CN107037154A - The chiral LC MS/MS high-flux detection methods of Pantoprazole in human plasma - Google Patents
The chiral LC MS/MS high-flux detection methods of Pantoprazole in human plasma Download PDFInfo
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Abstract
The present invention relates to a kind of chiral LC MS/MS high-flux detection methods of Pantoprazole in human plasma；This method is to take test plasma sample to be mixed into inner mark solution, using the pre-treating method of precipitation of protein, and 500 μ L inner mark solutions are added in plasma sample as protein precipitant, after being vortexed and centrifuging, supernatant is taken, testing sample is obtained, LC MS/MS analyses are carried out；The advantage of the invention is that：This method meets the demand of clinical batch samples analysis, compared with prior art with simple to operate, pre-treatment time and chromatography time it is short, be adapted to high flux sample pretreatment the characteristics of.
Description
Technical field
The present invention relates to Pantoprazole in a kind of biological sample and its analyzing detecting method of enantiomter, more particularly to
The chiral LC-MS/MS high-flux detection methods of Pantoprazole in a kind of human plasma.
Background technology
Chiral drug refers to introduce a pair obtained after chiral centre pair in kind with mirror image each other in drug molecular structure
Isomers is reflected, the physicochemical property of chiral drug enantiomter is substantially similar, only optical activity is different, but their pharmacodynamics,
Pharmacokinetics and toxicology there may be very big difference, such as proton pump inhibitor class medicine has similar architectural feature, point
There is the mixture that a chiral sulfur atom center is a pair of enantiomer compositions, the pharmacodynamics and pharmacokinetics table of such medicine in son
Reveal the otherness of enantiomer.Prior art common problem is that most of technology only suppresses to proton pumps such as Pantoprazoles
Agent raceme is quantified, seldom quantitative to its single enantiomter, and most of technology does not use deuterated internal standard, this shadow
The accuracy and reappearance of analysis are rung, the method also having employs liquid chromatogram-ultraviolet corona detector, this sensitivity
The requirement of low dosage human body medicine dynamics research can not be met, there is a small number of document reports Chiral liquid chromatography-tandem mass spectrometry
(LC-MS/MS) detection technique, but due to the structural difference very little of correspondence isomers, chromatographic isolation usually needs longer time,
The efficiency of analysis is greatly reduced, is not suitable for analyzing large batch of clinical samples thousands of easily.
S- (-)-Pantoprazole and the technology of R- (+)-Pantoprazole at present in chiral detection blood plasma is mainly two kinds, i.e.,
Liquid chromatogram-ultraviolet technology and liquid chromatography-tandem mass spectrometry technology, because liquid chromatogram-ultraviolet technology needs the color grown very much
Disengaging time is composed, analysis sample size is less than 50 daily, greatly limit analysis throughput, and sensitivity is poor, and lower limit of quantitation is led to
Often in more than 100ng/mL, it is impossible to the need for meeting current Pantoprazole clinical pharmacokinetics research, it would therefore be highly desirable to develop a kind of letter
Just the high sample-pretreating method of quick, accuracy, with reference to LC-MS/MS chromatographic techniques, is realized high while shortening run time
Flux.
The content of the invention
Present invention aims at high there is provided the easy quick, precision of one kind, it is adapted in the human plasma that batch samples are detected
The chiral LC-MS/MS high-flux detection methods of Pantoprazole.
