CN112986459A

CN112986459A - LC-MS/MS detection method for linezolid in human plasma

Info

Publication number: CN112986459A
Application number: CN202110403272.6A
Authority: CN
Inventors: 王小董; 许杨; 孙珍珍; 曾虹
Original assignee: Anhui Wanbang Pharmaceutical Technology Co ltd
Current assignee: Anhui Wanbang Pharmaceutical Technology Co ltd
Priority date: 2021-04-15
Filing date: 2021-04-15
Publication date: 2021-06-18

Abstract

The invention discloses an LC-MS/MS detection method of linezolid in human plasma. Firstly, treating a human plasma sample to be detected by a protein precipitation method; then, LC-MS/MS analysis was performed using an appropriate organic solvent and buffer solution as a mixed mobile phase. The invention has high sensitivity, strong specificity and high stability, the recovery rate is up to more than 95 percent, the requirement of the recovery rate can be met, and the detection efficiency can be improved. The method has good reproducibility and short analysis time, can meet the detection requirement of linezolid concentration in human plasma, and can be used for linezolid pharmacokinetic research and bioequivalence research.

Description

LC-MS/MS detection method for linezolid in human plasma

Technical Field

The invention relates to the technical field of linezolid concentration detection in human plasma, in particular to an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) detection method for linezolid in human plasma.

Background

Linezolid is a novel clinical oxazolidinone antibacterial drug, has a unique pharmacological mechanism, has no cross-resistance with other drugs, and has a good treatment effect on infection caused by most gram-positive bacteria including multidrug-resistant enterococcus, staphylococcus, streptococcus pneumoniae and the like. Due to the existence of various factors (such as edema, fluid therapy, pleural effusion, burns, etc.) affecting the distribution and excretion of drugs in the body of an infected patient under pathophysiological conditions, the changes of the in vivo processes of drugs may be affected. Therefore, a simple, convenient, quick, sensitive and reliable method for monitoring the blood concentration of linezolid is established clinically, so that the effective treatment of the medicament can be better ensured, the clinical use of the linezolid is optimized, and the generation of drug-resistant strains and adverse reactions are reduced.

At present, methods for measuring linezolid in human plasma mainly comprise HPLC, LC-MS/MS, UPLC-MS/MS and the like. However, these methods have limitations such as insufficient sensitivity, complicated sample processing, and high requirements for instruments.

Therefore, the development of an LC-MS/MS method with good sensitivity, high accuracy, wide measurement range and simple sample processing to detect the drug concentration of linezolid in human plasma is urgently needed, thereby meeting the needs of clinical blood drug concentration monitoring and pharmacokinetic research.

Disclosure of Invention

Aiming at the problems in the prior art, the LC-MS/MS detection method for linezolid in human plasma provided by the invention comprises the following steps of firstly, processing a human plasma sample to be detected by a protein precipitation method; then, LC-MS/MS analysis was performed using an appropriate organic solvent and buffer solution as a mixed mobile phase.

Preferably, the organic solvent is methanol and the buffer solution is 5mM ammonium acetate in water.

The method specifically comprises the following steps:

and S1, preparing a standard working solution to obtain a linezolid working solution, a linezolid parallel working solution, an internal standard working solution, a correction standard sample and a quality control sample.

Further, adding methanol into linezolid to prepare a linezolid stock solution with the concentration of 2.000mg/mL, and diluting the linezolid stock solution into a plurality of linezolid working solutions with the volume ratio of methanol-water solution of 1:1, wherein the concentration range of the linezolid working solutions is 0.600-600.000 mug/mL; repeating the steps once to prepare a plurality of linezolid parallel working solutions with the concentration range of 0.600-450.000 mug/mL; adding methanol into the internal standard to prepare internal standard stock solution with the concentration of 1.00mg/mL, and diluting the internal standard stock solution into internal standard working solution with the volume ratio of 1:1 by using methanol-water solution with the volume ratio of 2.500 mu g/mL; all stock solutions and working solutions are stored at-20 ℃ for later use; taking blank blood plasma of a healthy person, respectively adding the linezolid working solution, uniformly mixing in a vortex mode, and preparing a plurality of calibration standard samples with corresponding concentrations, wherein the concentration range is 30-30000 ng/mL; and (3) taking blank plasma of a healthy person, respectively adding the linezolid parallel working solution, and uniformly mixing by vortex to prepare a plurality of quality control samples with corresponding concentrations, wherein the concentration range is 30.000-22500.000 ng/mL. The internal standard adopts linezolid-d 3.

