CN109682915A - A kind of method that LC-MS measures pyrazinamide concentration in blood plasma - Google Patents
A kind of method that LC-MS measures pyrazinamide concentration in blood plasma Download PDFInfo
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- CN109682915A CN109682915A CN201910040165.4A CN201910040165A CN109682915A CN 109682915 A CN109682915 A CN 109682915A CN 201910040165 A CN201910040165 A CN 201910040165A CN 109682915 A CN109682915 A CN 109682915A
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/26—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
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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
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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
- G01N30/26—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
- G01N2030/342—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient fluid composition fixed during analysis
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
Abstract
The invention discloses the methods of pyrazinamide concentration in a kind of LC-MS measurement blood plasma first to take sample to be tested using LC-MS system measurement, a certain amount of mixed organic solvents are added to be extracted twice, after pretreatment, through chromatography post separation, detected with mass detector.The method of the present invention quickly, it is accurate, high sensitivity, easy to operate, provide foundation for the determination of plasma concentration of pyrazinamide；The plasma standard curve linear range of this method is 0.1~20 μ g/mL, and in batch and betweenrun precision RSD is respectively less than ± 15%, is suitable for measuring the concentration of pyrazinamide in blood plasma.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of measuring method of drug, in particular to a kind of liquid matter connection
With the method for pyrazinamide concentration in measurement blood plasma.
Background technique
Tuberculosis is to threaten the serious infectious diseases of human health, and the drug currently used for first-line treatment has pyrazinamide, different
Cigarette hydrazine, rifampin and ethylamin ethanol.Wherein, pyrazinamide is white or off-white color crystalline powder, odorless or almost odorless, taste
Slight bitter, slightly molten in water, soluble,very slightly in ethanol.Fusing point is 188~192 DEG C, has preferable antibacterial to Bacillus tuberculosis
Effect, in pH5~5.5, bactericidal effect is most strong.The 12.5 μ g/ml of Mlc in human body can kill tuberculosis up to 50 μ g/ml
Bacillus.Mechanism of action is related with pyrazine acid, and pyrazinamide infiltrates through after phagocyte and enters in tubercle bacillus thallus, in thallus
Amidase so that it is sloughed amide groups, be converted into pyrazine acid and play antibacterial action.Separately because of pyrazinamide in chemical structure with
Niacinamide is similar, and by replacing niacinamide to interfere dehydrogenase, prevention dehydrogenation interferes utilization of the tubercle bacillus to oxygen, and
The eubolism for influencing bacterium, causes death.Pyrazinamide only has killing effect to tubercle bacillus, living without antibacterial to other bacteriums
Property, structural formula are as follows:
Currently, the document report not yet about pyrazinamide measuring method.
Summary of the invention
The purpose of the present invention is to provide the methods of pyrazinamide concentration in a kind of LC-MS measurement blood plasma, and this method can
Improve speed, the accuracy and sensitivity of detection.
