CN106950302A - The quantitative analysis method of naproxen in a kind of human plasma - Google Patents
The quantitative analysis method of naproxen in a kind of human plasma Download PDFInfo
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Abstract
The invention discloses a kind of quantitative analysis method of naproxen in human plasma, comprise the following steps：（1）The preparation of standard working solution；（2）Sample treatment；（3）Standard curve making；（4）Quantitative analysis：Test specimen is taken according to step（3）Method processing, and by step（3）The calibration curve equation of gained is calculated, and obtains the concentration of naproxen in testing sample.Present invention application LC-MS technology (HPLC MS/MS) determines the concentration of naproxen in human plasma, pass through the optimization of chromatographic condition and Mass Spectrometry Conditions, extraction recovery is improved, matrix effect is reduced, enhances selectivity, improves detection sensitivity, so that whole quantitative detecting method is practical, easy to operate, repeatability is high, and accuracy is good, it is workable, it can directly apply to sample detection in bioequivalence and analyze.
Description
Technical field
The invention belongs to field of bioanalysis, more particularly, to one kind application LC-MS technology (HPLC-MS/MS)
Determine the quantitative analysis method of naproxen in human plasma.
Background technology
Naproxen is crucial antipyretic-antalgic composition in antiphlogistic naproxen sodium, is PG synthetase inhibitors.Oral absorption
It is rapid and complete, 2~4 hours plasma concentration peakings after 1 administration, more than 99% is combined with plasma protein in blood, for
Rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, ventilation, the chronic degenerative disease of kinematic system and light moderate pain
Deng effective in cure.U.S. sanitary research shows that the anti-inflammatory drug such as brufen (refined peacekeeping Merrill Lynch) that it may be more conventional than other is right
It is safer for heart.
At present, for naproxen the quantitative detection and method applied to clinical phase Bioequivalence Test is also present not
Enough fast and accurate problems.To accelerate the listing of China's imitation medicine, and the situation of the current Drug shortage of China is solved, need badly and build
A kind of vertical quick, highly sensitive naproxen content analysis method.
The content of the invention
There is provided a kind of quantitative analysis side of naproxen in human plasma for above-mentioned deficiency of the prior art by the present invention
Method, naproxen is extracted by the sample handling characteristics of liquid-liquid extraction from human plasma, and the sample extracted is diluted, gone forward side by side
Row LC-MS is analyzed.
The above-mentioned purpose of the present invention is achieved by the following technical programs.
The quantitative analysis method of naproxen, comprises the following steps in a kind of human plasma：
(1) preparation of standard working solution：Naproxen is weighed, methanol is added, concentration is configured to for 2.00~3.00mg
mL-1Naproxen storing solution, by the naproxen storing solution volume ratio 1:1 methanol-water solution is diluted to gradient concentration
0.0250~25.0 μ gmL-1Naproxen working solution；It is repeated once by above-mentioned steps, it is 0.025 to be configured to gradient concentration
~200 μ gmL-1Naproxen multiple operation solution；Weigh orinase, add methanol be configured to concentration for 0.200~
0.300mg·mL-1Internal standard storing solution, then with volume ratio 1:1 methanol-water solution is diluted to 80~120 μ gmL-1It is interior
Mark working solution；All storing solutions and working solution are preserved at 0~10 DEG C, standby；
Blank people's normal plasma is taken, the naproxen working solution is separately added into, mixes, is configured to the correction of corresponding gradient
Standard specimen, its concentration range is 0.200~200 μ gmL-1；Blank people's normal plasma is taken, the parallel work of the naproxen is separately added into
Make solution, mix, be configured to the quality-control sample of corresponding gradient, its gradient concentration scope is 1.00~8000 μ gmL-1；
(2) sample treatment：Isometric the proofreaded sample, quality-control sample, test specimen, blank human plasma and water are taken, toward school
The internal standard working solution is separately added into positive standard specimen, quality-control sample, test specimen, is separately added into blank human plasma and water
Volume ratio 1:1 methanol-water solution；Ultra-pure water and methyl tertiary butyl ether(MTBE) are added into above-mentioned each mixed liquor, is mixed, centrifugation,
Shift supernatant and dried up with nitrogen, each extract is preserved；
(3) standard curve making：Step (2) is obtained into the proofreaded sample, the extract of quality-control sample carries out LC-MS/MS points
Analysis is determined, and the chromatographic peak area ratio using naproxen and internal standard orinase is ordinate, with the concentration of naproxen in human plasma
Standard curve is made for abscissa；
(4) quantitative analysis：Test specimen is taken to be handled according to the method for step (3), and the standard curve as obtained by step (3)
Equation is calculated, and obtains the concentration of naproxen in testing sample.
