development of lc/ms techniques for plant and drug metabolism...

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ISBN 978-951-42-9440-2 (Paperback)ISBN 978-951-42-9441-9 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)

U N I V E R S I TAT I S O U L U E N S I SACTAA




OULU 2011

A 574

Aleksanteri Petsalo

DEVELOPMENT OFLC/MS TECHNIQUESFOR PLANT AND DRUG METABOLISM STUDIES

UNIVERSITY OF OULU,FACULTY OF SCIENCE,DEPARTMENT OF CHEMISTRY,FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

A 574

ACTA

Aleksanteri Petsalo

A C T A U N I V E R S I T A T I S O U L U E N S I SA S c i e n t i a e R e r u m N a t u r a l i u m 5 7 4

ALEKSANTERI PETSALO

DEVELOPMENT OF LC/MS TECHNIQUES FOR PLANT AND DRUG METABOLISM STUDIES

Academic dissertation to be presented with the assent ofthe Faculty of Science of the University of Oulu for publicdefence in Auditorium F100, Futura, Joensuu Campus, on4 June 2011, at 2 p.m.

UNIVERSITY OF OULU, OULU 2011

Copyright © 2011Acta Univ. Oul. A 574, 2011

Supervised byDocent Ari TolonenDocent Miia Turpeinen

Reviewed byProfessor Janne JänisDocent Tiia Kuuranne

ISBN 978-951-42-9440-2 (Paperback)ISBN 978-951-42-9441-9 (PDF)http://herkules.oulu.fi/isbn9789514294419/ISSN 0355-3191 (Printed)ISSN 1796-220X (Online)http://herkules.oulu.fi/issn03553191/

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2011

Petsalo, Aleksanteri, Development of LC/MS techniques for plant and drugmetabolism studies. University of Oulu, Faculty of Science, Department of Chemistry, P.O. Box 3000, FI-90014University of Oulu, Finland, Faculty of Medicine, Institute of Biomedicine, Department ofPharmacology and Toxicology, P.O. Box 5000, FI-90014 University of Oulu, FinlandActa Univ. Oul. A 574, 2011Oulu, Finland

Abstract

Liquid chromatography (LC) combined with mass spectrometry (MS) is a powerful tool forqualitative and quantitative analytics of organic molecules from various matrices, and the use ofthis hyphenated technique is very common in bioanalytical laboratories. In this study, LC/MSmethods and the required sample preparation applications were developed for plant flavonoid anddrug metabolism studies. The main focus was in developing methods to be used duringcytochrome P450 (CYP) -specific drug interaction studies. Traditional high performance liquidchromatography (HPLC) and new, more efficient and faster ultra-performance liquidchromatography (UPLC) were utilized together with time-of-flight (TOF) and triple quadrupole(QqQ) mass spectrometry. In the flavonoid study, collision-induced radical cleavage of flavonoidglycosides was tested and observed to be a suitable tool for the structure elucidation of the 15flavonol glycosides extracted from the medicinal plant Rhodiola rosea. Ten of these glycosideswere previously unreported in the plant.

Several unreported in vivo bupropion metabolites were identified from human urine whendeveloping the method for the new and more extensive in vitro and in vivo N-in-one interactioncocktail assays. The qualified analysis methods developed here enable faster analysis for the N-in-one cocktail assays, in turn enabling a more efficient screening of drugs that affect CYP-enzyme activities. In the case of the human in vitro cocktail assay, fourteen compounds wereanalyzed using a single LC/MS/MS run. The method has proven to be very reliable and has beenused in several interaction studies utilizing different sample matrices. The in vivo cocktail assaythat was developed enables totally non-invasive sample collection from the patients, the urinesample being sufficient for the UPLC/MS/MS analysis of all target compounds. The last part ofthe study consisted of developing a specific and very sensitive UPLC/MS/MS method for theanalysis of one of the in vivo cocktail analytes, the antidiabetic drug repaglinide, from humanplacenta perfusates.

Keywords: cytochrome P450 enzyme system, drug metabolism, flavonoids, liquidchromatography, mass spectrometry, urine analysis

Petsalo, Aleksanteri, LC/MS-menetelmien kehittäminen kasvi- ja lääkeaineidenmetabolian tutkimukseen. Oulun yliopisto, Luonnontieteellinen tiedekunta, Kemian laitos, PL 3000, 90014 Oulunyliopisto, Lääketieteellinen tiedekunta, Biolääketieteen laitos, Farmakologia ja toksikologia, PL5000, 90014 Oulun yliopistoActa Univ. Oul. A 574, 2011Oulu

Tiivistelmä

Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on tehokas työväline kvalita-tiivisessa ja kvantitatiivisessa analytiikassa, ja tätä tekniikkaa käytetään erityisesti bioalan labo-ratorioissa. Tässä väitöskirjatyössä kehitettiin ja sovellettiin LC/MS- ja näytteenkäsittelymene-telmiä kasvien flavonoidimetabolian ja lääkeaineiden metaboliatuotteiden tutkimukseen keskit-tyen erityisesti sytokromi P450 (CYP) -entsyymispesifisten lääkeaineiden interaktiotutkimuk-siin tarvittaviin menetelmiin. Työssä hyödynnettiin perinteistä korkean erotuskyvyn nestekroma-tografiaa (HPLC) ja uutta, suorituskyvyltään vielä tehokkaampaa ja nopeampaa nestekromato-grafiaa (UPLC) yhdessä lentoaika- (TOF) ja kolmoiskvadrupolimassaspektrometrian (QqQ)kanssa. Tutkimustyön flavonoidimetaboliaan keskittyneessä osuudessa havaittiin törmäyksenaiheuttaman (CID) radikaalipilkkoutumisen soveltuvan lääkinnällisenä kasvina käytetystä ruusu-juuresta (Rhodiola rosea) uutettujen viidentoista flavonoliglykosidin rakennemääritykseen.Kymmentä näistä löydetyistä glykosideista ei oltu aiemmin raportoitu ruusujuuresta. Tutkimus-työn keskeisimpänä tavoitteena kehitettiin kvalifioidut LC/MS -analyysimenetelmät käytettäväk-si aikaisempaa kattavampien in vitro ja in vivo -olosuhteiden N-in-one -tyyppisten CYP-entsyy-mi-interaktiotutkimusten analyyttisenä työkaluna. Näitä analyysimenetelmiä kehitettäessä löy-dettiin ja tunnistettiin ihmisen virtsasta aiemmin raportoimattomia metaboliitteja CYP2B6 -ent-syymin malliaineena käytetyn bupropionin annostelun jälkeen. Kyseisten kehitettyjen analyysi-menetelmien avulla CYP-entsyymien toimintaan vaikuttavien lääkeaineiden tutkiminen onaiempaa nopeampaa ja antaa yhdellä kertaa samasta tutkimuksesta entistä laaja-alaisempaa tie-toa. In vitro -tutkimusta varten kehitetty LC/MS/MS -analyysimenetelmä on osoittautunut erit-täin käyttökelpoiseksi lukuisissa interaktiotutkimuksissa, ja in vivo -tutkimusta varten kehitettyUPLC/MS/MS -analyysimenetelmä mahdollistaa täysin ei-invasiivisen näytteenoton potilaista.Tutkimustyön viimeisessä vaiheessa kehitettiin erittäin herkkä ja spesifinen UPLC/MS/MS -ana-lyysimenetelmä CYP2C8-entsyymin toiminnan malliaineena käytetyn repaglinidin analysoimi-seksi koejärjestelystä, jossa tutkitaan yhdisteiden kulkeutumista raskausaikana äidin ja sikiönverenkierron välillä istukan kautta.

Asiasanat: aineenvaihdunta, analyysimenetelmät, flavonoidit, lääkeaineet,massaspektrometria, nestekromatografia, sytokromit

To my loved ones

Acknowledgements

This work was carried out during the years 2005–2011. Most of the laboratory workwas performed at Novamass facilities for which I am very grateful to Jouko Uusitalo(the founder of Novamass) and my supervisor Docent Ari Tolonen for giving me theopportunity to do my PhD studies there.

I wish to express my warmest thanks to my supervisors, Docents Ari Tolonen andMiia Turpeinen, for introducing me to the fields of LC/MS and drug metabolism. Yourguidance, knowledge and understanding have been a great support during these years.

I am grateful to Professor Janne Jänis and Docent Tiia Kuuranne for their carefulreview of this thesis. Your constructive criticism and comments were valuable. I alsowant to thank Dr. Roy Goldblatt for revising the language of this thesis.

I would like to thank all my co-authors, and especially professor Olavi Pelkonen forhis whole-hearted contribution to our studies. I warmly thank all my colleagues andco-workers at Novamass and at the Structural Elucidation Chemistry Division of theDepartment of Chemistry for their collaboration. Particularly the laboratory staff isgratefully acknowledged.

Finally, I would like to thank all my loved ones. I am deeply grateful to my parentsfor their never-ending love and support. I thank my brother for being nothing but himself.You have helped me in so many ways. I thank my sister and her family for the fun timeswe have had. I also want to thank PK for the everlasting support and believe in me. Isincerely thank my godparents and relatives for their financial support during these years.With your support I have been able to do things I like. My dear friends, you all are mostwarmly acknowledged. You make my journey worth of living and I can never expresshow much you all mean to me. And last but not least, I would like to thank my owncoach for her love and understanding. Everything would be so different without you.

Funding provided by the Orion-Farmos Research Foundation and Tauno TönningFoundation is gratefully acknowledged.

Joensuu, April 2011

Aleksanteri Petsalo

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Abbreviations

ADMET absorption, distribution, metabolism, excretion, toxicityANR anthocyanidin reductaseANS anthocyanidin synthaseAPCI atmospheric pressure chemical ionizationAPI atmospheric pressure ionizationCHI chalcone isomeraseCHS chalcone synthaseCID collision induced dissociationCRM charge residue modelCYP cytochrome P450DFR dihydroflavonol 4-reductaseESI electrospray ionizationF3H flavanone 3-hydroxylaseFNS flavone synthaseGSH glutathioneHPLC high-performance liquid chromatographyIEM ion evaporation methodIFS isoflavone synthaseIS internal standardLC liquid chromatographyLLE liquid-liquid extractionLoD limit of detectionMDF mass defect filterMRM multiple reaction monitoringMS mass spectrometryMS/MS tandem mass spectrometrym/z mass-to-charge ratioNADPH nicotinamide adenine dinucleotide phosphateNAT N-acetyltransferaseNMR nuclear magnetic resonancePAH polycyclic aromatic hydrocarbon

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PAPS 3’-phosphoadenosine-5’-phosphosulfatePBS phosphate buffered salinePDA photodiode arrayPP protein precipitationQqQ triple quadrupoleRP reversed phaseSAA single analyte assaySAM S-adenosylmethionineSFE supercritical fluid extractionSPE solid phase extractionSRM selective reaction monitoringTOF time-of-flightUDP uridine diphosphateUDPGA uridine-5’-diphosphoglucuronic acidUGT UDP-glucuronosyltransferaseUPLC ultra-performance liquid chromatography

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List of original articles

This thesis is based on the following publications, which are referred to in the text bytheir Roman numerals [I–V]:

I Petsalo A, Jalonen J & Tolonen A (2006) Identification of flavonoids of Rhodiola rosea byliquid chromatography-tandem mass spectrometry. Journal of Chromatography A 1112:224–231.

II Petsalo A, Turpeinen M & Tolonen A (2007) Identification of bupropion urinary metabolitesby liquid chromatography-mass spectrometry. Rapid Communications in Mass Spectrometry21: 2547–2554.

III Tolonen A, Petsalo A, Turpeinen M, Uusitalo J & Pelkonen O (2007) In vitro interactioncocktail assay for nine major cytochrome P450 enzymes with thirteen probe reactions anda single LC/MS/MS run; analytical validation and further testing with monoclonal P450antibodies. Journal of Mass Spectrometry 42: 960–966.