To achieve the above object, a kind of method that LC-MS measures pyrazinamide concentration in blood plasma, plasma sample is through pre-
Detect its concentration through high performance liquid chromatography-tandem mass after processing, specific method the following steps are included:
(1) plasma sample pre-processes:
With K2EDTA is anti-coagulants, using pyrazinamide-d3 as internal standard；The accurate blood plasma that 100uL is added in 96 deep-well plates
Sample, the volume ratio that 5uL is added is the acetonitrile solution of 1:1, and the internal standard pyrazinamide-of the 0.01ug/uL of 5uL is added after mixing
1000uL acetonitrile is added in 96 deep-well plates in d3 solution after mixing, vortex mixed 1min is centrifuged in 20 DEG C with 3000rpm
10min takes supernatant liquor 50uL into 96 deep-well plates that 1000uL mixed organic solvents are housed, and mixed organic solvents are water: first
Alcohol: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.1 is vortexed and mixes, after 20 DEG C are centrifuged 5min with 3000rpm
It is to be detected as test sample；
(5) Specimen Determination:
It takes in 10uL test sample injection high performance liquid chromatography-tandem mass instrument, pyrazinamide and internal standard in test sample
The chromatographic peak of pyrazinamide-d3, and the pyrazinamide concentration in the plasma sample is calculated accordingly；
(6) liquid phase chromatogram condition:
Chromatographic column: SynergiTM4um Polar-RP 80A, column specification are 50 × 2mm；Chromatographic column temperature: 40 DEG C；Mobile phase
A: water/formic acid volume ratio is 100/0.1；Mobile phase B: methanol/formic acid volume ratio is 100/0.1；Washing lotion: acetonitrile/water
Volume ratio is 50/50；Autosampler temperature is 15 DEG C；Gradient elution, flow velocity 0.4mL/min, sample volume 10uL, analysis
Time 3.5min；
(7) Mass Spectrometry Conditions:
Ion source is electric spray ion source, and spray voltage 4500V, atomization temperature is 450 DEG C, and spray pressure power is
30Psi, auxiliary heating atmospheric pressure are 30Psi, and gas curtain atmospheric pressure is 30Psi, and collision atmospheric pressure is 8Psi, go the cluster voltage to be respectively
The pyrazinamide and pyrazinamide-d3 of 20eV；Collision cell entrance potential is respectively the pyrazinamide and pyrazinamide-d3 of 13eV；
Collision voltage is respectively the pyrazinamide and pyrazinamide-d3 of 25eV；Collision cell exit potential be respectively 8eV pyrazinamide and
Pyrazinamide-d3；Positive ion mode detection；Scanning mode is multiple-reaction monitoring MRM；
Ionic reaction for quantitative analysis is respectively as follows: m/z124.0 → m/z80.8, is pyrazinamide；And m/
Z127.0 → m/z83.7 is pyrazinamide-d3.
Preferably, in the step (3) gradient elution program are as follows:
| Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 98 | 2 |
| 1 | 98 | 2 |
| 1.01 | 40 | 60 |
| 2.01 | 40 | 60 |
| 2.02 | 98 | 2 |
| 3.50 | 98 | 2 |
Preferably, in the step (2), using internal standard method, with the peak area ratio of pyrazinamide and internal standard pyrazinamide-d3
Value brings the concentration for the pyrazinamide that calibration curve equation calculates in the plasma sample into.
Preferably, the calibration curve equation foundation the following steps are included:
10 parts of 100uL blank plasma are taken to be placed in 96 deep-well plates, adding 5uL concentration respectively in the form of stock solution is respectively
0.002μg/μL、0.004μg/μL、0.01μg/μL、0.02μg/μL、0.04μg/μL、0.1μg/μL、0.24μg/μL、0.4μg/
The pyrazinamide solution of μ L is fixed to minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest
It measures in upper limit sample, adds acetonitrile solution that 5 μ l volume ratios are 1:1 respectively to blank sample and zero-dose sample, after mixing
Respectively to minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest upper limit of quantification sample,
Internal standard pyrazinamide-d3 the solution of the 0.01ug/uL of 5uL is added in zero-dose sample, the volume of 5uL is added into blank sample
Than the acetonitrile solution for 1:1,1000uL acetonitrile is added into 10 parts of samples in 96 deep-well plates respectively after mixing, vortex mixed
1min is centrifuged 10min in 20 DEG C with 3000rpm, takes supernatant liquor 50uL to 96 deep holes that 1000uL mixed organic solvents are housed
In plate, mixed organic solvents are water: methanol: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.1, are vortexed and mix, in
20 DEG C are centrifuged 5min with 3000rpm, to be detected respectively as 10 parts of standard samples；
It is taken in 10uL standard sample injection high performance liquid chromatography-tandem mass instrument respectively, the pyrazinamide in test sample
With the chromatographic peak of internal standard pyrazinamide-d3, and standard curve is obtained accordingly, for calculating the pyrazinamide in the blood plasma
Concentration.
