CN107064389A - The UPLC MS/MS detection methods of free paclitaxel in a kind of blood plasma - Google Patents
The UPLC MS/MS detection methods of free paclitaxel in a kind of blood plasma Download PDFInfo
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- 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
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- G01N30/06—Preparation
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The present invention relates to a kind of UPLC MS/MS detection methods of free paclitaxel in blood plasma；This method is balanced dialysis to plasma sample by the dialysis membrane of activation, inner mark solution, saltout auxiliary reagent and acetonitrile are added in the equilibrium dialysis solution of acquisition, progress is saltoutd auxiliary liquid-liquid extraction, take supernatant, after nitrogen drying, add redissolution liquid to be redissolved, obtain testing sample, carry out UPLC MS/MS analyses；The advantage of the invention is that：Accuracy is high, sensitivity is high, favorable reproducibility, is adapted to the UPLC MS/MS detection methods of free paclitaxel in the blood plasma of batch samples detection.
Description
Technical field
The present invention relates to a kind of analyzing detecting method of free paclitaxel in biological sample, more particularly to a kind of blood plasma middle reaches
From the UPLC-MS/MS detection methods of taxol.
Background technology
Taxol is widely used in oophoroma, breast cancer, prostate cancer, cancer of the esophagus, cervical carcinoma and carcinoma of endometrium etc.
The treatment of polytype cancer, it is water-soluble in order to increase it because taxol soluble is poor, need to add polyoxyethylene castor in preparation
Sesame oil and absolute ethyl alcohol, these additives usually cause serious allergic reaction.In blood plasma, taxol and plasma protein height
With reference to, it is not the major part for playing drug effect and producing toxic reaction with protein bound free paclitaxel, there are some researches prove
The concentration of free paclitaxel is influenceed by form of administration, such as castor oil, which has wrapped up taxol protomere is formed in blood plasma, to be subtracted
The small concentration of free paclitaxel, free drug concentration of injection taxol (albumin combination type) preparation in blood plasma also may be used
It can be influenceed by its preparation.
The method common problem of free paclitaxel concentration is most of detection side in detection blood plasma in the prior art
Method uses the detection techniques such as liquid chromatogram-ultraviolet method and liquid chromatography-mass spectrometry, most in these detection methods
The total concentration of mating type and free paclitaxel in detection human or animal's blood plasma is applied, the lower limit of quantitation of detection method is in ng/mL
Level, because taxol and plasma protein are highly combined, Cf, well below total concentration in blood plasma, is pg/mL grades, thus this
The sensitivity of a little detection methods can not meet the requirement for the paclitaxel concentration that dissociates in detection blood plasma.It would therefore be highly desirable to develop a kind of letter
Just it is quick, sample-pretreating method, with reference to UPLC-MS/MS chromatographic techniques, realized while shortening run time high flux and
High sensitivity.
The content of the invention
Present invention aims at there is provided a kind of accuracy is high, sensitivity is high, favorable reproducibility, it is adapted to batch samples detection
Blood plasma in free paclitaxel UPLC-MS/MS detection methods.
To achieve these goals, the invention provides a kind of UPLC-MS/MS detection sides of free paclitaxel in blood plasma
Method, comprises the following steps：
S1, plasma sample pre-treatment：Dialysis is balanced to plasma sample by the dialysis membrane of activation, in the balance of acquisition
Inner mark solution, saltout auxiliary reagent and acetonitrile are added in dialysis solution, auxiliary liquid-liquid extraction of saltouing is carried out, takes supernatant, nitrogen blows
After dry, add redissolution liquid and redissolved, obtain testing sample；
S2, UPLC-MS/MS testing conditions：
I.UPLC conditions：Eclipse Plus C18 posts：50mm × 2.1m, 1.8 μm；Sampling volume：20μL；Mobile phase A：
0.1mM lithium carbonate aqueous solutions；Mobile phase B：Acetonitrile, uses volume ratio for 50:50 A:B phase Gradient elutions, the stream of mobile phase
Speed is 0.5mL/min；
II.MS conditions：Ion gun：Electron spray ionisation source ESI；Ionization voltage：5500V；Temperature：450℃；Ion source gas
1：N2Pressure is 75psi；Ion source gas 2：N2Pressure is 80psi；Curtain gas：N2Pressure is 28psi；Collide atmospheric pressure：
10psi；Remove cluster voltage：80V；Scan pattern：Positive ion mode；
S3, standard curve drafting：Weigh Taxol Standard and prepare standard series working solution, with above-mentioned UPLC-MS-MS
Condition detects the titer respectively, and draws corresponding standard curve according to testing result.
