Research Collection

Doctoral Thesis

Development of chromatographic methods and their applications for the analysis of chiral drug metabolism by enzymes altered during pregnancy

Author(s): Korner-Wyss, Sara

Publication Date: 2015

Permanent Link: https://doi.org/10.3929/ethz-a-010361935

Rights / License: In Copyright - Non-Commercial Use Permitted

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ETH Library DISS. ETH NO. 22444

DEVELOPMENT OF CHROMATOGRAPHIC METHODS AND THEIR APPLICATIONS FOR THE ANALYSIS OF CHIRAL DRUG METABOLISM BY ENZYMES ALTERED DURING PREGNANCY

A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH Zurich

(Dr. sc. ETH Zurich)

presented by

SARA KORNER-WYSS

Msc ETH Pharm. Sc, ETH Zurich

born on 25.02.1981

citizen of Bern

accepted on the recommendation of

Prof. Dr. Karl-Heinz Altmann, examiner Prof. Dr. Ursula Quitterer, co-examiner Dr. Irmgard A. Werner, co-examiner

2015

An expert is a person who has made all the mistakes that can be made in a very narrow field. Niels Bohr (1885-1962)

Dank

Ich bedanke mich herzlich bei allen, die mich in den letzten Jahren unterst¨utzthaben und zum Gelingen dieser Arbeit beigetragen haben.

Ganz besonders bedanke ich mich bei Herrn Prof. Karl-Heinz Altmann f¨urdas Vertrauen und die M¨oglichkeit unter seiner Leitung zu promovieren. Frau Prof. Ur- sula Quitterer bin ich sehr dankbar, dass sie bereit war das Korreferat f¨urdiese Arbeit zu ¨ubernehmen. Frau Dr. Irmgard A. Werner danke ich herzlich, dass sie mir die M¨oglichkeit gegeben hat in ihrer Gruppe die Doktorarbeit zu machen und sie in mir die Faszination zur Gaschromatographie wecken konnte. Ich bedanke mich f¨ur die Unterst¨utzungund Ratschl¨age,wenn ich nicht weiter wusste und daf¨ur,dass ich meine Arbeit mit grosser Unabh¨angigkeit ausf¨uhrendurfte. Ein weiteres Dankesch¨ongeht an die ¨ubrigeAnalytikgruppe mit Danielle L¨uthi, Ruth Alder und Philipp Manser, f¨urihre grosse Unterst¨utzung und Hilfe in allen Belangen sowie die interessanten Diskussionen. Zudem danke ich auch meinen Mas- terstudenten Luca Castelnovo, Hulda Brem und Angela Tschabold, die mit grossem Einsatz mein Projekt weitergebracht haben.

Auch der Firma Brechb¨uhler AG, davon insbesondere Urs Hofstetter, Benjamin Oberlin und Ren´eWaldner danke ich f¨urden stets prompten und hilfreichen tech- nischen Support. Wobei ich Urs herzlich f¨ur seine ausgezeichnete Unterst¨utzungbei GC Fragen und Problemen und die guten Tipps und Tricks danke. Der Firma BGB Analytik AG und davon im Speziellen Georg Hottinger und Bernhard Fischer danke ich f¨urszur Verf¨ugung stellen von zahlreichen chiralen S¨aulenund Material f¨urdie GC-Analysen.

Der Gruppe Radiopharmazie unter Prof. Roger Schibli spreche ich ebenfalls ein herzliches Dankesch¨onaus, da ich bei Seminaren, Vortr¨agenund anderen Gruppen- aktivit¨atenstets willkommen war und mich als einen erweiterten Teil dieser Gruppe f¨uhlendurfte. Dr. Selena Sephton danke ich f¨urdie gute Zusammenarbeit und dass ich bei chemischen Fragen ein offenes Ohr bei ihr fand. Auch bei PD Dr. Stefanie Kr¨amerbedanke ich mich f¨urdie Unterst¨utzungbei Fragen zu biopharmazeutischen Themen. Ausserdem danke ich Monica Langfritz f¨urihren IT-Support.

e Ganz herzlich danke ich auch der Mittagsrunde, die Dr. Cindy Fischer, Roger Slavik, Romana Meletta, Dr. Adrienne Herde, PD Dr. Stefanie Kr¨amer,Dr. Linjing Mu, Claudia Keller, Dr. Thomas Betzel und Silvan Boss einschliesst, f¨urdie vielen guten und motivierenden Gespr¨ache ¨uber wissenschaftliche, aber auch allt¨agliche Themen und nat¨urlich auch f¨urdie auflockernden Abenden und Unternehmungen.

Ein zus¨atzlicher grosser Dank geht an meine Familie, meine Freunde, das gesamte T¨odi-Apotheke Team, und ganz speziell an meinen Malala Lukas Korner. F¨urihre Unterst¨utzung,Geduld und Aufmunterung zu jeder Zeit bin ich sehr dankbar. Abstract

During pregnancy, the female body undergoes important changes not only anatomi- cally, but also physiologically. These changes can lead to alterations in drug response and drug metabolism or even the emergence of new biological targets. Due to the scarcity of pharmacokinetic and pharmacodynamic data on newer and particularly chiral drugs, mainly ”old drugs” are used during gestation, even though these com- pounds are not often the first choice treatment. In light of this situation, in vitro methods for the assessment of chiral drug metabolism during pregnancy is of significant interest and importance. With newly developed gas chromatographic (GC) methods for chiral and also non-chiral ap- plications, separation of drug enantiomers and their metabolites were successfully performed. Two model compounds, with poor knowledge of safe use in pregnancy, were selected to establish in vitro methods, methylphenidate (MPH), a commonly used drug for the treatment of the attention deficit hyperactivity disorder (ADHD) in children and adults, and tramadol (TMD), a weak µ-receptor agonist for the treatment of moderate-severe pain. Both compounds are commercially available as racemic mixtures. For a few years, methylphenidate is also approved in enantiomeric form. Because samples with high water content or improper handling of chiral GC columns rapidly lead to column impairment and unreliable results, an option for chiral GC column state and performance check was needed. Since no test mixture for such conditions was available, a new chiral test mixture for cyclodextrin based (CD) stationary phases with versatile applicability was developed. The mixture consists of twelve enantiomer pairs with different functional groups and was tested on 14 chiral CD-columns. Successful analysis was obtained and no co-elution of any compound was observed. Generally best enantiomeric separation was achieved with β-CD capillary columns. Thereof, on one column all enantiomers were separated (BGB-176SE, BGB Analytics). On the column Beta DexTM 325 (Supelco), besides methylphenidate, every enantiomer pair was separated. On other columns (α-, β-, γ-CD) at least one enantiomer pair was baseline separated (Rs ≥ 1.5). The elution of the enantiomers still occurred in random order. Since different mechanisms are responsible for chiral recognition on cyclodextrins, separation efficiency and elution order are difficult to predict. However, the two enantiomeric forms of γ-valerolactone showed the same elution order on all columns. It is assumed to be the result of a

i predominant inclusion-interaction with the cyclodextrin molecules. For automated, highly sensitive and robust detection and for quantitative anal- ysis of chiral drug molecules and their metabolites, new analytical protocols were established. Such a method consists of automated sample preparation coupled to GC-MSD procedures with large volume injections (LVI > 10 µL) and pre-column backflushing (BKF) for mild evaporation conditions. This allows the analysis of thermo-labile compounds like methylphenidate. Furthermore, the sample matrix with potential risk to contaminate the analytical column or disturbing the analysis, is removed with BKF and extends the column lifetime. Additionally, high boiling substances are analyzable with successful removal of solvent prior to reaching the analytical column and enhanced analyte sensitivity is obtained. This is of particular interest for chiral GC columns, susceptible to solvent-induced harm. Efforts to develop liquid chromatographic methods (HPLC) to complement the GC methods did not lead to advantages, because sensitivity and screening capability using the same method were reduced with HPLC. Automated sample preparation was performed using the miniaturized extraction method micro extraction by packed sorbent (MEPS), where total solvent volumes of about 1 mL and minimal analyte volumes of 10 µL allowed successful extractions from a broad range of different matrices like water or buffer (saliva, blood or plasma would also be possible). Sample preparation with MEPS was beneficial in analyte enrichment but also in exchange of solvent, since water has to be avoided for di- rect GC analyses. The resulting MEPS-LVI-BKF-GC-MSD methods for chiral and non-chiral applications exhibited high sensitivity for the model compounds methyl- phenidate and tramadol. Other compounds (amphetamine, ibuprofen, cocaine, pen- tobarbital, tolperisone) also were successfully analyzed with the same method and highly sensitive mass selective detection. With a set of enzymes, whose expression levels are known to be important dur- ing pregnancy (e.g. carboxylesterase, cytochrome P450 2D6, 3A4), the model com- pounds methylphenidate and tramadol were analyzed. Carboxylesterase 1 (CES1), a serine hydrolase, was in a first attempt investigated for stability and optimal activity conditions, which are already known for cytochrome P450 enzymes (CYP). In metabolic experiments with chiral MEPS-LVI-BKF-GC-MSD analysis, racemic methylphenidate (MPH) was mainly stereoselectively metabolized by CES1 to ri- talinic acid (RA), the predominant metabolite of MPH. Also esterification could be demonstrated by formation of the parent compound in presence of ritalinic acid, methanol and CES1. To our knowledge it is the first time to be demonstrated. Methylphenidate was just weakly metabolized by CYP2D6, but no metabolite for- mation was detected and no stereoselectivity could be determined. With CYP3A4 no metabolism was observed. Tramadol showed low CES1 mediated metabolism, but without the appearance of formed metabolites or stereopreference. In contrast, CYP2D6 or CYP3A4 mediated TMD metabolism was stereoselective and also for- mation of the metabolites O-desmethyltramadol (ODT, M1) by CYP2D6, which