Further, the concentration of the linezolid working solution is: 0.600. mu.g/mL, 1.200. mu.g/mL, 6.000. mu.g/mL, 30.000. mu.g/mL, 120.000. mu.g/mL, 240.000. mu.g/mL, 480.000. mu.g/mL, and 600.000. mu.g/mL; the concentration of the linezolid parallel working solution is as follows: 0.600. mu.g/mL, 1.800. mu.g/mL, 18.000. mu.g/mL, 180.000. mu.g/mL, and 450.000. mu.g/mL; the calibration standard sample and the quality control sample are prepared fresh; the concentrations of the calibration standards were: 30.000ng/mL, 60.000ng/mL, 300.000ng/mL, 1500.000ng/mL, 6000.000ng/mL, 12000.000ng/mL, 24000.000ng/mL, 30000.000 ng/mL; the concentration of the quality control sample is as follows: 30.000ng/mL, 90.000ng/mL, 900.000ng/mL, 9000.000ng/mL, 22500.000 ng/mL.

S2, taking a calibration standard sample, a quality control sample, a human plasma sample to be detected and blank human plasma which are equal in volume, respectively adding the internal standard working solution into the calibration standard sample, the quality control sample, the human plasma sample to be detected and the blank human plasma, adding a methanol-water solution with the volume ratio of 1:1 into the blank human plasma, uniformly mixing in a vortex manner, centrifuging, and taking supernatant. By adding the methanol precipitator into the human plasma sample, compared with the traditional solid phase extraction pretreatment method, the method has the advantages of simpler operation, high treatment speed, small introduced error, good separation effect and high detection efficiency.

Further, the vortex mixing time is 10 min; the centrifugation conditions were: the temperature during centrifugation is 4 ℃, the centrifugation speed is 4200r/min, and the centrifugation time is 10 min.

And S3, determining the supernatants of the calibration standard sample and the quality control sample processed in the step S2 by an LC-MS/MS method, and preparing a standard curve by taking the chromatographic peak area ratio of linezolid to an internal standard as a vertical coordinate and the concentration of linezolid in human plasma as a horizontal coordinate. The standard curve is determined by correcting the standard sample and the accompanying quality control sample, so that the precision and the accuracy of detection are improved.

Further, chromatographic conditions: a chromatographic column: 4.6X 50mm, 5 μm; column temperature: 40 ℃; sample introduction volume: 2 mu L of the solution; mobile phase A: 5mM ammonium acetate in water containing 0.1% formic acid; mobile phase B: methanol, elution, autosampler temperature 4 ℃, mobile phase flow rate 0.8 mL/min.

Further, the column was used Welch Ultimate XB-C18.

Still further, the elution procedure is:

mass spectrum conditions: an ion source: electrospray ionization source ESI; the spraying voltage is 5500V; ion source temperature: 500 ℃; and (4) CUR: 30.00 psi; scanning mode: positive ion multi-reaction monitoring + MRM, linezolid and linezolid-d 3 ion reactions were m/z 338.2 → 195.1 and m/z 342.3 → 298.2, respectively, and collision energies CE were 30 and 26V, respectively. Qualitative accuracy and method sensitivity can be effectively improved by adopting a multi-reaction monitoring mode for qualitative and quantitative determination.

S4, calculating the human plasma sample to be detected processed in the step S2 according to the standard curve equation obtained in the step S3 to obtain the concentration of linezolid in the human plasma sample to be detected.

