CN111665302A

CN111665302A - Kit for detecting antiviral drugs in serum

Info

Publication number: CN111665302A
Application number: CN202010483951.4A
Authority: CN
Inventors: 成晓亮; 李美娟
Original assignee: Nanjing Pinsheng Medical Technology Co ltd
Current assignee: Nanjing Pinsheng Medical Technology Co ltd
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2020-09-15

Abstract

The invention relates to a kit for detecting antiviral drugs in serum, which can detect 3 common antiviral drugs at one time, has high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the antiviral drugs in the serum within 5.0 minutes, basically meets the requirements on accuracy and precision, can be used for the quantitative analysis of the antiviral drugs in the serum in clinic, and provides a simple and rapid detection method for the concentration monitoring of the antiviral drugs in the serum in clinic.

Description

Kit for detecting antiviral drugs in serum

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a kit for detecting an antiviral drug in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the specific drugs are as follows: ganciclovir (Ganciclovir, GCC), Tenofovir (Tenofovir, TNF) and Efavirenz (Efavirenz, EFV).

Background

Viruses are an acellular form of nucleic acid molecules and proteins, living parasitically between living and non-living organic species. Viruses can infect almost all living organisms with cellular structures. Efavirenz is a noncompetitive inhibitor of HIV-1 virus reverse transcriptase, is a non-nucleoside drug, is mainly combined with other antiviral drugs, is suitable for people infected by HIV-1 virus, and is considered as a first-line antitubercular drug with the highest compatibility; the level of efavirenz in vivo determines whether the treatment is successful and whether central nervous system toxicity is produced, and the blood concentration changes more when the combination is used, so that the concentration needs to be monitored. The nucleotide antiviral medicine is the first choice medicine for treating AIDS, hepatitis and other viral diseases clinically, and has the action target of inhibiting DNA polymerase of DNA virus or reverse transcriptase of RNA virus. Tenofovir is a novel nucleoside reverse transcriptase inhibitor, is a first-line antiviral drug for treating HIV and hepatitis B virus infection, has great advantages in safety, effectiveness and tolerance, but has poor bioavailability and needs to monitor the concentration in real time. Ganciclovir is also a nucleotide analogue, and oral ganciclovir can be used for treating infections such as hepatitis virus, human Cytomegalovirus (CMV), Herpes Simplex Virus (HSV), and the like. With the increasing of drug resistance, toxic and side effects and other drug diseases caused by antiviral drugs, the required concentration of a patient needs to be monitored when the drugs are used, so that the curative effect is ensured, the toxic and side effects are reduced, and personalized administration and treatment are realized.

At present, methods for detecting the in vivo concentration of antiviral drugs mainly comprise high performance liquid chromatography and ultra-high performance liquid-tandem mass spectrometry, and patent reports (patent number CN 110361483A) disclose 'a method for detecting tenofovir in human serum by UPLC/MS-MS combined use', the sample size of the method needs to be large, the detection is only carried out on one drug tenofovir, and the defects of long analysis time of a single sample, unsuitable linear range and the like exist.

Disclosure of Invention

The invention aims to provide a kit for detecting antiviral drugs in serum on the basis of the prior art.

The invention also aims to provide application of the kit in detecting antiviral drugs in serum by using an ultra performance liquid chromatography tandem mass spectrometry technology.

The technical scheme of the invention is as follows:

a kit for detecting antiviral drugs in serum,

the antiviral drugs are respectively: ganciclovir (GCC), Tenofovir (TNF) and Efavirenz (EFV);

the internal standard substances corresponding to the antiviral drugs are respectively as follows: ganciclovir-d 5(GCC-d5), tenofovir-d 6(TNF-d6) and efavirenz-d 5(EFV-d 5);

the kit comprises the following reagents:

(1) eluent:

eluent A: 0.01 to 0.2 percent of formic acid-1 to 5mM ammonium acetate aqueous solution; eluent B: acetonitrile;