ii is pharmacologically active, or N -desmethyltramadol (M2, NDT) by CYP3A4, an inactive metabolite, was detected. Also HPLC analyses demonstrated tendencies for same results. Concluding, a reduced metabolic risk is stated for a medical treatment with methylphenidate during pregnancy. Nevertheless, enantiomeric methylpheni- date and lowest effective doses are to be chosen, for minimal drug and metabolite exposition for the fetus and prevention of possible, but still unknown adverse events. Concerning tramadol, no long-term administration is recommended, because of in- creased formation of active metabolite M1 with risk for withdrawal symptoms in a newborn, due to up-regulated CYP2D6 expression. The development of these new chromatographic methods and in vitro protocols for enzymatic studies can now also be applied for other drug compounds of interest, since particularly for new drugs knowledge about safe use during pregnancy is not available. The obtained results contribute in making predictions about the risk of undesired metabolic effects during pregnancy for a particular drug molecule, and are to be considered as addition to follow-up studies after drug approval.

iii Zusammenfassung

W¨ahrendder Schwangerschaft ver¨andertsich der weibliche K¨orper nicht nur ana- tomisch, auch physiologische Ver¨anderungenfinden statt. Dies kann zu ungewohnten Medikamentenwirkungen oder einem ver¨andertenMetabolismus f¨uhren.Ein Wirk- stoff kann sogar an neue Zielstrukturen binden. Weil kaum Daten ¨uber die Verwen- dung und den Metabolismus von neueren und / oder chiralen Arzneistoffen w¨ahrend der Schwangerschaft zur Verf¨ugung stehen, werden in der Praxis vorwiegend alt- bew¨ahrte Arzneistoffe bei Schwangeren eingesetzt. Dies, obwohl diese Arzeneistoffe in vielen F¨allennicht die therapeutisch bevorzugten Molek¨uledarstellen. Mit diesem Hintergrund wird die Bedeutung der Notwendigkeit f¨ur in vitro- Methoden deutlich. In der vorliegenden Arbeit wurden neue gaschromatographische Methoden (GC) entwickelt, um Arzneistoffenantiomere auf chiralen S¨aulenzu tren- nen, zu identifizieren und zu quantifizieren. Zwei chirale Modell-Molek¨ule,deren Verwendung in der Schwangerschaft wenig belegt ist, wurden ausgew¨ahlt,um die in vitro-Methoden zu entwickeln. Methylphenidat (MPH) ist ein h¨aufig eingesetz- ter Arzneistoff f¨urdie Behandlung der Aufmerksamkeits-Defizit-Hyperaktivit¨atsSt¨o- rung (ADHS) bei Kindern und Erwachsenen. Tramadol (TMD) ist ein schwacher µ-Rezeptor Agonist, der f¨urdie Behandlung von mittelstarken bis starken Schmerzen eingesetzt wird. Beide Verbindungen sind in razemischer Form als Spezialit¨aterh¨alt- lich, das Methylphenidat seit einigen Jahren auch als enantiomerenreines Dexmethyl- phenidat. Weil Proben mit hohem Wasseranteil oder unsachgem¨asserGebrauch von chiralen S¨aulenrasch zu Einbussen bei der S¨aulentrennleistung und zu schwer repro- duzierbaren Resultaten f¨uhrt,wurde ein Mittel zur Uberwachung¨ des GC Systems ben¨otigt. Zudem stand keine geeignete Testmischung zur Verf¨ugung. Aus diesen Gr¨undenwurde eine neue Testmischung f¨urchirale Kapillar-GC-S¨aulenentwic- kelt, die aber auch f¨urnicht-chirale Zwecke eingesetzt werden kann. Die Mischung besteht aus zw¨olf Enantiomerenpaaren mit verschiedenen funktionellen Gruppen und wurde auf 14 chiralen GC Kappilars¨aulenmit Cyclodextrin-basierten (CD) sta- tion¨arenPhasen getestet (CSPs). Es wurde auf keiner S¨auleeine Koelution von zwei Substanzen beobachtet. Die erfolgreichsten Trennungen wurden im Allgemeinen mit β-CD-Kapillars¨aulenerreicht, wobei auf einer S¨aule alle Enantiomere getrennt eluierten (”BGB-176SE”, BGB Analytics). Auf der S¨aule”Beta DexTM 325” (Su- pelco) wurden, abgesehen von Methylphenidat, ebenfalls alle Enantiomere getrennt. Auf weiteren S¨aulen(α-, β-, γ-CD) waren mindestens ein Enantiomerenpaar basis-

iv liniengetrennt (Rs ≥ 1.5). Die Elution und Trennleistung der Enantiomere zeigten eine randomisierte Reihenfolge, weil verschiedene Mechanismen f¨ur die chirale Erken- nung mit Cyclodextrinen verantwortlich sind. Dennoch, γ-Valerolacton zeigte auf allen getesteten S¨aulendie gleiche Reihenfolge bei der Enantiomerenelution. Es wird angenommen dass γ-Valerolacton haupts¨achlich durch einen Einschluss-Komplex mit Cyclodextrin inter-agiert, und die ¨ubrigenMechanismen aus diesem Grund weniger bedeutend sind f¨urdie Enantiomerentrennung auf einer chiralen CD-GC- S¨aule. Um automatisierbare, hoch sensitive und zuverl¨assigeAnalysen von chiralen Arzneistoffen und deren Metaboliten f¨urquantitative und qualitative Zwecke durch- zuf¨uhren,wurden neue analytische Methoden entwickelt. Eine solche Methode baut sich auf durch automatisierte Probenaufbereitung und anschliessende GC- MSD-Analyse mit Gross-Volumen-Injektion (LVI > 10 µL) und Vors¨aulen-Back- flushing (BKF) auf, was schonende Verdampfunsbedingungen zur Folge hat. Da- durch k¨onnenauch thermolabile Substanzen wie Methylphenidat analysiert werden. Mit Hilfe von BKF wird das L¨osungsmittelunter milden Bedingugen abgetrennt, bevor es die analytische S¨auleerreicht. Zudem kann durch die Probenmatrix ent- fernt werden, welche die S¨aulebesch¨adigenoder die Messungen beeintr¨achtigen k¨onnte. Dadurch wird die Lebensdauer einer analytischen S¨auleverl¨angert. Auch h¨ohersiedende Substanzen k¨onnenmit diesem Verfahren analysiert werden und die Sensitivit¨atder Methode gegen¨uber den Analyten wird verbessert. Insbeson- dere f¨urchirale S¨aulenist Backflushing ein Vorteil, da diese S¨aulengegen¨uber verschiedenen L¨osungsmittelnempfindlich sind. Ans¨atze, um die GC Methode mit Fl¨ussigchromatogrphie (HPLC) zu erweitern, zeigten keine Vorteile, weil die Empfindlichkeit mit HPLC reduziert war und f¨urdie Modellsubstanzen je individu- elle Analysenmethoden ben¨otigtwurden. Die automatische Probenaufbereitung wurde mit MEPS (micro extraction by packed sorbent) durchgef¨uhrt.Mit dieser Methode k¨onnen Mikroextraktionen aus- gef¨uhrtwerden, die f¨urden gesamten Prozess ein maximales L¨osungsmittelvolumen von nur rund 1 mL ben¨otigen und Extraktionen aus Volumina bis zu 10 µL erlauben. MEPS kann f¨urverschiendene Probenmatrices verwendet werden, wie Puffer oder Wasser, jedoch auch K¨orperfl¨ussigkeiten (Speichel, Blut, Plasma) w¨arenverwend- bar. MEPS war n¨utzlich in der Anreicherung von Analyten und dem L¨osungsmittel- austausch, da Wasser f¨urdirekte gaschromatographische Messungen zu vermeiden ist. Die entwickelte MEPS-LVI-BKF-GC-MSD-Methode zeigte f¨urchirale und nicht- chirale Anwendungen eine hohe Sensitivit¨atf¨urdie Modellsubstanzen Methylpheni- dat und Tramadol, aber auch andere chirale Substanzen (Amphetamin, Ibuprofen, Kokain, Pentobarbital, Tolperison) wurden mit dieser Methode erfolgreich und mit hoher Sensitivit¨atanalysiert. Ausgew¨ahlteEnzyme, deren Expression w¨ahrendeiner Schwangerschaft als be- deutsam bekannt ist, wie etwa die Carboxylesterase oder Cytochrom P450 En- zyme 2D6 und 3A4, wurden verwendet, um die Modellmolek¨ulezu untersuchen.

