CN110243962A - A kind of method for detecting perphenazine concentration in human plasma of ultra-high sensitive - Google Patents
A kind of method for detecting perphenazine concentration in human plasma of ultra-high sensitive Download PDFInfo
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Abstract
The present invention relates to the methods of perphenazine concentration in a kind of detection human plasma of ultra-high sensitive, comprising the following steps: step 1: plasma sample pre-treatment；Step 2: chromatographic isolation；Step 3: Mass Spectrometer Method.Detection method sensitivity is higher, and the minimum concentration that can be quantified can reach 10.0pg/mL, is existing maximum sensitivity perphenazine detection technique, and more existing report technology improves 5 times.
Description
Technical field
The present invention relates to the methods of perphenazine concentration in a kind of detection human plasma of ultra-high sensitive.
Background technique
Perphenazine is antipsychotics, clinically for treating the symptoms such as mental disease, Nausea and vomiting, neural anxiety.Its
Pharmacological action blocks mesolimbic system and midbrain-cortical pathway dopamine receptor (DA2) with it and blocks on reticular structure
The alpha-2-adrenoceptor of line activating system is related.It includes: 1, quality research that the technology of detection perphenazine, which is mainly used in, at present
Perphenazine content in middle measurement drug；2, measure perphenazine drug concentration in blood sample, be applied to clinical toxicology detection with
And clinical drug pharmacokinetic studies and bioequivalence Journal of Sex Research etc..In quality research, test sample drug concentration or content are higher,
It is of less demanding to detection sensitivity, usually using high performance liquid chromatography (HPLC) detection technique.Perphenazine concentration in blood sample
Detection technique include HPLC method, gas-chromatography (GC) method, radioactive immunoassay and liquid chromatography-tandem mass spectrometry connection
Usage (LC-MS/MS).Since perphenazine clinical dosage is lower and bioavilability is not high, perphenazine concentration is logical in blood sample
It is often lower, it detects perphenazine concentration in blood sample and usually requires that the high sensitivity of detection method.Above-mentioned perphenazine inspection at present
Survey technology sensitivity is poor to be only able to satisfy clinical poisonous substance screening, drug abuse etc., and is unable to satisfy clinical pharmacokinetics or life
The demand of the equivalent Journal of Sex Research of object.Clinical data, population take perphenazine 4mg or so, and drug reaches Cmax (C in blood plasmamax) only
For 200pg/mL or so.According to " pharmaceutical preparation human bioavailability and bioequivalence Journal of Sex Research skill in 2015 editions pharmacopeia annex
Art guideline ", in order to obtain complete blood concentration-time curve, need detection method sensitivity that can measure 3-5 half
Phase or the blood concentration of declining are Cmax1/10-1/20.Therefore, the sensitivity needs for detecting the detection technique of perphenazine in blood sample reach
To the level of 10.0pg/mL.
The sensitivity for paying the GC method that the HPLC hair established for effect 2011 and Cooper in 1979 are established respectively reaches
27.0ng/mL and 5.00ng/mL is only applicable to the application ranges such as poisonous substance screening and clinical monitoring, is not able to satisfy clinical pharmacokinetics
Research Requirements.The perphenazine analysis method that Midha is established in radioactive immunoassay measurement blood plasma is quantitatively limited to
50.0pg/mL but radioactive immunoassay is not strong to small molecule determinand separating capacity, this method is by drug metabolite
Interference, cause measure concentration it is obviously higher.
Slawson established the perphenazine in LC-MS/MS method measurement serum or blood plasma in 2016.100 μ L sample of blood are through albumen
Through Mass Spectrometer Method after precipitation process.The analysis is mainly used for situations such as clinical detection and overdose use, and lower limit of quantitation is
200pg/mL.Zhao Meng established perphenazine and other four kinds of phenothiazines medicines in LC-MS/MS method measurement whole blood sample in 2013
The concentration of object, 1mL whole blood sample detect after albumen precipitation is handled through LC-MS/MS method, and the lower limit of quantitation of method is 50.0pg/
mL.Above-mentioned two detection method is only limited the use of in poisonous substance screening and clinical monitoring, and sensitivity is not able to satisfy clinical pharmacokinetics research
Demand.In addition the analysis method blood sample dosage of Zhao Meng is excessive, and plasma sample collection capacity is usually less than 2mL in pharmacokinetic studies, such as
Fruit sample usage amount is excessive, and the sample that can not carry out conventional reinspection and laws and regulations requirement analyzes test again.In pharmacokinetic studies
The blood sample at nearly 20 time points is usually acquired in a short time, if increasing sample acquisition volume is unfavorable for the strong of subject
Health easily causes ethics problem.Therefore detection consumption sample volume is usually less than 200 μ L in pharmacokinetic studies.
