阅读说明：本技术 抗病毒药物临床研究血浆样本中替诺福韦艾拉酚胺及代谢物替诺福韦浓度的生物分析方法 (Biological analysis method for clinical research of tenofovir alafenamide and tenofovir metabolite concentration in plasma sample of antiviral drug ) 是由 陈云辉 钟勘 肖涛 杨勇 史中杰 温凤娇 刘旭凌 周林芳 姜金方 周茂金 于 2020-12-30 设计创作，主要内容包括：本发明涉及一种抗病毒药物临床研究血浆样本中替诺福韦艾拉酚胺及代谢物替诺福韦浓度的生物分析方法,通过LC-MS/MS对血浆中替诺福韦艾拉酚胺及代谢物替诺福韦进行分析,采用蛋白沉淀预处理方法,以替诺福韦艾拉酚胺-d5和替诺福韦-d7为内标,采用Eclipse Plus C-(18)色谱柱,梯度洗脱,电喷雾电离源(ESI)串联质谱检测。本发明操作简便、分析速度快、灵敏度高、满足临床研究大批量样品分析需求。(The invention relates to a bioanalysis method for tenofovir alafenamide and tenofovir metabolite concentration in a plasma sample in clinical research of antiviral drugs, which analyzes tenofovir alafenamide and tenofovir metabolite in plasma through LC-MS/MS (liquid chromatography-mass spectrometry), adopts a protein precipitation pretreatment method, takes tenofovir alafenamide-d 5 and tenofovir-d 7 as internal standards, and adopts Eclipse Plus C 18 Chromatographic column, gradient elution, electrospray ionization source (ESI) tandem mass spectrometry detection. The invention has simple operation, fast analysis speed and high sensitivity, and can meet the analysis requirement of clinical research on a large number of samples.)

1. A bioanalysis method for the concentration of tenofovir alafenamide and a metabolite tenofovir in a plasma sample in clinical research of antiviral drugs is characterized by comprising the following steps:

pretreating, adding plasma into a 96-well plate, adding an internal standard solution, adding methanol and acetonitrile in a volume ratio of 50:50, v/v, performing vortex flow and centrifugation, taking supernate into another clean 96-well plate, adding a formic acid aqueous solution containing 0.1%, performing vortex flow and uniform mixing, and injecting 20.0 mu L of sample;

chromatography, separating the sample to be detected by liquid chromatography, and adopting Eclipse Plus C₁₈Performing gradient elution on a chromatographic column, wherein a mobile phase A is an aqueous solution containing 0.1% of formic acid and 2mM of ammonium acetate, a mobile phase B is acetonitrile, and the chromatographic running time is 5.3 min;

mass spectrometry using an electrospray ion source, positive ion detection, ejection voltage 5500V, Gas 1(Gas1)60psi, Gas 2(Gas2)60psi, Curtain Gas (Curtain Gas)35psi, ion source temperature 550 ℃, collision induced dissociation 9psi, residence time 100ms, Tenofovir alafenamide quantitative ion pair m/z477.2 → 346.1, Collision Energy (CE)60eV, declustering voltage (DP)80V, Tenofovir quantitative ion pair 288.1 → 176.1, Collision Energy (CE)35eV, declustering voltage (DP)80V, Tenofovir alafenamide-d 5 quantitative ion pair 482.3 → 351.2, Collision Energy (CE)60eV, declustering voltage (DP)80V, Tenofovir-d 7 quantitative ion pair 295.2 →.1, Collision Energy (CE)35eV, declustering voltage (DP) 80V.

2. The bioanalytical method of tenofovir alafenamide and the metabolite tenofovir in clinical study plasma samples of antiviral drugs according to claim 1, wherein the bioanalytical method comprises the following steps: the pretreatment is carried out, wherein 100 mu L of plasma sample is taken, 50.0 mu L of internal standard solution is added, 300 mu L of methanol: acetonitrile (50:50, v/v) is added, 100 mu L of supernatant is taken to be put into another clean 96-well plate after vortex and centrifugation, 300 mu L of aqueous solution containing 0.1 percent of formic acid is added, and vortex and uniform mixing are carried out.

