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Copyright© ABE&M todos os direitos reservados. 844 DOI: 10.1590/0004-2730000003347 Accepted on21/Ago/2014 Received on11/Mar/2014 [email protected] 04344-010 –SãoPaulo, SP, Brazil Av. General Valdomiro deLima,508 José GilbertoHenriques Vieira Correspondence to: 1 Fleury, SãoPaulo, SP, Brazil original article original SteroidLaboratories,Grupo with the RIA for salivary cortisolwith theRIAforsalivary measurement,withtheadditionalpossibilityofconcomitantcor massas (HPLC-MS/MS)paraamedidadecortisol ecortisona salivares. cia nodesenvolvimento demetodologiabaseadaemcromatografia líquidaeespectrometriade screening coleta epresençalimitadade interferentes têmoriginadomúltiplas publicações, emespecialno no estudodafunçãoadrenocortical. Suaaltacorrelação sérica,facilidadede comafraçãolivre cortisol andlinearityfrom24to1929 ng/dL.Salivacortisol valuesobtainedinthe181 samplespre mass spectrometry method liquid chromatography-tandem inhuman salivacortisone by a and Determination ofcortisol Objetivo: RESUMO tisone measurementandtheevaluationof11 β (r =0.820,P<0.0001). ng/dL, with 5-95% percentile of 100 and 1,133 ng/dL. Correlation with cortisol values was significant high correlation (r=0.8312, P<0.0001). measuredin159 samplesshowedamedian of 278 86, 25and436ng/dL,respectively, and withvaluesforRIAbeingsignificantlyhigher(P<0.0001) sented amedianof52ng/dLwith5-95%percentile24and374 ng/dL. With theRIAresultswere was cortisol usedforsalivary resultscomparison. mine transitionsmonitoredincludedcortisol andcortisone. An in-houseradioimmunoassay(RIA) and cortisone measurement. sure liquidchromatography cortisol tandemmassspectrometry(HPLC-MS/MS)methodforsalivary of adrenal hyperfunction. In this paper we present our experience in the development of a high pres interfering , generated multiple recent studies of its application, in special in the screening tion. Itshighcorrelation withfreeserumcortisol, theeasyofsamplingandlimitedpresence Objective: ABSTRACT José Gilberto Henriques Vieira em cromatografia líquidaeespectrometria demassasem tandem nasaliva ecortisona Determinação por método decortisol baseado sona e avaliação da atividade daenzima11sona eavaliaçãodaatividade β para amedidadecortisol salivar, comapossibilidadeadicional damedidaconcomitantedecorti Concluímos queométodobaseado emHPLC-MS/MScompara-sefavoravelmente comoRIE ng/dL. A correlação comosvaloresdecortisol (r=0,820, P <0,0001). foisignificativa medida em159 amostras, mostroumedianade278ng/dL,compercentis5-95%entre100 e1.133 e alta correlação Cortisona, mais elevados (P < 0,0001), (r = 0,8312, significativamente P < 0,0001). comosvaloresobtidosnoRIE Com oRIE,osresultadosforam86,25e436ng/L,respectivamente, dos nas181 amostras apresentaram medianade52ng/dL,compercentis5-95%24e374 ng/dL. noensaio (RIE)inhousefoiempregado paracomparação. ção com hidroxilamina, as transições monitoradas incluíram cortisol e cortisona. Um radioimu HPLC-MS/MS baseou-senumespectrômetrodemassas Waters Quattro Premier. Após derivatiza Para denossarotinadiagnóstica. esteestudoutilizamos181 amostrasdesaliva A metodologiade Premier tandemmassspectrometerwithanelectrosprayprobe. After withhydroxyla derivatization from ourroutinediagnosticlaboratory. The HPLC-MS/MSmethodwas basedona Waters Quattro Salivary cortisol; salivary cortisone; mass spectrometry; radioimmunoassay; hypercortisolismscreening cortisone;massspectrometry; cortisol;salivary Salivary Keywords Cortisol salivar;cortisona salivar;espectrometriademassa; radioimunoensaio; triagemdehipercortisolismo Descritores Valdemir Melechko Carvalho para cortisol foide24ng/dL,comlinearidade entre24e1,929ng/dL.Osvaloresdecortisol obti A dosagem de cortisol salivar é uma metodologia que vem tendo crescente aceitação A dosagemdecortisol éumametodologiaquevemtendocrescenteaceitação salivar de pacientessuspeitoshiperfunção.Nestetrabalhoapresentamos nossaexperiên­ Salivary cortisol measurementplaysanimportant roleintheevaluationofadrenalfunc Conclusion: Materials andmethods: We concludethattheHPLC-MS/MSmethodcomparesfavorably 1

