CN115932105A - Method for analyzing concentration of pregabalin in plasma sample by using liquid chromatography-tandem mass spectrometry - Google Patents

Abstract

The invention provides a method for analyzing pregabalin concentration in a plasma sample by using liquid chromatography-tandem mass spectrometry. It includes: adding an internal standard solution and acetonitrile into the plasma sample for vortex, centrifuging and collecting supernatant to obtain a pretreated sample to be detected; and (3) performing liquid chromatography-tandem mass spectrometry detection on the sample to be detected, performing mass spectrometry detection, drawing a standard curve based on the detection peak area ratio to obtain a regression equation, and finally calculating to obtain the concentration of the pregabalin in the sample to be detected. The method has the characteristics of simple and convenient pretreatment operation and short analysis time, and is suitable for analyzing large-batch clinical research samples; in addition, the sensitivity is higher, and the lower limit of the pregabalin quantification is 10.0ng/mL.

Description

Method for analyzing concentration of pregabalin in plasma sample by using liquid chromatography-tandem mass spectrometry

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for analyzing the concentration of pregabalin in a plasma sample by using liquid chromatography-tandem mass spectrometry, wherein the pregabalin is used for treating diabetic neuralgia and herpes zoster neuralgia.

Background

Pregabalin is an analog of the neurotransmitter gamma-aminobutyric acid (GABA) with high affinity to the α 2- δ site (a complementary subunit of voltage-gated calcium channels) in the central nervous system. Pregabalin was developed by Warner-Lambert (now incorporated into the pharmaceutical company of fevereie), FDA approved in 2004 for the treatment of diabetic and herpes zoster neuralgia, and marketed in the united states in 9 months 2005, which was the first approved drug in both the united states and europe for the treatment of both pains. Pregabalin is currently approved for the treatment of neuropathic pain in 40 countries, europe, canada, mexico, and the united states. In order to accelerate the clinical application, a simple, accurate, rapid and sensitive biological analysis method is needed.

At present, the liquid chromatography-tandem mass spectrometry technology is a main method for analyzing pregabalin in human plasma. Huangjie et al also did not have high enough sensitivity in 2016's studies of pregabalin bioequivalence. Liuyan et al, 2017, developed a method for determining pregabalin in human plasma with high sensitivity, but adopted sulfamethoxazole as an internal standard, and the sample pretreatment step was tedious, the vortex flow required twice, the supernatant required to pass through a microporous filter membrane, and the filtrate was used for analysis, which resulted in high experimental cost. Xufenghua et al developed a method for determining pregabalin in human plasma in 2017, the plasma consumption of a subject was 200 μ L, the analysis time was 6min, the plasma consumption was more, the analysis time was long, the method was not suitable for analysis of a large number of clinical samples, the sensitivity of the method was not high enough, and the lowest limit of quantitation LLOQ was 30.0ng/mL.

In summary, the prior art methods for detecting the concentration of pregabalin in a plasma sample cannot give consideration to both sensitivity and analysis speed, and some technical pre-treatments are complex. These disadvantages are disadvantageous for accurate analysis of the concentration of pregabalin in clinical tests in large batches of plasma samples.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a method for analyzing the concentration of pregabalin in a plasma sample by using liquid chromatography-tandem mass spectrometry.

The purpose of the invention is realized by the following technical scheme:

in one aspect, the present invention provides a method for analyzing the concentration of pregabalin in a plasma sample by liquid chromatography-tandem mass spectrometry, comprising the steps of:

adding an internal standard solution and acetonitrile into the plasma sample for vortex, centrifuging and collecting supernatant to obtain a pretreated sample to be detected;

and (3) adopting liquid chromatography-tandem mass spectrometry detection, firstly carrying out liquid chromatography separation on a sample to be detected, then carrying out mass spectrometry detection, drawing a standard curve based on the detection peak area ratio to obtain a regression equation, and finally calculating to obtain the concentration of the pregabalin in the sample to be detected.

In the above method, preferably, the internal standard solution is Pregabalin-d4 (Pregabalin-d) ₄ )。

In the above method, preferably, the specific method of the pretreatment includes:

adding 50.0 mu L of plasma sample into a 96-well plate, then adding 50.0 mu L of internal standard solution and 300 mu L of acetonitrile, mixing uniformly by vortex, centrifuging, collecting supernatant, and placing the supernatant into another clean 96-well plate to obtain a sample to be detected.

