CN103529160B - UPLC-MS/MS method measures human plasma Chinese and Western not for the concentration of Buddhist nun - Google Patents
UPLC-MS/MS method measures human plasma Chinese and Western not for the concentration of Buddhist nun Download PDFInfo
- Publication number
- CN103529160B CN103529160B CN201310469995.1A CN201310469995A CN103529160B CN 103529160 B CN103529160 B CN 103529160B CN 201310469995 A CN201310469995 A CN 201310469995A CN 103529160 B CN103529160 B CN 103529160B
- Authority
- CN
- China
- Prior art keywords
- buddhist nun
- concentration
- western
- west
- chinese
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to Ultra Performance Liquid Chromatography tandem mass spectrometry (UPLC-MS/MS) and measure human plasma Chinese and Western not for the concentration of Buddhist nun.The invention provides and detect blood plasma Chinese and Western not for the method for Buddhist nun's concentration, wherein by UPLC-MS/MS method, blood plasma Chinese and Western is not analyzed for the concentration of Buddhist nun.Method of the present invention preferably adopts the Methanol Protein precipitation method to carry out Sample pretreatment, is interior mark with erlotinid hydrochloride, and adopt ZORBAX SB-C8 chromatographic column as analytical column, electro-spray ionization (ESI+) tandem mass spectrum detects.The extraction recovery of the sample of the inventive method is greater than 90%, has no matrix effect.The specificity of the inventive method, multigelation stability, Long-term Cryopreservation stability and dilution effect are all verified, and have been successfully applied to and give hydrochloric acid west and detect for the blood concentration of Buddhist nun's sheet treatment patient.
Description
Technical field
The present invention relates to blood plasma Chinese and Western not for the method that the concentration of Buddhist nun is analyzed.In the method for the invention, adopt Ultra Performance Liquid Chromatography tandem mass spectrum (UPLC-MS/MS) not analyze for the concentration of Buddhist nun blood plasma Chinese and Western, achieve blood plasma Chinese and Western not for quick, sensitive, the special analysis of Buddhist nun's concentration.
Background technology
Lung cancer is the main cause of current global tumour associated death, and nearly 1.4 million peoples die from lung cancer [1] every year.Non-small cell lung cancer (non-small cell lung cancer, NSCLC) accounts for 85% [2] of lung cancer toatl proportion, and wherein gland cancer is main histological typing.Most NSCLC is still based on chemotherapy, and effective [3].
EGF-R ELISA (Epidermal growth factor receptor, EGFR), a kind of tyrosine kinase receptor (receptor tyrosine kinase, RTK), play a significant role in the Cell Differentiation of NSCLC, propagation, Angiogenesis and apoptosis.EGFR sudden change is in 15% white people [4], 39.5% (593/1503) Korean [5], express in 26% (15/58) Japanese [6] and 47.6% (89/187) Chinese, prompting EGFR mutation rate is higher than west crowd in asian population.The patient carrying EGFR sudden change benefits larger [4,7] in EGFR TKI class medicine is as Gefitinib and Tarceva treatment, and bad reaction is lighter.The long-term Progression free survival (prolonged progression free survival, PFS) that EGFR TKI treats and disease control rate are all higher than traditional line platinum class treatment [8,9].Along with people are to the understanding of EGFR at promotion angiogenesis function, develop multiple EGFR TKIs in recent years.
At present, one has three Small molecular TKI obtains the approval of FDA for oncotherapy, and comprise Tarceva, Gefitinib and Lapatinib [10], wherein Tarceva and Gefitinib are used for the treatment of NSCLC.Hydrochloric acid Conmana is a kind of Small molecular TKI [11] being reached medicine company independent research by Zhejiang shellfish, obtains the treatment that the approval of Chinese food Drug Administration is used for NSCLC in 2011.The clinical testing of current Conmana is carried out [12-14].
Hydrochloric acid west is not for Buddhist nun, N-(the chloro-4-fluorophenyl of 3-)-7-methoxyl-6-[2-(5,8-dioxa-10-azepine two spiral shell [2.0.4.3] undecane) ethoxy] quinazoline-4-amine hydrochlorate (Fig. 1), a kind of new small molecule tyrosine kinase inhibitors (tyrosine kinase inhibitor, TKI), this molecule is synthesized by AdvenchenLaboratories LLC (Northridge, USA).Preclinical study shows that west is not equivalent to or is better than positive control medicine erlotinid hydrochloride for the effect of Buddhist nun in suppression HEP system A431, and significantly can suppress A431 tumor model (not delivering result).Rat and beasle dog not for Buddhist nun's well-tolerated, are respectively 20mg/kg and 6mg/kg without visible adverse reaction Dosages to west, have no sudden change and are formed and distortion toxicity (not delivering result).In a nearest tolerance single dose administration clinical research, Chinese healthy volunteers also shows good tolerance for Buddhist nun to hydrochloric acid west, and the highest safe administration dosage reaches 500mg (not delivering data).Based on above preclinical data and clinical research, hydrochloric acid west does not launch (ClinicalTrials.gov identifier:NCT01772732) in January, 2013 at Cancer Hospital of Chinese Academy of Medical Sciences for successive administration security in Buddhist nun's sheet late NSCLC patient, tolerance, pharmacokinetics Ib clinical trial phase.