IV Petsalo A, Turpeinen M, Pelkonen O & Tolonen A (2008) Analysis of nine drugs and theircytochrome P450 specific probe metabolites from urine by liquid chromatography-tandemmass spectrometry utilizing sub 2 µm particle size column. Journal of Chromatography A1215: 107–115.

V Tertti K, Petsalo A, Niemi M, Ekblad U, Tolonen A, Rönnemaa T, Turpeinen M, Heikkinen T& Laine K (2011) Transfer of repaglinide in the dually perfused human plancenta and the roleof organic anion transporting polypeptides (OATPs). Manuscript.

Some unpublished results are also included.

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Contents

AbstractTiivistelmäAcknowledgements 9Abbreviations 11List of original articles 13Contents 151 Introduction 172 Literature review 19

2.1 Plant flavonoid secondary metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.2 Mass spectrometric methods in flavonoid studies . . . . . . . . . . . . . . . . . . . . . . . . 212.3 Xenobiotic (drug) metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.4 Cytochrome P450 enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252.5 LC/MS methods in metabolite profiling and quantitation . . . . . . . . . . . . . . . . . 272.6 Drug interaction studies with N-in-one cocktails . . . . . . . . . . . . . . . . . . . . . . . . 29

3 Aims of the study 334 Experimental procedures 35

4.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354.2 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.3 Matrix effect tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

5 Results and Discussion 395.1 Structure analysis of flavonoids and drug metabolites [I–II] . . . . . . . . . . . . . . 39

5.1.1 Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395.1.2 Bupropion metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445.1.3 TOF vs. QqQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

5.2 Quantitative analysis of CYP specific probe substrates and theirmetabolites (in vitro and in vivo) [III - V] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505.2.1 Method development and validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505.2.2 Comparison of N-in-one assay and single analyte analysis . . . . . . . . . 63

6 Conclusions 65References 67Original articles 77

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1 Introduction

Liquid chromatography (LC) combined with mass spectrometry (MS) is a powerfultool in, inter alia, pharmaceutical and plant metabolism analytics, and the use of thishyphenated technique is now commonplace in bioanalytical laboratories [1, 2]. This is aresult of recent developments in separation sciences and instrumentations in general,and leads to the use of modern ultra-performance liquid chromatography (UPLC)together with the high selectivity and performance of mass spectrometry. The enhancedseparation of analytes in faster analysis time, yielding better throughput with superiorresults, is leading bioanalytical laboratories to shift from traditional high-performanceliquid chromatography (HPLC) to UPLC [3, 4].

Knowledge of the metabolic characteristics of new drug candidates plays a veryimportant role in the selection of suitable compounds for the later stages of drugdevelopment projects, and helps reduce the amount of drugs withdrawn from clinicaltrials and market. The most common reasons for the disqualification of drug candidatesmay include the formation of reactive metabolites as well as interaction problemsdirected at the drug metabolizing cytochrome P450 (CYP) enzymes [5, 6]. Carefuland comprehensive drug screening and identification of the metabolites thus formsan integral part of the ADMET (absorption, distribution, metabolism, excretion andtoxicology) data in the development of drugs.

Liver CYP enzyme interaction studies have traditionally been performed separatelyusing specific in vitro assays for each enzyme, utilizing HPLC or LC/MS/MS methodsfor analysis [7]. However, in recent years, cocktail / N-in-one assays have also beendeveloped for simultaneous screening of several enzyme interactions [8–10] and similarassays have also been developed for in vivo drug-drug interaction studies and CYPactivity and phenotyping purposes [11–13].

This thesis describes the utilization of traditional HPLC and improved performanceUPLC techniques combined with time-of-flight (TOF) and triple quadrupole (QqQ) massspectrometry for studies of plant and drug metabolism. Using the developed qualitativeand quantitative LC/MS methods, compounds were identified and analyzed from plantextracts and human liver microsome homogenates, urine and placental perfusates. Thegreat variety of sample matrices in the study emphasizes sample preparation with respectto accuracy of the results and thus various methods, such as protein precipitation (PP)

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and solid phase extraction (SPE), were used to process the biomatrices prior to LC/MSanalysis.

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2 Literature review

2.1 Plant flavonoid secondary metabolism

Flavonoids are polyphenolic compounds with a wide variety of biological activities.They are plant pigments that color most flowers and also protect plants against UV-light and phytopathogens [14, 15]. Their beneficial effects on human health are alsowidely-known. Flavonoids reduce the risk of cardiovascular diseases [16–18] and cancer[19, 20]. Their ability to act as antioxidants [21–23] and their anti-inflammatory andantimicrobial effects are favorable in the treatment of illnesses such as diabetes andrheumatic diseases [23–25].

The chemical structure of flavonoids is based on the C6−C3−C6 skeleton, which isformed by a series of biosynthesis reactions (Figure 1). The main dietary flavonoidsubclasses are flavanones, flavones, isoflavones, flavonols, anthocyanidins and flavan-3-ols [26]. The initial material, 4-coumaroyl-CoA, in the flavonoid pathway is derivedfrom phenylalanine via cinnamic acid and p-coumaric acid through the shikimatepathway [27, 28]. The flavonoid biosynthetic pathway starts with the chalcone synthase(CHS) catalyzed reaction of one molecule of 4-coumaroyl-CoA and three moleculesof malonyl-CoA. This condensation reaction yields naringenin-chalcone. Chalconeisomerase (CHI) catalyzes the isomerisation reaction from chalcone to flavanone,naringenin. From flavanone the flavonoid pathway diverges into side branches resultingin flavone, isoflavone and dihydroflavonol products. These reactions are catalyzed byflavone synthase (FNS), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H)enzymes, respectively. The F3H catalyzed reaction is a stereospecific hydroxylationreaction from flavone to dihydroflavonol. The first step in converting the dihydroflavonol(dihydroquercetin) into anthocyanidin is the reduction of the C-ring of dihydroquercetinwith the catalyzing enzyme dihydroflavonol 4-reductase (DFR) [29]. Anthocyanidinsynthase (ANS) converts the intermediate leucocyanidin into cyanidin, which in turn isconverted to (-)-epicatechin (flavan-3-ol) by anthocyanidin reductase (ANR).

Flavonoids are mostly present in plants as glycosides, O-glycosides and C-glycosides.O-glycosides have a sugar moiety bound to the hydroxyl group of the aglycone, and inthe case of C-glycosides the sugars are linked to the aglycone with a carbon-carbonbond. The most typical monosaccharides are glucose and rhamnose, but in rare cases

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Fig 1. Part of the flavonoid pathway and key enzymes involved in the biosynthe-sis of the flavonoid subclasses. Enzyme abbreviations: CHS, chalcone synthase;CHI, chalcone isomerase; FNS, flavone synthase; IFS; isoflavone synthase; F3H,flavanone 3-hydroxylase; FLS, flavonol synthase; F3’H; flavonol 3’-hydroxylase;DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; ANR, antho-cyanidin reductase [26, 28].

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arabinose and xylose also appear [30].Flavonols (kaempferol in the Figure 1) are most widespread of the flavonoids and are

found most frequently as O-glycosides, where one or more hydroxyl groups are replacedby a glycosyl group with a C–O glycosidic bond. The glycosylation reaction is catalyzedby glycosyltransferase. The most common hydroxyl groups for glycosylationin flavonolsare at the 3- and 7-positions [1].

2.2 Mass spectrometric methods in flavonoid studies

A structure analysis of flavonoids and other plant secondary metabolites is essentialwhen searching for new biologically active compounds. Structural determination is acomplicated task, especially when analyzing flavonoids from complex plant matrixextracts. Nuclear magnetic resonance (NMR) and X-ray crystallography are advancedanalytical techniques for structure elucidation, but these techniques require a largeamount of purified sample isolated from plant extracts. Hyphenated techniques such asliquid chromatography-mass spectrometry (LC/MS) is one useful option for overcomingthe problem of a limited amount of sample.

A large number of papers concerning the methods for identifying flavonoid structureby tandem mass spectrometric (MS/MS) methods have been published [1, 31–39].Mass spectrometric methods provide information about flavonoid aglycones and theirglycosidic moieties. The nomenclature system for MS fragment ions for flavonoidaglycones and glycosides [40, 41] is widely accepted and used. The nomenclaturesystem for flavonoids is presented in Figure 2.

Glycosides can be differentiated by their positive and negative ionization spectra.Positive ionization is more commonly used in the structure analysis of flavonoids [1]although, negative ionization is more sensitive to flavonoids [32, 42, 43]. Fragmentscontaining the aglycone part of the glycoside are labeled i, jXk, Yk and Zk, the subscriptindicating the number of the interglycosidic bond broken (counted from the aglycone),whereas the supercripts of the ion X indicate the cleavages within the sugar rings (Figure2). Application of low or medium fragmentation energy in collision induced dissociation(CID) for O-glycosides results in the heterolytic cleavage of their glycosidic bonds,which yields characteristic Y+

k fragments containing the aglycone part of the molecule[34, 44–46]. Low fragmentation energy does not provide adequate fragmentation forC-glycosides. Medium fragmentation energy, however, causes intraglycosidic cleavagesfor C-glycosides resulting in i, jX+ fragments that may cause interpretation difficulties

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Fig 2. Fragment nomenclature used for flavonol glycosides [40, 41].

since the higher fragmentation energy also yields Yk fragments for C-glycosides. Bothfragments, i, jX+ and Y+

k , were detected for C,O-glycosides [47].The sugar type of the glycan part of the flavonols can easily be determined by

the fragments appearing at different m/z values for hexoses (glucose), deoxyhexoses(rhamnose) and pentoses (arabinose/xylose). The characteristic mass losses for theglycan parts of O-glycosides are -162 Da, -146 Da and -132 Da, respectively.

Sugar units can be attached directly to the hydroxyl groups in the aglycone orto another sugar moiety. Di-O-glycosides (two sugars at different positions formingmonosaccharides) or two sugars at same position (O-diglycosides, forming disaccharide)can be differentiated by their Y1, Y0 and Z1 ions in the product ion spectra. In the caseof O-diglycosides, fragmentation of the inner glucose residue is also possible usinglow-energy CID in the positive ionization mode. The irregular Y∗ ion formed indicatesthe internal residue loss and may have a very high relative abundance and thus yieldsfalse information about the glycan sequence [34]. Loss of inner sugar from the tworing-sequence of O-diglycosides cannot occur in the negative ion mode [1] and thusdetection of two independent losses of one sugar moiety from the [M – H]� ion may beinterpreted as fragmentations from a di-O-glycoside.

Radical fragment ion formation has been used for the elucidation of the glycosylationposition in the flavonol aglycone [38, 48–52]. The glycan part fragments by homolyticcleavage and forms a radical ion. This homolytic cleavage occurs more easily fromthe 3-position than the 7-position [48]. The attachment site of the glycan part can bedifferentiated using the relative abundances of the radical fragment ions and ions fromthe heterolytic cleavages obtained from the product ion spectra [38, 49]. However,

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when using homolytic cleavage as a tool for structure elucidation of flavonoids, oneshould be aware that the instrumentation used [38, 51] as well as the length of thesaccharide chain at the 3-position of the flavonol glycosides [52] affect the homo- andheterolytic cleavages. The validation of the fragmentation rules is therefore considered aprerequisite when different types of instruments are used to identify glycosylation sites[51].

The aglycone part of flavonoid O-glycosides can be identified by means of MS3

experiments of the Y0 fragment. Flavonol and flavone aglycones with the samemolecular mass can be differentiated from the spectra since the fragmentation pathwaysare different, corresponding to the substitution pattern of the molecule [40, 43]. The0,2A+ fragment is charasteristic for flavonols, whereas the abundance of 1,3A+ and0,2B+ ions is high for both groups of compounds [39, 40].