In the above method, preferably, the centrifugation time is 10min, the centrifugation temperature is 4 ℃, and the centrifugation speed is 3900rpm.

In the above method, preferably, the concentration of the internal standard solution is 500ng/mL.

In the above method, preferably, the chromatographic column used for the liquid chromatography is:

dC18 column, 3 μm, 4.6X 100mm; the mobile phases used were: phase A: 0.3% formic acid and 10mM ammonium acetate in water, phase B: and (3) acetonitrile.

In the above method, preferably, the elution conditions for performing liquid chromatography are as follows:

gradient elution:

time (min)	Flow rate (mL/min)	Mobile phase A/%)	Mobile phase B/%)
				0.00～0.60	0.7	75	25
0.80～2.00	0.7	45	55
				2.10～3.00	0.7	75	25

Elution time: 3.00min;

sample introduction amount: 5.00 mu L;

autosampler temperature: 4 ℃;

column temperature: at 40 deg.c.

In the above method, preferably, the mass spectrometric conditions for performing mass spectrometric detection are:

an ion source: electrospray ion source (ESI);

ejection voltage: 5000V;

gas spray (Gas 1): 45psi;

auxiliary Gas (Gas 2): 45psi;

the detection mode is as follows: a positive ion;

ion source temperature: 500 ℃;

collision induced dissociation (CAD): 10psi;

air Curtain Gas (Curtain Gas): 25psi;

residence time: 200ms.

In the above method, preferably, the mass spectrometric detection is performed by using a quantitative analysis ion pair, the quantitative analysis ion pair is:

pregabalin m/z 160.1 → 142.1, collision Energy (CE) 20eV, declustering voltage (DP) 50V;

pregabalin-d4 m/z 164.2 → 146.1, collision Energy (CE) 20eV, declustering voltage (DP) 50V.

In the above method, preferably, the standard curve is specifically made as follows:

and (3) taking the theoretical concentration of the sample to be detected as a horizontal coordinate and the peak area ratio of the sample to be detected and the internal standard substance as a vertical coordinate, and performing regression analysis calculation to obtain a linear regression equation.

In another aspect, the present invention also provides the use of the method described above for the analysis of the concentration of pregabalin in a plasma sample; the pregabalin is used for treating diabetic neuralgia and herpes zoster neuralgia.

The invention has the beneficial effects that:

(1) The method has the characteristics of simple and convenient pretreatment operation, can perform analysis only by one-step extraction, has high analysis speed, and only needs 3min of analysis time, so the method is suitable for analyzing large-batch clinical research samples.

(2) The method has high sensitivity, the lower limit of pregabalin quantification is 10.0ng/mL, the amount of the substance to be detected on the column is 6.25pg according to the dilution multiple of 8 and the sample injection amount of 5 mu L, the linear range of the method is reasonably selected, and the concentration of the medicine can be more accurately analyzed.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

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

FIG. 1 is a ion scanning mass spectrum of a pregabalin product;

FIG. 2 shows Pregabalin-d ₄ Scanning mass spectrogram of product ion;

FIG. 3 is an MRM chromatogram of pregabalin in a blank plasma sample;

FIG. 4 shows the quantitation of Pregabalin (left) and Pregabalin-d in the lower limit samples ₄ (right) MRM chromatogram;

fig. 5 is a standard graph of pregabalin.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. 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

1. Pretreatment:

the method uses the protein precipitation method, has high recovery rate of the pregabalin, and has the advantages of simple operation, short extraction time and no time-consuming concentration step. The kit is matched with a 96-well plate for use, and is suitable for pretreatment of high-flux samples in clinical research.

The specific pretreatment method comprises the following steps:

1. to a 96-well plate, 50.0. Mu.L of a plasma sample and 50.0. Mu.L of an internal standard solution (Pregabalin-d) were added ₄ Concentration 500 ng/mL), 300. Mu.L acetonitrile;

2. mixing by vortex, and centrifuging for 10min (4 deg.C, 3900 rpm);

3. the supernatant was taken to another clean 96-well plate.