Preferably, the condition of work of the analyses of LC-MS/MS described in step (3) continuous mode is：
A. chromatographic condition：Chromatographic column is Μ ItimateXB C18；Mobile phase A：The aqueous solution of 0.1% formic acid；Mobile phase B：
The acetonitrile solution of 0.1% formic acid；Column oven temperature：30℃；Flow velocity：1.20mL/min；Post pressure during chromatographic column poised state：
11.5~12.5MPa；Type of elution is gradient elution；
B. Mass Spectrometry Conditions：Ion gun is ESI sources, using positive ion mode, multiple-reaction monitoring pattern；Electron spray voltage：
4500V；Vortex ionspray temperature：650℃；Gas curtain gas species：30psi；Collision cell gaseous species：Middle rank；Atomization gas species
Gas1：60psi；Aid in gas Gas 2：60psi；Data collection time：1.0min.
Preferably, the concentration of naproxen storing solution described in step (1) is 2.50mgmL-1, the internal standard storing solution
Concentration is 0.250mgmL-1, the gradient concentration of the naproxen working solution is respectively 0.0250,0.0500,0.250,
1.00、2.50、12.5、22.5、25.0μg·mL-1, the concentration gradient of the naproxen multiple operation solution is 0.025,
0.075、1.125、18.75、200μg·mL-1, the concentration of the internal standard working solution is 100 μ gmL-1。
Preferably, the gradient concentration of step (1) described quality-control sample is respectively 1.00,3.00,45.0,750,8000 μ g
mL-1；The quality-control sample is in -15~-90 DEG C of storages；The gradient concentration of the proofreaded sample is respectively 0.200,0.400,
2.00、8.00、20.0、100、150、200μg·mL-1；The proofreaded sample is Fresh.
Preferably, the condition of step (2) described centrifugation is with 17000 × g centrifugation 5min.
Preferably, step (1) is also toward the naproxen storing solution, internal standard storing solution, internal standard working solution, naproxen work
It is 1 that volume ratio is added in solution:1 methanol-water solution is diluted, and preparation obtains the degree of accuracy/estimation of stability solution.
Preferably, step (3) described chromatographic condition also includes：The μ L of sampling volume 10.0；Automatic sampler cleaning solution is
Methanol；Automatic sampler washes needle body product：200μL；Pressure foot lifting amount：50mm；Soak time 1s when auto injection pin is cleaned；Automatically
Injector cleaning model：Cleaned before sample introduction after cleaning and sample introduction；Automatic sampler temperature：4.0℃.
Preferably, the monitoring ion pair of naproxen is 229.0/ in step (3) the LC-MS/MS analyses continuous mode
185.0, one ion pair when institute residence time 200ms of scanning, removes cluster voltage 50V every time, and collision energy 45eV, mass spectrum retains
0.25~0.30min of time.
Preferably, the monitoring ion pair of internal standard orinase is in step (3) the LC-MS/MS analyses continuous mode
271.1/91.0；One ion pair when institute residence time 50ms of scanning, removes cluster voltage 37V, collision energy 44eV, mass spectrum every time
0.18~0.22min of retention time.
Compared with prior art, beneficial effect of the present invention is：(1) extraction recovery is improved, up to more than 70%.(2)
Matrix effect is reduced, absolute matrix factors have been reached between 0.85~1.15.(3) selectivity is enhanced, is reduced to analyte
With the interference of uantitative analytical.(4) detection sensitivity is improved, it is determined that quantification range so that expected lower limit of quantitation sample
Product meet quantitative analysis requirement.Naproxen is dense in present invention application LC-MS technology (HPLC-MS/MS) measure human plasma
The concentration of naproxen, passes through the reasonable choosing of chromatographic column, lysate, mobile phase, Mass Spectrometry Conditions etc. in degree, accurate quantitative analysis human plasma
Select, the optimization of the process conditions such as flow velocity, type of elution, Mass Spectrometry Conditions so that whole quantitative detecting method is practical, operation letter
Just, automation equipment degree is high, and repeatability is high, and accuracy is good, workable, can directly apply to sample in bioequivalence
Product are tested and analyzed.