v Carboxylesterase 1 (CES1) ist eine Serinhydrolase. Dieses Enzym wurde in er- sten Untersuchungen auf seine Stabilit¨atund Aktivit¨atbei verschiedenen Bedingun- gen getestet. F¨urCytochrom P450 Enzyme (CYP) waren solche Angaben bereits verf¨ugbar. In enzymatischen Experimenten mit anschliessender MEPS-LVI-BKF-GC-MSD Analyse, wurde razemisches Methylphenidat (MPH) durch CES1 stereoselektiv zu Ritalins¨aure(RA) abgebaut, dem Hauptmetaboliten von MPH. Enzymatische Ver- esterung konnte ebenfalls in vitro best¨atigt werden, indem aus Ritalins¨aurein Gegenwart von Methanol und CES1 die Ausganssubstanz Methylphenidat gebildet wurde. Gem¨assunsrem Wissen, wurde die enzymatische Bildung von Methylphenidat aus Ritalins¨aurebisher noch nicht beschrieben. Durch das Enzym CYP2D6 wurde MPH nur schwach metabolisiert und kein Metabolit konnte detektiert werden, wie auch keine enantioselektive Tendenz f¨urden CYP2D6 Metabolismus beobachtet werden. Auch f¨urCYP3A4 konnte kein Metabolismus mit MPH festgestellt wer- den. Tramadol zeigte schwachen CES1-Metabolismus, jedoch ohne Detektion von Metaboliten oder einer enantioselektiven Tendenz. Dagegen wurden f¨urdie En- zymversuche mit CYP2D6 und 3A4 Stereopr¨aferenzenund Metaboliten detektiert. Tramadol, inkubiert mit CYP2D6, zeigte eine deutliche Bildung von O-Desmethyl- tramadol (ODT, M1), dem aktiven Metaboliten von Tramadol. Inkubation mit CYP3A4 erlaubte die Bildung von N-Desmethyltramadol (NDT, M2), einem inak- tiven Haptmetaboliten von Tramadol. Auch HPLC Analysen zeigten diese Tendenz. Aufgrund der Resultate darf angenommen werden, dass f¨urdie therapeutische An- wendung von Methylphenidat w¨ahrendder Schwangerschaft ein geringes metabolis- ches Risiko besteht. Dennoch sind, um allf¨alligeheute unbekannte Ereignisse zu vermeiden, die tiefst m¨oglichen effektiven Dosen zu w¨ahlenund ausschliesslich eine enantiomerenreine Formulierung, damit der F¨otuseiner geringen Arzneistoff- und Metaboliten-Konzentration ausgesetzt ist. Im Bezug auf Tramadol wird keine Lang- zeittherapie w¨ahrend der Schwangerschaft empfohlen, weil die Bildung von aktivem M1 Metaboliten erh¨oht ist, bedingt durch die st¨arkere Exprimierung von CYP2D6, und daher Entzugssymptome bei einem Neugeborenen erwartet werden m¨ussen. Die Entwicklung dieser chromatographischen Methoden und in vitro Protokolle f¨urmetabolische Studien k¨onnennun f¨urweitere Arzneistoffe verwendet werden, weil insbesondere bei neueren Arzneistoffen keine Informationen ¨uber eine sichere Ver- wendung w¨ahrendder Schwangerschaft verf¨ugbarsind. Die daraus resultierenden Informationen zu einem Arzneistoff unterst¨utzendie Voraussage zu unerw¨unschten metabolischen Arzneimittelwirkungen bei einer kurzzeitigen oder dauerhaften Ein- nahme w¨ahrendder Schwangerschaft. Trotzdem sind fortlaufende Studien zur Erken- nung von seltenen Ereignissen und zur Uberwachung¨ auch nach Zulassung eines Arzneimittels wie bis anhin unerl¨asslich.

vi Contents

1 Introduction1 1.1 Chirality - Same Things, but Different...... 1 1.2 Pregnancy...... 3 1.3 Pharmacokinetics and Pharmacodynamics...... 5 1.4 Enzymes and Metabolism...... 8 1.4.1 Cytochrome P450 Enzymes...... 9 1.4.2 Esterases and Hydrolases - Carboxylesterase...... 9 1.4.3 Conjugation Enzymes...... 10 1.5 Model Compounds Methylphenidate and Tramadol...... 12 1.5.1 Methylphenidate...... 12 1.5.2 Tramadol...... 16 1.6 Chromatography...... 20 1.6.1 Gas Chromatography...... 21 1.6.2 Liquid Chromatography...... 28 1.7 Sample Preparation...... 29

2 Aims and Scope 33

3 Results and Discussion 35 3.1 Monitoring of GC Systems...... 35 3.1.1 Test Mixture for Chiral Cyclodextrin Capillary GC Columns. 35 3.2 Method Development for Automated Sample Preparation and Chro- matography...... 45 3.2.1 MEPS Sample Preparation...... 46 3.2.2 Large Volume Injection with GC...... 48 3.2.3 Automated Sample Preparation and Large Volume Injection. 61 3.2.4 Chiral MEPS-LVI-BKF-GC...... 66 3.2.5 Derivatization for GC...... 71

vii 3.2.6 Liquid Chromatography...... 72 3.2.7 Stability Investigations...... 76 3.3 Enzyme Experiments...... 78 3.3.1 Stability and Performance Investigations on Carboxylesterase 78 3.3.2 Carboxylesterase Metabolism of Model Compounds...... 83 3.3.3 Cytochrome P450 Metabolism of Model Compounds..... 88

4 Conclusions and Outlook 92

5 Experimental Section 95 5.1 General Information...... 95 5.2 Identity of Methylphenidate and Tramadol...... 96 5.3 Chromatography...... 99 5.3.1 Gas Chromatography...... 99 5.3.2 HPLC-UV...... 101 5.3.3 Chromatographic Methods...... 102 5.3.4 Chiral Test Mixture...... 118 5.3.5 LVI-BKF-GC-MSD...... 125 5.3.6 MEPS-LVI-BKF-GC-MSD...... 125 5.3.7 Chiral LVI-BKF-GC-MSD...... 126 5.3.8 Chiral MEPS-LVI-BKF-GC-MSD...... 127 5.3.9 Derivatization for GC...... 128 5.3.10 Stability of Methylphenidate with Non-Chiral HPLC Analysis 129 5.3.11 Chiral Column Performance Test...... 130 5.3.12 Chiral HPLC of Methylphenidate...... 130 5.3.13 Chiral HPLC of Tramadol...... 131 5.4 Enzyme Experiments...... 132 5.4.1 Stability of CES...... 132 5.4.2 Methylphenidate Metabolism with CES and GC-MSD Analysis134 5.4.3 Methylphenidate Metabolism with CES and Chiral Chromato- graphic Analysis...... 134 5.4.4 Tramadol Metabolism with CES and Chiral Chromatographic Analysis...... 135 5.4.5 CYP2D6 Metabolism of Methylphenidate with Chiral Chro- matographic Analysis...... 135 5.4.6 CYP2D6 Metabolism of Tramadol with Chiral Chromatographic Analysis...... 136

viii 5.4.7 CYP3A4 Metabolism of Methylphenidate with Chiral Chro- matographic Analysis...... 137 5.4.8 CYP3A4 Metabolism of Tramadol with Chiral Chromatographic Analysis...... 138

Appendices 140 A HPLC Experiments...... 140 A.1 Saturation of MEPS BIN with 5x MEPS Sampling of TMD. 140 A.2 CES Metabolism of MPH and Chiral HPLC Analysis..... 141 A.3 CES Metabolism of TMD and Chiral HPLC Analysis..... 142 A.4 CYP2D6 Metabolism of MPH and Chiral HPLC Analysis... 142 A.5 CYP2D6 Metabolism of TMD and Chiral HPLC Analysis.. 143 A.6 CYP3A4 Metabolism of MPH and Chiral HPLC Analysis... 144 A.7 CYP3A4 Metabolism of TMD and Chiral HPLC Analysis.. 144