Further, inner mark solution described in the step S1 is matched somebody with somebody by weighing docetaxel with methanol dissolving and constant volume
Internal standard stock solution processed, draws internal standard stock solution in right amount, and it is 50 to add volume ratio:50 methanol：Water dilutes, and obtains concentration and is
5.00ng/mL inner mark solution.
Further, the ion pair in the step S2 for quantitative analysis is 860.4 → m/z of taxol m/z respectively
814.0 → m/z of 291.8, docetaxel m/z 287.9, collision energy is 32eV, and sweep time is 100ms.
Further, the auxiliary reagent of saltouing in the step S1 is 6M ammonium acetate solution.
Further, it is that volume ratio is 50 that liquid is redissolved described in the step S1:50 water:Methanol solution.
Further, equilibrium dialysis solution described in the step S1 add the inner mark solution and saltout auxiliary reagent it
Before also need to add with the isometric methanol of the equilibrium dialysis solution, be vortexed 1min, ultrasonic 10min.
Further, the equilibrium dialysis solution, the volume ratio of saltout auxiliary reagent and acetonitrile are 1:8:16.
Further, UPLC conditions described in the step S2 are also 50 DEG C including column temperature temperature and sample introduction room temperature is
5℃。
Further, the concentration range of taxol is 0.100- in standard series working solution described in the step S3
250ng/mL。
The present invention sets up in the UPLC-MS/MS detection methods of free paclitaxel in detection blood plasma, pre-treating method and adopted first
Goldstandard equilibrium dialysis used is determined with protein binding rate and separates free paclitaxel, and accuracy is high, shadow that is being adsorbed without film
Ring, secondly using the liquid-liquid extraction method for auxiliary of saltouing, using the relatively low acetonitrile of volatility as extracts reagent, than traditional ethers thing
Matter is more environmentally friendly, nonhazardous, safer, completes a collection of sample total time-consuming and is less than 1 hour, is more suitable for the clinical sample of detection high-volume
Product；The chromatography time is short in the detection of UPLC-MS/MS instruments, only 1.5 minutes, and 100 samples can be completed within 4 hours
The analysis of product, and sample consumption is low, and sample consumption is only 50 μ L, and sample detection sensitivity is high, and lower limit of quantitation is
0.100ng/mL, sensitivity is promoted to pg/mL grades from ng/mL compared with traditional sensing techniques；Mass signal stability is strong, uses
0.1mM lithium carbonates detect signal stabilization, are difficult by instrument body as Mobile Phase Additives, the mass spectrum response of enhancing taxol
The influence of sodium salt, favorable reproducibility in system.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, described in detail below with presently preferred embodiments of the present invention as after.
Brief description of the drawings
Fig. 1 is the ion scan mass spectrogram of taxol in embodiment one；
Fig. 2 is the scanning mass spectrogram of paclitaxel product ion in embodiment one；
Fig. 3 is the ion scan mass spectrogram of docetaxel in embodiment one；
Fig. 4 is the scanning mass spectrogram of docetaxel product ion in embodiment one；
Fig. 5 is taxol and docetaxel MRM chromatograms in blank plasma samples in embodiment one；
Fig. 6 is the MRM chromatograms of the interior target LLOQ samples of addition in embodiment one；
Fig. 7 is to add taxol to 0.100ng/ml and docetaxel in the equilibrium dialysis liquid of blank plasma in embodiment one
To 5.00ng/ml MRM chromatograms；
Fig. 8 is the MRM that rat intravenous gives injection taxol (albumin combination type) plasma sample afterwards in embodiment one
Chromatogram；
Fig. 9 is the canonical plotting of taxol in embodiment one；
6 rat single intravenous injections in Figure 10 embodiments twoBe averaged free paclitaxel afterwards
Concentration time curve.
Embodiment
With reference to embodiment, the embodiment to the present invention is described in further detail.Following examples are used for
Illustrate the present invention, but be not limited to the scope of the present invention.