Brief description of the drawings
Fig. 1 is the standard curve of the quantitative analysis method of naproxen in the present inventor's blood plasma.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.Unless stated otherwise, the reagent of the invention used, method and apparatus is the art conventional reagent, methods
And equipment.
Using following street drug, experiment consumptive material and instrument in embodiment.
Reference substance：Naproxen (Naproxen), Beijing lark prestige Science and Technology Ltd. (lot number：LBC0O18).
Internal standard：Orinase (lot number：LS80O27).
Matrix：Human plasma (anti-coagulants is liquaemin), people's whole blood (anti-coagulants is liquaemin).
Reagent：Acetonitrile：Chromatographically pure, Fisher Scientific；Methanol：Chromatographically pure, Fisher Scientific；First
Acid：Analyze pure, Chemical Reagent Co., Ltd., Sinopharm Group.
Ultra-pure water：Make (pure water meter model and producer by oneself：Master-S15 Μ V, company of Shanghai Hitech Instruments Co., Ltd.).
Assay balance：BAS124S a ten thousandth electronic balances, Sai Duolisi scientific instrument (Beijing) Co., Ltd.
Centrifuge：Sorvall Legend Micro 17R supercentrifuges, Thermo Scientific.
Vortex mixed instrument：MTV-100, Hangzhou Ao Sheng Instrument Ltd..
Ultrasonic cleaner：KQ5200E, Kunshan Ultrasonic Instruments Co., Ltd..
Pipettor：0.5-10 μ L, 2-20 μ L, 20-200 μ L, 100-1000 μ L, 0.5-5mL, Eppendorf.
Continuous liquid-moving machine：M μ ltipipette Pl μ s, Eppendorf.
Glass sample bottle and screw lid：3.0mL, 5.0mL, 7.0mL, 10.0mL, 20.0mL, and U.S. laboratory apparatus science and technology
Net.
96 hole depth orifice plates：Like the company that pursues progress in P-DW-20-C, 2.00mL, the U.S..
96 hole depth pore plate by sealing pads：Like the company that pursues progress in P-DW-20-C, 2.00mL, the U.S..
Polypropylene centrifuge tube：1.5mL, 2.0mL, 5.0mL, 10.0mL, 15.0mL, 50.0mL, extra large Peter Krass experiment equipment
Co., Ltd.
Polypropylene cryopreservation tube：2.0mL, extra large Peter Krass experiment equipment Co., Ltd.
Chromatographic column：Μ ltimate XB C18,2.1 × 50.0mm, 5.0 μm, scientific and technological (Shanghai) limited company of the moon rising sun.
Efficient liquid-phase chromatographic pump：LC-20AD XR, Shimadz μ companies.
Automatic sampler：SIL-20ACXR, Shimadz μ companies.
Column oven：CTO-20AC, Shimadz μ companies.
Mass spectrograph：QTRAP 4500, AB Sciex companies.
The quantitative analysis method of naproxen, comprises the following steps in the human plasma that the present invention is set up：
(1) preparation of standard working solution：Naproxen is weighed, methanol is added, concentration is configured to for 2.00~3.00mg
mL-1Naproxen storing solution, by the naproxen storing solution volume ratio 1:1 methanol-water solution is diluted to gradient concentration
0.0250~25.0 μ gmL-1Naproxen working solution；It is repeated once by above-mentioned steps, it is 0.025 to be configured to gradient concentration
~200 μ gmL-1Naproxen multiple operation solution；Weigh orinase, add methanol be configured to concentration for 0.200~
0.300mg·mL-1Internal standard storing solution, then with volume ratio 1:1 methanol-water solution is diluted to 80~120 μ gmL-1It is interior
Mark working solution；All storing solutions and working solution are preserved at 0~10 DEG C, standby；
Blank people's normal plasma is taken, the naproxen working solution is separately added into, mixes, is configured to the correction of corresponding gradient
Standard specimen, its concentration range is 0.200~200 μ gmL-1；Blank people's normal plasma is taken, the parallel work of the naproxen is separately added into
Make solution, mix, be configured to the quality-control sample of corresponding gradient, its gradient concentration scope is 1.00~8000 μ gmL-1；
(2) sample treatment：Isometric the proofreaded sample, quality-control sample, test specimen, blank human plasma and water are taken, toward school
The internal standard working solution is separately added into positive standard specimen, quality-control sample, test specimen, is separately added into blank human plasma and water
Volume ratio 1:1 methanol-water solution；Ultra-pure water and methyl tertiary butyl ether(MTBE) are added into above-mentioned each mixed liquor, is mixed, centrifugation,
Shift supernatant and dried up with nitrogen, each extract is preserved；
(3) standard curve making：Step (2) is obtained into the proofreaded sample, the extract of quality-control sample carries out LC-MS/MS points
Analysis is determined, and the chromatographic peak area ratio using naproxen and internal standard orinase is ordinate, with the concentration of naproxen in human plasma
Standard curve is made for abscissa；
(4) quantitative analysis：Test specimen is taken to be handled according to the method for step (3), and the standard curve as obtained by step (3)
Equation is calculated, and obtains the concentration of naproxen in testing sample.
