determination of methotrexate in human urine at trace levels by solid phase extraction and...

Determination of methotrexate in human urine attrace levels by solid phase extraction and high-performance liquid chromatography/tandemmass spectrometry

Roberta Turci*, Maria Lorena Fiorentino, Cristina Sottani and Claudio MinoiaLaboratory of Environmental Hygiene and Industrial Toxicology, Salvatore Maugeri Foundation, via Alzaia 29, Pavia, Italy

For biological monitoring of hospital personnel occupationally exposed to antineoplastic agents, highlysensitive and specific methods are required. In order to detect trace MTX urinary concentrations, a preciseand accurate high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS)procedure, incorporating solid phase extraction, has been developed. Urine samples were purified by solidphase extraction (SPE) on octadecyl bonded, endcapped silica SPE columns. After eluting with methanol,the solvent was evaporated obtaining a 25-fold concentration of the analyte. This procedure was validatedby using 7-OHMTX as internal standard. Calibration curves had correlation coefficients always higher than0.999, and the limit of detection was assessed at 0.2mg Lÿ1. High specificity of the HPLC-MS/MS techniqueassures that no interfering substances are detected rather than the analyte of interest. Copyright# 2000John Wiley & Sons, Ltd.

Received 5 November 1999; Revised 6 December 1999; Accepted 9 December 1999

Methotrexate (MTX, 4-amino-N10-methylpteroyl glutamicacid, amethopterine) acts as an antimetabolite and is widelyused for the treatment of various malignant diseases,including acute leukaemia, osteosarcoma, non-Hodgkinlymphoma and breast carcinoma, as well as non-neoplasticdisorders.1–4 MTX is converted to polyglutamates, whichtake part in the cytostatic action of the drug, and to 7-hydroxymethotrexate (7-OHMTX), which is about 200-foldless effective than the parent compound.5,6

During preparation and administration, hospital person-nel may absorb antineoplastic drugs.7–9 Although theInternational Agency for Research on Cancer (IARC) hasincluded MTX in Group 3 (inadequate evidence ofcarcinogenicity in humans), MTX is a mutagen and ateratogen.10 Thus, all contact with this drug should bereduced to the lowest possible level. In particular, it hasbeen shown that MTX does not penetrate human skin,11 sothat, if no accidental events occur, the main exposure routeseems to be inhalation. Furthermore, biological safetycabinets (BSC) and personal protective equipment providefor worker safety and reduce exposure levels. Consequently,MTX urinary concentrations in occupationally exposedsubjects are very low, even less than 1mg Lÿ1.

Previously available analytical methods are mostlydesigned for pharmacokinetic studies and clinical monitor-ing,12–14and therefore, they lack the required sensitivity andcannot be used to assess professional exposure. Among thefew authors taking into account occupational hazards,Mader et al. developed an HPLC method with columnswitching which can determine nanomolar levels of urinary

MTX after purifying samples by solid phase extraction.15

Nevertheless, the limit of detection was 4mg Lÿ1 and nointernal standard was used. In addition, column switchingcan be considered to be a time-consuming procedure.

In this paper, a sensitive, specific and reliable HPLCmethod with tandem mass spectrometry detection ispresented. The procedure is based on sample cleanup andconcentration, using solid phase extraction (SPE), and isvalidated by using 7-OHMTX as internal standard. Highsensitivity and specificity, together with precision andaccuracy even at very low concentrations, allow applicationof the present method to the evaluation of occupationalexposure.

EXPERIMENTAL

Reagents and chemicals

Methotrexate and 7-hydroxymethotrexate were supplied by

Table 1. SPE columns, sorbent types and corresponding retentionefficiencies

SPE column Sorbent typeMTX retained bythe sorbent (%)

Isolute C18 Octadecyl bonded, endcapped silica 100Isolute C8 Octyl bonded, endcapped silica 100Isolute CN Cyanopropyl bonded, endcapped silica 99.32Isolute ENV� Styrene-divinylbenzene resin 100Isolute SAX Quaternary amine bonded silica with Clÿ

counterion85.91

LC-Diol Diol bonded silica 69.67LC-Si Silica gel with no bonded phase 99.1ENVI-Chrom P Styrene-divinylbenzene resin 100

*Correspondence to: R. Turci, Laboratory of Environmental Hygieneand Industrial Toxicology, S. Maugeri Foundation, via Alzaia 29,Pavia, Italy.E-mail: [email protected]

CCC 0951–4198/2000/030173–07 $17.50 Copyright# 2000 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom.14, 173–179 (2000)

Sigma-Aldrich, Inc. (St. Louis, MO, USA). HiPerSolv forHPLC2 ammoniumacetate(purity >98%), glacial aceticacid and methanol (purity >99.8%) were obtainedfromBDH Laboratory Supplies(Poole, UK). LichroSolv1 ethylacetate (purity >99.8%) was purchased from Merck(Darmstadt,Germany). Distilled and deionisedwater wasproduced from a Milli- Q Pluswaterpurification system.

