CN111665309A - Kit for detecting nucleoside antiviral drugs in serum and application thereof - Google Patents

Kit for detecting nucleoside antiviral drugs in serum and application thereof Download PDF

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CN111665309A
CN111665309A CN202010704142.1A CN202010704142A CN111665309A CN 111665309 A CN111665309 A CN 111665309A CN 202010704142 A CN202010704142 A CN 202010704142A CN 111665309 A CN111665309 A CN 111665309A
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entecavir
tenofovir
ganciclovir
solution
mixed
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成晓亮
李美娟
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Nanjing Pinsheng Medical Technology Co ltd
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Nanjing Pinsheng Medical Technology Co ltd
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    • G—PHYSICS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

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.

Description

Kit for detecting nucleoside antiviral drugs in serum and application thereof
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
Figure BDA0002594030760000051
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.
Example 1
First, experimental material and instrument
The samples for the kit research experiments were obtained from serum samples collected from the 8 month clinic in 2019 of the Nanjing drummer 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 instruments (ELGALabWater, 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); MS grade acetonitrile (Fisher, usa); HPLC grade acetonitrile (Honeywell, usa); MS grade formic acid (Fisher, usa); HPLC grade methanol (Honeywell, usa); column ACQUITY UPLC BEH C18 (2.1X 50mm,1.7 μm).
3. And (3) standard substance: the standards and their corresponding internal standards are shown in table 7 below.
TABLE 7 Standard and internal standards
Serial number Name of Chinese Manufacturer of the product
1 Tenofovir TRC
2 Tenofovir-d 6 TRC
3 Ganciclovir TRC
4 Ganciclovir-d 5 TRC
5 Entecavir TRC
6 Entecavir-d 3 TRC
4. Quality control product: the non-interference serum matrix solution containing nucleoside antiviral drugs has three concentrations of low, medium and high, namely QC (L), QC (M) and QC (H), which are shown in Table 1.
The upper and lower peripheries of the kit are coated, the shockproof and heat preservation are carried out, mobile phases A and B are placed at the upper left part, 11 ampoule bottles are respectively placed at the lower left part, and the standard solution, the quality control product, the mixed internal standard solution and the quality control product are respectively contained; to the right, 25mL of protein precipitant was placed.
Second, liquid condition
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, 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: in an electrospray ionization detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; 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 was monitored simultaneously with the isotope internal standard. The mass spectrometric acquisition parameters for each target analyte are shown in table 6.
Third, the experimental process
1. Preparing a standard substance:
respectively mixing the nucleoside antiviral drug mother liquor with the following concentrations: GCC 20mg/mL, TNF 10mg/mL and ETC0.1mg/mL, mixed standard solutions containing GCC 200000ng/mL, TNF40000ng/mL and ETC 200ng/mL were prepared with 80% methanol aqueous solution (see Table 3 for details).
The mixed standard solution and a blank serum matrix solution are prepared into calibration solution with seven different concentration points, the concentration of each calibration point is listed in table 4, and 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/mLETC：0.05ng/mL、0.1ng/mL、0.25ng/mL、0.5ng/mL、2.5ng/mL、5ng/mL、10ng/mL
2. preparation of mixed internal standard solution
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 (see Table 2 for details).
3. Preparing a quality control product:
the mixed standard solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without nucleoside antiviral drugs, and the details are shown in a table 1.
QC (L) includes: GCC 100ng/mL, TNF 20ng/mL, ETC0.1 ng/mL
QC (M) comprises: GCC 1000ng/mL, TNF 200ng/mL, ETC 1ng/mL
QC (H) includes: GCC 4000ng/mL, TNF 800ng/mL, ETC 4ng/mL
4. Sample processing
(1) Pretreatment of a standard product: 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.
(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 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.
(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 8.
TABLE 8 preparation of nucleoside antiviral drug assay kit Components (100 persons)
Figure BDA0002594030760000111
Figure BDA0002594030760000121
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 substance of the nucleoside 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 substance of the nucleoside antiviral drug; FIG. 2 is a chromatogram of extracted ion current of nucleoside antiviral drugs 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 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 9.
TABLE 9 linear regression equation and linear correlation coefficient for nucleoside 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 ETC 1.56 0.05-10 y＝0.522084*x+0.00881642 0.9976
3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample is prepared, low, medium and high 3-concentration standard substances are respectively added, the same steps are repeatedly used for processing and measuring for 5 times, the result shows that the adding standard recovery rate of the nucleoside antiviral drugs 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 a table 10.
TABLE 10 Standard recovery results (unit: ng/mL) for nucleoside antiviral drugs
Figure BDA0002594030760000122
Figure BDA0002594030760000131
4. And (3) precision test: 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 11.
TABLE 11 results of inter-batch precision measurements (unit: ng/mL)
Figure BDA0002594030760000132
Figure BDA0002594030760000141
The kit is used for detecting the nucleoside antiviral drugs in human serum, the serum dosage is small (only 50 mu L), the pretreatment is simple, and one-needle analysis of various substances only needs 3.5min, and the kit is simple and rapid.
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 precision of the nucleoside antiviral drug in batches is 1.47-11.02%, the precision between batches is 2.96-9.24%, and the method has good reproducibility.
In a word, the kit disclosed by the invention is high in sensitivity, strong in specificity, accurate and simple in pretreatment process, completes 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 drugs, and provides a reliable detection method for related drug concentration monitoring.
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 (10)