The inventive method is further described by taking specific implementation condition as an example below.
The preparation of the experimental solutions of embodiment 1 and working solution
Mobile phase A (aqueous solution for containing 0.1% formic acid) [being denoted as MA1]：Add in the vial equipped with 1000mL ultra-pure waters
Enter 1.00mL formic acid, vibration is mixed.Condition of storage：Room temperature.The term of validity：2 weeks.
Mobile phase B (acetonitrile for containing 0.1% formic acid) [being denoted as MB1]：Added in the vial equipped with the pure acetonitriles of 1000mL
1.00mL formic acid, vibration is mixed.Condition of storage：Room temperature.The term of validity：2 weeks.
Methanol：Water (50:50, v：V) [it is denoted as R01]：500mL methanol and 500mL water, vibration are added in a vial
Mix.Condition of storage：Room temperature.The term of validity：1 month.
Blood plasma [being denoted as HE] containing 2% whole blood cells dissolved matter：Melt freezing people's whole blood containing anticoagulant sodium heparin.
If, can be in frozen fresh whole blood in the refrigerator at a temperature of -15~-35 DEG C or -60~-90 DEG C without ready-made freezing whole blood
Or in the acetone added with dry ice snap frozen.Then, by the people's whole blood melted in the centrifuge being set under the conditions of 4 DEG C
In with least 3000 × g centrifugation 15 minutes.[HE] per 2.00mL, by the supernatant of 40.0 μ L whole blood cells dissolved matters
It is formulated, gently shakes up with 1960 μ L human plasmas.Condition of storage：- 60~-90 DEG C.The term of validity：1 year.
Note：The dose volume of solution can be scaled according to composition reagent used or liquor capacity.
The storing solution of naproxen is weighed by independent twice, prepares obtain respectively.The storing solution of all preparations should all be preserved
In 0~10 DEG C of refrigerator.The term of validity is set as 1 year, but can evaluate its stability in 1 year, with the practical stability of measure
Property data are defined.The working solution for preparing the proofreaded sample and quality-control sample must be from the independent storing solution weighed twice.
Naproxen storing solution [is denoted as S01], 2.50mg/mL：A certain amount of naproxen is weighed respectively and is positioned over two glass
In glass bottle, weight is recorded, S01-1, S01-2 is respectively labeled as.According to the purity of naproxen, moisture, whether into salt and its form
Calculate the actual weight of the analyte etc. information, add appropriate methanol, with the ultimate density of storing solution is 2.50mg/
ML, vortex mixed.If necessary can the dissolving of ultrasonic wave added compound.
Internal standard storing solution [is denoted as I01], 0.250mg/mL.A certain amount of orinase is weighed in vial, is recorded
Weight, labeled as I01.According to the purity of orinase, moisture, whether truly weighed into salt and its form calculus interior target
Amount.Add appropriate methanol, with the ultimate density of storing solution is 0.250mg/mL, vortex mixed.If necessary can ultrasonic wave added
Compound dissolves.
Internal standard working solution (100 μ g/mL) [being denoted as I02].49980 μ L R01 is added in vial, liquid relief is then used
Device is made I02, is stored in 0~10 DEG C of ice toward the orinase internal standard storing solution I01 that 20.0 μ L are added in bottle, vortex mixed
In case, validity date is set as two months.
