ELSEVIER
`
`Journal of Chromatography B 705 1998 159164
`
`Short communication
`
`JOURNAL OF
`CHROMATOGRAPHY
`
`13
`
`Determination of paclitaxel
`
`in human plasma using single solvent
`extraction prior to isocratic reversed phase highperformance
`chromatography with ultraviolet detection
`
`liquid
`
`Alex Sparreboom Peter de Bruijn Kees Nooter Walter J Loos Gerrit Stoter
`Jaap Verweij
`
`Institute Daniel
`Laboratory of Experimental Chemotherapy and Pharmacology Department of Medical Oncology Rotterdam Cancer
`den Hoed Kliniek and University Hospital Rotterdam PO Box 5201 3008 AE Rotterdam Netherlands
`
`Received
`
`1 July 1997 received
`
`in revised form 4 September
`
`1997 accepted 7 October 1997
`
`Abstract
`
`at 230 nm has
`An isocratic
`reversed phase highperformance liquid chromatographic method with ultraviolet detection
`in human plasma Plasma samples were prepared by a selective onestep
`been developed for the determination of paclitaxel
`liquidliquid extraction involving a mixture of acetonitrilenbutyl chloride 14 vv Paclitaxel and the internal standard
`using a column packed with ODS80A material and a mobile phase consisting of
`watermethanoltetrahydrofuranammonium
`docetaxel were separated
`hydroxide 375602501 vv The calibration graph for paclitaxel was linear in
`the range 10500 ngml with a lower
`limit of quantitation of 10 ngml using
`1 ml plasma samples The extraction
`to drug free human plasma were 896±852 and 937±50 respectively
`recoveries of spiked paclitaxel and docetaxel
`Validation data showed that
`is sensitive selective accurate and reproducible The assay has been
`the assay for paclitaxel
`0 1998
`used in a single pharmacokinetic
`the applicability of the method in vivo
`experiment in a patient to investigate
`Elsevier Science By
`
`Keywords Paclitaxel
`
`1 Introduction
`
`is a highly functionalized di
`Paclitaxel Taxol
`terpene amide from the bark of the Pacific yew tree
`i a 1 having
`Taxis brevifol
`a bulky fused taxane
`ring system with a rare four membered oxetane ring
`at C13 Fig 1 The
`and an ester
`side chain
`inhibitor of cell replication in
`
`compound is a potent
`
`malignant tissues a property attributed to its ability
`
`Corresponding
`
`author
`
`to stabilize microtubules and block the transit of
`
`cycling cells from the G2 phase to the M phase 23
`con
`In clinical studies paclitaxel has demonstrated
`siderable activity against a variety of malignancies
`including platinum refractory ovarian cancer breast
`cancer and non small cell
`reviewed in
`lung cancer
`
`Over
`numerous methods
`few decades
`the last
`for the determination of pa
`have been developed
`in human plasma samples including bio
`clitaxel
`on
`based
`tubulinformation
`procedures
`
`56 competitive inhibition enzyme immunoassays
`
`chemical
`
`0 1998 Elsevier Science BV All rights reserved
`0378434798$1900
`I S0378434797005021
`P I
`
`Abraxis EX2026
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`160
`
`A Sparreboom et al
`
`J Chromatogr
`
`B 705 1998 159164
`
`accurate pharmacokinetic monitoring of paclitaxel
`
`trials because of its rapidity selectivi
`during clinical
`ty and sensitivity The available HPLC methods have
`generally employed ultraviolet UV absorption de
`tection techniques and a variety of sample cleanup
`procedures Table 1 1020 To date all analytical
`procedures with sufficient sensitivity to justify appli
`in pharmacologic
`cation
`studies
`involving
`serial
`blood sampling have employed tedious and expen
`sive solidphase extraction SPE techniques
`either
`alone 1520 or in combination with protein pre
`liquidliquid extraction LLE
`cipitation 11 or
`1314 In addition recent experiments
`have shown
`vehicle Cremophor
`the paclitaxelformulation
`EL which is present
`in clinical samples at con
`centrations up to 10 vv can have
`a profound
`by affecting the SPE
`effect on assay performance
`for paclitaxel 21
`extraction efficiency
`In this report we describe an alternative HPLC
`method with UV detection for the determination of
`in human plasma with a lower
`limit of
`paclitaxel
`quantitation LLQ of 10 ngml which avoids the
`use of SPE for sample cleanup
`The method is a
`modification of our procedure
`routinely applied for
`
`that
`
`A
`
`H3C
`
`H3C
`
`0
`
`OH
`
`CH3
`
`c=o
`
`CH3
`
`NH CHCH C0
`41 OH
`
`0 C
`
`H3C C H3
`9 p
`
`H3C
`
`113c
`
`0
`
`NH CH CH C0
`
`OH
`
`OH
`
`OH
`
`CH3
`
`C=O
`
`C= 0
`
`CH3
`
`Fig 1 Chemical
`
`structures of paclitaxel A and the internal
`standard docetaxel B
`
`78 and capillary electrophoresis 9 Lately how
`ever
`highperformance
`HPLC has emerged as the technique of choice for
`
`chromatography
`
`liquid
`
`selective
`
`Taxotere
`
`compound
`docetaxel
`the related
`the analysis of
`22 and involves
`a mpid and highly
`onestep LLE A pilot pharmacokinetic
`the drug was
`in a cancer patient
`receiving
`study
`included to investigate the suitability of the method
`for clinical use
`
`Table
`
`1
`
`Highperformance
`
`liquid chromatographic methods
`
`available
`
`for the analysis of paclitaxel
`
`in human plasma
`
`Sample pretreatment
`
`Internal standard
`
`Detection
`
`wavelength nm
`
`LLQ
`ngml
`
`Reference
`
`PP acetonitrile
`PP acetonitrile+SPE C13
`LLE ethylacetate+gadient
`elution
`LLE ethylacetate+columnswitching
`LLE tertbutylmethylether+SPE Cis
`SPE nitrile
`SPE nitrile
`SPE Ci3
`SPE C18
`SPE C3
`SPE Ci3
`
`nCyclohexylbenzamide
`
`Cephalomannine
`
`2Methylpaclitaxel
`
`Cephalomannine
`
`Cephalomannine
`
`nHexylphydroxybenzoate
`
`nNitrosodiphenylamine
`
`227
`
`227
`
`227
`
`229
`
`227
`
`227
`
`227
`
`230
`
`230
`
`227
`
`227
`
`Not reported
`
`25
`
`25
`
`10
`
`85
`
`10
`
`10
`
`11
`
`20
`
`15
`
`10
`
`Wiernik et al 10
`Grem et al 11
`et al 12
`Longnecker
`Song and Au 13
`Rizzo et al 14
`Willey et al 15
`Huizing et al 16
`JamisDow et al 17
`Mase et al 18
`Ohtsu et al 19
`ElYazigi et al 20
`
`Abbreviations
`
`LLQ=lower
`
`limit of quantitation PP=plasma protein precipitation
`
`LLE=liquidliquid
`
`extraction SPE=solidphase
`
`extraction
`
`