In conclusion subject's medication dose is small since perphenazine tablet Bioequivalence or Pharmacokinetic are studied,
Perphenazine is extremely low in Plasma, must be reached according to the sensitivity of laws and regulations requirement bioanalytical method can measure drug up to peak it is dense
The sensitivity of the plasma sample of the 1/10-1/20 of degree, the prior art is low, is unable to satisfy above-mentioned requirements.In addition the prior art is also deposited
It is excessively high in plasma sample usage amount, cause sample can not repetition measurement the problem of, drug and metabolite cannot be distinguished measurement is caused to be tied
The disadvantages of fruit is higher.
Summary of the invention
In order to solve the above technical problems, the object of the present invention is to provide perphenazine in a kind of detection human plasma of ultra-high sensitive
The method of concentration.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of method for detecting perphenazine concentration in human plasma of ultra-high sensitive, comprising the following steps:
Step 1: plasma sample is added in plasma sample pre-treatment in 1.5mL centrifuge tube, and methanol, whirlpool is added in inner mark solution
Stream oscillation, centrifugation are taken out supernatant and are transferred in 96 orifice plates, using concentration is dried with nitrogen, double solvents are added to redissolve, eddy oscillating, into
Row LC-MS/MS analysis；
Step 2: chromatographic isolation；
Chromatographic condition, chromatographic column: Triart-C18,3.0 μm, 2.0*100mm；Sample volume: 20 μ L；Column temperature: 40 DEG C；Flowing
Phase A phase selects 1mM formic acid aqueous ammonium containing 0.5%, 0.1% formic acid acetonitrile of Mobile phase B Xiang Weihan, under condition of gradient elution,
Gradient elution: 0-0.50min, 30%B phase；2.50min, 60%B phase；2.51min~3.50min, 30%B phase, automatic sampling
Device temperature is 15 DEG C, the flow velocity of mobile phase are as follows: 0.5mL/min；
Step 3: Mass Spectrometer Method；
Mass Spectrometry Conditions: electrospray ionisation source；Voltage is 5500V；Ion source temperature is 550 DEG C；Curtain Gas and CID
Pressure is respectively 30 and 8psi；Scan pattern is+MRM；Monitoring ionic reaction is respectively as follows: perphenazine, 404.2 → m/z of m/z
171.2；408.2 → m/z of perphenazine-d4, m/z 171.2；CE is respectively 32 and 31.5eV；DP is respectively 100V and 81V；It sweeps
Retouching the time is 150ms.
Preferably, step 1 plasma sample pre-treatment takes 100 μ L plasma samples, be separately added into 25.0 μ L internal standard working solutions,
300 μ L methanol are vortexed 2min, are centrifuged 5min with 14000rpm revolving speed, take out 300 μ L of supernatant and are transferred in 96 orifice plates, N2Drying,
120 μ L double solvents acetonitrile-waters are added to redissolve, eddy oscillating 5min takes 20.00 μ L to carry out LC-MS/MS analysis.
According to the above aspect of the present invention, the present invention has at least the following advantages:
1, the present invention handles plasma sample using precipitation of protein, compares and liquid-liquid extraction and solid phase extraction, albumen
The precipitation method have the higher rate of recovery to perphenazine, can utmostly improve the concentration of perphenazine in sample after extracting, and then are promoted
The sensitivity of detection method.
2, detection method sensitivity is higher, can quantitatively minimum concentration can reach 10.0pg/mL, for it is existing most
Highly sensitive perphenazine detection technique, more existing report technology improve 5 times.In addition the amount of samples of analysis method of the present invention is only
100μL.The present invention is suitble to use in perphenazine tablet bioequivalence or Pharmacokinetic research.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the product ion full scan mass spectrogram of perphenazine of the present invention；
Fig. 2 is the product ion full scan mass spectrogram of internal standard perphenazine-d4 of the invention；
Fig. 3 be perphenazine of the invention the hollow white man's blood plasma of human plasma (on), lower limit of quantitation sample (in) and subject
After oral 4mg perphenazine tablet plasma sample (under) MRM chromatogram；
Fig. 4 be perphenazine-d4 of the present invention the hollow white man's blood plasma of human plasma (on), lower limit of quantitation sample (in) and subject
After oral 4mg perphenazine tablet plasma sample (under) MRM chromatogram；
Fig. 5 is that 1 subject of the present invention takes orally pharmaceutical concentration-time curve after perphenazine tablet.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not intended to limit the scope of the invention for illustrating the present invention.
