CN111830162A

CN111830162A - Method for detecting concentration of nucleoside antiviral drug in serum

Info

Publication number: CN111830162A
Application number: CN202010705327.4A
Authority: CN
Inventors: 成晓亮; 李美娟
Original assignee: Nanjing Pinsheng Medical Laboratory Co ltd
Current assignee: Nanjing Pinsheng Medical Laboratory Co ltd
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2020-10-27

Abstract

The invention discloses a method for detecting the concentration of nucleoside antiviral drugs in serum, belonging to the technical field of drug analysis. The method specifically comprises the steps of pretreating a serum sample through protein precipitation, separating a drug from a serum matrix by using 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 to an 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, and calculating the content of the target drug in the serum. The method can be used for detecting 3 commonly used antiviral drugs of ganciclovir, tenofovir and entecavir at one time, has high sensitivity and good accuracy, can be well applied to clinic, and provides a reliable basis for antiviral drug combination research.

Description

Method for detecting concentration of nucleoside antiviral drug in serum

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting the concentration of nucleoside antiviral drugs in serum.

Background

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 (TNF) 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 real-time monitoring on the concentration. Ganciclovir (GCC) 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), etc. Entecavir (Entecavir, ETC) is a highly effective nucleoside analog with low drug-resistant mutation rate and side effects, has now become the first line of choice in treatment guidelines for Chronic Hepatitis B (CHB) patients and has become an indispensable part due to its powerful inhibitory effect on viral replication and a huge genetic barrier to drug resistance. However, the antiviral therapeutic effect is related to the dosage form, absorption, age, sex, body mass index, blood lipid level, etc., resulting in complication of the treatment. Therefore, studies monitoring their concentration have important clinical value for optimizing therapy. 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, for example, patent CN 109900819A discloses a UPLC/MS-MS combined method for detecting tenofovir in human serum, and patent CN 109239215A discloses an isotope dilution ultra-high performance liquid chromatography mass spectrometry combined method for detecting entecavir in serum or plasma, but the methods have the defects of large sample size, detection only for one drug, long analysis time of a single sample, unsuitable linear range and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for detecting the concentration of nucleoside antiviral drugs in serum, 3 commonly used antiviral drugs can be detected at one time by adopting an LC-MS/MS method, the sensitivity is high, the accuracy is good, the method can be well applied to clinic, and a reliable basis is provided for the research of antiviral drug combination.

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

a method for detecting the concentration of nucleoside antiviral drugs in serum comprises the steps of taking a preprocessed serum sample, separating a target drug from a serum matrix by using ultra-high performance liquid chromatography, obtaining the mass-to-charge ratio of the target drug and an isotope internal standard substance by mass spectrometry, quantifying by using an isotope internal standard method, and respectively calculating the concentration of the target drug according to an established calibration curve;

the target drugs are tenofovir, ganciclovir and entecavir;

the isotope internal standard substances are ganciclovir-d 5(GCC-d5), tenofovir-d 6(TNF-d6) and entecavir-d 3(ETC-d 3).

Further, the ultra-high performance liquid chromatography conditions are as follows:

the mobile phase A is 0.01 to 0.2 percent of formic acid aqueous solution, and the mobile phase B is 0.01 to 0.2 percent of formic acid methanol solution;

the chromatographic column is ACQUITY UPLC BEH C18;

the method adopts a mode that the mobile phase A and the mobile phase B are mixed mobile phases to carry out gradient elution, and the initial ratio of the mobile phase A to the mobile phase B is 95: 5, the volume ratio of the mobile phase A to the mobile phase B is within 0-1.0 minutes from 95: 5, gradually changing to 70:30 at a constant speed; 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 1.0-2.1 minutes; the volume ratio of mobile phase a to mobile phase B was changed from 10:90 to 95: 5, the flow rate is 0.2-0.5 mL/min, the column temperature is 30-50 ℃, and the sample injection volume is 0.2-5 muL.

Further, the mobile phase a is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid methanol solution.

Further, the flow rate was 0.3mL/min, the column temperature was 45 ℃ and the injection volume was 1. mu.L.

Further, the mass spectrometry conditions are: under an electrospray ionization positive ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spray voltage was 3.0kV (ESI +); the source temperature is 150 ℃; the temperature of the atomizing gas is 500 ℃, the airflow speed of the atomizing gas is 800L/h, and the airflow speed of the taper hole is 150L/h.