4. The injection volume was 5.0. Mu.L.

2. And (3) chromatographic analysis:

chromatography, separating the sample to be tested by liquid chromatography

dC18 column, gradient elution, mobile phase A containing 0.3% formic acid and 10mM ammonium acetate in water solution, mobile phase B acetonitrile.

The chromatographic separation of the invention adopts

The dC18 chromatographic column has better retention to an object to be detected and an internal standard, and has a sharp peak shape. In order to realize the elution of two types to be detected in a short time, a rapid gradient elution mode is adopted. The instrument analysis flux is high in the mode, the chromatographic running time is only 3.0min, the detection is rapid, and the method is suitable for analyzing a large number of samples in clinical research.

3. Mass spectrometry analysis:

electrospray ion source, positive ion detection, spray voltage 5000V, gas1 (Gas 1) 45psi, gas2 (Gas 2) 45psi, curtain Gas (Curtain Gas) 25psi, ion source temperature 500 deg.C, collision induced dissociation 10psi, residence time 200ms, pregabalin quantitative analysis ion pair m/z 160.1 → 142.1, collision Energy (CE) 20eV, declustering voltage (DP) 50V, pregabalin-d4 quantitative analysis ion pair 164.2.2 → 146.1, collision Energy (CE) 20eV, declustering voltage (DP) 50V.

The invention is illustrated in detail below by means of specific examples:

example 1:

description of abbreviations:

1. material

1.1 Instrument for measuring the position of a moving object

Chromatograph: LC-20AD flash liquid chromatography system, shimadzu, japan.

Mass spectrometry: AB Sciex API 4000 triple quadrupole tandem mass spectrometer equipped with electrospray ionization source (Turbo Ion Spray) Sciex, canada.

The data processing adopts software: analyst (version 1.6.3), sciex, canada.

A centrifuge: model Her μ Le Z2326K bench centrifuge, haemomer, germany.

Analytical balance: analytical balance model CD225D, beijing sidoris instruments ltd.

1.2 control and reagents

Pregabalin (content 99.7%) was purchased from the chinese institute for food and drug testing. Pregabalin-d4 was purchased from TLC. Methanol (HPLC grade), acetonitrile (HPLC grade) were purchased from Sigma company, usa. Formic acid (HPLC grade) was purchased from Sigma company, usa. Ammonium acetate (HPLC grade) was purchased from ROE corporation, usa. Deionized water (18.2 m Ω, TOC. Ltoreq.50 ppb) was prepared from a Milli-Q ultrapure water system.

2. Method of producing a composite material

2.1 Preparation of solutions and samples

Standard series of samples: accurately weighing appropriate amount of each reference substance, dissolving with methanol respectively, and metering volume to obtain a stock solution with pregabalin concentration of about 1.00 mg/mL. Precisely sucking appropriate amount of respective stock solutions, diluting with human blank plasma step by step to obtain mixed standard series samples, wherein the concentration range of pregabalin is 10.0-5000 ng/mL.

Quality control of the sample: 4 concentration level mixed quality control samples of pregabalin are prepared by a method similar to that of a standard series of samples. The lower limit concentration of the quantification is 10.0ng/mL, the Low Quality Control (LQC) concentration is 30.0ng/mL, the secondary medium Quality Control (AMQC) concentration is 250ng/mL, the Medium Quality Control (MQC) concentration is 1500ng/mL, and the High Quality Control (HQC) concentration is 4000ng/mL.

Internal standard solution: quantitatively transferring Pregabalin-d4 reference substance, dissolving with methanol and fixing volume to prepare internal standard stock solution with the concentration of about 1.00 mg/mL. Precisely absorbing a proper amount of the internal standard stock solutions, adding acetonitrile: water (50, v/v) dilution to obtain an internal standard solution with Pregabalin-d4 concentration of 500ng/mL.

2.2 plasma sample treatment

2.3 chromatographic and Mass Spectrometry conditions

Chromatographic conditions are as follows:

mass spectrum conditions:

2.4. methodology validation

The methodology of the method is verified according to the guidance principle of Chinese pharmacopoeia 9012, and the contents comprise stability, selectivity, linearity, accuracy, precision, recovery rate matrix effect and the like.