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.
CN202010704142.1A 2020-07-21 2020-07-21 Kit for detecting nucleoside antiviral drugs in serum and application thereof Pending CN111665309A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112834670A (en) * 2020-12-30 2021-05-25 苏州海科医药技术有限公司 Biological analysis method for clinical research of tenofovir alafenamide and tenofovir metabolite concentration in plasma sample of antiviral drug

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1845745A (en) * 2003-09-05 2006-10-11 安那迪斯药品股份有限公司 Administration of TLR7 ligands and prodrugs thereof for treatment of infection by hepatitis c virus
JP2010509566A (en) * 2006-10-10 2010-03-25 ロス アラモス ナショナル セキュリティー，エルエルシー Advanced drug development and manufacturing
US20150099270A1 (en) * 2013-10-04 2015-04-09 Thomas C. DOWLING Method of screening pharmaceuticals for drug interactions and nephrotoxicity
CN109900819A (en) * 2019-02-22 2019-06-18 南京海纳医药科技股份有限公司 A kind of method that UPLC/MS-MS combination detects tenofovir in human serum

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1845745A (en) * 2003-09-05 2006-10-11 安那迪斯药品股份有限公司 Administration of TLR7 ligands and prodrugs thereof for treatment of infection by hepatitis c virus
JP2010509566A (en) * 2006-10-10 2010-03-25 ロス アラモス ナショナル セキュリティー，エルエルシー Advanced drug development and manufacturing
US20150099270A1 (en) * 2013-10-04 2015-04-09 Thomas C. DOWLING Method of screening pharmaceuticals for drug interactions and nephrotoxicity
CN109900819A (en) * 2019-02-22 2019-06-18 南京海纳医药科技股份有限公司 A kind of method that UPLC/MS-MS combination detects tenofovir in human serum

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMEDEO DE NICOLÒ 等: "UPLC–MS/MS method for the simultaneous quantification of anti-HBV nucleos(t)ides analogs: Entecavir, lamivudine, telbivudine and tenofovir in plasma of HBV infected patients", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 *
HÉLÈNE GOUGET 等: "UPLC–MS/MS method for the simultaneous quantification of bictegravir and 13 others antiretroviral drugs plus cobicistat and ritonavir boosters in human plasma", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 *
李力等: "高效液相色谱质谱联用法同时测定人血浆中5种核苷类抗病毒药物浓度", 《中国临床药理学杂志》 *
陈枳潓 等: "LC-MS/MS法测定人血浆中替诺福韦质量浓度及其生物等效性研究", 《四川大学学报（医学版）》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112834670A (en) * 2020-12-30 2021-05-25 苏州海科医药技术有限公司 Biological analysis method for clinical research of tenofovir alafenamide and tenofovir metabolite concentration in plasma sample of antiviral drug

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Application publication date: 20200915