To achieve these goals, the invention provides a kind of chiral LC-MS/MS high fluxs of Pantoprazole in human plasma
Detection method, comprises the following steps：
S1, plasma sample pre-treatment：50.0 μ L blood plasma are taken, 500 μ L inner mark solutions are added as protein precipitant, vortex
And after centrifuging, take supernatant, obtain testing sample；
S2, using LC-MS/MS methods determine testing sample in S- (-)-Pantoprazole, R- (+)-Pantoprazole, S- (-)-dissolve
Support draws azoles-D6 and R- (+)-Pantoprazole-D6 concentration：
I.LC conditions：Lux cellulose-4 posts：250mm × 4.6m, 5 μm；Column temperature：40℃；Sampling volume：2μL；Stream
Dynamic phase A：10mM ammonium acetate aqueous solutions；Mobile phase B：Acetonitrile, uses volume ratio for 10：90 A：B phase Gradient elutions, mobile phase
Flow velocity be 1.3mL/min；
II.MS conditions：Ion gun：Atmosphere pressure chemical ion source APCI；It is atomized electric current：3.0μA；Ion source temperature：550
℃；Ion source gas N2：55psi, curtain gas N2：40psi；Scan pattern：Cation multiple-reaction monitoring+MRM；
S3, standard curve drafting：S- (-)-Pantoprazole is weighed with methanol dissolving and constant volume, S- (-)-dissolve support is configured to
Draw azoles concentration be about 1.00mg/mL stock solution, weigh R- (+)-Pantoprazole with methanol dissolving and constant volume, be configured to R- (+)-
The stock solution that Determination of pantoprazole is about 1.00mg/mL, is drawn stock solution and is diluted to obtain mixing step by step with people's blank plasma and marked
Quasi- series of samples, detects the titer, and draw corresponding standard according to testing result respectively with above-mentioned LC-MS-MS conditions
Curve.
Further, the time being vortexed in the step S1 is 2min, and rotational speed of eddy current is 4500rpm.
Further, inner mark solution described in the step S1 is-Pantoprazole-D6 and R- (+)-dissolves respectively with S- (-)
Support drawing azoles-D6 standard items are dissolved using methanol and constant volume is prepared in internal standard stock solution of the internal standard concentration as 0.8mg/mL, precision absorption
State each internal standard stock solution appropriate, plus dilution in acetonitrile, obtain S- (-)-Pantoprazole-D6 and R- (+)-Pantoprazole-D6 concentration
It is 50.0ng/mL mixing inner mark solution.
Further, the elution time of chromatographic column is 5.5min in the step S2.
Further, the step S2 is used and shunted after post, and the mobile phase flowed out after chromatographic column is split into the first flowing
Mutually with the second mobile phase, the first mobile phase enters mass spectrum with 0.4mL/min flow velocity, and the second mobile phase is with 0.9mL/min stream
Speed enters waste discharge pipeline.
Further, it is room temperature that the LC conditions in the step S2, which also include automatic sampler temperature,.
Further, in the step S2 cation multiple-reaction monitoring S- (-)-Pantoprazole and R- (+)-Pantoprazole
After being ionized through APCI produce precursor ion m/z be 384.1, main fragment ion be m/z 200.1, S- (-)-Pantoprazole-
The precursor ion that D6 and R- (+)-Pantoprazole-D6 is produced is m/z 390.2, and main fragment ion is m/z 206.1, impact energy
Amount is 18eV.
Further, S- (-)-Pantoprazole and R- (+) in hybrid standard series of samples blank plasma in the step S3-
The concentration range of Pantoprazole is 0.0150-15.0 μ g/mL.
The present invention sets up Chiral liquid chromatography-string of S- (-)-Pantoprazole and R- (+)-Pantoprazole in detection human plasma
Join mass spectrometry method, the precipitation of protein that pre-treating method is used is compared with the liquid-liquid extraction often used in prior art, pre- place
Manage that step is few, flux is high, more environmentally friendly, be more suitable for detecting high-volume clinical sample, using the method shunted after high flow rate and post, together
When meet high-throughout chromatographic isolation, and stable Mass Spectrometer Method, the chromatography time is short, and the final chromatographic isolation time is only
For 5.5min, about half of prior art or so, S- (-)-Pantoprazole and R- (+)-Pantoprazole retention time are respectively
3.9 and 4.5min, two isomers reach baseline separation, and instrument detection flux lifts 1 times, and 200 clinical samples can be analyzed daily
Product, greatly reduce the time cost of detection while improving instrument service efficiency.