(2) calibration solution:

a mixed standard stock solution containing Ganciclovir (GCC)200000ng/mL, Tenofovir (TNF)40000ng/mL and Efavirenz (EFV)200000ng/mL was formulated in a blank serum matrix as a calibrator solution at seven different concentration points:

the concentrations of Ganciclovir (GCC) and Efavirenz (EFV) are the same, with seven concentration points in order: 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL, 10000 ng/mL;

seven concentration points of Tenofovir (TNF) are in order: 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000 ng/mL;

(3) mixing internal standard solutions:

contains a methanol solution of ganciclovir-d 55000 ng/mL, tenofovir-d 610000 ng/mL and efavirenz-d 515000 ng/mL;

(4) protein precipitant:

a mixed solution of methanol, acetonitrile and isopropanol;

(5) quality control product:

blank serum matrix containing antiviral drugs comprises low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,

QC (L) is the above mixed standard stock solution diluted 2000 times with blank serum matrix;

QC (M) is the above mixed standard stock solution diluted to 200 times with blank serum matrix;

qc (h) was a 50-fold dilution of the above mixed standard stock solution in blank serum matrix.

In a preferred embodiment, the eluent A is 0.1% -0.15% formic acid-1-2.5 mM ammonium acetate aqueous solution, preferably 0.1% formic acid-2 mM ammonium acetate aqueous solution.

In one scheme, the volume ratio of methanol to acetonitrile to isopropanol in the protein precipitant is 1-5: 1: 1-2; preferably, the volume ratio of methanol, acetonitrile to isopropanol in the protein precipitant is 1:1: 1.

In one embodiment, the serum blank matrix is serum blank without an antiviral drug of interest.

The mixed standard stock solutions mentioned in the present invention were prepared as follows:

the antiviral drug is prepared into standard mother liquor with the following concentration: ganciclovir 20mg/mL, tenofovir 10mg/mL and efavirenz 20 mg/mL;

respectively transferring mother liquor of each standard product: ganciclovir 10 μ L, tenofovir 4 μ L and efavirenz 10 μ L; add to 976. mu.L of methanol to give 1mL of mixed standard stock solution.

The mixed internal standard solution mentioned in the invention is prepared according to the following method:

preparing the following isotope internal standard mother liquor by using methanol: ganciclovir-d 51 mg/mL, tenofovir-d60.5mg/mL and efavirenz-d 51 mg/mL;

respectively transferring the internal standard mother liquor of each isotope: ganciclovir-d 55 μ L, tenofovir-d 620 μ L and efavirenz-d 515 μ L; then adding into 960 mu L methanol to obtain 1mL mixed internal standard solution; wherein, ganciclovir-d 55000 ng/mL, tenofovir-d 610000 ng/mL and efavirenz-d 515000 ng/mL;

the serum mentioned in the invention is human or animal serum.

In a preferred embodiment, a kit for detecting an antiviral drug in serum comprises the following reagents:

(1) eluent:

eluent A: 0.1% formic acid-2 mM ammonium acetate in water; eluent B: acetonitrile;

(2) calibration solution:

respectively transferring mother liquor of each standard product: ganciclovir 10 μ L, tenofovir 4 μ L and efavirenz 10 μ L; then adding the mixture into 976 mu L of methanol to obtain 1mL of mixed standard stock solution; the concentrations are given in table 1 below.

Table 1 stock solutions of mixed standards

The invention prepares the stock solution of the mixed standard substance into the solutions of the calibrator with seven different concentration points by using a blank serum substrate (blank serum without antiviral target drugs), and the preparation process is as follows:

adding 10 mu L of mixed standard stock solution into 190 mu L of blank serum matrix to serve as a first high-value concentration point; taking the first high-value concentration point, and diluting the first high-value concentration point with an equal volume of blank serum matrix to obtain a second high-value concentration point; diluting the first high-value concentration point with 3 times volume of blank serum substrate to obtain a third high-value concentration point; diluting the second high-value concentration point with 9 times volume of blank serum substrate to obtain a fourth high-value concentration point; diluting the third high-value concentration point with 9 times volume of blank serum substrate to obtain a fifth high-value concentration point; diluting the fourth high-value concentration point with a blank serum substrate with 4 times of volume to obtain a sixth high-value concentration point; and (4) diluting the fifth high-value concentration point with blank serum substrate with 4 times of volume to obtain a seventh high-value concentration point.