Bibliography 145

Publications 160

ix Lists of Abbreviations and Terms

abbreviation explanation L,W,d system size (length in cm, width, diameter) A drug fraction ACN acetonitrile ADHD attention/deficit hyperactivity disorder ADME pharmacokinetic processes of a drug in an organism, divided into absorption, distribution, metabolism and excretion AGP α1-glycoprotein (chiral HPLC column) Amm. Ac. buffer ammonium acetate buffer As symmetry factor ATR attenuated total reflection AUC area under the curve BBB blood brain barrier BGB-15 15% phenyl-, 85% methylpolysiloxane matrix by BGB Ana- lytics BGB-1701 14% cyanopropylphenyl -, 86% methylpolysiloxane matrix by BGB Analytics BIN barrel insert and needle of a MEPS syringe BKF backflush C concentration C2 modified silica stationary phase with an ethyl-side chain for LC or SPE C8 modified silica stationary phase with an octyl-side chain for LC or SPE C18 modified silica stationary phase with an octadecyl-side chain for LC or SPE CD cyclodextrin CES carboxylesterase CO cardiovascular output CSP chiral stationary phase CYP cytochrome P-450 enzyme

x abbreviation explanation d distance between the perpendicular dropped from the peak maximum and the leading edge of the peak at one-twentieth of the peak height D injected drug dose or volume of distribution DCM dichloromethane DOE design of experiments df film thickness in micrometer DPTMDS 1,3-diphenyl-1,1,3,3-tetramethyldisilazane EI electron ionization ER endoplasmic reticulum eV electron volt F bioavailability as the AUC comparison of AUC intra- and extravascular application FDA US Food and Drug Administration fa fraction of absorbed drug in the plasma fe unchanged renal extracted drug fraction ffp fraction of absorbed drug in the plasma after first-pass metabolism FFNSC flavors and fragrances of natural and synthetic compounds database FID flame ionization detector FTIR fourier transform infrared spectroscopy fu unbound drug fraction in plasma GC gas chromatography GF R glomerular filtration rate GIT gastrointestinal tract GST glutathion S-transferase H height equivalent to a theoretical plate or peak height for de- termination of S/N h noise of noise in a chromatogram HETP height equivalent to a theoretical plate HPLC high performance liquid chromatography I.D. inner diameter of a column IR infrared spectroscopy k10 drug elimination rate KM constant of Michaelis-Menten, describing the substrate con- centration at the half-maximal velocity of metabolic clearance KS association constant for chiral selector and (S)-enantiomer KR association constant for chiral selector and (R)-enantiomer LLE liquid-liquid extraction LOD limit of detection LogP partition coefficient LOQ limit of quantification LVI largen volume injection xi abbreviation explanation MeOH methanol MEPS microextraction by packed sorbent MPA methyl phenylacetate MPH methylphenidate MS or MSD mass spectrometry MSTFA N-methyl-N-trimethylsilyl-trifluoro-acetamide Mw molecular mass m/z mass-to-charge ratio N theoretical plate number of a chromatographic column NADPH nicotinamide adenine dinucleotide phosphate NDT N-desmethyl tramadol, pharmacologic inactive M2-meta- bolite of tramadol formed by CYP3A4 NMEs new molecular entities NP nitrophenol NPA nitrophenyl acetate ODT O-desmethyl tramadol, pharmacologic active M1-metabolite of tramadol formed by CYP2D6 P-buffer phosphate buffer PES polyethersulfone pI Isoelectric point pKa negative logarithm of the acid dissociation constant PBPK model physiologically based pharmacokinetic model Ph.Eur. Pharmacopoeia Europaea RA ritalinic acid RC regenerated cellulose RP column reversed phase column in liquid chromatography PTV programmed temperature vaporization injector Rs resolution of two adjacent peaks SE-52 5% phenyl-, 95% methylpolysiloxane matrix by BGB Ana- lytics SIM selected-ion monitoring S/N signal-to-noise ratio SPB-20 poly 20% phenyl, 80% dimethylsiloxane matrix by Supelco SPB-35 poly 35% diphenyl, 65% dimethylsiloxane matrix by Supleco SPE solid-phase extraction SPME solid-phase microextraction SSL split-splitless injector SULT sulfotransferase TBDMS tert - butyldimethylsilyl ether TIC total ion current TMD tramadol Torr unit of pressure (used in software of the MS detector) 1 Torr = 133.3224 Pa = 1.333 mbar

xii abbreviation explanation t1/2 half-life of a certain drug compound tR retention time of a peak UDP uridine diphosphate UDPGA uridine diphosphate glucuronic acid (UGT cofactor) UGT UDP-glucuronosyl transferase enzyme USP United States Pharmacopoeia UV-VIS ultraviolet-visible spectrophotometry V volume of distribution for a certain drug compound w0.05 width of a peak at one-twentieth peak height w0.5 width of a peak at half peak height

term explanation achiral molecule being superimposable with its mirror image simply by rotation in a plane acromegaly endocrinological disease with overexpression of growth hormone chiral nature of a molecule containing an asymmetric atom with four different substituents, mostly a carbon atom chiral center atom with four different substituents, commonly a car- bon atom Chiral separation reversible formation of diastereoisomers between enan- tiomers and a chiral selector with resulting separation of the enantiomers (KR 6= KS) chiral switch redevelopment of a marketed racemic drug to a single enantiomeric drug diastereoisomer stereoisomers with more than one chiral center and not being enantiomers of each other distomer pharmaceutically inactive or less active enantiomer enantiomer molecule existing in two different configurations, being mirror images, with at least one chiral center and sha- ring same physico-chemical properties in a non-chiral environment eutomer pharmaceutically active enantiomer first-pass metabolism after oral administration, metabolism of absorbed drug prior to reaching systemic circulation

xiii term explanation isomers molecules with same molecular mass because of equal atom number qualitatively and quantitatively, dif- fering in constitution and in physico-chemical pro- perties metabolism transformation of endo- and exogenous substances in a biological system into physiologically active or in- active compounds that are often more hydrophilic narcolepsy disease with imperative urge to sleep during the day optical activity ability of a molecule to rotate polarized light in a plane by compound specific amounts pharmacodynamics description of drug response and pharmacological ef- fects in a body pharmacokinetics description of concentration changes, metabolite for- mation and drug transport within a body prodrug inactive drug compound with optimized pharmaco- kinetic properties (enhanced absorption), that is en- zymatically metabolized after systemic availability to its active metabolite racemate mixture of enantiomers in equimolar amounts and with absence of optical activity stereoisomers molecules with same molecular formula, but with dif- ferent three-dimensional arrangement. Enantiomers and diastereoisomers are stereoisomers stereoselectivity specific preference for one stereoisomer over another xenobiotic artificial compound found in the body like drugs, pol- lutants, flavors, preservative agents and others with- out physiological function

xiv Chapter 1

Introduction

1.1 Chirality - Same Things, but Different

The physicist Sir William Thomson (Lord Kelvin) was probably not aware in 1883 that he would give name to an essential dimension in nature and sciences. Chira- lity, as he called it, describes the state of geometrically identical figures not to be superimposable by any rotation, only by being mirror images [1]. The meaning of chirality comes from the Greek root ”χιρ” (”cheir”), which means handed. Hands show mirror images of each other. Chirality is omnipresent and important in biological systems, mainly observed as carbon-atoms bonded to four different substituents. A molecule having one chiral center has one mirror image called enantiomer or optical isomer. Enantiomers have same properties, but are able to polarize light in the opposite direction (optical activity). They may exert different properties in an asymmetric environment. Dia- stereoisomers are isomers, having two or more chiral centers (C-, P-, S-atom with four different substituents). They differ in physicochemical properties. Today, chiral conformation of a compound is denoted with (S)- or (R)-descriptors by the Cahn- Ingold-Prelog priority rules [2]. For sugars and amino acids, the Fisher-convention (d-, l-forms) is still popular and also the optical rotation with (+)- or (-)-notation (not related to Cahn-Ingold-Prelog priority rules) is often used [3]. In nature commonly one enantiomeric form is formed or preferred in synthesis and biological function, for example l-amino acids or d-sugars (glucose, fructose, cel- lulose). Biological receptors recognize and discriminate in most cases chiral isomers. For example volatile enantiomers may smell differently. An exemplary compound, but rare with that clear distinction, is carvone (figure 1.1). The monoterpene (S)- carvone has a typically caraway flavor, whereas (R)-carvone smells like spearmint [4]. Racemates consist of equimolar enantiomer amounts and have no optical activity. Many drugs on the market are chiral. The rising awareness of differing enan- tiomeric properties in asymmetrical environment has led chirality to become a major theme in design, discovery, development, patenting and marketing of new drugs since the early 1980s [5,6]. Drug chirality is distinguished in achiral, racemate and single

1 Chapter 1. Introduction

Figure 1.1: Carvone enantiomers are stereoselectively recognized by olfactory receptors. (S)-carvone smells like caraway, where (R)-carvone has a spearmint odor.