3. The bioanalytical method in clinical study of tenofovir alafenamide and the metabolite tenofovir in plasma samples of antiviral drugs according to claim 1 or 2, characterized in that: the internal standard solution is tenofovir alafenamide-d 5 solution and tenofovir-d 7 solution.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a bioanalysis method for the concentrations of tenofovir alafenamide and a metabolite tenofovir in a plasma sample in clinical research of antiviral drugs.

Background

Tenofovir alafenamide is a precursor drug of Tenofovir, is a novel nucleotide reverse transferase inhibitor, and is used for treating chronic hepatitis B by entering DNA reverse transferase of hepatitis B virus to inhibit DNA replication. Propofol fumarate Tenofovir tablets were developed by Gilidde, USA and approved by the FDA for the treatment of adult chronic hepatitis B in 2016 under the trade name Vemlidy. Tenofovir alafenamide is hydrolyzed into tenofovir as a lipophilic drug under the action of carboxylesterase in a human body. Currently, few technical reports exist for simultaneously analyzing tenofovir alafenamide and tenofovir in plasma. In imitation pharmaceutical clinical studies and therapeutic drug monitoring, it is necessary to determine the concentrations of tenofovir alafenamide and tenofovir in the plasma of a subject or patient to study their pharmacokinetic behavior. In order to accelerate the clinical application, a simple, accurate, rapid and sensitive biological analysis method is needed.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a biological analysis method for clinically researching concentrations of tenofovir alafenamide and a metabolite tenofovir in a plasma sample by using an antiviral drug.

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

a bioanalysis method for tenofovir alafenamide and metabolite tenofovir in a plasma sample in clinical research of antiviral drugs comprises the following steps:

pretreating, adding plasma into a 96-well plate, adding an internal standard solution, adding methanol, namely acetonitrile (50:50, v/v), carrying out vortex and centrifugation, taking supernate into another clean 96-well plate, adding a 0.1% formic acid aqueous solution, carrying out vortex mixing, and carrying out sample injection of 20.0 mu L;

chromatography, separating the sample to be detected by liquid chromatography, and adopting Eclipse Plus C₁₈Performing gradient elution on a chromatographic column, wherein a mobile phase A is an aqueous solution containing 0.1% of formic acid and 2mM of ammonium acetate, a mobile phase B is acetonitrile, and the chromatographic running time is 5.3 min;

mass spectrometry using an electrospray ion source, positive ion detection, ejection voltage 5500V, Gas 1(Gas1)60psi, Gas 2(Gas2)60psi, Curtain Gas (Curtain Gas)35psi, ion source temperature 550 ℃, collision induced dissociation 9psi, residence time 100ms, Tenofovir alafenamide quantitative ion pair m/z477.2 → 346.1, Collision Energy (CE)60eV, declustering voltage (DP)80V, Tenofovir quantitative ion pair 288.1 → 176.1, Collision Energy (CE)35eV, declustering voltage (DP)80V, Tenofovir alafenamide-d 5 quantitative ion pair 482.3 → 351.2, Collision Energy (CE)60eV, declustering voltage (DP)80V, Tenofovir-d 7 quantitative ion pair 295.2 →.1, Collision Energy (CE)35eV, declustering voltage (DP) 80V.

Preferably, in the biological analysis method for concentrations of tenofovir alafenamide and a metabolite tenofovir in a plasma sample of clinical research of the antiviral drug, in the pretreatment step, 100 mu L of the plasma sample is taken, 50.0 mu L of an internal standard solution is added, 300 mu L of methanol: acetonitrile (50:50, v/v) is added, 100 mu L of supernatant is taken to be put into another clean 96-well plate after vortex flow and centrifugation, 0.1% formic acid aqueous solution is added, and vortex flow mixing is carried out.

Preferably, the bioanalytical method for the concentration of tenofovir alafenamide and the metabolite tenofovir in the clinical research plasma sample of the antiviral drug comprises tenofovir alafenamide-d 5 and tenofovir-d 7 solutions.