The invention has the beneficial effects that: 1. the invention adopts a multi-reaction monitoring mode for qualitative and quantitative determination, can effectively improve the qualitative accuracy and the sensitivity of the method, has the lowest detection limit reaching the level of nanogram, short analysis time and less required sample amount; 2. compared with the traditional solid phase extraction pretreatment method, the method has the advantages that the methanol precipitator is added into the human plasma sample, the operation is simpler, the treatment speed is high, the introduced error is small, the separation effect is good, and the detection efficiency is high; 3. the invention utilizes the calibration standard sample and the accompanying quality control sample to determine the standard curve, thereby improving the precision and the accuracy of detection; 4. the detection method has the advantages of strong specificity, high stability and high recovery rate of more than 95 percent, can meet the requirement of the recovery rate, and can improve the detection efficiency.

Drawings

Fig. 1 is an ion scanning mass spectrum of linezolid product in the LC-MS/MS detection method for linezolid in human plasma provided in example 1;

FIG. 2 is a product ion scanning mass spectrum of linezolid-d 3 in the LC-MS/MS detection method for linezolid in human plasma provided in example 1;

FIG. 3 is a MRM chromatogram of linezolid (left) and linezolid-d 3 (right) from an empty plasma sample of example 1;

FIG. 4 is a MRM chromatogram of linezolid (left) and linezolid-d 3 (right) from a LLOQ plasma sample from example 1.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.

Example 1:

as shown in fig. 1 to 4, a method for detecting linezolid in human plasma by LC-MS/MS.

1. Preparation of solutions and samples

1.1 Standard series samples: precisely weighing a proper amount of linezolid irradiation, dissolving with methanol and fixing the volume, precisely absorbing a proper amount of each stock solution, and adding methanol: diluting the standard series working solution with water (1:1, v/v) step by step to prepare a standard curve sample linezolid with the concentration range of 30.000-30000.000 ng/mL for drawing a standard curve.

1.2 quality control samples: the linezolid quality control samples with five concentration levels are prepared by a method similar to a standard series of samples, the lower limit of quantitation (LLOQ) concentration is 30.000ng/mL, the Low Quality Control (LQC) concentration is 90.000ng/mL, the medium 1 quality control (M1QC) concentration is 900.000ng/mL, the medium 2 quality control (M2QC) concentration is 9000.000ng/mL, and the High Quality Control (HQC) concentration is 22500.000 ng/mL.

1.3 internal standard solution: precisely weighing a proper amount of linezolid-d 3 standard, correcting the standard by a mass correction coefficient, dissolving the standard in methanol to obtain a stock solution with the final concentration of 1.000mg/mL, precisely sucking a proper amount of internal standard stock solution, and adding methanol: and diluting with water (1:1, v/v) to obtain internal standard working solutions with linezolid-d 3 concentration of 2.500 mu g/mL respectively.

2. Sample pretreatment

Samples thawed at room temperature or newly formulated samples were vortexed and mixed. Adding 50 mu L of sample (standard curve, quality control sample, system applicability sample or human plasma sample to be detected, and adding 50 mu L of blank matrix sample for double blank sample or zero sample) into the hole of a 96-well plate; for the double blank sample or ULOQWithout IS sample, add 50 μ L of 50% methanol solution, for the other samples add 50 μ L of internal standard working solution (2.500 μ g/mL), vortex and mix; adding 400 mu L of methanol into each sample hole, sealing the plate, and uniformly mixing for 10 min; centrifuging the sample at 4 deg.C and 4200r/min for 10 min; taking 50 μ L of centrifuged supernatant to another 96-well collection plate, adding 450 μ L of 0.5% formic acid, sealing the plate, and mixing for 10 min; the sample was centrifuged at 4 ℃ and 4200r/min for 10min before being injected.

