阅读说明：本技术 一种检测血清中核苷类抗病毒药物的试剂盒及其应用 (Kit for detecting nucleoside antiviral drugs in serum and application thereof ) 是由 成晓亮 李美娟 于 2020-07-21 设计创作，主要内容包括：本发明公开了一种检测血清中核苷类抗病毒药物的试剂盒及其应用,属于药物分析技术领域。采用本发明的试剂盒,可以一次性对3种常用的抗病毒药物更昔洛韦、替诺福韦和恩替卡韦进行检测,前处理过程简单,成本低,且灵敏度高、特异性强,3.5 min之内完成核苷类抗病毒药物的分离和检测,准确度与精密度基本满足要求,可用于临床上核苷类抗病毒药物的定量分析,为临床上核苷类抗病毒药物的治疗浓度监测提供一种可靠的检测方法。(The invention discloses a kit for detecting nucleoside antiviral drugs in serum and application thereof, belonging to the technical field of drug analysis. The kit can be used for detecting 3 commonly used antiviral drugs, namely ganciclovir, tenofovir and entecavir, at one time, has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, can finish the separation and detection of the nucleoside antiviral drugs within 3.5min, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of the clinical nucleoside antiviral drugs, and provides a reliable detection method for monitoring the treatment concentration of the clinical nucleoside antiviral drugs.)

1. A kit for detecting nucleoside antiviral drugs in serum is characterized in that: the nucleoside antiviral drugs comprise ganciclovir, tenofovir and entecavir;

the kit comprises: removing liquid, calibrating solution, mixed internal standard solution, protein precipitator and quality control product;

the eluent comprises eluent A and eluent B; eluent A is 0.01-0.2% formic acid water solution, and eluent B is 0.01-0.2% formic acid methanol solution;

the calibrator solution comprises a series of mixed solutions of ganciclovir, tenofovir and entecavir prepared from blank serum with known concentrations;

the mixed internal standard solution is a mixed solution of ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3 which is prepared by methanol water solution and has known concentration;

the quality control product comprises a mixed solution of ganciclovir, tenofovir and entecavir prepared by blank serum with known concentration;

the protein precipitator is a mixed solution of methanol and acetonitrile.

2. The kit of claim 1, wherein: the eluent A is 0.1% formic acid water solution, and the eluent B is 0.1% formic acid methanol solution.

3. The kit of claim 1, wherein: the calibrator solution comprises the following seven mixed solutions:

mixing liquid I: the concentrations of the ganciclovir, the tenofovir and the entecavir are respectively 50ng/mL of the ganciclovir, 10 ng/mL of the tenofovir and 0.05 ng/mL of the entecavir;

and (2) mixing liquid II: the concentrations of the ganciclovir, the tenofovir and the entecavir are 100ng/mL, 20ng/mL and 0.1ng/mL respectively;

and (3) mixing liquid III: the concentrations of ganciclovir, tenofovir and entecavir are 250 ng/mL, 50ng/mL and 0.25 ng/mL respectively;

and (4) mixing liquid IV: the concentrations of the ganciclovir, the tenofovir and the entecavir are respectively 500 ng/mL of the ganciclovir, 100ng/mL of the tenofovir and 0.5 ng/mL of the entecavir;

and (5) mixing liquid: the concentrations of ganciclovir, tenofovir and entecavir are 2500 ng/mL, 500 ng/mL and 2.5 ng/mL respectively;

and (6) mixing liquid six: the concentrations of ganciclovir, tenofovir and entecavir are 5000ng/mL of ganciclovir, 1000ng/mL of tenofovir and 5 ng/mL of entecavir respectively;

and (5) mixed liquid seven: the concentrations of ganciclovir, tenofovir and entecavir are 10000ng/mL of ganciclovir, 2000 ng/mL of tenofovir and 10 ng/mL of entecavir respectively.