About the detection method of the concentration of generation TKIs in human plasma, currently reported [15-21], also has methodology report [22-26] simultaneously detecting multiple TKI class medicine in recent years.The result of study of prior art shows, when carrying out pre-treatment to TKI medicine as Conmana with precipitation of protein, there is very large matrix effect, and the recovery low (26.5%), even if adopt liquid-liquid extraction (liquid-liquidextraction, LLE) method, also needs repeatedly to dilute and could eliminate matrix effect.[27]。The concentration of Tarceva in the detection method analysed for plasma sample of document [29,30], its lower limit of quantitation is respectively 80ng/mL [29] and 50ng/mL [30], and the detection time of single sample is respectively 15min [29] and 12min [30].Document [20] adopts the mode of hexane and ethyl acetate liquid-liquid extraction to carry out pre-treatment to Tarceva plasma sample, and its recovery of extraction is only 79-88%, and lower limit of quantitation is 10ng/mL, and the detection time of single sample is 6min.Also document is had to adopt LLE method to detect Sutent [19] in blood plasma, the concentration of Tarceva [20] and Conmana [27].The pretreatment mode complex operation of liquid-liquid extraction is consuming time, can not meet the detection of large sample in Clinical pharmacokinetics.96 orifice plate Solid-Phase Extraction 9 kinds TKI medicines also have report recently, but the method is very expensive and program is complicated [25].At present, pertinent literature is not also had to report biological specimen Chinese and Western not for the methodology of Buddhist nun's Concentration Testing.
Because sample size when Clinical pharmacokinetics detects is usually very large, therefore need a kind of quick, sensitive, special, quantitative analysis method that sample requirement is few.
List of references:
[1]A.Jemal，M.M.Center，C.DeSantis，E.M.Ward，Cancer EpidemiolBiomarkers Prev19(2010)1893.
[2]S.S.Ramalingam，T.K.Owonikoko，F.R.Khuri，CA Cancer J Clin61(2011)91.
[3]O.S.Breathnach，B.Freidlin，B.Conley，M.R.Green，D.H.Johnson，D.R.Gandara，M.O′Connell，F.A.Shepherd，B.E.Johnson，J Clin Oncol19(2001)1734.
[4]V.L.Keedy，S.Temin，M.R.Somerfield，M.B.Beasley，D.H.Johnson，L.M.McShane，D.T.Milton，J.R.Strawn，H.A.Wakelee，G.Giaccone，J Clin Oncol29(2011)2121.
[5]Y.L.Choi，J.M.Sun，J.Cho，S.Rampal，J.Han，B.Parasuraman，E.Guallar，G.Lee，J.Lee，Y.M.Shim，PLoS One8(2013)e56011.
[6]J.G.Paez，P.A.Janne，J.C.Lee，S.Tracy，H.Greulich，S.Gabriel，P.Herman，F.J.Kaye，N.Lindeman，T.J.Boggon，K.Naoki，H.Sasaki，Y.Fujii，M.J.Eck，W.R.Sellers，B.E Johnson，M.Meyerson，Science304(2004)1497.
[7]Ont Health Technol Assess Ser10(2010)1.
[8]X.Chen，Y.Liu，O.D.Roe，Y.Qian，R.Guo，L.Zhu，Y.Yin，Y.Shu，PLoS One8(2013)e59314.
[9]E.Juhasz，J.H.Kim，G.Klingelschmitt，S.Walzer，Eur J Cancer49(2013)1205.
[10]T.M.Brand，M.Iida，D.L.Wheeler，Cancer Biol Ther11(2011)777.
[11]F.Ciardiello，G.Tortora，Clin Cancer Res7(2001)2958.
[12]F.Tan，X.Shen，D.Wang，G.Xie，X.Zhang，L.Ding，Y.Hu，W.He，Y.Wang，Y.Wang，Lung Cancer76(2012)177.
[13]H.P.Wang，L.Zhang，Y.X.Wang，F.L.Tan，Y.Xia，G.J.Ren，P.Hu，J.Jiang，M.Z.Wang，Y.Xiao，Chin Med J(Engl)124(2011)1933.
[14]Q.Zhao，J.Shentu，N.Xu，J.Zhou，G.Yang，Y.Yao，F.Tan，D.Liu，Y.Wang，J.Zhou，Lung Cancer73(2011)195.
[15]J.Qian，Y.Wang，J.Cao，J.Li，J Pharm Biomed Anal80(2013)173.
[16]R.W.Sparidans，D.Iusuf，A.H.Schinkel，J.H.Schellens，J.H.Beijnen，J Chromatogr B Analyt Technol Biomed Life Sci877(2009)4090.
