RAPID COMMUNICATIONS IN MASS SPECTROMETRY
`Rapid Commun Mass Spectrom 2006 20 21832189
`Published online in Wiley InterScience wwwintersciencewileycom
`
`DOI 101002rcm2567
`
`Measurement of paclitaxel and its metabolites in human
`plasma using liquid chromatographyion trap mass
`spectrometry with a sonic spray ionization interface
`
`Henrik Green Karin Vretenbrant Bjorn Norander and Curt Peterson
`Link6ping University SE581 85 Link6ping
`Department of Medicine and Care Faculty of Health Sciences
`
`Division of Clinical Pharmacology
`Sweden
`
`Received 1 March 2006 Revised 9 May 2006 Accepted
`
`11 May 2006
`
`A quantitative
`liquid chromatographyion trap mass spectrometry method for the simultaneous
`in human plasma has
`determination of paclitaxel 6ahydroxypaclitaxel and p3hydroxypaclitaxel
`been developed and validated 6ap3Dihydroxypaclitaxel was also quantified using paclitaxel as a
`reference and docetaxel as an internal standard The substances were extracted from 0500 mL plasma
`using solid phase extraction The elution was performed with acetonitrile and the samples were
`reconstituted in the mobile phase Isocratic highperformance liquid chromatography analysis was
`performed by injecting 50 p1 of reconstituted material onto a 100 X 300 mm C12 column with a
`methano11 trifluoroacetic acidammonium trifluoroacetate in H20 7030 mobile phase at 350 p11
`min The MPHJ+ ions generated in the sonic spray ionization interface were isolated and frag
`mented using two serial mass spectrometric methods one for paclitaxel transition 854 > 569
`551
`and the dihydroxymetabolite transition 886 > 585
`567 and one for the hydroxy metabolites
`551 and docetaxel N+Nar transition
`transition 870 > 585
`567 transition 870 > 569
`830 > 550 Calibration curves were created ranging between 05 and 7500 ngmL for paclitaxel 05
`and 750 ngmL for 6ahydroxypaclitaxel and 05 and 400 ngmL for p3hydroxypaclitaxel Adduct
`ion formation was noted and investigated during method development and controlled by mobile
`phase optimization In conclusion a sensitive method for simultaneous quantification of paclitaxel
`and its metabolites suitable for analysis in clinical studies was obtained Copyright
`2006 John
`Sons Ltd
`
`Wiley
`
`Paclitaxel Taxo11 is an anticancer drug with a broad
`spectrum of antitumor activity including breast ovarian
`It was originally isolated from the
`skin and lung cancer
`Pacific yew tree Taxus brevifolia2 and together with the drug
`taxanes The taxanes
`represents the antineoplastic
`docetaxel
`have a unique mechanism of action among chemotherapeu
`the drugs facilitate the formation and
`tic agents in that
`suppress the depolymerization of microtubules Exposed
`cells are blocked in the G2 M phase in the cell cycle and
`undergo
`apoptosis31
`eventually
`this may be a result of different
`apoptosis directly but
`mechanisms at different drug concentrations5
`in the liver by the
`is primarily metabolized
`P450 CYP enzymes CYP3A4 and CYP2C8
`cytochrome
`CYP3A4 metabolizes
`to p3hydroxypaclitaxel
`paclitaxel
`and CYP2C8 converts the drug into 6ahydroxypaclitaxel
`can be further oxidized to 6ap3
`and these metabolites
`dihydroxypaclitaxe16 Other metabolites have been identified
`
`Paclitaxel also induces
`
`Paclitaxel
`
`to H Green Engineering Biology Division of
`Correspondence
`and Care
`of Medicine
`Clinical Pharmacology Department
`Faculty of Health Sciences Linkoping University SE 581 85
`Linkoping Sweden
`Email henrikgreenimvliuse
`Contractgrant sponsor Swedish Cancer Society Gunnar Nils
`sons Cancer Foundation and the County Council
`in Ostergot
`land
`
`in smaller quantities Studies have shown that
`the major
`and its metabolites
`portion of unchanged
`paclitaxel
`excreted in the feces indicating extensive nonrenal clear
`ance8 However only paclitaxel has been shown to be toxic
`and to have an antitumor activity while its metabolites are
`considered to be inactive9
`
`are
`
`Since
`
`the 1990s when Taxol was
`the beginning of
`as an anticancer drug1° an extensive number
`introduced
`studies have been done on the substance
`of pharmacokinetic
`These studies have resulted in the development
`different methods
`for quantification of paclitaxel
`and its
