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Mass Spectrometry3 Has LC/MS/MS becomethe technique of choicefor corticosteroid analysis?9 ASMS 2013 Preview© pjmorley / Shutterstock.comSPONSORS11 Q&A: ionisation methods,detection limits andion mobility13 MS-based multi-analyteassays for the targetedquantification ofendogenous compoundsEuropean Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com 1 Volume 18 | Issue 2 | 2013

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© pjmorley / Shutterstock.comIN-DEPTH FOCUSCorticosteroids andmass spectrometry; latestapplications using LC/MS 3András SzeitzFaculty of Pharmaceutical Sciences, University of British ColumbiaThe adrenals were identified in man more than 400 years ago, but in the followingfew hundred years, not much progress was made to learn about their role 1 . This delaywas certainly made up for by the remarkably quick development of thecorticosteroids (CSs), the hormones of the adrenals, beginning with their isolationfrom the adrenal glands in the late 1920s to their widespread use as therapeuticagents in the present day. CSs can be analysed by several methodologies, and thisbrief review is focused on the mass spectrometry techniques for the determination ofCSs in a variety of matrices.IntroductionThe adrenal glands were identified by Eustachi,in 1563 1 , and their physiological importancewas discovered by Addison in 1855 2 , and laterproven by Brown-Séquard in 1856 3 . By 1927-30,numerous adrenal cortex extracts had beenisolated 4-8 , and in 1937, Reichstein synthesised11-desoxycorticosterone, the first syntheticCS 9,10 . As of 1942, 28 steroids were extracted fromthe adrenal cortex 11,12 , and in 1949, Hench andKendall demonstrated the dramatic effect ofcortisone and adrenocorticotropic hormone inrheumatoid arthritis 13-15 . In 1950, Hench, Kendalland Reichstein received the Nobel Prize forPhysiology or Medicine for their long andsuccessful research on the hormones of theadrenal cortex 9,12,13 . Aldosterone, an effective CSregulating the electrolyte balance, wassynthesised by Simpson et al. in 1954 16,17 .CSs are a class of steroid hormones naturallysynthesised in the adrenal cortex from cholest -erol. Based on their physiological effects, theyare divided into mineralocorticoids andglucocorticoids. The mineralocorticoids regulatethe electrolyte and water balance and the bloodvolume, and the glucocorticoids are responsiblefor the carbohydrate, fat, protein metabolism,the glycogenesis, the immune functions and theinhibition of inflammatory reactions. Certain CSshave only mineralocorticoid activity, such asaldosterone, others have both mineralocorticoidand glucocorticoid activity, i.e.,corticosterone, fludrocortisone and somecompounds have mainly glucocorticoid and lowmineralocorticoid activity, such as cortisol,prednisolone, etc 18,19 .CSs are mobilised from the adrenal cortex asa reaction to stress and while there are differentviews as to whether they play an indirect ordirect role in initiating the stress response orthey suppress the stress response itself 20 , CSs arenow extensively used in many areas of medicine.Corticosterone (Figure 1, page 4), whichhas both mineralocorticoid and glucocorticoidactivity, may be used to demonstratethe pharmacological activities of the CSs. Thedouble bond at C4-5, oxygen at C3, C20, C21 andmethyl at C10 are present in every pharma -cologically active CS.Substitution with methyl at C13, andoxygen at C11 are necessary for the antiinflammatoryactivity, which increases with ahydroxyl substitution at C17 (hydrocortisone),and a double bond at C1-2 (prednisolone).Further enhancement in glucocorticoid activityis achieved by a substitution with a fluorine atC9, with a hydroxyl at C16 (triamcinolone) ormethyl at C16 (dexamethasone) 21 .The CSs are among the strongest pharma -ceutical agents used in a variety of inflammatoryEuropean Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com 3 Volume 18 | Issue 2 | 2013

IN-DEPTH FOCUS: MASS SPECTROMETRYand allergic disorders, such as rheumatoidarthritis, asthma, systemic lupus erythematosus,ulcerative colitis, hepatitis, Crohn’s and Addison’sdisease, etc. Their use is not recommendedin diabetes mellitus, osteoporosis, epilepsyand in different psychotic states (anxiety,depression, etc.) 22 .For their pharmacological importance andwidespread use in many fields of medicineand health care, there has been a great demandfor reliable, fast and cost-effective analyticalmethods for the measurement of CSs in a varietyof matrices.Figure 1: CorticosteroneEarly methods and immunoassaysThe early methodologies for the analysis of CSsincluded high-performance thin-layer chrom -ato graphy with fluorescence detection 23 ,liquid chromatography (LC) with UV 24,25 andflorescence detection 26 . Immunoassays areconvenient and their use has gained popularity,particularly in clinical laboratories. CSs weredetermined in the plasma of patients withrhinitis by radioimmunoassay 27 and by electro -chemiluminescence immunoassay 28 , inplasma and urine of patients with primarybiliary cirrhosis by fluorescence polarisationimmunoassay 29 , in urine of patients withhypercholesterolemia by enzyme-linkedimmunosorbent assay 30 , and in the salivaof chimpanzees by enzyme immunoassay 31 .These methods are rapid and can be automatedfor high-throughput routine analysesbut on the negative side, immunoassayssometimes lack sensitivity and can sufferfrom cross-reactivity 30,31 .GC/MSWith the appearance of the first synthetic CSs astherapeutic agents in the late 1940s, mainlybecause of their anti-inflammatory activity,these drugs quickly became used in the world ofsports. Now, synthetic CSs are listed as dopingagents and are banned by the InternationalOlympic Committee 30 . Fast, sensitive andselective GC/MS methods have been adoptedfor the routine screening of CSs used as dopingsubstances. As an example, a GC/MS methodusing electron impact ionisation (EI) withoutsample derivatisation was used to determine18 endogenous or synthetic CSs in humanurine 30 . A sensitive and reliable GC/MS EI methodwas also utilised for the simultaneous determ -ination of several CSs in human plasma 32 . GC/MSpositive chemical ionisation with isobutene as areagent gas was employed for the simultaneousanalysis of free CSs in human urine 33 . GC/MSmethods may be sensitive