2.3 Xenobiotic (drug) metabolism

Chemicals foreign to the normal composition of the human body are called xenobiotics.These chemicals include a wide variety of drugs, pollutants, pesticides and foodadditives. In the human body xenobiotics are transformed into a more hydrophilicform for excretion via kidneys or biliary system. The aim of this metabolism processis to eliminate xenobiotics and to control the levels of the desirable compounds [5].Biotransformation reactions convert drug molecules to inactive, active and toxicmetabolites. In most cases the resulting product of an active substrate is an inactivemetabolite. Active metabolites are often formed from an active drug, but in some casesbiotransformation reactions convert an inactive substrate to an active metabolite [53].This phenomenon is utilized, for example, with prodrugs. The metabolic product mayalso be toxic [54]. An example of a toxic metabolite is N-acetyl-p-benzoquinone imine(Figure 3f), which is a reactive liver toxin resulting from the metabolic modification ofacetaminophen [55, 56].

Metabolism reactions are usually divided into phase I and phase II reactions[57]. In the phase I a functional group, such as a hydroxyl group, is added to thesubstance or in some cases the functional group is exposed from the structure bybiotransformation reactions [58]. The cytochrome P450 is the most important phaseI enzyme system (for more information, see chapter 2.4). The phase II reactions areconjugation reactions, where an endogenic product such as a glucuronic acid, sulfate orglutathione is transferred to the functional group formed in the phase I metabolism.

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Sometimes the conjugation takes place directly with the unmodified substrate. Thephase II reactions are catalyzed by different conjugative enzymes (Table 1). Thesemetabolites are rarely pharmacologically active.

Table 1. Phase II reactions.

Reaction Enzyme Conjugate

Glucuronidation UDP-Glucuronosyltransferase Glucuronide

Sulfation Sulfotransferase -SO3

Glutathione conjugation Glutathione-S-transferase Glu-Cys-Gly

Methylation Methyltransferase -CH2

Acetylation Acetyltransferase -OCCH2

Amino acid conjugation Various Various

Glucuronidation is the most important conjugation reaction in mammals [59]. Glu-curonic acid, originating from UDPGA (uridine-5’-diphosphoglucuronic acid), isconjugated via a glycosidic bond to the substrate. The reaction is catalyzed by UDP-glucuronosyltransferase (UGT). UGTs are a superfamily consisting of a number ofisoenzymes [60, 61]. The formed glucuronide is very polar and thus more easilyexcreted.

ROH +UDPGA→ RO−GA+UDP (1)

Sulfate conjugation is also a significant metabolic route in mammals. The sulfate donorin this reaction is PAPS (3’-phosphoadenosine-5’-phosphosulfate) and the reaction iscatalyzed by the sulfotransferase enzyme. The sulfate conjugate is anionic and thusmuch more soluble than the parent compound. In the Equation 2, S=SO3.

ROH +PAPS→ RO−S+PAP (2)

Glutathione (GSH) is a tripeptide formed from glutamate, cysteine and glycine. Theglutathione conjugation occurs easily with electrophilic groups and is an importantmetabolic route, especially for reactive metabolites. It is a defence mechanism againstcarcinogens. Glutathione binds to substrate through the cysteine nucleophilic thiolgroup.

RH +GSH→ R−S−G (3)

The conjugate formed is modified further as the glutamyl and glycinyl groups areremoved while the remaining cysteinyl group is acetylated. The product of the yield, a

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mercapturic acid derivative (=N-acetyl-cysteine conjugate), is excreted into the urine.Other phase II reactions are methylation and acetylation. These reactions do not

improve solubility but rather reduce pharmacological activity [57]. Methylation is notvery common for exogenous drug molecules but it is a conventional metabolism routefor endogenic substances. The methyl group from the cofactor S-adenosylmethionine(SAM) is transferred to the substrate by methyltransferases. In acetylation the acetylgroup donor is Acetyl-CoA and the reaction is catalyzed by N-acetyltransferase (NAT).

X +AcetylCoA→ Acetyl−X +CoA (4)

Amino acid conjugations are also possible phase II metabolism reactions, in particularwith the carboxylic acids [5]. The amino acid in the conjugation reactions is usuallyglutamine or glycine. The formation of hippuric acid through the glycine conjugation ofbenzoic acid is an example of amino acid conjugations.

The metabolism of drugs and other xenobiotics is affected by matters such asgenetic, non-genetic and environmental factors [62, 63], which lead to a wide inter-individual variability of drug metabolism. The most important exogenous factorsaffecting xenobiotic metabolism are substances with inhibitive or inductive effectstowards drug metabolizing enzymes. Polycyclic aromatic hydrocarbons (PAH) are oneof the best known substances with inductive properties. Hypericum perforatum (St.John’s wort) also has drug interaction tendencies through the induction of CYP3A4and CYP2C9 enzymes [64, 65]. In addition, many drugs, for example, ketoconazole(CYP3A4), fluvoxamine (CYP1A2) and quinidine (CYP2D6), are known to be inhibitors[6, 66, 67].

2.4 Cytochrome P450 enzymes

The cytochrome P450 (CYP) enzymes are a superfamily of hemoproteins which catalyzea wide range of biotransformation reactions. The CYP enzymes convert foreignsubstances into a more suitable, i.e. hydrophilic, form for excretion. The cytochromeP450 enzymes are undoubtedly the most important metabolizing enzyme system inhumans since the CYP enzymes are responsible for approximately 70-80% of the phaseI (oxidative) metabolism of drugs and other xenobiotics [68]. The cytochrome P450enzymes are distributed in almost every human organ, but the highest concentrations ofCYP enzymes are found in the liver. The liver is the center of xenobiotic metabolismand is capable of catalyzing both oxidation and reduction pathways [69]. Cytochrome

25

P450-catalyzed reactions can be broadly classified into four categories: hydroxylations,epoxidations, reductions and heteroatom oxidations [70]. Examples of some CYPcatalyzed phase I reactions are shown in Figure 3.

Fig 3. Some CYP catalyzed phase I reactions [57, 62].

The CYP metabolic system consist of the CYP enzyme, molecular oxygen and NADPH-cytochrome reductase, which serve to transfer electrons. The generic oxidative reactionfor a given substrate R may be presented as follows:

RH +O2 +NADPH +H+→ ROH +NADP++H2O (5)

The cytochrome P450 superfamily is divided into a number of families and subfamiliesaccording to the amino acid composition of the enzymes. Genes in the same subfamilyhave a similarity greater than 55% in their structures [5]. The CYP1-, CYP2- and CYP3families are mainly responsible for xenobiotic metabolism; the most important enzymesare CYP1A1/2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1and CYP3A4 [71]. In the human liver the most important drug metabolizing enzyme isCYP3A4, whereas the members of other CYP families than mentioned here are largerly

26

responsible for the biotransformation of endogenic components such as steroids andfatty acids [72].

2.5 LC/MS methods in metabolite profiling andquantitation

Nowadays LC/MS techniques have clearly become the most widely-used tool inanalyzing drugs and their metabolites [2, 73]. Typical steps in these analyses consist ofsample preparation, chromatographic separation, mass spectrometric detection and dataprocessing.

The sample preparation is an important step in all analyses, and especially whenanalyzing biological fluids [3, 74]. The analyte must be extracted from the samplematrix components and other impurities that may reduce the specificity of detection orinterfere with the mass spectrometric ionization process by suppression or enhancement.A commonly used sample preparation method in LC/MS analysis is still traditionalliquid-liquid extraction (LLE), although the method is time-consuming and producesgreat amounts of solvent waste [3]. Another commonly used method, solid phaseextraction (SPE), uses a sorbent material containing a solid phase and a flowing liquidphase to isolate a compound or certain type of compounds from a liquid sample. Thesample is loaded into cartridge, undesired compounds are washed away, and the analytesare eluted with a different solvent [75]. Numerous SPE cartridges containing differentpacking materials are available and can be used for extracting acidic, basic and neutralcompounds from different kind of matrices and also use the ion exchange principle [76].It is worth noting that when using extraction methods the discarding of metaboliteswith different recoveries can occur and thus modify the metabolic profile of the studycompound. Therefore, in the case of metabolite profiling, it is recommended thatunspecific sample preparation methods such as protein precipitation with acetonitrile ormethanol rather than SPE or LLE be used.

Chromatographic separations are most commonly obtained using high-performanceliquid chromatography (HPLC) with reversed-phase columns. In the early phase ofmetabolite profiling rapid generic chromatographic methods are often utilized, applying5-80% acetonitrile or methanol and aqueous phase in a gradient run of some minutes.The optimization of gradient strength and aqueous phase pH using parent compound,however, also improve peak shapes and thus lead to better chromatographic resolution

27

and detection sensitivity [73]. As column properties strongly affect separation efficiency,it is most convenient to use short columns with a narrow inner diameter (2 mm x 50 mm)packed with small particles (≤ 3 µm). Techniques using columns with a particle size ofsub-2 µm and an instrumentation capable of working with elevated back pressures areusually called ultra-performance liquid chromatography (UPLC) [77–80]. The smallerparticle size enables a shorter analysis time and better resolution.

For metabolic studies the most common ionization methods in LC/MS applicationsare electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).The ion sources are referred to as API sources, as the ionization does not take place in avacuum but in atmospheric pressure. ESI is suitable for almost all drug-like moleculeswith at least one easily ionizable functional group [81], whereas for steroids and otherless polar compounds, APCI is often utilized [82]. Of these two methods, APCI isgenerally less susceptible to matrix effects [83, 84]. The negative ionization polarityis considered more specific, whereas the positive ionization mode is more vulnerableto ion suppression [84, 85]. In ESI, the analyte is ionized within the solution andthus the chemistry of the liquid phase has a great impact to ionization efficiency. Thesolution containing the analyte is directed through the high voltage capillary (2 - 5kV), where the capillary voltage gathers the oppositely charged ions to the sides ofthe capillary and thus the droplets spraying from the capillary are excessively chargedwith the same polarity as that in the capillary. The liquid flow and gas flow parallelto the capillary aid the formation of droplet mist. The droplets diminish by meansof vaporization (aided by drying gas flow) until the repulsion forces of the similarlycharged ions are larger than the cohesive strengths of the surface tension, when theindividual ions vaporize to gas phase (ion evaporation method, IEM). In the chargeresidue model (CRM) the vaporization of the solvent continues until only single iondroplets remain. The IEM is believed to be dominant with the small molecules, whereasin the case of large molecules, such as proteins, CRM occurs [86]. The ion suppression /enhancement by the co-eluting component may occur due its effect to droplet formationor the availability of charge (proton), or co-precipitation of the analyte from the spray asa neutral molecule [87–89].

From the MS analyzer point of view, quadrupole instruments are most used inquantitative analysis due to their high sensitivity, good specificity and high linear range,whereas TOF instruments are used in screening type analyses, such as profiling ofunknown metabolites, because they offer high sensitivity wide mass range scan withhigh mass resolution (>40 000 FWHM) and high mass accuracy. However, modern

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time-of-flight mass spectrometers could be used for quantitative work as their linearrange has significantly improved recently due to the introduction of dynamic rangeenhancement systems [90]. TOF instruments are capable to very high data acquisitionrates and thus ideal for UPLC. Ion trap mass spectrometers are most commonly used forscreening type analysis because of their wide mass range and relatively high sensitivity[91] and capability to MSn in structure characterization, but their use with UPLC islimited by their relatively slow data acquisition rate.