`logical samples The concentrations
`of paclitaxel
`in human plasma are measured preferably by
`metabolites
`highperformance liquid chromatography HPLC or liquid
`chromatography mass spectrometry LCMS although
`
`of several
`
`in bio
`
`other
`
`techniques have also been reported Immunological
`methods
`such as enzyme linked immunosorbent assay
`ELISA12 and radioimmunoassay13
`have been used to
`screen human plasma but
`these methods have a major
`drawback of cross reactions Most frequently used are HPLC
`systems with UV detection although other chromatography
`eg capillary electrophoresis14 and methods
`techniques
`
`tWILEY
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`MN
`
`Copyright 0 2006 John Wiley
`
`Sons Ltd
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`

`

`2184 H Green et al
`
`using other detectors eg fluorescence detectors15 have also
`been reported
`
`Several developments
`
`in the use of paclitaxel have led to
`of the drug and the need for
`lower plasma concentrations
`more sensitive assays First of all clinicians
`have begun to
`investigate weekly therapy as an alternative
`to the every
`three week administration schedule with a reduction
`of
`dose as a result16 In the development of new chemother
`apeutic regimens where paclitaxel
`is used in combination
`with other drugs the dose is often less than the recom
`mended
`phase II dose In addition the investigation
`of
`has also led to the need
`individualization of chemotherapy
`The relative lack of sensitivity
`for more accurate techniques
`of the HPLC methods with UVdetection17
`and specificity
`of methods
`using mass
`has
`led to the development
`and multiple MS for detection of
`the sub
`Recent reports have described paclitaxel quanti
`based on LCMSMS instrumentation Several
`columns
`different extraction methods mobile phases
`interfaces and MS instruments have been used However
`most of
`these methods were developed
`for paclitaxel only
`and not for the metabolites For our purposes of trying to
`predict individual elimination of paclitaxel
`in cancer patients
`
`spectrometry
`
`stances1731
`
`fications
`
`factors affecting the extent of conversion by
`by investigating
`each metabolic enzyme we needed an accurate and sensitive
`method for quantification of paclitaxel and its metabolites in
`human plasma
`The aim of our study was to develop an LCMS method
`for simultaneous quantification of paclitaxel and its metab
`olites in human plasma To our knowledge this is the first
`method
`for quantification of paclitaxel 6ahydroxypacli
`and p36adihydroxypacli
`taxel p3hydroxypaclitaxel
`in human plasma using ion trap mass spectrometry
`ITMS with a sonic spray ionization SSI interface
`
`taxel
`
`EXPERIMENTAL
`
`Materials
`Paclitaxel C47H5iN014 was kindly supplied by BristolMyers
`CT USA p3 Hydroxypaclitaxel
`Squibb Wallingford
`C47H51N015 and 6ahydroxypaclitaxel C47H51N015 were
`purchased from Gentest Woburn MA USA through BD
`Biosciences Docetaxel C431153N014 was kindly supplied by
`Aventis Pharma Vitry Alforville France and used as an
`and
`internal standard HPLCgrade methanol acetonitrile
`hexane were from LabScan Analytical Sciences Dublin
`Ireland Ethanol was from Kemetyl Stockholm Sweden
`Water was obtained from a Milli Q Biocell station Millipore
`AB Stockholm Sweden Trifluoroacetic acid TFA was from
`Sigma Stockholm Sweden Human plasma was delivered
`from the blood bank of Linkoping University Hospital
`
`Instrumentation
`The LCMS system consisted of a LaChrome chromatograph
`pump model L7100 autosampler L7200 UV detector L
`7400 and an interface model D7000 with a Merck Hitachi
`M8000 LCMSn ion trap equipped with a SSI
`interface
`from Merck KgaA Darmstadt Germany The system was
`controlled using a computer equipped with the Chromatog
`raphy Data Station software LC3DQ MS System Manager
`
`Copyright © 2006 John Wiley
`
`Sons Ltd
`
`Instruments
`
`from Merck Darmstadt Germany and Hitachi
`Inc San Jose CA USA An infusion pump model 11
`Harvard Apparatus Holliston MA USA was connected
`to
`the system during some steps in the optimization The
`4 p Max RP 80A
`column
`was
`a Synergi
`100 x 300 mm Phenomenex CA USA and a guard column