and provide goodseparation of the compounds 30 , but they requirevolatile analytes, occasional sample derivatisa -tion 32 , and their selectivity may be limited by thesingle quadrupole GC/MS instrumentation usedfor the CSs analyses 30,32 .LC/MSAtmospheric pressure ionisation techniques,such as electrospray ionisation (ESI) andatmospheric pressure chemical ionisation(APCI), have been extensively used in LC/MS, andare highly applicable to the analysis of themoderately polar CSs 33,34 . Using low concen -trations of organic acids as modifiers in the LCsolvent, CSs form the positively chargedmolecular ion [M+H] + and also the negativelycharged adduct ion with the conjugated basemoiety of the organic acid [M+base] - .Accordingly, these positively or negativelycharged ions can be used for detection in LC/MS,or can serve as precursor ions to undergofragmentation using LC coupled with triplequadrupole mass spectrometry (LC/MS/MS) 33 .Single quadrupole LC/MS methods for CSsanalysis are convenient and relatively cheap touse. For example, in a comprehensive study, aLC/MS method using ESI, positive ion mode, wascompared to the GC/MS analysis of 18 CSsduring the screening of doping substances inhuman urine 30 . This study found that GC/MS had‘‘Single quadrupole LC/MS methodsfor CSs analysis are convenient andrelatively cheap to use.’’an overall better sensitivity, in particular for theendogenous CSs, but LC/MS had comparable orsometimes better sensitivity for the syntheticCSs 30 . In another study, an LC/MS ESI, positive ionmode method was employed in a qualitativeanalysis to characterise the mass spectra ofbudesonide and structurally related CSs in theidentification of budesonide breakdownproducts 35 . In this study, the mass spectralproperties were successfully correlated with theMass spectrometry (MS)In recent years, MS technology has advancedsignificantly, and when coupled with gaschromatography (GC) or LC, GC/MS and LC/MStechniques have become the preferredmethodologies for the determination of tracelevels of CSs in many applications.Figure 2: The schematic view and the principle of operation of the hybrid linearion trap triple quadrupole mass spectrometerCopyright: AB SciexEuropean Pharmaceutical ReviewVolume 18 | Issue 2 | 2013 4

PUSHING THE LIMITS IN SENSITIVITYExceedingly sensitive.Sharply focused.THE 6500 SERIES WITH IONDRIVE TECHNOLOGYSee what couldn’t be seen. Until now.The new 6500 LC/MS/MS series with multi-component IonDrive technology is AB SCIEX’s most sensitive triple quadrupole, improvingsensitivity up to 10X and detector dynamic range by 20X over thebest selling high performance triple quad – with no compromisein mass range.When merged with the Eksigent ekspert microLC 200 system,the functionally stackable design reduces lab space by 100%, whileminimizing maintenance costs and reducing mobile phase costs byup to 95%.The new AB SCIEX 6500 Series. It’s the farsighted successor to a longline of leading AB SCIEX mass spec systems.Explore visionary sensitivity atwww.absciex.com/6500-epr© 2013 AB SCIEX. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB SCIEX Pte. Ltd. or their respective owners.

IN-DEPTH FOCUS: MASS SPECTROMETRYadministered by inhalation or intraocularinjection, the resulting plasma concentrationscould be very low. For such applications, a highlysensitive capillary μLC-MS/MS ESI, positive ionmode method was developed for the analysisof four clinically important synthetic CSs inporcine plasma achieving a LLOQ of 5 pg/mLusing 1 mL of plasma 40 .Food products and edible tissuesAmong the many LC/MS/MS methods reportedfor the analysis of CSs in biological fluids, thereare but a limited number of assays for CSs in foodsamples of animal origin. LC/MS/MS ESI,negative ion mode, was used for the simul -taneous determination of 17 glucocorticoids inmilk and eggs 41 , while positive ionisationmode was employed for the analysis of13 glucocorticoids residues in Pasteurisedbovine milk 42 . ESI, negative ion mode, wasutilised to measure nine synthetic CSs inbovine liver 19 , and 16 glucocorticoids in pigmuscle, liver, and kidney achieving a LLOQranging from 0.10 to 1.00 μg/kg requiring fivegrams of tissue sample 43 .Environmental samplesCSs, and other steroidal hormones, are con -sidered to be endocrine disruptor chemicalswith regards to their potential hazards to aquaticwildlife. CSs are used in veterinary medicine andmay be found in agricultural sewage runoffand wastewater treatment effluents 44 . Six CSswere determined simultaneously using an APCI,negative ion mode method for samples of riverand drinking water. The limit of detectionFigure 4: A representative LC/MS 3 chromatogram of (a) hydrocortisone, (b) budesonide, and (c)dexamethasone, in a sample containing a mixture of 50 ng/mL of each compound. The MS3 transitions areindicated under the names of the compoundsranged from 0.06 to 0.2 ng/L and 0.01 to0.21 ng/L for 100 mL and 1000 mL samplesvolumes, respectively 44 . Several CSs wereidentified and their concentrations quantified inwastewater extracts prior to sewage treatmentusing LC-high-resolution Orbitrap MS/MS in ESI,positive ion mode 45 . Cortisol, cortisone andcortisol glucuronide were investigated in bovinefaecal matter using a validated quadrupole iontrap MS method with ESI, negative ion mode 46 .LC/ MS 3Nowadays, multiple reaction monitoring (MRM)is the standard mode of MS operation usedfor the quantification of many analytes ofinterest 39,41,43 . The method represents a significantimprovement in the detection selectivity of MScompared to single quadrupole mode ofoperations, however, MRM assays can still sufferfrom interferences 37 . The latest developmentsof the triple quadrupole instrumentationinclude the hybrid linear ion trap (LIT) tech -nology, which further improves the selectivityof detection by MS 47 .Similar to the conventional triple quad -rupole operation, the precursor ions in LIT areselected in the first quadrupole followed byfragmentation in the second quadrupole orcollision cell via collision activated dissociation.First generation product ions are19. Dusi, G., Gasparini, M., Curatolo, M., Assini, W., Bozzoni,E., Tognoli, N., Ferretti, E., Development and validation ofa liquid chromatography–tandem mass spectrometrymethod for the simultaneous determination of ninecorticosteroid residues in bovine liver