CN112834659A

CN112834659A - Method for determining concentration of adefovir in blood plasma by liquid chromatography-mass spectrometry

Info

Publication number: CN112834659A
Application number: CN202110118657.8A
Authority: CN
Inventors: 范志鸿
Original assignee: Xuzhou Lishun Kangda Medical Technology Co ltd
Current assignee: Xuzhou Lishun Kangda Medical Technology Co ltd
Priority date: 2021-01-28
Filing date: 2021-01-28
Publication date: 2021-05-25

Abstract

The invention discloses a method for determining the concentration of adefovir in plasma by liquid chromatography-mass spectrometry, which comprises the steps of firstly taking a sample to be determined, adding a certain amount of organic solvent for extraction, pretreating, separating by a chromatographic column, and detecting by a mass spectrometer. The method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of the adefovir dipivoxil; the linear range of the plasma standard curve of the method is 1-50 ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of adefovir in plasma.

Description

Method for determining concentration of adefovir in blood plasma by liquid chromatography-mass spectrometry

Technical Field

The invention belongs to the technical field of medicines, particularly relates to a method for measuring a medicine, and particularly relates to a method for measuring the concentration of adefovir in blood plasma by liquid chromatography-mass spectrometry.

Background

Adefovir dipivoxil is rapidly converted to adefovir in vivo, and adefovir has no inhibitory effect on any of the following common human CYP450 enzymes at concentrations significantly higher than those observed in vivo (> 4000 fold): CYP1a2, CYP2C9, CYP2C19, CYP2D6 and CYP3a 4. Adefovir is not a substrate for the action of these enzymes. However, it is not clear whether adefovir induces CYP450 enzymes. Depending on the results of in vitro experiments and the renal route of clearance of adefovir, adefovir as an inhibitor or substrate has a low probability of interacting with other drugs mediated by CYP 450. Adefovir is excreted via the kidney by means of glomerular filtration and active secretion from the renal tubules. The combination of 10mg of adefovir dipivoxil and other drugs secreted by the renal tubules or drugs for changing the secretory function of the renal tubules can increase the serum concentration of the adefovir dipivoxil or the combined drugs. 10mg of adefovir dipivoxil was combined with 100mg of lamivudine, and the pharmacokinetic properties of both drugs were not changed. Use of 10mg adefovir dipivoxil with drugs actively secreted by the renal tubules should be cautious because two drugs compete for the same elimination pathway and may cause elevated serum levels of adefovir or the combined drugs.

At present, the speed, the precision, the sensitivity and the selectivity of the existing adefovir determination method need to be improved.

Disclosure of Invention

The invention aims to provide a method for determining the concentration of adefovir in plasma by liquid chromatography-mass spectrometry, which can improve the sensitivity, precision, selectivity and speed of detection.

In order to achieve the aim, the invention provides a method for determining the concentration of adefovir in plasma by liquid chromatography-mass spectrometry, wherein a plasma sample is pretreated and then the concentration of the plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:

(1) plasma sample pretreatment:

plasma with K₂EDTA as anticoagulant, adefovir-d 4 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of a volume ratio of 1: 1, adding 5 mu L of 0.4 ng/mu L of internal standard adefovir dipivoxil-d 4 solution after uniformly mixing, adding 400 mu L of methanol after uniformly mixing, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 100 mu L of supernatant into another 96 deep-well plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging at 20 ℃ at 3000rpm for 5min, and taking the supernatant as a test sample to be detected; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution;

(2) and (3) sample measurement:

and (3) injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of adefovir in the sample and adefovir-d 4 in the internal standard, and calculating the concentration of the adefovir in the plasma sample according to the chromatographic peaks.