Selectivity is selected

Taking six blank blood plasmas with different sources and respectively prepared quantitative lower limit samples, and then carrying out sample injection analysis. The peak area of the chromatogram co-outflow interferent is required to be less than 20% of the peak area of the quantitative lower limit analyte and less than 5% of the peak area of the internal standard.

Standard curve of

Linear regression equation (weight factor W = 1/x) calculated by regression analysis with the concentration of the physical theory to be measured as abscissa (x) and the peak area ratio of the substance to be measured to the internal standard as ordinate (y) ² ). The method verifies that each analysis batch is analyzed against a double sample of standard curve samples.

Precision and accuracy

The method verifies that each analysis batch determines six samples of five concentration quality control samples. Quantitative lower limit intra-and inter-batch precision is acceptable at less than 20% as calculated by Relative Standard Deviation (RSD) and accuracy is acceptable at between-20% and 20% as calculated by relative deviation (RE). The precision of the QC samples of other concentration levels in each component batch and between batches is required to be less than 15 percent to be acceptable, and the precision is between-15 percent and 15 percent to be acceptable.

Stability of

And (3) when the stability of each object to be detected in the plasma sample is inspected, placing the LQC and the HQC in different temperatures and environments, and performing three-sample analysis after the placement is finished. A total of four placement conditions were examined, which were: standing at room temperature for 42h, extracting, standing in a sampler for 69h, and performing 5 times of freeze-thaw cycles (from-75 + -10 deg.C to room temperature), and standing at 75 + -5 deg.C for 120 days.

Recovery rate

Taking blank plasma 50.0 mu L, adding the solution to be measured and the internal standard solution after extraction (without adding the internal standard solution) to make the final concentration be the same as LQC, MQC and HQC, and carrying out sample injection and measurement. And 6 parts of each of LQC, MQC and HQC are extracted, and the sample injection and the determination are carried out. The extraction recovery rate was calculated from the peak area ratios of the 2 treatments.

Matrix effect

Taking blank plasma of 6 different sources, extracting (without adding internal standard solution), adding to-be-detected substance solution and internal standard solution with the same concentration as LQC and HQC, mixing by vortex, and measuring. And treating with deionized water instead of blood plasma by the above method. Matrix factors were calculated as peak area ratios obtained by both methods and matrix effects were assessed by RSD of internal standard normalized matrix factors, less than 15% being acceptable.

2.5 clinical study

The established method is applied to analyze the concentration of the pregabalin in a clinical research plasma sample, and is used for human pharmacokinetics research of the pregabalin. 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. Collecting venous blood at different time points before (0 h) and after administration, collecting venous blood, collecting each 4mL, placing in a heparin anticoagulation centrifuge tube, centrifuging, separating plasma, and storing at-75 + -10 deg.C.

3. Results and discussion

3.1 Methodology validation

Selectivity of the process

As shown in FIGS. 3 and 4, pregabalin and Pregabalin-d ₄ The retention time is about 1.85 min and 1.84min respectively, and no co-outflow interference peak exists at the retention time.

Standard curve of

Ion scanning mass spectrum and Pregabalin-d of Pregabalin product ₄ The product ion scanning mass spectrograms are respectively shown in figure 1 and figure 2; measurement of Pregabalin in plasma samples from clinical studies of Pregabalin, the linear range of Pregabalin was 10.0-5000 ng/mL, as shown in FIG. 5. The typical linear regression equations of the standard curves of the objects to be measured are respectively as follows:

pregabalin y =0.00153x + -0.000143;

detection limit

The concentration of pregabalin in the lower limit sample of the quantification is 10.0ng/mL, and the amount of the substance to be detected on the column is 6.25pg according to the dilution multiple of 8 and the sample injection amount of 5 mu L.

Precision and accuracy of the method

Precision accuracy results were all in accordance with accepted standards and are shown in table 1.

Table 1 shows the precision and accuracy of pregabalin determination in human plasma

Table 1:

recovery rate of treatment

LQC, MQC and HQC concentration levels: the extraction recovery rates of pregabalin are respectively 88.0%, 97.6% and 93.9%; pregabalin-d ₄ The recovery of (b) was 106.3%.