MS data processing is nowadays highly automated and the development of post-acquisition data mining techniques are strongly enabled by high mass resolutiondata. Metabolite screening is assisted by software that compares the acquired databetween the sample and negative control and suggests biotransformations and calculatedelemental compositions for the found metabolites using accurate mass measurementsand information about parent compound. Some software includes features like thedealkylation tool [92] and are capable of fragment ion data interpretation [93, 94]and mass defect filtering (MDF) [95, 96]. Fragment ion data is used to identifybiotransformation sites in the parent molecule by the use of computer algorithms thatcompare mass shifts of the diagnostic fragment ions to the parent molecule and detectedmetabolites. A dealkylation tool is used to identify bonds having the potential to cleaveby biotransformation reactions, whereas MDF removes biological background usingthe observation that biotransformations usually change relatively little the decimalcomponent of the m/z observed [92]. In addition, using current in silico methods togetherwith accurate mass measurements for predicting metabolism and mass fragmentation isa solution to differentiate parent molecules and metabolites with identical molecularformulae when there is an absence of reference standards or spectra [97].

2.6 Drug interaction studies with N-in-one cocktails

Drug-induced inhibition of CYP enzymes is known to be the most common reason fordrug-drug interactions and therefore studying the inhibitory effect of new chemicalentities is extremely important when selecting lead compounds and for minimizing thenumber of drugs being withdrawn from clinical studies [6, 98, 99].

N-in-one assays can be used to study a number of different CYP enzymes at the sametime, increasing the amount of data obtained from a single experiment, and thus increas-ing throughput and saving time. The three to six most usually included CYP isoenzymesin the N-in-one assays are CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and

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CYP3A4. Generally, the cocktails lack the probe metabolites for isoenzymes CYP2A6,CYP2B6 and CYP2C8.

A number of papers describing analytical methods for monitoring several CYP-specific probe metabolites from human urine or plasma after a single or N-in-oneadministration ("cocktail/cassette dosing") have been published for in vivo interactionstudies or cytochrome P450 activity and phenotyping purposes [11, 12, 100–112].

The cytochrome P450 interaction studies have been traditionally conducted withseparate specific assays for each single CYP isoform, which offers the possibility tooptimize the analysis for only one compound at a time. This provides the best possibleperformance for the analysis. For example, the effect of HPLC eluent pH can beoptimized for the best chromatographic peak shape and for optimum detection response,i.e. for mass spectrometric ionization. The requirements for the analytical method withN-in-one assay are clearly of a higher level, as it is often the case that many substanceswith chemically very diverse properties (acids or bases with high or low hydrophilicity)should be analyzed as fast as possible, and preferably from a single analysis from asingle sample. Therefore the set-up of a functional analytical method is most often thesearch for the best compromise in the analytical performance between the completelydifferent analytical conditions, which are optimum for each compound.

Thus far, almost all published in vitro N-in-one assays for CYP activity screeninghave relied on reversed-phase liquid chromatography (RP-HPLC) as an analyticalmethod, and most commonly with mass spectrometric detection [8, 9, 13, 113–118].Only few in vitro and in vivo N-in-one assays have utilized ultra performance liquidchromatography [10, 111, 119].

The need for throughput is high in the drug industry due to the large amount oflead compounds produced by combinatorial chemistry and the samples obtained frommetabolism studies. Analysis time is thus an important factor in CYP interactionscreening. In the published assays analysis time (gradient elution and column equi-libration) varies from less than 1 min to 15 min cycle time. The time used for eachindividual sample is not only affected by sample preparation and chromatographicmethods, but also by the mass analyzer used. LC/MS detection with triple quadrupolemass spectrometers is usually preferred over other types of instruments not only becauseof their best linear range of detection, but also due to the highly specific multiple reactionmonitoring (MRM, sometimes also called selective reaction monitoring, SRM). InMRM, two mass filters are used for ion selection; first the precursor (parent) ion isselected (Q1) to collision cell (q2) and, after the collision with gas atoms (CID, collision

30

induced dissociation), the specific fragment ions of the parent are selected with thesecond mass filter (Q3). The use of MRM enables fast chromatrographic runs due to areduced need for complete chromatographic separation for most of the analytes.

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3 Aims of the study

The aim of the study was to develop faster and more sensitive LC/MS techniquesfor the identification and quantitation of plant and drug metabolism products. Anumber of cocktail-type N-in-one-assays have been developed for screening CYPinhibition or induction for different CYP enzymes. These assays have, unfortunately,not been extensive enough or the analysis methods used have been laborious to perform.Therefore, the main focus was in developing LC/MS analysis methods for CYP-specificdrug interaction studies, i.e. for N-in-one in vitro and in vivo cocktail assays.

The detailed aims of the study were

– to identify flavonoids of Rhodiola rosea [I] by liquid chromatography-mass spec-trometry and to test the suitability of collision-induced radical cleavage of flavonoidglycosides as a tool for glycosylation site identification

– to identify LC/MS urinary metabolites of bupropion [II] for the development of an in

vivo cocktail assay– to develop and validate LC/MS/MS analysis methods for an in vitro interaction

cocktail assay [III] and for nine drugs and their cytochrome P450-specific probemetabolites from human urine [IV]

– to develop a specific and sensitive analysis method for the quantitation of repaglinidefrom human placental perfusates [V]

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4 Experimental procedures

This section briefly presents the sample preparation methods, instrumentation and matrixeffect tests used in this study. More detailed descriptions of the sample preparationmethods, experimental parameters, columns and additional instruments can be found inoriginal publications I–V.

4.1 Sample preparation

The sample matrices used in this study were dried plant material, urine and samplesfrom liver microsomal incubations and placenta perfusions.

The plant material used for Rhodiola rosea flavonoid analysis in study I was materialgrown at the Department of Biology and Botanical Gardens at the University of Oulu.The sample preparation methods used for the plant material were supercritical fluidextraction (SFE) and liquid extraction with aqueous methanol in an ultrasonic bath.

Human urine samples in studies II and IV were incubated in the presence of β -glucuronidase for the hydrolysis of glucuronide conjugates of the phase I metabolites. Inaddition, solid phase extraction with different sorbents was tested on the urine samples(unpublished data); the sorbents were Oasis HLB (macroporous copolymer, 10mg),MAX (strong anion exchange, 30 mg), MCX (strong cation exchange, 30 mg), WAX(weak anion exchange, 30 mg) and WCX (weak cation exchange, 30 mg) from Waters(Waters Corp., Milford, MA, USA). The urine sample was loaded into the cartridgewithout any pH modifications after glucuronide hydrolysis. The wash and elution stepswere performed according to the manufacturer’s standard instruction protocol as follows:MCX/WAX and WCX/MAX cartridges were washed with 2% formic acid in waterand 5% ammonium hydroxide in water, respectively, while the HLB cartridge waswashed with 5% aqueous methanol. Acetonitrile:methanol (1:1) mixture was used forthe elution of analytes from HLB cartridge, and methanol was used for the first elution(wash step 2) with the MCX/WAX and WCX/MAX cartridges. For the final elution ofthe analytes from MCX/WAX and WCX/MAX cartridges, 5% ammonium hydroxide inmethanol and 2% formic acid in methanol were used, respectively. After SPE, sampleswere evaporated to dryness and reconstituted to 300 µl 1:5 mixture of acetonitrile andphosphate buffered saline (PBS), which was used for retaining good peak shape. The

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trial for dosing and collecting human urine samples was designed and monitored inaccordance with Good Clinical Practice and the Declaration of Helsinki.

The human in vitro liver microsomal incubations in study III were performed asdescribed by Turpeinen et al. [114] using 0.5 mg/ml microsomal protein content andNADPH as a cofactor.

In the repaglinide study V, solid phase extraction using Oasis MAX anion-exhange96-well plates was utilized for the placenta perfusion samples, and for antipyrineanalysis, the samples were used as such, without any pre-treatment.

4.2 Instrumentation

The LC/MS instrumentation used in this study is presented in Table 2. The liquidchromatographic separations were performed using the HPLC and UPLC systems. Theinitial screening and accurate mass measurements of the compounds and metabolitespresent in the sample matrices were carried out using an LCT TOF mass spectrometerequipped with a LockSpray ion source. The more detailed structure determinationsand quantitative analysis were performed using triple quadrupole mass spectrometers.All three Waters Quattro mass spectrometers were equipped with Z-spray ion sources.Electrospray ionization with positive and negative polarities was used in all studies.

Several chromatographic columns and mobile phase eluents were tested whendeveloping the LC/MS methods used in the studies. The LC columns and eluents chosenfor the studies are presented in Table 3.

4.3 Matrix effect tests

The effects of matrix components on the ionization response of analytes were tested bytwo different test approaches [89, 120–122]: a) comparing the LC/MS/MS peak intensityobtained from samples spiked to low analyte concentration (ng/ml) to blank samplematrix and blank solvent (sample matrices processed similar to the real samples) and b)injecting a blank matrix sample (processed similar to the real samples) and monitoringthe suppression or enhancement of the baseline for the analyte compounds achieved bypost-column infusion of the analyte compound solution into the flow directed to the ionsource.

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Table 2. LC/MS instruments used in studies I–V.

Liquid chromatographs Mass spectrometersWaters 2690 Alliance HPLC system (Waters Cor-poration, Milford, USA) [I]

Micromass Quattro II triple quadrupole mass spec-trometer (Micromass, Altrincham, UK) [I]LCT TOF time-of-flight mass spectrometer withLockSpray ion source (Micromass, Altrincham,UK) [I, II, IV]

Waters 2695 Alliance HPLC system (Waters Cor-poration, Milford, USA) [II-IV]

Micromass Quattro Micro triple quadrupole massspectrometer (Micromass, Altricham, UK) [II-IV]

Finnigan Surveyor HPLC (Thermo Finnigan, SanJose, USA) [III]

TSQ Quantum Discovery MAX triple quadrupolemass spectrometer (Thermo Finnigan, San Jose,USA) [III]

Waters Acquity UPLC system (Waters Corpora-tion, Milford, USA) [IV, V]

Micromass Quattro Premier triple quadrupolemass spectrometer (Micromass, Altrincham, UK)[IV, V]

Table 3. LC columns and mobile phase eluents used in the studies I–V.

Study Columns

I Waters Symmetry C18 (2.1 mm × 100 mm, 3.5 µm)

II Phenomenex Gemini C18 (2.0 mm × 150 mm, 5.0 µm)

III Waters Sunfire RP18 (2.1 mm × 100 mm, 5.0 µm)

IV Waters BEH Shield RP18 (2.1 mm × 50 mm, 1.7 µm) (UPLC)

Waters XBridge Shield RP18 (2.1 mm × 50 mm, 3.5 µm)

V Waters BEH Shield RP18 (2.1 mm × 50 mm, 1.7 µm) (UPLC)

Study Mobile phase eluents

I A) 0.1 % formic acid B) acetonitrile

II A) 0.1 % acetic acid (ESI+), 2 mM ammonium acetate (ESI−) B) methanol

III A) 1% formic acid + 10 mM ammonium acetate (pH 2.4) B) methanol

IV A) 0.1 % acetic acid B) acetonitrile

V A) 0.1 % acetic acid B) acetonitrile

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5 Results and Discussion

The main results of the study are described and discussed in this section. Someunpublished data is also presented in the method development section (5.2.1). Moredetailed information can be found in the original publications I-V.

5.1 Structure analysis of flavonoids and drugmetabolites [I–II]

5.1.1 Flavonoids

LC/TOF-MS was used for screening flavonoids from the Rhodiola rosea extracts. Atotal of 15 flavonol glycosides were detected by LC/MS from the ethanol extracts of theaerial parts of the R. rosea. The LC/TOF-MS chromatograms of the detected flavonoidsare presented in Figure 4 and the molecular weights and retention times collected fromthe analysis together with the identifications of the detected flavonoids are shown inTable 4; the structures of all the detected flavonoids are presented in Figure 6.

The initial identifications of the flavonoids were obtained using accurate massmeasurements and detailed identifications were carried out using tandem mass spec-trometric methods. Five of the detected flavonoids (7, 9, 11, 13 and 15) were knownfrom the literature [123–126] and the remainder (1-6, 8, 10, 12 and 14) were previouslyunreported from Rhodiola rosea. Most of the flavonols were detected with both positiveand negative electrospray ionization (Figure 4); flavonols 9 and 10 were detected usingnegative ionization mode since negative electrospray ionization is more sensitive toflavonoids [32, 42].