[17]Y.Hsieh，G.Galviz，Q.Zhou，C.Duncan，Rapid Commun MassSpectrom23(2009)1364.
[18]Z.Iqbal，M.Elliott，D.G.Watson，T.Holyoake，H.Jorgensen，Pak JPharm Sci24(2011)285.
[19]P.Minkin，M.Zhao，Z.Chen，J.Ouwerkerk，H.Gelderblom，S.D.Baker，J Chromatogr B Analyt Technol Biomed Life Sci874(2008)84.
[20]A.R.Masters，C.J.Sweeney，D.R.Jones，J Chromatogr B AnalytTechnol Biomed Life Sci848(2007)379.
[21]D.Luethi，S.Durmus，A.H.Schinkel，J.H.Schellens，J.H.Beijnen，R.W.Sparidans，J Chromatogr B Analyt Technol Biomed Life Sci934C(2013)22.
[22]L.Gotze，A.Hegele，S.K.Metzelder，H.Renz，W.A.Nockher，ClinChim Acta413(2012)143.
[23]I.Andriamanana，I.Gana，B.Duretz，A.Hulin，J Chromatogr BAnalyt Technol Biomed Life Sci926(2013)83.
[24]A.Davies，A.K.Hayes，K.Knight，S.J.Watmough，M.Pirmohamed，R.E.Clark，Leuk Res34(2010)702.
[25]S.Bouchet，E.Chauzit，D.Ducint，N.Castaing，M.Canal-Raffin，N.Moore，K.Titier，M.Molimard，Clin Chim Acta412(2011)1060.
[26]L.Couchman，M.Birch，R.Ireland，A.Corrigan，S.Wickramasinghe，D.Josephs，J.Spicer，R.J.Flanagan，Anal Bioanal Chem403(2012)1685.
[27]D.Liu，J.Jiang，P.Hu，F.Tan，Y.Wang，J Chromatogr B AnalytTechnol Biomed Life Sci877(2009)3781.
[28]J.Qian，Y.Wang，J.Cao，J.Li，J Pharm Biomed Anal80(2013)173.
[29]L.Faivre，C.Gomo，O.Mir，F.Taieb，A.Schoemann-Thomas，S.Ropert，M.Vidal，D.Dusser，A.Dauphin，F.Goldwasser，B.Blanchet，JChromatogr B Analyt Technol Biomed Life Sci879(2011)2345.
[30]Y.Zhen，A.Thomas-Schoemann，L.Sakji，P.Boudou-Rouquette，N.Dupin，L.Mortier，M.Vidal，F.Goldwasser，B.Blanchet，J Chromatogr BAnalyt Technol Biomed Life Sci928(2013)93.
Summary of the invention
The present invention establishes quick, sensitive Ultra Performance Liquid Chromatography tandem mass spectrum (ultra highperformance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS) technology for detection human plasma Chinese and Western not for the concentration of Buddhist nun.
With prior art instruct contrary, result of study of the present invention display can adopt precipitation of protein to carry out Sample pretreatment in the conditions of the invention, and obtains surprising high-recovery, thus can avoid loaded down with trivial details Liquid-liquid Extraction Processes consuming time.In the present invention, albumen precipitation process can be carried out by such as methyl alcohol or acetonitrile.Preferably, methyl alcohol can be adopted in the conditions of the invention to carry out albumen precipitation.Result display Methanol Protein of the present invention precipitates the extraction recovery that can obtain higher than acetonitrile, and extraction ratio up to 90%, and can avoid matrix effect.Consider that existing bibliographical information precipitation of protein process similar TKI medicine exists very large matrix effect and the unacceptable low recovery (26.5%), and existing document adopts extraction to the recovery of similar medicine also only 79-88%, present invention obtains the excellent effect be all beyond one's expectations.And, the present invention adopts albumen precipitation easy, quick as the method for pre-treatment, plasma sample amount used can be 30 μ l-100 μ l, such as 35 μ l-90 μ l, 40 μ l-80 μ l, 45 μ l-70 μ l, 50 μ l-60 μ l, therefore this be specially adapted to the object of plasma sample measure analysis less in clinical testing significantly lower than the sample size of bibliographical information.Although precipitation of protein is preferred, method of the present invention also can adopt liquid-liquid extraction method to carry out pre-treatment.The present inventor is to liquid-liquid extraction as t-butyl methyl ether process is studied, and result display can obtain and precipitate more consistent result with Methanol Protein.