Matrix effect

The internal standard normalized matrix factors of pregabalin at LQC, HQC concentration levels were 104.1% and 100.4%, respectively, and RSD was 1.1% and 0.4%, respectively. The results show that the matrix effect does not interfere with the accuracy of the analyte analysis.

Plasma stability study

The results of the plasma stability test are shown in table 2, and the results show that pregabalin is stable under the investigation conditions.

Wherein Table 2 is the stability of pregabalin in human plasma (n = 6)

Table 2:

4 human pharmacokinetics study

The validated method was used to simultaneously analyze pregabalin in plasma to evaluate pregabalin pharmacokinetic characteristics. The detection method has the advantages that the sensitivity can completely describe the pharmacokinetic characteristic of the pregabalin, the selection of the linear range is close to the concentration level of an actual sample, and the determination accuracy is high.

The principle and the implementation mode of the invention are explained by applying specific embodiments in the invention, and the description of the embodiments is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims (10)

1. A method for analyzing the concentration of pregabalin in a plasma sample by liquid chromatography-tandem mass spectrometry comprises the following steps:

adding an internal standard solution and acetonitrile into the plasma sample for vortex flow, centrifuging and collecting supernatant to obtain a preprocessed sample to be detected;

and (3) performing liquid chromatography-tandem mass spectrometry detection on the sample to be detected, performing mass spectrometry detection, drawing a standard curve based on the detection peak area ratio to obtain a regression equation, and finally calculating to obtain the concentration of the pregabalin in the sample to be detected.

2. The method according to claim 1, wherein the internal standard solution is pregabalin-d 4.

3. The method of claim 1, wherein the specific method of pre-processing comprises:

adding 50.0 mu L of plasma sample into a 96-well plate, then adding 50.0 mu L of internal standard solution and 300 mu L of acetonitrile, carrying out vortex mixing, centrifuging, collecting supernatant, and placing the supernatant into another clean 96-well plate to obtain a sample to be detected; preferably, the centrifugation time is 10min, the centrifugation temperature is 4 ℃, and the centrifugation speed is 3900rpm.

4. The method according to claims 1-3, wherein the concentration of the internal standard solution is 500ng/mL.

5. The method of claim 1, wherein the liquid chromatography is performed using a column comprising:

dC18 column, 3 μm, 4.6X 100mm; the mobile phases used were: phase A: 0.3% formic acid and 10mM ammonium acetate in water, phase B: and (3) acetonitrile.

6. The method of claim 5, wherein the elution conditions for performing the liquid chromatography separation are:

gradient elution:

time (min) Flow rate (mL/min) Mobile phase A/%) Mobile phase B/%) 0.00～0.60 0.7 75 25 0.80～2.00 0.7 45 55 2.10～3.00 0.7 75 25

Elution time: 3.00min;

sample introduction amount: 5.00 mu L;

autosampler temperature: 4 ℃;

column temperature: at 40 ℃.

7. The method of claim 1, wherein the mass spectrometric conditions for performing the mass spectrometric detection are:

an ion source: electrospray ion source (ESI);

ejection voltage: 5000V;

mist spray (Gas 1): 45psi;

auxiliary Gas (Gas 2): 45psi;

the detection mode is as follows: a positive ion;

ion source temperature: 500 ℃;

collision induced dissociation (CAD): 10psi;

air Curtain Gas (Curtain Gas): 25psi;

residence time: 200ms.

8. The method of claim 1 or 6, wherein performing mass spectrometric detection employs quantitative analysis ion pairs of:

pregabalin m/z 160.1 → 142.1, collision Energy (CE) 20eV, declustering voltage (DP) 50V;

pregabalin-d4 m/z 164.2 → 146.1, collision Energy (CE) 20eV, declustering voltage (DP) 50V.

9. The method according to claim 1, wherein the standard curve is specifically made as:

and (3) taking the theoretical concentration of the sample to be detected as a horizontal coordinate and the peak area ratio of the sample to be detected and the internal standard substance as a vertical coordinate, and performing regression analysis calculation to obtain a linear regression equation.

10. Use of the method of any one of claims 1 to 9 for analyzing the concentration of pregabalin in a plasma sample; the pregabalin is used for treating diabetic neuralgia and herpes zoster neuralgia.

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