`4 x 30 mm with the same stationary phase was used
`
`analytical
`
`coefficients
`
`Preparation of stock solutions and plasma
`samples
`Paclitaxel was dissolved in methanol
`to a concentration of
`20 mg mL and further diluted in methanol
`to give stock
`solutions in the range of 02200 tigmL using glass pipettes
`The internal standard docetaxel was dissolved in ethanol
`to a
`concentration of 20 mg mL and diluted to 20 lagmL p3
`Hydroxypaclitaxel and 6ahydroxypaclitaxel were dissolved
`and methanol respectively to give approxi
`in acetonitrile
`mate concentrations of 20 lagmL Due to the high inaccuracy
`the manufacturers quantity determination the concen
`of
`trations were determined using absorption
`spectroscopy
`The absorbance was measured at 227 nm for paclitaxel and
`230 nm for
`The following molar
`the other
`substances
`were used 298 mM1 cm 1 for
`extinction
`358 mM1 cm 1
`paclitaxel 148 m141 cm 1 for docetaxel
`and 262 mM1 cm 1 for 6a
`for p3hydroxypaclitaxel
`hydroxypaclitaxel according to the manufacturers docu
`mentation
`Standards were prepared by diluting the stock solutions in
`human drug free plasma Nine different standards denoted
`STD1STD9 were prepared with the following concen
`trations 05 2 5 10 50 250 1000 4000 and 7500 ngmL of
`paclitaxel 05 2 5 10 25 50 100 250 and 750 ngmL of 6a
`and 05 2 5 10 25 50 100 200 and
`hydroxypaclitaxel
`400 ngmL of p3hydroxypaclitaxel The standard concen
`trations were based on the concentrations found in human
`plasma after a dose of 175 mg m2 paclitaxel
`infused during 3 h
`Five different quality control QC samples denoted QC1
`QC5 were prepared with the following concentrations
`8
`100 300 1500 and 5000 ngmL of paclitaxel 8 2040 80 and
`400 ngmL of 6ahydroxypaclitaxel
`and 8 20 40 80 and
`300 ngmL of p3hydroxypaclitaxel
`
`to
`
`Solidphase extraction
`The solid phase extraction SPE procedure was adopted
`from Huizing et al32 with modifications Prior to extraction
`the tips in the vacuum box were washed in acetonitrile
`The CN solid phase 100 mg 1 mL
`minimize crossover
`Isolute 1ST UK columns were conditioned
`with 2 mL
`methanol and 2 mL 10 mM ammonium acetate buffer pH 50
`Then 500 µ1 of spiked plasma were mixed with an equal
`volume of 02 M ammonium acetate pH 50 and 50111 of
`internal standard 20 lagmL and applied to the column The
`columns were washed with 2 mL of 10 mM ammonium
`acetate and 1 mL of methanol 10 mM ammonium acetate
`2080 and finally with 1 mL of hexane Prior to and after the
`hexane wash the columns were dried for a minimum of
`2 min The samples were eluted in 2 mL of acetonitrile and
`evaporated under a stream of nitrogen at 35°C The analytes
`were reconstituted in 200 µ1 mobile phase and sonicated for
`5 min to ensure that
`the compounds dissolved completely
`
`Rapid Commun Mass Spectrom 2006 20 21832189
`DOT 101002rcm
`
`

`

`The samples were then placed in the autosampler and 50 pL
`were injected twice from each vial
`
`LCMS optimization and settings
`Several mobile phases were tested to yield a sensitive and
`stable chromatography and MS detection The mobile phase
`finally used for chromatographic
`separation was methanol
`1 TFA ammonium trifluoroacetate
`in H20 pH 77030 at a
`flow rate of 350 laL min and the chromatography
`ran for
`9 min
`The SSI settings were as follows plate temperature 200°C
`aperture 1 temperature 150°C aperture 2 temperature 120°C
`chamber voltage 06 kV drift voltage 35 V and focus voltage
`34V
`In the ion trap mass spectrometer positive ion polarity was
`used the multiplier voltage being set to 400 V and the peak
`threshold to 10 The automatic sensitivity control ASC was
`turned on and for each run three microscans were centroided
`and averaged The mass spectrometer was run in MS2 mode
`using two alternating methods The first MS2 method was
`used for detection of paclitaxel and dihydroxypaclitaxel a
`reference substance was not available for this metabolite so
`paclitaxel was used as reference In the ion accumulation
`step for this method the low mass cutoff LMC was set to 80
`the accumulation mass range was set to 750940 mz the
`voltage was set to 003 V and the sensitivity
`accumulation
`was 200 The settings for the isolation step were as follows
`and 881891 LMC 750
