11S
`
`Journal of Chromatography B 709 1998 281288
`
`JOURNAL OF
`CHROMATOGRAPHY
`
`13
`
`Assay of paclitaxel Taxol
`in plasma and urine by high
`performance liquid chromatography
`N Martinb J Catalin MF Blachonb A Durand
`
`aLaboratoire de Toxicocinetique et de Pharmacocinetique Faculte de Pharmacie 27 Boulevard
`Cedex 5 France
`bLaboratoire de Pharmacodnetique et Toxicocinetique CHU Timone 27 Boulevard Jean Moulin 13385 MariIIe Cedex 5 France
`
`Jean Moulin 13385 Marille
`
`Received
`
`23 October 1997 received
`
`in revised form 16 January 1998 accepted 27 January
`
`1998
`
`Abstract
`
`A new rapid and sensitive highperformance liquid chromatographic
`method for the analysis of paclitaxel Taxol
`in
`human plasma and urine was developed and validated After addition of an internal standard paclitaxel was extracted from
`plasma or urine by a liquidliquid extraction using diethyl ether Extraction efficiency averaged 90 Chromatography was
`times were 77 and 67 min for
`performed isocratically on a reversed phase column monitored at 227 inn Retention
`paclitaxel and docetaxel respectively and the assay was linear in the range 251000 ngml The limits of quantification for
`paclitaxel were 25 and 40 ngml
`in plasma and urine respectively The assay was shown to be suitable for phannacokinetic
`0 1998 Elsevier Science BV All rights reserved
`studies of children involved in a phase I clinical
`
`trial
`
`Keywords Paclitaxek
`
`Taxol
`
`1 Introduction
`
`Paclitaxel Taxol
`
`tax11en9one5320
`
`103 13i hexahydroxy 410 diacetate 2
`
`epoxy
`
`benzoate136phenylhippurate is an antineoplastic
`to a new group of cytotoxic
`agent which belongs
`agents the taxanes Fig 1 Their main mechanism
`of action is mediated by the stabilization of cellular
`have now demonstrated
`microtubules Investigators
`taxol activity against adult epithelial ovarian cancer
`breast cancer
`in adults Taxol was used as
`In clinical
`trials
`single 3 6 24 72 or 96 h infusions ranging from 15
`to 390 mgm2 1271 In a phase I study carried out
`
`lung cancer and melanoma 1
`
`paclitaxel
`
`C6HC0
`
`docetaxel
`
`tBuOCO
`
`OH
`
`RI NH
`
`Corresponding
`
`author
`
`Fig 1 Structures of paclitaxel and docetaxel
`
`internal standard
`
`0 1998 Elsevier Science RV All rights reserved
`0378434798$1900
`PIT S0378434798000607
`
`Abraxis EX2027
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`282
`
`N Martin et al
`
`J Chromatogr
`
`B 709 1998 281288
`
`1
`
`Table
`Published HPLC methods
`
`for determination of paclitaxel
`
`n plasma and urine
`
`Author
`
`Extraction
`
`Column
`
`Elution
`
`Internal standard
`
`LOD or LOQ of paclitaxel
`
`Waters
`
`C18
`
`Whatman Partisil
`
`10 pm
`Radial Pak
`10 pm ODS3 column
`Varian C18 reversed phase 10 gm column
`C column MOS hypersil 5 pm
`APEX octyl 5
`C column hypersil
`
`pin
`
`Gradient
`
`Gradient
`
`Isocratic
`
`Isocratic
`
`Isocratic
`
`Isocratic
`
`Ncyclohexylbenzamide
`
`LODun=50
`
`nM 427 ngm
`
`No
`
`No
`
`No
`
`No
`
`No
`
`LODa=100
`
`nM 80 mg fml
`LOQa=11 7 nM 10 ngml
`
`Longnecker et al 87 4
`et al 87 10
`Wiemik
`Grem et al 87 11
`Rizzo et al 90 12
`Willey etal 93 13
`Sonnischen et al 94 9