The liquid chromatography determination conditions were: the chromatographic column is Agilent ZORBAX Eclipse XDB-C18, and the specification of the column is 4.6 multiplied by 50 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 and the mobile phase B is methanol; the washing liquid is methanol: water is mixed according to the volume ratio of 1: 1 mixing the obtained mixture; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 4.5 min;

the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the spraying voltage is 3200V, the atomizing temperature is 550 ℃, the spraying air pressure is 85Psi, the auxiliary heating air pressure is 50Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the declustering voltages of adefovir and the internal standard adefovir-d 4 are both 50 eV; the collision chamber inlet voltages of the adefovir and the internal standard adefovir-d 4 are both 5 eV; the collision voltage of the adefovir and the internal standard adefovir-d 4 is 41 eV; the outlet voltages of the collision chambers of the adefovir and the internal standard adefovir-d 4 are both 20 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 274.0 → m/z162.1, which is adefovir; and m/z 278.1 → m/z 166.2, which is adefovir-d 4.

Preferably, the gradient elution in step (2) is performed by the following procedure:

preferably, in the step (2), the concentration of adefovir in the plasma sample is calculated by introducing the peak area ratio of adefovir and the internal standard adefovir-d 4 into a standard curve equation by using an internal standard method.

Preferably, the establishment of the standard curve equation comprises the following steps:

placing ten 100 mu L blank plasma in a 96-deep-well plate, and sequentially naming the blank plasma as a lowest quantitative lower limit sample, a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a highest quantitative upper limit sample, a zero-concentration sample and a blank sample to total ten samples, wherein the zero-concentration sample contains an internal standard adefovir-d 4 solution and does not contain the adefovir solution, and the zero-concentration sample is used for eliminating the interference of the internal standard adefovir-d 4 solution on the detection result; the blank sample does not contain an adefovir solution and an internal standard adefovir-d 4 solution, and is used for eliminating the interference of the used blank plasma on the detection result;

adding 10 mul of Adefovir solutions with the concentrations of 0.02 ng/mul, 0.04 ng/mul, 0.1 ng/mul, 0.2 ng/mul, 0.3 ng/mul, 0.4 ng/mul, 0.6 ng/mul and 1 ng/mul to the lowest quantitative limit sample, the standard samples 1-6 and the highest quantitative limit sample in the form of stock solution, respectively adding 5 mul of Adefovir solutions with the volume ratio of 1: 1, respectively mixing the ten samples, respectively adding 5 mu L of 0.4 ng/mu L of internal standard adefovir-d 4 solution into nine samples except for blank samples, and adding 5 mu L of volume ratio of 1: 1, respectively and uniformly mixing the ten samples, respectively adding 400 mu L of methanol into the ten samples, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 100 mu L of supernatant liquid into another 96 deep-hole plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging at 20 ℃ for 5min at 3000rpm, and taking the supernatant liquid as a test sample to be detected; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution;

and respectively injecting 10 mu L of standard samples into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of adefovir in the samples and adefovir-d 4 as an internal standard, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of adefovir in the plasma.

Further, the liquid chromatography determination conditions in the step (3) further include: the volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 3 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.

Compared with the prior art, the invention has the following advantages:

(1) the pretreatment method is simple and convenient, and the protein is precipitated and is suitable for conventional determination;

(2) the specificity is strong: under the chromatographic conditions adopted in the experiment, the retention time of the adefovir is about 1.017min, the retention time of the internal standard adefovir-d 4 is about 1.013min, the peak shapes of the adefovir and the internal standard adefovir-d 4 are good, the determination is free of the interference of miscellaneous peaks, and the base line is stable;

(3) the sensitivity is high: the minimum limit of quantitation of the plasma is 1ng/mL, the concentration of the adefovir dipivoxil in the plasma can be accurately determined, the sensitivity is high, and the specificity is strong;

(4) the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of the adefovir dipivoxil. The linear range of the plasma standard curve of the method is 1-50 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.