`

`A Sparreboom et al
`
`J Chromatogr
`
`B 705 1998 159164
`
`161
`
`2 Experimental
`
`21 Materials
`
`internal
`
`standard
`
`powder batch 484034 purity 983
`Paclitaxel
`by reversed phase HPLC and commercially avail
`able paclitaxel formulated in Cremophor ELdehy
`drated ethanol USP 11 vv Taxol were kindly
`donated by BristolMyers Squibb Woerden Nether
`The
`lands
`batch
`docetaxel
`14PR0C92320 purity 980 by reversed phase
`HPLC was obtained
`from RhonePoulenc
`Rorer
`VitrysurSeine Cedex France Reference standards
`of the paclitaxel metabolites 6a3pdihydroxypa
`and 6ahydroxy
`clitaxel 3phydroxypaclitaxel
`paclitaxel were isolated
`and purified from feces
`samples from a patient as described in detail previ
`ously 23 The structures of
`the metabolites were
`confirmed by online photodiode array detection and
`fast atom bombardment mass spectrometry Stock
`solutions of paclitaxel docetaxel and the metabolites
`1 mgml and
`were prepared in dimethylsulfoxide at
`were stored in glass at 20°C Cremophor EL was
`purchased from Sigma St Louis MO USA All
`other chemicals were of the highest grade available
`and were from Rathburn Walkerburn UK Water
`was purified and deionized by the MilliQUF system
`Millipore Milford MA USA and was
`used
`throughout Human plasma was obtained from heal
`thy volunteers
`
`22 Sample preparation
`
`A 100µ1 volume of
`the internal standard 10
`µgm1 in methanolwater 11 vv and 5 ml of
`acetonitrilenbutylchloride 14 vv were added to
`1 ml of human plasma in a 12 ml glass tube and
`closed with a PTFEfaced cap The tube was rocked
`on a multitube vortex mixer for 5 min and cen
`trifuged at 4000 g for 5 min Next the organic layer
`was transferred to a clean glass tube with a pasteur
`pipette and evaporated to dryness under a stream of
`nitrogen at 60°C taking care to discontinue evapora
`tion immediately after a residue appeared in the
`bottom approximately 45 min A 125µ1Nolume of
`methanolwater 11 vv was added to the residue
`by ultrasonication for 1 min The
`and reconstituted
`contents of the tube was transferred to a low volume
`
`insert of glass and a 100µ1 aliquot was subjected to
`chromatography
`
`23 Chromatography
`
`staMetric
`
`system consisted of a con
`The chromatographic
`pump LDC Analytical
`3200
`Rivera
`Beach FL USA a Waters 717plus
`autosampler
`Milford MA USA a model SpH99 column oven
`Spark Holland Meppel Netherlands and a Spectra
`Physics UV2000 detector San Jose CA USA The
`stationary phase was 5 µm Inertsil ODS80A materi
`in a 150X46 mm ID stainless steel
`al packed
`column GL Science Tokyo Japan protected by a
`Lichrospher 100 RP18 guard column 40X40 mm
`5 µm particles The mobile phase consisted of
`hy
`watermethanoltetrahydrofuranammonium
`droxide 375602501 vv with the pH adjusted
`to 60 formic acid The flow rate of the mobile
`phase was set at 10 mlmin and the eluent was
`monitored at ambient room temperature at an absorp
`tion wavelength of 230 nm The column temperature
`was 60°C
`
`With each
`run duplicate cali
`chromatographic
`in blank human
`bration standards were prepared
`plasma by serial dilution at concentrations of 10 25
`50 100 250 and 500 ngml Sets of quality control
`QC samples were prepared in batch in the same
`manner at 10 40 200 400 and 15 000 ngml The
`QC sample containing the highest concentration was
`used to investigate
`the effect of sample dilution
`andor
`limited volume injection Acquisition
`and
`integration of data were performed with the Chrom
`to an ICW
`Card data analysis
`system connected
`chromatographic work station Fisons Milan Italy
`Calibration graphs were calculated by weighted 1
`X2 least squares linear
`analysis of the
`regression
`peak area ratio of paclitaxel and the internal standard
`ordinate versus the drug concentration of the nomi
`nal standard abscissa
`
`24 Validation
`
`Method validation was performed according to the
`report on
`recorded
`in the conference
`guidelines
`Analytical Methods Validation Bioavailability Bio
`equivalence and Pharmacokinetic Studies 24 with
`minor modifications as described previously 22 All
`
`