enantiomer. Since racemic drugs are considered as two different drugs administered at the same time, a chiral switch to single enantiomeric drugs has gained importance and has also been supported by the US Food and Drug Administration (FDA). In the development of new molecular entities (NMEs) single enantiomers are preferred nowadays. As it is known that in many cases the preferred therapeutic effect re- sides in one enantiomer only. Therefore, it was predicted that the development and approval for racemates will vanish rapidly and only single enantiomers be of further interest. Annual publication of the distribution of worldwide-approved drugs shows instead a still persisting constant number of marketed NMEs in racemic form [5,7]. Not only commercial or marketing strategies are the rationale for rejecting the chiral-switch strategy. Certain enantiomer pairs show synergistic or antagonistic properties or even inversion towards the other form occurs. One example is the weak opioid agonist tramadol, which is approved in racemic form only. Tramadol enantiomers demonstrate increased adverse effects after enantiomeric administra- tion and analgesia is significantly reduced in comparison to racemic drug adminis- tration [8]. A second example is methylphenidate. This compound is used for the treatment of attention deficit hyperactivity disorder in children and adults. Chiral switch strategy has led to the occurrence of racemic and enantiopure formulations on the market [9]. A third drug is ibuprofen, a non-steroidal analgesic, also marketed in racemic and single enantiomeric form. Although the analgesic activity resides just in the eutomer (S)-ibuprofen, the racemic drug is still preferred because in vivo the distomer, inactive drug enantiomer ((R)-ibuprofen) is slowly converted to the eutomer (S)-ibuprofen [10–15]. This inversion is particularly considered in the formulation of long acting drugs, where the presence of the racemate is preferred. In contrast, for rapid analgesic onset the intake of the single enantiomer is chosen, but because of short half-life, the analgesic effect is of short duration. With this background, extra regard is needed for drug intake during pregnancy, where new pharmacokinetic and pharmacodynamics conditions appear and chiral (particularly racemic) drugs may elicit unexpected responses. The following section gives an insight to changes and risks of drug administration during pregnancy.

2 1.2. Pregnancy

1.2 Pregnancy

A woman undergoes in pregnancy profound changes that affect the anatomy of a woman by obvious alterations (total body weight gain, pelvis dilatation etc.). Also the physiology is involved with dynamic changes. Comparing those physiological changes with the physiology of non-pregnant women, quite unhealthy conditions have to be suggested. However, the body of pregnant women generally nicely toler- ates those drastic changes, but in case of drug treatment, dose adjustments have to be considered, as the following part explains. To ensure an optimal development of the fetus, many physiological parameters are changed reversibly during pregnancy. Most changes initiate at early pregnancy. In table 1.1 relevant parameters are reported. The interpolated values are calculated from functions based on measured data from Abduljalil et. al. [16].

Table 1.1: Calculated relative changes of anatomical and physiological parameters during pregnancy on the basis of published reference data [16]. anatomical, physiological relative changes in pregnancy com- and biological parameters pared to non-pregnant average volume (%) non- unit end of first end of second end of third pregnant trimester trimester trimester average (week 13) (week 27) (week 40-42) value total body weight 61.1 kg 6.2 15.2 27.5 total fat mass 17.14 kg 10.7 24.0 40.2 total body water 31.67 L 12.6 29.0 49.8 cardiac output 301 L/h 20.6 31.8 31.0 plasma volume 2.5 L 10.6 43.3 51.4 red blood cell volume 1.49 L 8.6 17.8 27.6 plasma protein concentration 69.7 g/L -1.6 -6.9 -0.8 total lipid concentration 6.0 g/L 21.7 45.0 70.1 CYP1A2 activity 100 activity -38.2 -60.6 -63.1 CYP2D6 activity 100 activity 23.6 35.9 33.9 CYP3A4 activity 100 activity 26.3 26.5 0.7 glomerular filtration rate 114 mL/min 28.4 40.1 30.7 effective renal blood flow 53.1 L/h 44.1 44.6 1.3 estradiol concentration 0.062 ng/mL 3977 14540 33085 progesterone concentration 1.42 ng/mL 2278 6079 15408

The cardiovascular system has an increased cardiovascular output (CO) as a re- sult of increased heart rate and stroke volume. In contrast, systemic and pulmonary vascular resistances decrease. The resulting physiologic hypotension is present at weeks 14 - 24. While maternal blood volume increases, a hemodilutional anemia and decrease in serum colloid osmotic pressure occurs with maximal values at weeks 30 - 32. As albumin and erythrocyte concentration drop, protein binding is reduced. The decrease of erythrocyte concentration and risk of deficient oxygen transport is counterbalanced by erythrocyte size growth. Leucocyte production is increased and coagulation as well, since fibrinolysis is altered leading to a hypercoagulable state.

3 Chapter 1. Introduction

This explains the higher risk for pregnancy-induced thromboembolism [16–18]. Also the respiratory system undergoes mechanical and functional changes. The sudden and enormous increase in estradiol concentration leads to hypervascularity in the respiratory system. The tidal volume increases, but the respiratory rate and vital capacity remain unchanged. This is still the case even though the increasing intra-abdominal pressure moves the diaphragm upward by up to 4 - 5 cm and the alveoli at the bases of the lungs collapse [18–20]. The renal system shows at the end of the first trimester a significant elevation in the glomerular filtration rate (GF R) of approximately 30 - 50%. Creatinine and blood urea concentrations decrease consequently. Natriuresis is favored by the ele- vated progesterone concentration, but elevated levels of aldosterone compensate the loss of sodium. This leads to a net sodium and water retention and therefore to the increase in total body water. Until end of pregnancy about 6 liters extracellular and 2 liters intracellular water are accumulated. In parallel, renal clearance augments until the third trimester to about doubled value compared to pre-pregnant state and normalizes until delivery [17, 18, 21]. In pregnancy, the gastrointestinal system has prolonged gastric emptying and intestine transit. The motility of the gall bladder is reduced, leading to a delayed absorption and to a certain risk for cholelithiasis. Also the endocrine system is affected by presenting a diabetogenic state, where increased insulin resistance is observed. Other hormones as well, not discussed in more detail, show a different secretion pattern in pregnancy [18, 19]. Metabolic changes occur as a result of altered enzyme expression in the liver or extra hepatic. These proteins generally transform xenobiotics into inactive com- pounds. Also transformation of xenobiotics to toxic metabolites or active metabo- lites may occur [22]. There are studies implying cases where fetal metabolism in animals led to higher metabolite concentrations in the fetus compared to the mother [23]. As described earlier, during pregnancy all pharmacokinetic events like absorp- tion, distribution, metabolism and excretion (ADME) are concerned. These changes have a significant impact on pharmacokinetics and pharmacodynamics. This implies the need for drug dose considerations and adaptions for compounds used in preg- nancy. In particular drugs, mainly unchanged renally eliminated, require a dosage adjustment to maintain pre-pregnant plasma concentrations. Most drugs employed during pregnancy are not developed or tested for use in gestation [24–26]. To assure maternal and fetal safety, information about appropri- ate treatment, dosing (amount, interval) and efficacy must be provided. But still, particularly old drugs are used for treatment in pregnancy. These compounds are often applied without official approval for pregnant women. This ”off-label use” generates only little data or case reports and it is the reason, why preferably old compounds are employed. The development of new drugs generally does not include studies with pregnant women, unless it is indicated for that purpose. This is due to ethical concerns or difficult study designs.

4 1.3. Pharmacokinetics and Pharmacodynamics

However, with growing insight and knowledge of diseases a new focus in clinical treatment of pregnant women has been developed. Nowadays women under medical long-term treatment are able to get pregnant even if the medical treatment needs to be continued. It is known that more than 90% of pregnant women need medical long-term treatment or a short-term medication [27–30]. The wellbeing of a mother is important, since health of the mother also influences the development of the fetus. Consequently, an appropriate balancing of benefit and risk is needed. In many cases the risk of health threatening conditions for the mother by withdrawing a treatment is higher estimated than possible harming of the fetus by continuation of a treatment. Efforts are undertaken to identify harmful drug compounds and their risk covering multiple- (e.g. isotretinoin, statins, ACE inhibitors) or single-use of drugs (e.g. thalidomide). The understanding of drug risk in pregnancy requires knowledge about the phys- iology of pregnant women [17, 21, 31]. Not only physical malformations, but also neuro-developmental disorders representing long-time effects can happen. The first trimester is the most vulnerable period for physical malformations. In the second and third trimester neuronal differentiation and organ maturation takes place with an increased risk for neuro-developmental retardation [32–34]. The following section will focus on the physiological changes during pregnancy.