3. Detection apparatus and analysis conditions

3.1 Main Equipment instruments Table 2 below

Table 2: main equipment instrument

3.2 conditions of analysis

3.2.1 chromatographic conditions: the chromatographic column was Welch Ultimate XB-C184.6X 50mM 5 μm, the column temperature was 40 ℃, the autosampler was set to 4 ℃, mobile phase A was 5mM ammonium acetate aqueous solution containing 0.1% formic acid, mobile phase B was methanol, the needle wash: 80% methanol (containing 0.1% formic acid); plunger washing liquid: 10% methanol, flow rate 0.8mL/min, sample size 2 uL.

3.2.2 Mass Spectrometry conditions: electrospray ionization source ESI; the spraying voltage is 5500V; ion source temperature: 500 ℃; and (4) CUR: 30 psi; scanning mode: positive ion multi-reaction monitoring + MRM, ion reactions of linezolid and linezolid-d 3 were monitored as m/z 338.2 → 195.1 and m/z 342.3 → 298.2, respectively, and collision energy CE was 30 and 26V, respectively.

4. Methodology validation

The methodology of the method is verified according to Chinese pharmacopoeia and American FDA guiding principles, and the contents comprise stability, selectivity, linearity, accuracy, precision, residual effect, recovery rate, matrix effect and dilution reliability.

4.1 Selectivity

Processing six blank plasmas from different sources and LLOQ samples prepared respectively, and then carrying out sample injection analysis to obtain an MRM chromatogram map 3 of linezolid in the blank plasma samples and an MRM chromatogram map 4 of the linezolid in the LLOQ samples, wherein peak areas of chromatographic co-outflow interferents are both less than 20% of peak area of LLOQ to-be-detected substance and less than 5% of peak area of internal standard.

4.2 precision and accuracy

The method verifies and analyzes six samples of each quality control sample for measuring five concentrations in each batch, continuously measures three batches, calculates the precision and the accuracy between the batches, the precision between the LLOQ batches and the batches is acceptable when the precision between the LLOQ batches and the batches is calculated by Relative Standard Deviation (RSD) and is less than 20 percent, the accuracy is acceptable when the precision between the LLOQ batches and the batches is calculated by relative deviation (RE), the precision between the QC samples of other concentration levels is acceptable when the precision between the LLOQ batches and the batches is less than 15 percent, the accuracy is acceptable when the precision between the LLOQ batches and the batches is 15 percent, and the result is shown in a table 3.

Table 3: precision and accuracy for determination of linezolid in human plasma

4.3 Standard Curve

The method comprises the steps of taking the concentration of a physical theory to be measured as an abscissa (X), taking the peak area ratio of the substance to be measured to an internal standard substance as an ordinate (y), carrying out regression operation by using a weighted (W1/X2) least square method, obtaining a linear regression equation which is a standard curve, verifying that double-sample analysis is carried out on a standard curve sample by each analysis batch, and obtaining a linezolid plasma sample standard curve linear equation y 0.00104X +0.00269(r 0.9981) which shows that the linear relationship of linezolid in the concentration range of 30.000-30000.000 ng/mL is good.

4.4 residual Effect

The residual effect was verified as the response of the instrument at the time of the peak of linezolid and internal standard was analyzed by sampling a blank sample after high concentration sample detection. And (3) sampling a blank plasma sample after the upper limit sample is quantified, wherein chromatographic peak areas at the retention time of the blank sample linezolid are all smaller than 20% of the quantitative lower limit peak area of the batch standard curve, and chromatographic peak areas at the retention time of the internal standard are all smaller than 5% of the quantitative lower limit internal standard peak area of the batch standard curve.

4.5 extraction recovery

Preparing low, medium and high concentration quality control plasma samples, performing 6 sample analysis on each concentration, performing the same treatment on blank plasma except that an internal standard is not added, adding a control solution with a certain concentration into the obtained supernatant to ensure that the final concentrations of a substance to be detected and the internal standard are respectively the same as the theoretical concentrations of the low, medium and high quality control samples after treatment, performing 6 sample analysis on each concentration, calculating the recovery rate by using the peak areas of two treatment methods of each concentration, and respectively setting the recovery rates of the low, medium and high concentrations of linezolid to be 96.8%, 98.2% and 100.9%. The extraction recovery of the internal standard was 100.7%.