The foundation of the UPLC-MS/MS detection methods of free paclitaxel in the blood plasma of embodiment one
1st, the preparation of solution and sample
1.1 standard series samples：Precision weighs taxol in right amount, and with methanol dissolving and constant volume, precision is configured to taxol
The stock solution that concentration is about 1.10mg/mL, absorption stock solution is diluted step by step with methanol obtains standard series working solution, taxol
Concentration range is 0.100-250ng/mL；
1.2 quality-control sample：Matter is mixed using four concentration levels of taxol are prepared with standard series sample identical method
Sample is controlled, lower limit of quantitation LLOQ concentration is 0.100ng/mL, and low-quality control LQC concentration is 0.300ng/mL, middle Quality Control MQC's
Concentration is 10.0ng/mL, and high Quality Control HQC concentration is 200ng/mL；
1.3 inner mark solution：Precision weighs docetaxel reference substance in right amount, with methanol dissolving and constant volume, prepares internal standard concentration
About 1.00mg/mL internal standard stock solution, draws internal standard stock solution with methanol：Water (50：50, v/v) dilute, obtaining concentration is
5.00ng/mL inner mark solution.
2nd, plasma sample pre-treatment
2.1 equilibrium dialysis：
A) board-washing：Each cleaning equilibrium dialysis plate of PBS orders by methanol, deionized water, containing 0.02% polysorbas20 is each twice；
B) dialysis membrane is activated：Dialysis membrane is installed in 96 hole equilibrium dialysis plates, respectively added in donor sides and receiver sides
Enter deionized water, activate 10min, take out dialysis membrane, be placed in dust-free paper and get on after moisture removal with the PBS of 0.02% polysorbas20
96 hole equilibrium dialysis plates 2 times, dialysis membrane is reinstalled；
C) it is loaded：100 μ L plasma samples are added in donor sides, 300 μ L phosphate buffers are added in receiver sides；
D) equilibrium dialysis is quickly hatched：In the CO2gas incubator that equilibrium dialysis plate is placed in 37 DEG C, shake 4 hours；
E) sample：Go out sample from receiver side draws, preserve equilibrium dialysis solution to be measured.
2.2 saltout auxiliary liquid-liquid extraction：
Isometric methanol is added in equilibrium dialysis solution, 1min, ultrasonic 10min is vortexed, sample after 50.0 μ L processing is taken,
200 μ L 6M ammonium acetates, 25.0 μ L inner mark solutions and 400 μ L acetonitriles are separately added into, 5min is vortexed, 1min, rotating speed is centrifuged
14000rpm, takes supernatant in 40 DEG C of N entirely2Drying is flowed down, residue adds 150 μ L methanol：Water (50:50, v/v), vortex is mixed,
20 μ L are taken to carry out LC-MS/MS analyses.
3rd, detecting instrument and analysis condition
3.1 Japan's Shimadzu Corporation LC-30AD fast liquid chromatography systems, series connection is furnished with electron spray ionisation source (Turbo
Ion Spray) Canadian Sciex companies provide 6500 type triple quadrupole bar tandem mass spectrometers.
3.2 analysis condition
3.2.1UPLC condition：Eclipse Plus C18 posts：50mm × 2.1m, 1.8 μm；Column temperature：50℃；Sample introduction room temperature
Degree：5℃；Sampling volume：20μL；Mobile phase A：0.1mM lithium carbonate aqueous solutions；Mobile phase B：Acetonitrile, uses volume ratio for 50:50
A:B phase Gradient elutions, the flow velocity of mobile phase is 0.5mL/min；
3.2.2MS condition：Ion gun：Electron spray ionisation source ESI；Ionization voltage：5500V；Temperature：450℃；Ion gun gas
Body 1：N2Pressure is 75psi；Ion source gas 2：N2Pressure is 80psi；Curtain gas：N2Pressure is 28psi；Collide atmospheric pressure：
10psi；Remove cluster voltage：80V；Scan pattern：Positive ion mode；Collision energy 32eV, the ionic reaction point for quantitative analysis
Not Wei 860.4 → m/z of taxol m/z 291.8,814.0 → m/z of docetaxel m/z 287.9, sweep time is
100ms；The scanning mass spectrogram 1 of taxol is obtained, Product ion scans are carried out to the ions of m/z 860.4, main fragment ion is
M/z 291.8, obtains the scanning mass spectrogram 2 of paclitaxel product ion；The scanning mass spectrogram 3 of docetaxel, main fragment ion
For m/z287.9, the scanning mass spectrogram 4 of docetaxel product ion is obtained.