Control human urine, used to preparedaily standard

calibrationcurvesand quality control (QC), was obtainedfrom non-exposedvolunteers.

Supplies

Isolute1 C18, C8, CN, ENV� andSAX columnsfor SPEwereused(IST House,Duffryn Industrial Estate,UK). LC-Diol, ENVI 2ChromP andLC-Si SPEtubes wereobtained

Figure 1. Efficiency of MTX elution dependingon sorbenttype.

Figure 2. Extractionefficiency dependingon sorbenttypeandappliedsamplevolume(elutionvolume= 3 mL;pH of thesample= 5).

Rapid Commun.MassSpectrom.14, 173–179(2000) Copyright# 2000JohnWiley & Sons,Ltd.

174 SPE-LC/MS/MS OF METHOTREXATE IN HUMAN URINE

from Supelco (Bellefonte, PA, USA). HPLC columns:Discovery2 C18,150� 4.6mm,5 mm (Supelco)andguardcolumn H50DS-10C� 5 (Hypersil, Cheshire,UK).

Preliminary study

SinceMTX is practically insoluble in all water-immisciblesolvents,SPEis more efficient thanliquid-liquid extractionfor sampleconcentration and cleanup. In addition, SPEallows changingthe matrix of the analyte if needed forsubsequent analyses. In order to obtain high extractionefficienciesandaslow a limit of quantitation aspossible, anumber of variables must be considered: sorbent type,elution solvent, samplevolume,elution volume,pH of thesample, interference from the elution solvent. Duringmethod development, each step was first optimised byloading pureaqueous solutionson SPEcolumns.Then,theprocedurewasappliedto urine samples.

The first stepis to selectthe properSPEtube,that is tosay the sorbentthat canbind the compound of interest. Inorder to measurethe analyteamountretainedon the SPEpacking, 3-mL aliquots of a 100ngmLÿ1 MTX solutionwere addedto eight different SPEtubes corresponding toeight differentsorbent types (Table1). Fromtheanalysisoftheeffluent, it appearedthatonly four tubescanretain100%MTX. Table1 showsthe MTX percentagesobtained afteradding spiked samples at pH 5. Each tube had beenpreviously conditionedwith 6 mL of methanol followedby6 mL of water. Theanalytewasfinally elutedwith 3 mL ofmethanol and evaporated to dryness. The residue wasreconstituted with 500mL of mobile phase (methanol/acetate buffer, 0.02 M, pH 4.0, 30:70, v/v) and 10-mLaliquots wereinjectedontotheHPLC column.Recovery ofMTX from eachtubeis shown in Fig. 1. Fromthese results,it seemsthatthebestSPEtubefor MTX extractionis ENVIChrom P. Nevertheless,when increasing samplevolume,Isolute C18 yields 85% MTX vs. 66% obtainedby usingENVI ChromP,asreportedin Fig. 2. Sinceloadinga larger

sample volume results in enhanced sensitivity, C18 waschosen asthemost suitableSPEtubefor MTX cleanupandconcentration.

The following stepwasaimedat evaluatingtheeffectofdifferent elution volumeson extraction efficiency. To thisend,10-mL aliquotsof a 100ngmLÿ1 MTX solutionwereadjustedto pH 5, addedto theSPEcolumnandelutedwith1, 3, 6 and 10mL of MeOH. Although no significant dif-ferences are observed (Fig. 3), elution with 3 mL ofmethanol ensuresthe highestanalyteextraction efficiency(81%).

In orderto prevent ionisationof theanalyte,thepH of thesample must beadjustedsoasto enhance retention on SPEtubes.An experimentin duplicatewascarriedout to controltheinfluenceof thesamplepH on recovery.Figure 4 showsthat pH values from 3 to 5 can maximise the extractionefficiencyof MTX. Consequently, theintermediatevalueof4, corresponding to a MTX mean recoveryof 88.2%, waschosen asthe optimum pH of the sample.

Extraction procedure

A Visiprep2-DL SPEvacuummanifold (Supelco)wasusedto simultaneouslyprocessup to 24 samples. Isolute C18(packing 500mg/ 10mL column reservoir) solid phaseextraction tubes were conditioned with 6 mL of methanoland6 mL of 0.02M ammonium acetatebuffer(pH 4.0).Thecolumns were preventedfrom drying beforeapplying theanalytesolutions.A 5-mL urinesamplewasdiluted1:1withacetate buffer and,after vortex mixing, the entire volume(10mL) wasapplied to theC18columnswith a flow rateof1 mL minÿ1. Thereafter,the cartridges were washedwith6 mL of ethylacetate,whichcanremoveurineimpurities, asreportedby Mader etal.15 Theanalytewaselutedwith three1-mL aliquotsof methanol. In thisstep, dropwiseflow ratesare required so as to increase recovery. In addition, eachaliquotshouldremain in contactwith thesorbent for at least

Figure 3. Extraction efficiency dependingon elution volume (applied samplevolume= 10mL; pH of thesample= 5).