The invention adopts a gradient dilution method to prepare standard yeast, after a standard solution is taken out from a refrigerator at the temperature of 20 ℃ below zero, the standard solution is vortexed for 10s, the highest concentration point of the standard yeast is prepared by using the standard solution within 2min, and the standard yeast is stored at the temperature of 80 ℃ below zero after the standard yeast is prepared, and the specific process is shown in the following table 2 (unit: ng/mL).

TABLE 2 Standard koji preparation

(3) Mixing internal standard solutions:

respectively transferring the internal standard mother liquor of each isotope: ganciclovir-d 55 μ L, tenofovir-d 620 μ L and efavirenz-d 515 μ L; then adding into 960 mu L methanol to obtain 1mL mixed internal standard solution; the concentrations refer to table 3 below.

TABLE 3 Mixed internal standard solution preparation

(4) Protein precipitant:

a mixed solution of methanol, acetonitrile and isopropanol, wherein the volume ratio of the methanol to the acetonitrile to the isopropanol is 1:1: 1;

(5) quality control product:

the mixed standard stock solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by blank serum without antiviral target drugs, wherein the three different concentrations are specifically shown in table 4.

TABLE 4 corresponding concentration of antiviral drugs (unit ng/mL)

QC (L) includes: 100ng/mL Ganciclovir (GCC), 20ng/mL Tenofovir (TNF) and 100ng/mL Efavirenz (EFV).

QC (M) comprises: ganciclovir (GCC)1000ng/mL, Tenofovir (TNF)200ng/mL and Efavirenz (EFV)1000 ng/mL.

QC (H) includes: ganciclovir (GCC)4000ng/mL, Tenofovir (TNF)800ng/mL and Efavirenz (EFV)4000 ng/mL.

When the kit provided by the invention is used for detecting the antiviral drugs in serum, the mixed internal standard solution and the protein precipitator are mixed to prepare the protein precipitator containing the internal standard. In a preferable scheme, the volume ratio of the mixed internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7. In a more preferred embodiment, the volume ratio of the mixed internal standard solution to the protein precipitant is 0.2:19.8(1: 99). For example, the internal standard-containing protein precipitant is prepared by mixing a mixed internal standard solution and a protein precipitant (methanol, acetonitrile and isopropanol in a volume ratio of 1:1:1) in a volume ratio of 1: 99.

The application of the kit in detecting the antiviral drugs in the serum by using the ultra-performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.

The specific detection method comprises the following steps:

a method for detecting the concentration of antiviral drugs in serum,

after a serum sample is subjected to protein precipitation, oscillating and centrifuging to take a supernatant for sample injection, detecting the antiviral drug in pretreated serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating an object to be detected and a serum matrix by using the ultra-high performance liquid chromatography, establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance and an internal standard substance as an X axis and the peak area ratio of the standard substance and the internal standard substance as a Y axis, and calculating the content of the antiviral drug, wherein the specific chromatographic conditions are as follows:

(1) ultra-high performance liquid chromatography conditions:

mobile phase A: 0.01 to 0.2 percent of formic acid-1 to 5mM ammonium acetate aqueous solution; mobile phase B: acetonitrile;

the type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 100mm,1.7 μm);

a mixed mobile phase A and a mixed mobile phase B are adopted for gradient elution, and the initial ratio of the mobile phase A to the mobile phase B is 90-100: 10-0; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from the initial ratio to 70:30 at a constant speed within 0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 70:30 to 10:90 at a constant speed within 1.0-3.0 minutes; in 3.0-5.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 10:90 to the initial ratio at a constant speed; the collection time of each sample was 5.0 minutes;

(2) mass spectrum conditions:

under an electrospray ionization positive ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring, wherein the spray voltage is 3.0kV (ESI +); the source temperature is 150 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; each target and its corresponding isotope internal standard were monitored simultaneously.