Note：The dose volume of solution can be adjusted according to actual needs, but final aimed concn must be consistent, and
Record faithfully.
The preparation of working solution：In vial or polypropylene centrifuge tube, the R01 of the volume as specified by following table is added,
Then pipettor is used toward source solution of the addition in each bottle such as specified volume in following table, vortex mixed.The work of all preparations
Make solution to be all stored in 0~10 DEG C of refrigerator.Before the extended storage stability data of working solution are obtained, its term of validity is set
It is set to two months.
Note：S01-1 is naproxen storing solution, is subsequently used for preparing the proofreaded sample；S01-2 is the parallel storing solution of naproxen,
It is subsequently used for preparing quality-control sample；C01-C13 represents working solution.
The preparation of the proofreaded sample：It is determined that before the long-time stability that analyte is stored in refrigerator in matrix, correcting mark
Sample should on the day of sample treatment Fresh.After stability data is obtained, the proofreaded sample of freezen protective can be used,
The proofreaded sample of Fresh can also be used.During if necessary to Fresh the proofreaded sample, its compound method is as shown in the table.
Note：The dose volume of the proofreaded sample can be adjusted according to actual needs, but final aimed concn must keep one
Cause, and record faithfully.
The preparation of quality-control sample：In polypropylene centrifuge tube, appropriate blank human plasma is added with pipettor.Then, such as
Shown in following table, corresponding source solution is added, is vortexed and mixes (about 30 seconds), be configured to corresponding quality-control sample.In order to just
Number of freezing and thawing is used and reduced in later, quality-control sample is divided into some equal portions.It is specific as follows：Appropriate volume is shifted (it is recommended that 200
μ L) quality-control sample into the polypropylene cryopreservation tube that has marked in advance, store for future use.The quality-control sample of all preparations is stored in item
In the refrigerator of mesh proved recipe case specific temperature.
Note：The dose volume of quality-control sample can be adjusted according to actual needs, but final aimed concn must keep one
Cause, and correctly record.* QC-8000 is dilution QC (DQC) sample, for diluting reliability evaluation.
The preparation of the degree of accuracy/estimation of stability solution：Using pipettor, added such as into vial or polypropylene centrifuge tube
The source solution of designated volume in following table, then adds appropriate specified dilute solution, makes the degree of accuracy or steady of setting concentration
Qualitative evaluation solution, vortex mixed.Then by the solution storage in 0~10 DEG C of refrigerator, the term of validity is 1 week.Also fresh it can match somebody with somebody
Analysis test is carried out after system immediately.The solution for being compared or investigating is needed all to prepare to every part a individually for comparing
The solution of estimation of stability.
Note：The dose volume of solution can be adjusted according to actual needs, but final aimed concn must be consistent, and
Record faithfully.The concentration or weighing accuracy that SC3 solution is used for two parts of storing solutions of the parallel preparation of analyte compare, it can also be used to
The estimation of stability of analyte storing solution.ISC2 solution is used for the estimation of stability of internal standard storing solution.HIC2 solution is used to analyze
The estimation of stability of thing maximum concentration working solution.IIC solution is used for the estimation of stability of internal standard working solution.
Note：Analyte maximum concentration working solution refer to for prepare the proofreaded sample working solution in concentration highest
Working solution, not including the working solution for preparing quality-control sample.LIC solution is used for analyte least concentration working solution
Estimation of stability.
Note：Analyte least concentration working solution refer to for prepare the proofreaded sample working solution in concentration it is minimum
Working solution, not including the working solution for preparing quality-control sample.
Embodiment 2
The quantitative analysis method of naproxen in a kind of human plasma, it is characterised in that comprise the following steps：
(1) preparation of standard working solution：The gradient concentration of naproxen working solution is respectively 0.0250,0.0500,
0.250、1.00、2.50、12.5、22.5、25.0μg·mL-1, the concentration gradient of naproxen multiple operation solution is 0.025,
0.075、1.125、18.75、200μg·mL-1；The gradient concentration of quality-control sample is respectively 1.00,3.00,45.0,750,8000 μ
g·mL-1, the gradient concentration of the proofreaded sample is respectively 0.200,0.400,2.00,8.00,20.0,100,150,200 μ gmL-1；
The concentration of internal standard working solution is 100 μ gmL-1；The degree of accuracy/estimation of stability solution D IS, DA, SC3, ISC2,
HIC2, IIC are 25.0 μ gmL-1, SC1 is 25000 μ gmL-1, ISC1, HIC1 are 2500 μ gmL-1, SC2 is 250 μ
g·mL-1, LIC is 12.5 μ gmL-1；
As long as the preparation of above-mentioned solution ensures final concentration, its compound method can the system based on each solution in embodiment 1
Preparation Method, or other method.