Drawings

FIG. 1 is a standard graph of adefovir in human plasma by HPLC-MS/MS;

FIG. 2 is a HPLC-MS/MS graph of human blank plasma;

FIG. 3 is a HPLC-MS/MS graph of human blank plasma added with adefovir-d 4;

FIG. 4 is a HPLC-MS/MS graph of human blank plasma with Adefovir and Adefovir-d 4 added;

FIG. 5 is a HPLC-MS/MS chart showing the addition of an internal standard adefovir-d 4 to a plasma sample after the administration of adefovir dipivoxil to healthy subjects.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example (b): human K₂Determination of Adefovir concentration in EDTA plasma

First, experimental material and analytical equipment

Adefovir (analytes) Toronto Research Chemicals or the same, higher-grade standard Adefovir-d 4 (internal standard) TLC Pharmaceutical Standards or the same, higher-grade standard reagents used are listed in Table 1 below:

TABLE 1 details of reagents

Name of reagent	Rank of	Manufacturer(s)
			Methanol	HPLC	J.T.Baker
Ammonium acetate	HPLC	J.T.Baker
			Ammonium hydroxide	ACS	J.T.Baker
Dimethyl sulfoxide	HPLC	Adamas-beta

Note: the same or higher level of reagents may also be used

The analytical equipment used is shown in table 2 below:

TABLE 2 details of the devices used

Assembly	Model number	Manufacturer(s)
			Binarypump (binary pump)	ACPump	ABSCIEX
Degasser (deaerator)	Degasser	ABSCIEX
			Columnoven (constant temperature column box)	ACColumnoven	ABSCIEX
Autosampler (automatic sampler)	ACAutosampler	ABSCIEX
			Samplerack (sample holder)	RackChanger	ABSCIEX
Massspectrometer (Mass spectrometer)	TRIPLEQTRAP^TM6500+	ABSCIEX
			Dataprocessor (data processor)	Analyst1.6.3(software)	ABSCIEX

The same LC/MS system may also be used.

Second, liquid condition

1. Conditions of liquid chromatography

The chromatographic column is AgilentZORBAXeclipse XDB-C18, and the specification of the column is 4.6 x 50 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 and the mobile phase B is methanol; the washing liquid is methanol: water is mixed according to the volume ratio of 1: 1 mixing the obtained mixture; the autosampler temperature was 15 ℃; gradient elution, flow rate of 0.4mL/min, sample size of 10 μ L, analysis time of 4.5 min.

The volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 3 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.

TABLE 3 gradient elution procedure

2. Conditions of Mass Spectrometry

The ion source is an electrospray ion source, the spraying voltage is 3200V, the atomizing temperature is 550 ℃, the spraying air pressure is 85Psi, the auxiliary heating air pressure is 50Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the declustering voltages of adefovir and the internal standard adefovir-d 4 are both 50 eV; the collision chamber inlet voltages of the adefovir and the internal standard adefovir-d 4 are both 5 eV; the collision voltage of the adefovir and the internal standard adefovir-d 4 is 41 eV; the outlet voltages of the collision chambers of the adefovir and the internal standard adefovir-d 4 are both 20 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 274.0 → m/z162.1, which is adefovir; and m/z 278.1 → m/z 166.2, which is adefovir-d 4.

Third, the experimental process

1. Preparation of standard solution of adefovir

The standard solution (containing stock solution and working solution) for the adefovir standard curve is weighed and prepared by the following steps:

weighing weight (mg)	Dissolved volume (μ l)	Final concentration (ng/. mu.l)
			1.041	10206	100

The preparation process adopts dimethyl sulfoxide as diluent: methanol: water: ammonia water according to a volume ratio of 50: 25: 22.5: 2.5, obtaining 100 ng/mu L of adefovir dipivoxil stock solution, and then mixing the adefovir dipivoxil stock solution with the volume ratio of 1: 1, sequentially diluting the methanol aqueous solution to prepare an adefovir standard solution, wherein the specific dilution concentration is shown in the following table 4:

TABLE 4 Adefovir Standard solution preparation concentration

a: prepared directly from adefovir (analyte)

The standard adefovir solution is stored in plastic container and refrigerator (4 deg.C) when it is not used, and its volume can be proportionally increased or decreased according to the requirement.