Further, the pretreated serum sample is prepared according to the following method: taking a serum sample, adding a protein precipitator containing an internal standard into the serum sample, centrifuging and taking a supernatant.

Further, the pretreated serum sample is prepared according to the following method: taking 50 mu L of serum sample, adding 200 mu L of protein precipitator containing internal standard, oscillating at high speed for 5min, centrifuging at 14000r/min and 4 ℃ for 5min, and taking supernatant.

Further, the protein precipitant containing the internal standard is obtained by mixing a mixed internal standard solution and the protein precipitant; the mixed internal standard solution is a mixed solution of ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3 prepared by methanol, and the concentrations of ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3 are 5 mug/mL, 2.5 mug/mL and 0.25 mug/mL respectively; the protein precipitant is one or two of methanol and acetonitrile.

Further, the protein precipitant is a mixed solution of methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 2: 1; the volume ratio of the mixed internal standard solution to the protein precipitant is 1: 99.

further, the calibration curve takes 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, and the concentrations of the adopted standard substances are as follows:

GCC：50ng/mL、100ng/mL、250ng/mL、500ng/mL、2500ng/mL、5000ng/mL、10000ng/mL；

TNF：10ng/mL、20ng/mL、50ng/mL、100ng/mL、500ng/mL、1000ng/mL、2000ng/mL；

ETC：0.05ng/mL、0.1ng/mL、0.25ng/mL、0.5ng/mL、2.5ng/mL、5ng/mL、10ng/mL。

has the advantages that: the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the nucleoside antiviral drugs in the serum within 3.5min, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of the nucleoside antiviral drugs in the serum in clinic, and provides a simple and rapid detection method for monitoring the concentration of the nucleoside antiviral drugs in the clinic.

Drawings

FIG. 1 is the ion flow chart of the nucleoside antiviral drug standard of example 1.

FIG. 2 is an extracted ion flow chart of the antiviral drug nucleoside in serum of example 1.

Detailed Description

The invention provides a method for detecting the concentration of a nucleoside antiviral drug in serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology, which specifically comprises the following steps: pretreating a serum sample, oscillating, centrifuging, taking supernatant for sample injection, separating the nucleoside antiviral drug from a serum matrix by using ultra-high performance liquid chromatography, and establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and 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 to calculate the content of the nucleoside antiviral drug.

The nucleoside antiviral drugs comprise tenofovir, ganciclovir and entecavir;

the isotope internal standard substances are GCC-d5, TNF-d6 and ETC-d 3.

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 aqueous solution; mobile phase B: 0.01 to 0.2 percent of formic acid methanol solution;

the type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm,1.7 μm);

the method adopts a mode that the mobile phase A and the mobile phase B are mixed mobile phases to carry out gradient elution, and the initial ratio of the mobile phase A to the mobile phase B is 95: 5, the volume ratio of the mobile phase A to the mobile phase B is within 0-1.0 minutes from 95: 5, gradually changing to 70:30 at a constant speed; 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 1.0-2.1 minutes; the volume ratio of mobile phase a to mobile phase B was changed from 10:90 to 95: 5, the flow rate is 0.3mL/min, the collection time of each sample is 3.5min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;

(2) mass spectrum conditions:

performing positive ion scanning in an electrospray ionization (ESI) mode by using Multiple Reaction Monitoring (MRM); the spray voltage was 3.0kV (ESI +); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes 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 formic acid is added into the mobile phase A, so that the ionization efficiency of certain target compounds can be effectively improved, under the coordination of other conditions, compared with the prior art that an LC-MS/MS method is adopted to detect the nucleoside antiviral drugs in serum, the method has the advantages of higher sensitivity, simple pretreatment process, low cost, high sensitivity and strong specificity, and the separation and detection of the nucleoside antiviral drugs can be completed within 5 min. In a preferable embodiment, the mobile phase a is 0.01% to 0.2% formic acid aqueous solution, and the mobile phase B is 0.01% to 0.2% formic acid methanol aqueous solution, without affecting the effect of the present invention. In a more preferred embodiment, mobile phase A is 0.1% formic acid in water and mobile phase B is 0.1% formic acid in methanol.