samples,Analytica Chimica Acta, 700, 49-57, 201120. Sapolsky, R. M., Romero, L. M., Munck, A. U., How DoGlucocorticoids Influence Stress Responses? IntegratingPermissive, Suppressive, Stimulatory, and PreparativeActions, Endocrine Reviews, 21, 55-89, 200021. Haynes, Jr., R. C., Larner, J., Adrenocorticotropichormone: Adrenocortical steroids and their syntheticanalogs; inhibition of adrenocortical steroidsbiosynthesis, In The Pharmacological Basis ofTherapeutics, fifth edition, pp. 1473-1506, MacMillanPublishing Co., Inc., New York, 197522. Poetker, D. M., Reh, D. D., A Comprehensive Review ofthe Adverse Effects of Systemic Corticosteroids,Otolaryngologic Clinics of North America, 43,753-768, 201023. Vanoosthuyze, K. E., Van Poucke, L. S. G., Deloof, A. C. A.,Van Peteghem, C. H., Development of a highperformancethin-layer chromatographic method forthe multi-screening analysis of corticosteroids,Analytica Chimica Acta, 215, 177-182, 199324. Cham, B. E., Sadowski, B., O'Hagan, J. M., de Wytt, C. N.,Bochner, F., Eadie, M. J., High performance liquidchromatographic assay of dexamethasone inplasma and tissue, Therapeutic Drug Monitoring, 2,373-37, 198025. Diamandis, E. P., D’Costa M., Selective determination ofurinary cortisol by liquid chromatography aftersolid-state extraction, Journal of Chromatography, 426,25-32, 198826. Neufeld, E., Chayen, R., Stern, N., Fluorescencederivatisation of urinary corticosteroids forhighperformance liquid chromatographic analysis,Journal of Chromatography B, 718, 273-277, 199827. Möller, C., Ahlström, H., Henricson, K.-Å., Malqvist,L.-Å., Åkerlund, A., Hildebrand, H., Safety of nasalbudesonide in the long-term treatment of children withperennial rhinitis, Clinical and Experimental Allergy, 33,816-822, 200328. Algorta, J., Pena, M. A., Francisco, S., Abajo, Z., Sanz, E.,Randomised, crossover clinical trial, in healthyvolunteers, to compare the systemic availability of twotopical intranasal budesonide formulations, Trials, 9, 34(www.trialsjournal.com/content/9/1/34), 200829. Hempfling, W., Grunhage, F., Dilger, K., Reichel, C.,Beuers, U., Sauerbruch, T., Pharmacokinetics andPharmacodynamic Action of Budesonide in Early- andLate-Stage Primary Biliary Cirrhosis, Hepatology, 38,196-202, 200330. Pujos, E., Flament-Waton, M. M., Paisse, O., Grenier-Loustalot, M. F., Comparison of the analysis ofcorticosteroids using different techniques, Analyticaland Bioanalytical Chemistry, 381, 244-254, 200531. Kutsukake, N., Ikeda, K., Honma, S., Teramoto, M., Mori, Y.,Hayasaka, I., Yamamoto, R., Ishida, T., Yoshikawa, Y,Hasegawa, T., Validation of Salivary Cortisol andTestosterone Assays in Chimpanzees by LiquidChromatography-Tandem Mass Spectrometry,American Journal of Primatology, 71, 696-706, 200932. Shibasakia, H., Nakayama, H., Furuta, T., Kasuyaa, Y.,Tsuchiyab, M., Soejimab, A., Yamadab, A., Nagasawa, T.,Simultaneous determination of prednisolone,prednisone, cortisol, and cortisone in plasma by GC–MS:Estimating unbound prednisolone concentration inpatients with nephrotic syndrome during oralprednisolone therapy, Journal of Chromatography B,870, 164-169, 2008References continued overleafEuropean Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com 7 Volume 18 | Issue 2 | 2013

IN-DEPTH FOCUS: MASS SPECTROMETRYproduced, which are then transferred to thethird quadrupole, and after a specific filltime, the ions are trapped. The trapped firstgeneration product ions undergo resonanceexcitation whereby they collide with oneanother and produce the second generation ofproduct ions which are then detected. Thisprocedure improves the MS/MS process toMS/MS/MS (MS 3 ) adding a new dimension to thedetection selectivity in triple quadrupole MSinstrumentation. This procedure can be used for‘‘Since the advent of CSs in medicalscience and practice, there has been asteady demand for reliable, time andcost-effective methodologies forthe determination of these drugs inmany types of matrices’’quantitation as the current advancements intechnology allow very fast scan times, so thatmultiple MS3 transitions can be monitored anddetected at the same time. The schematic viewand the principle of operation of a hybrid LIT MSis presented in Figure 2 on page 4.In our laboratory, we recently developed aLC/MS 3 method for the quantification ofhydrocortisone (HC), and budesonide (BUD)using dexamethasone (DEX) as an internalstandard. A representative first and secondgeneration production ion mass spectraof HC, BUD and DEX are shown in Figure 3on page 6. The MS 3 transitions monitoredfor HC were m/z 363.4/327.2/309.1, forBUD m/z 431.3/413.3/147.1, and for DEX m/z393.3/355.1/237.2. The instrumentation usedwas an Agilent 1290 Infinity UHPLC connectedto an AB Sciex QTrap® 5500 hybrid LIT MS/MSsystem equipped with a Turbo Spray sourceoperated in positive ionisation mode (AB Sciex,Concord, Ontario, Canada). For the chromato -graphy, we used a Waters Acquity UPLC BEH C18,1.7 μm, 2.1 x 50 mm column with LC solventsand gradient programming as follows: solvent Awater with 2.5 mM ammonium formate (AF),Solvent B methanol with 2.5 mM AF. The mobilephase initial conditions were Solvent A (40 percent) and Solvent B (60 per cent) ramped toSolvent A (five per cent) by two minutesand held for one minute, followed by anequilibration cycle to the initial conditions. Theflow rate was 0.2 mL/min; injection volume was15 μL with a total run time of four minutes.A representative LC/MS 3 chromatogramis shown in Figure 4 on page 7 (unpublisheddata).ConclusionsSince the advent of CSs in medical science andpractice, there has been a steady demand forreliable, time and cost-effective methodologiesfor the determination of these drugs inmany types of matrices. With the significantadvancement in MS technology in the pastdecade, LC/MS/MS has become the techniqueof choice for CSs analysis. WhileLC/MS/MS remains in the mainstream ofCSs determ ination, the future challenges forthe technique may not only be to establishincreasingly sensitive methods, but increasinglyspecific methods having less interference.A possible answer to this challenge could bethe introduction of LC/MS 3 technology for thequantitative analysis of low concentrations ofCSs for a diversity of applications.AcknowledgementsI wish to thank Dr. Frank S. Abbott, Professor andDean Emeritus, Faculty of PharmaceuticalSciences, The University