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 to dissolve Tuo La in the chiral LC-MS/MS high-flux detection methods of Pantoprazole in the human plasma that provides of the present invention
The ion scan mass spectrogram of azoles；
Pantoprazole in the chiral LC-MS/MS high-flux detection methods of Pantoprazole in the human plasma that Fig. 2 present invention is provided
The mass spectrogram of mass spectrometry fragmentation mode；
In the human plasma that Fig. 3 present invention is provided in the chiral LC-MS/MS high-flux detection methods of Pantoprazole in isotope
Mark thing Pantoprazole-D6 ion scan mass spectrogram；
In the human plasma that Fig. 4 present invention is provided Tuo La is dissolved in the chiral LC-MS/MS high-flux detection methods of Pantoprazole
The mass spectrogram of azoles-D6 mass spectrometry fragmentation modes；
Fig. 5 is the MRM chromatograms of Pantoprazole and Pantoprazole-D6 in blank plasma samples in embodiment one；
Fig. 6 is the MRM chromatograms of LLOQ samples and the interior target LLOQ samples of addition in embodiment one；
Fig. 7 is target MRM chromatograms in only being added in blank plasma in embodiment one；
Fig. 8 is the MRM chromatograms of blood sample and the interior target blood sample of addition in embodiment one；
Fig. 9 be subject's drip-feed 80mg Pantoprazole Sodiums in embodiment two blood sample in mean drug concentration-
Time graph；
Figure 10 be subject's drip-feed 40mg Levpantoprazole Sodiums in embodiment two blood sample in average drug
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 chiral LC-MS/MS assay methods of S- (-)-Pantoprazole and R- (+)-Pantoprazole in the human plasma of embodiment one
Foundation
1st, the preparation of solution and sample
1.1 standard series samples：S- (-)-Pantoprazole is weighed with methanol dissolving and constant volume, S- (-)-Pan Tuola is configured to
The stock solution that azoles concentration is about 1.00mg/mL, weighs R- (+)-Pantoprazole with methanol dissolving and constant volume, is configured to R- (+)-dissolve
Support draws the stock solution that azoles concentration is about 1.00mg/mL, draws stock solution and is diluted step by step with people's blank plasma and obtain hybrid standard
Series of samples, S- (-)-Pantoprazole and R- (+)-Determination of pantoprazole scope are 0.0150-15.0 μ g/mL, with above-mentioned LC-
MS-MS conditions detect the titer respectively, and draw corresponding standard curve according to testing result；
1.2 quality-control sample：Four concentration level mixing quality-control samples are prepared using the method similar with standard series sample
With enantio-selectivity quality-control sample, lower limit of quantitation LLOQ concentration is 0.150 μ g/mL, and low-quality control LQC concentration is 0.450 μ g/
ML, middle Quality Control MQC concentration is 2.25 μ g/mL, and high Quality Control MQC concentration is 11.5 μ g/mL, enantio-selectivity quality-control sample R-
(+)-Pantoprazole/S- (-)-Determination of pantoprazole is respectively 1.00/0.100 and 0.100/1.00 μ g/mL；
1.3 inner mark solution：Precision weighs S- (-)-Pantoprazole-D6 and R- (+)-Pantoprazole-D6 internal standards control respectively
Appropriate product, are dissolved and constant volume with methanol, prepare the internal standard stock solution that internal standard concentration is about 0.8mg/mL, and precision is drawn above-mentioned each interior
Mark stock solution is appropriate, plus dilution in acetonitrile, and the concentration for obtaining S- (-)-Pantoprazole-D6 and R- (+)-Pantoprazole-D6 is
50.0ng/mL mixing inner mark solution.
2nd, plasma sample pre-treatment
Take 50.0 μ L blood plasma, add 500 μ L inner mark solutions as protein precipitant, be vortexed 2min, under 4500rpm from
After heart 10min, supernatant is taken, testing sample is obtained, is placed in automatic sampler, LC-MS/MS analyses are carried out, sampling volume is
2.00μL。
3rd, detecting instrument and analysis condition
3.1 Japan's Shimadzu Corporation LC-30AD fast liquid chromatography systems, series connection is furnished with atmosphere pressure chemical ion source (APCI)
Canadian Sciex companies provide 5500 type triple quadrupole bar tandem mass spectrometers.