1 , Odete Hirata Nakamura HSD2. HSD2 activity. Results: Arq BrasEndocrinolMetab. 2014;58(8):844-50 For this study we used 181 saliva samples For samples thisstudyweused181 saliva Functional sensitivity was 24 ng/dL for Functionalwas sensitivity 24ng/dLfor Resultados: Arq BrasEndocrinolMetab. 2014;58(8):844-50 Arq Bras Metab. Endocrinol 2014;58/8 1 ,

A sensibilidade funcional A sensibilidadefuncional Materiais emétodos: Conclusão: ------

Arq Bras Metab. Endocrinol 2014;58/8 oftech specificity (13-15).Theintroduction necessary toachievethe inorder extraction andchromatography metabolites, demands inspecialcortisol fering steroids, ofseveralendogenousinter case inurine,thepresence (1,2).Inthe steroids trations ofendogenousinterfering concen andthelow relative molecular weightprotein coupledtoahigh againstcortisol-3-oxime produced vation isvaliddespitethehighspecificityofantisera enzymaticdefects.Thisobser in patientswithadrenal and use(e.g.) with syntheticcorticosteroids mainly cortisol, immunoassaysfortotalserum routine within laboratories,ishighlydesirable(12). method,comparable ofareference The introduction (HPA) conditions. andstress axisactivityundernormal abouthypothalamic-pituitary-adrenal tant information impor canprovide cortisol it wasshownthatsalivary assessment.Actually inabiomarkerforstress cortisol salivary samplecollectionturns (10,11). Thestress-free pathologies,inspecialCushing’s syndrome of adrenal forthescreening frequently andmore being usedmore insalivais cortisol intervals, atshort can berepeated is easy, bythepatientathome,and canbeperformed (9). Because saliva sample collection high correlation showed fractioninserum withfree when compared successfullyadapted (7,8)and several methodswere fractionisthemaincomponent, and since thefree fluid, insalivaorparotid ofcortisol the measurement Yet promising. anotherpossibilitywas very were results (6), and fractionisfiltered urine, sinceonlythefree equilibrium dialysis(5). the availableRIAsandserum describedwithassaysmostlybasedon CBG levels,were of excludingtheinterference fraction,therefore the free ceptives use.Theneedofmethodsfortheevaluation contra andsteroid conditions likepregnancy clinical deficiencies (hypoCBGnemias)(4),tofrequent inCBGlevelsandaffinity,fere familialCBG rare from S INTRODUCTION dependent on CBG levels. Several circumstances inter dependent onCBGlevels.Severalcircumstances strongly orplasmaare inserum levels oftotalcortisol (~4%) and, consequently,to (~6%) or free the notboundtoCBGiseitherweakly (3). Cortisol andspecificity withhighaffinity endogenous globulin (CBG), an bound to -binding mostly (90%) circulates laboratories (1,2). Cortisol availableindiagnosticandresearch assays toberoutinely Analytical specificity shows punctual problems for Analytical specificityshowspunctualproblems in cortisol was to measure Another alternative assay (RIA) was one of the first hormone hormone assay (RIA)wasoneofthefirststeroid byradioimmuno measurement totalcortisol erum ------convenience anddiagnosticspecificityofsalivacorti evidenceforthe strong provides Cushing’s syndrome specifictechniques. formore driving theinterest (14), of5to10%withcortisone shown cross-reactivity 3-oximederivatives,eventhebestones, against cortisol Theantiseradeveloped much higherthaninserum. sequently, are levelsofcortisone/cortisol therelative binding.Con cortisol (MR)from receptors corticoid 11β epitheliumcellsexpress RIA-based techniques.Salivary forthespecificityof tisone poseapotentialproblem inurine,thehighlevelsofcor metabolites) present (inspecialcortisol steroids majority oftheinterfering levels. cortisone concomitantly free measure to theopportunity (16,17), andadditionallyprovided ofthegeneralcharacteristicsassays improvement inthe adefinitivestepforward were cortisol urinary offree forthemeasurement tandem massspectrometry associated with niques based in liquid chromatography dian variation as wellincludesmall number ofphar quently, expectedtocomprisecirca valueswere cortisol bytheattendingphysician.Conse to theprescription according of collectionandthenumber ofsampleswere 23:00 to 24:00; time 16:00 to 17:00 and from 9:00, from 8:00 to timewindows:from three liva samplesobserved stableintheseconditions(24).Collectionofsa tents are at -20°C. Saliva and its con stored samples were erwise at 4°C until analysis thattook place in 24 upto 48h, oth centrifugedandsalivastored were rived atthelaboratory andassoontheyar Germany) (Sarstedt, Numbrecht, nostic laboratory. Salivawascollected usingSallivettes diag ourroutine For thisstudyweusedsamplesfrom Samples MATERIALS ANDMETHODS RIA. nique withofatime-validateddirect andthecomparisonofnewtech sol andcortisone corti ofsalivary MS/MS methodforthemeasurement ourexperiencein the developmentofaHPLC- present methods (HPLC-MS/MS)(20-23).Inthispaperwe based tography coupledtotandemmassspectrometry liquidchroma ofhigh-pressure with theintroduction assays ofsalivacortisol for thepotentialimprovement stimulus (10,11,18,19). Thesepublicationsprovided withemphasisforlate nightsamples sol measurements, that converts cortisol to cortisone, protecting mineralo protecting tocortisone, cortisol that converts Recent literature concerning the screening for the screening concerning Recent literature In salivasamples,inspiteoftheabsence -hydroxysteroid dehydrogenase type2(11β dehydrogenase -hydroxysteroid Salivary cortisol and cortisone by HPLC-MS/MS andcortisone Salivary cortisol -HSD2) -HSD2) 845 ------