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 0.01 to 0.2 percent formic acid water solution and acetonitrile as mobile phases, and the types of chromatographic columns are as follows: ACQUITYUPLCBEH C18(2.1 × 50mm,1.7 μm), under the coordination of other conditions, the endogenous substance does not interfere with the determination of the sample, the sensitivity is high, the specificity is strong, the cost is low, the pretreatment process is simple, the separation and the detection can be completed within 3.5min, and both the precision and the accuracy 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 entecavir-d 3(ETC-d3) are respectively adopted as internal standards, the deuterated internal standards 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 nucleoside antiviral drugs in serum are better.

In one embodiment, the flow rate is 0.1-0.4 mL/min, preferably 0.3 mL/min.

Further, the column temperature is 35-50 ℃, preferably 45 ℃.

Furthermore, the injection volume is 1-5 μ L, preferably 1 μ L.

In a preferred scheme, when the ultra-high performance liquid chromatography tandem mass spectrometry technology is adopted to detect the nucleoside antiviral drugs in the serum, the specific chromatographic conditions are as follows:

(1) high performance liquid chromatography conditions:

mobile phase A: 0.1% formic acid-water solution;

mobile phase B: 0.1% formic acid-methanol solution;

the gradient elution mode is adopted, and is shown in the table 1; the flow rate is 0.3mL/min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;

TABLE 1 mobile phase gradient elution parameters

Time of day	Flow rate (mL/min)	％A	％B	Curve
						0	0.3	95	5	-
1	0.3	70	30	6
					2.1	0.3	10	90	6
3.5	0.3	95	5	1

(2) Mass spectrum conditions:

performing positive ion mode scanning in an electrospray ionization (ESI) mode by using Multiple Reaction Monitoring (MRM); the spray voltage was 3.0kV (ESI +); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; simultaneously, nucleoside antiviral drugs and corresponding isotope internal standards thereof are monitored, and the mass spectrum acquisition parameters of each target object to be detected are shown in table 2.

TABLE 2 Mass Spectrometry Source parameters

The mass spectral parameters of each target are shown in table 3.

TABLE 3 detection of Mass Spectrometry parameters for nucleoside antiviral drugs

The serum samples referred to in the present invention are human or animal sera.

The pretreated serum mentioned in the present invention is prepared as follows: adding a protein precipitator containing an internal standard into the serum, and taking supernatant after vortex centrifugation; the protein precipitator containing the internal standard is prepared by mixing a mixed internal standard solution and a protein precipitator, wherein the protein precipitator is a mixed solution of methanol and acetonitrile.

Preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1-5: 1, for example, the volume ratio of methanol to acetonitrile in the protein precipitant is 2:1, without affecting the effect of the present invention.

In a preferred embodiment, the pre-treated serum is prepared as follows: 50 mu L of serum is taken and put into a 1.5mL centrifuge tube, 200 mu L of protein precipitator (the volume ratio of methanol to acetonitrile is 1-5: 1) containing internal standard is added into the centrifuge tube, and 60 mu L of supernatant is taken after centrifugation is carried out for 4-10 min at 12000-15000 r/min and 1-5 ℃. The protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and a protein precipitant, wherein the ratio of the mixed internal standard solution to the protein precipitant is 0.1-0.3: 19.7 to 19.9.

In a more preferred embodiment, the pre-treated serum 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 to acetonitrile is 2:1), and oscillating at high speed (maximum oscillation 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 in a 1. mu.L sample volume. The protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the ratio of the mixed internal standard solution to the protein precipitant is 0.2: 19.8, the same as below.

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

GCC-d 51 mg/mL, TNF-d60.5mg/mL and ETC-d 30.05mg/mL were combined with 80% aqueous methanol to form a mixed internal standard solution containing GCC-d 55000 ng/mL, TNF-d 610000 ng/mL and ETC-d 350 ng/mL.

In a preferred embodiment, the mixed internal standard solution is prepared as follows:

accurately transferring a certain volume of isotope internal standard mother liquor of the nucleoside antiviral drugs respectively, adding 959 mu L of 80% methanol aqueous solution, and uniformly mixing to obtain 1mL of mixed internal standard solution, wherein the concentration is shown in Table 4.