of British Columbia,Vancouver, B.C., for his comments.BiographyDr. András Szeitz received his BSc degreein Pharmaceutical Sciences at the Faculty ofPharmaceutical Sciences, SemmelweisMedical University, Hungary, in 1981 and hisDoctorate degree in 1988. After working inthe pharmaceutical industry, and clinicalresearch, he joined the Faculty of Pharma -ceutical Sciences (FPS) at The University of British Columbia (UBC),Vancouver, B.C., Canada, in 1990. Following UBC, he spent severalyears with biotechnology and contract research organisations inVancouver and in the United States working under GLP compliance.His broad experience includes setting up LC/MS/MS laboratories,developing and validating LC/MS/MS methods in a research orregulatory-compliant environment. Currently, he is the Manager ofthe Mass Spectrometry Facility at the FPS, UBC, where he is involvedin research and contract work, and teaching a graduate course onanalytical research techniques, such as GC, HPLC and LC/MS/MS.References continuted33. Cuzzola, A., Petri, A., Mazzini, F., Salvadori, P., Applicationof hyphenated mass spectrometry techniques for theanalysis of urinary free glucocorticoids, RapidCommunications in Mass Spectrometry, 23, 2975-2982,200934. Antignac, J.-P., Monteau, F., Négriolli, J., André, F., LeBizec, B., Application of Hyphenated MassSpectrometric Techniques to the Determination ofCorticosteroid Residues in Biological Matrices,Chromatographia, Supplement, 59, S13-S22, 200435. Hou, S., Hindle, M., Byron, Peter R., Chromatographicand mass spectral characterization of budesonide anda series of structurally related corticosteroids usingLC–MS, Journal of Pharmaceutical and BiomedicalAnalysis 39, 196-205, 200536. DiFrancesco, R., Frerichs, V., Donnelly, J., Hagler, C.,Hochreiter, J., Tornatore, K. M. J., Simultaneousdetermination of cortisol, dexamethasone,methylprednisolone, prednisone, prednisolone,mycophenolic acid and mycophenolic acidglucuronide in human plasma utilizing liquidchromatography–tandem mass spectrometry, Journalof Chromatography B, 859, 42-51, 200737. Ionita, I. A., Fast, D. M., Akhlaghi, F. J., Development of asensitive and selective method for the quantitativeanalysis of cortisol, cortisone, prednisolone andprednisone in human plasma, Journal ofChromatography B, 877, 765-772, 200938. McWhinney, B. C., Briscoe, S. E., Ungerer, J. P. J., Pretorius,C. J., Measurement of cortisol, cortisone, prednisolone,dexamethasone and 11-deoxycortisol with ultra highperformance liquid chromatography–tandem massspectrometry: Application for plasma, plasmaultrafiltrate, urine and saliva in a routine laboratory,Journal of Chromatography B, 878, 2863-2869, 201039. Taylor, R. L., Grebe, Stefan S. K., Singh, R. J., Quantitative,Highly Sensitive Liquid Chromatography–TandemMass Spectrometry Method for Detection of SyntheticCorticosteroids, Clinical Chemistry, 50, 2345-2352, 200440. Qu, J., Qu, Y., Straubinger, R. M., Ultra-SensitiveQuantification of Corticosteroids in Plasma SamplesUsing Selective Solid-Phase Extraction and Reversed-Phase Capillary High-Performance LiquidChromatography/Tandem Mass Spectrometry,Analytical Chemistry, 79, 3786-3793, 200741. Cui, X., Shao, B., Zhao, R., Yang, Y., Hu, J., Tum, X.,Simultaneous determination of seventeenglucocorticoids residues in milk and eggs byultraperformance liquid chromatography/electrospraytandem mass spectrometry, Rapid Communications inMass Spectrometry, 20, 2355-2364, 200642. Caretti, F., Gentili, A., Ambrosi, A., Rocca, L. M., Delfini,M., Di Cocco, M. E., D’Ascenzo, G., Residue analysis ofglucocorticoids in bovine milk by liquid chromato -graphy–tandem mass spectrometry, Analytical andBioanalytical Chemistry, 397, 2477-2490, 201043. Shao, B., Cui, X., Yang, T., Zhang, J., Wu, Y., Validation of aSolid-Phase Extraction and Ultra-Performance LiquidChromatographic TandemMass Spectrometric Methodfor the Detection of 16 Glucocorticoids in Pig Tissues,Journal of AOAC International, 92, 604-611, 200944. Tölgyesi, Á., Verebey, Z., Sharma, V. K., Kovacsics, L.,Fekete, J., Simultaneous determination ofcorticosteroids, androgens, and progesterone in riverwater by liquid chromatography–tandem massspectrometry, Chemosphere, 78, 972-979, 201045. Schriks, M., Van Leerdam, J. A., Van der Linden, S. C., Vander Burg, B., Van Wezel, A. P., de Voogt, P., High-Resolution Mass Spectrometric Identification andQuantification of Glucocorticoid Compounds inVarious Wastewaters in The Netherlands, Environ -mental Science & Technology, 44, 4766-4774, 201046. Arioli, F., Fidani, M., Casati, A., Fracchiolla, Maria L.,Pompa, G., Investigation on possible transformations ofcortisol, cortisone and cortisol glucuronide in bovinefaecal matter using liquid chromatography–massspectrometry, Steroids, 75, 350-354, 201047. Cesari, N., Fontana, S., Montanari, D., Braggio, S.,Development and validation of a high-throughputmethod for the quantitative analysis of d-amphetamine in rat blood using liquidchromatography/MS3 on a hybrid triple quadrupolelinearion trap mass spectrometer and its application toa pharmacokinetic study, Journal of ChromatographyB, 878, 21-28, 2010European Pharmaceutical ReviewVolume 18 | Issue 2 | 2013 8

ShowPREVIEWDate: 9-13 June 2013 · Location: Minneapolis, MN, USAThe 61st ASMS Conference on Mass Spectrometry and Allied Topics will take placefrom 9 – 13 June at the Minneapolis Convention Center, Minnesota, USA. TheAmerican Society for Mass Spectrometry sponsors the conference, which willbe attended by more than 6,500 scientists. Approximately 3,000 papers will bepresented as posters and talks.The program begins on Sunday 9 June withtutorial lectures at 5pm from Andrew Hoofnagle,University of Washington, entitled ‘A WideSpectrum: Clinical Diagnostics for the Masses’and John Engen, Northeastern University,entitled ‘The Nuts and Bolts of Protein HydrogenExchange MS’. The opening session and plenarylecture from Michael L. Gross, WashingtonUniversity of St. Louis ‘The First Fifty Tears ofMS: Building A Foundation’ will follow this.A Welcome Reception in the poster / exhibit hallwill conclude the first day of the conference.Monday through to Thursday will be fullprogram days of concurrent oral sessions, postersessions, and workshops. These will includetopics such as: High Mass Accuracy in Drug Discovery andDevelopment Biotherapeutics and