1.3 Pharmacokinetics and Pharmacodynamics

Pharmacokinetics describes kinetic processes of a drug in the body. These processes are divided into four parts, called ADME, where absorption (A) reflects the uptake of a drug compound through the gastrointestinal tract (GIT) or the skin. Distribution (D) designates the dispersion of the drug in the body. Metabolism (M) and excretion (E) specify the elimination of the drug from the body by modifying the parent compound to metabolites or by withdrawing the parent drug through the kidneys or other organs [35]. Pharmacodynamics is of particular interest with new molecular entities (NMEs) and unknown compounds. It describes the effects of a molecule or a metabolite in the body. Generally, pharmacokinetic core-parameters (t1/2, V , fe, fu, F ) with constant values under standard conditions are used to calculate plasma concentrations. In case of co-medications for an individual person, the parameters allow to calculate dose corrections. These core-parameters are also important to predict drug response in the body as a pharmacokinetic profile. The parameters of approved drug com- pounds are listed in literature [36]. In pregnancy, pharmacokinetic core-parameters change and literature core-para- meters are imprecise. This explains the risk for changed drug response or changed metabolite formation. Figure 1.2 illustrates the kinetic in vivo processes, which in most cases can be

5 Chapter 1. Introduction

Aa(t)

ka

A(t) A(t)

C(t) C(t)

k10 k10

Ael(t) Ael(t)

Figure 1.2: Schematic drawing of a commonly used one-compartment model, describing intravascular (i.v.) and extravascular (e.v.) application. The drug is rapidly absorbed and distributed in the body (case). A(t) is the drug fraction at time t and C(t) the plasma concentration at time t. Elimination of the compound occurs with a constant elimination rate k10 to Ael(t), the amount of eliminated drug at time t. Aa(t) describes the amount of non-absorbed drug at time t with extravascular application and ka the constant absorption rate. described with a one-compartment model. The body is depicted as a kinetically homogenous entity. This simplified model is allowed to use for molecules with rapid distribution in the body (7 - 10 min) after absorption or injection, but needs distinc- tion between intravascular and extravascular application. In the following, kinetic processes are summarized according to Langguth et. al. [35]. The terminal half-life (t1/2) of a drug compound is dependent on the elimina- tion (metabolism and excretion) of a drug. In most cases the therapeutic plasma concentration is far below the saturation constant of Michaelis-Menten and phar- macokinetics of first order describes the half-life of a drug (Cther. < 0.1 KM ).

A(t) = A(0)e−k10t (1.1)

lnC(t) = lnC(0) − k10t (1.2)

6 1.3. Pharmacokinetics and Pharmacodynamics

ln2 t1/2 = (1.3) k10

The resulting value is dose independent. Also in pregnancy, half-life (t1/2) is a constant value but it is changing in magnitude throughout gestation as a result of varying enzyme expression and plasma-protein binding [17]. The volume of distribution (V ) does not reflect the physiologic effective body volume. It is an artificial volume respecting lipophilicity and plasma-protein binding of a compound. Hydrophilic drugs tend to small V and lipophilic drugs to increased V . It is determined by intravenous application (i.v.), where the value is dependent on the injected dose (D) and initial plasma concentration (C(0)).

D V = (1.4) C(0) Alternatively, the volume of distribution (V ) can be described by the sum of the plasma-volume (VP ) and the tissue-volume (VT ) with respect to the fraction of unbound drug in the plasma (fu) and tissue (fuT ). The tissue-volume (VT ) is dependent on the drug distribution between fat and water.   fu V = Vp + VT (1.5) fuT The increase in total body-water during pregnancy leads to a reduced plasma- protein concentration. As the volume of distribution (D) is also directly dependent on the fraction of unbound drug in the plasma (fu), a changed volume of distribution for highly protein bound drugs is the result [18]. Bioavailability (F ) is a core-parameter with relation to drug formulation. It is the product of the fraction of absorbed drug in the plasma (fa) and the fraction of absorbed drug in the plasma after first-pass metabolism (ffp). With changing enzyme activity during pregnancy, also F can be affected for drugs with high first- pass metabolism.

F = faffp (1.6) In general the unbound part (fu) of drug in the plasma is a constant value that is the quotient of the unbound drug fraction (Cu) and the total drug concentration (C) in plasma.

[A] Cu fu = = (1.7) [Atot] C The unbound drug fraction has mainly low impact on drug response, except for high protein binding drugs (fu < 0.1), where the unbound drug fraction may increase significantly in case of changed protein-binding. Still, the problem is of minor risk because increased fu generally also causes increased drug clearance. In

7 Chapter 1. Introduction

pregnancy, plasma-protein concentration is changing dynamically, as table 1.1 shows. Particularly drugs with low fu and narrow therapeutic window, need a continued monitoring of plasma-concentration to prevent unwanted concentration levels. The unchanged renally excreted drug fraction (fe) is the fifth core-parameter. It describes the renal fraction of parent drug clearance and is the quotient of renal clearance (CLR) and total body clearance (CL).

CL f = R (1.8) e CL As clearance is dependent on the glomerular filtration rate (GF R) it has to be taken into account with pregnant women. GF R is elevated and therefore higher drug doses might be indicated. The dose of drugs with high renally excreted drug fraction needs to be adapted in case of renal insufficiency [20, 24]. Besides consideration of pharmacokinetics and pharmacodynamics also another point is worthy to mention. Generally, the fetus is not much influencing the kinetics of the mother, but a certain difference in fetus and mother has to be respected. One important example is the risk for fetal drug capturing. The pH of fetal blood is slightly lower than the pH of maternal blood. Weak bases are ionized in the fetal circulation and may accumulate in the fetus, called ion-trapping phenomenon [17, 37].

1.4 Enzymes and Metabolism

The dramatic increase of steroids (estradiol, progesterone) in pregnant women causes changes in the expression and activity of different enzymes [38]. Already at early stage of development, fetus and placenta show transporter and enzyme expression, due to important endogenous functions in the development or the elimination of waste [39]. The contribution of feto-placental metabolism is only a minor part in the total metabolism and will therefore be touched only briefly [40]. Metabolism or biotransformation is driven by catalyzing enzymes that consist of two major groups, the group of functionalization enzymes and the group of conjugat- ing enzymes. Functionalization is characterized by hydrolysis, oxidation and reduc- tion reactions. Conjugation reactions involve acetylation, glucuronidation, methy- lation and sulfation. The transformation of a parent drug by functionalization often precedes an enzymatic conjugation reaction. For this reason the functionalization reactions tend to be the rate-determining steps of overall metabolism and are of primary interest in metabolic studies [41, 42]. The fraction of enzymatically metabolized drugs in an adult person is described by cytochrome P450 enzymes (CYP, 72%), UDP-glucuronosyl transferase enzyme (UGT, 16%) and esterases (8%). Other enzymes make only 4% in total drug metabolism [41, 43]. Selected enzymes with relation to pregnancy are discussed in more detail in the following part.

8 1.4. Enzymes and Metabolism

Figure 1.3: Reaction equation of cytochrome P450 catalyzed metabolism of a substrate (R).

1.4.1 Cytochrome P450 Enzymes The membrane-bound enzyme superfamily cytochrome P450 (CYP) is a group of oxidative hemoproteins, consisting of approximately 500 amino acids (corresponding to about 57 kDa) [44]. This group is involved in the transformation of xenobiotics and endogenous substances and its enzymes are mainly present in the endoplasmic reticulum (ER) of mammalian liver cells, facing with the active site the cytosolic side of ER. The catalytic activity of a CYP enzyme requires the interaction of the redox partners NADPH and CYP450-oxidoreductase. The mechanism of action is nicely described in literature [45, 46] and summarized as reaction equation in figure 1.3. In pregnant women mainly three CYP enzymes appear to significantly change their protein expression [16, 18]. CYP1A2 decreases in gestational week 38 dra- matically to about 35% of pre-pregnant activity. CYP2D6 increases until end of pregnancy to about 136% of pre-pregnant status and the activity of CYP3A4, the predominant CYP enzyme in humans, is also highly increased (137% of pre-pregnant status) until week 16, but decreases again to 109% until week 38 of gestation and to pre-pregnant state at week 40. Enzyme expression normalizes after delivery to pre-pregnant state. Twelve weeks after delivery, most changes are rebuilt to pre- pregnant conditions. In fetal liver, CYP3A7 is the most abundant enzyme and has same substrate specificity as CYP3A4 [47]. It is known to be responsible for about half of fetus metabolizing enzyme activity. Other CYP enzymes like CYP1A2, 2D6, 2C9, 2C19 contribute in fetal drug elimination [48–51]. Enzyme activity is dependent on genetic, physiologic and environmental condi- tions. Particular attention should be paid to CYP2D6 which has genetic polymor- phism [16, 17, 19, 21, 52]. Polymorphisms exhibit different phenotypes in enzyme activity, defined as poor metabolizer, extensive metabolizer and ultra-rapid metabo- lizer [53].