Copyright© ABE&M todos os direitos reservados. Copyright© ABE&M todos os direitos reservados. acquired from CambridgeIsotopeLaboratories.Anali from acquired and standards, usedasinternal code DLM-9142)were (2,2,4,6,6,12,12-D7,98%, DLM-2218) andcortisone 846 minicolumns (Phenomenex, Torrance, CA,USA).After (20/80, v/v)andthe­ ized witha0.2mol/Lzincsulphate/methanol solution firstdeprotein Saliva samples(0.5mL)were processing. of each(inwater)wasadded tothesalivasamplesprior to2ng corresponding quot of2µLeachstandard, (9,11,12,12-D4, UK). Deuteratedcortisol (Waters/Micromass, probe an electrospray Manchester, Waters with tandemmassspectrometer Premier Quattro The assaywasbasedona2777Waters autosampler anda HPLC-MS/MS and inter-assay of8.7and10.7%,respectively. precision 25to750ng/dL, andintra-assay linearity rangingfrom bound phases.Functionalsensitivitywas25ng/dL,with solutiontoseparateboundandun tran-coated charcoal land Nuclear, Boston,USA)wasusedastracer, anddex (20 µL) addeddirectly, were (NewEng tritiatedcortisol RI,USA.Salivasamples Steraloids Inc.,Newport, from (F9-1) purchased were isdepictedintable1.Allsteroids samples (9).Thespecificitystudyfortheantiserum liva albumin(2), modifiedforsa coupled tobovineserum derivative by immunizingrabbitswithacortisol-3-oxime describedinouroriginalpublication, produced antiserum Radioimmunoassay: theinhouseRIAmethodused procedures Assay PA,(David RoadsAssoc.,KennettSquare, USA). cision points and method validation was EP Evaluator used for extrapolatingde CA, USA)andtheprogram non-parametric (Prism6,GraphPadInc,SanDiego, The statisticalmethodsusedforassaycomparisonwere Statistical methods to2.76nmol/L). 100 ng/dLcorresponds To tonmol/Lmultiplyby0.0276(e.g. cortisone. convert MS/MS. Values and inng/dLforcortisol expressed are byHPLC- alsomeasured levelswere samples, cortisone evaluatedusingbothmethodsandin159ofthese were the newHPLC-MS/MSmethod.Atotalof181samples with contentsmeasured andcortisone had theircortisol RIAmethod,samples usingtheroutine cortisol salivary included).Afteranalysisfor Cushing’s diseasepatientsare twoknown and pathologicalconditions(samplesfrom macological inducedvariation(post-Dexametasoneuse), by HPLC-MS/MS andcortisone Salivary cortisol supernatant extracted in Strata X extractedinStrataX supernatant