4. The kit of claim 1, wherein: the concentrations of the ganciclovir-d 5, the tenofovir-d 6 and the entecavir-d 3 in the mixed internal standard solution are ganciclovir-d 55000 ng/mL, tenofovir-d 610000ng/mL and entecavir-d 350 ng/mL respectively.

5. The kit of claim 1, wherein: the volume ratio of methanol to acetonitrile in the protein precipitant is 1-5: 1.

6. The kit of claim 5, wherein: the volume ratio of methanol to acetonitrile in the protein precipitant is 2: 1.

7. The kit of claim 1, wherein: the quality control product comprises mixed solutions with the following three concentrations:

low concentration quality control product: the concentrations of the ganciclovir, the tenofovir and the entecavir are 100ng/mL, 20ng/mL and 0.1ng/mL respectively;

medium concentration quality control: the concentrations of the ganciclovir, the tenofovir and the entecavir are respectively 1000ng/mL of the ganciclovir, 200ng/mL of the tenofovir and 1ng/mL of the entecavir;

high concentration quality control product: the concentrations of ganciclovir, tenofovir and entecavir are 4000ng/mL, 800ng/mL and 4ng/mL respectively.

8. The use of the kit of claim 1 in detecting antiviral drugs that are nucleosides in serum using ultra performance liquid chromatography tandem mass spectrometry.

9. Use according to claim 8, characterized in that: the mixed internal standard solution in the kit is firstly mixed with the protein precipitator to prepare the protein precipitator containing the internal standard and then used for detection, and the volume ratio of the mixed internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7.

10. Use according to claim 9, characterized in that: the volume ratio of the mixed internal standard solution and the protein precipitant is 0.2: 19.8.

Technical Field

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

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 the kit for detecting the nucleoside antiviral drugs in the serum, which can detect 3 common antiviral drugs at one time by adopting an LC-MS/MS method, has high sensitivity and good accuracy, can be well applied to clinic and can provide reliable basis for the research of the antiviral drug combination.

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

a kit for detecting nucleoside antiviral drugs in serum, wherein the nucleoside antiviral drugs comprise ganciclovir, tenofovir and entecavir;

the kit comprises: removing liquid, calibrating solution, mixed internal standard solution, protein precipitator and quality control product;

the eluent comprises eluent A and eluent B; eluent A is 0.01-0.2% formic acid water solution, and eluent B is 0.01-0.2% formic acid methanol solution;

the calibrator solution comprises a series of mixed solutions of ganciclovir, tenofovir and entecavir prepared from blank serum with known concentrations;

the mixed internal standard solution is a mixed solution of ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3 which is prepared by methanol water solution and has known concentration;

the quality control product comprises a mixed solution of ganciclovir, tenofovir and entecavir prepared by blank serum with known concentration;

the protein precipitator is a mixed solution of methanol and acetonitrile.

Further, the eluent A is 0.1% formic acid aqueous solution, and the eluent B is 0.1% formic acid methanol solution.

Further, the calibrator solution includes mixed solutions of seven series concentrations.

Further, the concentrations of ganciclovir-d 5, tenofovir-d 6 and entecavir-d 3 in the mixed internal standard solution are ganciclovir-d 55000 ng/mL, tenofovir-d 610000ng/mL and entecavir-d 350 ng/mL respectively.

Further, the volume ratio of methanol to acetonitrile in the protein precipitator is 1-5: 1.

Further, the volume ratio of methanol to acetonitrile in the protein precipitant is 2: 1.

Further, the quality control product comprises a mixed solution with three concentrations.

The kit is applied to the detection of the nucleoside antiviral drugs in the serum by using the ultra-high performance liquid chromatography tandem mass spectrometry technology.

Has the advantages that: when the kit is used for detecting the nucleoside antiviral drugs in the serum, the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and the detection of the nucleoside antiviral drugs are completed within 3.5min, the accuracy and the precision basically meet the requirements, the kit can be used for quantitative analysis of the nucleoside antiviral drugs in clinic, and a reliable detection method is provided for monitoring the treatment concentration of the nucleoside antiviral drugs in 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 kit for detecting nucleoside antiviral drugs in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the nucleoside antiviral drugs are respectively as follows: ganciclovir (Ganciclovir, GCC), Tenofovir (Tenofovir, TNF), Entecavir (Entecavir, ETC).