`isolation mass range 849859
`isolation voltage 015 V and isolation time 10 ms During
`the collision induced dissociation CID step the following
`and 876896
`settings were used CID resonance 849863
`LMC 420V CID time 50 ms and CID voltage 015V The
`second MS2 method was used for the detection of both
`and docetaxel The settings
`step were
`as
`follows
`the
`the ion accumulation
`for
`to 766940 mz the
`accumulation mass range was set
`voltage was set to 003 V and the sensitivity
`accumulation
`was 200 During the isolation step the following settings
`were used isolation mass range 825835 and 865875 LMC
`770 isolation voltage 012 V and isolation time 10 ms In the
`CID step LMC was set to 420 CID resonance to 825835 and
`865881 mz CID time to 50 ms and CID voltage to 02 V
`During both methods the MS spectra were scanned for a
`mass to charge ratio between 500 and 600 mz Chromato
`from the MS
`substance were extracted
`for each
`grams
`ions as indicated
`
`chromatograms
`Table 1
`
`using the product
`
`in
`
`hydroxypaclitaxel metabolites
`
`Validation procedure
`The precision and accuracy of the method were evaluated by
`analyzing paclitaxel p3hydroxypaclitaxel and 6a hydro
`
`Measurement of paclitaxel using LC SSIITMS
`
`2185
`
`STD1STD9
`
`xypaclitaxel at five different concentrations QC1QC5 on
`three different days Six QC samples at each concentration
`were used to evaluate within day variation and six were
`used for between day variation To determine the concen
`tration of the analytes in the QC samples one calibration
`of nine different concentrations
`curve consisting
`were analyzed each day Calibration was performed
`peak in the chromato
`using the area of the corresponding
`gram and the calibration curve was created using a cubic fit
`regression model forced through zero
`The precision of the assay was expressed as a coefficient of
`variation CV at each
`concentration
`the
`by calculating
`as a percentage of the mean calculated
`standard deviation
`concentrations while accuracy of the method was evaluated
`the mean calculated
`concentration
`as a
`by expressing
`the nominal concentration
`The percentage
`percentage of
`extraction
`recoveries
`of paclitaxel p3hydroxypaclitaxel
`for the QC samples were deter
`and 6ahydroxypaclitaxel
`mined by comparing extracted blank samples in which the
`amount of
`SPE eluate had been spiked with a corresponding
`
`checked
`
`substance to those obtained by injections of extracted spiked
`plasma samples The stability of paclitaxel p3hydroxypa
`clitaxel and 6ahydroxypaclitaxel
`in the samples was
`in three different experiments one for
`room
`temperature stability one for stability after
`reconstitution
`in the mobile phase and one experiment for freeze thaw
`stability The stability at room temperature was evaluated by
`two different
`letting eight samples of spiked plasma at
`
`concentrations paclitaxel 50 and 1000 ng mL p3hydro
`xypaclitaxel 50 and 200 ng mL and 6ahydroxypaclitaxel 50
`and 200 ng mL stand at room temperature for 24h and
`another set of eight samples for 48 h The concentrations were
`then compared with the QC samples with corresponding
`To check the stability after reconstitution ten
`concentrations
`QC samples were left on the bench after
`injection and re
`injected 4 days later The areas of
`the peaks were then
`compared to the first
`injection Eight plasma samples at the
`same two concentrations
`the room temperature
`stability test were frozen to 70°C and thawed for three
`cycles and then extracted and analyzed The areas of
`the
`peaks were compared to the QC samples spiked at the same
`concentrations
`
`as for
`
`RESULTS AND DISCUSSION
`Method development
`this assay was to
`The major goal
`in the development
`and its
`quantify low concentrations
`of both
`paclitaxel
`hydroxy metabolites in human plasma Due to the fact that
`we only had access to small amounts of the metabolites the
`and then the
`method was first
`for paclitaxel
`
`developed
`
`of
`
`ions and isolation mz values in MS and retention times Rt for the analytes
`Rt min
`Isolation mz
`
`Product
`
`ions
`
`Method
`
`1 2 2 1 2
`
`550+551+568+569
`
`550+551+568+569
`
`566+567+583586
`
`566+567+583586
`
`548551
`