In order to obtain better peak type and chromatographic resolution, the present invention have extensively studied the wider conventional C of different pH value range
18reverse-phase chromatographic column: ZORBAX Eclipse Plus C
18(50mm × 4.6mm and50mm × 2.1mm, 3.5 μm, Agilent), Acquity UPLC BEH C
18(50mm × 2.1mm, 1.7 μm, Waters) and Acquity BEH Shield RP18 (100mm × 2.1mm, 1.7 μm, Waters), but there is serious hangover in these chromatographic columns, and residual serious.In the conditions of the invention, find chromatographic column ZORBAX SB-C8 (100mm × 2.1mm, 3.5 μm, Agilent) surprisingly, under constant gradient condition, west is not symmetrical for Buddhist nun peak type, is suitable for the present invention.Therefore, in the conditions of the invention, ZORBAX SB-C8 or kin chromatographic column are preferred, and such chromatographic column is known in the art.The result that the present invention studies further shows preferably to adopt formic acid-ammonium formate and methyl alcohol to be mobile phase.More preferably, when methyl alcohol and formic acid-ammonium formate solution ratio are 80:20 (v:v), the peak type be not separated for Buddhist nun west is best, and its Chinese and Western does not replace the appearance time of Buddhist nun to be 1.8min, and the appearance time of Tarceva is 1.9min.And methyl alcohol has higher signal to noise ratio (S/N ratio) (signal-to-noise, S/N) compared with acetonitrile.
Conmana can be comprised in the method for the invention or Tarceva is interior mark material standed for.This result of study shows preferably using Tarceva as interior mark, and it is not noiseless for Buddhist nun to west.
West of determining of the present invention does not replace the range of linearity of Buddhist nun to be 1-1000ng/mL, and result shows in this scope internal linear better.R
2all be greater than 0.995.
Therefore, the invention provides and detect blood plasma Chinese and Western not for the method for Buddhist nun's concentration, wherein by Ultra Performance Liquid Chromatography tandem mass spectrum, blood plasma Chinese and Western is not analyzed for the concentration of Buddhist nun.
In a preferred embodiment of the invention, before described Ultra Performance Liquid Chromatography tandem mass spectrum does not replace the concentration of Buddhist nun to analyze to blood plasma Chinese and Western, pre-treatment can be carried out by precipitation of protein to plasma sample.
In a preferred embodiment of the invention, precipitation of protein can be undertaken by methyl alcohol or acetonitrile, carries out preferably by methyl alcohol.
In a preferred embodiment of the invention, before Ultra Performance Liquid Chromatography tandem mass spectrum does not replace the concentration of Buddhist nun to analyze to blood plasma Chinese and Western, pre-treatment can be carried out by liquid-liquid extraction method to plasma sample.
In a preferred embodiment of the invention, liquid-liquid extraction method can be undertaken by t-butyl methyl ether.Method of the present invention can adopt lower sample volume to carry out.In the conditions of the invention, find that lower sample can meet analysis needs completely surprisingly.The verified remarkable plasma sample volume lower than bibliographical information in the conditions of the invention of method of the present invention is as 30 μ l-100 μ l, such as 35 μ l-90 μ l, 40 μ l-80 μ l, 45 μ l-70 μ l, namely the volume of 50 μ l-60 μ l can meet analysis needs, is therefore particularly suitable for the object of plasma sample measure analysis less in clinical testing.
In a preferred embodiment of the invention, Ultra Performance Liquid Chromatography tandem mass spectrum can adopt ZORBAX SB-C8 chromatographic column or kin chromatographic column to carry out, and such chromatographic column is known in the art.
In a preferred embodiment of the invention, Ultra Performance Liquid Chromatography tandem mass spectrum adopts A: formic acid water-ammonium formate, B: methyl alcohol is mobile phase.In especially preferred mobile phase, the volume ratio of A:B is 20/80.
In a preferred embodiment of the invention, Ultra Performance Liquid Chromatography tandem mass spectrum comprises interior mark, preferred erlotinid hydrochloride.
The present invention establishes quick, sensitive UPLC-MS/MS technology for detection mammal and comprises rat, mouse, human plasma Chinese and Western not for the concentration of Buddhist nun.In a preferred embodiment of the invention, the Methanol Protein precipitation method can be adopted to carry out Sample pretreatment, be interior mark with erlotinid hydrochloride.In a preferred embodiment of the invention, ZORBAX SB-C8 chromatographic column (2.1mm × 100mm, 3.5 μm) or kin chromatographic column can be adopted as analytical column, electro-spray ionization (ESI+) tandem mass spectrum detects.Preferred mobile phase is formic acid-ammonium formate, methyl alcohol (20/80, v/v), and flow velocity is 0.2mL/min.
The analysis time that The inventive method achieves single sample is only 4min, and the extraction recovery of sample is greater than 90%, and has no obvious matrix effect.West is not good in 1 ~ 1000ng/mL scope internal linear relation for Buddhist nun; In a few days, day to day precision (RSD) is all less than 10.4%.
The specificity of the inventive method, multigelation stability, Long-term Cryopreservation stability and dilution effect are all verified.Method of the present invention has been successfully applied to and has given hydrochloric acid west not for the blood concentration detection of Buddhist nun's sheet treatment patient.