Compared with prior art, the invention has the following advantages that
(1) preprocess method is easy, two step organic extractant solutions, is suitable for conventional determining；
(2) specificity is strong: under chromatographic condition used by this experiment, pyrazinamide retention time is 1.134min left
The right side, internal standard pyrazinamide-d3 retention time is in 1.113min or so.The peak shape of pyrazinamide and internal standard pyrazinamide-d3 are good,
Without miscellaneous peak interference measurement, baseline is steady；
(3) high sensitivity: blood plasma is minimum to be quantitatively limited to 0.1ug/mL, the concentration of pyrazinamide in energy Accurate Determining blood plasma,
High sensitivity, high specificity；
(4) the method for the present invention quickly, it is accurate, high sensitivity, easy to operate, provided for the determination of plasma concentration of pyrazinamide
Foundation.The plasma standard curve linear range of this method is 0.1~20ug/mL, be in a few days respectively less than with day to day precision RSD ±
15%.
Detailed description of the invention
Fig. 1 is canonical plotting of the pyrazinamide that measures of HPLC-MS/MS method in human plasma；
Fig. 2 is that the HPLC-MS/MS of people's blank plasma schemes；
Fig. 3 is that pyrazinamide and the HPLC-MS/MS figure of pyrazinamide-d3 is added in people's blank plasma；
Fig. 4 adds the HPLC- of internal standard pyrazinamide-d3 for plasma sample after the oral pyrazinamide drug of health volunteer
MS/MS figure.
Specific embodiment
Invention is further described in detail with reference to embodiments.
Embodiment: mankind K2The measurement of pyrazinamide concentration in edta plasma
One, experimental material and analytical equipment
Pyrazinamide (analyte): U.S.Pharmacoeial Convention or identical, greater degree standard items
Pyrazinamide-d3 (internal standard): TLC Pharmaceutical Standards or identical, greater degree standard items
It see the table below 1 using reagent:
1 reagent detail of table
| Reagent name | Rank | Manufacturer |
| Acetonitrile | HPLC | J.T.Baker |
| Methanol | HPLC | J.T.Baker |
| Formic acid | ACS | Adamas |
Note: also the reagent of same levels or higher level can be used to see the table below 2 using analytical equipment:
Table 2 uses device inventory
| Component | Type | Manufacturer |
| Binary pump (binary pump) | AC Pump | AB SCIEX |
| Degasser (degasser) | Degasser | AB SCIEX |
| Column oven (thermocolumn case) | AC Column oven | AB SCIEX |
| Autosampler (Autosampler) | AC Autosampler | AB SCIEX |
| Sample rack (sample rack) | Rack Changer | AB SCIEX |
| Mass spectrometer (mass spectrograph) | TRIPLE QUADTM6500+ | AB SCIEX |
| Data processor (data processor) | Analyst1.6.3(software) | AB SCIEX |
Identical LC/MS/MS system can also be used.
Two, liquid matter condition
1, liquid phase chromatogram condition
SynergiTM4um Polar-RP 80A, column specification are 50 × 2mm；Chromatographic column temperature: 40 DEG C；Mobile phase A: water/first
The volume ratio of acid is 100/0.1；Mobile phase B: methanol/formic acid volume ratio is 100/0.1；Washing lotion: the volume ratio of acetonitrile/water is
50/50；Autosampler temperature is 15 DEG C；Gradient elution, elution program see the table below 3；Flow velocity is 0.4mL/min, and sample volume is
10uL, analysis time 3.5min.
3 gradient elution program of table
| Step | Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 1 | 0 | 98 | 2 |
| 2 | 1 | 98 | 2 |
| 3 | 1.01 | 40 | 60 |
| 4 | 2.01 | 40 | 60 |
| 5 | 2.02 | 98 | 2 |
| 6 | 3.50 | 98 | 2 |
2, Mass Spectrometry Conditions
Ion source is electric spray ion source, and spray voltage 4500V, atomization temperature is 450 DEG C, and spray pressure power is
30Psi, auxiliary heating atmospheric pressure are 30Psi, and gas curtain atmospheric pressure is 30Psi, and collision atmospheric pressure is 8Psi, go the cluster voltage to be respectively
The pyrazinamide and pyrazinamide-d3 of 20eV；Collision cell entrance potential is respectively the pyrazinamide and pyrazinamide-d3 of 13eV；
Collision voltage is respectively the pyrazinamide and pyrazinamide-d3 of 25eV；Collision cell exit potential be respectively 8eV pyrazinamide and
Pyrazinamide-d3；Positive ion mode detection；Scanning mode is multiple-reaction monitoring MRM；Ionic reaction for quantitative analysis is distinguished
Are as follows: m/z124.0 → m/z80.8 is pyrazinamide；It is pyrazinamide-d3 with m/z127.0 → m/z83.7.