2. Preparation of standard solution of adefovir-d 4 internal standard

The standard solution of adefovir-d 4 internal standard is weighed and prepared as follows:

weighing weight (mg)	Dissolved volume (μ l)	Final concentration (ng/. mu.l)
			1.212	10117	100

The diluent adopted in the preparation process is dimethyl sulfoxide: methanol: water: ammonia water according to a volume ratio of 50: 25: 22.5: 2.5, obtaining 100 ng/mu L adefovir-d 4 stock solution, and then mixing the two solutions according to the volume ratio of 1: 1, diluting the methanol aqueous solution to prepare an adefovir dipivoxil-d 4 internal standard solution with the concentration of 0.4 ng/. mu.L, wherein the specific dilution concentration is shown in the following table 5:

TABLE 5 Adefovir-d 4 Standard solution preparation concentration

a: prepared directly from adefovir-d 4 (internal standard)

b: for sample preparation procedures

Standard solutions of adefovir-d 4 internal standard were stored in plastic containers and refrigerators (4 ℃) when not in use, with volumes that could be scaled up or down as needed.

3. Linear test

Putting the blank plasma into a water bath at room temperature for unfreezing; transferring 10 parts of 100 μ L of blank plasma to a 96-well plate (each standard curve sample, blank sample-00 and zero concentration sample-0), respectively and precisely adding 5 μ L of adefovir standard solution or diluted solution with different concentrations to prepare each sample according to the list in the following table 6, mixing uniformly to prepare the drug-containing plasma with different concentrations, and carrying out the operation according to the 'plasma sample pretreatment'. The ratio Y (Y ═ As/Ai) of the peak area As of adefovir and the peak area Ai of adefovir-d 4 was calculated, and the blood concentration X was subjected to regression calculation using the peak area ratio Y, and the results are shown in FIG. 1 and Table 7. The average ratio Y is used for carrying out regression calculation on the blood concentration X to obtain a regression equation Y which is 0.0456X +0.0097, r which is 0.9986, and a weight coefficient W which is 1/X²The lowest quantitative limit of the blood concentration of the adefovir dipivoxil measured by the method is as follows: 1 ng/mL.

TABLE 6 Adefovir Standard Curve for the preparation of the concentration

TABLE 7 Standard Curve of adefovir in human plasma by HPLC-MS/MS method (n ═ 11)

b: diluted solution of analyte: MeOH/H₂O＝50/50

4. Accuracy and precision

Putting the blank plasma into a water bath at room temperature for unfreezing; the appropriate volume of blank plasma was transferred to the appropriate container and Adefovir standard solution was added to prepare 5 drug-containing plasma quality control samples (LLOQ, QL, QLM, QM, QH) of different concentrations and a follow-up standard curve, which were processed as "plasma sample pretreatment", and the quality control sample preparation was as shown in Table 8 below. Making one batch and one following standard curve every day, continuously making 3 days for three batches, respectively making 6 samples for each concentration of the first batch and the second batch, respectively making 14 samples for each concentration of the third batch, calculating the ratio Y of the peak area As of the adefovir dipivoxil and the peak area Ai of the peak area of the internal standard adefovir-d 4, substituting the ratio Y into the standard curve on the day to obtain the actually measured concentration, calculating the precision between the batches and the precision between the batches according to the actually measured concentration, and determining the ratio of the actually measured concentration to the added concentration As the accuracy, wherein the result is shown in Table 9. The result shows that the precision and accuracy of the adefovir plasma sample in batch and between batches are less than +/-15 percent and meet the requirements.

TABLE 8 quality control sample preparation concentration

a: final volume is source solution volume + plasma volume

Sufficient volume was dispensed into the labeled sample vials as required for each assay batch and stored at the theoretical temperature-80 ℃. The volume may be scaled up or down as desired.

TABLE 9 determination of Intra-batch, inter-batch precision and accuracy of Adefovir in plasma by HPLC-MS/MS method

Note: the data of the evaluation results are from the data of 3 batches of 26 groups of quality control samples in table 9.

5. Interference

Nine different blank plasma samples are respectively from different healthy human bodies, and the nine different blank plasma samples are prepared and analyzed in the same analysis batch according to the sample preparation steps to evaluate the interference of the different blank plasma on the adefovir analyte and the internal standard adefovir-d 4.