98%, code 98%, code ------

Vac) andderivatizedwith0.3mLofa1.5mol/Lhy evaporated (Speed methanol elution, the samples were steroids listed in table 1, were studied,showingno in listedintable1,were steroids solutions of 5,000 ng/dL of each of the study were software. AnalysisemployedtheQuanLynx cortisone). and398>167398>149(D7- 391>149 (cortisone) 391>167and sol), 397>136and397>91(D4-cortisol), m/z393>136and393>91(corti were tions monitored positivemode.Thetransi MS operatedinelectrospray analysedbytheMS/ minutes. Theeluatewasdirectly 60% methanolofthemobilephasewasappliedin3.25 2to nol at0.3mL/min.Alineargradientrangingfrom andmetha eluted with0.5mol/Lammoniumformate tem thatconsistsinaOnyxC1825x4.6mmcolumn in the HPLC sys injected directly of the solution were min at70°C.Aftercoolingandcentrifugation,200µL (50:50, v/v) solution for 90 droxylamine/methanol reference fortheHPLC-MS/MSmethod quantification 24ng/dLforcortisoland100cortisone)as with(limitof (AUC) percentage (%)intermsofareaunderdecurve percentage (%), accordingto Abraham (24)andthesamesteroidsas Table 1. Cross-reactivitylevelsofantibodyF79-1, ascalculatedin Triancinolone 6α-metilprednisolone Prednisone Dexametasone Betametasone 5β- 5α-tetrahydrocortisol 6β-hydroxycortisol 20β-dihydrocortisol 20α-dihydrocortisol 5β-dihydrocortisol 5α-dihydrocortisol 21-deoxycortisol Cortisol-21-sulphate Cortisol-21-glucoronate 5β- DOC Cortisone Cortisol Steroid The validationoftheassayincludedspecificity < 0.012 < 0.012 < 0.012 < 0.012 RIA (%) 0.075 Arq Bras Metab. Endocrinol 2014;58/8 17.7 52.0 0.03 0.82 0.09 10.0 12.2 0.37 0,26 0.42 100 3.1 5.0 2.5 2.8 1.2 8.5 Cross reactivity HPLC-MS/MS (AUC) < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 100 100 (%) ------Arq Bras Metab. Endocrinol 2014;58/8 with theRIAand theHPLC-MS/MSmethods. Figure 1. cortisolmeasurementsobtained Correlationbetweensalivary 1. isdepictedinfigure measures sentation ofthepaired than theobtainedwithHPLC-MS/MS.Agraphicpre two methods;valueswiththeRIAmethodbeinghigher results obtainedwiththe between significant difference a testshowedaPvalueof<0.0001,confirming matched-pairssigned The applicationoftheWilcoxon a high positive value of r = 0.8312 (P < 0.0001). provided correlation ≤24ng/dL.Spearman 48/181 sampleswere 5%24ng/dLand95%374ng/dL; ng/dL, percentile median52 HPLC-MS/MS method,thenumberswere: the ≤24ng/dL. With 95% of436ng/dL;17/181were 5%of25ng/dLand of 86ng/dL,withthepercentile In the181samplesRIAmethodshowedamedian RESULTS value of 578 and 1453 ng/dL for cortisone. value of578and1453ng/dLforcortisone. respectively, and9.16.8%forsampleswithmean and 5.3%forasamplewithmeanvalueof413ng/dL, 7.9% forasamplewithmeanvalueof84ng/dLand4.7 5.6and were forcortisol Intra andinterassayprecision and90.3to101.3%forcortisone. 107.1%, forcortisol studiesvariedbetween90,4and recovery for cortisone; 100to2,400ng/dL 24to1920ng/dLforcortisol, from and100ng/dLforcortisone. 24 ng/dLforcortisol (AUC),was underthecurve ofthearea in thereading Functional sensitivity, definedasaCVoflessthan20% solutionforassayusewas1%DMSO/water.standard based onDMSOsolutionsof1mg/mL.Thefinal inhouse prepared were standards Allsteroid terference.

Salivary cortisol HPLC-MS/S ng/dL Linearity, studiedby sequentialdilution,ranging 1000 100 10 10 p <0.0001 n =181 Salivary cortisol RIAng/dL Salivary 100 1000 -