Three, experimentation
1, the preparation of pyrazinamide standard solution
The preparation of pyrazinamide standard solution: precision weighs pyrazinamide (analyte) 0.02g, is placed in 10mL volumetric flask
In, the acetonitrile solution that volume ratio is 1:1 is added and dissolves and be settled to scale, shakes up, both obtains the pyrazinamide reserve of 2ug/uL
Liquid, then pyrazinamide standard solution is prepared for the molten successively dilution of acetonitrile solution of 1:1 with volume ratio, specific diluted concentration is seen below
Table 4:
4 pyrazinamide standard solution compound concentration of table
| Source solution (ug/uL) | Source liquor capacity (uL) | Final volume (mL) | Ultimate density (ug/uL) |
| 2a | 2000 | 10 | 0.4 |
| 2a | 1200 | 10 | 0.24 |
| 2a | 500 | 10 | 0.1 |
| 2a | 200 | 10 | 0.04 |
| 2a | 100 | 10 | 0.02 |
| 2a | 50 | 10 | 0.01 |
| 0.1 | 400 | 10 | 0.004 |
| 0.1 | 200 | 10 | 0.002 |
| 0.1 | 600 | 10 | 0.006 |
A: it is directly prepared from pyrazinamide (analyte)
For pyrazinamide standard solution in being stored in plastic containers and (4 DEG C) of refrigerator preservations when not in use, volume can be optionally
It increases or decreases to scale.
2, the preparation of pyrazinamide-d3 internal standard standard solution
The preparation of pyrazinamide-d3 internal standard standard solution: precision weighs pyrazinamide-d3 (internal standard) 0.002g, is placed in
In 10mL volumetric flask, the acetonitrile solution that volume ratio is 1:1 is added and dissolves and be settled to scale, shakes up, has both obtained 0.2ug/uL's
Pyrazinamide-d3 stock solution, then for the acetonitrile solution dilution of 1:1, to be configured to concentration be 0.01ug/uL pyrazine acyl with volume ratio
Amine-d3 inner mark solution, specific diluted concentration see the table below 5:
5 pyrazinamide-d3 standard solution compound concentration of table
| Source solution (ug/uL) | Source liquor capacity (uL) | Final volume (mL) | Ultimate density (ug/uL) |
| 0.2a | 5000 | 100 | 0.01b |
A: it is directly prepared from pyrazinamide-d3 (internal standard)
B: sample preparation steps are used for
For pyrazinamide-d3 internal standard standard solution in being stored in plastic containers and (4 DEG C) of refrigerator preservations when not in use, volume can
Optionally increase or decrease to scale.
3, linear test
Blank plasma is put into water-bath in room temperature environment to thaw；10 parts of blank plasma of transfer 100uL are into 96 deep-well plates
(each standard curve sample, blank sample -00 and zero-dose sample -0), according to listed by the following table 6, accurate addition is different dense respectively
The pyrazinamide standard solution 5uL or dilute solution of degree prepare each sample and mix, and are made into the plasma containing drug of various concentration,
It is operated by " plasma sample pretreatment ".Calculate the ratio Y (Y of pyrazinamide peak area As and internal standard pyrazinamide-d3 peak area Ai
=As/Ai), blood concentration X is made with peak area ratio Y to return calculating, the result is shown in Figure 1 and table 7.With mean ratio Y to blood medicine
Concentration X does recurrence and calculates, and obtains regression equation Y=3.91X-0.00952, r=0.9996, weight coefficient W=1/X2, in this way
The minimum quantitative limit of the blood concentration of the pyrazinamide measured are as follows: 0.1ug/mL.