After nine blank healthy human plasma samples from different sources were prepared and analyzed, the peak interference responses at adefovir retention times were all less than 20.0% of the adefovir response of the lower quantitative limit sample in the standard curve of the assay batch, and the results are shown in table 10. The result shows that the analysis method has specificity to the analysis of adefovir.

After nine different sources of blank healthy human plasma samples were prepared and analyzed, the interference peak responses at retention times consistent with the internal standard adefovir-d 4 were all less than 5.0% of the internal standard adefovir-d 4 response of the quantitation lower limit sample in the standard curve of the assay lot, see table 11 in the appendix. The results show that the assay is selective for the assay of the internal standard adefovir-d 4.

TABLE 10 comparison of interference data of blank healthy human plasma from nine different sources on Adefovir analysis

a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve). times.100.0% to 20.0%

b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".

TABLE 11 comparison of interference data of blank healthy human plasma from nine different sources against the internal standard adefovir-d 4

a: analyte peak area (selective sample)/internal standard peak area (LLOQ of standard curve) x 100.0% ≦ 5.0% b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".

As can be seen from tables 10 and 11, the blank plasma of different human bodies did not interfere with the detection results of Adefovir. Therefore, the method can be used for detecting the concentration of the adefovir in the plasma of different human bodies.

6. Detection of human plasma samples

(1) Taking human blank plasma without adefovir, accurately adding 100 mu L of blank plasma sample into a 96-deep-well plate, and adding 10 mu L of blank plasma sample with the volume ratio of 1: 1, adding 400 mu L of methanol into the methanol aqueous solution, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 100 mu L of supernatant into another 96-deep-hole plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging at 20 ℃ at 3000rpm for 5min, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 2; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution.

(2) Taking human blank plasma without adefovir, accurately adding 100 mu L of blank plasma sample into a 96-deep-well plate, and adding 5 mu L of blank plasma sample with the volume ratio of 1: 1, adding 5 mu L of an internal standard adefovir dipivoxil-d 4 solution with the concentration of 0.4 ng/mu L after uniformly mixing, adding 400 mu L of methanol after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 3000rpm at 20 ℃, taking 100 mu L of supernatant into another 96 deep-well plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging for 5min at 3000rpm at 20 ℃, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the result of a representative map is shown in figure 3; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution.

(3) Taking human blank plasma without adefovir, precisely adding 100 mu L of blank plasma sample into a 96 deep-well plate, adding 5 mu L of adefovir standard solution, uniformly mixing, adding 5 mu L of 0.4 ng/mu L of internal standard adefovir-d 4 solution, uniformly mixing, adding 400 mu L of methanol, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 100 mu L of supernatant into another 96 deep-well plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging at 20 ℃ for 5min at 3000rpm, taking 10 mu of sample, carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 4; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution.

(4) Collecting plasma of healthy subjects after oral administration of adefovir dipivoxil or pharmaceutical salts thereof, precisely adding 100 mu L of collected human plasma samples into a 96-deep-well plate, and adding 5 mu L of a mixture of the human plasma samples with a volume ratio of 1: 1, adding 5 mu L of 0.4 ng/mu L of an internal standard adefovir dipivoxil-d 4 solution after uniformly mixing, adding 400 mu L of methanol after uniformly mixing, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 100 mu L of supernatant into another 96 deep-well plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging at 20 ℃ for 5min at 3000rpm, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the result of a representative map is shown in figure 5; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution.

In conclusion, the invention provides a simple and convenient method for determining the concentration of adefovir in plasma by a pretreatment method, adopts a two-step organic solution extraction method, and is suitable for conventional determination; meanwhile, under the chromatographic conditions adopted in the experiment, the retention time of the adefovir is about 1.017 minutes, the retention time of the internal standard adefovir-d 4 is about 1.013 minutes, the peak shapes of the adefovir and the internal standard adefovir-d 4 are good, the determination is free of the interference of miscellaneous peaks, and the base line is stable; the method has higher specificity, can accurately determine the concentration of the adefovir dipivoxil in the blood plasma, has higher sensitivity, and has the minimum limit of quantitation of the blood plasma of 1 ng/mL; meanwhile, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of the adefovir dipivoxil. The linear range of the plasma standard curve of the method is 1-50 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.