`Song and Au 95 14
`
`LLE ethylacetate
`
`LLE acetonitrile
`
`L LE +SPE
`
`L LE +SPE
`
`SPE
`
`L LE +SPE
`
`LLE+SPE Nova pack C1
`Waters column switching
`
`Gianni et al 95 5
`Sparreboom etal 95 15
`
`SPE isocratic
`
`LLE+ SPE
`
`Reversed phase Backerbond
`
`Isocratic
`
`Cephalomannine
`
`LOD°=585 nIVI 5 ngml
`
`octadecyl C18 5 pm
`Superspher C1 column
`APEX octyl 5 gm
`
`Isocratic
`
`Isocratic
`
`Cephalomannine
`2 Methyl paclitaxel
`
`LOD°=175 nM 15 nem°
`
`LOQ°=29 nM 25 ngml
`LODa=94 8 nem
`nM 10 ngml
`LOQa=117
`
`Huizing et al 95 16
`
`SPE or LLE
`
`APEX octyl 5 pm
`
`Isocratic
`
`LLE liquidliquid extraction SPE solid phase extraction
`
`LOD limit of detection un=undefined a=defined as the concentration of compound giving a signaltonoise ratio greater than 3 1
`
`LOQ limit of quantification
`
`a
`
`=defined
`
`as the lowest concentration that
`
`can be measured with
`
`accuracy
`
`and precision 20 17
`
`clitaxel
`
`in children Taxol doses 200420 mgm2 were
`delivered as single 24h infusions 89
`Several publications have appeared in which pa
`is assayed by HPLC Characteristics of the
`analytical methods are given in Table 1 45916
`Some assays employ solidphase extraction SPE
`others use liquidliquid extraction LLE and a few
`liquid and solid extraction
`use
`a combination of
`Some assays do not use an internal standard or a
`extraction procedure Some techniques
`complicated
`sensitivity or were not validated
`show inadequate
`
`according to current
`
`requirements
`a rapid simple and robust
`This paper describes
`HPLC method with internal standard
`to FDA requirements 17 for the de
`according
`in human plasma and urine
`termination of paclitaxel
`The assay
`is suitable for clinical pharmacokinetic
`studies
`
`validated
`
`2 Experimental
`
`21 Materials
`
`Acetonitrile RS grade ethanol HPLC grade
`and diethyl ether RPE grade were obtained from
`Carlo Erba Milan Italy Paclitaxel Taxon and
`from Sigma St
`Cremophor EL were purchased
`
`Louis MO USA Docetaxel used as internal stan
`dard IS was a gift
`from Rhone Poulenc Rorer
`France All other chemicals were of analytical
`grade Deionized water was used throughout
`the
`human plasma collected on citrate
`study Pooled
`was obtained from the Centre de Transfusion San
`35 mM
`The buffer
`guine Marseilles France
`ammonium acetate pH 50 was prepared by adding
`27 g ammonium acetate
`and acetic acid to 1
`deionized water
`
`1
`
`22 HPLC instrumentation and conditions
`
`A solvent delivery pump Waters 600E system
`controller was used with a UV detector Kontron
`HPLC 432 Data output was monitored using a NEC
`advanced personal computer software MAXIMA 820
`Samples were injected with an a Waters 717 chro
`ambient
`temperature
`matographic
`autosampler
`Separation was performed at ambient temperature on
`
`a 250X46 mm Nucleosil 5 vim particle C column
`Macherey Nagel The mobile phase consisted of
`acetonitrile35 mM ammonium acetate buffer pH
`5tetrahydrofuran 45505 vvv and was filtered
`system Millipore 045 vim under
`vacuum and
`separation was moni
`degassed Chromatographic
`tored at 227 nm using a flow rate of 18 mlmin
`
`