(2) sample treatment：Each sample is taken out from refrigerator, melted in room temperature, the sample in each tubule is distinguished into whirlpool
Rotation is mixed；Using pipettor, 50.0 μ L correcting mark is separately added into the polypropylene centrifuge tube or 96 orifice plates marked in advance
Sample, quality-control sample, test specimen, blank human plasma and water, are separately added into the proofreaded sample, quality-control sample, test specimen
25.0 μ L I02,25.0 μ L R01 are separately added into blank human plasma and water；
100 μ L ultra-pure waters, vortex mixed 5min are added into above-mentioned each mixed liquor, then is added into above-mentioned each mixed liquor
Enter 800 μ L methyl tertiary butyl ether(MTBE)s, vortex mixed 5min, with 17000 × g centrifugation 5 minutes, transfer in 4 DEG C of centrifuges
500 μ L of supernatant liquid are dried up into 96 orifice plates, and with 96 hole Nitrogen evaporators；Before sample introduction analysis, by all extractions after sample treatment
At a temperature of thing is stored in automatic sampler or in 0~10 DEG C of refrigerator；
(3) standard curve making：Step (2) is obtained into the proofreaded sample, the extract of quality-control sample carries out following LC-MS/
MS analyses are determined, and each analysis batch prepares a standard curve being made up of 8 concentration level the proofreaded samples, while utilizing Quality Control
Sample is detected.
Chromatographic condition：
Chromatographic column：UltimateXB C18,2.1 × 50.0mm, 5.0 μm, Welch；
Mobile phase A (MA1)：The aqueous solution of 0.1% formic acid；
Mobile phase B (MB1)：The acetonitrile solution of 0.1% formic acid；
Automatic sampler cleaning solution：Methanol；
Column oven temperature：30℃；
Flow velocity：1.20mL/min；
Substantially post pressure during chromatographic column poised state：12.0Mpa；
Automatic sampler temperature：4℃；
Sampling volume：10.0μL；
Pressure foot lifting amount：50mm；
Automatic sampler cleaning is set：Only rinse；
Automatic sampler cleaning model：Before sample introduction and after sample introduction；
Automatic sampler washes needle body product：200μL；
Soak time when automatic sampler sample introduction needle is cleaned：1s；
Chromatogram gradient：
Note：The sample introduction analytical cycle (Cycle Time) of single sample：About 1.50 minutes (since a sample sample introductions
Time difference when next sample starts sample introduction).
Mass Spectrometry Conditions：
Ion gun：ESI；
Ionization mode：Positive ion mode；
Detection pattern：Multiple-reaction monitoring；
Electron spray voltage：4500V；
Vortex ionspray temperature：650℃；
Gas curtain gas species：30psi；
Collision cell gaseous species：Middle rank；
Atomization gas species (Gas1)：60psi；
Aid in gas species (Gas 2)：60psi；
Data collection time：1.0min.
* indicate：Residence time, referred to herein as when monitoring ion pair, stopped every time during one ion pair of scanning
Time.
The proofreaded sample, quality-control sample are detected by above-mentioned chromatogram and Mass Spectrometry Conditions, chromatogram collection and chromatographic peak product
Divide and handled by software Analyst 1.6.2 (AB Sciex), with naproxen and the chromatographic peak area of internal standard orinase
Than for ordinate, the concentration of naproxen is abscissa using in human plasma, with weighting (W=1/x2) least square method is with naphthalene in blood plasma
The concentration (X) of general life carries out linear regression with peak area ratio (Y), and the regression equation (Y=a+bX) of gained is standard curve, tool
Body is y=0.00807x+0.000506 (r=0.9997), concentration unit μ g/mL, is as a result shown, naproxen is 0.200~200
Linear good making standard curve in the range of μ g/mL, as shown in Figure 1.