1.4.2 Esterases and Hydrolases - Carboxylesterase Esterases and hydrolases are important mammalian enzymes. Carboxylesterase (CES), a member of serine hydrolases, catalyzes the hydrolysis of ester and amide containing compounds. This enzyme family is present in many tissues and located in the endoplasmic reticulum (ER). CES are important in the detoxification of poten- tially harmful compounds and the activation of prodrugs [54]. There are five major groups to classify carboxylesterases: CES1 to CES5. Most members are assigned to group CES1 or CES2. Members of CES1 preferentially metabolize ester-compounds

9 Chapter 1. Introduction

with small residues. CES2 prefers ester-compounds with larger residues. Interest- ingly, incidence of those two enzyme groups is in different organs. CES1 enzymes are mainly expressed in liver and lung, whereas CES2 enzymes are found predominantly in small intestine and kidney [55, 56]. CES1 is also found in fetal liver, but it seems to be of only limited interest for metabolism in pregnant women [57]. Focused on group CES1, it consists of a trimer with 60 kDa subunits [58], the two subgroups CES1A and CES1B differ in one residue at the N-terminus. Both enzymes are therefore considered to have similar enzyme activity [59]. The catalytic center of this enzyme family is highly conserved, consisting of a serine (Ser203), histidine (His450) and glutamic acid (Glu336), the catalytic triad. Two glycine residues (Gly123, Gly124) are part of the oxyanion hole for intermediate stabilizing. The proposed mechanism by Hosokawa et al. [56] is illustrated in figure 1.4. Simplified, the first step (i) starts with a nucleophilic attack to the carbonyl group of the substrate and positioning of the substrate for further reaction. Hydrolysis of the ester bond is induced by formation of a tetrahedral intermediate on the serine residue. Two glycine residues aid to stabilize the intermediate by forming H-bridges. In the second step (ii) the ester bond breaks, the leaving group gets a proton from the imidazolium ion (His450). The acyl group is still bonded to the serine as an acyl-enzyme complex and the alcohol leaves the catalytic site. A water molecule in exchange enters the active pocket (iii), attacks the acyl-enzyme intermediate leading again to a tetrahedral intermediate (iv). In the last step, the formed acid (acyl-product) diffuses away (v) and the enzyme is ready for a new catalysis (vi). CES1A is known to catalyze also transesterification in presence of an alcohol in vivo and in vitro [60]. In catalytic step iii the enzyme accepts an alcohol (e.g. methanol or ethanol) instead of water as a second substrate, leading to transesteri- fication but not hydrolysis. A competing alcohol (light grey) is shown in figure 1.4 (step iii). In presence of ethanol methylphenidate is metabolized via CES1A to ritalinic acid (RA) and to a certain amount to ethylphenidate (ritalinic acid ethyl ester). The same is true for cocaine, also substrate for CES1A, where cocaethylene (benzoylecgonine ethyl ester) is formed under same conditions. Transesterification happens less distinctive than hydrolysis, but resulting transesterified products arise in detectable range for in vivo samples. Those findings are interesting in forensic investigations, because those metabolites often demonstrate increased toxicity and are found in autopsy of overdosed subjects [58, 61–67].

1.4.3 Conjugation Enzymes Among conjugation enzymes, like SULT (sulfotransferase), NAT (N-acetyltrans- ferase) or UDP-glucuronosyl transferase (UGT), UGT is the most abundant metabo- lizing enzyme. It is an important instrument in the detoxification or elimination of xenobiotics. Glucuronidation occurs in low amounts already at early stage of fetal development. The fetal expression of UGT represents 1 - 10% of adult level [22, 51]. Other conjugation enzymes like GST (glutathione S-transferase) and SULT also

10 1.4. Enzymes and Metabolism

Figure 1.4: Proposed mechanism of action for carboxylesterase (CES1A) adapted from Hosokava et al. [56]. This enzyme catalyzes the hydrolysis of ester- or amide-groups. In presence of an alcohol, the enzyme is also able to catalyze transesterification where the alcohol (R” = -CH3, -CH2CH3) is accepted as second substrate instead of water. contribute in fetal metabolism, but to minor amounts [49, 50, 68]. UGTs are membrane-bound enzymes in the lumen of the ER, mainly expressed in liver and intestine. These enzymes catalyze the conjugation of glucuronic acid (Mw 176) to various functional groups like free hydroxyl-, carbonyl- or amine- residues. UGT-metabolism generally succeeds a CYP-mediated functionalization of a drug compound. The formed metabolites usually are more polar and ionized at physiological pH. Those conjugates are basically eliminated via the kidneys or the biliary pathway. The UGT-family unites three major sub-families: UGT1, UGT2A and UGT2B. All forms contain a highly conserved N-terminal region with the bind- ing site for the enzyme co-substrate uridine diphosphate glucuronic acid (UDPGA),

11 Chapter 1. Introduction

which is indispensable for UGT activity [41]. In pregnant women UGT metabolism also plays an important role [21, 38]. Par- ticularly UGT1A1 expression is up-regulated in pregnancy as a result of elevated estrogen blood levels [69]. Xenobiotics being inactivated or renally eliminated via glucuronidation tend to exert higher clearance rates in pregnant women. In certain cases toxic metabolites are formed by glucuronidation, for example acyl-glucuronides with immunogenic properties [43]. Special attention is therefore also to be paid on drug administration with UGT transformation, where dose adjustments or even drug avoidance may be considered.

1.5 Model Compounds Methylphenidate and Tra- madol

Models for the prediction of drug metabolism require knowledge about the mecha- nism of action, pharmacokinetics and pharmacodynamics of the target molecule [70]. The widely recognized Lipinski ”rule of 5” describes required properties for a drug compound considering absorption or permeability. A drug candidate should meet less than 10 hydrogen-bond acceptor sites, not more than 5 hydrogen-bond donors, a maximal molecular weight (Mw) of 500 and a calculated partition coefficient (LogP ) below 5 [71]. The interest and use of models is increasing in drug development. Particularly in obstetrics molecular modeling is preferred to reduce the gap of lacking animal mod- els. Available pregnancy physiologically based pharmacokinetic models (pPBPK) are promising but still not represent the effective state of pregnancy [16, 31, 72]. Model compounds serve in in vitro assays for elucidation of drug metabolism dur- ing pregnancy. In this project two model compounds were chosen for enzymatic studies. Both compounds are commonly described with an one-compartment pharmacokinetic model.

1.5.1 Methylphenidate A chemist of Chemische Industrie Basel (CIBA) Leandro Panizzon published in 1944 the synthesis of pyridil- and piperidine-compounds. One of the compounds was the methyl ester of methyl 2-phenyl-2-(piperidin-2-yl)acetate hydrochloride (figure 1.5), known today as methylphenidate hydrochloride (abbreviated as MPH or MPH HCl in this work) [73]. This compound appeared to have stimulating properties. Also the wife of Leandro Panizzon, Margherita Panizzon, felt beneficial effects in tennis performance with methylphenidate. And so it is reported, she gave name with her nickname Rita to the patented brand name Ritalin R . The compound was mar- keted in Europe in 1957 by CIBA for the treatment of fatigue and psychological disorders [74].

12 1.5. Model Compounds Methylphenidate and Tramadol

Figure 1.5: (a) Threo-enantiomers of methylphenidate with chiral carbons at position 2 and 7: (R,R)-methylphenidate hydrochloride in petrol-blue and (S,S)-methylphenidate hydrochloride in bordeaux-red. Therapeutical activity resides only in the (R,R)-enantiomer; (b) amphetamine structure indicating structural similarity with methylphenidate.

Nowadays MPH is mainly used for the treatment of attention/deficit hyper- activity disorder (ADHD) in children and adults, but is also approved by American and European authorities (FDA, EMEA) for narcolepsy and since 2012 as orphan drug for the treatment of acromegaly [9]. Besides Ritalin R , there are other MPH- products on the Swiss market such as Concerta R , Medikinet R or Equasym XR R and Focalin XR R . The ADHD syndrome is characterized by a predominantly dis- rupted neurotransmission of dopamine, resulting in hyperactivity/impulsivity and inattention [75]. About 5 - 10% of children and adolescents are diagnosed to be affected by ADHD and about 30 - 70% of them still have symptoms into adult age, corresponding to about 3 - 5% of adults [76, 77]. Methylphenidate hydrochloride is a central nervous system stimulant. The effec- tiveness of methylphenidate is due to enhancement of dopaminergic neurotransmis- sion in the brain, mainly in striatal tissue and prefrontal cortex [78]. MPH binds to dopamine transporters and inhibits the reuptake of dopamine into dopaminergic neurons. It also binds to noradrenaline transporters inhibiting the reuptake of nor- adrenaline. Other neurotransmitters like serotonin or acetylcholine are contributing to the stimulating effect [78–83]. MPH is attributed to the group of amphetamines because of characteristic sim- ilarities (figure 1.5), although its mechanism of action differs slightly from that of amphetamine. Amphetamine mainly enhances the release of dopamine into the synaptic cleft, but MPH preferably inhibits the reuptake of dopamine into neu- rons [84]. Its potency is comparable to cocaine, but dependence on per-oral MPH is not claimed, because it shows different pharmacokinetic properties with slower onset of action [85, 86]. The two asymmetric carbon atoms C-2 of the piperidine ring and C-7 at the methyl phenylacetate moiety allow four possible stereoisomers of methylphenidate. Ritalin R is the racemate of the (R,R)- and (S,S)-enantiomers (threo-form), but also the enantiopure (R,R)-enantiomer (dexmethylphenidate, Focalin XR R ) is com- mercially available. Therapeutic benefit exclusively results from the threo-methyl- phenidate, in particular the (R,R)-enantiomer. The (R,S)- and (S,R)-enantiomers (erythro-form) are assumed as impurity in the Pharmacopoeia (Ph.Eur. impurity