4th, Method validation
Method validation is carried out to this method according to U.S. FDA guideline, content includes selective, linear, accurate
Degree, precision, stability, rate of recovery matrix effect.
4.1 selectivity
Take receiver sides solution and the LLOQ each prepared after the blank rat plasma equilibrium dialysis of six separate sources
Sample introduction is analyzed after sample treatment, obtain taxol and docetaxel MRM chromatograms 5 in blank plasma samples, blank plasma it is flat
Weigh and taxol is added in dialyzate to 0.100ng/ml and docetaxel to 5.00ng/ml MRM chromatograms 6, blank plasma
Taxol is added in equilibrium dialysis liquid to 0.100ng/ml and docetaxel to 5.00ng/ml MRM chromatograms 7 and rat intravenous
Give the MRM chromatograms 8 of injection taxol (albumin combination type) plasma sample afterwards, taxol and docetaxel retention time
Common outflow Interference Peaks are had no near respectively 1.1 and 1.0min, retention time, chromatogram flows out determinand in blank plasma and retained altogether
Chromatographic peak area is not higher than in the 20% of LLOQ chromatographic peak areas, blank plasma chromatographic peak area at internal standard retention time at time
Not higher than the 5% of internal standard chromatographic peak area.
4.2 standard curve
Using taxol theoretical concentration as abscissa (x), the peak area ratio of taxol and internal standard docetaxel is ordinate
(y) linear regression equation (the weight factor W=1/x of regression analysis calculating, is carried out2).Method validation is double to standard curve sample
Sample analysis, LLOQ samples measured value is with theoretical value relative deviation within ± 20%, and other concentration samples are within ± 15%；
At least 75% standard curve sample should be met must be extremely in above-mentioned condition, and the LLOQ and upper limit of quantification double sample sample of standard curve
Ensure that a sample meets above-mentioned condition less；The square value r of the coefficient correlation of standard curve2>0.99。
Determine each concentration point measured value of paclitaxel standard curve in equilibrium dialysis liquid and be shown in Table 1, obtain UPLC-MS/MS methods and survey
Determine standard curve Fig. 9 of taxol in solution, standard curve linear regression equation：Y=0.206x+0.00329, shows in blood plasma
Free paclitaxel it is linear good in 0.100-250ng/mL concentration ranges, r2=0.996.
The UPLC-MS/MS methods of table 1 determine each concentration point measured value of paclitaxel standard curve in dialyzate
4.3 preci-sion and accuracy
Method validation determines four each six samples of concentration Quality Control sample, and LLOQ precision is calculated with relative standard deviation RSD to be less than
20% side can receive, and the degree of accuracy calculates RE with relative deviation can receive between ± 20%, the QC samples of remaining each concentration level
Each composition precision of product, which need to be less than 15% side, to be received, and the degree of accuracy can receive between ± 15%, taxol LLOQ precision
For 9.4%, the degree of accuracy is -2.2%, and the precision RSD of the QC samples of remaining concentration level taxol of taxol is less than 6.5%,
Degree of accuracy RE is between -0.8~4.3%, and QC sample tests meet the related request and testing program of Determination of Biological Samples
Receive standard, the results are shown in Table 2.
The UPLC-MS/MS methods of table 2 determine the veracity and precision of taxol in dialyzate
4.4 stability
When investigating stability of the taxol in plasma dialysis liquid sample, LQC and HQC quality-control samples are placed in different temperatures
And in environment, placement carries out six sample analyses after terminating.4 kinds of placement conditions are investigated altogether, are respectively：Room temperature places 6h, 4 after extraction
DEG C place 40 days, 3 freeze-thaws of experience circulation (arriving room temperature from -20 ± 5 DEG C), -20 ± 5 DEG C of placements 40 days.Dialysate samples
40 days determinand stabilizations are placed after sample pretreatment for 4 DEG C, and taxol RE is between -4.7~2.0%, dialysate samples room temperature is put
Put 6h determinands stable, taxol RE is between -1.4~-1.7%, 3 freeze-thaw circulation determinands of experience are stable, Japanese yew
Alcohol RE is between -2.2~8.2%, and it is stable that 40 days determinands are placed in the freezing of -20 DEG C of dialysate samples sample, taxol RE -
Between 2.6~-1.7%, as a result show that taxol is stablized under these conditions.