The isotope internal standard substances corresponding to the nucleoside antiviral drugs are respectively as follows: ganciclovir-d 5(GCC-d5), tenofovir-d 6(TNF-d6), entecavir-d 3(ETC-d 3).

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.

The kit comprises the following reagents:

(1) eluent:

eluent A: 0.01% -0.2% aqueous formic acid; eluent B: 0.01% -0.2% formic acid methanol solution;

(2) calibration solution:

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

(3) mixing internal standard solutions:

methanol aqueous solution containing GCC-d 55000 ng/mL, TNF-d610000ng/mL and ETC-d 350 ng/mL;

(4) protein precipitant:

a mixed solution of methanol and acetonitrile;

(5) quality control product:

the non-interference serum matrix solution containing nucleoside antiviral medicine has low, medium and high concentration, which are QC (L), QC (M) and QC (H), respectively, wherein,

QC (L) is QC (M) diluted to 10 times with non-interfering serum matrix solution,

QC (M) is the mixed standard solution diluted to 200 times by using the interference-free serum matrix solution,

QC (H) is the above mixed standard solution diluted to 50 times with non-interfering serum matrix solution.

In a preferred embodiment, the eluent A is 0.1% formic acid water solution; eluent B was 0.1% formic acid in methanol.

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

The mixed standard solution mentioned in the invention is prepared according to the following method: 20mg/mL of nucleoside antiviral drug mother liquor GCC, 10mg/mL of TNF and 0.1mg/mL of ETC are respectively prepared into mixed standard solutions containing 200000ng/mL of GCC, 40000ng/mL of TNF and 200ng/mL of ETC by using methanol aqueous solution.

The mixed internal standard solution mentioned in the invention is prepared according to the following method: GCC-d 51 mg/mL, TNF-d60.5mg/mL and ETC-d 30.05mg/mL mother liquors were prepared into mixed internal standard solutions containing GCC-d 55000 ng/mL, TNF-d610000ng/mL and ETC-d 350 ng/mL with aqueous methanol.

When preparing a mixed standard solution and a mixed internal standard solution, the adopted methanol aqueous solution is 50-95% methanol aqueous solution; preferably 70 to 90 percent of methanol aqueous solution; more preferably 80% aqueous methanol.

When preparing the calibrator solution and the quality control product, the non-interference serum matrix is blank serum without nucleoside antiviral drugs.

The concentration of the aqueous methanol solution referred to in the present invention generally means a volume concentration.

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

In a preferred scheme, the kit for detecting the nucleoside antiviral drugs in the serum by the ultra-high performance liquid chromatography tandem mass spectrometry technology comprises the following reagents:

(1) eluent:

eluent A: 0.01 to 0.2 percent of formic acid aqueous solution; eluent B: 0.01 to 0.2 percent of formic acid methanol solution;

(2) calibration solution:

respectively preparing the nucleoside antiviral drug mother liquor with the concentration 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, and preparing the mixed standard solution into seven calibrator solutions with different concentration points by blank serum without the nucleoside antiviral drug;

(3) mixing internal standard solutions:

preparing mixed internal standard solutions containing GCC-d 55000 ng/mL, TNF-d610000ng/mL and ETC-d 350 ng/mL by using mother liquor of GCC-d 51 mg/mL, TNF-d60.5mg/mL and ETC-d 30.05mg/mL with methanol aqueous solution;

(4) protein precipitant:

the volume ratio of methanol to acetonitrile is 1-5: 1

(5) Quality control product:

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 drug.