`849859
`
`865875
`
`865875
`
`881891
`
`825835
`
`61
`36
`51
`
`3 7
`
`Rapid Commun Mass Spectrom 2006 20 21832189
`DOT 101002 rcm
`
`Table 1 Product
`
`Substance
`
`Paclitaxel
`
`p3Hydroxypaclitaxel
`
`6aHydroxypac1itaxe1
`
`6ap3Dihydroxypac1itaxe1
`Docetaxel
`
`Copyright 0 2006 John Wiley
`
`Sons Ltd
`
`

`

`2186 H Green et al
`
`settings were adopted to fit
`metabolites and thereafter
`
`the quantification of the available
`finetuned for the best perform
`
`paclitaxel
`
`either
`
`ance
`In most instruments and previously reported methods
`forms the M±H1+ ion or the JM±Nal
`molecular ion To suppress the formation of the Na+ adduct
`in favor of the M±H1 + ion an acidic mobile phase would be
`required However
`in our system we could not get a
`reproducible formation of the JM±H1+ ion between different
`batches of acidic mobile phase due to the formation of a
`lower or higher amount of JM±Nar To stabilize the system
`to form
`the mobile phase was changed to push paclitaxel
`other ions instead of JM±H1+ By adding positive or negative
`ions to the mobile phase the following adducts could be
`detected in the LCMS M±K1+ 2 mM KC1 Me0H 3070
`JM±Nal ± 2 mM NaC1Me0H 3070 and JM+Acetater
`20 mM ammonium acetatepH
`50 Me0H 3070 The
`highest sensitivity of these adducts was reached with the
`the M±H1+ ion showed a higher
`JM±K1+ ion However
`sensitivity although unstable than any of
`the other
`ions
`probably due to fragmentation of the latter in the ion source
`We then tried to stabilize the formation of the M±H1+ ion by
`suppressing the JM+Nal+ ion This was done using crown
`ethers or TFA the latter giving the highest sensitivity and the
`lowest amount of JM±Nal +adducts Although TFA caused a
`the signaltonoise SN
`suppression of the absolute signal
`ratio for paclitaxel was increased and a stable JM±H1+ ion
`formation was achieved
`The mobile phase was then
`and pH Optimum MS2
`optimized for TFA concentration
`conditions for the analyte response were established using a
`continuous
`infusion of a stock solution containing paclitaxel
`while running the chosen mobile phase components Note
`forms the JM±Nal + adduct using this mobile
`that docetaxel
`phase and instrument
`interfaces were
`Several
`to evaluate
`the
`response The M8000 LCMS ion trap was equipped with
`both an SSI
`interface as well as an electrospray
`ionization
`ESI interface The SSI interface was about
`four times more
`
`also tested
`
`efficient
`
`than the ESI one We also had
`in ionizing paclitaxel
`the ionization on a Perkin Elmer
`the opportunity to test
`SCIEX equipped with both an ESI and an atmospheric
`ionization APCI interface In this system
`pressure chemical
`interface was more efficient
`the ESI
`in ionizing paclitaxel
`than the APCI
`interface The SSI
`interface was
`then
`
`paclitaxel
`
`considered the best choice
`The HPLC column was chosen to retain the analytes
`sufficiently to avoid
`ion suppression
`by early eluting
`substances in the sample extract and separate the analytes
`since some of them have identical mass to charge ratios for
`have the
`some fragments The monohydroxy metabolites
`same molecular mass but they fragment into ions with a
`different mz However p3hydroxypaclitaxel dissociates
`into fragments with the same mz as
`from
`fragments
`and 6ahydroxypaclitaxel
`dissociates into frag
`ments with the same mz as dihydroxypaclitaxel Fig 1 To
`two methods were
`achieve single peak mass chromatograms
`run in parallel one for paclitaxel and dihydroxypaclitaxel
`and one for the monohydroxy metabolites Table 1 By
`the HPLC conditions
`making sure that
`yielded peak
`between the analytes was mini
`separation interference
`retention times were 605 min for paclitaxel
`mized Typical
`365 min for p3hydroxypaclitaxel 50 min for 6ahydro
`and 68 min for
`xypaclitaxel 33 min for dihydroxypaclitaxel
`docetaxel After each injection the chromatography was run
`9 min Figure
`2 shows
`chromatograms
`for
`for
`typical
`paclitaxel and its hydroxylated metabolites generated from
`the product ions formed in MS2 mz values shown in Table
`1 Docetaxel was used as an internal standard however it
`did not increase the precision or accuracy of the method and
`was therefore only used to control