In order to enable those skilled in the art to better understand the solution of the present invention, below in conjunction with attached in the embodiment of the present invention
Figure, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is only this
Invention a part of the embodiment, instead of all the embodiments.Embodiments of the present invention, which are generally described and illustrated herein in the accompanying drawings
Component can arrange and design with a variety of different configurations.Therefore, the implementation of the invention to providing in the accompanying drawings below
The detailed description of example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiment of the present invention, those skilled in the art are obtained all without making creative work
Other embodiments shall fall within the protection scope of the present invention.
Embodiment
A kind of method for detecting perphenazine concentration in human plasma of ultra-high sensitive, comprising the following steps:
Step 1: plasma sample is added in plasma sample pre-treatment in 1.5mL centrifuge tube, and methanol, whirlpool is added in inner mark solution
Stream oscillation, centrifugation are taken out supernatant and are transferred in 96 orifice plates, using concentration is dried with nitrogen, double solvents are added to redissolve, eddy oscillating, into
Row LC-MS/MS analysis；
Step 2: chromatographic isolation；
Chromatographic condition, chromatographic column: Triart-C18,3.0 μm, 2.0*100mm；Sample volume: 20 μ L；Column temperature: 40 DEG C；Flowing
Phase A phase selects 1mM formic acid aqueous ammonium containing 0.5%, 0.1% formic acid acetonitrile of Mobile phase B Xiang Weihan, under condition of gradient elution,
Gradient elution: 0-0.50min, 30%B phase；2.50min, 60%B phase；2.51min~3.50min, 30%B phase, automatic sampling
Device temperature is 15 DEG C, the flow velocity of mobile phase are as follows: 0.5mL/min.
Step 3: Mass Spectrometer Method；
Mass Spectrometry Conditions: electrospray ionisation source；Voltage is 5500V；Ion source temperature is 550 DEG C；Curtain Gas and CID
Pressure is respectively 30 and 8psi；Scan pattern is+MRM；Monitoring ionic reaction is respectively as follows: perphenazine, 404.2 → m/z of m/z
171.2；408.2 → m/z of perphenazine-d4, m/z 171.2；CE is respectively 32 and 31.5eV；DP is respectively 100V and 81V；It sweeps
Retouching the time is 150ms.
In order to improve detection method sensitivity in above-mentioned chromatography, chromatographic condition must utmostly improve determinand when optimizing
Mass signal, and evade Interference Peaks, 3.0 μm of Triart-C18 chromatographic column compared with the chromatographic column that other types are investigated, 2.0*
100mm can get very sharp chromatographic peak profile, improve detection sensitivity, and acid additives can promote perphenazine ionization,
The optimized interference for efficiently separating perphenazine monitoring signals of chromatography gradient elution program, and most sharp chromatographic peak profile is obtained,
Sample volume is optimized for 20 μ L, not influence the maximum sample volume in the case of chromatographic peak profile, makes to enter the maximization of instrument sample amount, into
And promote detection signal.
Embodiment one
Abbreviation explanation
1 material
1.1 instrument
Japanese Shimadzu Corporation LC-30AD ultra performance liquid chromatography system and the triple level four bars series connection of 6500 type of AB Sciex
Mass spectrograph is furnished with the source ESI.
Data processing uses Analyst 1.6.3 software.
Beijing Sai Duolisi Instrument Ltd. CP225D type analysis balance.
Japanese Hitachi company CT15RE type centrifuge.
1.2 standard items and reagent
Perphenazine (purity 98%) and perphenazine-d4 (chemical purity 97.5%, isotopic purity 99.9%) are purchased from plus take
Big TRC company；Methanol and acetonitrile buying are in Sigma Co., USA；Formic acid is purchased in Japanese TCI company；Ammonium formate is purchased in beauty
ROE company, state；Deionized water is prepared by Millipore pure water meter.Mentioned reagent is chromatographically pure.
2 methods
The preparation of 2.1 solution
Standard curve sample preparation
Perphenazine reference substance is weighed, with methanol dissolution and constant volume, concentration is about 1.00mg/mL.Draw above-mentioned perphenazine storage
Standby liquid is appropriate, is diluted step by step with methanol-water (50/50, v/v) and obtains series of concentrations standard solution, perphenazine concentration difference 200,
400,800,1600,3200,4800,10000,12000pg/mL.Above-mentioned working solution, which is diluted, with blank plasma obtains perphenazine
The standard curve sample of concentration difference 10.0,20.0,40.0,80.0,160,240,500,600pg/mL.
Internal standard working solution
Perphenazine-d4 reference substance is weighed, with methanol dissolution and constant volume, the internal standard stock solution that concentration is about 0.100mg/mL.