3.2 analysis condition
3.2.1 chromatographic condition：Chromatographic column use Phenomenex companies of the U.S. Lux cellulose-4 posts, 250mm ×
4.6mm, 5 μm, column temperature：40 DEG C, pre-column use Phenomenex companies of the U.S. Lux cellulose-4 guard columns, 4.0 ×
3.0mm, automatic sampler is set to room temperature, sampling volume：2μL；Mobile phase A：10mM ammonium acetate aqueous solutions；Mobile phase B：Second
Nitrile, uses volume ratio for 10：90 A：B phase Gradient elutions, the flow velocity of mobile phase is 1.3mL/min, is shunted using after post, will
The mobile phase flowed out after chromatographic column splits into the first mobile phase and the second mobile phase, and the first mobile phase is with 0.4mL/min flow velocity
Into mass spectrum, the second mobile phase enters waste discharge pipeline with 0.9mL/min flow velocity；
3.2.2 Mass Spectrometry Conditions：Ion gun：Atmosphere pressure chemical ion source APCI, is atomized electric current：3.0 μ A, ion source temperature：
550 DEG C, ion source gas N2：55psi, curtain gas N2：40psi, scan pattern is cation multiple-reaction monitoring (+MRM), is swept
Retouch scope：M/z 50~400, S- (-)-Pantoprazole and R- (+)-Pantoprazole produce precursor ion [M+ after being ionized through APCI
H]+Peak mass-to-charge ratio (m/z) is 384.1, obtains the scanning mass spectrogram 1 of Pantoprazole, and product ion is carried out to the ions of m/z 384.1
CID is scanned, main fragment ion is m/z 200.0, obtains the mass spectrogram 2 of Pantoprazole mass spectrometry fragmentation mode；Isotopic Internal Standard
The precursor ion that thing S- (-)-Pantoprazole-D6 and R- (+)-Pantoprazole-D6 is produced is m/z 390.2, obtains scanning mass spectrum
Fig. 3, main fragment ion is m/z 206.1, obtains the mass spectrogram 4 of Pantoprazole-D6 mass spectrometry fragmentation modes.
4th, Method validation
Method validation is carried out to this method according to U.S. FDA guideline, content includes stability, selectivity, line
Property, the degree of accuracy, precision, rate of recovery matrix effect.
4.1 selectivity
Take the blank plasma and a hemolytic plasma of six separate sources and the LLOQ sample treatments each prepared after sample introduction
Analysis, chromatogram flows out in blank plasma that chromatographic peak area is not higher than LLOQ chromatographic peak areas at determinand retention time altogether
20%, chromatographic peak area is not higher than the 5% of internal standard chromatographic peak area at internal standard retention time in blank plasma, obtains blank plasma
The MRM colors of Pantoprazole and Pantoprazole-D6 MRM chromatograms 5, LLOQ samples and the interior target LLOQ samples of addition in sample
Target MRM chromatograms 7 and subject's blood sample and the interior target blood sample of addition in only being added in spectrogram 6, blank plasma
MRM chromatograms 8, S- (-)-Pantoprazole and R- (+)-Pantoprazole retention time respectively may be about 3.9min and 4.5min, protect
Stay and common outflow Interference Peaks are had no near the time.
4.2 standard curve
Using determinand theoretical concentration as abscissa (x), the peak area ratio of determinand and internal standard compound is ordinate (y), is carried out
Linear regression equation (the weight factor W=1/x that regression analysis is calculated2).The each analysis batch of method validation is to standard curve sample
Two-sample analysis, LLOQ samples measured value and theoretical value relative deviation within ± 20%, other concentration samples ± 15% it
Interior, at least 75% standard curve sample should be met in above-mentioned condition, and the LLOQ and upper limit of quantification double sample sample of standard curve
It must at least ensure that a sample meets above-mentioned condition, the square value (r of the coefficient correlation of standard curve2)>0.99.Obtain and determine human blood
The average regression equation (n=3) of determinand standard curve straight line is respectively in slurry：
S- (-)-Pantoprazole:Y=(2.26 ± 0.087) x+ (0.000585 ± 0.00195) (r=0.9996 ±
0.0004),
R- (+)-Pantoprazole：Y=(2.21 ± 0.102) x+ (0.000659 ± 0.001351) (r=0.9996 ±
0.0002),
Represent that S- (-)-Pantoprazole and R- (+)-Pantoprazole are linear in 0.0150-15.0 μ g/mL concentration range
Relation is good.