`the SPE and autosampler
`Paclitaxel adheres strongly to most surfaces carryover
`effects and ghost peaks were major problems during early
`method development The problems were traced to the SPE
`procedure and solved by replacing the nylon tips in the SPE
`vacuum box with steel needles which were washed between
`
`all extractions
`
`Paclitaxel+1 854 mz
`OHPac+1 870 mz
`
`0
`
`Paclitaxel
`
`6aOHPac
`p30HPac
`
`R1
`H
`H
`OH
`
`R2
`H
`OH
`H
`
`Paclitaxel 569 raiz
`p30HPac+1 569 mz
`585 mz
`6aOHPac+1
`
`H20
`
`Paclitaxel 551 mz
`p30HPac+1 551 mz
`567 mz
`6aOHPac+1
`
`Figure 1 Molecular structure and fragmentation of paclitaxel and its mono
`hydroxy metabolites
`
`Copyright © 2006 John Wiley
`
`Sons Ltd
`
`Rapid Commun Mass Spectrom 2006 20 21832189
`DOT 101002rcm
`
`

`

`1111Mffil
`
`Bann 0
`
`a00110
`
`700176
`
`604104
`
`btUOtl
`
`4119111f
`
`31113011
`
`21113130
`
`111000
`
`ct
`
`2rg
`
``c
`
`eaa
`
`g
`
`3
`
`Tinle min
`
`1000008
`
`900000
`
`100000
`
`700000
`
`600000
`
`500000
`
`4001100
`
`300000
`
`200000
`
`10000C1
`
`Figure 2 Typical
`hydroxy metabolites
`
`of
`
`its
`
`receiving
`
`for
`
`and
`chromatograms
`for paclitaxel
`isolated from plasma 15 min post end
`infusion from a patient with ovarian cancer
`175 mgm2 of paclitaxel Taxo18 The chromatograms were
`ions formed in MS2
`generated using the sum of the product
`550+551+568+569 mz for paclitaxel 550+551+568+569
`mz for p3hydroxypaclitaxel
`and 566+567+583586 mz
`In the chromatogram the baseline
`for 6ahydroxypaclitaxel
`is elevated with 5000 AU and that
`for 6ahydroxypaclitaxel
`paclitaxel with 100 000 AU for
`illustrative purposes In this
`method the ion trap works with two serial methods one
`generating the paclitaxel and the dihydroxymetabolite chro
`matogram and one the hydroxyl metabolites and the internal
`the peak at 61 min in the p3hydro
`standard Note that
`xypaclitaxel chromatogram is from paclitaxel more than 100
`fold intensity in the correct method and that the peak at 7 min
`in the 6ahydroxypaclitaxel chromatogram is from the internal
`standard which has a 13C fragment with the same mz as the
`metabolite
`
`Measurement of paclitaxel using LCSSIITMS
`
`2187
`
`in the trap at high ion concentrations Others
`charge transfer
`have solved this nonlinearity by diluting the samples33
`However we found that in the range presented in this assay
`the use of nonlinear calibration curves worked just as well
`The calibration curves for the metabolites were linear over
`the whole calibration
`range Figure 3 shows a calibration
`curve for paclitaxel obtained during one of the validation
`runs
`The final method
`
`for several
`
`reasons First of all
`
`concentrations
`
`conditions were chosen
`low
`they allow the quantification
`of
`<10 ng mL of
`in human
`the analytes
`plasma Second they allow the determination of paclitaxel
`and its metabolites simultaneously The method presented
`here has some special features especially the use of TFA as a
`Naadduct
`suppressor and the SSI interface both of which
`of the assay
`improved the performance
`
`analyzing six replicates of
`
`of
`
`Precision and accuracy
`the method was evaluated
`The reproducibility
`by
`five different QC samples on
`three different days Extracted standard curves obtained in
`the range of 057500 ngmL showed excellent curve fitting
`with a coefficient of correlation greater than 099 The within
`day and between day variations are reported in Table 2 The
`method was found to be acceptably precise CV <14 and
`accurate range 83113 except for the within day variation
`The mean
`the lowest
`concentration
`of paclitaxel
`extraction recovery assessed at the same concentrations
`range 6374 p3
`the QC samples was a bit low paclitaxel
`hydroxypaclitaxel 6571 and 6ahydroxypaclitaxel 72
`85 The
`and its hydroxy
`of paclitaxel
`quantification
`metabolites was possible within the range of the standard
`curves The lower limit of quantification was set to 05 ngmL
`for paclitaxel within day precision <12 n = 4 and
`between day accuracy 103122 n = 3 and to 2 ngmL
`for the hydroxy metabolites withinday variation <20
`n = 4 and between day accuracy 107125 n = 3
`
`at
`
`as
`
`Each
`sample was injected twice due to spiking of