Biomarkers:Advances in Quantitative Analysis Antibodies and Antibody-Drug Conjugates Microorganisms: Identification andCharacterisation Biomarkers of Drug Response, Efficacy andToxicity: Novel MS Approaches Principles of Protein Identification andCharacterisation Ambient Ionisation: Instrumentationand Applications Top-Down and Middle-DownProtein Analysis Nucleic AcidsFundamentals of Peptide FragmentationBiomarkers in Drug Discoveryand DevelopmentProteomics: Infectious DiseasesRegulated Bioanalysis and Diagnosticsusing High Resolution LC/MSGlycoproteins and Glycans:New MS ApproachesInstrumentation: New Developmentsin High Resolution and Mass AccuracyIon SpectroscopyMetabolomics / Lipidomics: New MSTechnologies and ApplicationsIntegrated Qualitative and QuantitativeLC-MS for Small Molecule AnalysisFundamentals of Ion Activationand DissociationPoster sessions are scheduled from 10.30am to2.30pm to allow four hours of viewing. Theprogram will conclude on Thursday 13 June witha plenary lecture followed by a Closing Gala atthe Convention Center (ticket required).Short courses will precede the annualconference on 8 and 9 June 2013. Two day shortcourses include: Bioinformatics for Protein Identification Experimentation and Data Analysisin DMPK Introduction to High Resolution MassSpectrometry for Qualitative andQuantitative AnalysisAmerican Society for Mass SpectrometryThe American Society for Mass Spectrometry (ASMS) was formed in 1969 to promote and disseminateknowledge of mass spectrometry and allied topics. ASMS is a non-profit 501 c 3 corporation.Membership includes over 8,500 scientists involved in research and development. Members comefrom academic, industrial and governmental laboratories. Their interests include advancement oftechniques and instrumentation in mass spectrometry, as well as fundamental research in chemistry,geology, forensics, biological sciences and physics.Mass Spectrometry of Glycansand GlycoproteinsIon Mobility in Mass SpectrometryLC-MS: Techniques of Electrospray, APCI andAPPI: Understanding and Optimising toDevelop Successful LC-MS MethodsPractical LC-MS: Fundamentals,Techniques and ApplicationsMALDI Imaging Mass Spectrometry:Basic Tools and TechniquesMS-MS: An IntroductionMass Analysers: Everything You Wanted toKnow about Common Mass SpectrometersBut Didn’t Know Who to AskMass Spectrometry of Peptides and ProteinsPractical Mass SpectrometricCharacterisation and Quantitation ofProtein TherapeuticsProtein Structural Analysis by MassSpectrometry: Hydrogen Exchange andCovalent LabellingQuantitative Mass SpectrometryCase Studies in Quantitative Proteomics100 years of mass spectrometryASMS will contemplate and celebrate 100 yearsof mass spectrometry through special exhibits,sessions and posters. Every registrant attendingthe conference will receive a memorable gift.Further informationFor further information ASMS, please visit:www.asms.orgEuropean Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com 9 Volume 18 | Issue 2 | 2013

© ilolab / Shutterstock.comIN-DEPTH FOCUSMulti-analyte LC-MS/MS assaysfor the quantification of endogenouscompounds during the developmentof drugs and companion diagnostics– the beauty and the beastUwe Christians, Jelena Klawitter and Jost KlawitteriC42 Integrated Solutions for Systems Biology in Clinical Research and Development, Department of Anesthesiology, University of Colorado DenverPersonalised medicine is an intriguing concept and constitutes a paradigm shift inmedicine. Abandoning the blockbuster drug concept will not only require thedevelopment of safer and more efficacious drugs that are focused on the individualpatient’s genotype and/or phenotype, it also requires the development of newcontext-specific diagnostic tools that provide biological or clinical informationthat allows for better decision-making about the development and use of a drug,so-called context dependent biomarkers and companion diagnostics 1-3 . Onceapproved together with a drug, companion diagnostics are used to predict if anindividual patient will benefit from this drug or not 1 .multi-analyte assays lack the manufacturingand batch-to-batch reproducibility challengesof many antibody-based assays. Mass spectro -metry can be used for non-targeted analyticalstrategies such as metabolomics, lipidomics andproteomics. Here, a multi-analyte assay isdefined as a targeted assay for the simultaneousquantification of two or more clearly identifiedcompounds.Changes of metabolites, peptides and proteinsin body fluids, if mechanistically linked todisease processes and drug effects in tissues andorgans, have the potential to serve as surrogatemarkers or biomarkers 3,4 . Modern analyticaltechnologies allow for the identification ofpatterns that confer significantly more infor -mation than the measurement of a singleparameter, just as a barcode contains moreinformation than a single number. Well-qualifiedmolecular marker patterns yield more detailedand mechanistically relevant informationtranslating into good specificity. The betterthe specificity of a molecular marker pattern, themore this will reduce non-specific backgroundnoise and reduced background noise may alsoresult in better sensitivity.Multi-analyte bioanalytical technologies toassess the phenotype, including but not limitedto arrays, bead immunoassays and massspectrometry, allow for the assessment ofmolecular marker panels in body fluids, ideally ina single run. Among these technologies, massspectrometry is attractive due to its sensitivity,specificity and flexibility. It allows for absolutequantification and assays can relatively easily bevalidated. Whereas antibodies are derived frombiological sources, mass spectrometry-basedMulti-analyte assaysin drug development andregulatory considerationsMulti-analyte assay strategies can be usedthroughout the drug development process toidentify unknown molecular mechanisms, fordrug discovery, pre-clinical and clinical drugdevelopment and, after regulatory approval, asclinical diagnostic tools 2,3,5 (Figure 1, page 14).The utilisation of molecular marker strategies toimprove drug development efficiency andsafety has been encouraged by drug regulatoryagencies such as the United States Food andDrug Administration (FDA) 6 .European Pharmaceutical Reviewwww.europeanpharmaceuticalreview.com 13 Volume 18 | Issue 2 | 2013