Preferably, said one antiviral drug is in a clinical study plasma sampleA biological analysis method of tenofovir alafenamide and tenofovir metabolite concentration, and the chromatographic step and the chromatographic column type are Eclipse Plus C₁₈。

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

1. the method has the characteristics of simple and convenient pretreatment operation, can be used for analysis only by one-step extraction and one-step dilution, is easy to store the extracted sample, has high analysis speed and analysis time of 5.3min, and is suitable for analyzing large-scale clinical research samples.

2. The invention has higher sensitivity, the lower limit of the quantification of the tenofovir alafenamide and the tenofovir is 1.25 ng/mL and 0.300ng/mL respectively, and the detection limit is 0.0380 ng/mL and 0.0340ng/mL respectively through calculation.

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 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.

Figure 1 is an ion scan mass spectrum of the tenofovir alafenamide product.

Figure 2 is a tenofovir product ion scan mass spectrum.

Figure 3 is an ion scanning mass spectrum of the product tenofovir alafenamide-d 5.

Figure 4 is an ion scanning mass spectrum of the product tenofovir-d 7.

FIG. 5 is an MRM chromatogram of tenofovir alafenamide (left) and tenofovir alafenamide-d 5 (right) from a blank plasma sample.

FIG. 6 is an MRM chromatogram of tenofovir (left) and tenofovir-d 7 (right) from a blank plasma sample.

FIG. 7 is an MRM chromatogram of tenofovir alafenamide (left) and tenofovir alafenamide-d 5 (right) in a quantitative lower limit sample.

FIG. 8 is an MRM chromatogram of tenofovir (left) and tenofovir-d 7 (right) in a quantitative lower limit sample.

Fig. 9 is a drug concentration-time curve of tenofovir alafenamide after a single oral administration of 25mg of propineb fumarate tenofovir tablets to 1 healthy subject.

Fig. 10 is a drug concentration-time curve of tenofovir after a single oral administration of 25mg of propiophenol fumarate tenofovir tablets in 1 healthy subject.

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

Pretreatment

The method uses the plasma with the dosage of only 100 mu L, is suitable for clinical research and biological analysis work, adopts a protein precipitation method, has higher recovery rates for tenofovir alafenamide with weaker polarity and tenofovir with stronger polarity, 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-throughput samples in clinical research.

The specific pretreatment method comprises the following steps:

1. add 100. mu.L of plasma sample, 50.0. mu.L of internal standard solution (20.0/3.00ng/mL), 300. mu.L of methanol to acetonitrile (50:50, v/v) to 96-well plate;

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

3. taking 100 mu L of supernatant to another clean 96-well plate;

4. the mixture was diluted with 300. mu.L of a 0.1% formic acid aqueous solution.

5. The injection volume was 20.0. mu.L.

Mass spectrometry

As shown in fig. 1 to 4, electrospray ion source, positive ion detection, ejection voltage 5500V, Gas 1(Gas1)60psi, Gas 2(Gas2)60psi, Gas Curtain Gas (Curtain Gas)35psi, ion source temperature 550 ℃, collision induced dissociation 9psi, residence time 100ms, tenofovir alafenamide quantitative analysis ion pair m/z477.2 → 346.1, Collision Energy (CE)60eV, declustering voltage (DP)80V, tenofovir quantitative analysis ion pair 288.1 → 176.1, Collision Energy (CE)35eV, declustering voltage (DP)80V, tenofovir alafenamide-d 5 quantitative analysis ion pair 482.3 → 351.2, Collision Energy (CE)60eV, declustering voltage (DP)80V, tenofovir-d 7 quantitative analysis ion pair 295.2 183.1, Collision Energy (CE)35eV, declustering voltage (DP)80V → DP).