Accompanying drawing explanation
Fig. 1: molecular structure and the second order ms figure of Buddhist nun (left side) and interior mark (right side) are not replaced in west.
Fig. 2: west is not for Buddhist nun (left side) and interior mark Tarceva (right side) characteristic chromatogram: two blank (Double-blank), blank (Blank sample), 0.5h plasma sample (concentration is 11ng/mL) after light basis weight lower limit (LLOQ) and the administration of experimenter's first day.
Fig. 3: west is not for Buddhist nun's typical curve.
Fig. 4: experimenter west for Buddhist nun's first day single-dose (100mg), 8-10 days and the 15th day multiple dosing (100mg/ days) blood concentration-time curve.
Embodiment
1. method and result
1.1. medicine and reagent
West is not provided (purity 99.8%) by Jiangsu first sign pharmaceutical Co. Ltd for Buddhist nun's standard items, and interior mark Tarceva standard items are provided (purity 99.3%) by Jiangsu first sign pharmaceutical Co. Ltd.Chromatographic Pure Methanol is purchased from Fisher company, and formic acid and ammonium formate available from Sigma, experimental water is ultrapure water.
1.2. equipment and analysis condition
1.2.1 chromatographic condition:
Mobile phase: A:0.1% formic acid water, 10mM ammonium formate B: methyl alcohol
Flow velocity: 0.2mL/min
Sample size: 2 μ L
Column temperature: 25 DEG C
Temperature in injector: 5 DEG C
Eluent gradient is arranged:
1.2.2 Mass Spectrometry Conditions:
Electro-spray ionization source (ESI), MRM multiion reaction monitoring, positive ion mode, spray voltage 5500v, temperature 450 DEG C, GAS1 flow velocity is 25mL/min, GAS2 flow velocity is 30mL/min, CUR is 15mL/min, DP, EP and CXP value is respectively 135V, 7V, 35V.Under ESI Ionization mode, west does not mainly generate [M+H]+quasi-molecular ion peak for Buddhist nun, and be m/z501.2, main fragmention is m/z182.2, CE is 35 (Fig. 1).[M+H]+quasi-molecular ion peak that erlotinid hydrochloride mainly generates is m/z394.1, and main fragmention is m/z278.1, CE is 35 (Fig. 1).Fig. 1 shows molecular structure and the second order ms figure that Buddhist nun (left side) and interior mark (right side) are not replaced in west.
1.3. standard solution and the preparation of interior mark working fluid
Determination of plasma west is not for the preparation of Buddhist nun's standard solution: get west and replace Buddhist nun's reference substance not appropriate, accurately weighed, take methyl alcohol as dissolution with solvents, quantitatively be mixed with the standard solution storing solution that west is not 1mg/mL for Buddhist nun's concentration, quantitatively be diluted to 10 with methyl alcohol again, 30,100,300,1000,3000, the typical curve working solution of 10000ng/mL, and 20,400, the Quality Control solution of 8000ng/mL.
The preparation of erlotinid hydrochloride (interior mark) solution: get erlotinid hydrochloride reference substance appropriate, accurately weighed, take methyl alcohol as dissolution with solvents, quantitatively be mixed with the standard solution storing solution that erlotinid hydrochloride concentration is 1mg/mL, be more quantitatively diluted to the standard working solution of 50ng/mL with methyl alcohol.
Above solution is all placed in-20 DEG C and saves backup.
1.4. plasma sample process
Precision measures plasma sample 50 μ L, puts in the Ep pipe of 1.5mL, and precision adds interior mark standard working solution 20 μ L, vortex mixing 10s, adds methyl alcohol 400 μ L, vortex 10s, in the centrifugal 10min of 12000rpm, draw 50 μ L supernatants in sample injection bottle, get 2 μ L and carry out LC-MS/MS analysis.
1.5. method validation
With reference to FDA detection of biological samples governing principle [28], Method validation comprises the aspects such as the sensitivity of method, specificity, typical curve and the range of linearity, precision and accuracy, sample stability, extraction recovery, matrix effect, dilution effect, methodology Quality Control.
1.5.1. specificity and sensitivity
Get the blank plasma 50 μ L of 6 different experimenters respectively, except not adding interior mark, operate by under " 1.4 plasma sample process " item in accordance with the law, carry out LC-MS/MS analysis, obtain blank plasma samples chromatogram (Fig. 2).
Get the blank plasma 45 μ L of 6 different experimenters respectively, add the west of 10ng/mL not for Buddhist nun's standard solution 5 μ L, operate by " process of plasma sample " in accordance with the law, carry out LC-MS/MS analysis, obtain blank plasma samples+west not for Buddhist nun+interior mark reference substance chromatogram (Fig. 2).
Fig. 2 shows west not for Buddhist nun (left side) and interior mark Tarceva (right side) characteristic chromatogram: two blank (Double-blank), blank (Blank sample), 0.5h plasma sample (concentration is 11ng/mL) after light basis weight lower limit (LLOQ) and the administration of experimenter's first day.