`

`N Martin et al
`
`J Chromatogr
`
`B 709 1998 281288
`
`283
`
`23 Preparation of stock solutions
`
`26 Liquidliquid extraction
`
`stock solutions were prepared by dis
`Paclitaxel
`in 10 ml of ethanol The
`solving 5 mg of paclitaxel
`exact concentration was determined by UV spectro
`photometry after appropriate dilution 629 800 at
`227 nm The paclitaxel stock solutions were stored
`in 200R1 aliquots at 20°C until needed Paclitaxel
`stock solutions were stable for at
`least 5 months
`standard solutions of paclitaxel 10 ngjd
`Working
`and 1 ngjd were prepared by appropriate dilutions
`of the stock solutions in ethanol These solutions
`were made daily Docetaxel
`stock solutions internal
`10 mg of
`standard were made by dissolving
`in 10 ml of ethanol Working standard
`docetaxel
`solutions 10 jtgm1 were obtained by dilution of
`the stock solution in ethanol and all solutions were
`stored at 20°C
`
`24 Preparation of standard and quality control
`solutions
`
`ELethanol
`
`an
`For preparation
`of
`the
`calibration curves
`appropriate volume of working standard solution was
`added to 1 ml aliquots of blank human plasma or
`blank urine The plasma standards ranged from 25
`to 1000 ngml 25 50 100 250 500 1000
`ngml
`ngml Urine was stabilized with Cremophor
`11 wv according
`to the method of
`Huizing et al 16 Urine standards ranged from 40
`to 1000 ngml 40 100 250 500 1000 ngml
`Quality control solutions QC were
`prepared
`from plasma or urine in the same manner using a
`different fresh stock solution at concentrations of 44
`440 and 750 ngml and stored at 20°C for at least
`2 months
`
`25 Clinical samples
`
`Blood samples 5 ml of patients were collected
`into heparinized tubes stored at 4°C and centrifuged
`900 g within 2 h of collection The resultant plasma
`samples were stored at 20°C until assayed
`Fresh urine samples stabilized by adding 05 ml
`of Cremophor ELethanol 11 wv to 95 ml urine
`16 were also stored at 20°C until assayed
`
`For analysis 1 ml of standard
`quality control
`solution or 50 j11 ml of plasma or urine sample
`were placed in glass tubes A 50j1 volume of
`jd of 35 mM ammonium
`100
`internal standard
`acetate buffer pH 5 and 7 ml of diethyl ether were
`The tubes were vortexed for 12
`added successively
`s shaken for 15 min on a Rotmix RK Heto 35
`rp m and then centrifuged
`at 2500 g for 5 mm
`+4°C to separate aqueous and organic layers The
`organic layers were placed in glass tubes and dried
`room temperature The residues
`under nitrogen at
`in 200j1 aliquots of HPLC
`were
`reconstituted
`mobile phase These samples were mixed for 20 s
`at 10 000 g for 3 min The solution
`and centrifuged
`was transferred to a microvial and the autosampler
`
`at
`
`programmed to inject 140 jd into the chromatogmph
`
`27 Quantification
`
`Using MAXIMA 820 software Millipore the peak
`to internal standard were
`ratios of paclitaxel
`height
`used to constmct
`the calibration curve The cali
`bration curves were analysed by weighted 1x least
`squares linear regression analysis The concentration
`of each unknown
`substance was calculated from this
`
`calibration curve
`
`28 aability study
`
`in plasma was assessed
`The stability of paclitaxel
`from spiked samples 44 ngml and 750 ngml
`after storage of aliquots at 20°C for 10 weeks The
`samples were brought
`to room temperature well
`vortexed and analysed immediately Each determi
`nation was performed in duplicate
`
`3 Results
`
`31 Chromatography
`
`extracts
`
`Fig 2 shows the chromatographic
`separation of
`from blank plasma spiked with
`prepared
`from blank plasma and from plasma of a
`paclitaxel
`receiving paclitaxel The approximate reten
`patient
`tion times for the internal standard and taxol were
`
`