IN-DEPTH FOCUS: MASS SPECTROMETRYphysico-chemical properties into a single assay.This potentially affects all aspects of an assayfrom sample handling to mass spectrometrybasedquantification, assay validation and qualitycontrol strategies. The major steps of mostmass spectrometry-based analytical workflowsare sample collection, handling and storage,sample extraction, high-performance liquidchromatography (HPLC) and mass spectrometryanalysis including ionisation. Each of thesesteps contributes to the specificity and sensitivityof the assay.Figure 1: Development and regulatory review of drugs and context- dependent molecular markers andcompanion diagnostic devices in parallel to drug developmentThe regulatory review process is based on the United States Federal Drug Administration pilot process forbiomarker qualification 12 . In general, the review processes proposed or established by other regulatory agenciesfollow a similar flow. The FDA biomarker review process includes the following steps 12 : (A) Submission of an initialletter defining the biomarker, its context and data sources for its qualification, and review by an Initial ProposalReview Group (IPRG), (B) Decision to proceed to a full qualification by the Biomarker Qualification ReviewTeam (BQRT), (C) Full submission of qualification data for review by the BQRT. (C) Voluntary eXploratory DataSubmission (VXDS) meeting to go over the qualification data and to identify potential information gaps before a fullreview can be completed for the qualification package. (D) Review drafted by BQRT, (E) internal review at FDA, and(F) communication of decision to sponsorRegulatory agencies have establishedreview structures as well as guidance thatoutlines the biomarker qualification process andsubmission 1,2,7-12 (Figure 1).Translation of a molecular marker from thediscovery stage into pre-clinical testing andclinical development greatly depends on theavailability of robust, precise and sensitiveassays for the measurement of a larger numberof samples 13 . While during the discovery phase apartial validation following ‘fit-for-purpose’principles will be sufficient, assay validationmust become more stringent if drug develop -ment strategies, regulatory approval and clinicaldecisions will depend on such a molecularmarker 2 . During later clinical development andespecially when developed as a clinicaldiagnostic tool, absolute quantification of theanalytes and complete validation followingapplicable regulatory guidance is critical 1,2 .If appropriately qualified and based onadequately validated assays, molecular markerscan support primary outcomes 2,14 ; they may helpto understand and monitor mechanisms oftoxicity, drug interactions, disease-druginteractions and the effects of genotypes,gender and age 2,5 (Figure 1). They can be used tostratify patient populations, guide subgroupanalyses to bridge safety and efficacy databetween different populations such as adults topaediatric patients, and among different ethnicgroups. However, diagnostic devices used tomake treatment decisions in a clinical trial ofa therapeutic product will be consideredinvestigational devices, unless employed for anintended use for which the device is alreadycleared 1 . If used to make critical treatmentdecisions, such as patient selection, treatmentassignment or treatment arm, a diagnosticdevice will generally be required to comply withthe investigational device exemption (IDE)regulations 1 . Although mass spectrometry is anattractive technology for the development ofcompanion diagnostic devices, as of today nomass spectrometry-based companion diag -nostic device has been approved yet 15 .Challenges of mass spectrometry-basedmulti-analyte assaysTraditionally in clinical diagnostics, a specificassay has been used to measure a specific analyte.Multi-analyte assays open up new opportunitiesby measuring combinatorial markers allowingfor pattern analysis, but are also associated withgreater challenges as they require the integrationof endogenous analytes with often differentSample integrityMaintenance of sample integrity is an oftenunderestimated problem. Sample integrity isdefined as stability of the analyte(s) in thebiological matrix throughout variableenvironments spanning from sample collection,storage, shipping and further storage up to thelast sample analysis 13 . The analytical results andthe conclusions drawn from the results can onlybe valid if the sample that reaches the laboratoryat the moment of processing for analyticalanalysis is of sufficient quality. The greater thenumber of analytes that are included in a multianalyteassay, the greater the difficulty inestablishing sample handling protocols that willensure the stability of all compounds of interest.Stability testing is an important component ofthe validation of a multi-analyte assay.Ion suppression / ion enhancementDuring the extraction procedure, potentiallyinterfering compounds are eliminated fromthe resulting extract. HPLC separates thecompounds of interest from remaining inter -ferences. The problem is that although massspectrometry detection can be highly specific,using concepts such as multiple reactionmonitoring and/or exact mass, the ionisationprocedure is not. If samples are analysed byLC-MS/MS, it should be kept in mind thatespecially in the case of multiple analytesthat are quantified simultaneously, the likeli -hood of significant interactions during theionisation process resulting in ion suppressionor enhancement is increased 16 . A good exampleis 17 showing that during profiling of high-energyphosphates using LC-MS, the analytes supp -ressed each other’s ionisation if not completelyseparated by HPLC. The extraction procedureand HPLC separation are critical to reduce thenumber of potentially interfering analytes thatare in the ion source at any given time and thusEuropean Pharmaceutical ReviewVolume 18 | Issue 2 | 2013 14