4.3 preci-sion and accuracy
The each analysis batch of method validation is determined in four each six samples of concentration Quality Control sample, LLOQ batches, betweenrun precision is with phase
Standard deviation (CV) is calculated can receive less than 20% side, and deviations in accuracy calculates (RE) with relative deviation can between ± 20%
Receive, each composition of QC samples of remaining each concentration level is in a few days, day to day precision need to be less than 15% side to receive, deviations in accuracy
It can receive between ± 15%, S- (-)-Pantoprazole LLOQ betweenrun precisions are respectively less than 6.8%, and withinrun precision is respectively less than
3.3%, deviations in accuracy is in the range of -11.0~3.1%, and remaining concentration QC samples withinday precision is no more than 3.0%, day
Between precision no more than 5.4%, deviations in accuracy the results are shown in Table 1 in the range of -8.9~2.0%；R- (+)-Pantoprazole
LLOQ day to day precision is respectively less than 6.1%, and withinrun precision is respectively less than 3.8%, and deviations in accuracy is in -7.0~6.3% scope
Interior, remaining concentration QC samples withinday precision is no more than 2.3%, and day to day precision is no more than 6.1%, deviations in accuracy
In the range of -7.5~6.4%, 2 are the results are shown in Table.
Table 1. determines S- (-)-Pantoprazole preci-sion and accuracy in human plasma
Table 2. determines R- (+)-Pantoprazole preci-sion and accuracy in human plasma
4.4 enantio-selectivity
Each six sample of enantio-selectivity Quality Control sample is determined, each constituent concentration of QC samples of each concentration level is than batch interior essence
Density, which need to be less than 15% side, to be received, and the degree of accuracy can receive between ± 15%.Measurement result shows that enantio-selectivity is dense
Degree is respectively less than 1.5% than CV, and deviations in accuracy the results are shown in Table 3 in the range of -4.3~0.7%.
The preci-sion and accuracy of the enantio-selectivity quality-control sample of table 3. detection
4.5 the rate of recovery
Basic, normal, high three concentration Quality Control plasma sample is taken, wherein S- (-)-Pantoprazole and R- (+)-Pantoprazole is in blood
Concentration in slurry is respectively 0.450,2.25 and 11.5 μ g/mL, and each concentration carries out six sample analyses, takes blank plasma according to reality
The plasma sample pre-treatment operation in the step 2 of example 1 is applied, internal standard is substituted with acetonitrile, it is parallel to prepare many parts, blank supernatant will be obtained
Basic, normal, high three concentration rate of recovery sample is prepared after merging, wherein S- (-)-Pantoprazole and R- (+)-Pantoprazole is in blood plasma
Concentration be respectively 0.450,2.25 and 11.5 μ g/mL, internal standard plasma concentration is 5.00 μ g/mL, and six samples point are carried out per concentration
Analysis, the rate of recovery is calculated with the peak area ratio of each two kinds of processing methods of concentration.The internal standard rate of recovery is with institute under two kinds of processing methods
The internal standard peak area ratio for having sample is calculated, LQC, MQC and HQC concentration level：The extraction recovery of S- (-)-Pantoprazole point
Not Wei 106%, 105% and 105%, S- (-)-Pantoprazole-D6 be 107%；The extraction recovery of R- (+)-Pantoprazole point
Not Wei 105%, 102% and 108%, R- (+)-Pantoprazole-D6 be 106%.