`the
`chromatograms We were not able to get rid of the spiking
`phenomenon by adjusting the MS conditions or cleaning of
`the mass spectrometer although improving the water quality
`in the mobile phase helped some The spiking was however
`infrequent and readily identified and if one of
`rather
`the
`contained interference the other
`chromatograms
`injection
`was used With more advanced software it would be rather
`easy just to exclude a single data point caused by spiking
`instead of having to run the samples twice
`Due to the wide range of paclitaxel concentrations found in
`plasma the calibration curve for paclitaxel was divided into
`a low 05250 ngmL and high range 2507500 ngmL
`calibration curve The calibration curve was also created
`regression model due to nonlinearity at high
`using a cubic fit
`The response is linear from 05 up
`paclitaxel concentrations
`to 1000 ngmL Proposed causes of nonlinearity at the high
`end of
`the calibration curve have been the formation of
`paclitaxel dimers and trimers at the interface33 as well as
`
`Copyright C 2006 John Wiley
`
`Sons Ltd
`
`30
`
`kr 20
`D
`
`10
`
`0
`
`0
`
`2000
`4000
`6000
`8000
`Paclitaxel concentration ngmL
`
`Figure 3 A nonlinear calibration curve for the higher
`range
`obtained during one of the vali
`of paclitaxel concentrations
`dation runs The concentration of paclitaxel
`in spiked standard
`samples is shown on the xaxis and the response of the mass
`spectrometer as the area of the peak is presented on the y
`axis A cubic fit
`regression model was used for calibration of
`The responses of the metab
`the paclitaxel concentrations
`olites were linear within the calibration ranges
`
`Rapid Commun Mass Spectrom 2006 20 21832189
`DOT 101002rcm
`
`

`

`2188 H Green et al
`
`Table 2 Precision and accuracy of the quantification of paclitaxel and its monohydroxy metabolites
`reconstitution in the mobile phase
`represents the stability of the substances after
`
`in plasma The last column
`
`Concentration
`
`Accuracy
`
`Precision
`
`Reinj
`
`Within day
`
`Between day
`
`Within day
`
`Between day
`
`4 days later
`
`CV
`
`Mean
`
`100
`103
`116
`106
`88
`127
`106
`131
`117
`86
`120
`108
`113
`115
`92
`
`13
`
`68471
`
`0
`10
`10
`
`41
`
`451
`
`251
`
`0
`11
`
`CV
`
`497472697997939
`
`Mean
`
`151b
`84
`87
`96
`92
`106
`108
`95
`86
`93
`113
`113
`105
`102
`103
`
`Mean
`
`93
`91
`100
`100
`87
`95
`97
`83
`84
`97
`101
`99
`90
`93
`92
`
`Substance
`
`Sample
`
`Paclitaxel
`
`6aOHpac
`
`p30Hpac
`
`QS1
`QS2
`Q53
`Q54
`QS5
`QS1
`QS2
`Q53
`Q54
`QS5
`
`QS1
`QS2
`0S3
`Q54
`QS5
`
`ngmL
`
`Plasma
`
`8
`
`100
`
`300
`
`1500
`
`5000
`
`8
`
`20
`
`40
`
`80
`
`400
`
`8
`
`20
`
`40
`
`80
`
`300
`
`a For each concentration six samples were tested for the within day variation for the between day variation two samples on three different days
`n = 6 were used for each concentration
`and the re injection was done on the within day variation samples
`b The within day accuracies on the following three days were 80 103 and 88
`
`Stability experiments
`The results of the stability tests on the samples are presented
`in Tables 2 and 3 Freeze thaw cycling did not affect
`the
`precision or accuracy of the assay The substances in plasma
`were also stable at room temperature for at least 48 h and
`in the mobile phase the substances were
`after reconstitution
`stable for at least 4 days
`
`Discussion
`Several mass spectrometry methods have been developed for
`in different matrixes1731
`of paclitaxel
`the quantification
`In
`the M+Hr ion has been used for MS
`most methods
`analysis which we found to have the highest sensitivity
`as M+NH41+242534
`other
`adducts
`such
`although
`N4 ±Nai +232534 M+M+Na +23 and M+Acetate126 have
`of paclitaxel and its
`been used In the early characterizations
`
`Table 3 The stability
`of paclitaxel p3hydroxypaclitaxel
`and 6ahydroxypaclitaxel during three cycles of freezethaw
`ing to 70°C and room temperature RT stability for 24 and
`48 h
`
`Concentration
`
`RT
`
`RT
`
`ngmL
`
`Plasma
`
`1000
`
`50
`
`200
`
`50
`
`200
`
`50
`
`Freezethawing
`
`24 h
`
`48 h
`
`Mean
`
`103
`117
`89
`117
`116
`115
`
`Mean
`
`Mean
`
`97
`110
`96
`112
`104
`105
`
`98
`116
`91
`114
`109
`101
`
`Substance
`
`Paclitaxel