(4) quantitative analysis：Naproxen is extracted from human plasma by the sample handling characteristics of liquid-liquid extraction, is diluted, then
LC-MS/MS LC-MS analyses are carried out according to the condition in step (3)；The anti-coagulants of naproxen is liquaemin, test sample used
The volume of product is 50.0 μ L.Calibration curve equation as obtained by step (3) is calculated, and obtains the concentration of naproxen in test specimen.
Embodiment 3
The present embodiment determines the degree of accuracy of naproxen concentration, precision in human plasma to LC-MS/MS methods in embodiment 2
Evaluated.Each portion of quality-control sample of above-mentioned tetra- concentration of QC-1, QC-3, QC-45, QC-750 of 20.0 μ L is taken, by above-mentioned side
Method is operated, in each sample replication 6/18 time of concentration 3 in 1 day, and respectively in not 3 analyses batch of METHOD FOR CONTINUOUS DETERMINATION on the same day,
Precision is calculated, it is as a result as shown in the table：
Batch in and batch between veracity and precision evaluation result
Note：Batch in batch between veracity and precision calculating when, n is respectively 6 and 18.Concentration value, average and SD take three
Effective digital, RSD, deviations in accuracy take one decimal place.
Rate of recovery test result such as following table：
Analyte working solution prepares 13 concentration gradients, and this concentration gradient is by lot of experiments and cleverly set meticulously
Meter, optimization has reached accuracy and precision.QC-8000 is used to dilute reliability evaluation in Quality Control solution, can evaluate line
Property outer 10 times of the maximum concentration of scope in analyte concentration.
Weighing accuracy and estimation of stability solution are prepared in this method research, the rate of recovery and matrix effect evaluate solution
Prepare the degree of accuracy for ensureing sample preparation, particularly stability, the rate of recovery.Sample recovery rate is realized using liquid-liquid extraction to reach
More than 90%, fully meet the realization of quantitative analysis.The preparation program of analyte working solution and quality-control sample solution be through
Cross careful consideration, it is sufficient to ensure quantifying for naproxen mass concentration in whole experiment process.
Implementation of the invention described in detail above, still, the present invention are not limited to specific thin in above-mentioned embodiment
Section, in the range of the technology design of the present invention, can carry out a variety of simple variants to technical scheme, these simple changes
Type belongs to protection scope of the present invention.
Claims (9)
1. the quantitative analysis method of naproxen in a kind of human plasma, it is characterised in that comprise the following steps：
（1）The preparation of standard working solution：Naproxen is weighed, methanol is added, concentration is configured to for 2.00 ~ 3.00 mg mL-1's
Naproxen storing solution, by the naproxen storing solution volume ratio 1:It is 0.0250 that 1 methanol-water solution, which is diluted to gradient concentration,
~25.0 μg•mL-1Naproxen working solution；It is repeated once by above-mentioned steps, is configured to gradient concentration for 0.025 ~ 200 μ g
mL-1Naproxen multiple operation solution；Orinase is weighed, methanol is added and is configured to concentration for 0.200 ~ 0.300 mg mL-1Internal standard storing solution, then with volume ratio 1:1 methanol-water solution is diluted to 80 ~ 120 μ g mL-1Internal standard working solution；Institute
There are storing solution and working solution to be preserved at 0 ~ 10 DEG C, it is standby；
Blank people's normal plasma is taken, the naproxen working solution is separately added into, mixes, is configured to the correcting mark of corresponding gradient
Sample, its concentration range is 0.200 ~ 200 μ g mL-1；Blank people's normal plasma is taken, the naproxen multiple operation is separately added into molten
Liquid, mixes, is configured to the quality-control sample of corresponding gradient, and its gradient concentration scope is 1.00 ~ 8000 μ g mL-1；
（2）Sample treatment：Isometric the proofreaded sample, quality-control sample, test specimen, blank human plasma and water are taken, toward correcting mark
The internal standard working solution is separately added into sample, quality-control sample, test specimen, volume is separately added into blank human plasma and water
Than 1:1 methanol-water solution；Ultra-pure water and methyl tertiary butyl ether(MTBE) are added into above-mentioned each mixed liquor, is mixed, is centrifuged, transfer
Supernatant is simultaneously dried up with nitrogen, and each extract is preserved；
（3）Standard curve making：By step（2）Obtain the proofreaded sample, the extract of quality-control sample carries out LC-MS/MS analyses and surveyed
Fixed, the chromatographic peak area ratio using naproxen and internal standard orinase is ordinate, and the concentration of naproxen is horizontal stroke using in human plasma
Coordinate makes standard curve；
（4）Quantitative analysis：Test specimen is taken according to step（3）Method processing, and by step（3）The calibration curve equation of gained
Calculate, obtain the concentration of naproxen in testing sample.
