle, sucking 10mL methanol by using a 10mL transfer pipette, adding the methanol into the glass bottle, shaking up, calculating the amount of methanol required to be supplemented when the concentration reaches 1mg/mL according to the actual weighed value, the perindopril content and the content of removed tert-butylammonium salt, supplementing the methanol by using a transfer pipette until the perindopril concentration reaches 1mg/mL, and shaking up. Thus obtaining a standard reference substance stock solution containing perindopril with the concentration of 1mg/mL, and storing the standard reference substance stock solution at the temperature of minus 20 ℃.

Preparation of Perindoprilat Standard control Stock solution (PDPLL STD Stock,1mg/mL)

Weighing perindopril A reference substance about 10mg, placing in self-made cup-shaped aluminum foil paper, and accurately recording the weight. Placing the aluminum foil paper in a 10mL brown wide-mouth glass bottle, sucking 10mL DMSO by using a 10mL pipette, adding the DMSO into the glass bottle, shaking up, calculating the required DMSO amount for supplementing when the concentration reaches 1mg/mL according to the actual weighing value and the perindopril content, supplementing the DMSO amount by using the pipette until the perindopril content reaches 1mg/mL, and shaking up. Thus obtaining a standard reference substance stock solution containing perindopril A with the concentration of 1mg/mL, and storing the standard reference substance stock solution at the temperature of minus 20 ℃.

Preparation of standard series working solution:

preparation of Perindopril standard curve working solution

Preparation of working solution of perindopril A Standard Curve

Preparation of Perindopril and Perindoprilat mixed standard curve working solution

*: the working solution is STD Spike 1-8 correspondingly coded by PDPLL

Preparation of SST sample and standard curve plasma sample

3. Preparation of internal standard solution

Internal standard stock solution preparation

Perindopril-d₄Reference stock solution preparation (PDPL-d)₄ IS Stock,10mg/mL)

Directly adding 978 μ L DMSO, dissolving 10mg reference substance, shaking to obtain 10mg/mL reference substance stock solution, and storing at-20 deg.C.

Perindoprilat-d₄Reference stock solution formulation (PDPLL-d)₄ IS Stock,10mg/mL)

Directly adding 860 μ L DMSO, dissolving 10mg reference substance, shaking to obtain 10mg/mL reference substance stock solution, and storing at-20 deg.C.

Preparation of mixed internal standard working solution

Taking PDPL-d₄IS Stock 100. mu.L to 900. mu.L DMSO, and mixed well to obtain 1mg/mL PDPL IS Spike-1. Taking PDPLLd₄IS Stock 100. mu.L to 900. mu.L DMSO, and mixed well to obtain 1mg/mL PDPLL IS Spike-1.

Mixing PDPL IS Spike-150 μ L and PDPLL IS Spike-15 μ L to 945 μ L in 50% acetonitrile water to obtain IS Spike-1(50/5 μ g/mL)

IS Spike-1 (50/5. mu.g/mL) was taken in a 50. mu.L to 50mL volumetric flask, diluted to the mark with 50% acetonitrile water and mixed to obtain IS Spike (50/5 ng/mL).

4. Preparation of quality control standard solution

Preparing a quality control reference substance stock solution:

preparation of Perindopril quality control reference Stock solution (PDPL QC Stock,1mg/mL)

Weighing perindopril tert-butylamine salt reference substance about 10mg, placing in self-made cupped aluminum foil paper, and accurately recording the weight. Placing the aluminum foil paper in a 10mL brown wide-mouth glass bottle, sucking 10mL methanol by using a 10mL transfer pipette, adding the methanol into the glass bottle, shaking up, calculating the amount of methanol required to be supplemented when the concentration reaches 1mg/mL according to the actual weighed value, the perindopril content and the content of removed tert-butylammonium salt, supplementing the methanol by using a transfer pipette until the perindopril concentration reaches 1mg/mL, and shaking up. Thus obtaining a quality control reference substance stock solution containing perindopril with the concentration of 1mg/mL, and storing the quality control reference substance stock solution at the temperature of minus 20 ℃.

Preparation of Perindoprilat quality control reference Stock solution (PDPLL QC Stock,1mg/mL)

Weighing perindopril A reference substance about 10mg, placing in self-made cup-shaped aluminum foil paper, and accurately recording the weight. Placing the aluminum foil paper in a 10mL brown wide-mouth glass bottle, sucking 10mLDMSO by using a 10mL transfer pipette, adding the sucked 10mLDMSO into the glass bottle, shaking up, calculating the required additional DMSO amount when the concentration reaches 1mg/mL according to the actual weighing value and the perindopril content, adding the DMSO amount by using the transfer pipette until the perindopril content reaches 1mg/mL, and shaking up. Thus obtaining a quality control reference substance stock solution containing perindopril with the concentration of 1mg/mL, and storing the quality control reference substance stock solution at the temperature of minus 20 ℃.