In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid and ammonium acetate into the mobile phase A, can effectively improve the ionization efficiency of certain target compounds, has higher sensitivity for detecting the antiviral drugs in the serum by adopting an LC-MS/MS method in the prior art under the coordination of other conditions, has simple pretreatment process, low cost, high sensitivity and strong specificity, and completes the separation and detection of the antiviral drugs within 5 minutes. In a preferable embodiment, the mobile phase A is 0.1% -0.15% formic acid-1-2.5 mM ammonium acetate aqueous solution, and preferably, the mobile phase A is 0.1% formic acid-2 mM ammonium acetate aqueous solution without affecting the effect of the invention.

In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts acetonitrile and 0.01-0.2% formic acid-1-5 mM ammonium acetate aqueous solution as mobile phase, the type of chromatographic column is ACQUITY UPLC BEH C18(2.1 multiplied by 100mM,1.7 mu m), endogenous substances do not interfere the determination of a sample under the coordination of other conditions, the sensitivity is high, the specificity is strong, and the accuracy and the precision basically meet the requirements.

When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. According to the invention, ganciclovir-d 5(GCC-d5), tenofovir-d 6(TNF-d6) and efavirenz-d 5(EFV-d5) are respectively adopted as internal standards, the deuterated internal standard and the substance to be tested have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy in the determination of the antiviral drug in serum are better.

In a preferable scheme, the initial ratio of the mobile phase A to the mobile phase B is 95-99: 5-1. Further preferably, the initial ratio of mobile phase a and mobile phase B is 97: 3.

In a preferred embodiment, the flow rate is 0.2-0.5 mL/min, preferably 0.3 mL/min.

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

In one embodiment, the injection volume is 0.2-5 μ L, preferably 1 μ L.

In a preferred scheme, when the antiviral drugs in the pretreated serum are detected by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, the specific chromatographic conditions are as follows:

(1) ultra-high performance liquid chromatography conditions:

mobile phase A: 0.1% formic acid-2 mM ammonium acetate in water; mobile phase B: acetonitrile;

the type of the chromatographic column: a CQUITY UPLC BEH C18 (2.1X 100mm,1.7 μm);

adopting a mode of gradient elution by taking a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the initial ratio of the mobile phase A to the mobile phase B is 97: 3; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 97:3 to 70:30 at a constant speed within 0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 70:30 to 10:90 at a constant speed within 1.0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 10:90 to 97:3 at a constant speed within 3.0-5.0 minutes; the collection time of each sample was 5.0 minutes; the gradient elution pattern is detailed in table 5; the flow rate was 0.3mL/min, the column temperature was 45 ℃ and the injection volume was 1. mu.L.

TABLE 5 mobile phase gradient elution parameters

(2) Mass spectrum conditions:

under an electrospray ionization positive ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring, wherein the spray voltage is 3.0kV (ESI +); the source temperature is 150 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; the mass spectrum source parameters are shown in table 6, and the mass spectrum parameters of each target and the corresponding isotope internal standard thereof are simultaneously monitored and shown in table 7.

TABLE 6 Mass Spectrometry Source parameters

TABLE 7 antiviral drug detection Mass Spectrometry parameters

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

The pretreated serum mentioned in the present invention is prepared as follows: adding a protein precipitator containing an internal standard into the plasma, and then oscillating and centrifuging to obtain a supernatant; wherein the protein precipitator is a mixed solution of methanol, acetonitrile and isopropanol; preferably, the volume ratio of methanol to acetonitrile to isopropanol in the protein precipitant is 1-5: 1: 1-2. More preferably, the volume ratio of methanol, acetonitrile to isopropanol in the protein precipitant is 1:1: 1.

In a preferred embodiment, the pretreated serum of the present invention is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing internal standard (the volume ratio of methanol to acetonitrile to isopropanol in the protein precipitator is 1-5: 1: 1-2), oscillating for 3-5 min, centrifuging for 4-10 min at 1-5 ℃ at 12000-15000 r/min, transferring 60 mu L of supernatant in the centrifuge tube into a plastic liner tube, and feeding the sample; the protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the volume ratio of the mixed internal standard solution to the protein precipitant is 0.1-0.3: 19.9-19.7.