The in-source generated aglycone [M + H–glycose]+ fragment-ions were chosen forfurther MS/MS experiments ( = "pseudo MS3") to identify the aglycone parts of thedetected flavonoids. All aglycones were identified by comparing their spectra withthose of standard compounds and the literature [36, 127]. The data from the "pseudoMS3" experiments is presented in Table 3 in original publication I. The aglycones wereidentified as kaempferol, herbacetin, gossypetin, quercetin and hydroxyl-gossypetin, inwhich the hydroxylation sites in the B-ring were identified as propably being 3’-, 4’- and

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5’-hydroxylated, as in flavonoid hibiscetin.

Table 4. Detected flavonoids in R. rosea extracts.

Flavonoid RT (min) MW Identification

1 11.8 642 Gossypetin-di-O-glucoside

2 13.4 642 OH-gossypetin-7-O-rha-8-O-glu

3 14.3 626 Herbacetin-di-O-glucoside

4 15.1 610 Kaempferol-3-O-glu-7-O-glu

5 15.1 610 Quercetin-3-O-rha-7-O-glu

6 15.5 642 Gossypetin-di-O-glucoside or O-di-glucoside

7 16.3 626 Gossypetin-7-O-rha-8-O-glu = rhodiolgidin

8 16.5 612 Gossypetin-3-O-glu-7-O-xylo/ara

9 17.6 596 Herbacetin-8-O-xylo-3-O-glu = rhodalidin

10 17.6 594 Kaempferol-3O-rha-7-O-glu

11 18.7 610 Herbacetin-7-O-rha-8-O-glu = rhodionidin

12 18.8 596 Herbacetin-3-O-glu-7-O-xylo/ara

13 27.8 464 Gossypetin-7-O-rha = rhodiolgin

14 31.0 448 Quercetin-3’/4’-rha

15 32.4 448 Herbacetin-7-O-rha = rhodionin

ara: arabinose; glu: glucose; rha: rhamnose; xylo: xylose

The glycosylation sites of the detected flavonols were tentatively identified by comparingthe intensities of the CID fragment ions from [M + H]+ and according to the radicalfragment ion intensities produced in negative electrospray ionization. The glycosesfrom different sites of the protonated flavonol di-O-glycosides cleave more easily thanothers, producing a more intense fragment peak, the cleaving order being: 5 > 3 >3’ ≥ 5’ > 4’ > 7 [1]. Yet, the intensity of radical aglycone [Y0 – H]•− ion, formedby homolytic cleavage of glycose from the aglycone in the MS/MS of [M – H]−

ions is dependent on the position in which the cleaving glycoside is attached to theaglycone; the phenomenon was also tentatively discussed by Hvattum & Ekeberg [48]and Cuyckens & Claeys [38] at the very time we were studying the issue. However, theinterpretation of the data should be carried out very carefully, and only after comparingthe data with fragmentations of known standard flavonoids obtained with the verysame instrument and the same parameters. Therefore, we used six different flavonol

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Fig 4. LC/MS sum ion chromatograms of detected flavonoids acquired at ESI+

(a) and ESI� (b) from the ethanol extract of R. rosea aerial parts. The chro-matograms are amplified 24× 1) 11.8 min Gossypetin-di-O-glucoside, 2) 13.4min OH-gossypetin-7-O-rha-8-O-glu, 3) 14.3 min Herbacetin-di-O-glucoside, 4)15.1min Kaempferol-3-O-glu-7-O-glu, 5) 15.1 min Quercetin-3-O-rha-7-O-glu, 6)15.5 min Gossypetin-di-O-glucoside or O-di-glucoside, 7) 16.3 min Gossypetin-7-O-rha-8-O-glu = rhodiolgidin, 8) 16.5 min Gossypetin-3-O-glu-7-O-xylo/ara, 9)17.6 min Herbacetin-8-O-xylo-3-O-glu = rhodalidin, 10) 17.6 min Kaempferol-3O-rha-7-O-glu, 11) 18.7 min Herbacetin-7-O-rha-8-O-glu = rhodionidin, 12) 18.8 minHerbacetin-3-O-glu-7-O-xylo/ara, 13) 27.8 min Gossypetin-7-O-rha = rhodiolgin,14) 31.0 min Quercetin-3’/4’-rha, 15) 32.4 min Herbacetin-7-O-rha = rhodionin [I].

glycosides with glycosylation sites in the 3-, 7-, 8- and 4’-positions for the comparisonexperiments (Table 1 in original publication I). Three of the flavonol standards usedwere mono-O-glycosides, two were O-diglycosides (the saccharides were rutinose andneohesperidose) and one was di-O-glycoside, with robinin and rhamnose as the sugarunits. The aglycone fragment ions obtained from the standard compounds, as well as thefragment ions due to losses of each glycose moiety from robinin, are presented in Figure5.

When comparing the losses of glycosides from the 3-position (Figure 5a and 5b) andthe loss of glucose in the 4’-position (Figure 5c), the cleavages of the glycosides fromthe 3-position produced very intense [Y0 – H]•� ions, as was reported in earlier studies[38, 48], and thus were distinguishable from the cleavage from the 4’-position. The

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cleavages from the 7- and 8-positions (Figure 5d and 5e) were also clearly distinguishablefrom 3- and 4’-positions, and the relative intensities between the produced [Y0 – H]•−

ions compared to the [Y0 ]− ions were much lower than the intensities produced by thecleavages from the 3- and 4’-positions. However, it was not possible to distinguishbetween the fragmentation of flavonols glycosylated in 7- and in 8-positions. Thecleavage of rhamnose from the 7-position of robinin did not produce a radical ion, butonly a [M–H–146]− ion at m/z 593, whereas the loss of robinose (6-rhamnosyl-galactose)from the 3-position produced a very intense radical fragment ion at m/z 430 (Figure 5f).This known phenomenon of an intense radical ion produced from the 3-position offlavonol glycosides has also been corroborated in more recent studies [50, 52].

Fig 5. [Y0 – H]•− and [Y0 ]− ions from MS/MS spectra of [M – H]− acquired froma) quercitrin (rhamnoside in 3-position); b) rutin (rutinoside in 3-position); c) spi-raeoside (glucose in 4’ -position); d) kaempferol-7-neohesperidose; e) gossypin(glucose in 8-position); and f) [M – H – rha]− and [M – H – rob]•− ions acquiredfrom robinin (rhamnose in 7-position and robinose in 3-position). Radical frag-ment ions marked with circles [I].

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Fig 6. Flavonoids identified from the R. rosea extract [I].

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The experiments for the two known 7-O-rhamnopyranose-8-O-glucopyranosidesfrom the R. rosea, rhodiolgidin (7) and rhodionidin (11), showed that the presence ofanother glycan moiety in the 7-position (here, rhamnose) strongly increases radicalfragment ion formation in the cleavage from the 8-position (Figure 3 and Table 2 inI). Thus, the differentiation of 7-,8-substituted and 3-,7-substituted flavonoid di-O-glycosides is very difficult, the radical ion formation due to cleavage from the 8-positionbeing very similar to that from the 3-position. In addition, the fragmentation behaviourof glycans from the 3- and 8-position in these kinds of di-O-glycosides is also verymuch alike with positive ionization (Table 2 in I), making the identification of the 3- and8-positions even more difficult. However, if the aglycone of an unknown flavonoidglycoside does not have hydroxyl groups in both the 7- and 8-positions, the suggestiveidentification of glycosylation sites may be obtained with correct instrument adjustmentsand comparison with known standards.

After identifying the correct aglycones, compounds 9 (rhodalidin), 13 (rhodiolgin)and 15 (rhodionin), in addition to rhodiolgidin (7) and rhodionidin (11), were identifiedas the known flavonoids of the plant. The other detected flavonoids were identified usingthe methods described above (data presented in Tables 2 and 3 in I).

The results of this study suggest that radical ion fragmentation could be used as astructure elucidation tool for flavonol glycosides, although, the obtained data should beanalyzed very carefully and always in comparison with known standards. This study is,to our knowledge, the only one containing the homolytic fragmentation behaviour offlavonol 8-O-glycosides. It was also the first to report the identification of compounds1-6, 8, 10, 12, and 14 for Rhodiola rosea.

5.1.2 Bupropion metabolites

Drug metabolites may have some structural features which may enable the formationof reactive species in the human body; thus the structure elucidation of even minormetabolites is important from toxicological point of view. In this study, 20 bupropionmetabolites were detected by LC/TOF-MS from human urine samples. Sixteen ofthe detected metabolites were conjugates, including 12 glucuronides, three sulfatesand a glycine conjugate; the other four metabolites were phase I metabolic products.Biotransformations were screened using accurate mass measurements and the detailedidentification of the detected metabolites was performed using tandem mass spectro-metric methods (data shown in Table 1 in II). The LC/TOF-MS chromatograms of the

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detected metabolites are presented in the Figures 7 and 8.The tandem mass spectrometric experiments confirmed the identifications of hydrox-

ybupropion (M1), threo- and erythrohydrobupropion (M2 and M3) and m-chlorohippuricacid (M5). The M2 was identified as threohydrobupropion as its concentration is knownto be much higher than the other detected amino alcohol metabolite, M3 [128, 129].The M4 was also identified as the previously known metabolite with butyl-grouphydroxylation and keto-group reduction to an alcohol [130, 131].

Figure 8 shows the presence of four isobaric glucuronide conjugates for threo- anderythrohydrobupropion (a, M6 - M9), as well as four glucuronidated hydroxymetabo-lites (b, M10 - M13), three glucuronides for a metabolite with hydroxylation andhydrogenation (c, M14 - M16) and a glucuronide for dihydroxylated bupropion (d,M17), in addition to sulfate conjugates for hydroxylation metabolite (f, M18) and forhydroxylations with hydrogenation (e, M19 and M20). The sulfates were detected onlyin negative ion mode, whereas all the other metabolites were detected in positive ionmode.

The high stability of M6 - M9 to β -glucuronidase activity suggested N-glucuronidation.As the N-glucuronidation may form a new chiral center in the molecule and lead to theformation of a total of four glucuronide-conjugated amino alcohol metabolites, metabo-lites there were assigned as N-glucuronides of threo- / and erythrohydrobupropion. Theconfiguration of nitrogen can be tetrahedral and the lone nitrogen electron pair canbe considered as the fourth group providing chirality, especially if racemization bypyramidal inversion is restricted by glucuronidation. The M12 and M13 (glucuronida-tions of hydroxybupropion) and M17 (glucuronide of dihydroxylation metabolite)were also resistant to hydrolysis and thus suggest N-glucuronidation. In addition, theformation of two diastereoisomers of the glucuronides M12 and M13 also pointed toN-glucuronidation. In the case of M10, M11 and M14 - M16, the glucuronides werethought to be O-glucuronidations as the hydrolysis rates obtained were similar to theO-glucuronide standards of 6-OH-chlorzoxazone and 1-OH-midazolam.

The aglycone parts of the conjugated metabolites were identified by pseudo-MS3 ex-periments where the in-source generated [M + H –glucuronide]+ and [M – H –sulfate]−

fragments were chosen for the collision cell CID. The hydroxylation sites of M10 - M13were identified by comparing the fragmentation patterns of the aglycone part of themetabolite with those of bupropion and hydroxybupropion (Figure 6 in II). The M10and M11 were identified as glucuronides of aromatically hydroxylated stereoisomers,whereas the aglycone parts of M12 and M13 were identified as morpholino hydroxy-

45

Fig 7. LC/TOFMS ion chromatograms for bupropion and its phase I metabolites,generated using [M + H]+ ions: a) bupropion; b) threo- / erythrohydrobupropion(M2 and M3); c) hydroxybupropion (M1); d) hydroxylation with hydrogenation (M4);and e) m-chlorohippuric acid (M5) [II].