TABLE 4 Mixed internal standard solution preparation

The calibration curve is established 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 standard substance is prepared according to the following steps:

respectively preparing nucleoside antiviral drug mother liquor with the concentrations of 20mg/mL GCC, 10mg/mL TNF and 0.1mg/mL ETC to obtain mixed standard solution containing 200000ng/mL GCC, 40000ng/mL TNF and 200ng/mL ETC;

preparing the mixed standard solution into calibration solution with seven different concentration points by using a blank serum matrix solution, wherein the seven concentration points of the calibration solution are as follows:

GCC：50ng/mL、100ng/mL、250ng/mL、500ng/mL、2500ng/mL、5000ng/mL、10000ng/mL，

TNF：10ng/mL、20ng/mL、50ng/mL、100ng/mL、500ng/mL、1000ng/mL、2000ng/mL，

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 into the centrifuge tube, and then carrying out high-speed oscillation for 5min, 14000r/min and centrifugation for 5min at 4 ℃; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube for injection.

In a more preferred embodiment, the calibrator solution is prepared as follows:

accurately transferring a certain volume of nucleoside antiviral drug standard mother liquor, adding 984 μ L of 80% methanol aqueous solution, and mixing well to obtain 1mL of mixed standard solution with concentration shown in Table 5.

TABLE 5 Mixed Standard solution

Numbering	Components	Mother liquor concentration (μ g/mL)	Volume removal (mu L)	Total volume (μ L)	Concentration of Mixed Standard solution (ng/mL)
						1	GCC	20	10	1000	200000
2	TNF	10	4	1000	40000
						3	ETC	0.1	2	1000	200

Preparing standard yeast by gradient dilution method, taking out standard solution from refrigerator at-20 deg.C, vortex for 10s, preparing maximum concentration point of standard yeast with standard solution within 2min, and storing at-80 deg.C. The specific procedure is shown in Table 6 (unit: ng/mL):

TABLE 6 Standard preparation

Standard song	Pipetting solution (mu L)	Blank serum (mu L)	GCC(ng/mL)	TNF(ng/mL)	ETC(ng/mL)
						S7	Standard solution 10	190	10000	2000	10
S6	S7 50	50	5000	1000	5
						S5	S7 40	120	2500	500	2.5
S4	S6 20	180	500	100	0.5
						S3	S5 20	180	250	50	0.25
S2	S4 50	200	100	20	0.1
						S1	S3 50	200	50	10	0.05

Taking the mixed standard solution and preparing the mixed standard solution into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without the nucleoside antiviral drugs as quality control products. Wherein, the corresponding concentration of the nucleoside antiviral drug quality control products in QC (L), QC (M), QC (H) is shown in Table 7.

TABLE 7 nucleoside antiviral drugs quality control corresponding concentration (unit: ng/mL)

Numbering	Components	QC(L)	QC(M)	QC(H)
					1	GCC	100	1000	4000
2	TNF	20	200	800
					3	ETC	0.1	1	4

QC (L) includes: 100ng/mL of GCC, 20ng/mL of TNF and 0.1ng/mL of ETC;

QC (M) comprises: GCC 1000ng/mL, TNF 200ng/mL, ETC 1 ng/mL;

QC (H) includes: GCC 4000ng/mL, TNF 800ng/mL, ETC 4 ng/mL.

The technical solution of the present invention will be further described with reference to the following specific examples, which will be better understood. 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

1. Material

Methodology samples from the study were obtained from serum samples collected from the 8 month clinic of drugstore hospital, Nanjing, 2019.

(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 (Vortex genie2, 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); MS grade formic acid (Fisher, usa); column ACQUITYUPLC BEH C18 (2.1X 50mm,1.7 μm) (Waters Corporation).

(3) Quality control product: the serum solution containing nucleoside antiviral drug molecules has low, medium and high concentrations of QC (L), QC (M) and QC (H), respectively, as shown in Table 7.

2. Method of producing a composite material

(1) Chromatographic conditions are as follows: mobile phase A: 0.1% formic acid-water solution; mobile phase B: 0.1% formic acid-methanol solution; the type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm,1.7 μm), using gradient elution; 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; the source parameters of the mass spectra are shown in table 2, and the mass spectra parameters of each target are shown in table 3.