6 pyrazinamide standard curve compound concentration of table
B: the dilute solution of analyte: ACN/H2O=50/50
Standard curve (n=10) of the pyrazinamide that table 7HPLC-MS/MS method measures in human plasma
4, accuracy and precision
Blank plasma is put into water-bath in room temperature environment to thaw；Shift proper volume blank plasma to container appropriate simultaneously
Add the plasma containing drug quality-control sample (LLOQ, QL, QLM, QM, QH) and one that pyrazinamide standard solution prepares 5 kinds of various concentrations
Item retinue standard curve, is operated by " plasma sample pretreatment ", and quality-control sample preparation is as shown in table 8 below.A batch and one are daily
Item retinue standard curve is continuously done 3 days, and totally three batches, first and each concentration of second batch make 6 parts of samples respectively, and third batch is every
A concentration makees 16 parts of samples respectively, calculates the ratio Y of pyrazinamide peak area As and internal standard pyrazinamide-d3 peak area Ai, generation
Measured concentration is acquired in standard curve on the day of entering, batch interior and betweenrun precision, measured concentration and addition are calculated by measured concentration
The ratio of concentration is accuracy, the results are shown in Table 9.The result shows that in pyrazinamide plasma sample batch, it is betweenrun precision, accurate
Degree is less than ± 15% and meets the requirements.
8 quality-control sample compound concentration of table
A: final volume=source liquor capacity+Plasma volumes
According to needed for each analysis batch, enough volumes are dispensed into the specimen bottle bottle indicated and are stored in theoretical temperatures-
80℃.Volume can be increased or decreased optionally to scale.
Table 9HPLC-MS/MS method measure blood plasma in pyrazinamide batch in, batch between precision and accuracy
5, interference
Six different blank plasma samples distinguish source difference healthy human body, by six different blank plasma samples in same
Analysis batch is prepared and is analyzed according to sample preparation steps, to evaluate different blank plasmas to pyrazinamide analyte and internal standard pyrazine acyl
The interference of amine-d3.
After six separate sources blank healthy human body plasma sample preparation analyses, meeting at pyrazinamide retention time
Interference peak response is below the 20.0% of the pyrazinamide response of lower limit of quantitation sample in the standard curve of the analysis batch, as a result sees
Table 10.The result shows that the analysis method has specificity to the analysis of pyrazinamide.
After six separate sources blank human normal plasma sample preparation analyses, meeting the Interference Peaks at internal standard retention time
Response is below the 20.0% of the internal standard response of lower limit of quantitation sample in the standard curve of the analysis batch, sees the table 11 in annex.
The result shows that the internal target analysis of the analysis method has selectivity.
Interference data comparison table of the 10 6 separate sources blank healthy human body blood plasma of table to pyrazinamide analyte
A: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve) × 100.0%≤
20.0%
B: when " no significant peak value can be integrated (or without peak) " or " retention time of peak area, which is not met in sample, divides
Analyse the retention time of object ", which is considered zero.
Interference data comparison table of the 11 6 separate sources blank healthy human body blood plasma of table to pyrazinamide-d3
A: analyte peak area (selective sample)/internal standard peak area (LLOQ of standard curve) × 100.0%≤
20.0%
B: when " no significant peak value can be integrated (or without peak) " or " retention time of peak area, which is not met in sample, divides
Analyse the retention time of object ", which is considered zero.
As can be seen that the blank plasma of different human body does not cause the testing result of pyrazinamide from table 10 and table 11
Interference.Therefore, this method can be used for detecting the pyrazinamide concentration in different human body blood plasma.
6, human plasma sample detects
(1) the people's blank plasma for being not given to pyrazinamide is taken, the accurate blank blood that 100uL is added in 96 deep-well plates
Slurry samples, the volume ratio that 10uL is added is the acetonitrile solution of 1:1,1000uL acetonitrile is added after mixing in 96 deep-well plates, whirlpool
Rotation mixing 1min, is centrifuged 10min in 20 DEG C with 3000rpm, takes supernatant liquor 50uL to being equipped with 1000uL mixed organic solvents
In 96 deep-well plates, mixed organic solvents are water: methanol: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.1, are vortexed
It mixes, takes 10uL sample to carry out LC-MS/MS analysis after being centrifuged 5min in 20 DEG C with 3000rpm, as a result as shown in Figure 2.