Preparing a quality control working solution:

preparation of Perindopril quality control working solution

Preparation of Perindoprilat quality control working solution

Preparation of perindopril and perindopril A mixed quality control working solution

*: working solution is QC Spike 1-5 correspondingly coded by PDPLL

Preparing a quality control plasma sample:

respectively corresponding to HOQ QC, MOQ QC, LOQ QC, LLOQ and AQL.

5. Methodology validation

5.1 Linear Range and lower quantitative limits

Taking 10 mu L of each standard series working solution, diluting the standard series working solutions into blank plasma to ensure that the total volume is 0.2mL, and preparing standard curve plasma samples with perindopril and perindopril ralac concentration values as follows: 1/0.4 ng/mL.

Add 200. mu.L of precipitant to the sample wells (96 well plates).

Add 50. mu.L of 50% acetonitrile water to Double blank, carryover, RB wells, add 50. mu.L of internal standard (IS Spike, 50/5ng/mL) to corresponding sample wells for SST samples, blank samples, standard curve plasma samples, QC plasma samples.

Blank plasma samples (Double blank, carryover), ultrapure water (RB), SST samples, standard curve plasma samples (STD 1-8), and QC plasma samples were each pipetted 50. mu.L into corresponding sample wells.

Seal plate, vortex for 5 min.

Centrifuge, 2490g, 10 min.

15 μ L of sample was introduced into the chromatography system for analysis.

And (4) conclusion: the linear curve chart of the obtained standard curve sample is shown in 1-2, wherein the standard curve equations of perindopril and perindopril A measured by LC-MS/MS method in human plasma are respectively y 0.017626x +7.093819E-004 (perindopril), y 0.246053x-0.003765 (perindopril A), and R is²0.99797166 (perindopril) and 0.99374085 (perindopril); average of 6 measurements, R²Respectively, SD of 0.00055068 (perindopril) and 0.00232886 (perindopril A), CV of 0.1 (perindopril) and 0.2 (perindopril A); the slopes SD were 0.001071 (perindopril) and 0.028376 (perindopril a), CV were 6.2 (perindopril) and 12.5 (perindopril a), respectively; the linear range of perindopril is 1-100 ng/mL, the linear range of perindopril is 0.4-20 ng/mL, the lowest quantitative lower limit of blood concentration of perindopril is 1ng/mL, the lowest quantitative lower limit of blood concentration of perindopril is 0.4ng/mL, and the linear relation in the linear range is good.

5.2 precision and accuracy

Taking four quality control blood plasma samples with different concentrations, wherein the concentrations of perindopril and perindopril A quality control blood plasma sample liquid are 75/15ng/mL, 16/2ng/mL, 3/1ng/mL and 1/0.4ng/mL respectively; the concentrations of HOQ QC, MOQ QC, LOQ QC and LLOQ are respectively corresponded to and detected. As a review of accuracy and precision within and between batches. Each concentration was prepared in 6 replicates and tested at least three times. The mean values were used to assess batch accuracy and precision. The results are given in the following table:

table 1: LC-MS/MS method for determining precision and accuracy of perindopril in plasma in batches and in batches

Table 2: LC-MS/MS method for measuring precision and accuracy of perindopril in plasma in batches and in batches

The results show that: plasma samples of perindopril and perindopril A have precision and accuracy deviation less than 15% in batches and in batches. The measured values for the low, medium and high concentration levels of the standard plasma sample should be within 15% of the indicated values, with an intra-and inter-batch precision (RSD) of less than 15%. It is demonstrated that the method of the present invention has good precision and accuracy for the detection of perindopril and perindopril A in plasma.