1.5.2. preci-sion and accuracy
Preparation blood plasma west does not replace Buddhist nun's concentration to be 2ng/mL, 40ng/mL, the sample of basic, normal, high 3 concentration of 800ng/mL, each concentration prepares 6 parts, as a collection of preci-sion and accuracy sample, operate by under " process of 2.4 plasma samples " item, within one day, prepare a collection of preci-sion and accuracy sample, different 3 days, measure three batches of preci-sion and accuracy samples, record chromatogram, calculates the ratio f of medicine peak area As and interior mark peak area Ai, the typical curve substituting into the same day tries to achieve measured concentration, calculates batch interior and betweenrun precision and accuracy.Preci-sion and accuracy represents with RSD% and RE% respectively, and its absolute value is less than 15% for qualified, and low Quality Control point is less than 20% for qualified.The results are shown in Table 1.
Table 1. west is not for Buddhist nun's preci-sion and accuracy
1.5.3. typical curve and light basis weight lower limit
Get blank plasma 45 μ L, add west respectively not for Buddhist nun's standard solution 5 μ L, be mixed be equivalent to blood plasma Chinese and Western not for Buddhist nun's concentration be 1ng/mL, 3ng/mL, 10ng/mL, 30ng/mL, 100ng/mL, the plasma sample of 300ng/mL and 1000ng/mL, by same operation from " precision adds inner mark solution 20 μ L " under " process of 1.4 plasma samples " item, record chromatogram; To the west of be not horizontal ordinate for Buddhist nun's concentration and interior mark concentration proportion (X), west does not replace the peak area ratio f of Buddhist nun and internal standard compound to be ordinate, carry out regressing calculation by weighting (W=1/X2) least square method, the linear regression equation of trying to achieve is typical curve.Result (Fig. 3) shows that west is not good in 1 ~ 1000ng/mL concentration range internal linear relation for Buddhist nun.
With legal system for six groups of lower limit of quantitation samples, try to achieve measured concentration according to typical curve on the same day, prepare one batch every day, detect for three days on end.
Fig. 3 shows west not for Buddhist nun's typical curve.
1.5.4. residual effect
Residual effect is verified as with dummy sample introduction after high concentration pattern detection, analyzes west does not mark peak time place instrument response for Buddhist nun and Nei.In high concentration west not for sample introduction blank plasma samples after Buddhist nun, not going out Feng Chu for Buddhist nun in west has peak area to be equivalent to the peak appearance of 30-40%LLOQs, show to there is certain remaining, then when entering blank sample, this peak-to-peak area is 15% of LLOQ.Therefore need into blank sample to reduce residual effect after high concentration sample sample introduction.
1.5.5. extraction recovery and matrix effect
Group 1: accurate absorption blank plasma 45 μ L, puts in the Ep pipe of 1.5ml, adds methyl alcohol 400 μ L, vortex 1min, in the centrifugal 10min of 12000rpm, draw whole supernatant in another Ep pipe, precision adds concentration and is respectively 20ng/mL, 400ng/mL, the west of 8000ng/mL is not for Buddhist nun's standard working solution 5 μ L, and precision adds inner mark solution 20 μ L, after vortex mixing, in the centrifugal 5min of 12000rpm, get 2 μ L and carry out LC-MS/MS analysis.Each concentration respectively prepares 6 parts, record chromatogram, records each concentration west not for Buddhist nun and Nei target peak area.
Group 2: be mixed with blood plasma west by " plasma sample typical curve " assay method and do not replace Buddhist nun's concentration to be 2ng/mL, 40ng/mL, each 6 parts of the plasma sample of basic, normal, high three kinds of variable concentrations of 800ng/mL, operate equally by above-mentioned " 2.4 plasma sample disposal route ", carry out LC-MS/MS analysis, record chromatogram, records each concentration west not for Buddhist nun and Nei target peak area.
Group 3: precision adds the standard solution 5 μ L that concentration is 20ng/mL, 400ng/mL, 8000ng/mL in Ep pipe, precision adds inner mark solution 20 μ L, adds water 45 μ L and methyl alcohol 400 μ L, and vortex mixes, each concentration samples respectively prepares three parts, gets supernatant 2 μ L and carries out LC-MS/MS analysis.Record chromatogram, records each concentration west not for Buddhist nun and Nei target peak area.
The results are shown in Table 2, show that west is not all greater than 90% for the extraction recovery of Buddhist nun at variable concentrations, and have no matrix effect, interior mark extraction recovery is not consistent for Buddhist nun with west with matrix effect.
Table 2. west is for Buddhist nun and Nei target extraction recovery and matrix effect (n=6)
1.5.7 stability
Stability assessment covers the testing process of whole sample, arrange basic, normal, high by (2.00,40.0 and 800ng/mL) three concentration, each concentration repeats five samples, and after investigating the room temperature 12h stability of plasma sample ,-80 DEG C of two months stability, three multigelation stability, extraction, sample is in injector internal stability and storing solution long-time stability.The results are shown in Table 3.