Precision absorption internal standard stock solution is appropriate, with methanol-water (50/50, v/v) stepwise dilution, obtains the internal standard that concentration is 5.00ng/mL
Working solution.
The processing of 2.2 plasma samples
100 μ L blood plasma are taken, 25.0 μ L internal standard working solutions, 300 μ L methanol are separately added into, are vortexed 2min, are turned with 14000rpm
Speed centrifugation 5min, takes out 300 μ L of supernatant and is transferred in 96 orifice plates, N2Drying, adds 120 μ L double solvents acetonitrile-waters (30:70, v/v)
It redissolves, eddy oscillating 5min.20.00 μ L are taken to carry out LC-MS/MS analysis.
2.3 chromatographies and Mass Spectrometry Conditions
Chromatographic condition
Gradient program elution:
0-0.50min, 30%B phase；
2.50min, 60%B phase；
2.51-3.50min, 30%B phase；
Mass Spectrometry Conditions
As depicted in figs. 1 and 2, product ion mass spectrogram and the dissociation pathways of supposition.Electrospray ionisation source
(TurboIonSpray)；Voltage is 5500V；Ion source temperature is 550 DEG C；Curtain Gas and CID pressure is respectively 30 Hes
8psi；Scan pattern is+MRM；Monitoring ionic reaction is respectively as follows: perphenazine, 404.2 → m/z of m/z 171.2；Perphenazine-d4,
m/z 408.2→m/z 171.2；CE is respectively 32 and 31.5eV.DP is respectively 100V and 81V；Sweep time is 150ms.
2.4 methodology validation
Selectivity
Plasma sample after taking blank human plasma, LLOQ sample and subject to take 4mg perphenazine respectively handles analysis, obtains
Corresponding chromatogram (Fig. 3 and Fig. 4).The retention time of perphenazine and internal standard perphenazine-d4 are 2.2min, in blank plasma spectrogram
Noiseless peak at the retention time.
Standard curve
Using perphenazine theoretical concentration as abscissa (x), perphenazine and perphenazine-d4 peak area ratio are ordinate (y), are carried out
Linear regression calculates (weight factor W=1/x2), the classic regression equation of perphenazine is y=0.00789x-1.54 × 10-4, r=
0.9976, perphenazine is obvious linear within the scope of 10.0~600 product pg/mL.
Accuracy, precision and stability
With people's blank plasma, perphenazine stock solution is diluted, being configured to LQC, MQC, HQC, (perphenazine concentration is 30.0,100
And 450pg/mL) and LLOQ (perphenazine concentration is 10.0pg/mL), method validation four concentration Quality Control samples of each analysis batch measurement
This each six sample.In LLOQ days, day to day precision can be received with relative standard deviation (RSD) < 20% side, accuracy (RE)-
It can receive between 20%~20%.Each ingredient of QC sample of remaining each concentration level in a few days, day to day precision (RSD) < 15%
It can receive, accuracy (RE) can receive between -15%~15%.
It is shown in Table 1, the results showed that the preci-sion and accuracy that this method measures perphenazine is acceptable, and the LLOQ of perphenazine is
10.0pg/mL。
Table 1:
Measure the preci-sion and accuracy of perphenazine in human plasma
Take LQC and HQC sample, investigate its be placed at room temperature for 15 DEG C of placement 126h after 25h, processing, multigelation 6 times and-
The stability of 80 DEG C of placements 60 days.
It is shown in Table 2, the results showed that, perphenazine is stable under the above conditions, and RE is between -7.4%~-1.2%.
Table 2:
Perphenazine plasma stability
The rate of recovery and matrix effect
Analyze QC six samples of sample of basic, normal, high concentration.100 μ L of blank plasma is separately taken simultaneously, at plasma sample
Reason, is added perphenazine contrast solution and internal standard working solution into supernatant, vortex mixing, sample introduction measurement, in 2 kinds of samples put forth energy be
Quiet average peak area is than being the rate of recovery.The result shows that three concentration level extraction recoveries are all larger than 60.0%.The internal standard rate of recovery
It is 57.0%.
Separate sources people blank plasma (n=6) is taken, (being added without internal standard) is handled by plasma sample, is added into supernatant
Contrast solution and mixing internal standard working solution, sample introduction measurement.100 μ L deionized waters separately are taken, are handled according to the above method, sample introduction measurement.
Matrix factors are calculated with two kinds of sample perphenazine peak area ratios.Perphenazine matrix factors under LQC and HQC concentration are respectively
98.0% and 99.1%, RSD is respectively less than 4.8%, shows under this experimental condition, and matrix effect can be ignored and measure to perphenazine
Influence.