4.5 the rate of recovery
Basic, normal, high three concentration quality-control samples are prepared, the concentration of wherein taxol is respectively 0.300,10.0 and 200ng/
Ml, is operated, each concentration carries out six sample analyses, while separately taking rat by the plasma sample pre-treatment of step 2 in embodiment one
Receiver sides solution after blank plasma equilibrium dialysis, in addition to internal standard is not added with, remaining presses the plasma sample of step 2 in embodiment one
Pre-treatment is operated, and the μ L of control Quality Control solution 25.0 and the μ L of inner mark solution 25.0 of respective concentration are added into the supernatant of acquisition,
After vortex mixing, in 40 DEG C of N2Drying is flowed down, 150 μ L methanol are added：Water (50：50, v/v), vortex is mixed, and takes 20 μ L to carry out
UPLC-MS/MS is analyzed, and obtains corresponding peak area, and the rate of recovery is calculated with the peak area ratio of each two kinds of processing methods of concentration.
LQC, MQC and HQC concentration level：The extraction recovery of taxol is respectively 96.1%, 98.9% and 98.9%；Docetaxel
Extraction recovery is 99.1%, and the results are shown in Table A in 3 and table 4, table 3 is to add to compare matter with QC isoconcentrations after blank plasma is handled
Control the peak area that solution is obtained, B be in the peak area that obtains after quality-control sample is handled, table 4 A be add after blank plasma processing with
The peak area that QC isoconcentrations control Quality Control solution is obtained, B is to control the peak area obtained after sample treatment.
The UPLC-MS/MS methods of table 3 determine the taxol treatment rate of recovery (%) in dialyzate
The UPLC-MS/MS methods of table 4 determine the docetaxel processing rate of recovery (%) in dialyzate
4.6 matrix effect
Receiver sides solution after the blank plasma equilibrium dialysis of 6 rats is taken respectively, and in addition to internal standard is not added with, remaining is by real
The plasma sample pre-treatment operation of step 2 in example one is applied, the control Quality Control solution of respective concentration is added into the supernatant of acquisition
The 25.0 μ L and μ L of inner mark solution 25.0, after vortex mixing, in 40 DEG C of N2Drying is flowed down, it is 50 to add 150 μ L volume ratios:50
Water:Methanol solution, vortex is mixed, and takes 20 μ L to carry out UPLC-MS/MS analyses, obtains corresponding peak area；Simultaneously blank is replaced with water
Blood plasma, with UPLC-MS/MS analyses are carried out after method operation, calculates taxol and internal standard docetaxel under two kinds of processing modes respectively
Matrix factors, and calculate with this matrix factors after internal normalization.To normalize the degree of variation evaluation point of matrix factors
The matrix effect of analysis method, matrix factors, which are less than 15% side, to be received.The matrix factors of taxol point under LQC, HQC concentration level
Not Wei 102% and 101%, RSD be less than 3.6%, the results are shown in Table 5 and table 6, upper result shows, matrix do not disturb determinand quantify
Analysis.
The UPLC-MS/MS methods of table 5 determine the matrix effect (%) of taxol in dialyzate
The UPLC-MS/MS methods of table 6 determine the matrix effect (%) of internal standard docetaxel in dialyzate
Embodiment two determines S- (-)-Pantoprazole and R- (+)-Pantoprazole in blood plasma
The UPLC-MS/MS detection methods of free paclitaxel are used to quantitatively detect big in the blood plasma that will be set up in embodiment one
Free paclitaxel in mouse blood plasma, is moved with evaluating free paclitaxel medicine of injection taxol (albumin type) preparation in rat body
Learn.
6 rats are provided by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences's Experimental Animal Center, body weight 180-220g, male and female are each
Half, 6 rat injections give 45mg/kg'sPlasma sample is gathered after different time points, and it is saturating by balance
Analysis method obtains receiver side samples and tested, and the average free paclitaxel concentration time curve of acquisition is as shown in Figure 10.
The paclitaxel concentration that dissociates after drug administration by injection, in blood plasma is rapidly reached peak value, CmaxThe detection side provided for 326ng/mL, the present invention
Method sensitivity is high, can measure the plasma free paclitaxel concentration after being administered 24 hours, average value is 0.212ng/mL.