46

Fig 8. LC/TOF-MS ion chromatograms for conjugated bupropion metabolites,generated using the [M + H]+ and [M – H]− ions: a) [M + H]+ glucuronides ofthreo- / erythrohydrobupropion (M6 - M9); b) [M + H]+ glucuronides of hydroxy-lated metabolite (M10 - M13 ); c) [M + H]+ glucuronides of hydroxylation with hy-drogenation (M14 - M16). Peaks at retention times of 7.9 min, 9.9 min, 11.2 min and11.6 min are due to 37Cl isotope of glucuronide conjugates of hydroxymetabolites(m/z 432); d) [M + H]+ glucuronide of dihydroxylation (M17); e) [M – H]− sulfates ofhydroxylation with hydrogenation (M19 and M20) and f) [M – H]− sulfate of hydrox-ylation (M18) [II].

47

bupropion. The cleavage of the butyl-group from the glucuronide conjugate (Figure 9c)at m/z 376 and the fragment at m/z 200 indicate the loss of an intact butyl group from theprotonated aglycone part of the molecule. The fragment at m/z 200 is analogous to theion at m/z 184 rising from the loss of the butyl-group in bupropion and morpholinobupropion (Figure 6a and 6b in II). The fragment at m/z 216 in the spectrum (Figure9g) of the glucuronide conjugate of the dihydroxylation metabolite (M17) is againanalogous to the ion at m/z 184 of the glucuronide conjugate of hydroxybupropion(Figure 9d), indicating that the hydroxylation was not located in the butyl group of thesubstrate, as was previously reported for the dihydroxymetabolite [132]. The aglyconepart of the sulfate conjugate M18 was identified to be the metabolite with aromatichydroxylation and the aglycone of M19 and M20 was identified as a hydroxylated andhydrogenated metabolite. The structures of the detected bupropion metabolites arepresented in Figure 4 in II.

In this study, one previously unreported new phase I metabolite, dihydroxylation,was detected. In addition, previously unpublished glucuronide conjugates for dihydroxy-bupropion, hydroxylation with hydrogenation and for two different hydroxy metabolites,together with the sulfate conjugates of aromatic hydroxylation and hydroxylation withhydrogenation, were reported for the first time.

5.1.3 TOF vs. QqQ

When comparing the time-of-flight and triple quadrupole mass spectrometers, the TOFinstrument proved to be a powerful tool for screening purposes; the QqQ instrumentwas used for more detailed elucidation tasks due to a lack of Q-TOF-instrument; TOFmay, however, yield very similar results to triple quadrupole MS/MS when using highcone voltages (in-source CID). TOFs offer excellent sensitivity in the high mass rangedetection mode with very good resolution and mass accuracy. This enables accuratemass measurement and thus identification of the metabolic biotransformation withrespect to the parent compounds. Although the triple quadrupole instruments operatingwith different scan modes, such as neutral loss mode, could be applied to screen flavonolglycosides and conjugation metabolites from plant extracts and in vitro / in vivo samples,respectively, the detection probability of unexpected metabolites is far better with TOFthan with triple quadrupole instruments.

48

Fig 9. MS/MS spectra for glucuronide conjugates of a) threohydrobupropion,(M2); b) erythrohydrobupropion, (M3); c) aromatic hydroxylation, (M11); d) hydrox-ybupropion, (M13); e) hydroxylation with hydrogenation, (M14); f) hydroxylationwith hydrogenation, (M16); and g) dihydroxylation, (M17) [II].

49

5.2 Quantitative analysis of CYP specific probesubstrates and their metabolites (in vitro and in vivo)[III - V]

5.2.1 Method development and validation

N-in-one cocktail assay [III, IV]

In recent years, several in vitro and in vivo cocktail / N-in-one assays have beendeveloped for CYP enzyme interaction screening purposes. These assays contain up toseven most important CYP isoforms, but usually exclude the probe metabolites forthe isoenzymes CYP2A6, CYP2B6 and CYP2C8, and thus the activity of all drugmetabolizing enzymes is not studied [8, 12, 108, 133]. In addition, the analytical methodperformances in these assays have been somewhat deficient, low sensitivity leading toa need for high parent drug dose levels, which is a drawback when dosing multipledrugs at the same time. In some cases more than one analysis was also required todetect all the metabolites due to the analysis of CYP2E1 probe drug chlorzoxazone[116, 118, 134] or the need to analyze both urine and plasma samples [12, 135].

Here an LC/MS/MS method was developed [III] for our in vitro cocktail assay toextent the linearity and enhance the sensitivity of the earlier LC/TOF-MS method [114].The in vitro cocktail consisted of ten CYP-selective probe substrates, which lead to theanalysis of 13 probe reactions in a single run. In addition, an UPLC/MS/MS analysismethod was developed for the in vivo N-in-one cocktail assay [IV] that consisted of ninedrugs and their CYP-specific metabolites and utilized a non-invasive sample collectionfrom the patients.

The sample matrices used in the N-in-one cocktail assays enabled simple samplepreparation steps before LC/MS analysis, and thus the method development in thesestudies mainly focused on chromatography and mass spectrometry. Only centrifugationwas needed for the incubation samples [III] prior to analysis and for the urine samplesin the study IV the dilution of the matrix (hydrolysis of the glucuronide conjugates)was considered sufficient to keep the matrix effect at an acceptable level. Solid phaseextraction, however, was tested for the N-in-one assay urine samples (subsection below).

In both N-in-one cocktail assays, a number of probe metabolites, with a greatvariation in structures (Figure 10 shows the structures of the in vivo cocktail compounds,IV) and chemical properties had to be separated by liquid chromatography prior to mass

50

spectrometric detection. The effect of different modifiers (i.e. acetic acid, ammoniumacetate, ammoniun formate and formic acid), eluent pH and LC columns with differentstationary phases were tested for the best chromatographic separation of the analytes.The pH of the eluents also affects to the ionisation efficiency of each analyte andthus the overall sensitivity of LC/MS methods. Here, we had to compromise whenchoosing the best LC/MS conditions for the cocktail assays. In study III, a mixture of 10mM ammonium acetate and 1% formic acid was considered to be the best aqueousphase, especially for the strongly tailing desethylamodiaquine, although the best generalsensitivity was obtained when the 0.1% acetic acid was used as the aqueous phaseand methanol as the organic phase eluent. The use of methanol or acetonitrile mayhave a different effect to the ionization efficiency, and in some cases the selectivity ofthe column and thus separation efficiency as well may be improved by changing theacetonitrile to methanol. However, the use of acetonitrile leads to decreased columnback pressures in comparison to methanol gradients.

In study IV, the very poor ionization efficiency of repaglinide and losartan and theirmetabolites in basic conditions and the weak retention of nicotine and its metabolitecotinine in acidic conditions were the starting points for the development of LC methodfor the analytes. We sought to improve the retention of cotinine by using buffer solutionssuch as ammonium formate or ammonium acetate, but these ion pairing reagentsdecreased the detection sensitivity of the losartan and repaglinide metabolites. Thebest LC/MS performance was achieved with 0.1% acetic acid as the aqueous phaseand using acetonitrile as organic phase eluent instead of methanol. The Waters BEHShield RP18 (and the corresponding XBridge Shield RP18 with the HPLC system)column was substantially better than the other columns tested. In the case of in vivo

cocktail, weak retention of cotinine required a low organic solvent content at thebeginning of the gradient, whereas a high proportion of organic eluent was applied forthe effective elution of losartan and repaglinide metabolites. In both studies the slope ofthe gradient was mainly determined by the separation of three isobaric metabolites ofomeprazole. Human in vitro omeprazole hydroxylations to the 5- and 3- positions andsulfone metabolites responded with the same LC/MS/MS detection reaction optimizedto hydroxymetabolites, so an adequate chromatographic separation step is needed todetect of all three metabolites. As the formation of 5-hydroxylation is activated byCYP2C19, whereas the 3-hydroxylation and sulfonation are formed via CYP3A4, itis of very high importance that these three metabolites are chromatographically wellseparated before the MS/MS-detection. The need for this separation may increase

51

Fig 10. The structures of the in vivo cocktail drugs and their CYP-specific metabo-lites.

52

the analysis time per sample in the assays utilizing omeprazole metabolites as CYPactivity probes; this was the case when using HPLC in urine sample analysis in study IV.In a recent assay [134] the 5-hydroxyomeprazole was used as a probe metabolite forCYP2C19 from human liver microsomes with a very fast chromatographic method, andonly one LC/MS/MS peak was detected with the MRM reaction from m/z 362 to m/z

214, suggesting that the responses of all three metabolites were mixed into a single peak,leading to the summarized response from activities of CYP3A4 and CYP2C19.

In the N-in-one assays, the differences in concentrations between various CYPspecific substrates and their probe metabolites may be as high as hundred fold and thusthe need for high linear detection response is vital. However, the CYP interaction assaysare typically run using only relative chromatographic responses for the same probemetabolites (or responses between the analyte and internal standard) from run to run,without testing the linear range of the method’s response by creating calibration curveswith spiked standard samples. The method must also be capable for giving a specificresponse for each analyte and the result obtained must specifically reflect the activity ofeach CYP isoform, with no interference from the background or the probe metabolitespresent for other enzymes. Therefore, two different test approaches were utilized toobtain the effect of matrix components on the ionization response of the urine analytes,whereas only post-column infusion method was used for the in vitro probe metabolites.Another method employed with the urine matrix was the comparison of the LC/MS/MSpeak intensities obtained from the samples spiked to same analyte concentration forurine and blank water. The observed enhancement or suppression of the ionizationwas between 6% and 25% with the urine matrix, and with the in vitro cocktail matrixthe decrease in the detected baseline was no more than 10%. The results suggest thematrix effect is not significant in either case. The dilution of the urine samples wasconsidered sufficient to keep the matrix effect at an acceptable level, whereas in the caseof in vitro cocktail samples an even higher matrix effect would have been acceptable, asthe method was used for comparing the peak areas of the samples with a similar matrix.

Knowing the limits of the analytical method performance is highly important forestimating the reliability of the obtained results. In most of the in vitro N-in-one CYPscreening assays the analytical methods used are not adequately validated and, evenmore alarmingly, some of the publications describing the assays give no informationwhatsoever regarding analytical method performance, decreasing the credibility of thefunctionality of the assays. In our studies, the analytical methods developed for cocktailassays were validated for linear range, detection limit, accuracy and precision for each

53

Table 5. Performance of the LC/MS/MS assay A, in vitro cocktail [III].

Compound* LoD (nM) Range (nM) Accuracy (%) Precision (%)Desethylamodiaquine 10 10-2000 (R2 = 0.992) 90-106 4-11Hydroxymelatonin 2 2-4000 (R2 = 0.992) 93-107 3-15Dextrorphan 1 1-1000 (R2 = 0.985) 85-110 1-16Hydroxycoumarin 2 2-4000 (R2 = 0.998) 89-108 1-9Hydroxybupropion 0.4 1-2000 (R2 = 0.993) 87-115 2-15Hydroxychlorzoxazone 15 30-6000 (R2 = 0.994) 88-105 2-155-Hydroxyomeprazole 2 2-2000 (R2 = 0.992) 94-109 2-10Hydroxytolbutamide 0.6 1.5-3000 (R2 = 0.999) 93-113 1-131-Hydroxymidazolam 0.2 1-1000 (R2 = 0.999) 93-108 1-146β -hydroxytestosterone 6 15-1500 (R2 = 0.985) 98-116 4-14Omeprazole sulfone 0.4 2-1000 (R2 = 0.990) 88-107 4-11* DeM-OME and 3-OH-OME were not available as standards.

metabolite. Precision and accuracy were calculated at 7-11 and 6-8 concentrationswithin the specified ranges, in III and IV, respectively. The amount of parallel sampleswas three (n=3) in both of the studies.