4.6 matrix Effect

Matrix effects were examined at three QC concentration levels, low, medium 2, and high. Taking 6 blank plasmas from different sources, operating according to a plasma sample pretreatment method except that no internal standard is added, adding a certain concentration of control solution into obtained supernatant to enable final concentrations of an object to be detected and the internal standard to be respectively the same as theoretical concentrations of low, medium and 2 high QC samples after treatment, performing 3 sample analysis, simultaneously preparing pure solution samples with the final concentrations of the object to be detected and the internal standard to be respectively the same as the theoretical concentrations of the low, medium and 2 high QC samples after treatment, performing 6 sample analysis after the operation of the same method, respectively calculating internal standard normalized matrix factors under two treatment modes, evaluating matrix effects through precision of the matrix factors, and being acceptable when RSD of the internal standard normalized matrix factors from different sources is less than 15%.

The mean values of the internal standard normalized matrix effect factors of linezolid at low, medium and high concentrations are 0.960, 0.997 and 0.990 respectively, the RSD of the internal standard normalized matrix factors of plasma from different sources is less than 4.2 percent, and the results show that the matrix does not interfere with the determination of linezolid.

4.7 stability

The stability test covers the whole detection process of the sample, two concentrations of low concentration 90.000ng/mL and high concentration 22500.000ng/mL are set, 6 samples are repeated at each concentration, the stability of the whole blood in ice bath and the room temperature for 2 hours, the stability of the plasma sample in room temperature for 20 days at-70 ℃, the stability of the plasma sample in repeated freeze-thaw for 5 times and the stability of the extracted sample in 4 ℃ are examined, the relative deviation (%) is acceptable within +/-15%, and the results are shown in Table 4.

Table 4: linezolid plasma stability

4.8. Dilution reliability

Preparing a pure solution sample with linezolid concentration of 900.000 mug/mL, preparing a plasma sample with linezolid concentration of 45000.000ng/mL, diluting twice with blank plasma, pretreating 50.0 muL of plasma, performing sample injection analysis after the pretreatment is finished, preparing 6 samples at each concentration, and measuring that the precision and the accuracy of the linezolid dilution quality control sample are 94.6% and 5.4% respectively.

It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in the art and related arts based on the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in the art and related arts based on the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention.

Claims

1. An LC-MS/MS detection method of linezolid in human plasma is characterized in that: firstly, treating a human plasma sample to be detected by a protein precipitation method; then, LC-MS/MS analysis was performed using an appropriate organic solvent and buffer solution as a mixed mobile phase.

2. The LC-MS/MS detection method of linezolid in human plasma according to claim 1, characterized in that: the organic solvent is methanol, and the buffer solution is 5mM ammonium acetate aqueous solution.

3. The LC-MS/MS detection method of linezolid in human plasma according to claim 2, characterized in that: the method comprises the following steps:

s1, preparing a standard working solution to obtain a linezolid working solution, a linezolid parallel working solution, an internal standard working solution, a correction standard sample and a quality control sample;

s2, taking a calibration standard sample, a quality control sample, a human plasma sample to be detected and blank human plasma which are equal in volume, respectively adding internal standard working solution into the calibration standard sample, the quality control sample, the human plasma sample to be detected and the blank human plasma, adding methanol-water solution with the volume ratio of 1:1 into the blank human plasma, uniformly mixing in a vortex manner, centrifuging, and taking supernatant;

s3, determining the supernatants of the calibration standard sample and the quality control sample processed in the step S2 by an LC-MS/MS method, and preparing a standard curve by taking the chromatographic peak area ratio of linezolid to an internal standard as a vertical coordinate and the concentration of linezolid in human plasma as a horizontal coordinate;