and 95 percentile of 1,133 ng/dL; 16 samples were of1,133ng/dL;16 samples were and 95percentile of100ng/dL median of278ng/dL,with5%percentile a lection showed the expected (Figure 3). rhythm(Figure lection showedtheexpectedcircadian valuesagainsttimeofsamplecol Plottingcortisol result. and X to theRIA sponds to the HPLC-MS/MS result Ycorre sulted intheequationY=0.85X-11-4,were values with the two methods re analysis of the cortisol valuesofsamples.Regression withincreasing increased higherandthatthedispersion tained withtheRIAare showclearlythatthevaluesob samples. Bothfigures average ofthetwoindividualvaluesforeach181 (HPLC-MS/MS–RIA)againstthe two measurements (ng/dL)obtainedbetweenthe senting thedifferences shown as a Bland-Altman plot pre 2 data are In figure man correlation of r = 0.820 (P < 0.0001) (Figure 4). ofr=0.820 (P<0.0001)(Figure man correlation asignificant Spear values,butpresenting than cortisol significantly higher valueswere ≤ 100ng/dL.Cortisone 23:00 to24:00h. the threewindowsofsamplecollection: 8:00to9:00, 16:00to17:00 and Figure 3. Median, 25, cortisolobtainedin and75percentilesofsalivary against theaverageoftwoindividualvaluesfor181samples. cortisolmethods(HPLC-MS/MS–RIA)plotted between thetwosalivary Figure 2. Bland-Altman presentation of the differences (ng/dL) obtained

Cortisone values obtained in 159 samples showed valuesobtainedin159samplesshowed Cortisone Cortisol HPLC-MS/MS ng/dL Difference 100 200 300 -400 -200 200 400 0 0 4 8 Salivary cortisol and cortisone by HPLC-MS/MS andcortisone Salivary cortisol 100 Time ofcollection 12 16 20 1000 Average 24 847 ------