Chromatography, separating the sample to be detected by liquid chromatography, and adopting Eclipse Plus C₁₈Chromatographic column, gradient elution, mobile phase A of 0.1% formic acid and 2mM ammonium acetate water solution, and mobile phase B of acetonitrile. The tenofovir alafenamide has weaker polarity and is obviously retained on a conventional reversed-phase chromatographic column, and the tenofovir has stronger polarity than the tenofovir alafenamide and weaker retention due to containing phosphonic acid groups.

The chromatographic separation of the invention adopts Eclipse Plus C₁₈The chromatographic column has good retention to the object to be detected and the internal standard, and has sharp peak shape. 0.1% formic acid is added into a mobile phase A adopted by the chromatogram, so that the ionic tenofovir can be converted into a molecular state, and the polarity of the tenofovir is reduced to enhance the chromatographic retention. Because the polarity of the tenofovir and the tenofovir alafenamide is different, in order to realize the elution of two types to be tested in a short time, the fast elution method is adoptedMode of rapid gradient elution. The instrument analysis flux is high in the mode, the chromatographic running time is 5.3min, the detection is rapid, and the method is suitable for clinical research and analysis of large-batch samples.

The first embodiment is as follows:

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^TM 6500⁺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

5810R Multi-function bench centrifuge, Eppendorf, Germany

1.2 control and reagents

Tenofovir alafenamide (98% pure) was purchased from TRC; tenofovir (99.7% pure, 93.4% content) was purchased from TLC company; tenofovir alafenamide-d 5 (98% pure) was purchased from TRC; tenofovir-d 7 (99.8% chemical purity, 99.6% isotopic purity) was purchased from TLC. Methanol (HPLC grade), acetonitrile (HPLC grade) were purchased from Sigma company, usa. Formic acid (HPLC grade) was purchased from TCI company. Ammonium acetate (HPLC grade) was purchased from ROE corporation. Deionized water (18.2m Ω, TOC. ltoreq.50 ppb) was prepared from a Milli-Q ultrapure water system.

2 method

2.1 preparation of solutions and samples

Standard series of samples: precisely weighing a proper amount of a tenofovir alafenamide reference substance, dissolving the reference substance by using methanol and fixing the volume, precisely weighing a proper amount of the tenofovir reference substance, dissolving the reference substance by using methanol and water (10:90, v/v) and fixing the volume to prepare a stock solution with the concentrations of the tenofovir alafenamide and the tenofovir being 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 ranges of tenofovir alafenamide and tenofovir are 1.25-500ng/mL and 0.300-15.0ng/mL respectively.

Quality control of the sample: the mixed quality control samples of tenofovir alafenamide and tenofovir with 4 concentration levels are prepared by adopting a method similar to that of the standard series of samples. The lower limit concentration of the quantification is 1.25/0.300ng/mL, the concentration of Low Quality Control (LQC) is 3.75/0.900ng/mL, the concentration of sub-Middle Quality Control (AMQC) is 25.0/2.00ng/mL, the concentration of Medium Quality Control (MQC) is 250/7.50ng/mL, and the concentration of High Quality Control (HQC) is 400/12.0 ng/mL.

Internal standard solution: precisely weighing a tenofovir alafenamide-d 5 reference substance, dissolving the reference substance by methanol and fixing the volume, precisely weighing a tenofovir-d 7 reference substance, dissolving the reference substance by methanol and water (10:90, v/v) and fixing the volume, and preparing an internal standard stock solution with the concentration of about 1.00 mg/mL. Precisely sucking appropriate amount of the internal standard stock solutions, adding methanol: water (50:50, v/v) to obtain internal standard solutions with tenofovir alafenamide-d 5 and tenofovir-d 7 concentrations of 20.0 and 3.00ng/mL, respectively.

2.2 plasma sample treatment

2.3 chromatographic and Mass Spectrometry conditions

Chromatographic conditions

Conditions of Mass Spectrometry

2.4 methodological 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

Six blank blood plasma with different sources and respectively prepared quantitative lower limit samples are taken for processing and then sample injection analysis is carried out. 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

Linear regression equation (weight factor W is 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 substance 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 analyzing six samples after the placing is finished. A total of four placement conditions were examined, which were: placing in ice water bath for 9h, extracting, placing in sample injector for 101h, performing 5 times of freezing-thawing cycle (from-75 + -10 deg.C to ice water bath), and placing at 75 + -5 deg.C for 40 days.