4.6 matrix effect
6 separate sources blank plasmas and 1 part of haemolysis blank plasma are taken respectively, take blank plasma according to the step 2 of embodiment 1
In plasma sample pre-treatment operation, with acetonitrile substitute internal standard, it is parallel to prepare many parts, will obtain blank supernatant merge after prepare
Low, high two concentration matrix sample, wherein S- (-)-Pantoprazole and R- (+)-Determination of pantoprazole are respectively 0.0450 and 11.5 μ
G/mL, internal standard plasma concentration is 5.00 μ g/mL, and each concentration level in each source carries out three sample analyses, while using water generation
For blank plasma, with three sample analyses are carried out after same method operation, determinand is calculated under two kinds of processing modes respectively and interior
Respective matrix factors are marked, and the matrix factors after internal normalization are calculated with this, are commented with the degree of variation for normalizing matrix factors
The matrix effect of valency analysis method, matrix factors CV, which is less than 15% side, to be received.S- (-)-Pan Tuola under LQC, HQC concentration level
The matrix factors of azoles are respectively 102% and 103%, and CV is no more than 1.7%；The matrix factors of R- (+)-Pantoprazole are respectively
101% and 102%, RSD no more than 2.0%.Result above shows that matrix does not disturb determinand quantitative analysis.
Embodiment two determines S- (-)-Pantoprazole and R- (+)-Pantoprazole in human plasma
The chiral LC-MS/MS high-flux detection methods of Pantoprazole are quantitatively examined in the human plasma that Application Example one is set up
S- (-)-Pantoprazole and R- (+)-Pantoprazole surveyed in blood plasma, the pharmacokinetics for evaluating injection Levpantoprazole Sodium
Feature, health volunteer distinguishes the Levpantoprazole Sodium and positive control Pantoprazole Sodium of drip-feed various dose, respectively
In 10min after (0h) before administration, administration, 20min, 30min, 35min, 45min, 60min, 90min, 2.0h, 3.0h, 4.0h,
Blood sample is gathered before 5.0h, 6.0h, 8.0h and 12h and 2-4 days every mornings of administration, venous blood sampling 5mL is placed in heparin and resisted
In solidifying centrifuge tube, centrifuged at 3500rpm and 4 DEG C, separated plasma after 5min, in 75 ± 10 DEG C of preservations, Application Example one
The chiral LC-MS/MS high-flux detection methods of Pantoprazole are detected in the human plasma of foundation, obtain typical health volunteer
Average drug-time-concentration curve Fig. 9 and health volunteer's vein drop in the blood sample of drip-feed 80mg Pantoprazole Sodiums
Note average drug-time-concentration curve Figure 10 in the blood sample of 40mg Levpantoprazole Sodiums.
The chiral LC-MS/MS high-flux detection methods of Pantoprazole and existing liquid phase in the human plasma that the present invention is provided
Chromatogram-ultraviolet corona detector and liquid chromatography-tandem mass spectrometry detection technique are compared, and analysis ability is substantially improved, in addition, at present
The detection method of document report, more using liquid-liquid extraction as sample processing method, the operation of this method is more complicated, extracts examination
Agent can not directly detect that this greatly reduces sample process efficiency, and then influences sample, it is necessary to which extracts reagent is concentrated and solution is redissolved
The detection speed of product；Because Pantoprazole is clinical more with intravenous administration approach, and dosage is higher, and its internal exposed amount is big, medicine
Plasma concentration it is higher therefore relatively low to the sensitivity requirement of detection, generally can meet requirement, sample extraction at ng/mL grades
When need not be concentrated, therefore, using precipitation of protein only must onestep extraction process, by the Sample pretreatment time by original
That comes shorten to present 30min or so in more than 1 hours, and sample processing throughput is greatly improved.