In the case of the in vitro cocktail assay [III], the validation was made withtwo different LC/MS/MS systems from different manufacturers, and showed a goodapplicability of the method. The performance of the LC/MS/MS method in the assay A(Waters instruments) is presented in Table 5 and the performance of assay B (ThermoFinnigan instrumentation) is presented in Table 3 in III. Fourteen compounds wereanalyzed from a single LC/MS/MS run. However, the Finnigan TSQ Quantum DiscoveryMAX triple quadrupole instrument was incapable of switching polarities, and thus the in

vitro cocktail samples in assay B were analyzed with two different runs, as the negativeion mode had to be used for analyzing the hydroxychlorzoxazone alone. The detectionlimits were between 0.2 and 30 nM and the linear ranges obtained were 3 or 4 orders ofmagnitude with both of the instrumentations used. The accuracies were 85-116% andobtained standard deviations for precision were below 16%, thus at an acceptable levelfor screening-type analysis. The results obtained with both of the tested instrumentationswere somewhat similar and no clear difference in the general detection sensitivity wasobtained. However, the sensitivity for the metabolites with the lowest abundance wasbetter with the Waters instrumentation and the capability of polarity switching made theWaters instrumentation (assay A) a better choice for the analysis described in study III.

In study IV, twelve CYP-specific probe metabolites and their nine parent drugs fromhuman urine were analyzed. The performance of the LC/MS/MS method utilizing UPLCis presented in Table 6. The HPLC method (assay B) was developed only for comparison

54

purposes, and thus only the detection limits for the analytes were estimated with theassay. The detection limits obtained with the UPLC/MS/MS method (assay A) weregenerally 0.4 ng/ml or less in urine, the only exception being hydroxychlorzoxazoneand its substrate chlorzoxazone, for which the detection limits were 2.0 and 1.0 ng/ml,respectively. The highest detection sensitivity was obtained for 1-hydroxymidazolam,only 0.05 ng/ml in urine. The detection limits with the traditional HPLC method were 2- 5 times higher than with the method utilizing UPLC, the LoDs being between 0.4 and4.0 ng/ml. When taking into account the variables affecting non-chromatography-baseddetection sensitivity, such as injection volume used and the difference in sensitivities ofthe mass spectrometers used, about 1.5 - 3 times higher sensitivities were obtained usingUPLC instead of HPLC.

When comparing the performances of the UPLC and HPLC systems in the in vivo

cocktail assay (Figure 11), superior chromatographic peak shapes were obtained whenUPLC was utilized, leading to clearly increased chromatographic separation and higherdetection sensitivity. The most significant change in peak shape was obtained forlosartan metabolite E3174, while the increase in detection sensitivity was most strikingfor omeprazole sulfone. The signal-to-noise ratio was over 100 times better compared toHPLC, which is probably due to the decreased matrix effect. In addition, the analysisspeed was three times faster with UPLC in comparison to the corresponding methodbased on traditional HPLC.

When increasing the speed of chromatographic run, the data acquisition speed (datapoints/s) of the detector must be taken into account, so that at least 8-10 time points perchromatographic peak should be collected to obtain the adequate shape and correctintensity (peak area) for the chromatographic peak. When performing exact quantitativework, the data acquisition rate requirement is normally stated as 12-15 data points perpeak. When analyzing data concerning, for example, ten MRM-reactions at the sametime, with dwell time and inter-scan delay being 100 ms and 20 ms, respectively, thiswould mean one data point for each reaction every 1.2 s, and if 10 data points overa chromatographic peak is desired, the rate is not fast enough unless the peak widthis 12 s or more. In modern ultra fast chromatography, the peak widths may well be2-3 s, thus requiring a clearly faster data acquisition rate than that calculated above.However, some modern triple quadrupole or TOF mass spectrometers can easily collectdata points in times such as 10 ms per compound. Therefore, data acquisition must beoptimized so that the number of data points / chromatographic peaks meets the criteriaset above. If not, this can be improved by increasing the chromatographic run time to

55

Table6.P

erformance

oftheLC

/MS

/MS

assays[IV

](forassay

B(H

PLC

)onlythe

detectionlim

itwas

estimated).

Precision

andaccuracy

arecalculated

at6-8concentrations

(n=3)within

thespecified

range.

Com

poundA

ssayA

Assay

BR

etentiontim

eR

angeA

ccuracyP

recisionLoD

Retention

time

LoD(m

in)(ng/m

l)(%

)(std.

dev%

)(ng/m

l)(m

in)(ng/m

l)M

elatonin2.03

1-100096-103

1-90.4

5.840.5

Hydroxym

elatonin1.58

1-200086-115

1-80.2

4.450.5

Nicotine

0.620.4-1000

86-1134-14

0.20.83

2.0C

otinine0.78

0.4-200099-105

2-130.1

0.932.0

Bupropion

1.940.4-1000

88-1041-11

0.24.20

1.0H

ydroxybupropion1.67

1-100090-115

1-140.4

3.391.0

Repaglinide

3.880.4-1000

84-1134-15

0.110.30

0.4H

ydroxyrepaglinide3.01

a)a)

a)a)

9.62a)

Losartan2.87

0.4-100087-117

2-150.1

9.480.5

E3174

3.070.4-1000

92-1071-7

0.210.15

1.0O

meprazole

2.100.4-1000

90-1031-11

0.16.15

0.4D

emethylom

eprazole1.38

a)a)

a)a)

3.66a)

5-hydroxyomeprazole

1.760.4-2000

87-1031-12

0.25.35

0.53-hydroxyom

eprazole1.98

a)a)

a)a)

6.05a)

Om

eprazolesulphone

2.510.4-2000

95-1121-8

0.26.25

0.8D

extromethorphan

2.170.4-1000

88-1013-9

0.25.41

0.4D

extrorphan1.58

1-100090-114

1-90.2

3.910.4

Chlorozoxazone

2.514-1000

91-1181-12

1.07.30

2.06-hydroxychlorzoxazone

1.664-2000

80-1171-15

2.04.74

4.0M

idazolam2.37

1-100082-107

6-130.4

5.861.0

1-hydroxymidazolam

2.340.2-1000

90-1151-8

0.056.89

0.2a) notavailable

asstandard

56

Fig 11. SRM chromatograms for the analyzed metabolites with a method basedon a) HPLC and b) UPLC. The data is acquired from urine sample collected fromone study person dosed with nine drugs simultaneously. 3-Hydroxyomeprazole isnot included as it was not present in the samples, and not available as a standard[IV].

obtain wider peaks, reducing the scan time (dwell time) used for one data point, ordecreasing the number of overlapping MRM-detection reactions. The requirements forfast data acquisition are even stricter when analyzing both positive and negative ions atthe same time. The reason for using this so-called polarity switching in our N-in-onecocktail assays was the analysis of CYP2E1 probe metabolite hydroxychlorzoxazone,which is not ionized in positive ion mode ESI but required analysis in negative ion mode[116, 118, 134, 136, 137]. Although, switching the voltages between opposite polaritiesled to additional delays between the scans, the amount of data points collected per

57

chromatographic peak was increased by decreasing the simultaneous MRM-reactions byusing separate detection time windows for each analyte. This enabled detection of 11-14analytes from each run in a short analysis time.

The UPLC/MS/MS method developed for the analysis of twelve common probemetabolites for the nine most important drug metabolizing CYP isoforms enablescompletely non-invasive sample collection from the patients since the urine sample issufficient for the analysis. The obtained detection limits were very low, suggesting thepossibility of reducing the N-in-one administered dose levels even lower than those usedhere. The LC/MS/MS method developed for the in vitro metabolic screening assay hasproven to be a very robust and reliable method and has been used in a large number ofCYP-inhibition screening studies with many different matrices.

Solid phase extraction tests for urine N-in-one assay

Different solid phase extraction methods were tested (unpublished data) in the process ofdeveloping a urinary based in vivo cytochrome P450 interaction / phenotyping cocktail.Figure 12 presents the recoveries obtained by combining the two elutions of the ionexchange cartridges together with the only elution from HLB.

All ten probe metabolites (two probes for CYP3A4 and one for each of the remainingprimary CYP isoenzymes) were detected solely from the HLB extract when only thelast elution was collected. The recoveries from the HLB extract were 49 - 62%, withthe exception of cotinine, hydroxymelatonin and hydroxybupropion, whose recoverieswere 16%, 34% and 41%, respectively. Adjustment of elution solvent pH might haveimproved recoveries of some compounds, but probably also decreased recoveriesfor other compounds. When the cation exchange cartridge MCX was used and onlythe last elution was collected, the recoveries were 36 - 67%. The exceptions werecotinine, hydroxychlorzoxazone and hydroxymelatonin. Recoveries for cotinine andhydroxychlorzoxazone were 14% and 1%, respectively, whereas hydroxymelatonin wasnot detected. When two elution steps (wash step 2 using methanol and pH-adjustedfinal elution) were combined (Figure 12), the recoveries for the hydroxylations ofchlorzoxazone and melatonin increased substantially, whereas for E3174 (losartan acid)the recovery increased less, and for the rest of the compounds the recoveries remainedabout the same. With WCX, the weak cation exchange cartridge, dextrorphan was theonly metabolite with a decent recovery (55%), as it was the most alkaline compound.Most of the compounds were not even detected and only 1% recoveries were obtained

58

for the detected compounds. However, when the two elutions were combined, therecoveries were 21 - 94%, except for hydroxychlorzoxazone and hydroxymelatonin,whose recoveries were 6% and 5%, respectively.

As expected, the best recoveries were obtained for the acidic compounds withthe anion exchange cartridge, MAX. When only the last elution was collected, therecoveries were 52 - 124%, except for cotinine, dextrorphan and the hydroxylations ofmelatonin, midazolam and bupropion, for which the recoveries were below 3%. Again,the same trend of quite equal recoveries within the compounds was obtained when thetwo elutions were combined. E3174 (losartan acid) seemed to be the only compoundwith strong acidic properties since it had the best recovery (71%) with WAX, when onlythe last elution was collected. Demethylomeprazole had 14% recovery, whereas therecoveries for the rest of the detected compounds were below 5%. When the elutionswere combined, the recoveries were 42 - 76%, except for cotinine, dextrorphan andhydroxybupropion, whose recoveries were 4 - 21%.

In general, the function of SPE cartridges was as expected. Recoveries of the basiccompounds were higher when the cation exchange cartridges were used, and for theacidic compounds, the anion exchange cartridges were better. Some of the compoundswith both acidic and basic properties, for example, omeprazole sulfone, E3174 andhydroxyrepaglinide, had recoveries of 50 - 80% in both cation (MCX) and anion (MAX)exchange cartridges. In general, the best SPE sorbent for the ten different kinds ofmetabolites was the universal sorbent HLB, the extraction procedure being simple andfast. The ion exchange cartridges also proved to be usable for most of the compoundswhen both of the elutions were collected for analysis. However, overall sensitivity withSPE was somewhat poor, the recoveries of the probe metabolites with HLB being only16 - 62%, and thus suggesting the use of urine as such or diluted with ultra-pure waterwhen analysing the N-in-one in vivo cocktail samples.

Single analyte assay [V]

In the last part of the study, a UPLC/MS/MS method was developed for analysis of anantidiabetic drug repaglinide from the samples collected to study its perfusion acrossplacenta membranes. Repaglinide was one of the drugs used in the N-in-one in vivo

cocktail study [IV] and thus the chromatography and mass spectrometric conditions forrepaglinide were rather well-known from the earlier study and only some adjustments

59

0255075100

125

150

Cotinin

e (2

A6)O-d

em-D

XM (2

D6)OH-C

LZ (2E1)

OH-Mela

(1A2)

OH-MDZ (3

A4)SO2-

Ome

(3A4)

E3174

(2C9)

OH-Rep

a (2

C8) O-dem

-Om

e (2

C19)

OH-Bup (2

B6)13

62

7671

52

73

42

68

21

4

46

124

7679

5852

56

65

3

5553

17

464643

94

66

31

5656

6564

56

5358

1721

4339

36

6664

6056

53

6467

50

69

5862

14

3941

57

6462

6057

5959

34

4949

1416

Recovery, %HLB

MCX

WCX

MAX

WAX

Fig 12. The SPE recoveries obtained from the two combined elutions.