Table 3. west is not for Buddhist nun's stability (n=5, average (% deviation))
1.5.8. dilution effect
Ten times of dilutions: preparation west does not replace Buddhist nun's concentration to be the plasma sample of 1000ng/mL, after diluting five times with blank plasma, get 50 μ L plasma containing drugs, operate with method from " precision adds inner mark solution 20 μ L " by under " process of 2.4 plasma samples " item, sample introduction analysis after being disposed, each concentration prepares 6 samples.Retinue typical curve and Quality Control calculate measured concentration.
2. pharmacokinetics application
Screening previously accepted invalid or recurrence after one or more platiniferous standard chemotherapy regimen, or without the Locally Advanced of standard regimens and/or metastatic NSCLC patient, gave hydrochloric acid west not for Buddhist nun's sheet, every day 2 (t of health volunteer's single-dose
1/2for 6.08-16.01h), continuous 28 days.Dosage is 100mg.Experimenter 1h, 2h, 3h, 6h, 9h, 12h after (0.05h) and administration before (0.05h) and d28 administration before 1h, 2h, 3h, 6h, 9h, 12h, 24h and d8, d15, d22 administration in morning after (0.05h) and administration before d1 administration, respectively gather ulnar vein blood 2ml.Blood specimen collection is placed on and has posted label in vitro in advance, centrifugal (3000rpm, 10min) separated plasma in 10 minutes, and blood plasma is transferred to EP and manages mid--80 DEG C of Refrigerator stores.The pharmacokinetic parameter of single-dose and multiple dosing calculates acquisition by software WinNolin6.3 with non-compartment model, and Fig. 4 shows the oral west of this experimenter for the Drug-time curve after Buddhist nun's sheet.
Fig. 4 show experimenter west for Buddhist nun's first day single-dose (100mg), 8-10 days and the 15th day multiple dosing (100mg/ days) blood concentration-time curve.
Claims (7)
1. detect blood plasma Chinese and Western not for the method for Buddhist nun's concentration, wherein by Ultra Performance Liquid Chromatography tandem mass spectrum, blood plasma Chinese and Western is not analyzed for the concentration of Buddhist nun, wherein before described Ultra Performance Liquid Chromatography tandem mass spectrum does not replace the concentration of Buddhist nun to analyze to blood plasma Chinese and Western, by precipitation of protein, pre-treatment is carried out to plasma sample, wherein said precipitation of protein is undertaken by methyl alcohol, and wherein said Ultra Performance Liquid Chromatography tandem mass spectrum adopts ZORBAX SB-C8 chromatographic column to carry out, volume wherein for the plasma sample analyzed is 35 μ l-90 μ l, its Chinese and Western does not replace the range of linearity of Buddhist nun to be 1-1000ng/mL, wherein said Ultra Performance Liquid Chromatography tandem mass spectrum adopts A: formic acid water-ammonium formate, B: methyl alcohol is mobile phase.
2. method according to claim 1, the volume wherein for the plasma sample analyzed is 40 μ l-80 μ l.
3. method according to claim 1, the volume wherein for the plasma sample analyzed is 45 μ l-70 μ l.
4. method according to claim 1, the volume wherein for the plasma sample analyzed is 50 μ l-60 μ l.
5. method according to claim 1, wherein the volume ratio of A:B is 20/80.
6. the method according to any one of claim 1-4, wherein said Ultra Performance Liquid Chromatography tandem mass spectrum comprises interior mark.