Claims

1. A method for determining the concentration of adefovir in plasma by liquid chromatography-mass spectrometry is characterized in that: the plasma sample is pretreated and then the concentration of the plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:

(1) plasma sample pretreatment:

plasma with K₂EDTA as anticoagulant, adefovir-d 4 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of a volume ratio of 1: 1, adding 5 mu L of 0.4 ng/mu L adefovir dipivoxil-d 4 solution after uniformly mixing, adding 400 mu L of methanol after uniformly mixing, carrying out vortex mixing for 1min, centrifuging at 20 ℃ and 3000rpm for 10min, taking 100 mu L of supernatant liquid to another 96 deep-hole plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging at 20 ℃ and 3000rpm for 5min, and taking the supernatant liquid as a test sample to be detected; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution;

(2) and (3) sample measurement:

injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of adefovir and adefovir-d 4 in the sample, and calculating the concentration of adefovir in the plasma sample according to the chromatographic peaks;

the liquid chromatography determination conditions were: the chromatographic column is Agilent ZORBAX Eclipse XDB-C18, and the specification of the column is 4.6 multiplied by 50 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixture; the mobile phase B is methanol; the washing liquid is methanol: water is mixed according to the volume ratio of 1: 1 mixing the obtained mixture; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 4.5 min;

the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the spraying voltage is 3200V, the atomizing temperature is 550 ℃, the spraying air pressure is 85Psi, the auxiliary heating air pressure is 50Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the declustering voltages of adefovir and the internal standard adefovir-d 4 are both 50 eV; the collision chamber inlet voltages of the adefovir and the adefovir-d 4 are both 5 eV; the collision voltage of both adefovir and adefovir-d 4 is 41 eV; the outlet voltages of the collision chambers of the adefovir and the adefovir-d 4 are both 20 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 274.0 → m/z162.1, which is adefovir; and m/z 278.1 → m/z 166.2, which is adefovir-d 4.

2. The method for determining the concentration of adefovir in plasma by LC-MS according to claim 1, wherein: the gradient elution procedure in the step (2) is as follows:

3. the method for determining the concentration of adefovir in plasma by LC-MS according to claim 1 or 2, wherein: in the step (2), an internal standard method is adopted, and the concentration of adefovir in the plasma sample is calculated by substituting the peak area ratio of adefovir and the internal standard adefovir-d 4 into a standard curve equation.

4. The method for determining the concentration of adefovir in plasma by LC-MS according to claim 3, wherein: the establishment of the standard curve equation comprises the following steps:

adding 10 mul of Adefovir solutions with the concentrations of 0.02 ng/mul, 0.04 ng/mul, 0.1 ng/mul, 0.2 ng/mul, 0.3 ng/mul, 0.4 ng/mul, 0.6 ng/mul and 1 ng/mul to the lowest quantitative limit sample, the standard samples 1-6 and the highest quantitative limit sample in the form of stock solution, respectively adding 5 mul of Adefovir solutions with the volume ratio of 1: 1, respectively mixing the ten samples, respectively adding 5 mu L of 0.4 ng/mu L of internal standard adefovir-d 4 solution into nine samples except for blank samples, and adding 5 mu L of volume ratio of 1: 1, respectively and uniformly mixing the ten samples, respectively adding 400 mu L of methanol into the ten samples, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 100 mu L of supernatant liquid into another 96 deep-hole plate filled with 600 mu L of mixed solvent, carrying out vortex mixing, centrifuging at 20 ℃ for 5min at 3000rpm, and taking the supernatant liquid as ten standard samples to be detected; wherein: the mixed solvent is water: 1M ammonium acetate: ammonia water according to the volume ratio of 100: 0.05: 0.04 mixing the obtained mixed solution;

5. The method for determining the concentration of adefovir in plasma by LC-MS according to claim 1 or 2, wherein: the conditions for the liquid chromatography determination in the step (2) further include: the volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 3 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.