In a more preferred embodiment, the pretreated serum of the present invention is prepared as follows: putting 50 μ L of serum into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol, acetonitrile and isopropanol is 1:1:1), and shaking at high speed for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transferring 60 mu L of supernatant in the EP tube to a plastic lining tube for sample injection, wherein the protein precipitator containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitator, and the volume ratio of the mixed internal standard solution to the protein precipitator is 0.2:19.8 (namely 1: 99).

In one embodiment, the protein precipitant containing the internal standard is prepared as follows:

preparing the following isotope internal standard mother liquor by using methanol: ganciclovir-d 5(GCC-d5)1mg/mL, tenofovir-d 6(TNF-d6)0.5mg/mL and efavirenz-d 5(EFV-d5)1 mg/mL;

respectively transferring the internal standard mother liquor of each isotope: ganciclovir-d 5(GCC-d5)5 μ L, tenofovir-d 6(TNF-d6)20 μ L and efavirenz-d 5(EFV-d5)15 μ L; then adding into 960 mu L methanol to obtain 1mL mixed internal standard solution; wherein, ganciclovir-d 55000 ng/mL, tenofovir-d 610000 ng/mL and efavirenz-d 515000 ng/mL.

And adding 200uL of the mixed internal standard solution into 19.8mL of protein precipitant to obtain the internal standard-containing protein precipitant.

In one scheme, the protein precipitating agent is a mixed solution of methanol, acetonitrile and isopropanol; preferably, the volume ratio of methanol to acetonitrile to isopropanol in the protein precipitant is 1-5: 1: 1-2. More preferably, the volume ratio of methanol, acetonitrile to isopropanol in the protein precipitant is 1:1: 1.

In one embodiment, the standard solution is prepared as follows:

the antiviral drug is prepared into standard mother liquor with the following concentration: ganciclovir (GCC)20mg/mL, Tenofovir (TNF)10mg/mL and Efavirenz (EFV)20 mg/mL;

respectively transferring mother liquor of each standard product: ganciclovir (GCC)10 μ L, Tenofovir (TNF)4 μ L and Efavirenz (EFV)10 μ L; add to 976. mu.L of methanol to give 1mL of mixed standard stock solution. The mixed standard stock solution comprises: ganciclovir (GCC)200000ng/mL, Tenofovir (TNF)40000ng/mL, and Efavirenz (EFV)200000 ng/mL.

Preparing the mixed standard stock solution into a calibrator solution with seven different concentration points by using a blank serum matrix (blank serum without an antiviral target drug), wherein the seven concentration points of the calibrator solution are as follows:

seven concentration points of Tenofovir (TNF) are in order: 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000 ng/mL.

50 mu L of sample at each concentration point is taken and put into a 1.5mL centrifuge tube, 200 mu L of protein precipitant containing internal standard (the volume ratio of methanol, acetonitrile and isopropanol in the protein precipitant is 1:1:1) is added into the centrifuge tube, the mixture is shaken for 5min, and after the centrifuge is carried out for 5min at 14000r/min and 4 ℃, 60 mu L of supernatant in the centrifuge tube is transferred into a plastic lining tube for sample injection, and the sample injection amount is 1 mu L.

The invention also comprises a quality control product prepared by the following method: preparing the mixed standard stock solution into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without an antiviral target drug, wherein,

qc (l) was a 2000-fold dilution of the above mixed standard stock solution in blank serum matrix.

Qc (m) was a 200-fold dilution of the above mixed standard stock solution in blank serum matrix.

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

when the kit provided by the invention is used for detecting the antiviral drugs in the serum, 3 common antiviral drugs can be detected at one time, the sensitivity is high, the specificity is strong, the accuracy is high, the pretreatment process is simple, the separation and the detection of the antiviral drugs in the serum can be completed within 5.0 minutes, the accuracy and the precision basically meet the requirements, the kit can be used for the quantitative analysis of the antiviral drugs in the serum in clinic, and a simple and rapid detection method is provided for the concentration monitoring of the antiviral drugs in the serum in clinic.