In a more preferable scheme, the kit for detecting the nucleoside antiviral drugs in the serum by the ultra-high performance liquid chromatography tandem mass spectrometry technology comprises the following reagents:

(1) eluent:

eluent A: 0.1% aqueous formic acid; eluent B: 0.1% formic acid in methanol;

(2) calibration solution:

respectively preparing the nucleoside antiviral drug mother liquor with the concentrations of 20mg/mL GCC, 10mg/mL TNF and 0.1mg/mL ETC, preparing mixed standard solution containing 200000ng/mL GCC, 40000ng/mL TNF and 200ng/mL ETC by using 80% methanol aqueous solution, and preparing the mixed standard solution into seven calibrator solutions with different concentration points by using blank serum without the nucleoside antiviral drug;

(3) mixing internal standard solutions:

preparing a mixed internal standard solution containing GCC-d 55000 ng/mL, TNF-d610000ng/mL and ETC-d 350 ng/mL by using 80% methanol aqueous solution to obtain mother liquor of GCC-d 51 mg/mL, TNF-d60.5mg/mL and ETC-d 30.05mg/mL;

(4) protein precipitant:

the volume ratio of methanol to acetonitrile is 2: 1;

(5) quality control product:

preparing the mixed standard solution into three different concentrations of QC (L), QC (M) and QC (H) by using blank serum without the nucleoside antiviral drugs, wherein the corresponding concentrations of the quality control products of the nucleoside antiviral drugs in the QC (L), the QC (M) and the QC (H) are shown in a table 1.

TABLE 1 nucleoside antiviral drug 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.

In a more 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 2.

TABLE 2 Mixed internal standard solution preparation

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 the concentration shown in Table 3.

TABLE 3 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 4 (unit: ng/mL):

TABLE 4 Standard koji 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

The application of the kit in detecting the nucleoside 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:

the method comprises the steps of detecting the nucleoside antiviral drugs in pretreated serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating a target object to be detected from interfering components in a serum matrix by utilizing the ultra-high performance liquid chromatography, establishing a calibration curve by utilizing 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 nucleoside antiviral drugs in the serum.

The nucleoside antiviral drugs are respectively: ganciclovir (Ganciclovir, GCC), Tenofovir (Tenofovir, TNF), Entecavir (Entecavir, ETC); the isotope internal standard substances corresponding to the nucleoside antiviral drugs are respectively as follows: ganciclovir-d 5(GCC-d5), tenofovir-d 6(TNF-d6), entecavir-d 3(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 0.1-0.4 mL/min, the collection time of each sample is 3.5min, the column temperature is 35-50 ℃, and the sample injection volume is 1-5 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: the method has the advantages that the method has ACQUITYUPLC BEH C18(2.1 multiplied by 50mm,1.7 mu m), endogenous substances do not interfere with the determination of samples under the coordination of other conditions, 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 3.5 min.

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

the gradient elution mode is adopted, see table 5; the flow rate is 0.3mL/min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;

TABLE 5 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 6.

TABLE 6 Mass Spectrometry parameters for nucleoside antiviral drugs

Compound (I)	Parent ion	Daughter ions	Declustering voltage (V)	Collision voltage (V)
					GCC	256.2	152	22	10
GCC-d5	261.1	152.1	6	12
					TNF	288.1	176	40	18
TNF-d6	294.2	182.1	40	24
					ETC	278.1	151.9	26	16
ETC-d3	281.1	155.1	28	15

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

In one protocol, pre-treated serum was 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, and 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-d610000ng/mL and ETC-d 350 ng/mL.

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

respectively mixing the nucleoside antiviral drug mother liquor with the following concentrations: GCC 20mg/mL, TNF 10mg/mL and ETC0.1mg/mL, and 80% methanol aqueous solution is used for preparing mixed standard solution containing GCC 200000ng/mL, TNF40000ng/mL and ETC 200 ng/mL.

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；

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

taking 50 mu L of each concentration point sample, putting the sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the 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.

In one scheme, the quality control product is prepared according to the following method: preparing the mixed standard solution into three different concentrations of QC (L), QC (M) and QC (H) by using blank serum of the nucleoside-free antiviral drug:

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 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.