Chromatographic isolation is carried out present invention uses ultra performance liquid chromatography system UPLC, compared with conventional chromatogram system,
While UPLC systems can ensure chromatographic isolation effect, the chromatographic isolation time is substantially reduced, and can obtain more sharp
Chromatographic peak and then the sensitivity for lifting analysis method, the retention time of taxol and docetaxel be respectively 1.2min and
1.1min, chromatographic peak width is only less than 0.2min, and chromatographic run total time is only 1.5min, in order to which the mass spectrum for stablizing determinand is believed
Number, the present invention with the addition of 0.1mM lithium carbonates in flow visualizing, and Li ions other metals can be added and ion with Reverse transcriptase
Signal, play a part of stabilization signal, the concentration of optimization lithium carbonate is 0.1mM, and excessive concentration is easily in ionization process
The accumulation of forming salt, influence ionization process simultaneously pollutes mass spectrum, when concentration is too low, the high standard curve sample of taxol without
Method obtains enough Li ions, produces the situation of response saturation, in turn results in the range of linearity and narrow；In addition, [M+Li]+Peak-to-peak signal
Intensity is high, is conducive to improving the sensitivity of detection method.
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Claims (9)
1. the UPLC-MS/MS detection methods of free paclitaxel in a kind of blood plasma, it is characterised in that：Comprise the following steps：
S1, plasma sample pre-treatment：Dialysis is balanced to plasma sample by the dialysis membrane of activation, in the equilibrium dialysis of acquisition
Inner mark solution, saltout auxiliary reagent and acetonitrile are added in solution, auxiliary liquid-liquid extraction of saltouing is carried out, supernatant is taken, after nitrogen drying,
Add redissolution liquid to be redissolved, obtain testing sample；
S2, UPLC-MS/MS testing conditions：
I.UPLC conditions：Eclipse Plus C18 posts：50mm × 2.1m, 1.8 μm；Sampling volume：20μL；Mobile phase A：
0.1mM lithium carbonate aqueous solutions；Mobile phase B：Acetonitrile, uses volume ratio for 50:50 A：B phase Gradient elutions, the stream of mobile phase
Speed is 0.5mL/min；
II.MS conditions：Ion gun：Electron spray ionisation source ESI；Ionization voltage：5500V；Temperature：450℃；Ion source gas 1：N2
Pressure is 75psi；Ion source gas 2：N2Pressure is 80psi；Curtain gas：N2Pressure is 28psi；Collide atmospheric pressure：10psi；
Remove cluster voltage：80V；Scan pattern：Positive ion mode；
S3, standard curve drafting：Weigh Taxol Standard and prepare standard series working solution, with above-mentioned UPLC-MS-MS conditions
The titer is detected respectively, and corresponding standard curve is drawn according to testing result.
2. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 1, it is characterised in that：It is described
Inner mark solution described in step S1 prepares internal standard stock solution by weighing docetaxel with methanol dissolving and constant volume, draws internal standard
Appropriate stock solution, it is 50 to add volume ratio:50 water：Methanol dilution, obtains the inner mark solution that concentration is 5.00ng/mL.
3. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 2, it is characterised in that：It is described
Ion pair in step S2 for quantitative analysis is 860.4 → m/z of taxol m/z 291.8, docetaxel m/z respectively
814.0 → m/z 287.9, collision energy is 32eV, and sweep time is 100ms.
4. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 1, it is characterised in that：It is described
The auxiliary reagent of saltouing in step S1 is 6M ammonium acetate solution.
5. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 1, it is characterised in that：It is described
It is that volume ratio is 50 that liquid is redissolved described in step S1:50 water:Methanol solution.
6. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 1, it is characterised in that：It is described
Equilibrium dialysis solution described in step S1 is also needed to add before adding the inner mark solution and auxiliary reagent of saltouing and put down with described
Weigh the isometric methanol of dialysis solution, is vortexed 1min, ultrasonic 10min.
7. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 6, it is characterised in that：It is described
Equilibrium dialysis solution, the volume ratio of saltout auxiliary reagent and acetonitrile are 1:8:16.
8. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 1, it is characterised in that：It is described
UPLC conditions described in step S2 are also 50 DEG C including column temperature temperature and sample introduction room temperature is 5 DEG C.
9. the UPLC-MS/MS detection methods of free paclitaxel in blood plasma according to claim 1, it is characterised in that：It is described
The concentration range of taxol is 0.100-250ng/mL in standard series working solution described in step S3.
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| CN111089915A (en) * | 2019-11-15 | 2020-05-01 | 沈阳和合医学检验所有限公司 | Method for measuring concentration of docetaxel drug in human plasma |
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