Drawings

FIG. 1 is an extracted ion flowgram of an antiviral drug standard;

FIG. 2 is an extracted ion flowgram of antiviral drugs in serum.

Detailed Description

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

Example 1:

first, experimental material and instrument

1. Material

The sample is from a serum sample collected from the outpatient clinic of 2019 months in Beijing 301 Hospital.

(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube vortex mixer (vortegenie 2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like.

(2) Reagent consumables: MS grade methanol (Fisher, usa); HPLC grade methanol (Honeywell, usa); MS grade acetonitrile (Fisher, usa); HPLC grade acetonitrile (Honeywell, usa); HPLC grade isopropanol (Honeywell, usa); MS grade formic acid (Fisher, usa); MS grade ammonium acetate (Sigma, usa); the type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 100mm,1.7 μm) (Waters Corporation).

(3) And (3) standard substance: the standards and their corresponding internal standards are shown in table 8 below.

TABLE 8 Standard and internal standards

(4) Quality control product: the blank serum containing antiviral drug substances has low, medium and high concentrations of QC (L), QC (M) and QC (H), respectively, as shown in Table 4.

Second, liquid condition

(1) Chromatographic conditions are as follows: chromatographic conditions are as follows: mobile phase A: 0.1% formic acid-2 mM ammonium acetate in water; mobile phase B: and (3) acetonitrile. The type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 100mm,1.7 μm), using gradient elution, as detailed in Table 5. The flow rate was 0.3mL/min, the column temperature was 45 ℃ and the injection volume was 1. mu.L.

(2) Mass spectrum conditions: under an electrospray ionization positive ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring, wherein the spray voltage is 3.0kV (ESI +); the source temperature is 150 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; the mass spectrum source parameters are shown in table 6, and the mass spectrum parameters of each target and the corresponding isotope internal standard thereof are simultaneously monitored and shown in table 7.

Second, the experimental procedure

(1) Preparing a standard substance:

the antiviral drug is prepared into standard mother liquor with the following concentration: ganciclovir (GCC)20mg/mL, Tenofovir (TNF)10mg/mL and Efavirenz (EFV)20 mg/mL.

Respectively transferring mother liquor of each standard product: ganciclovir (GCC)10 μ L, Tenofovir (TNF)4 μ L and Efavirenz (EFV)10 μ L; add to 976. mu.L of methanol to give 1mL of mixed standard stock solution. The mixed standard stock solution comprises: ganciclovir (GCC)200000ng/mL, Tenofovir (TNF)40000ng/mL, and Efavirenz (EFV)200000 ng/mL. See table 1 for details.

The mixed standard stock solution is prepared into a calibrator solution with seven different concentration points by using a blank serum matrix (blank serum without an antiviral target drug), which is detailed in table 2, wherein the seven concentration points of the calibrator solution are as follows:

the concentrations of Ganciclovir (GCC) and Efavirenz (EFV) are the same, with seven concentration points in order: 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL, 10000 ng/mL.

(2) Preparation of mixed internal standard solution

Preparing the following isotope internal standard mother liquor by using methanol: ganciclovir-d 5(GCC-d5)1mg/mL, tenofovir-d 6(TNF-d6)0.5mg/mL, and efavirenz-d 5(EFV-d5)1 mg/mL.

Respectively transferring the internal standard mother liquor of each isotope: ganciclovir-d 5(GCC-d5)5 μ L, tenofovir-d 6(TNF-d6)20 μ L and efavirenz-d 5(EFV-d5)15 μ L; additional addition to 960. mu.L methanol gave 1mL of mixed internal standard solution. Wherein the mixed internal standard solution comprises: ganciclovir-d 5(GCC-d5)5000ng/mL, tenofovir-d 6(TNF-d6)10000ng/mL, and efavirenz-d 5(EFV-d5)15000 ng/mL. See table 3 for details.