(2) the people's blank plasma for being not given to pyrazinamide is taken, the accurate blank blood that 100uL is added in 96 deep-well plates
Slurry samples are added the pyrazinamide standard solution that 5uL concentration is 0.01ug/uL, the interior of the 0.01ug/uL of 5uL are added after mixing
Mark pyrazinamide-d3 solution, after mixing be added 1000uL acetonitrile in 96 deep-well plates, vortex mixed 1min, in 20 DEG C with
3000rpm is centrifuged 10min, takes supernatant liquor 50uL into 96 deep-well plates that 1000uL mixed organic solvents are housed, and mixes organic
Solvent is water: methanol: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.1, is vortexed and mixes, in 20 DEG C with 3000rpm
10uL sample is taken to carry out LC-MS/MS analysis after after centrifugation 5min, as a result as shown in Figure 3.
(3) acquisition health volunteer takes orally the blood plasma after pyrazinamide drug, accurate in 96 deep-well plates to be added 100uL's
The human plasma sample of acquisition, the volume ratio that 5uL is added is the acetonitrile solution of 1:1, and the 0.01ug/uL of 5uL is added after mixing
Internal standard pyrazinamide-d3 solution, after mixing be added 1000uL acetonitrile in 96 deep-well plates, vortex mixed 1min, in 20 DEG C with
3000rpm is centrifuged 10min, takes supernatant liquor 50uL into 96 deep-well plates that 1000uL mixed organic solvents are housed, and mixes organic
Solvent is water: methanol: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.1, is vortexed and mixes, in 20 DEG C with 3000rpm
10uL sample is taken to carry out LC-MS/MS analysis after centrifugation 5min, as a result as shown in Figure 4.
In conclusion the present invention provides the measurement sides of pyrazinamide concentration in a kind of blood plasma of preprocess method simplicity
Method is suitable for conventional determining using two step organic extractant solution methods；Above-mentioned analysis is the results show that the color used by this experiment
Under spectral condition, pyrazinamide retention time is 1.134min or so, and internal standard pyrazinamide-d3 retention time is on the left side 1.113min
The peak shape of the right side, pyrazinamide and internal standard pyrazinamide-d3 are good, no miscellaneous peak interference measurement, and baseline is steady；This method has higher
Specificity, can pyrazinamide in Accurate Determining blood plasma concentration, sensitivity is higher, and blood plasma is minimum to be quantitatively limited to 0.1ug/
mL；Meanwhile the method for the present invention quickly, it is accurate, high sensitivity, easy to operate, for pyrazinamide determination of plasma concentration provide according to
According to.The plasma standard curve linear range of this method is 0.1~20ug/mL, be respectively less than in batch with betweenrun precision RSD ±
15%.
Claims (4)
1. a kind of method of pyrazinamide concentration in LC-MS measurement blood plasma, it is characterised in that: plasma sample is after pretreatment
Detect its concentration through high performance liquid chromatography-tandem mass, specific method the following steps are included:
(1) plasma sample pre-processes:
With K2EDTA is anti-coagulants, using pyrazinamide-d3 as internal standard；The accurate plasma sample that 100uL is added in 96 deep-well plates,
The volume ratio that 5uL is added is the acetonitrile solution of 1:1, and the internal standard pyrazinamide-d3 that the 0.01ug/uL of 5uL is added after mixing is molten
Liquid, is added 1000uL acetonitrile in 96 deep-well plates after mixing, vortex mixed 1min is centrifuged 10min in 20 DEG C with 3000rpm, takes
For supernatant liquor 50uL into 96 deep-well plates that 1000uL mixed organic solvents are housed, mixed organic solvents are water: methanol: formic acid is pressed
It according to the mixture that volume ratio 95:5:0.1 is mixed to get, is vortexed and mixes, as test specimens after being centrifuged 5min using 3000rpm in 20 DEG C
Product are to be detected；
(2) Specimen Determination:
It takes in 10uL test sample injection high performance liquid chromatography-tandem mass instrument, pyrazinamide and internal standard pyrazine in test sample