Copyright# 2000JohnWiley & Sons,Ltd. Rapid Commun.MassSpectrom.14, 173–179(2000)

SPE-LC/MS/MS OF METHOTREXATE IN HUMAN URINE 175

20 seconds.A mild vacuumwasapplied to themanifold tocarryout thesampleloadingandinterferenceelution steps.

The eluate wasevaporated to drynessundera streamofnitrogen and the residue was redissolved in 200mL ofmobile phase and filtered with polypropylene syringemicrofilters before injection onto the HPLC column.

Instr umentation and chromatographic conditions

All thesampleswereanalysedonanHPLC-MS/MSsystem.It consistedof a triple quadrupole massspectrometerAPI300(Perkin Elmer-Sciex,Toronto, Canada)interfaced to anHPLCsystem(Perkin Elmer,Norwalk, CT, USA) incorpor-ating a Series200 LC quaternary pump. Separationwascarried out on a Discovery C18 (150� 4.6mm i.d., 5 mmfilm thickness)equippedwith a guard column H50DS-10C� 5 (samepackingmaterial).

The isocratic mobile phasewas methanol/ammoniumacetate,0.02M, pH 4.0(30:70,v/v), deliveredata flow rateof 1 mL minÿ1. A volumeof 10mL wasinjectedona 20mLloop. The HPLC column eluate was split 1:20 beforeentering the mass spectrometer ion source. The retentiontimes were 2.51 and 3.81 for MTX and 7-OHMTX,respectively.

Massspectrometry

The triple quadrupole mass spectrometer, operating inpositive-ion mode, was equipped with an atmosphericpressure ionisation (API) source and with an ionsprayinterface.An Apple2 Macintosh1 System7.5.3wasusedfor instrument control, dataacquisitionanddataprocessing.Polypropyleneglycol wasusedto calibrate the instrumentand to adjust the resolution to 0.7 m/z units over the m/zrange100–600for HPLC-MS/MSacquisition.

The ionsprayvoltagewas� 5800V. The [MH]� ions ofMTX (m/z 454.9) and 7-OHMTX (m/z 471.0) weretransmitted throughthe first quadrupole (Q1) and into the

collision cell (Q2). The productions for MTX (m/z308.4)and internal standard(m/z324.1)were monitoredthroughthe third quadrupole(Q3). Thedwell time perchannel was1500ms. Collision-activated dissociation (CAD) takesplace in the collision cell where the mass filtered ionscollide with CAD gas (Elab = 6 eV). The pressure ofnitrogenwas8 mTorr (1 Torr = 133.3Pa).

Validati on study

Calibrationsampleswerepreparedby spiking5 mL of urinefrom non-exposed subjects with 50mL of appropriatesolutionsin order to obtainMTX concentrationsof 0.5, 1,2,4,8 and16mg Lÿ1. To assessthereliability of themethod,7-OHMTX wasusedasinternalstandard andaddedto eachurine sample in order to obtain a final concentration of40mgLÿ1. The volume addedwasalwayslessthan 2% ofthe total volumeof the samples,so that the integrity of theurine was maintained. Then, the samples were seriallydilutedwith 5 mL of acetate buffer (pH 4.0)andpurified asdescribedin the “Extractionprocedure” section.

Theratiosof thepeakareaof MTX to thatof 7-OHMTXwere plotted against nominal concentrations. Standardcalibration curves were obtained from linear regressionanalysisof the data, andthe resulting slopesandinterceptswereusedto back-calculateconcentrationvaluesfor qualitycontrol samples. Peak areas were measured by a postacquisitiondataanalysisprogram(MacQuan2), which alsocreatedcalibration curvesandcalculatedthecoefficients ofcorrelation.

Both theintra- andinterdayprecisionandaccuracyof themethodweretested.To this aim, 5-mL urine aliquotswerespikedin quadruplicate at threeconcentrations, low (1.6mgLÿ1), middle (3.2mg Lÿ1) and high (6.4mg Lÿ1) on threesubsequentdays.Accuracywasevaluatedby calculating thepercentdifferencebetween the measured meanconcentra-tions and the corresponding theoretical ones.The limit ofquantitation (LOQ) is the lowestconcentration that canbe

Figure 4. Extractionefficiency dependingon pH (appliedsamplevolume= 10mL; elution volume= 3 mL).



determinedwith aninterday accuracyandprecision�20%.Thelimit of detection(LOD) wasdefinedasthelowestdrugconcentration that the analytical process can reliablydifferentiate from backgroundlevels, that is to say theconcentration yielding a signal intensity three times thebackgroundvalueexpressed as% of standard deviation.