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 (8)
1. the chiral LC-MS/MS high-flux detection methods of Pantoprazole in a kind of human plasma, it is characterised in that：Including following step
Suddenly：
S1, plasma sample pre-treatment：Take 50.0 μ L blood plasma, add 500 μ L inner mark solutions as protein precipitant, be vortexed and from
After the heart, supernatant is taken, testing sample is obtained；
S2, LC-MS/MS analysis condition：
I.LC conditions：Lux cellulose-4 posts：250mm × 4.6m, 5 μm；Column temperature：40℃；Sampling volume：2μL；Mobile phase
A：10mM ammonium acetate aqueous solutions；Mobile phase B：Acetonitrile, uses volume ratio for 10：90 A：B phase Gradient elutions, the stream of mobile phase
Speed is 1.3mL/min；
II.MS conditions：Ion gun：Atmosphere pressure chemical ion source APCI；It is atomized electric current：3.0μA；Ion source temperature：550℃；From
Source gas N2：55psi, curtain gas N2：40psi；Scan pattern：Cation multiple-reaction monitoring+MRM；
S3, standard curve drafting：S- (-)-Pantoprazole is weighed with methanol dissolving and constant volume, S- (-)-Pantoprazole is configured to
The stock solution that concentration is about 1.00mg/mL, weighs R- (+)-Pantoprazole with methanol dissolving and constant volume, is configured to R- (+)-dissolve support
The stock solution that azoles concentration is about 1.00mg/mL is drawn, stock solution is drawn and is diluted step by step with people's blank plasma and obtain hybrid standard system
Row sample, detects the titer, and draw corresponding standard curve according to testing result respectively with above-mentioned LC-MS-MS conditions.
2. the chiral LC-MS/MS high-flux detection methods of Pantoprazole, its feature in human plasma according to claim 1
It is：The time being vortexed in the step S1 is 2min, and rotational speed of eddy current is 4500rpm.
3. the chiral LC-MS/MS high-flux detection methods of Pantoprazole, its feature in human plasma according to claim 1
It is：Inner mark solution described in the step S1 is respectively with S- (-)-Pantoprazole-D6 and R- (+)-Pantoprazole-D6 standard
Product are dissolved using methanol and constant volume prepares internal standard stock solution of the internal standard concentration as 0.8mg/mL, and precision draws above-mentioned each internal standard stock solution
In right amount, plus dilution in acetonitrile, it is 50.0ng/mL's to obtain S- (-)-Pantoprazole-D6 and R- (+)-Pantoprazole-D6 concentration
Mix inner mark solution.
4. the chiral LC-MS/MS high-flux detection methods of Pantoprazole, its feature in human plasma according to claim 1
It is：The elution time of chromatographic column is 5.5min in the step S2.
5. the chiral LC-MS/MS high-flux detection methods of Pantoprazole, its feature in human plasma according to claim 1
It is：The step S2 is used and shunted after post, and the mobile phase flowed out after chromatographic column is split into the first mobile phase and the second flowing
Phase, the first mobile phase enters mass spectrum with 0.4mL/min flow velocity, and the second mobile phase enters waste pipe with 0.9mL/min flow velocity
Road.
6. the chiral LC-MS/MS high-flux detection methods of Pantoprazole, its feature in human plasma according to claim 1
It is：It is room temperature that LC conditions in the step S2, which also include automatic sampler temperature,.
7. the chiral LC-MS/MS high-flux detection methods of Pantoprazole, its feature in human plasma according to claim 1
It is：After S- (-)-Pantoprazole and R- (+)-Pantoprazole of cation multiple-reaction monitoring are ionized through APCI in the step S2
The m/z for producing precursor ion is 384.1, and main fragment ion is that m/z 200.1, S- (-)-Pantoprazole-D6 and R- (+)-dissolve
The precursor ion that support draws azoles-D6 to produce is m/z390.2, and main fragment ion is m/z 206.1, and collision energy is 18eV.
8. the chiral LC-MS/MS high-flux detection methods of Pantoprazole, its feature in human plasma according to claim 1
It is：In the step S3 in hybrid standard series of samples blank plasma S- (-)-Pantoprazole and R- (+)-Pantoprazole it is dense
It is 0.0150-15.0 μ g/mL to spend scope.
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