60

were needed for obtaining a competent LC/MS performance. Antipyrine was used as areference drug for passive diffusion-dependent placental perfusion, and due to highconcentration a UPLC/PDA method was developed to analyze antipyrine. In contrast,a very high detection sensitivity was required for repaglinide, and thus solid phaseextraction was used as a sample preparation step before the UPLC/MS/MS analysis.The analytical methods were validated for the linear range, detection limit, accuracy andprecision.

During method development, warfarin was chosen as an internal standard fromthe group of several tested compounds because of its similar behavior in solid phaseextraction with repaglinide. A Waters BEH Shield RP18 column was chosen for themethod and the eluents used were 0.1% acetic acid and acetonitrile. Ammonium formate(2 mM) was also tested as an aqueous phase, but better sensitivity was obtained forboth compounds with acetic acid. In addition, the retention time difference increasedusing ammonium formate as the retention of IS decreased, whereas the retention ofrepaglinide increased. A linear gradient elution with 5 - 80% acetonitrile in 0 - 2 minwas applied, followed by column equilibration with initial conditions for 1 minute.Different flow rates were tested for the optimum performance, the 0.7 ml/min flow ratebeing most suitable for the instrumentation used. When using UPLC columns packedwith small size particles (1.7 µm), the increase in the mobile phase flow rate does nothave a negative effect on the efficiency, as is the case with the HPLC columns packedwith larger diameter particles (e.g. 5 µm), and thus the higher flow rates are encouragedto use with UPLC. However, increasing the mobile phase flow rate may have a negativeinfluence on the detection sensitivity of ESI mass spectrometry, due to non-effectivemobile phase evaporation.

Selecting the right SPE sorbent depends strongly on the physicochemical propertiesof the analytes, and is thus the most important factor affecting the SPE performance. Inaddition, the sample matrix and the interactions with both the sorbent and analyte affectSPE performance [3]. The sample matrix used in the method development phase and forthe preparation of the standard and quality control (QC) samples should be exactly thesame or as close as possible to the analysis samples. However, this is not always possible,and some compromises have to be made when aiming at the closest alternative. Here,the matrix in the samples was a Krebs-Ringer solution containing BSA (bovine serumalbumine), which was prepared in-house for the method development and STD/QCsamples, and thus not exactly the same as in the real samples. The UV-chromatogramsof the unknown samples were very similar to those of the standards, suggesting the

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in-house prepared matrix solution was very close to the matrix of the real samples.Because of the assumption that the physicochemical properties of the substrate,

repaglinide, were somewhat similar than to its hydroxymetabolite, the two most suitableSPE sorbents for OH-repaglinide were chosen from the earlier in vivo tests (Figure12). Thus, Oasis HLB and MAX cartridges were tested with different wash and elutionsteps utilizing different combinations of organic solvents such as methanol, acetonitrile,isopropanol, ethyl acetate and acetone. The MAX anion-exchange sorbent was observedto be better than the universal sorbent HLB for both of the compounds, repaglinide andinternal standard warfarin.

The recovery of the MAX solid phase extraction method was 88 - 115% over theQC-range (50 - 5000 pg/ml, n=3 at each concentration) and for the warfarin (IS), therecovery was 105% (n=9). The selectivity of the UPLC/MS/MS method was confirmedwith the matrix effect tests by means of post-column infusion method at 1 and 10 ng/mlrepaglinide.

The performance of the developed UPLC/MS/MS method is presented in Table 7.The linear range was 2 - 50 000 pg/ml and the accuracies at each standard concentrationwere 90 - 113%. The precisions at each level were 3 - 12% and the accuracies obtainedfrom each individual QC samples were 90 - 112%. The lower limit of quantitation (2pg/ml) was the same as the limit of detection.

The developed and qualified UPLC/MS/MS method was a very sensitive and specificmethod for repaglinide analysis from the placenta perfusion samples. Despite ofthe competent results obtained with placenta samples, the suitability of the samplepreparation method must be tested before utilizing the SPE method for other samplematrices.

The UPLC/PDA method developed for antipyrine analysis was also very specificand sensitive: the limit of detection was 0.2 µg/ml and the obtained accuracies (n=3)and precisions (n=3) at each standard concentration were 92 - 113% and 1 - 10%,respectively, over the linear range 0.2 - 500 µg/ml.

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Table 7. Performance of the single assay analysis UPLC/MS/MS method [V]. Theaccuracy and precision were calculated at each standard concentration (n=3). Thecalibration curve was created using linear fitting and 1/x weighting, the obtainedcorrelation coefficient being > 0.997.

Concentration Accuracy Precision

(pg/ml) (%) (%)

2 98.3 2.8

5 93.2 3.5

10 103.6 7.5

20 104.4 11.5

50 109.6 6.6

100 113.4 3.5

200 92.4 5.8

500 102.0 2.9

1000 94.2 4.7

2000 90.2 5.3

5000 98.1 9.3

10000 101.3 8.9

20000 98.4 6.6

50000 101.1 5.5

5.2.2 Comparison of N-in-one assay and single analyteanalysis

When comparing the multi-analyte analysis i.e. N-in-one assay [IV] utilizing UPLCand the single analyte analysis (SAA) [V], the most striking difference was the ease ofmethod development phase for the SAA, as the sample preparation step, instrumentparameters and chromatography needed to be optimized for only one analyte (in additionto the internal standard). Furthermore, the analyte, repaglinide, was used in both of theassays, and thus made the method development even easier since the physicochemicalproperties of the analyte and the LC/MS conditions were already somewhat familiar.

The chromatography for repaglinide and warfarin (IS) in the single analyte analysis

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was adjusted without the boundaries created by the other analytes present in the cocktailsample. The weak retention of cotinine, the separation of three different omeprazolemetabolites and the tailing of losartan were avoided, and thus a relatively fast UPLCmethod was developed for repaglinide. Similarly, the MS parameters, such as capillaryvoltage and desolvation temperature, were tuned only for the repaglinide (and warfarin)without compromises made in the N-in-one cocktail assay, thus increasing the detectionsensitivity. The decreased number of simultaneous MRM reactions and, more precisely,the lack of polarity switching, also increased the sensitivity of the MS detection.

All in all, the analytic method developed for the single analyte assay was faster andmuch more sensitive than the method used in the N-in-one assay. The retention time ofrepaglinide was 3.9 min in the N-in-one assay, whereas the retention time in the SAAwas 1.7 min; thus the SAA method was more than twice as fast. The limit of detectionfor repaglinide in the SAA was 50 times lower than in the N-in-one assay, the LoDsbeing 2 pg/ml and 100 pg/ml, respectively. The much lower limit of detection in theSAA was due to the different sample preparation steps used, in addition to the betterLC/MS optimization.

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6 Conclusions

LC/MS was used for a qualitative and quantitative analysis of plant and drug metabolismproducts. Various LC/MS instrumentations were used in a versatile fashion and differentLC/MS/MS systems were compared to obtain the best possible analytical performance.Of the different chromatographic systems, the UPLC was confirmed to be a betterchoice for simultaneous analysis of several compounds from a cocktail, in comparison totraditional HPLC. Triple quadrupole mass spectrometers were used for quantitative workand for more detailed structure elucidation, whereas the TOF instrument proved to be apowerful tool for screening purposes. However, the ion trap and Q-TOF instrumentsare able to provide detailed MS/MS data and similarly specific MRM-detection, andthus could be utilized for detailed identification and in analysis of the N-in-one assay,although the ion trap instruments are not capable for fast data acquisition, and thusunsuitable for use with UPLC. In addition, modern time-of-flight mass spectrometerscould be used for quantitative work due to the introduction of dynamic range enhance-ment systems and improved ADC/TDC technology.

As a result of this study, new qualified analytical methods were obtained for thenew and more comprehensive in vitro and in vivo interaction cocktail assays. Withthese developed analytical methods, the studying of drugs and drug candidates affectingto CYP-enzyme activities is remarkably faster in comparison to older methods used.More extensive information about the factors affecting the whole CYP-enzyme family isalso obtained simultaneously from a single assay. In this study, several new in vivo

metabolites were also identified for the CYP2B6-selective probe drug, bupropion, afterhuman administration. In addition, a specific and very sensitive analytical method wasdeveloped for the analysis of the CYP2C8 activity model drug, repaglinide, in the thehuman in vitro placental perfusion samples from the study model describing the transferof compounds between the maternal and fetal blood flow through the placenta duringpregnancy. In the part of the study concentrating on flavonoid secondary metabolism,the collision-induced radical cleavage of flavonoid glycosides was observed to be asuitable tool for structure elucidation of glycosylated flavonols. However, there are a fewfactors, such as instrument parameters, to be considered when analyzing the obtainedMS/MS data. Most of the samples used in the study were complex mixtures and thus theutilization of different sample preparation methods such as SPE and PP prior to LC/MS

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analysis had a notable implication for the obtained results.Some additional studies could be performed to provide a deeper look in the observed

results or to improve the developed methods. For example, comparison of the bupropionmetabolite profile from plasma or serum with the observed urinary profile would beinteresting. The throughput of the in vitro interaction cocktail analysis could be improvedfurther, even to below one minute per run. This would be especially facilitated by chang-ing the CYP2C19 substrate omeprazole used, which forms three metabolites requiringclear chromatographic separation, to some other suitable substrate such as mephenytoin,producing only one target metabolite (4-hydroxymephenytoin). Moreover, the perfor-mance of the in vivo interaction cocktail could perhaps be further improved throughsample preparation utilizing different SPE treatments (both cation and anion exchange)for the same sample, even though clearly increasing time required for sample preparation.

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Original articles

I Petsalo A, Jalonen J & Tolonen A (2006) Identification of flavonoids of Rhodiola roseaby liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 1112:224–231.

II Petsalo A, Turpeinen M & Tolonen A (2007) Identification of bupropion urinary metabolitesby liquid chromatography-mass spectrometry. Rapid Communications in Mass Spectrometry21: 2547–2554.

III Tolonen A, Petsalo A, Turpeinen M, Uusitalo J & Pelkonen O (2007) In vitro interactioncocktail assay for nine major cytochrome P450 enzymes with thirteen probe reactions anda single LC/MS/MS run; analytical validation and further testing with monoclonal P450antibodies. Journal of Mass Spectrometry, 42: 960–966.

IV Petsalo A, Turpeinen M, Pelkonen O & Tolonen A (2008) Analysis of nine drugs and theircytochrome P450 specific probe metabolites from urine by liquid chromatography-tandemmass spectrometry utilizing sub 2 µm particle size column. Journal of Chromatography A1215: 107–115.

V Tertti K, Petsalo A, Niemi M, Ekblad U, Tolonen A, Rönnemaa T, Turpeinen M, Heikkinen T& Laine K (2011) Transfer of repaglinide in the dually perfused human plancenta and the roleof organic anion transporting polypeptides (OATPs). Manuscript.

Reprinted with permission of Elsevier (I, IV) & John Wiley and Sons (II, III).

Original publications are not included in the electronic version of the dissertation.

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OULU 2011

A 574

Aleksanteri Petsalo

DEVELOPMENT OFLC/MS TECHNIQUESFOR PLANT AND DRUG METABOLISM STUDIES

UNIVERSITY OF OULU,FACULTY OF SCIENCE,DEPARTMENT OF CHEMISTRY,FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

A 574

ACTA

Aleksanteri Petsalo

development of lc/ms techniques for plant and drug metabolism...

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