`

`162
`
`A Sparreboom et al
`
`J Chromatogr
`
`B 705 1998 159164
`
`validation runs were performed on four consecutive
`days and included
`a calibration curve processed in
`duplicate and a set of QC samples in quintuplicate
`analyzed with repeated cycles of freezing and thaw
`ing
`
`25 Pharmacokinetic
`
`study
`
`Blood samples 5 ml were obtained in heparin
`ized tubes from a patient who gave informed written
`before
`consent according
`to institutional guidelines
`treatment Samples were collected prior to dosing
`and at 150 300 308 325 350 375 400 500
`700 900 1100 1500 and 2700 h after administra
`tion of 225 mgm2 of paclitaxel by a 3h intravenous
`infusion Following centrifugation at 4000 g for 5
`min the plasma fraction was separated and stored
`frozen at 20°C until analysis within two weeks
`profile was fitted to a three
`The pharmacokinetic
`open model using the MWPharm
`compartment
`KinFit computer program MediWare Groningen
`Netherlands 25
`
`3 Results and discussion
`
`31 qoecificity
`
`contain
`
`Fig 2 displays typical chromatograms
`resulting
`from HPLC analysis of 1 ml human plasma extracts
`from a blank sample A and a sample spiked to
`50 ngml of paclitaxel B As shown
`paclitaxel t =75 min and the internal standard
`docetaxel t =85 min were well
`and
`resolved
`adequately separated from endogenous plasma com
`the optimum analytical
`under
`conditions
`ponents
`run time was
`used The overall chromatographic
`established at 30 min
`Certain drugs that are commonly used clinically in
`period were selected to
`the pre or postchemotherapy
`in the assay for
`check
`interference
`for potential
`paclitaxel These included acetaminophen
`codeine
`dexamethasone
`domperidon
`
`alizapride
`
`lorazepam
`and
`
`metoclopramide
`
`morphine
`
`paroxetine
`
`ranitidine After
`
`injection into the chromatograph
`post extraction only paroxetine was found to give a
`
`significant
`
`interfering peak during the analysis
`
`around the retention time of paclitaxel approximate
`
`A
`
`II
`
`0
`
`10
`
`20
`
`30
`
`retention
`
`time min
`
`Fig 2 Typical
`
`isocratic reversed phase HPLC chromatograms of
`a blank human plasma sample A and a sample
`of 50 ngml B Peaks corresponding to
`
`spiked
`
`at a
`
`standard
`
`docetaxel
`
`paclitaxel
`
`internal
`
`paclitaxel concentration
`tR=75 min and the
`tR=85 min are indicated by I and II
`
`respectively
`
`ly 70 min The selectivity and resolution of the
`HPLC system was also confirmed by co injection of
`paclitaxel with three known cytochrome P450 me
`diated metabolites ie 6a3pdihydroxypaclitaxel
`t =34 min 3phydroxypaclitaxel
`tR=40 min
`tR=59 min 23
`and 6ahydroxypaclitaxel
`
`32 Validation characteristics
`
`The assay was found to be linear over
`the range
`10500 ngml of paclitaxel
`in plasma with a mean
`regression correlation coefficient of 09998 n=4
`concen
`The percentage deviation of the predicted
`tration in each calibration sample calculated
`was 10 using the
`the nominal concentration
`
`from
`
`reciprocal
`
`of
`
`the concentration
`
`as
`
`the weighting
`
`factor
`In blank human plasma ten out of twenty samples
`spiked at 5 ng ml were outside the acceptable it 20
`10 ngml
`all were
`limits whereas at
`
`deviation
`
`