`
`6aOHpac
`
`p30Hpac
`
`n = 4
`
`Copyright © 2006 John Wiley
`
`Sons Ltd
`
`metabolites fast atom bombardment was used for ionization
`the compounds Most modern methods use the ESI
`of
`interface although the APC12631 and now the SSI interface
`have been shown to be useful Some of the more impressive
`methods developed are those published by Schellen et al26
`which has an analysis time of only 80 s and by Parise et al 1 7
`which has a range of almost 4 decades Although many
`methods have been published for paclitaxel only a few MS
`methods have been developed for the quantification of the
`metabolites283133 Royer et al31 used a MS1 method with an
`APCI interface and Mortier et a28 and Alexander et al3 used
`MS2 methods
`on triple quadrupole instrument with ESI
`
`interfaces as compared to our use of ion trap methodology
`and a SSI
`a method for
`interface Mortier et al developed
`quantification in oral fluids and human plasma with a liquid
`liquid extraction LLE and gradient chromatography The
`calibration ranges in plasma were 4100 ngmL for paclitaxel
`and 2100 ngmL for the metabolites The method developed
`by Alexander et al is used for quantification of the substances
`in dog and human plasma LLE was used for sample
`preparation and 13C6 labeled paclitaxel was used as an
`internal standard The calibration ranges in plasma were 01
`100 ngmL for all compounds We used SPE instead of LLE
`to minimize ion suppression we also had difficulties with
`using LLE by
`the metabolites when
`of
`the recovery
`unwanted
`compounds hopefully giving us a more robust
`method and isocratic chromatography since the mobile
`phase was shown to be of such importance
`for stable ion
`formation Our assay also has a rather wide calibration range
`of 057500 ngmL for paclitaxel 05750 ngmL for 6a
`hydroxypaclitaxel and 05400 ngmL for p3hydroxypa
`clitaxel which were chosen according to the concentrations
`found in human plasma after a 3h infusion of 175 mg m2
`Taxol® To our knowledge the use of TFA as a stabilizer of
`
`Rapid Commun Mass Spectrom 2006 20 21832189
`DOT 101002rcm
`
`

`

`M+111+ formation and an increase in SN ratio has not been
`presented before and the effect is probably due to the Na+
`binding effect of TFA The responses of the metabolites were
`regression model
`linear but in the case of paclitaxel a cubic fit
`was used due to nonlinearity Others have also found this
`nonlinearity and solved the problem by either diluting the
`sample33 or using nonlinear regression28
`
`CONCLUSIONS
`
`reported chromatographic method quantify
`This is the first
`in human plasma using
`ing paclitaxel and its metabolites
`LC SSIITMS methodology Comprehensive method devel
`opment with optimization of the mobile phase to yield as
`stable and marked formation of the M+111+ ion as possible
`led to an assay with a high response The development
`of a
`highly sensitive method with a wide calibration range for
`simultaneous quantification of paclitaxel and its metabolites
`in human plasma was a prerequisite for this project The
`The advantages of
`validation was performed successfully
`this method
`the simultaneous
`are the high sensitivity
`of all compounds and the use of a relatively
`quantification
`low cost SSI
`ion trap instrument compared with a triple
`quadrupole mass spectrometer the drawback is the spiking
`of the chromatograms which may call for reanalysis of single
`samples This analytical method has been used to quantify
`and its metabolites
`in more than 200 plasma
`paclitaxel
`samples from patients with ovarian cancer and will be used
`for individualized
`in the future to evaluate the conditions
`
`chemotherapy with paclitaxel
`
`Acknowledgements
`The authors wish to thank Martin Josefsson PhD National
`of Forensic Chem
`Board of Forensic Medicine Department
`istry Linkoping Sweden
`for fruitful discussions and for
`allowing us use of the Perkin Elmer SCIEX for an evaluation
`of ESI and APCI interfaces This study was supported by the
`Swedish Cancer Society Gunnar Nilssons Cancer Founda
`tion and the County Council in Ostergotland The authors
`also wish to thank Isaac Austin for proofreading the text
`
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