2. the quantitative analysis method of naproxen in a kind of human plasma according to claim 1, it is characterised in that step（3）
Described in LC-MS/MS analysis continuous mode condition of work be：
A. chromatographic condition：Chromatographic column is Μ ItimateXB C18；Mobile phase A：The aqueous solution of 0.1% formic acid；Mobile phase B：0.1%
The acetonitrile solution of formic acid；Column oven temperature：30℃；Flow velocity：1.20 mL/min；Post pressure during chromatographic column poised state：11.5~
12.5 MPa；Type of elution is gradient elution；
B. Mass Spectrometry Conditions：Ion gun is ESI sources, using positive ion mode, multiple-reaction monitoring pattern；Electron spray voltage：4500V；
Vortex ionspray temperature：650℃；Gas curtain gas species：30psi；Collision cell gaseous species：Middle rank；Atomization gas species Gas1：60
psi；Aid in gas Gas 2：60 psi；Data collection time：1.0 min.
3. the quantitative analysis method of naproxen in a kind of human plasma according to claim 1, it is characterised in that step（1）
Described in naproxen storing solution concentration be 2.50 mg mL-1, the concentration of the internal standard storing solution is 0.250 mg mL-1, institute
The gradient concentration for stating naproxen working solution is respectively 0.0250,0.0500,0.250,1.00,2.50,12.5,22.5,25.0
μg•mL-1, the concentration gradient of the naproxen multiple operation solution is 0.025,0.075,1.125,18.75,200 μ g mL-1,
The concentration of the internal standard working solution is 100 μ g mL-1。
4. the quantitative analysis method of naproxen in a kind of human plasma according to claim 1, it is characterised in that step（1）
The gradient concentration of the quality-control sample is respectively 1.00,3.00,45.0,750,8000 μ g mL-1；The quality-control sample in -15 ~
- 90 DEG C of storages；The gradient concentration of the proofreaded sample is respectively 0.200,0.400,2.00,8.00,20.0,100,150,200
μg•mL-1；The proofreaded sample is Fresh.
5. the quantitative analysis method of naproxen in a kind of human plasma according to claim 1, it is characterised in that step（2）
The condition of the centrifugation is with 17000 × g centrifugation 5min.
6. the quantitative analysis method of naproxen in a kind of human plasma according to claim 1, it is characterised in that step（1）
It is 1 also toward addition volume ratio in the naproxen storing solution, internal standard storing solution, internal standard working solution, naproxen working solution:1
Methanol-water solution be diluted, preparation obtain the degree of accuracy/estimation of stability solution.
7. the quantitative analysis method of naproxen in a kind of human plasma according to claim 2, it is characterised in that step（3）
The chromatographic condition also includes：The μ L of sampling volume 10.0；Automatic sampler cleaning solution is methanol；Automatic sampler washes needle body
Product：200 μL；Pressure foot lifting amount：50 mm；The s of soak time 1 when auto injection pin is cleaned；Automatic sampler cleaning model：Enter
Cleaned before sample after cleaning and sample introduction；Automatic sampler temperature：4.0℃.
8. the quantitative analysis method of naproxen in a kind of human plasma according to claim 2, it is characterised in that step（3）
The monitoring ion pair of naproxen is 229.0/185.0 in the LC-MS/MS analyses continuous mode, every time one ion pair of scanning
When 200 ms of institute's residence time, remove the V of cluster voltage 50, the eV of collision energy 45, the min of mass spectrum retention time 0.25 ~ 0.30.
9. the quantitative analysis method of naproxen in a kind of human plasma according to claim 2, it is characterised in that step（3）
The monitoring ion pair of internal standard orinase is 271.1/91.0 in the LC-MS/MS analyses continuous mode；Scanning one every time
The ms of institute's residence time 50 during ion pair, removes the V of cluster voltage 37, the eV of collision energy 44, mass spectrum retention time 0.18 ~ 0.22
min。
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