RESULTS AND DISCUSSION

HPLC/MS/MS

A typical chromatogramof urineblank samplespikedwith

3.2mg Lÿ1 of MTX and40mg Lÿ1 of 7-OHMTX (I.S.) isshown in Fig. 5. A lower sensitivity for 7-OHMTX wasobserved,aspreviously reported.16 Theretentiontimeswere2.51 and3.81for MTX andI.S. respectively. However, thechromatographic run lasted 8 min in order to have thecolumn properly washed,as recommendedwhen routineanalyses are performed. The selective MS/MS detectionassuresa high specificity sothatno interfering peaksaffectthequantitativeresults. In addition,becauseof its simplicityand sensitivity (LOD = 0.2mg Lÿ1), this procedure istimesavingandsuitablefor routineanalysesof urine fromhospital personneloccupationally exposedto antineoplastic

Figure 5. Representativechromatogramobtainedfrom theanalysisof blankhumanurinespikedwith 3.2mg Lÿ1

of MTX and40mg Lÿ1 I.S. (7-OHMTX).



drugs and working under protective measures. Injectiononto theHPLC systemcanalsobeautomated to saveextratime.

Validation study

Linearity was assessedby plotting peak area ratio (Y)againsttheoreticalMTX concentration(X) from 0.5to 16mgLÿ1. Theobtainedregression curves on threedifferentdaysare shown in Fig. 6. The correlation coefficients werealwayshigher than 0.999and intercepts were not statisti-cally different from zero.

Intra- andinterday precisionandaccuracyof themethodwere assessedby analysing in quadruplicate threequalitycontrol samples(1.6, 3.2, 6.4mg Lÿ1) on threesubsequentdays.Thewithin-dayCV% ranged from 6.8to 16.2%, whileday-to-dayCV wasalwayslessthan15%.The methodwasshown to beaccurate with a relativeerror<10 and12%forinter- and intraday experiments, respectively. The resultsarepresentedin Table2.

The limit of quantitation, determined on four replicateson threedifferent days,was0.5mg Lÿ1. At this level, day-to-day precision,expressedas the coefficient of variation(CV%), was less than 20%. Interday accuracywas alsoadequateandequalto 100.2%.Thelimit of detection (LOD)was 0.2mg Lÿ1. Most of the published methodswere not

validatedfor urinesamplesat low MTX concentrations. Theprocedureproposedby Mader15 hasbeenvalidated,but it isnot sensitive enoughto measure the expected low urineconcentrations.Moreover, column switching is somewhatcomplicatedandtime-consuming.

CONCLUSIONS

A very sensitive, specific and reliable high-performanceliquid chromatography/ionspraytandemmassspectrometrymethod (HPLC-MS/MS) has beendevelopedto quantifyeventracelevelsof methotrexate(MTX) in human urine.Inparticular, the solid phase extraction (SPE) procedureenablesa 25-fold preconcentration of the analyte,whichcan be further enhanced by increasing the appliedsamplevolume.Nevertheless, thiswouldrequireadditionaltime, sothat we prefer loading a sample volume of 5 mL, whichmakesthismethodsuitablefor routineanalyses. However, alimit of quantitation of 0.5mg Lÿ1 allows biologicalmonitoring of hospital personnelinvolved in preparationand administration of MTX. Moreover, the on-line chro-matographic separation with MS/MSdetection assuresbothhigh specificity andsensitivity, with a limit of detection of0.2mg Lÿ1. This procedure is alsosuitablefor the analysisof aqueous matrices (wipe samples,pads, air samples,

Figure 6. Regressioncurvesobtainedon threedifferent days:the arearatio wascalculatedby adding7-OHMTX asinternalstandard.

Table 2. Intra- and interday precision and accuracy of the method for determination of urinary MTX

MTX theoreticalconcentration(mg/L) n

MTX experimentalconcentration(mg/L)(mean� SD) CV (%) Accuracy(%)

Intraday precision 1.6 4 1.79� 0.29 16.2 111.93.2 4 3.21� 0.22 6.7 100.26.4 4 6.99� 0.59 8.4 109.3

Interday precision 1.6 3 1.72� 0.25 14.7 101.83.2 3 3.24� 0.25 10.7 101.46.4 3 7.03� 0.51 7.3 109.9



gloves) and can therefore be applied to environmentalmonitoring.

To summarise,the proposed methodis simple, fast andselective. Sinceevenvery low exposure levelsmayresultina health hazard,high sensitivityis required. In this respect,this procedurecanbesuccessfully usedto identify themainexposure routes and thus improve the safety of hospitalpersonnelhandlingantineoplastic drugs.

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determination of methotrexate in human urine at trace levels by solid phase extraction and...

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