`

`284
`
`N Martin et al
`
`J Chromatogr
`
`B 709 1998 281288
`
`A
`
`LS
`
`PACLITAXEL
`
`600
`
`400
`
`200
`
`NJ
`
`000
`000
`
`020
`
`040
`060
`x 101 minutes
`
`6001
`
`400 I
`
`2001
`
`0
`
`X
`
`t
`
`o1
`
`x1
`
`0
`
`000
`000
`
`020
`
`060
`040
`x 10 minutes
`
`080
`
`100
`
`6013
`
`tl
`
`0
`
`tcj
`
`X
`
`400
`
`200
`
`000
`000
`
`020
`
`PACUTAXEL
`
`LS
`
`000
`
`100
`
`060
`1040
`x 10 minutes
`
`Fig 2 Chromatograms of A an extracted blank plasma standard QC spiked with 500 ngml paclitaxel and 500 ngml docetaxel B an
`extracted blank plasma sample and C an extracted plasma sample from a patient
`
`C1=199 µgml sample=50
`
`10 mm after the end of a 350 mgm2 3h infusion
`
`

`

`N Martin et al
`
`J Chromatogr
`
`B 709 1998 281288
`
`285
`
`67 and 77 min Taxol and the internal standard
`eluted as sharp symmetrical peaks No significant
`endogenous
`could
`interfere with the
`peaks
`measurement of paclitaxel or internal standard were
`observed
`
`that
`
`32 Recovery
`
`in
`
`from plasma or urine
`of peak heights
`stan
`
`The recovery of paclitaxel
`was calculated by comparison
`extracted samples with those in corresponding
`of extraction from
`dard solutions The efficiency
`plasma was 82 and 92 at concentrations of 100
`respectively n=2 The
`ngml and 1000 ngml
`values for extraction from urine were 87 n=2
`and 97 n=3 at concentrations of 100 and 500
`ngml respectively
`
`33 Limit of detection and limit of quantification
`
`in
`
`The limit of detection LOD for paclitaxel
`plasma signaltonoise of 3 was 10 ngml 115
`nM The limit of quantification LOQ for paclitaxel
`in plasma was 25 ngml 29 nM n=6 mean=
`2463 ngml SD=190 ngml CV =772 The
`that can be mea
`LOQ is the lowest
`sured with accuracy and precision 20
`In urine the LOD was 15 ngml 18 nM and the
`LOQ was 37 ngml 43 nM n=6 mean=3721
`ngml SD=439 ngml CV 1180
`
`concentration
`
`34 Linearity
`
`In plasma calibration curves were linear over
`range 251000 ngml 291171 nM
`concentration
`The typical equation
`the standard line
`describing
`was y=398R522 An average correlation coeffi
`cient of r=09993 n=8 was obtained Table 2
`In urine calibration curves were linear over
`range 401000 ngml 591171 nM
`y=353R257 r=09993 n=8
`
`the
`
`the
`
`concentration
`
`35 I ntra and inter assay variabilities
`
`In plasma spiked with 44 440 and 750 ngml the
`were 634
`precision
`intraassay
`variabilities
`284 and 154 respectively
`The
`interassay
`
`variabilities at the same concentrations n=6 were
`1118 297 and 302 respectively Table 3
`The intraassay variabilities of stabilized urine
`spiked with 44 440 and 750 ngml were 898
`308 and 286 respectively
`The
`interassay
`the same conditions were
`139 204 and 29 at concentrations of 44 440
`respectively Table 4 Intmassay
`and 750 ngml
`and inter assay accuracies are reported in Tables 3
`and 4
`
`variabilities of urine under
`
`36 aability
`
`After extraction from spiked plasma the stability
`of paclitaxel 500 ngml and docetaxel
`in the
`mobile phase at room temperature was studied The
`ratio of paclitaxel vs docetaxel
`remained
`peak height
`for at least 48 h This is in agreement with
`constant
`the observations of Song and Au 14 who showed
`taxol was stable in 40 acetonitrile for 24 h
`in QC solutions
`low control 44 ngml
`Paclitaxel
`and high control 750 ngml stored in glass tubes at
`20°C was stable for at least 2 months
`
`that
`
`37 Interfering drugs
`
`several therapeutic
`
`I clinical
`
`trials
`
`Patients participating in a Phase
`were receiving
`compounds which
`we investigated
`inter
`for possible chromatographic
`ference The retention characteristics of these com
`pounds are given in Table 5 No interference
`from
`these drugs with the peaks of paclitaxel and docetax
`el was observed
`
`4 Discussion
`
`The
`
`suitable
`
`the best
`
`resolution of
`
`here is
`for
`the
`assay described
`in plasma and urine for
`determination of paclitaxel
`studies The mobile phase
`clinical pharmacokinetic
`was optimised to obtain
`paclitaxel and the internal standard docetaxel The
`
`quantity of acetonitrile 45 gave good resolution
`tetrahydrofuran 5 produced
`
`in a short
`
`run time The
`
`of
`
`the two compounds
`addition of
`
`sharp
`symmetrical peaks and enhanced the stability of the
`two compounds Storage for 1 week in the mobile
`phase without
`tetrahydrofuran caused a decreased in
`
`