`

`A Sparreboom et al
`
`J Chromatogr
`
`B 705 1998 159164
`
`163
`
`15
`
`20
`
`25
`
`30
`
`r1
`
`0
`
`5
`
`104
`
`10s
`
`102d
`
`10
`
`tagre
`
`paclitaxel
`
`time h
`Fig 3 Plasma
`in a human subject
`time profile
`concentration
`following administration of a 225 mgm2 dose of paclitaxel
`
`4 Conclusions
`
`In conclusion the method described for the de
`in human plasma is specific
`termination of paclitaxel
`accurate and precise and is selective and sensitive
`LLQ 10 ngml enough to be used in clinical trials
`As sample pretreatment
`consists of a rapid onestep
`
`solvent extraction it avoids the use of the compli
`cated and expensive SPE techniques
`that were used
`in previously reported
`
`In addition it
`
`procedures
`
`within 4 of the nominal concentration
`
`Based on
`these results the LLQ was established at 10 ngml
`limit of quantitation ie 500 ngml
`At the upper
`both the mean percentage deviation and the between
`
`run precision were also less than 5 The within run
`
`and between run variability expressed as the per
`by
`relative standard deviation
`calculated
`oneway analysis of variance were less than 3 at
`centage
`analyzed Table 2
`the four QC concentrations
`Similar results were obtained from the analysis of
`QC samples spiked to contain 1 vv Cremophor
`EL demonstrating
`in assay
`a lack of
`interference
`in the presence of this vehicle There
`performance
`fore accuracy and precision of the assay were found
`to be acceptable for the analysis of plasma samples
`studies The mean
`in support of pharmacokinetic
`overall extraction efficiencies were 896 85 for
`paclitaxel n=47 and 937 50 for docetaxel n
`47 which was used as internal standard and were
`independent of the spiked concentration
`The described analytical method was applied in
`our institute to a phase I and pharmacokinetic
`study
`of paclitaxel in combination with cisplatin in patients
`with advanced solid cancer A representative plasma
`concentration time profile of paclitaxel after an
`intravenous dose of 225 mgm2 is depicted in Fig 3
`to a three com
`The fitting of these concentrations
`partment model
`is in agreement with previous data
`26
`
`Table 2
`Accuracy within run and between run precision for the analysis of paclitaxel
`of 10 vv Cremophor EL
`Added ngml
`
`Recovered ngml
`
`DEV
`
`in spiked quality control samples in the absence and presence
`
`20
`
`20
`
`20
`
`20
`
`20
`
`3 3 3 2
`
`WRP
`
`222
`
`150
`
`206
`
`047
`
`299
`
`BRP
`
`276
`
`102
`
`067
`
`058
`
`191
`
`QC samples without Cremophor EL
`1014±047
`10
`4036±049
`1998±184
`4111±086
`15 080±2016
`
`40
`
`200
`
`400
`
`15 000
`
`QC samples containing
`40
`
`200
`
`400
`
`15 000
`
`EL
`
`+140
`+091
`012
`+277
`+054
`
`+706
`+091
`+413
`+420
`
`0 VvCremophor
`
`4304±377
`2018±671
`4165±898
`15630±3510
`
`Abbreviations DEV=percent
`deviation accuracy WRP=withinrun precision BRP=betweenrun
`observations within each validation
`
`precision
`
`n=number of replicate
`
`

`

`164
`
`A Sparreboom et al
`
`J Chromatogr
`
`B 705 1998 159164
`
`in SPEHPLC
`eliminates a pitfall
`subsequently
`methods associated with the presence of Cremophor
`EL in clinical samples causing decreased extraction
`for paclitaxel
`
`efficiency
`
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