5.3 System applicability:

taking a blood plasma sample of the pretreated perindopril and perindopril lat, wherein the concentration of the perindopril is 1ng/mL, and the concentration of the perindopril lat is 0.4 ng/mL; detecting by 15 mu L sample injection, and repeatedly analyzing for 5 times;

and respectively recording chromatographic peak area values and retention time of the object to be detected and the internal standard, and calculating the chromatographic peak area ratio of the object to be detected and the internal standard and the variation coefficient of the chromatographic peak retention time. The specific results are shown in the following tables 3-4:

table 3: perindopril system applicability

Table 4: perindoprilat System applicability

And (4) conclusion: the signal to noise ratio (S/N is not less than 5) of the substance to be detected, and the variation coefficient of the ratio of the area of the substance to be detected to the area of the internal standard chromatographic peak obtained by 5 times of repeated analysis is less than 5%; the variation coefficient of the retention time of the sample to be tested and the internal standard chromatographic peak obtained by 5 times of repeated analysis is less than 15.0%. Retention time coefficient of variation perindopril to be tested: 0.0%, perindopril: 0.0-0.2%, retention time variation coefficient of internal standard perindopril: 0.0%, perindopril: 0.0-0.2%, coefficient of variation of area ratio perindopril: 1.0-2.5%, perindopril A: 1.6-5.4%; it is shown that the method of the present invention is applicable to the determination of perindopril and perindopril A for the detection of drugs in blood plasma.

5.4 Selectivity

Selectivity refers to the ability to distinguish interference in a biological matrix when an analyte is measured by chromatographic methods. Before the method is validated, at least 6 different batches of blank substrates need to be screened for method validation.

Taking 50 mu L of blank plasma from different sources of 6 Chinese population, pretreating to obtain a Carryover sample, and then injecting 15 mu L of the sample into a chromatographic system for analysis.

Taking 50 mu L of 6 blank blood plasma from different sources, preparing perindopril with the concentration of the quality control blood plasma sample liquid of 1ng/mL, pretreating, and feeding 15L of the sample liquid into a chromatographic system for analysis;

taking 50 mu L of 6 blank blood plasma from different sources, preparing a solution of perindopril A quality control blood plasma sample liquid of 0.4ng/mL, pretreating, and feeding 15 mu L of sample into a chromatographic system for analysis;

the results of each blank sample and the standard plasma sample at the lower limit concentration level were recorded separately as follows.

Table 5: comparative table for selective investigation of perindopril analytes and internal standards by blank healthy human plasma from six different sources

Table 6: comparison table for selective investigation of perindopril A analytes and internal standards by blank healthy human plasma from six different sources

And (4) conclusion: the peak area of the chromatographic peak at the retention time of the analyte in at least 6 blank matrix samples from different sources is lower than 20.0% of the peak area of the to-be-measured substance at the corresponding blank matrix LLOQ concentration from different sources, and the peak area of the internal standard at the retention time of the internal standard is lower than 5.0% of the peak area of the internal standard at the corresponding blank matrix LLOQ concentration from different sources. The measured value of the LLOQ concentration prepared by 6 blank matrixes from different sources is in the range of 80.0-120.0% of the theoretical value. The interference range of the method of the invention for analytes in different sources of blank matrix samples is perindopril: 1.2-1.4%, perindopril A: 0.0-5.9%, the internal standard interference response value is perindopril: 0.0%, perindopril: 0.0 percent. It can be seen that blank blood plasma of different human bodies does not interfere with the detection results of perindopril and perindopril A, the method can be used for detecting perindopril and perindopril A in blood plasma of different human bodies, and blood plasma of different people has no influence on the detection method of the invention.

5.5 recovery

5.5.1 extraction recovery of analytes

Analyte recovery sample

Respectively taking perindopril/perindopril quality control blood plasma sample liquid with the concentration of 75/15, 16/2 and 3/1ng/mL, and parallelly preparing 6 parts;

and adding no mixed internal standard working solution, precipitating, taking a supernatant solution with a proper volume, adding 5ng/mL internal standard solution with the same volume as the supernatant, and measuring after vortex mixing.

Recovery reference sample:

the extraction process was the same as the analyte recovery sample, but the mixed internal standard working solution was added after precipitation, and the supernatant vortexed and then assayed.

5.5.2 extraction recovery of internal standard

Extraction recovery sample of internal standard

6 parts of blank plasma samples are prepared in parallel, mixed internal standard working solution is added, after precipitation, supernatant solution with proper volume is taken, and standard substance solution with concentration equivalent to that of MOQ QC quality control samples is added.

Recovery of internal standard reference sample:

the extraction process is the same as the extraction operation of the internal standard extraction recovery sample, but the MOQ QC quality control sample and the internal standard solution are added after precipitation.