Method validation is summarized
The method of the present invention passes through methodology validation, method sensitivity is high, selectivity is good, accurate, accurate, stability is good,
Linear good, indices meet Chinese Pharmacopoeia annex in 2015, and " 9012 biological sample quantitative analysis method verification guides are former
Then " require.The lower limit of quantitation of analysis method is up to 10.0pg/mL.
Pharmacokinetic studies
By the concentration of perphenazine in present invention measurement blood plasma, studied for perphenazine tablet Pharmacokinetic.Clinical research warp
Hospital Ethical Committee's approval is crossed, subject is apprised of empirical risk before the test, voluntarily signs informed consent form.Health by
Examination person gives 4mg perphenazine tablet.Different time points, venous blood sampling are set in anticoagulant centrifuge tube after (0h), administration before administration, from
Separated plasma is measured after the heart.As shown in figure 5, wherein subject's pharmaceutical concentration-time curve.The C of perphenazinemaxFor
140pg/mL, sensitivity of the invention meet Chinese Pharmacopoeia " pharmaceutical preparation human bioavailability and bioequivalence in 2015
Investigative technique guideline " it measures to 1/10~1/20CmaxRequirement.The present invention can be used for the research of perphenazine tablet Pharmacokinetic
And bioequivalence Journal of Sex Research.
The present invention has at least the following advantages:
1, the present invention handles plasma sample using precipitation of protein, compares and liquid-liquid extraction and solid phase extraction, albumen
The precipitation method have the higher rate of recovery to perphenazine, can utmostly improve the concentration of perphenazine in sample after extracting, and then are promoted
The sensitivity of detection method.
2, detection method sensitivity is higher, can quantitatively minimum concentration can reach 10.0pg/mL, for it is existing most
Highly sensitive perphenazine detection technique, more existing report technology improve 5 times.In addition the amount of samples of analysis method of the present invention is only
100μL.The present invention is suitble to use in perphenazine tablet bioequivalence or Pharmacokinetic research.
The above is only a preferred embodiment of the present invention, it is not intended to restrict 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 several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (2)
1. the method for perphenazine concentration in a kind of detection human plasma of ultra-high sensitive, which comprises the following steps:
Step 1: plasma sample is added in plasma sample pre-treatment in 1.5mL centrifuge tube, and methanol, vortex vibration is added in inner mark solution
It swings, is centrifuged, take out supernatant and be transferred in 96 orifice plates, using concentration is dried with nitrogen, double solvents is added to redissolve, eddy oscillating carries out LC-
MS/MS analysis；
Step 2: chromatographic isolation；
Chromatographic condition, chromatographic column: Triart-C18,3.0 μm, 2.0*100mm；Sample volume: 20 μ L；Column temperature: 40 DEG C；Mobile phase A
Mutually select 1mM formic acid aqueous ammonium contain 0.5%, 0.1% formic acid acetonitrile of Mobile phase B Xiang Weihan, under condition of gradient elution, gradient
Elution: 0-0.50min, 30%B phase；2.50min, 60%B phase；2.51min~3.50min, 30%B phase, autosampler temperature
Degree is 15 DEG C, the flow velocity of mobile phase are as follows: 0.5mL/min；
Step 3: Mass Spectrometer Method；
Mass Spectrometry Conditions: electrospray ionisation source；Voltage is 5500V；Ion source temperature is 550 DEG C；Curtain Gas and CID pressure
Respectively 30 and 8psi；Scan pattern is+MRM；Monitoring ionic reaction is respectively as follows: perphenazine, 404.2 → m/z of m/z 171.2；
408.2 → m/z of perphenazine-d4, m/z 171.2；CE is respectively 32 and 31.5eV；DP is respectively 100V and 81V；Sweep time
For 150ms.
2. the method for perphenazine concentration, feature exist in a kind of detection human plasma of ultra-high sensitive according to claim 1
In: step 1 plasma sample pre-treatment takes 100 μ L plasma samples, is separately added into 25.0 μ L internal standard working solutions, 300 μ L methanol, whirlpool
2min is flowed, 5min is centrifuged with 14000rpm revolving speed, 300 μ L of supernatant is taken out and is transferred in 96 orifice plates, N2Drying adds 120 μ L to redissolve
Agent acetonitrile-water redissolves, eddy oscillating 5min, and 20.00 μ L is taken to carry out LC-MS/MS analysis.
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| CN112834679A (en) * | 2020-12-31 | 2021-05-25 | 苏州海科医药技术有限公司 | Analysis method for clinically researching concentration of quetiapine in plasma sample |
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