Recovery rate

Taking 100 mu L of blank plasma, adding the solution to be detected and the internal standard solution after extraction (without adding the internal standard solution) to ensure that the final concentration is the same as LQC, MQC and HQC, and carrying out sample injection and determination. 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 from 6 different sources, extracting (without adding an internal standard solution), adding a solution to be measured and an internal standard solution with the same concentration as that of 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 concentrations of tenofovir alafenamide and tenofovir in clinical research plasma samples, and is used for human pharmacokinetics research of the Propofol fumarate tenofovir 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. 40 healthy subjects were given 25mg of Propofovir fumarate tablets. At different time points before (0h) and within 96h after administration, collecting venous blood, taking 4mL each, placing in an EDTA-K2 anticoagulation centrifuge tube, centrifuging (1700g, 4 ℃) for 10min, separating plasma, and storing at-75 +/-10 ℃.

3 results and discussion

3.1 methodological validation

Selectivity of the process

As shown in fig. 5 to 8, the retention times of tenofovir alafenamide, tenofovir alafenamide-d 5 and tenofovir-d 7 were about 4.12, 2.95, 4.11 and 2.95min, respectively, and there was no co-efflux interference peak at the retention time.

Standard curve

Determination of Tenofovir disoproxil fumarate clinical study the linear range of Tenofovir alafenamide and Tenofovir in plasma samples were 1.25-500ng/mL and 0.300-15.0ng/mL, respectively. The typical linear regression equation of the standard curve of the substance to be measured is respectively as follows:

tenofovir alafenamide, y 0.0319x + 0.00228;

tenofovir, y ═ 0.85x + 0.0297;

detection limit

The concentrations of tenofovir alafenamide and tenofovir in the lower limit samples were 1.25 and 0.300ng/mL, respectively, and the signal-to-noise ratios were 105 and 26.2, respectively. The detection limits were calculated as signal to noise ratio of 3 at 0.0380 and 0.0340ng/mL, respectively.

Precision and accuracy of the method

The results of precision accuracy were in accordance with the accepted standards and are shown in Table 1. Wherein, table 1 shows the precision and accuracy of the determination of tenofovir alafenamide and tenofovir in human plasma.

TABLE 1

Recovery rate of treatment

LQC, MQC and HQC concentration levels: the extraction recovery rates of tenofovir alafenamide are respectively 103.2%, 101.4% and 100.0%; the extraction recovery rates of tenofovir are respectively 101.1%, 101.2% and 98.9%; (ii) a The recovery rates of tenofovir alafenamide-d 5 and tenofovir-d 7 were 100.8% and 100.5%, respectively.

Matrix effect

The internal standard normalized matrix factors of tenofovir alafenamide at the concentration levels of LQC and HQC are 97.7% and 99.1% respectively, and the RSD is 1.4% and 1.4% respectively; the normalized prime factors of the tenofovir base internal standard are respectively 105.4% and 99.9%, and the RSD is respectively 2.6% and 2.5%. 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 tests are shown in table 2 and show that both tenofovir alafenamide and tenofovir are stable under the investigated conditions, wherein table 2 is the stability of tenofovir alafenamide and tenofovir in human plasma (n ═ 6).

TABLE 2

4 human pharmacokinetics study

The validated method was used to simultaneously analyze tenofovir alafenamide and tenofovir in plasma to evaluate its pharmacokinetic profile. 1 healthy subject orally takes 25mg of the propane fumarate tenofovir disoproxil fumarate tablet once, the plasma drug concentration-time curve is shown in fig. 9 and fig. 10, the detection method sensitivity can completely depict the pharmacokinetic characteristics of tenofovir alafenamide and tenofovir which is a metabolite of the tenofovir disoproxil fumarate, the selection of a linear range is close to the concentration level of an actual sample, and the determination accuracy is high.

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.