7. method according to claim 6, is designated as erlotinid hydrochloride in wherein said.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310469995.1A CN103529160B (en) | 2013-10-10 | 2013-10-10 | UPLC-MS/MS method measures human plasma Chinese and Western not for the concentration of Buddhist nun |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310469995.1A CN103529160B (en) | 2013-10-10 | 2013-10-10 | UPLC-MS/MS method measures human plasma Chinese and Western not for the concentration of Buddhist nun |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103529160A CN103529160A (en) | 2014-01-22 |
| CN103529160B true CN103529160B (en) | 2015-10-21 |
Family
ID=49931340
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310469995.1A Active CN103529160B (en) | 2013-10-10 | 2013-10-10 | UPLC-MS/MS method measures human plasma Chinese and Western not for the concentration of Buddhist nun |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103529160B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106990185B (en) * | 2017-05-12 | 2019-02-26 | 浙江省肿瘤医院 | Method that is a kind of while measuring six kinds of tyrosine kinase inhibitor concentration in blood plasma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245070A (en) * | 2008-03-26 | 2008-08-20 | 江苏先声药物研究有限公司 | Novel technique for synthesizing toroid quinazoline protein tyrosine kinase restrainer |
| CN101431894A (en) * | 2006-01-17 | 2009-05-13 | 陈国庆 | Spiro compounds and methods of use |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102040587A (en) * | 2009-10-26 | 2011-05-04 | 韩南银 | Preparation method of imatinib mesylate |
| WO2011095802A1 (en) * | 2010-02-02 | 2011-08-11 | Generics [Uk] Limited | Hplc method for analyzing sunitinib |
| US9315848B2 (en) * | 2010-08-18 | 2016-04-19 | Technion Research And Development Foundation Ltd. | Volatile organic compounds for detecting cell dysplasia and genetic alterations associated with lung cancer |
-
2013
- 2013-10-10 CN CN201310469995.1A patent/CN103529160B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101431894A (en) * | 2006-01-17 | 2009-05-13 | 陈国庆 | Spiro compounds and methods of use |
| CN101245070A (en) * | 2008-03-26 | 2008-08-20 | 江苏先声药物研究有限公司 | Novel technique for synthesizing toroid quinazoline protein tyrosine kinase restrainer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103529160A (en) | 2014-01-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Goldwirt et al. | Ibrutinib brain distribution: a preclinical study | |
| Couchman et al. | An automated method for the measurement of a range of tyrosine kinase inhibitors in human plasma or serum using turbulent flow liquid chromatography–tandem mass spectrometry | |
| Abdelhameed et al. | An LC–MS/MS method for rapid and sensitive high‐throughput simultaneous determination of various protein kinase inhibitors in human plasma | |
| Lopez-Flores et al. | A high-performance liquid chromatographic assay for determination of cisplatin in plasma, cancer cell, and tumor samples | |
| Dai et al. | Studies on oral bioavailability and first‐pass metabolism of withaferin A in rats using LC–MS/MS and Q‐TRAP | |
| Zhang et al. | LC–MS-MS determination of imatinib and N-desmethyl imatinib in human plasma | |
| Kappelhoff et al. | Simple and rapid method for the simultaneous determination of the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma using liquid chromatography | |
| CN109633063B (en) | Method for detecting concentration of ticagrelor and active metabolite thereof in human plasma | |
| Alajmi et al. | Interspecies Anticancer and Antimicrobial Activities of Genus Solanum and Estimation of Rutin by Validated UPLC‐PDA Method | |
| Bolandnazar et al. | Development and application of an HPLC method for erlotinib protein binding studies | |
| Zhang et al. | Evaluation of the inhibitory effect of quercetin on the pharmacokinetics of tucatinib in rats by a novel UPLC–MS/MS assay | |
| Li et al. | Effects of CYP2C19 inhibitors on mavacamten pharmacokinetics in rats based on UPLC-MS/MS | |
| Wu et al. | Cell-patterned glass spray for direct drug assay using mass spectrometry | |
| Wu et al. | Furmonertinib (Alflutinib, AST2818) is a potential positive control drug comparable to rifampin for evaluation of CYP3A4 induction in sandwich-cultured primary human hepatocytes | |
| Deng et al. | Determination of cepharanthine in rat plasma by LC–MS/MS and its application to a pharmacokinetic study | |
| Da Ruos et al. | Analytical methods for the determination of major drugs used for the treatment of COVID-19. A review | |
| Aboras et al. | A review on analytical strategies for the assessment of recently approved direct acting antiviral drugs | |
| Leenhardt et al. | Liquid chromatography–tandem mass spectrometric assay for the quantification of CDK4/6 inhibitors in human plasma in a clinical context of drug-drug interaction | |
| Gao et al. | Two validated liquid chromatography–mass spectrometry methods with different pretreatments for the quantification of an anti-CD47 monoclonal antibody in rat and cynomolgus monkey serum compared with an electrochemiluminescence method | |
| Ni et al. | Simultaneous determination of six tyrosine kinase inhibitors in human plasma using HPLC-Q-Orbitrap mass spectrometry | |
| Stanisławiak-Rudowicz et al. | Bidirectional pharmacokinetic drug interactions between olaparib and metformin | |
| CN103529160B (en) | UPLC-MS/MS method measures human plasma Chinese and Western not for the concentration of Buddhist nun | |
| Aditya et al. | Development of modified Spectrophotometric and HPLC method for simultaneous estimation of Ambroxol hydrochloride and Cetirizine hydrochloride in tablet dosage forms | |
| Fu et al. | Effects of voriconazole and fluconazole on the pharmacokinetics of almonertinib in rats by UPLC–MS/MS | |
| Valizadeh et al. | Single dose bioequivalence study of α-methyldopa tablet formulations using a modified HPLC method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211027 Address after: 210042 699 Xuanwu Road, Xuanwu District, Nanjing, Jiangsu -18 Patentee after: JIANGSU SIMCERE PHARMACEUTICAL Co.,Ltd. Address before: 100021 No. 17, South Lane, Chaoyang District, Beijing, Panjiayuan Patentee before: CANCER HOSPITAL, CHINESE ACEDEMY OF MEDICAL SCIENCES |
|
| TR01 | Transfer of patent right |