And (4) conclusion: 1) the extraction recovery rates of perindopril and perindopril A to be detected in the method of the invention in three concentration ranges of high, medium and low are respectively perindopril: 90.9%, 94.0%, 96.4%, perindopril a: 94.3%, 97.6%, 101.0%; the corresponding coefficient of variation CV value is perindopril: 0.7%, 3.0%, 1.0%, perindopril a: 1.4%, 1.9%, 2.4%; the total recovery was perindopril: 93.8%, perindopril A: 97.6 percent; the overall recovery variation CV value was perindopril peimine: 3.0%, perindopril A: 3.4 percent.

2) The extraction recovery rate of the internal standard substance of the method is perindopril-d₄: 99.9% of perindopril-d₄: 110.1 percent; the CV value of the recovery rate is perindopril-d₄: 2.2% of perindopril-d₄：2.0％。

5.6 matrix Effect

1) Matrix effects refer to the inhibition or enhancement of the ionization of an analyte by a component present in a biological matrix. Taking blank human plasma of 6 different batches respectively, preparing one part for each batch, and adding a standard solution and an internal standard solution with the same concentration as the low-concentration and high-concentration quality control samples after pretreatment for analysis.

The analysis was carried out in 6 replicates of pure solutions corresponding to the concentrations of the low-concentration and high-concentration quality control samples, in the absence of matrix.

2) Preparation of hemolysis matrix: taking 500 mu L of blank whole blood, carrying out ultrasonic destruction on the blood cells, taking 20 mu L of the blank whole blood, adding 980 mu L of normal blank plasma, and uniformly mixing to obtain 2% of hemolyzed plasma subjected to ultrasonic destruction, wherein the hemolyzed plasma is regarded as severe hemolysis.

Two samples of the analyte at two concentration levels (LOQ QC, HOQ QC) were prepared 6 times using simulated hemolyzed plasma, processed and then injected into a chromatographic system for analysis.

3) The low and high concentration level (LOQ QC, HOQ QC) samples of the test substance are prepared 6 parts each by using the blood plasma sample with high blood fat, and the processed samples are injected into a chromatographic system for analysis.

Table 11: test table for matric effect (Perindopril)

TABLE 11: test table for matrix Effect (Perindoprilat)

And (4) conclusion: the mean values of the matrix effect factors normalized by the internal standard are: perindopril: 0.996956-1.000131; perindopril A: 0.989354-0.989610; coefficient of variation CV% is: perindopril: 0.3-0.7%, perindopril A: 1.1-4.9%; the mean deviation in accuracy of the lysomatrix effect was: perindopril: -6.7 to-0.2%, perindopril a: -7.1 to-4.8%; the average accuracy deviation range of the hyperlipidemia matrix effect is as follows: perindopril: -4.6 to 2.6%, perindopril A: -2.9 to-2.8%; matrix effect: perindopril 0.996956, 1.000131, perindopril 0.989354, 0.989610; the method of the invention has no obvious matrix effect, and has no obvious hemolysis and hyperlipemia matrix effect.

5.7 stability

And (3) carrying out stability investigation on human plasma quality control samples with low and high concentrations (LOQ QC and HOQ QC) of the object to be detected under the following conditions:

1) repeated freeze thawing at-70 deg.C for 4 times.

2) Standing at room temperature for 20h for stabilization.

3) Is stable at 41 days at-20 ℃.

4) Whole blood stability: and (3) respectively inspecting the change of the ratio of peak areas of analytes containing low and high concentration levels (LOQ QC and HOQ QC) placed in ice-water bath in whole blood for (0h) and (0.5-2 h), after placing corresponding inspection time points, centrifugally separating the whole blood sample into plasma, adding an internal standard, and processing according to a corresponding plasma sample processing operation method, wherein each concentration level is parallel to 6 parts.

The samples after stability investigation and the samples prepared now are tested simultaneously, and the comparison result of each concentration is as follows:

table 7 stability at room temperature for 20 h-perindopril short term stability results:

table 7 stability at room temperature for 20 h-perindopril a short term stability results:

table 8-20 ℃ stability at 41 d-perindopril long term stability results:

table 8-20 ℃ long term stability results for 41d stable perindopril a:

table 9-70 ℃ repeated freeze-thaw 4 times perindopril stability results:

table 9-70 ℃ repeated freeze-thaw 4 times perindopril a stability results:

table 10 perindopril whole blood stability results:

table 10 perindopril a whole blood stability results:

the CV value of the concentration of each drug in the blood plasma measured by the method of the present invention under the above-mentioned examination conditions is less than 15%, which indicates that the method of the present invention has strong stability for the measurement of perindopril and perindopril A in whole blood and blood plasma under the above-mentioned examination conditions.