(3) Preparing a quality control product:

the mixed standard stock solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without antiviral target drugs, and the three different concentrations are specifically shown in Table 4.

(4) Sample processing

1) Pretreatment of a standard product: taking 50 mu L of each concentration point of seven different calibrator samples, putting the concentration points into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile to isopropanol is 1:1:1), and oscillating at a high speed for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube for injection.

2) Pretreatment of a serum sample: putting 50 μ L of serum into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol, acetonitrile and isopropanol is 1:1:1), and shaking at high speed for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube for injection.

3) Pretreatment of quality control products: the quality control solutions QC (L), QC (M), QC (H) are respectively taken and 50 μ L of each quality control solution QC (L), QC (M), QC (H) are respectively put into a 1.5mL centrifuge tube, and then the quality control solutions QC (L), QC (M), QC (H) are consistent with the pretreatment of the serum sample, and the details are not.

The components of the assay kit are shown in Table 9.

TABLE 9 preparation of antiviral drug concentration analysis kit Components

Remarking: the protein precipitant containing the internal standard is prepared by the following method, 200 mu L of the mixed internal standard solution is added into 19.8mL of protein precipitant to obtain the protein precipitant containing the internal standard.

Fourth, method verification

1. Extracting an ion current chromatogram: the peak shapes of the standard antiviral drug and the serum sample are symmetrical, and no interference of a foreign peak exists, which indicates that good detection can be obtained under the condition, and fig. 1 is an extracted ion flow chart of the standard antiviral drug; FIG. 2 is an extracted ion flowgram of an antiviral drug in a serum sample.

2. Calibration curve: and establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the serum by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of the antiviral drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, which is shown in Table 10.

TABLE 10 Linear regression equation and Linear correlation coefficient for antiviral drugs

Serial number	Compound (I)	Retention time (min)	Linear range (ng/mL)	Linear equation of equations	Coefficient of correlation (r)
						1	GCC	1.08	50-10000	y＝0.00247089*x-0.0129057	0.9981
2	TNF	1.07	10-2000	y＝0.000936988*x+0.0015083	0.9993
						3	EFV	2.16	50-10000	y＝0.0153918*x+0.0249297	0.9995

3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample is prepared, 3 concentrations of standard substances of low, medium and high are respectively added, the treatment is repeated by the same steps and 5 times of measurement are carried out, the result shows that the adding standard recovery rate of the antiviral drug is between 86.85% and 97.09%, the RSD of 5 times of repeated tests is in the range of 2.07% to 5.74%, and the statistical result is shown in a table 11.

TABLE 11 recovery of antiviral drug addition results

4. And (3) precision test: taking an interference-free blank serum sample, adding antiviral drug standard substances with different concentrations to obtain serum samples with low, medium and high concentrations, repeatedly processing 6 batches in one day for three days continuously, quantitatively determining the concentration of the antiviral drug by an isotope internal standard method, carrying out 3 batches of processing in three days, and calculating the batch precision to be 3.09-7.51%, wherein the results are shown in Table 12.

TABLE 12 results of the Intra-and Inter-batch precision measurements

Fifth, discuss

The invention establishes a method for simultaneously measuring the antiviral drugs in human serum by ID-UPLC-MS/MS. The serum dosage is less (only 50 mu L), the pretreatment is simple, and the analysis of various substances by one needle only needs 5 minutes, and the method is simple and quick.

The isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the result is not influenced by conditions such as a pretreatment process, instrument response fluctuation and the like, and accurate quantification can be achieved. The accuracy of the method is evaluated by a standard recovery test, and the result shows that the standard recovery of the antiviral drug is 86.85-97.09%, the RSD of 5 times of repeated tests is 2.07-5.74, and the accuracy is good.

The repeatability result of the method shows that the intra-batch precision of the antiviral drug is 1.47-10.50%, the inter-batch precision is 3.09-7.51%, and the repeatability of the method is good. The established serum sample pretreatment process is very simple, protein precipitation is completed in one step, and the serum dosage is only 50 mu L.