(3) Preparing a standard substance:

preparing nucleoside antiviral drugs into standard mother liquor by using pure methanol, wherein the concentrations are as follows: GCC 20mg/mL, TNF10mg/mL, ETC0.1mg/mL, then GCC 10. mu. L, TNF 4. mu. L, ETC 2. mu.L, respectively, was removed, and 984. mu.L of methanol was added to give 1mL of mixed standard solution.

Preparing an isotope internal standard substance into mother liquor by using pure methanol, wherein the concentrations are respectively as follows: GCC-d 51 mg/mL, TNF-d60.5mg/mL, ETC-d 30.05mg/mL, then respectively transferring GCC-d 55 mu L, TNF-d 620 mu L, ETC-d 31 mu L, adding 974 mu L of methanol to obtain 1mL of mixed internal standard solution, and adding 200 mu L of mixed internal standard solution to 19.8mL of protein precipitant to obtain the protein precipitant containing the internal standard.

(4) Preparation of quality control product

Low concentration quality control product: diluting the medium concentration quality control with mixed serum by 10 times;

medium concentration quality control: diluting the mixed standard solution by 200 times by using the mixed serum to obtain a medium-concentration quality control sample;

high concentration quality control product: diluting the mixed standard solution by 50 times by using the mixed serum to obtain a high-concentration quality control sample;

(5) sample processing

1) The preparation of a calibration curve is carried out by adopting a gradient dilution method, and the preparation process is as follows:

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

The seven concentration points of the calibrator solution were:

2) pretreatment of a serum sample: putting 50 mu L of serum sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing an internal standard into the centrifuge tube, then oscillating at high speed for 5min, 14000r/min, and centrifuging 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 are put into a 1.5mL centrifuge tube, and then the method is consistent with the pretreatment method of the serum sample, and the details are not repeated here.

Fourth, method verification

1. Extracting an ion flow spectrogram: the peak shapes of the standard substance of the nucleoside antiviral drugs and the serum sample are symmetrical, and no interference of a foreign peak exists, which indicates that the good detection can be obtained under the condition, and fig. 1 is an ion flow chart of the standard substance of the nucleoside antiviral drugs, and fig. 2 is an ion flow chart of the nucleoside antiviral drugs in the serum.

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 nucleoside antiviral drugs in respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, see table 8.

TABLE 8 Linear regression equation and Linear correlation coefficient for nucleoside antiviral drugs

3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. Preparing a mixed blank serum sample, respectively adding 3 quality control substance solutions with low, medium and high concentrations, and repeatedly processing and measuring for 5 times by the same steps, wherein the result shows that the adding standard recovery rate of the nucleoside antiviral drug is between 88.45% and 107.1%, the RSD of 5 times of repeated tests is in the range of 2.07% to 8.88%, and the statistical result is shown in Table 9.

TABLE 9 nucleoside antiviral drug addition recovery results

4. Taking an interference-free blank serum sample, adding nucleoside antiviral drug standard substances with different concentrations to obtain serum samples with low, medium and high concentrations, repeatedly processing 6 batches in one day, continuously processing for three days, quantitatively determining the concentration of the nucleoside antiviral drug by an isotope internal standard method, wherein the batch precision is 1.47-11.02%, processing 3 batches in three days, and calculating the batch precision to be 2.96-9.24%, and the result is shown in Table 10.

TABLE 10 results of precision test within and between batches (unit ng/mL)

The concentration of the nucleoside antiviral drug in human serum is measured by adopting an ID-UPLC-MS/MS method. Meanwhile, the method detects the peak time and the ion pair of the target object, has high sensitivity, can greatly eliminate matrix interference by adopting an isotope internal standard method for quantification, is not influenced by the conditions of pretreatment process, sample loading volume and flow and the like, and can achieve accurate quantification.

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 result of the accuracy of the method is evaluated by the standard recovery test, and shows that the standard recovery of the nucleoside antiviral drug is between 88.45 and 107.1 percent, the RSD of 5 times of repeated tests is between 2.07 and 8.88 percent, and the accuracy is good.