5.8 human plasma sample detection

200uL acetonitrile, 50uL IS Spike, 50uL perindopril and plasma samples of perindopril A, 20uL IS Spike, vortex mixing for 5min, centrifuging at 2490g for 10min at 4 ℃, and injecting 15uL into a chromatographic system for LC-MS/MS analysis, the results are shown in FIGS. 3-4.

And (4) conclusion: proved by methodology, the method has the advantages of high sensitivity, strong applicability, good precision and accuracy and good stability, the extraction recovery rate of the medicine is within an acceptable range, no obvious matrix effect is seen, and the method also has the advantages of high speed and high flux.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneous determination of perindopril and perindopril A concentration in human plasma, comprising the steps of:

2. Method for the simultaneous determination of perindopril and perindopril A concentration in human plasma according to claim 1, characterized in that the concentration of the standard control stock solution is 1 mg-mL^-1(ii) a The precipitant is 3% HClO₄And (3) solution.

3. A method for the simultaneous determination of perindopril and perindopril A concentration in human plasma according to claim 1, characterized in that the concentration range of the standard series of working solutions of perindopril is 40-4000 ng/mL; the concentration range of the perindopril A working solution of the standard series is 16-800 ng/mL; the concentration range of the perindopril working solution in the mixed standard curve working solution is 20-2000 ng/mL, and the concentration range of the perindopril working solution is 8-400 ng/mL.

4. A method for the simultaneous determination of perindopril and perindopril A concentration in human plasma according to claim 1, characterized in that the perindopril concentration in said standard curve plasma sample ranges from 1 to 100ng/mL and the perindopril A concentration ranges from 0.4 to 20 ng/mL.

5. Method for the simultaneous determination of perindopril and perindopril A concentration in human plasma according to claim 1, characterized in that said regression equation of the standard curve is obtained as follows: adding 200 μ L of precipitant into the sample well, adding 50 μ L of mixed internal standard working solution, 50 μ L of standard curve plasma sample, sealing the plate, vortexing for 5min, centrifuging, 2490g, and 10 min; 15 mu L of sample injection LC-MS/MS system to obtain a chromatogram; calculating the concentration of perindopril in blood plasma according to the ratio of the chromatographic peak area of perindopril to the peak area of the internal standard in the obtained chromatogram, and making a regression equation of a standard curve of perindopril; and calculating the concentration of perindopril A in blood plasma according to the ratio of the chromatographic peak area of perindopril A to the internal standard peak area in the obtained chromatogram, and establishing a standard curve regression equation of perindopril A.

6. A method for the simultaneous determination of perindopril and perindopril A concentration in human plasma according to claim 5, characterized in that said working solution of mixed internal standard is formulated as follows: weighing perindopril tert-butylamine salt-d₄And perindopril-d₄Respectively dissolving in DMSO in volumetric flasks to obtain 10mg/mL solutions^-1Perindopril-d of₄Internal standard reference stock solution and perindopril-d₄An internal standard control stock solution;

7. A method for the simultaneous determination of perindopril and perindopril A concentration in human plasma according to claim 1, characterized in that the conditions of the LC-MS/MS system are as follows:

chromatographic conditions

A chromatographic column: agilent ZORBAX SB-Aq;

protection of the column: ACE 5C18 ANALYTICL GRD CART (X5)

Mobile phase A: 0.5% aqueous formic acid; mobile phase B: methanol

Flow rate: 1 mL/min; stopping time: 5min

gradient elution is adopted;

conditions of Mass Spectrometry

the mass spectrum switching valve is set as follows:

Start Time(min) Div Valve 1 0 To Waste 2 2 To MS

the ion source parameters were set as follows:

。

8. a method for simultaneous determination of perindopril and perindopril a concentration in human plasma according to claim 7, characterized in that the mobile phase ratio of the gradient elution is set as follows:

。

9. a method for the simultaneous determination of perindopril and perindopril a concentration in human plasma according to claim 7, characterized in that the specifications of said chromatographic column are: 4.6X 150mm, 5 μm.

10. Method for the simultaneous determination of perindopril and perindopril A concentration in human plasma according to claim 1, characterized in that the regression equation of the standard curve is as follows:

the regression equation of the standard curve of perindopril measured in human plasma is: 0.246053x-0.003765, R²0.99374085; linear range of perindoprilThe circumference is 0.4-20 ng/mL, and the lowest quantitative lower limit of the blood concentration of perindopril is 0.4 ng/mL.