IN-DEPTH FOCUS: MASS SPECTROMETRYare important for assay performance includingsensitivity, specificity and quantification. Otherstrategies are the use of ion sources that are lessprone to ion suppression such as atmosphericpressure chemical ionisation (APCI) or nano -spray sources 18 . The use of appropriatedeuterated internal standards will not only helpin compensating for variability during extractionand degradation of the analyte, but willalso compensate for fluctuation in ionisationefficiencies. However, the development of multianalyteassays that quantify a range of analyteswith a variety of physico-chemical propertiesrequires compromises. With an increasingnumber of analytes, it becomes increasinglydifficult to find sufficiently selective extraction,HPLC and ionisation conditions that allow for thereduction of ion suppression / enhancementwhile allowing for the simultaneous extractionand quantification of a wide variety of analyteswith sufficient sensitivity.Mass spectrometryConcentration differences that can span severalorders of magnitude can become a problem forthe quantification of several endogenouscompounds in the same assay. Thus, thesensitivity and the dynamic range of the massspectrometry detector can become a limitingfactor. Multi-analyte mass spectrometry assayscan quantify dozens, if not hundreds ofcompounds. In such cases, especially whencombined with U-HPLC which results in narrowpeaks, the scan speed can become a limitingfactor. Insufficient scan speed can lead toindividual peaks being defined by only a fewdata points. This has a negative effect onintegration, quantification and reproducibility.Automatic peak integrationThe correctness and reproducibility of auto -matic integration depends on the peak shape.Broad peaks with shallow slopes usually make itmore difficult for the integration algorithms todecide where a peak starts and ends, especiallywhen the concentrations are close to the lowerlimit of quantification. As regulatory authoritiesfrown upon manual integrations, optimisingpeak shape is important, but it can be problem -atic to obtain satisfactory peak shapes for allanalytes included in a multi-analyte assay.Validation and quality controlDuring molecular marker development andafter the molecular marker of interest has beenidentified, the next step is to establish a targetedand validated assay that is capable of quanti -fying these specific compounds with acceptabletotal imprecision and sensitivity. Validation ofcomplex multi-analyte assays can be chall -enging. In most cases, such markers areendogenous compounds so an appropriateblank matrix may not be available. Sometimescharcoal stripping, dilution of the matrix withsaline or protein solutions, the use of corre -sponding matrices from other species orartificial surrogate matrices may provide asolution. In the case that blank matrices are notavailable, samples from healthy individuals oranimals, depending on the species relevant formolecular marker testing and preferably withlow concentrations of the compound ofinterest, have to be enriched with the referencecompounds and the endogenous signal has tobe subtracted later. In this case, the result is influ -enced by two measurements, the endogenoussignal and the signal of the added compound.If a molecular marker plays an important roleduring the pre-clinical development of a drug,quantification of the molecular marker may needto comply with the rules of good laboratorypractices (GLP). Validation of analytical assays forthe quantification of metabolic and proteinmolecular markers may have to follow applicableregulatory and other guidance and standards,including but not limited to, the United StatesFood and Drug Administration 19 , Clinical andDo you want toincrease your profile?Join our Pharma Supplier Directory andincrease your profile, drive traffic to your websiteand boost your search engine ranking.Listed are just a few of the hundreds of companiesthat are already benefitting. Adding your organisationis quick and easy, and you can now SAVE 20% on apremium listing when you enter the discount codeISSUE2 during sign-up.Visit: www.europeanpharmaceuticalreview.com/directory

IN-DEPTH FOCUS: MASS SPECTROMETRYLaboratory Standards Institute 20 and EuropeanMedicines Agency 21 . Based on most guidance,an assay is only acceptable if, except at thelower limit of quantitation, inter-day precisionis ≤15 per cent and inter-day accuracy iswithin ± 15 per cent of the nominal value.Accordingly, an analytical run of study samplesis accepted if at least two thirds of the qualitycontrol samples fall within ≤15 per centof their nominal value. For the quantification ofmacromolecules using immuno-based assays,regulatory agencies may accept limits of± 25 and ± 30 per cent 22 .Current regulatory guidance has beenwritten mostly with the quantification of singledrug compounds in mind and may be too rigidfor multi-analyte assays. The challenge withmulti-analyte assays is that several compoundsare quantified simultaneously and that, asaforementioned, it is not possible to optimisethe assay for each compound to the extent thatis possible for analysis of single compounds. Inaddition, the larger the number of simul -taneously measured compounds, the higher thestatistical probability that one accidentally failsto meet acceptance criteria.DiscussionIt has been suggested that molecular marker /endogenous compound multi-analyte assaysshould not be validated by the same guidingprinciples developed for drug analysis 13,22,23 .Today, it is common practice that allanalytes measured in the same assay have tomeet the same acceptance criteria and that ifone analyte fails to meet acceptance, the wholeanalytical run fails even if all other analytesare within predefined quality specifications.As discussed above, due to the potentiallyrelatively wide range of physico-chemical prop -erties of analytes included in a multi-analyteassay, the performance of specific analytesmay vary and some analytes may be morelikely to fail standard acceptance criteria thanothers may. Therefore, it has been sugg ested towiden the acceptance criteria from 15 to 25 percent or even 30 per cent for molecular markermulti-analyte assays 22 . Although this may beacceptable for some analytes, this may be tooliberal for others. A ‘one fits all’ approach to assayacceptance may not be a reasonable strategy formulti-analyte assays 24 .Overall, there is no question that massspectrometry based multi-analyte assays forthe targeted quantification of endogenouscom pounds are important tools for the develop -ment of context-dependent biomarkers andcompanion diagnostic devices 25 . However, thereare limitations to how many and which analytescan be combined in a single assay. A group ofshorter, more specialised assays may offer amore manageable and efficient