`

`286
`
`Table 2
`
`N Martin et al
`
`J Chromatogr
`
`B 709 1998 281288
`
`Statistics
`
`on parameters of paclitaxel standard curves in plasma and urine
`
`Analysis group
`
`Plasma
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`Mean
`SD
`RSD
`
`n
`
`Urine
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Slope
`
`3770
`
`3990
`
`3672
`
`4443
`
`3833
`
`3922
`
`3974
`
`4238
`
`3980
`
`2521
`
`633
`
`8
`
`3488
`
`3548
`
`3351
`
`3350
`
`3418
`
`Intercept
`
`+254
`326
`1494
`940
`799
`149
`109
`613
`522
`554
`
`8
`
`Correlation coefficient
`
`09960
`
`09996
`
`09997
`
`09997
`
`09999
`
`09999
`
`09999
`
`09997
`
`09993
`
`8
`
`09998
`
`09999
`
`09983
`
`09999
`
`09998
`
`6
`
`7
`
`8
`
`Mean
`SD
`RSD
`
`n
`
`3433
`
`3780
`
`3871
`
`3530
`
`1953
`
`553
`
`8
`
`SD standard deviation RSD =relative standard deviation
`
`Table 3
`
`Intraassay and inter assay precision and accuracy
`
`in plasma samples
`
`Quality
`
`Control
`
`Intraassay precision and accuracy
`Mean ngml
`SD ngml
`Precision RSD
`
`Accuracy
`
`n
`
`Inter assay precision and accuracy
`Mean ngml
`SD ngml
`Precision RSD
`
`Accuracy
`
`n
`
`Low
`44 ngml
`
`4135
`
`262
`
`634
`603
`6
`
`4189
`
`468
`
`1118
`48
`
`6
`
`SD standard deviation RSD =relative standard deviation
`
`815
`108
`2685
`1404
`40
`3935
`3892
`4012
`257
`1614
`
`8
`
`09992
`
`09995
`
`09986
`
`09993
`
`8
`
`Medium
`440 ngml
`
`High
`750 ngml
`
`47765
`
`1359
`
`284
`
`855
`
`6
`
`45274
`
`1345
`
`297
`
`061
`
`6
`
`75297
`
`1164
`
`154
`
`039
`
`6
`
`78232
`
`2363
`
`302
`
`43
`
`6
`
`