The reproducibility result of the method shows that the intra-batch precision of the nucleoside antiviral drugs is 1.47-11.02%, and the inter-batch precision is 2.96-9.24%. In the experiment, in order to obtain a target object signal which is more stable and has high sensitivity, the types and concentrations of different mobile phases and electrolytes are investigated, the baseline separation of compound and matrix interference is realized as far as possible, the established pretreatment process of a serum sample is very simple, protein precipitation is completed in one step, and the serum dosage is only 50 mu L.

In a word, the method has the advantages of high sensitivity, strong specificity, accuracy and simpler pretreatment process, can finish the separation and detection of the compound within 3.5min, meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical serum nucleoside antiviral drug concentration, and provides a reliable detection method for clinical nucleoside antiviral drug concentration treatment monitoring.

Claims

1. A method for detecting the concentration of nucleoside antiviral drugs in serum is characterized in that: taking a pretreated serum sample, firstly separating a target drug from a serum matrix by using ultra-high performance liquid chromatography, then obtaining the mass-to-charge ratio of the target drug and an isotope internal standard substance thereof by mass spectrometry, quantifying by using an isotope internal standard method, and calculating the concentration of the target drug according to an established calibration curve;

the target drugs are tenofovir, ganciclovir and entecavir;

the isotope internal standard substance is ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3.

2. The method of claim 1, wherein: the conditions of the ultra-high performance liquid chromatography are as follows:

the mobile phase A is 0.01-0.2% formic acid aqueous solution, and the mobile phase B is 0.01-0.2% formic acid methanol solution;

the chromatographic column is ACQUITY UPLC BEH C18;

the method adopts a mode that the mobile phase A and the mobile phase B are mixed mobile phases to carry out gradient elution, and the initial ratio of the mobile phase A to the mobile phase B is 95: 5, the volume ratio of the mobile phase A to the mobile phase B is within 0-1.0 minutes from 95: 5, gradually changing to 70:30 at a constant speed; 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 1.0-2.1 minutes; the volume ratio of mobile phase a to mobile phase B was changed from 10:90 to 95: 5, the flow rate is 0.2-0.5 mL/min, the column temperature is 30-50 ℃, and the sample introduction volume is 0.2-5 muL.

3. The method of claim 2, wherein: the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid methanol solution.

4. The method of claim 2, wherein: the flow rate was 0.3mL/min, the column temperature was 45 ℃ and the sample introduction volume was 1 μ L.

5. The method of claim 1, wherein: the mass spectrum conditions are as follows: under an electrospray ionization positive ion detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spray voltage was 3.0kV (ESI +); the source temperature is 150 ℃; the temperature of the atomizing gas is 500 ℃, the airflow speed of the atomizing gas is 800L/h, and the airflow speed of the taper hole is 150L/h.

6. The method of claim 1, wherein: the pretreated serum sample is prepared according to the following method: taking a serum sample, adding a protein precipitator containing an internal standard into the serum sample, centrifuging and taking a supernatant.

7. The method of claim 6, wherein: the pretreated serum sample is prepared according to the following method: and (3) taking 50 mu L of serum sample, adding 200 mu L of protein precipitator containing internal standard, oscillating at high speed for 5min, centrifuging at 14000r/min and 4 ℃ for 5min, and taking supernatant.

8. The method of claim 7, wherein: the protein precipitant containing the internal standard is obtained by mixing a mixed internal standard solution and the protein precipitant; the mixed internal standard solution is a mixed solution of ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3 prepared by methanol, and the concentrations of ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3 are 5 mu g/mL, 2.5 mu g/mL and 0.25 mu g/mL respectively; the protein precipitant is one or two of methanol and acetonitrile.

9. The method of claim 8, wherein: the protein precipitator is a mixed solution of methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 2: 1; the volume ratio of the mixed internal standard solution to the protein precipitant is 1: 99.

10. the method of claim 1, wherein: the calibration curve takes 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, and the concentrations of the adopted standard substances are as follows:

GCC：50 ng/mL、100 ng/mL、250 ng/mL、500 ng/mL、2500 ng/mL、5000 ng/mL、10000ng/mL；

TNF：10 ng/mL、20 ng/mL、50 ng/mL、100 ng/mL、500 ng/mL、1000 ng/mL、2000 ng/mL；

ETC：0.05 ng/mL、0.1 ng/mL、0.25 ng/mL、0.5 ng/mL、2.5 ng/mL、5 ng/mL、10 ng/mL。