The chromatographic peak of amide-d3, and the pyrazinamide concentration in the plasma sample is calculated accordingly；
(3) liquid phase chromatogram condition:
Chromatographic column: SynergiTM4umPolar-RP80A, column specification are 50 × 2mm；Chromatographic column temperature: 40 DEG C；Mobile phase A: water/first
The volume ratio of acid is 100/0.1；Mobile phase B: methanol/formic acid volume ratio is 100/0.1；Washing lotion: the volume ratio of acetonitrile/water is
50/50；Autosampler temperature is 15 DEG C；Gradient elution, flow velocity 0.4mL/min, sample volume 10uL, analysis time
3.5min；
(4) Mass Spectrometry Conditions:
Ion source is electric spray ion source, and spray voltage 4500V, atomization temperature is 450 DEG C, and spray pressure power is 30Psi, auxiliary
Helping heating atmospheric pressure is 30Psi, and gas curtain atmospheric pressure is 30Psi, and collision atmospheric pressure is 8Psi, and removing cluster voltage is respectively the pyrrole of 20eV
Carboxamide dihydrochloride and pyrazinamide-d3；Collision cell entrance potential is respectively the pyrazinamide and pyrazinamide-d3 of 13eV；Collision voltage
The respectively pyrazinamide of 25eV and pyrazinamide-d3；Collision cell exit potential is respectively the pyrazinamide and pyrazine acyl of 8eV
Amine-d3；Positive ion mode detection；Scanning mode is multiple-reaction monitoring MRM；
Ionic reaction for quantitative analysis is respectively as follows: m/z124.0 → m/z80.8, is pyrazinamide；With m/z127.0 →
M/z83.7 is pyrazinamide-d3.
2. the method for pyrazinamide concentration in a kind of LC-MS measurement blood plasma according to claim 1, it is characterised in that:
The program of gradient elution in the step (3) are as follows:
3. the method for pyrazinamide concentration, feature exist in a kind of LC-MS measurement blood plasma according to claim 1 or 2
In: in the step (2), using internal standard method, standard song is brought into the peak area ratio of pyrazinamide and internal standard pyrazinamide-d3
The concentration of pyrazinamide in plasma sample described in line equation calculation.
4. the method for pyrazinamide concentration in a kind of LC-MS measurement blood plasma according to claim 3, it is characterised in that:
The foundation of the calibration curve equation the following steps are included:
10 parts of 100uL blank plasma are taken to be placed in 96 deep-well plates, adding 5uL concentration respectively in the form of stock solution is respectively
0.002μg/μL、0.004μg/μL、0.01μg/μL、0.02μg/μL、0.04μg/μL、0.1μg/μL、0.24μg/μL、0.4μg/
The pyrazinamide solution of μ L is fixed to minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest
It measures in upper limit sample, adds acetonitrile solution that 5 μ l volume ratios are 1:1 respectively to blank sample and zero-dose sample, after mixing
Respectively to minimum lower limit of quantitation sample, standard specimen 1, standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6, highest upper limit of quantification sample,
Internal standard pyrazinamide-d3 the solution of the 0.01ug/uL of 5uL is added in zero-dose sample, the volume of 5uL is added into blank sample
Than the acetonitrile solution for 1:1,1000uL acetonitrile is added into 10 parts of samples in 96 deep-well plates respectively after mixing, vortex mixed
1min is centrifuged 10min in 20 DEG C with 3000rpm, takes supernatant liquor 50uL to 96 deep holes that 1000uL mixed organic solvents are housed
In plate, mixed organic solvents are water: methanol: the mixture that formic acid is mixed to get according to volume ratio 95:5:0.1, are vortexed and mix, in
20 DEG C are centrifuged 5min with 3000rpm, to be detected respectively as 10 parts of standard samples；
It is taken in 10uL standard sample injection high performance liquid chromatography-tandem mass instrument respectively, pyrazinamide in test sample and interior
The chromatographic peak of pyrazinamide-d3 is marked, and obtains standard curve accordingly, for calculating the dense of the pyrazinamide in the blood plasma
Degree.
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