`

`N Martin et al
`
`J Chromatogr
`
`B 709 1998 281288
`
`287
`
`Medium
`440 ngml
`
`High
`750 ngml
`
`42653
`
`1314
`
`308
`306
`
`6
`
`4269
`
`872
`
`204
`29
`
`6
`
`71955
`
`2061
`
`286
`406
`
`6
`
`70757
`
`2093
`29
`56
`
`6
`
`tate tertbutylmethyl ether only diethyl ether gave
`recovery 90 with good volatility
`excellent
`A useful
`detect
`
`feature of
`
`putative
`
`this assays is its ability to
`formed
`hydroxylated metabolites
`of paclitaxel with microsomal
`during incubation
`from different species rabbit rat dog
`preparations
`baboon macaques human No interference
`from
`was detected
`in the chromato
`these metabolites
`graphic profiles of paclitaxel and docetaxel 18 The
`performances of this analytical method were similar
`to those published previously Table 1 Only Huitz
`ing et al 16 and Willey et al 13 described a more
`their methods did not
`sensitive method but
`include
`an internal standard The use of LLE is particularly
`advantageous for routine therapeutic drug monitoring
`this method is rapid sensitive specific and
`uses a simple material This method is suitable for
`studies in human plasma and urine
`pharmacokinetic
`and could possibly be used to analyse metabolites
`
`Table 4
`
`Intraassay and inter assay precision and accuracy
`
`in urine samples
`
`Quality
`
`control
`
`Intraassay precision and accuracy
`Mean ngml
`SD ngml
`Precision RSD
`
`Accuracy
`
`Inter assay precidon and accuracy
`Mean ngml
`SD ngml
`Precision RSD
`
`Accuracy
`
`Low
`44 ngml
`
`3649
`
`328
`
`898
`17
`
`6
`
`446
`
`621
`
`139
`
`13
`
`6
`
`SD standard deviation RSD =relative standard deviation
`
`peak heights of the two compounds Variation in the
`pH used for liquidliquid extraction affected
`the
`recovery of paclitaxel with a pH of 5 giving the best
`efficiency of extraction at 500 ngml
`Docetaxel was chosen as the internal standard
`because of the similarities in structure and physical
`to paclitaxel Other possible internal stan
`properties
`dards were either not available or available only with
`LLE of paclitaxel with various
`solvents
`difficulty
`was examined as was SPE Some solvents caused
`compounds with pa
`of endogenous
`interference
`had an insufficient
`of
`efficiency
`application Of
`routine
`the
`extraction to permit
`different solvents examined chloroform ethyl ace
`
`clitaxel Others
`
`times tR of drugs tested for interference
`
`in paclitaxel
`
`because
`
`also
`
`5 Conclusion
`
`a rapid isocratic HPLC assay
`We have validated
`in human plasma and urine The assay
`for paclitaxel
`in the range 251000 ng and requires
`a
`is linear
`sample volume of 50 µ1 to 1 ml of plasma or urine
`The method involves UV detection at 227 nm and
`has a limit of quantification of 25 ngml 29 nM Its
`
`tR min
`
`15
`
`180
`
`2 2
`
`25
`38
`38
`390
`
`415
`
`481
`77
`89
`
`946
`
`Not detectable
`
`Table 5
`
`Retention
`
`analysis
`
`Drugs
`
`Cimetidine
`
`Acetaminophen
`
`Morphine
`
`Clonidine
`
`Bupivacaine
`
`Dexchlorpheniramine
`
`Clonazepam
`
`Hydroxyzine
`Dexamethasone
`
`Docetaxel
`
`Paclitaxel
`
`Chlorpromazine
`
`Prednisolone
`
`

`

`288
`
`N Martin et al
`
`J Chromatogr
`
`B 709 1998 281288
`
`simplicity allows the analysis of at least 30 samples
`
`day
`The method
`
`has been applied to a dose depen
`study in children involved in
`dence pharmacokinetic
`trial 19
`
`a Phase I
`
`Acknowledgements
`
`This work was supported in part by Bristol Myers
`Squibb France
`
`References
`
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`bara N Onetto B Winograd R Canetta J Clin Oncol
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`8 CA Hurwitz MY Relling SD Weitman Y Ravindranath
`TJ Vietti DR Strother AH Ragab CB Pratt J Clin
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`9 D Sonnichsen CA Hurwitz C Pratt JS Shuster MY
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`Relling J Clin Oncol
`10 PH Wiernik EL Schwartz JJ Strauman JP Dutcher
`RB Lipton E Paietta Cancer Res 47 1987 2486
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