4. The LC-MS/MS detection method of linezolid in human plasma according to claim 3, characterized in that: the preparation method of the standard working solution comprises the following steps: adding methanol into linezolid to prepare a linezolid stock solution with the concentration of 2.000mg/mL, and diluting the linezolid stock solution into a plurality of linezolid working solutions with the volume ratio of methanol-water solution of 1:1, wherein the concentration range of the linezolid working solutions is 0.600-600.000 mug/mL; repeating the steps once to prepare a plurality of linezolid parallel working solutions with the concentration range of 0.600-450.000 mug/mL; adding methanol into the internal standard to prepare internal standard stock solution with the concentration of 1.00mg/mL, and diluting the internal standard stock solution into internal standard working solution with the volume ratio of 1:1 by using methanol-water solution with the volume ratio of 2.500 mu g/mL; all stock solutions and working solutions are stored at-20 ℃ for later use; taking blank blood plasma of a healthy person, respectively adding the linezolid working solution, uniformly mixing in a vortex mode, and preparing a plurality of calibration standard samples with corresponding concentrations, wherein the concentration range is 30-30000 ng/mL; and (3) taking blank plasma of a healthy person, respectively adding the linezolid parallel working solution, and uniformly mixing by vortex to prepare a plurality of quality control samples with corresponding concentrations, wherein the concentration range is 30.000-22500.000 ng/mL.

5. The LC-MS/MS detection method of linezolid in human plasma according to claim 4, characterized in that: the concentration of the linezolid working solution is as follows: 0.600. mu.g/mL, 1.200. mu.g/mL, 6.000. mu.g/mL, 30.000. mu.g/mL, 120.000. mu.g/mL, 240.000. mu.g/mL, 480.000. mu.g/mL, and 600.000. mu.g/mL; the concentration of the linezolid parallel working solution is as follows: 0.600. mu.g/mL, 1.800. mu.g/mL, 18.000. mu.g/mL, 180.000. mu.g/mL, and 450.000. mu.g/mL; the calibration standard sample and the quality control sample are prepared fresh; the concentrations of the calibration standards were: 30.000ng/mL, 60.000ng/mL, 300.000ng/mL, 1500.000ng/mL, 6000.000ng/mL, 12000.000ng/mL, 24000.000ng/mL, 30000.000 ng/mL; the concentration of the quality control sample is as follows: 30.000ng/mL, 90.000ng/mL, 900.000ng/mL, 9000.000ng/mL, 22500.000 ng/mL.

6. The LC-MS/MS detection method of linezolid in human plasma according to claim 4, characterized in that: the internal standard adopts linezolid-d 3.

7. The LC-MS/MS detection method of linezolid in human plasma according to claim 3, characterized in that: in the step S2, the adopted vortex mixing time is 10 min; the centrifugation conditions used were: the temperature during centrifugation is 4 ℃, the centrifugation speed is 4200r/min, and the centrifugation time is 10 min.

8. The LC-MS/MS detection method of linezolid in human plasma according to claim 6, characterized in that: in step S3, the chromatographic conditions are: a chromatographic column: 4.6X 50mm, 5 μm; column temperature: 40 ℃; sample introduction volume: 2 mu L of the solution; mobile phase A: 5mM ammonium acetate in water containing 0.1% formic acid; mobile phase B: methanol, elution, the temperature of an autosampler is 4 ℃, and the flow rate of a mobile phase is 0.8 mL/min;

mass spectrum conditions: an ion source: electrospray ionization source ESI; the spraying voltage is 5500V; ion source temperature: 500 ℃; and (4) CUR: 30.00 psi; scanning mode: positive ion multi-reaction monitoring + MRM, linezolid and linezolid-d 3 ion reactions were m/z 338.2 → 195.1 and m/z 342.3 → 298.2, respectively, and collision energies CE were 30 and 26V, respectively.

9. The LC-MS/MS detection method of linezolid in human plasma according to claim 8, characterized in that: the chromatographic column was Welch Ultimate XB-C18.

10. The LC-MS/MS detection method of linezolid in human plasma according to claim 8, characterized in that: the elution is set as: