CN113917023B - Method for determining the concentration of brivudine in human plasma - Google Patents
Method for determining the concentration of brivudine in human plasma Download PDFInfo
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- 229960001169 brivudine Drugs 0.000 title claims abstract description 87
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- 238000000034 method Methods 0.000 title claims abstract description 58
- 239000000523 sample Substances 0.000 claims abstract description 53
- 238000003908 quality control method Methods 0.000 claims abstract description 38
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- 239000011550 stock solution Substances 0.000 claims abstract description 26
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 14
- 239000012472 biological sample Substances 0.000 claims abstract description 8
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- 238000002360 preparation method Methods 0.000 claims description 20
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- 229960001008 heparin sodium Drugs 0.000 description 3
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- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 3
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- JNTOCHDNEULJHD-UHFFFAOYSA-N Penciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(CCC(CO)CO)C=N2 JNTOCHDNEULJHD-UHFFFAOYSA-N 0.000 description 1
- 206010036376 Postherpetic Neuralgia Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- DRHKJLXJIQTDTD-OAHLLOKOSA-N Tamsulosine Chemical compound CCOC1=CC=CC=C1OCCN[C@H](C)CC1=CC=C(OC)C(S(N)(=O)=O)=C1 DRHKJLXJIQTDTD-OAHLLOKOSA-N 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 1
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- DCOPUUMXTXDBNB-UHFFFAOYSA-N diclofenac Chemical compound OC(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl DCOPUUMXTXDBNB-UHFFFAOYSA-N 0.000 description 1
- 229960001259 diclofenac Drugs 0.000 description 1
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- 238000001819 mass spectrum Methods 0.000 description 1
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- 208000004296 neuralgia Diseases 0.000 description 1
- 208000021722 neuropathic pain Diseases 0.000 description 1
- 229960001179 penciclovir Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
- G01N30/02—Column chromatography
-
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
-
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Abstract
The invention belongs to the technical field of medicine detection, and discloses a method for measuring the concentration of brivudine in human blood plasma, which comprises the following steps: preparing a brivudine standard curve stock solution, a quality control stock solution and an internal standard stock solution; preparing a standard curve plasma sample by using the prepared standard working solution and a blank matrix; preparing a brivudine quality control working solution by using a quality control stock solution, and preparing a quality control sample by using the prepared brivudine quality control working solution and a blank matrix; preparing an internal standard working solution by using an internal standard stock solution; determining a standard curve plasma sample, a quality control sample and an internal standard working solution by adopting an LC-MS/MS method to obtain a standard curve equation; and (3) determining the unknown biological sample by adopting an LC-MS/MS method, and determining the content of the brivudine in the unknown biological sample according to a standard curve equation. The invention overcomes the defect of accurate quantitative detection of lower concentration level, and can meet the requirement of content measurement in organisms when the dosage is smaller.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for measuring the concentration of brivudine in human blood plasma.
Background
Brivudine is a drug for the treatment of adult acute herpes zoster patients with normal immune function.
Shingles is an infectious skin disease caused by reactivation of latent varicella-zoster virus (VZV), which is well developed in the middle-aged and elderly, often manifested as unilateral rash along the sensory innervated area with neuropathic pain. With the increasing trend of ageing of the population in China, the incidence of herpes zoster is in a significant trend, and the prevention and treatment of the herpes zoster are already an important public health problem. Brivudine is a pharmacologically inactive substance that undergoes a series of phosphorylations, and the intracellular phosphate conversion process is catalyzed by viral thymidine kinase, ultimately forming brivudine triphosphate, which inhibits VZV replication.
The antiviral activity of the brivudine is 200-1000 times of that of acyclovir and penciclovir, and the brivudine is a single treatment scheme, has long half-life period and quick response, is used by patients with renal insufficiency without dosage adjustment, has high selectivity for herpes zoster virus, and can obviously reduce the occurrence risk of postherpetic neuralgia. Has great clinical advantages.
The existing published method for detecting the brivudine in the blood plasma has the limitations of overlong detection time, insufficient sensitivity and the like in the practical application process due to HPLC-UV or capillary electrophoresis.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for measuring the concentration of the brivudine in human blood plasma, which adopts an LC-MS/MS method to measure the concentration of the brivudine in human blood plasma, overcomes the defect of accurate quantitative detection of lower concentration level, can meet the requirement of content measurement in organisms when the dosage is smaller, meets the standard requirement in the signal-to-noise ratio in the detection process, has no interference of impurity peaks, and has high content accuracy of the measured medicine.
In order to solve the technical problems, the invention adopts the following technical scheme:
in one aspect, the present invention provides a method for determining the concentration of brivudine in human plasma comprising the steps of:
step one: preparing a brivudine standard curve stock solution, a quality control stock solution and an internal standard stock solution;
step two: preparing a standard curve plasma sample by using the prepared standard working solution and a blank matrix; preparing a brivudine quality control working solution by using a quality control stock solution, and preparing a quality control sample by using the prepared brivudine quality control working solution and a blank matrix; preparing an internal standard working solution by using an internal standard stock solution;
step three: measuring a bromof standard curve plasma sample, a quality control sample and an internal standard working solution by adopting an LC-MS/MS method to obtain a standard curve equation; and (3) determining the unknown biological sample by adopting an LC-MS/MS method, and determining the content of the brivudine in the unknown biological sample according to a standard curve equation.
Further, the preparation method of the brivudine standard curve stock solution comprises the following steps: and weighing the brivudine, and adding methanol for dissolution to obtain the tablet. Further, the concentration of the brivudine standard curve stock solution is 1mg/mL.
Further, the preparation method of the brivudine standard curve plasma sample is as follows: and preparing a standard curve plasma sample by using the prepared standard working solution and a blank matrix. Further, the concentration range of the brivudine standard working solution is 200-80000 ng/mL.
Further, the concentration range of the standard curve plasma sample is 10-4000 ng/mL.
Further, the preparation method of the quality control sample comprises the following steps: weighing brivudine, and adding methanol for dissolution to prepare a quality control stock solution; and preparing a quality control stock solution into a brivudine quality control working solution by adopting a 50% methanol aqueous solution, and preparing a quality control sample by using the prepared brivudine quality control working solution and a blank matrix. Further, the concentration range of the brivudine quality control working solution is 200-200000 ng/mL.
Further, the concentration range of the quality control sample is 10-10000 ng/mL.
Further, the preparation process of the internal standard working solution is as follows: and weighing hydrochlorothiazide, adding methanol for dissolution, and preparing an internal standard working solution by adopting a 50% methanol aqueous solution. Further, the concentration of the internal standard working solution is 1000ng/mL. The internal standard selects hydrochlorothiazide which is the same as negative ion and has close molecular weight, and the compound has good stability, high response and stability, and the retention time is similar to that of brivudine under the same gradient. Hydrochlorothiazide is less costly than other internal standards, such as deuterated brivudine; for example, diclofenac, the parent ion of hydrochlorothiazide is close to brivudine, the retention time is equal to that of brivudine under the same gradient, and the response value obtained by detection is high and stable.
Further, the pretreatment method of the standard curve plasma sample before detection is as follows: taking a standard curve plasma sample, swirling for 1min, taking acetonitrile precipitant sealing plate, swirling for 3min, centrifuging, carrying out 4 ℃,2450g,10min, sucking supernatant, adding ultrapure water, and carrying out sample injection detection after swirling.
Further, the conditions of the LC-MS/MS method are as follows:
chromatographic conditions
Chromatographic column: agilent poroshell EC-C182.7 μm (3.0.50 mm);
mobile phase a:0.1% formic acid solution; mobile phase B: acetonitrile;
flow rate: 0.400mL/min; stop time: 4.00min;
washing the needle washing liquid: 5s; sample injection amount: 5. Mu.L; column temperature: 40 ℃;
the mobile phase ratio is set as follows:
| Time | A | B | MaxPressure | |
| 1 | 0.00min | 90％ | 10％ | 1300bar |
| 2 | 0.20min | 90％ | 10％ | 1300bar |
| 3 | 2.00min | 10％ | 90％ | 1300bar |
| 4 | 3.00min | 10％ | 90％ | 1300bar |
| 5 | 3.01min | 90％ | 10％ | 1300bar |
| 6 | 4.00min | 90％ | 10％ | 1300bar |
mass spectrometry conditions
Adopting ESI source, negative ion MRM mode detection:
the ion source parameters were set as follows:
| Parameter | Value |
| IS | -4500 |
| Gas 1 | 50 |
| Gas 2 | 50 |
| CAD | 8 |
| CUR | 30 |
| Temp(℃) | 450 |
the mobile phase A and the mobile phase B adopted by the invention have great significance for improving response, can not pollute a mass spectrometer, and can not influence the service life of the mass spectrometer. The mobile phase adopts a gradient elution mode, and compared with isocratic elution, the gradient elution method is more stable and has no matrix effect. The invention adopts Agilentpore EC-C182.7 μm (3.0 x 50 mm) as chromatographic column, and the detected peak shape of the brivudine is more symmetrical and the responsivity is higher.
Further, the standard curve regression equation is as follows:
the standard curve regression equation for brivudine in human plasma was measured as: y=0.000608x+0.000562, r is 0.9995; the linear range of the brivudine is 10-4000 ng/mL, and the lowest quantitative lower limit of the blood concentration of the brivudine is 10ng/mL.
The invention selects chromatographic column: agilent poroshell EC-C182.7 μm (3.0.50 mm), the low concentration response to brivudine in the plasma measured in this application is higher, the peak shape is better, symmetrical and non-tailing, the peak time is not too early nor too late, just suitable; the to-be-detected object can be easily eluted, and the experimental time is effectively shortened.
The method selects the flow rate of 0.400mL/min and adopts the sample injection amount of 5 mu L to ensure the peak-out effect and the response value of the medicine, completely elute the medicine, avoid the pollution to the chromatographic column and simultaneously facilitate the improvement of the recovery rate and the improvement of the sensitivity of mass spectrum detection.
The invention adopts a gradient elution mode to detect the brivudine in the blood plasma, the elution proportion of a mobile phase changes at different time, and the problems of the plasma matrix effect and the like are comprehensively considered, so that the symmetry of the peak-to-peak shape of the medicine is perfect, the medicine can be fully eluted, the recovery rate is high, and the interference is small.
Compared with the prior art, the invention has the following beneficial effects:
compared with the existing liquid phase detection method, the method provided by the invention has the advantages that the lower detection limit can meet the requirement of content detection in organisms when the dosage is small, the signal-to-noise ratio in the detection process meets the standard requirement, no interference of impurity peaks exists, and the content accuracy of the detected medicine is high.
Under the chromatographic conditions adopted in the experiment, the retention time of the brivudine is about 1.93min, the retention time of the hydrochlorothiazide is about 1.77min, the peak shape is good, the measurement is not interfered by the impurity peak, and the baseline is stable; the method has higher specificity, can accurately measure the concentration of the brivudine in the blood plasma, has higher sensitivity, and has the lowest quantitative limit of 10.00ng/mL; the method provided by the invention is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of the brivudine. The linear range of the plasma standard curve of the brivudine is brivudine: 10.00-4000.00 ng/mL, and the precision (CV) of the batch and the batch is less than 5.0 percent. The method has good repeatability and high accuracy, is not interfered by matrixes, hyperlipidemia matrixes and hemolysis matrixes, and the plasma of different people has no interference phenomenon on the method.
The method can meet the requirements of human pharmacokinetics, and can be applied to clinical pharmacokinetics research.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 LC-standard graph of brivudine in human plasma measured by MS/MS method;
FIG. 2 is a graph showing the lower limit of quantitation of brivudine in human plasma as measured by the LC-MS/MS method;
FIG. 3 is a graph showing the lower limit of quantitation of hydrochlorothiazide in human plasma by the LC-MS/MS method;
FIG. 4 is a scan of a brivudine ion;
fig. 5 is a hydrochlorothiazide ion scan.
Detailed Description
The invention will be further illustrated with reference to specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
1. Main instrument
API 5500 type triple quadrupole liquid chromatograph-mass spectrometer (AB Sciex company) is provided with an electrospray ionization source and an analysis (version 1.6.3) data processing system, and is provided with an Agilent1290 automatic sampler, a liquid chromatograph and a column temperature box (Agilent, U.S.).
METTLER XPR type 2 parts per million plain (METTLER, switzerland).
2. Chromatographic conditions
Chromatographic column: agilent poroshell EC-C182.7 μm (3.0.50 mm);
mobile phase a:0.1% formic acid solution; mobile phase B: acetonitrile;
flow rate: 0.400mL/min; stop time: 4.00min;
washing the needle washing liquid: 5s; sample injection amount: 5. Mu.L; column temperature: 40 ℃;
the mobile phase ratio is set in table 1 below:
| Time | A | B | Flow | Max Pressure | |
| 1 | 0.00min | 90％ | 10％ | 0.400mL/min | 1300bar |
| 2 | 0.20min | 90％ | 10％ | 0.400mL/min | 1300bar |
| 3 | 2.00min | 10％ | 90％ | 0.400mL/min | 1300bar |
| 4 | 3.00min | 10％ | 90％ | 0.400mL/min | 1300bar |
| 5 | 3.01min | 90％ | 10％ | 0.400mL/min | 1300bar |
| 6 | 4.00min | 90％ | 10％ | 0.400mL/min | 1300bar |
3. mass spectrometry conditions
Negative ion MRM mode detection using ESI source is as shown in Table 2:
ion source parameters were set as follows in table 3:
| Parameter | Value |
| IS | -4500 |
| Gas 1 | 50 |
| Gas 2 | 50 |
| CAD | 8 |
| CUR | 30 |
| Temp(℃) | 450 |
4. anticoagulant: heparin sodium
5. Internal standard: hydrochlorothiazide
6. Data processing
6.1 atlas acquisition and concentration calculation method
Chromatographic retention time and chromatographic peak area were collected, processed and quantitatively analyzed by analysis (version 1.6.3). And performing linear least square regression calculation on the ratio of the analyte concentration to the internal standard concentration in the standard curve by using the ratio of the analyte peak area to the internal standard peak area, and calculating the actual measurement concentration of the analyte in the sample by using the obtained regression equation. The measured concentration of analyte in the sample is calculated by the instrument using the regression equation:
y＝ax+b
where y = analyte/internal standard peak area ratio
Slope of a = standard curve
x = drug concentration/internal standard concentration
b=intercept of standard curve
(weight factor 1/x) 2 )
6.2 concentration unit and decimal point retaining method
The decimal point number used for all calculations and statistical analyses is consistent with the software output. All concentration-related data retained 4 significant digits, chromatographic peak retention time 3 bits after the decimal point, percentage retained 1 decimal point, chromatographic peak area retained integer, peak area ratio retained 6 bits after the decimal point.
6.3 data transfer, calculation Process and use software
The data generated by the instrument is transferred to Microsoft Office Excel2013 which is the same version of the instrument to calculate CV values, deviations, average values and the like, and the calculation process is subjected to 100% audit by QA personnel.
Example 1
1. Solution preparation
Mobile phase a (0.1% formic acid aqueous solution)
2L of ultrapure water is measured, 2mL of formic acid is added, the mixture is uniformly mixed, and the ultrasonic treatment is carried out.
Mobile phase B (acetonitrile)
2L of acetonitrile (Merck) are measured and transferred to a suitable solvent bottle.
Needle washing solution (50% methanol water)
1000mL of methanol and 1000mL of ultrapure water were each transferred to an appropriate solvent bottle and mixed well.
Diluent solution (50% methanol water)
500mL of methanol and 500mL of ultrapure water were each transferred to an appropriate solvent bottle and mixed well.
Precipitant (acetonitrile)
200mL of acetonitrile was measured into an appropriate solvent bottle.
2. Preparing a standard solution:
preparation of standard reference stock solution
Weighing a certain amount of brivudine reference substance, placing the reference substance into self-made cup-shaped aluminum foil paper, recording the weight, placing the aluminum foil paper into a 20mL brown wide-mouth glass bottle, calculating the actual weight of an analyte according to the actual weighing amount and the brivudine content, adding a proper amount of methanol, and finally preparing the brivudine with the concentration of 1.000mg/mL, and shaking uniformly. Preserving at-20deg.C.
Table 4: preparation of standard curve working solution
Table 5: preparation of standard Curve plasma samples
3. Preparation of internal standard solution
Preparation of hydrochlorothiazide control Stock solution (QLSQ IS Stock,1.000 mg/mL)
Weighing a certain amount of hydrochlorothiazide reference substance, placing the reference substance into an aluminum foil cup, recording the weight, placing the aluminum foil cup into a 20mL brown wide-mouth glass bottle, calculating the actual weight of an analyte according to the actual weighing amount and the hydrochlorothiazide content, adding a proper amount of methanol, and finally preparing the hydrochlorothiazide with the concentration of 1.000mg/mL, and shaking uniformly. Preserving at-20deg.C.
Hydrochlorothiazide internal standard working solution preparation (IS Spike,1000 ng/mL)
Taking a volumetric flask with a volume of 100 mu L to 100mL of QLSQ IS Stock, fixing the volume to a scale with 50% methanol water, and uniformly mixing to obtain IS Spike (1000 ng/mL).
4. Quality control standard solution preparation
Preparing a quality control reference substance stock solution:
weighing a certain amount of brivudine reference substance, placing the reference substance into self-made cup-shaped aluminum foil paper, recording the weight, placing the aluminum foil paper into a 20mL brown wide-mouth glass bottle, calculating the actual weight of an analyte according to the actual weighing amount and the brivudine content, adding a proper amount of methanol, and finally preparing the brivudine with the concentration of 1.000mg/mL, and shaking uniformly. Preserving at-20deg.C.
Table 6: preparation of quality control working solution
Table 7: preparation of quality control plasma sample
Note that: the concentration of the follower quality control L/M/H QC is consistent with that of the L/M/HOQ QC, and the preparation method is the same as that of the follower quality control L/M/H QC. The H/LOQ QC samples were each split-packed at about 130. Mu.L and stored at-70℃with a portion of the split-packed samples being stored at-20 ℃.
The concentration of the follow-up quality control L/M/H QC is consistent with the concentration of the precision and the accuracy L/M/HOQ QC, and the preparation method is the same as that described above.
Pretreatment method before sample detection:
after thawing the blank plasma, standard plasma samples and quality control plasma samples were prepared according to "standard solution preparation", "quality control standard solution preparation" preparation standard curve.
Vortex 1min for taking the sample required for analysis of the batch.
Remove blank plasma (blank, caryover), ultrapure water (RB), SST sample, standard curve plasma sample, quality control plasma sample, 50. Mu.L each of each validation sample into the corresponding sample well.
50. Mu.L of each of the internal standard working fluid dilutions (50% methanol) and IS Spike was taken into the corresponding sample wells.
Take 300 μl of acetonitrile precipitant into the corresponding sample well.
Seal plate, vortex for 3min.
Centrifugation, at 4 ℃,2450g,10min.
mu.L of the supernatant was aspirated, 200. Mu.L of ultrapure water was added, and the mixture was vortexed for 3min.
Remarks: double blank samples are double blank samples for residual investigation, blank samples are single blank samples with only internal standard, and RB is reagent consumable acceptance blank sample.
5. Methodological verification
5.1 Linear Range and lower quantitative Limit
Standard curve samples were prepared using heparin sodium blank plasma formulation table. Taking 2 parts of blank plasma, wherein one part of the blank plasma is not added with the solution of the object to be detected and the internal standard reference substance, and the other part of the blank plasma is only added with the solution of the internal standard reference substance, and respectively recording 2 parts of blank human plasma chromatograms for evaluating the interference condition of the blank plasma.
Conclusion: the linear curve of the obtained standard curve sample is shown in fig. 1, wherein the standard curve equation of the brivudine in human plasma measured by an LC-MS/MS method is y=0.000608x+0.000562, and r is 0.9995; on average 6 determinations, SD of R was 0.0007 and CV was 0.1%; SD of slope was.0.000109, CV was 17.1%; the linear range of the brivudine is 10-4000 ng/mL, the lowest quantitative lower limit of the blood concentration of the brivudine is 10ng/mL, and the linear relation is good in the linear range.
5.2 precision and accuracy
Five quality control samples with different concentrations are taken, and the concentrations of the to-be-detected substances are LLOQ, LOQ QC, M1OQ QC, M2OQ QC and HOQ QC, and the to-be-detected substances are processed. As a study of intra-and inter-batch accuracy and precision. 6 parts of each concentration were prepared in parallel. The accuracy and precision between batches was assessed with 6 samples in parallel of five concentration level quality control samples of three independent analytical batches and performed for at least two days. The results are shown in the following table:
table 8: LC-MS/MS method for measuring precision and accuracy of brivudine in plasma
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* And (3) injection: this point is unresponsive.
The results show that: the LLOQ concentration level standard plasma sample should have a measured value in the range of 80.0% to 120.0% of the standard value, and should have an intra-and inter-batch precision (RSD) of no more than 20%. The measured values of the standard plasma samples with low, medium and high concentration levels are in the range of 85.0% -115.0% of the standard values, and the precision (RSD) between the batch and the batch is less than 15.0%. The relative deviation of the measured concentration and the theoretical concentration of at least 67.0% of the quality control samples per analysis batch should be within + -15.0% (the lower limit of quantification sample should be within + -20.0%), and at least 50.0% of the quality control samples per concentration should meet the above specifications. The method has good precision and accuracy for detecting the brivudine in the plasma, the precision between the batch and the batch is less than 5 percent, and the accuracy deviation between the batch and the batch is between-3.6 percent and 1.1 percent.
5.3 System applicability:
repeatedly analyzing the same standard plasma sample containing the bromvudine with the quantitative lower limit concentration (LLOQ) level for 5 times, respectively recording chromatographic peak area values and retention times of the sample and an internal standard, and calculating the chromatographic peak area ratio of the sample and the internal standard and the variation coefficient of the chromatographic peak retention time. Pure solution analysis batch system applicability can select solution samples with proper concentration to perform system balance so as to ensure instrument stability, and repeat analysis for 3 times. The specific results are shown in table 9 below:
table 9: LC-MS/MS method for determining applicability of brivudine system in plasma
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Conclusion: the signal to noise ratio (S/N is not less than 5) of the object to be detected, and the variation coefficient of the area ratio of the object to be detected and the internal standard chromatographic peak obtained by 5 times of repeated analysis is less than 15%; the coefficient of variation of retention time of the sample obtained by 5 times of repeated analysis and the internal standard chromatographic peak is less than 5 percent. The retention time variation coefficient of the object to be detected is 0.1-0.4%, the internal standard retention time variation coefficient is 0.1-0.6%, and the area ratio variation coefficient is 0.4-3.8%; the method of the invention is applicable to detection of brivudine in plasma.
5.4 Selectivity
Selectivity refers to the ability of chromatographic methods to distinguish between interferences in biological matrices when the analyte is assayed. At least 6 different batches of blank matrices need to be screened for method validation prior to method validation.
And taking blank plasma from different sources of 6 Chinese people, and respectively measuring without adding internal standard. Taking 6 parts of blank plasma from different sources, operating according to the method described in the section of sample pretreatment method to obtain 6 parts of standard plasma samples with the concentration of brivudine being the quantitative lower limit concentration level, treating the standard plasma samples according to the corresponding method, injecting treatment liquid into a chromatographic system for analysis, and respectively recording chromatograms of each part of blank sample and the standard plasma sample with the quantitative lower limit concentration level;
the results of each blank sample and standard plasma sample at the lower concentration level were recorded separately and quantified as follows.
Table 10: comparison table for selectively investigating analytes and internal standard by six blank healthy human blood plasma of different sources
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Conclusion: the chromatographic peak area at the retention time of the analytes in at least 6 blank matrix samples of different sources is lower than 20.0% of the peak area of the corresponding blank matrix LLOQ concentration to be detected of different sources, and the internal standard peak area at the retention time of the internal standard is lower than 5.0% of the internal standard peak area of the corresponding blank matrix LLOQ concentration of different sources. The LLOQ concentration measurement of 6 blank matrixes from different sources is in the range of 80.0-120.0% of the theoretical value. The interference range of the method for the analytes in the blank matrix samples from different sources is 6.5-7.4%, and the internal standard interference response value is 0-0.1%. Therefore, blank blood plasma of different human bodies does not cause interference on the detection result of the brivudine, the method can be used for detecting the brivudine in heparin sodium blood plasma of different human bodies, and blood plasma of different people has no influence on the detection method.
5.5 recovery rate
5.5.1 extraction recovery of analytes
Analyte recovery samples
Extraction recovery samples 6 aliquots were prepared in parallel from substrate samples at three concentration levels (LOQ QC, M2OQ QC, HOQ QC) of the test object.
Recovery reference sample: the extraction process is identical to the extraction operation of the sample at extraction recovery, but the analyte and internal standard solution do not participate in the extraction process.
In this analytical batch, the concentrations of the extracted recovery sample and the recovery reference sample were the same.
Acceptance criteria: the recovery rate of the extraction does not need to be high, but the results of the assay are reproducible. The extraction recovery rate RSD of 6 parts of each concentration level is less than or equal to 15.0 percent.
5.5.2 extraction recovery of internal Standard
The extraction recovery rate of the internal standard is inspected and carried out simultaneously with the analyte (medium concentration is generally selected, medium concentration is not applicable, low concentration and high concentration are selected), and the operation is consistent.
Acceptance criteria: the recovery rate of the extraction does not need to be high, but the results of the assay are reproducible. The extraction recovery rate RSD of 6 parts of each concentration level is less than or equal to 15.0 percent.
Table 11: recovery rate of internal standard
Conclusion: 1) The extraction recovery rate of the object to be detected-brivudine in the method is 96.1%, 96.3% and 94.4% in the high, medium and low concentration ranges respectively, and the corresponding variation coefficient CV values are 2.0%, 1.4% and 2.5%; the overall recovery was 95.6% and the overall recovery variation CV value was 2.1%.
2) The extraction recovery rate of the internal standard hydrochlorothiazide of the method is 103.8%, and the recovery rate CV value is 1.4%.
5.6 matrix Effect
1) Matrix effects refer to the inhibition or enhancement of the ionization of analytes by components present in a biological matrix. Taking 6 different batches of blank human plasma respectively, preparing one batch for each batch, and respectively adding standard substance solution and internal standard solution which are equivalent to the concentration of the treated low-concentration and high-concentration quality control samples after pretreatment for analysis.
Pure solutions, corresponding to the low and high concentration quality control sample concentrations, without matrix present, were analyzed in 6 replicates.
2) Hemolysis matrix effect: the validation method was evaluated using a simulated hemolyzed plasma sample (addition of 2.0% of sonicated whole blood to unhydrolyzed plasma, considered severely hemolyzed). Samples of the two concentration levels (LOQ QC, HOQ QC) of the test substance were prepared using simulated hemolyzed plasma, 6 parts each, and after processing, were injected into the chromatographic system for analysis.
3) In the verification method, a simulated hyperlipidemic plasma sample is used for evaluation (a 5.0% medium-long chain fat emulsion injection hyperlipidemic plasma sample is prepared), and 6 parts of low-concentration and high-concentration-level (LOQ QC and HOQ QC) samples of the to-be-detected substances are prepared from the simulated hyperlipidemic plasma sample and are treated and then injected into a chromatographic system for analysis.
Conclusion: the coefficient of variation of the internal standard normalized matrix factor must not be greater than 15.0%, the coefficient of variation cv% of the internal standard normalized matrix factor: 2.0 to 2.8 percent; the hemolysis control sample is evaluated by using a conventional quality control sample acceptance standard, the accuracy of average measured concentration and marked value of each concentration level is within 85.0-115.0%, RSD is less than or equal to 15.0%, and the average accuracy deviation range of the hemolysis matrix effect is as follows: -0.9-1.7%; the standard quality control sample is used for evaluating the hyperlipidemia quality control sample, the accuracy of the average measured concentration and the marked value of each concentration level is within 85.0-115.0%, the RSD is less than or equal to 15.0%, and the average accuracy deviation range of the hyperlipidemia matrix effect is as follows: 1.4 to 5.3 percent; the method of the invention has no obvious matrix effect and no obvious hemolysis and hyperlipidemia matrix effect.
5.7 stability
Human plasma quality control samples with low and high concentrations (LOQ QC and HOQ QC) of the object to be detected are subjected to stability investigation under the following conditions:
1) Repeated freeze thawing at-70 deg.c for 5 times for stabilization.
2) And the white light is stable at room temperature for 25 hours.
3) 4d stable at-20℃and 66d stable at-70 ℃.
4) Whole blood stability: the change of the ratio of peak areas of the analytes containing the low concentration level and the high concentration level (LOQ QC and HOQ QC) which are placed in the whole blood under an ice water bath for 0h and 2h is examined respectively, after the corresponding examination time point is placed, the whole blood sample is centrifuged to separate the plasma, an internal standard is added for treatment according to the corresponding plasma sample treatment operation method, and each concentration level is parallel to 6 parts.
The samples after stability investigation and the samples prepared at present are detected simultaneously, and the comparison result of each concentration is as follows:
table 12: results of 25h stability at room temperature-short term stability
Table 13: 4d stability-Long term stability results at-20℃
Table 14: 68d stability at 70℃and Long term stability results
Table 15: repeated freeze thawing at-70 ℃ for 5 times to obtain stability results
Table 16: whole blood stability results
The CV value obtained by the concentration of each drug in the blood plasma detected by the method is less than 5% under the above investigation condition, which indicates that the method has stronger stability to the brivudine in the whole blood and the blood plasma under the above investigation condition.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A method for determining the concentration of brivudine in human plasma comprising the steps of:
step one: preparing a brivudine standard curve stock solution, a quality control stock solution and an internal standard stock solution;
step two: preparing a standard curve plasma sample by using the prepared standard working solution and a blank matrix; preparing a brivudine quality control working solution by using a quality control stock solution, and preparing a quality control sample by using the prepared brivudine quality control working solution and a blank matrix; preparing an internal standard working solution by using an internal standard stock solution;
step three: measuring a bromv standard curve plasma sample and a quality control sample added with an internal standard working solution by adopting an LC-MS/MS method to obtain a standard curve equation; determining the unknown biological sample by adopting an LC-MS/MS method, and determining the content of the brivudine in the unknown biological sample according to a standard curve equation;
the pretreatment method of the unknown biological sample before detection comprises the following steps: taking an unknown biological sample, swirling for 1min, taking acetonitrile precipitant, sealing, swirling for 3min, centrifuging, absorbing supernatant fluid at 4 ℃,2450g for 10min, adding ultrapure water, and carrying out sample injection detection after swirling;
the conditions of the LC-MS/MS method are as follows:
internal standard: hydrochlorothiazide
Chromatographic conditions
Chromatographic column: agilent poroshell EC-C18,2.7 μm, 3.0X10 mm;
mobile phase a:0.1% formic acid solution; mobile phase B: acetonitrile;
flow rate: 0.400mL/min; stop time: 4.00min;
washing the needle washing liquid: 5s; sample injection amount: 5. Mu.L; column temperature: 40 ℃;
the mobile phase ratio is set as follows:
mass spectrometry conditions
Adopting ESI source, negative ion MRM mode detection:
the ion source parameters were set as follows:
the preparation process of the internal standard working solution is as follows: weighing hydrochlorothiazide, adding methanol for dissolution, and preparing an internal standard working solution by adopting a 50% methanol aqueous solution; the concentration of the internal standard working solution is 1000ng/mL.
2. The method of determining the concentration of brivudine in human plasma according to claim 1, wherein the method of preparing the brivudine standard curve stock solution is as follows: and weighing the brivudine, and adding methanol for dissolution to obtain the tablet.
3. The method of determining the concentration of brivudine in human plasma according to claim 2, wherein the brivudine standard curve plasma sample is formulated as follows: and preparing a standard curve plasma sample by using the prepared standard working solution and a blank matrix.
4. A method of determining the concentration of brivudine in human plasma according to claim 3, wherein the concentration of the brivudine standard curve stock solution is 1mg/mL, the concentration of the brivudine standard working solution is in the range of 200 to 80000ng/mL, and the concentration of the standard curve plasma sample is in the range of 10 to 4000ng/mL.
5. The method of claim 1, wherein the quality control sample is formulated as follows: weighing brivudine, and adding methanol for dissolution to prepare a quality control stock solution; preparing a quality control stock solution into a brivudine quality control working solution by adopting a 50% methanol aqueous solution, and preparing a quality control sample by using the prepared brivudine quality control working solution and a blank matrix; the concentration range of the brivudine quality control working solution is 200-200000 ng/mL; the concentration range of the quality control sample is 10-10000 ng/mL.
6. The method of determining the concentration of brivudine in human plasma according to claim 1, wherein the pretreatment of the standard curve plasma sample prior to detection is as follows: taking a standard curve plasma sample, swirling for 1min, taking acetonitrile precipitant sealing plate, swirling for 3min, centrifuging, carrying out 4 ℃,2450g,10min, sucking supernatant, adding ultrapure water, and carrying out sample injection detection after swirling.
7. The method of determining the concentration of brivudine in human plasma according to claim 1, wherein the standard curve regression equation is as follows:
the standard curve regression equation for brivudine in human plasma was measured as: y=0.000608x+0.000562, r is 0.9995; the linear range of the brivudine is 10-4000 ng/mL, and the lowest quantitative lower limit of the blood concentration of the brivudine is 10ng/mL.
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| CN1265741A (en) * | 1997-08-08 | 2000-09-06 | 新生物生物公司 | Method and compositions for overcoming resistance to biologic and chemotherapy |
| EP3792271A1 (en) * | 2019-09-13 | 2021-03-17 | Aurobindo Pharma Limited | A process for the preparation of brivudine |
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| CN1265741A (en) * | 1997-08-08 | 2000-09-06 | 新生物生物公司 | Method and compositions for overcoming resistance to biologic and chemotherapy |
| EP3792271A1 (en) * | 2019-09-13 | 2021-03-17 | Aurobindo Pharma Limited | A process for the preparation of brivudine |
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