Copyright© ABE&M todos os direitos reservados. Copyright© ABE&M todos os direitos reservados. 848 antibodies,aswaspatent forfree the bestanti-cortisol fortraditionalRIAmethods, evenemploying is true This steroids. concentration ofpotentially interfering ofahigher is thepresence measurements cortisol vary an canbeviewedastheinvivoequivalentto production, filtrationorsaliva CBG isabsent,sothatglomerular behind thiskindofmethodology(30). the assay, withallthepracticalandeconomiclimitations need ofequilibriumdialysisorultrafiltrationpriorto bemet,includingthe requirements imply thatcertain valid methods(5),that,tobetruly cortisol serum free fraction(29)andledtothedevelopmentof to thefree isrestricted plies thatthebiologicallyactivehormone hypothesisim hormone bound toCBG(3).Thefree circulates (CBG) levels, sinceupto 90% ofcortisol thefluctuationsofbinding protein are surements mea cortisol ofclinicalspecificityinserum problem real analyticallyspecificassaysisdoubtful. The for more low, are steroids of potentialinterfering andtheneed thelevels cortisol, ment isbeingmade.Fortotalserum themeasure pointisthemediawhere one important measurements, cortisol (27,28).Concerning Carvalho by recently than20yearsago(26)andreviewed more by Schackleton ology toHPLC-MS/MS,aspredicted RIA-basedmethod graduallyshiftingfrom tories are labora inclinicalandresearch measurements Steroid DISCUSSION calculation providedanr=0.820(P<0.0001). obtained by HPLC-MS/MS in 159 samples. Spearman correlation Figure 4. by HPLC-MS/MS andcortisone Salivary cortisol dialysis. One problem of free urinary orsali urinary offree in vitrodialysis.Oneproblem Cortisol is present in urine and saliva, media where inurineandsaliva,mediawhere ispresent Cortisol Salivary cortisone ng/dL 10000 1000 100 Correlation between salivary cortisol and cortisone values Correlation between salivary 0 p <0.0001 r =0.820 n =159 200 Salivary cortisolng/dL Salivary 400 600 800 ------points shouldbediscussed.First,salivaiseasytocol of HPLC-MS/MScomparabletechnology. assumingtheuse comparableresults, lower andmore the newmethodology(16).Higherspecificityimplies RIA techniques,madeclearthespecificityadvantageof and MS/MS, substitutingtraditionalchromatography usingHPLC- measurement cortisol urinary of free use.Ourexperiencewiththeintroduction laboratory primeforclinical nowadays, theymaybeconsidered of HPLCandMS/MSequipmenttechnologyand, use,paralleledthedevelopment itsroutine concerning ago (25).Thelongmaturationofthemethodology, than40years ofRIAmethodsmore to theintroduction is a landmark equivalent measurements clinical steroid methods (13,14). sincethefirstdescribed measurement cortisol urinary and the cumbersome 24h urine collection are circum and the cumbersome 24h urine collection are ortotal)sampling (free cortisol forserum venipuncture at home by the patient (11). The need of be performed severaltimesinsequence andcan lect, canberepeated our data is quite similar to one reported recently by recently our dataisquite similartoonereported methodology. equationgeneratedwith Theregression analytical specificityoftheHPLC-MS/MS the greater wasexpected infunctionof RIA technique.Thatresult nificantly lowerthantheones obtainedwiththedirect sig obtained withtheHPLC-MS/MS methodwere values cortisol (20-23).Salivary intheliterature ported inseveralcircumstances. potential interest theevaluationof11βHSD2activity,ples permits with inthesalivasam measuring concomitantlycortisone (32).Theadditionalpossibilityof of sodiumexcretion forthecontrol intestinal epithelium,tissuesresponsible epithelial tissues,mainlyinthekidney, sweatglandsand inother fortheMR(31).11βHSD2ispresent affinity thathasno tocortisone (MR) bymetabolizingcortisol receptor tector ofthespecificitymineralocorticoid of theenzyme11βHSD2.Thisactsasapro epitheliumofharboringsignificantquantities salivary essential. are collection inthehours before avoiding teethbrushing of oral washingand with anyoralbleeding.Instructions per, pointistoavoidsalivacontamination theonlyreal cial samplingdevices,liketheonedescribedinthispa theavailabilityofcommer long periods(24).With for insalivaisstableandcanbestored vented. Cortisol Concerning salivary cortisol measurements, several measurements, cortisol salivary Concerning ofHPLC-MS/MSmethodsto The introduction Our results are comparable to the ones recently re comparabletotheonesrecently are Our results Another pointtobediscussedisthepeculiarityof Arq Bras Metab. Endocrinol 2014;58/8 ------Arq Bras Metab. Endocrinol 2014;58/8 of thearticle. the development, draftingandapproval in theirparticipation Author contributions:allauthors confirm of thismethodologybyclinicians ingeneral(34). described inthispaper, theacceptance shouldincrease and specificity,methods of highprecision liketheone collection bythepatient,andnowmaturationof devices, theeasinessandoutofclinic/hospitalsample the lastdecades.Theavailabilityofsimplecollection liva isamethodologythathasbeeninmaturationfor MS/MS requires. lab based on HPLC- training, that a steroid and staff investments in installation, equipment the important havemade inlaboratoriesthatalready cost-effective only buttheyare cortisone, forsalivary surement betterspecificity,provide allowtheconcomitantmea method.HPLC-MS/MSme­ ple andaffordable a sim measurement cortisol salivary direct tive) turns labelingtechniques(non-radioac use ofalternative able, whencomparingtoLC-MS/MSmethods.The simpler,quality antisera)are cheaper and wildly avail (mainly whenusinghigh cortisol niques forsalivary thetubules. filtrateflowsthrough glomerular added as the is located in tubular cells, withcortisone saliva;inurinetheenzyme reaching duction directly pro glandacinarcells,withcortisone activity insalivary the11βHSD2 higher. concerns Thesecondobservation is of cortisone proportion (3), consequently thefiltered fractionofcortisol ishigherthanfree tion ofcortisone frac tions. Thefirstoneisbasedonthefactthatfree These findingscanbeexplainedmainlybytwoobserva 5.0. isaround of cortisol/cortisone the relation were theoneinserum, from completelydifferent bers are (17,33),andbothnum of0.28wasreported relation values using similar methods (33). In urine a reported to of0.19,whichisinaccordance cortisol/cortisone levels, showed a mean relation higher than the cortisol collected salivasamples,shouldbeexpected. withlatenight thods, inspecialforCushing’ssyndrome me­ forscreening Consequently betterperformance precision. valueswithmore the definitionofcut-off based inHPLC-MS/MSmethods,andshouldpermit theyare assaycomparison,provided realistic and more Thisopenthepossibilityofeasier saliva measurement. forthe precision more with HPLC-MS/MSprovides Additionally, ofvariationobtained thelower coefficient McWhinney andcols.(33),utilizingsimilarmethods. The analysis of , in special cortisol, insa inspecialcortisol, The analysisofhormones, a methodological point of view,From RIE tech valuesfoundinthisstudy, cortisone The salivary thods ------closure: José Gilberto Henriques Vieira, Odete Hirata Naka HenriquesVieira, JoséGilberto Disclosure: 7. 1 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. REFERENCES (FLRY3). po Fleury ownsstockofGru HenriquesVieira ry, SãoPaulo.JoséGilberto Fleu mura, Valdemir employees,Grupo are MelechkoCarvalho

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