In a word, the detection method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the compound within 5 minutes, meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical serum antiviral drugs, and provides a reliable detection method for monitoring related drug concentrations.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for detecting antiviral drugs in serum,

the antiviral drugs are respectively: ganciclovir, tenofovir and efavirenz;

the internal standard substances corresponding to the antiviral drugs are respectively as follows: ganciclovir-d 5, tenofovir-d 6 and efavirenz-d 5;

the kit comprises the following reagents:

(1) eluent:

(2) calibration solution:

preparing a mixed standard stock solution containing 200000ng/mL of ganciclovir, 40000ng/mL of tenofovir and 200000ng/mL of efavirenz into seven calibrator solutions with different concentration points by using a blank serum matrix, wherein the seven concentration points of the calibrator solutions are as follows:

the concentrations of ganciclovir and efavirenz are the same, and seven concentration points are as follows: 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 2500ng/mL, 5000ng/mL, 10000 ng/mL;

the seven concentration points of tenofovir are as follows: 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000 ng/mL;

(3) mixed internal standard solution

(4) protein precipitant:

a mixed solution of methanol, acetonitrile and isopropanol;

(5) quality control product:

2. The kit according to claim 1,

the eluent A is 0.1-0.15% formic acid-1-2.5 mM ammonium acetate aqueous solution, preferably 0.1% formic acid-2 mM ammonium acetate aqueous solution.

3. The kit according to claim 1,

the volume ratio of methanol to acetonitrile to isopropanol in the protein precipitant is 1-5: 1: 1-2; preferably, the volume ratio of methanol, acetonitrile to isopropanol in the protein precipitant is 1:1: 1.

4. The kit according to claim 1,

the blank serum matrix is blank serum without antiviral target drugs.

5. The kit according to claim 1, 2, 3 or 4,

the mixed standard stock solution was prepared as follows:

6. The kit according to claim 5,

the mixed internal standard solution is prepared according to the following method:

respectively transferring the internal standard mother liquor of each isotope: ganciclovir-d 55 μ L, tenofovir-d 620 μ L and efavirenz-d 515 μ L; additional addition to 960. mu.L methanol gave 1mL of mixed internal standard solution.

7. The kit according to claim 1,

the serum is human or animal serum.

8. The kit according to claim 6,

the kit comprises the following reagents:

(1) eluent:

(2) calibration solution:

respectively transferring mother liquor of each standard product: ganciclovir 10 μ L, tenofovir 4 μ L and efavirenz 10 μ L; then adding the mixture into 976 mu L of methanol to obtain 1mL of mixed standard stock solution; preparing the mixed standard stock solution into seven calibrator solutions with different concentration points by using blank serum without an antiviral target drug;

(3) mixing internal standard solutions:

respectively transferring the internal standard mother liquor of each isotope: ganciclovir-d 55 μ L, tenofovir-d 620 μ L and efavirenz-d 515 μ L; then adding into 960 mu L methanol to obtain 1mL mixed internal standard solution;

(4) protein precipitant

The volume ratio of methanol, acetonitrile to isopropanol in the protein precipitant is 1:1: 1;

(5) quality control product:

preparing the mixed standard stock solution into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without an antiviral target drug, wherein,

QC (L) includes: 100ng/mL of ganciclovir, 20ng/mL of tenofovir and 100ng/mL of efavirenz;

QC (M) comprises: 1000ng/mL of ganciclovir, 200ng/mL of tenofovir and 1000ng/mL of efavirenz;

QC (H) includes: ganciclovir 4000ng/mL, tenofovir 800ng/mL and efavirenz 4000 ng/mL.

9. The kit of claim 8, wherein the internal standard-containing protein precipitant is prepared by mixing the mixed internal standard solution and the protein precipitant in a volume ratio of 1:99, and is used for ultra high performance liquid chromatography detection.

10. Use of the kit of any one of claims 1 to 9 for detecting antiviral drugs in serum by ultra performance liquid chromatography tandem mass spectrometry.