alternative. Thereis a clear need to validate multi-analyte assays torealise their full potential in drug developmentand clinical diagnostics. However, the standardacceptance criteria that have mostly beendeveloped for single drug analyte assays are notappropriate for multi-analyte assays 23 . It seemsnecessary to develop novel concepts forquality control and acceptance criteria that areconsidered fit for GLP and clinical developmentas well as for clinical diagnostic tools takingthe characteristics of multi-analyte assaysinto account 23 . It seems reasonable to assumethat the lack of clear regulatory guidance is oneof the reasons preventing a more widespreaddevelopment and utilisation of quantitativemulti-analyte assays in drug development andclinical diagnostics.References1. United States Food and Drug Administration, Centerfor Drug Evaluation and Research. Draft Guidance forIndustry and Food and Drug AdministrationStaff In Vitro Companion Diagnostic Devices, versionJuly 14, 2011 http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm262292.htm (accessed March 24, 2013)2. United States Food and Drug Administration, Centerfor Drug Evaluation and Research. Guidance forIndustry. E16 Biomarkers Related to Drug orBiotechnology Product Development: Context,Structure, and Format of Qualification Submissions,version August 2011 www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM267449.pdf (accessed March 24, 2013)3. Christians U, Schmitz V, Klawitter J, Klawitter J. Proteometabolomicstrategies in the future of drugdevelopment. In: The Applicability of AnalyticalTechniques to Clinical Studies. Caroli S, Záray G (eds.)John Wiley and Sons, Inc. New York (2012), pp. 691-7744. Oresic M, Vidal-Puig A, Hänninen V. Metabolomicapproaches to phenotype characterization andapplications to complex diseases. Expert Rev MolDiagn 2006; 6: 575-5855. Amur S, Frueh FW, Lesko LJ, Huang SM. Integration anduse of biomarkers in drug development, regulation andclinical practice: a US regulatory perspective. BiomarkMed 2008; 2: 305-3116. U.S. Department of Health and Human Services, Foodand Drug Administration (2004) Challenge andopportunity on the critical path to new medicalproducts. http://www.nipte.org/docs/Critical_Path.pdf(accessed March 24, 2013)7. P.Y. Müller, F. Dieterle, Tissue-specific, non-invasivetoxicity biomarkers: translation from preclinical safetyassessment to clinical safety monitoring. Expert Opin.Drug Metab. Toxicol. 5 (2009) 1023-10388. United States Food and Drug Administration,Center for Drug Evaluation and Research. Guidancefor the Industry. Pharmacogenomic data submissions,version March 2005. http://www.fda.gov/downloads/RegulatoryInformation/Guidances/ucm126957.pdf (accessed March 24, 2013)9. United States Food and Drug Administration, Centerfor Drug Evaluation and Research. Guidance for theIndustry.Pharmacogenomic Data Submissions —Companion Guidance, version August 2007.www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM079855.pdf(accessed March 24, 2013)10. Goodsaid F, Frueh F. Biomarker qualification pilotprocess at the US Food and Drug Administration. AAPSJ 2007; 23: E105-10811. Goodsaid F, Frueh F. Implementing the US FDAguidance on pharmacogenomic data submissions.Environ Mol Mutagen 2007; 48: 354–35812. Goodsaid FM, Frueh FW, Mattes W. Strategic paths forbiomarker qualification. Toxicology 2008; 245: 219-22313. Lee JW, Weiner RS, Sailstad JM, Bowsher RR, Knuth DW,O'Brien PJ, Fourcroy JL, Dixit R, Pandite L, Pietrusko RG,Soares HD, Quarmby V, Vesterqvist OL, Potter DM, WitliffJL, Fritche HA, O'Leary T, Perlee L, Kadam S, Wagner JA.Method validation and measurement of biomarkers innonclinical and clinical samples in drug development: aconference report. Pharm Res 2005; 22; 499-51114. U.S. Department of Health and Human Services,Food and Drug Administration, Center for DrugEvaluation and Research. Guidance for Industry:Providing Clinical Evidence of Effectiveness for HumanDrug and Biological Products, version May 1998.http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm078749.pdf (accessed March 24, 2013)15. U.S. Department of Health and Human Services, Foodand Drug Administration. In Vitro CompanionDiagnostic Devices. http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm301431.htm (accessed March 24, 2013)16. Annesley TM. Ion suppression in mass spectrometry.Clin Chem 2003; 49: 1041–104417. Klawitter J, Schmitz V, Klawitter J, Leibfritz D, ChristiansU. Development and validation of an assay for thequantification of 11 nucleotides using LC/LCelectrosprayionization-MS. Anal Biochem 2007;365: 230-23918. Wickremsinhe ER, Ackermann BL, Chaudhary AK,Validating regulatory-compliant wide dynamic rangebioanalytical assays using chip-based nanoelectro -spray tandem mass spectrometry. Rapid Commun.Mass. Spectrom. 19 (2005) 47-5619. U.S. Department of Health and Human Services, Foodand Drug Administration, Center for Drug Evaluationand Research and Center for Veterinary Medicine.(2001) Guidance for the Industry. Bioanalytical MethodValidation. Version May 2001. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf (accessedMarch 24, 2013)20. Clinical and Laboratory Standards Institute,www.clsi.org (accessed March 24, 2013)21. European Medicines Agency, Committee forMedicinal Products for Human Use (CHMP).Guideline on bioanalytical method validation,EMEA/CHMP/EWP/192217/ 2009, version July 21, 2011www.ema.europa.eu/ema/pages/includes/document/open_document.jsp?webContentId=WC500109686(accessed March 24, 2013)22. Cummings J, Ward TH, Greystoke A, Ranson M, Dive C.Biomarker method validation in anticancer drugdevelopment. Br J Pharmacol 2008; 153: 646-65623. Williams PM, Lively TG, Jessup JM, Conley BA.Bridging the gap: moving predictive and prognosticassays from research to clinical use. Clin Cancer Res2012; 18: 1531-153924. Christians U, Klepacki J, Shokati T, Klawitter J, Klawitter J.Mass spectrometry-based multi-analyte for the analysisof biomarkers in drug development and clinicaldiagnostics- how much is too much? Microchem J2012; 105: 32-3825. Christians U, Klawitter J, Hornberger A, Klawitter J. Howunbiased is non-targeted metabolomics and istargeted pathway screening the solution? Curr PharmBiotechnol 2011; 12:1053-1066European Pharmaceutical ReviewVolume 18 | Issue 2 | 2013 16

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