13 Chapter 1. Introduction

Table 1.2: Dosage, formulations and analytical information about methylpheni- date [87–89]. parameter methylphenidate CAS base (racemate) 113-45-1 CAS HCl (racemate) 298-59-9 CAS HCl (enantiomer) 19262-68-1 (R,R)-MPH HCl Mw (base) 233.3 Mw (HCl) 269.8 pKa 8.8 LogP 2.31 UV maximum 210 nm daily dose (racemate) 10 - 60 mg daily dose (enantiomer) 5 - 20 mg (R,R)-MPH HCl therapeutical plasma concentration 10 ng/mL (= 40 nM) mass spectroscopy molecular fragment [M+H]+ m/z 234 main mass-fragment (MS) m/z 84 formulations capsule, regular tablet, extended- release tablet, chewable tablet, oral so- lution

B) [90]. They contribute, if present in higher amounts, by unwanted pharmacolog- ical side effects such as elevated blood pressure, insomnia or anxiety, as a result of non-specific binding. Same side effects are found with threo-methylphenidate in a dose dependent manner [91–99]. Table 1.2 shortly summarizes dosage, formulations and analytical information about methylphenidate. The molecule has good penetration properties (LogP = 2.31) [100]. It is well and fast absorbed from the gastrointestinal tract and penetrates easily through the blood brain barrier (BBB). Because of high first pass effect, the absolute bioavailability (F ) is about 22% for (R,R)-methylphenidate and 5% for (S,S)-methylphenidate. Two hours after oral administration, maximal plasma concentration is achieved (about 10 ng/mL corresponding to 40 nM); the volume of distribution (V ) reaches 2.6 L/kg and 1.8 L/kg for (R,R)-MPH and (S,S)-MPH re- spectively, with low protein binding (1 - fu) of about 10 - 33% for both enantiomers. The core-parameters are assembled in table 1.3. Human carboxylesterase 1A1 (CES1A1) is responsible for the transformation of MPH to ritalinic acid (RA), the main metabolite of methylphenidate (figure 1.6). The enzymatic hydrolysis occurs with stereopreference for the (S,S)-enantiomer, but no inversion toward the other enantiomer is observed. Ritalinic acid shows reduced lipophilicity (LogP = 1.8) compared to methylphenidate, and it is pharmacologically inactive [101, 102]. Other methylphenidate metabolites are formed by CYP-mediated transformations. About 80 - 90% of the total dose are renally eliminated in form of ritalinic acid with an elimination half-life (t1/2) of 6 hours for (R,R)-MPH and 3.6 hours for (S,S)-MPH. Only a very low part of unchanged methylphenidate (fe) is renally eliminated, 0.3% for (R,R)-MPH and 0.03% for (S,S)-MPH [36, 88, 95]. In presence of ethanol, as

14 1.5. Model Compounds Methylphenidate and Tramadol

Figure 1.6: Metabolism of methylphenidate: about 80 - 90% of the total dose are hydrolyzed via CES to the main metabolite ritalinic acid (RA) and re- nally eliminated. Other metabolites like 6-oxo-methylphenidate, p-hydroxy- methylphenidate or conjugates are formed but in small amounts only. In presence of ethanol, also formation of ethylphenidate occurs (adapted from Integrity database [9]).

Table 1.3: Pharmacokinetic core-parameters for methylphenidate enantiomers with oral administration in non-pregnant healthy individuals [95]. parameter (R,R)-MPH (S,S)-MPH t1/2 6 h 3.6 h V 2.6 L/kg 1.8 L/kg F 22% 5% fu 67-90% 67-90% fe 0.3% 0.03% earlier mentioned, ritalinic acid is metabolized via CES1A1 to ethylphenidate, an active compound similar to MPH, but with higher toxicity [62, 64]. In aqueous solution methylphenidate has reduced stability. Particularly at neu- tral or basic pH, hydrolysis of the ester is facilitated. After 24 hours already 30% of methylphenidate, dissolved in water, is hydrolyzed to ritalinic acid [103]. Methyl- phenidate hydrochloride is a white crystalline powder and stable for decades, when kept at dry and cold place. The compound has a maximal UV-absorption at 210 nm

15 Chapter 1. Introduction

in different solvents (table 1.2). There is only little information about use of methylphenidate in pregnancy. Methylphenidate is known to favor malformations in rabbits at high doses, but there are no signs for gestational malformations in rats [104]. Only a few case- reports of MPH use are available in human obstetrics. Certain malformations are reported, but because those women were polymedicated, no clear causal correlation is possible [105]. Owing to its high lipophilicity, methylphenidate easily penetrates the blood brain barrier (BBB), the placenta and reaches the fetus. This causes a certain risk for accumulation of methylphenidate or its metabolites in the fetus because fetuses have lower blood pH than the mother. Particularly basic substances can be trapped due to higher protonation [17].

1.5.2 Tramadol The synthetic centrally acting opioid receptor agonist tramadol hydrochloride (TMD or TMD HCl) was launched in Germany 1977 by Gr¨unenthal. It is derived from mor- phine by structural simplification but analgesic activity is maintained (figure 1.7). Besides opioid receptor affinity, also other mechanisms contribute to the analgesic potency. All commercially available tramadol products (c.f. Tramal R ) are racemates although the pain relieving action is attributed mainly to the (R,R)-enantiomer. Very surprisingly, De Waard et al. demonstrated in 2013 the occurrence of natu- ral tramadol. In the root bark of the plant Nauclea latifolia Sm., member of the rubiaceae-family, located in Cameroon, high amounts of racemic tramadol were found (about 0.4% (w/w) in dried root bark). This plant is also used in tradi- tional medicine for pain treatment. The findings of racemic natural tramadol are very particular, as in nature preferentially only one enantiomeric form exists [106]. In the meantime, a new publication [107] proclaims those tramadol findings in Nauclea latifolia to be anthropogenic contamination, because tramadol content in their plant samples varied enormously or was even absent, depending on the regional sample collection. Further investigations are indispensable to elucidate whether tramadol is natu- rally originated from Nauclea latifolia or occurring as anthropogenic contamination from a synthetic compound. Tramadol is a weak opioid receptor agonist, selectively binding to the µ-receptors. These G-protein receptors are present in the central and peripheral nervous system (brain, small intestine). The binding of an agonist like morphine leads to analgesia and sedation but also adverse effects occur (respiratory depression, constipation, excitation, euphoria, nausea, pupil constriction, tolerance). The analgesic potency of tramadol corresponds to about 10% of that of codeine and tramadol shows to be about 600-times less affine to the µ-receptor than morphine. By co-administration of naloxone, an opioid receptor antagonist, the analgesic effect is only partially blocked. (R,R)-tramadol has 20-times higher µ-receptor affinity than the (S,S)-tramadol.

16 1.5. Model Compounds Methylphenidate and Tramadol

Figure 1.7: (a) Enantiomers of cis-tramadol HCl: (R,R)-tramdol HCl in dark blue and (S,S)-tramadol HCl in light blue. The therapeutic effect resides mainly in the (R,R)-form, but tramadol preparations do not exist in enan- tiomeric form due to severe adverse reactions when using a single enantiomeric formulation; (b) morphin structure with indication to structural similarity with tramadol in blue.

In contrast, the receptor affinity for racemic tramadol is half of that of (R,R)- tramadol. Also a metabolite contributes to the opioid receptor derived analgesic action. (R,R)-O-desmethyltramadol (M1), which is CYP2D6 mediated, has 700- times higher µ-receptor affinity than its racemic parent compound. But it appears to be still 10-times less affine than morphine. Because of the active metabolite M1, tramadol may also be called a prodrug-like compound. In figure 1.8 most abundant human metabolites are illustrated. Until today 33 different tramadol derivatives are identified arising from functionalization and con- jugation reactions. Most of them are pharmacologically inactive. Besides M1, just for racemic M5 (N,O-didesmethyltramadol) high µ-receptor affinity was measured. But because of its high polarity, it does not cross the BBB and reveals no phar- macological activity. In addition to µ-receptor affinity, tramadol also inhibits the neuronal uptake of noradrenaline and serotonin, which are also involved in non- opioid analgesia. Whereby (R,R)-tramadol and O-desmethyltramadol show lower inhibition of noradrenaline reuptake than (S,S)-tramadol. In contrast, the (R,R)- enantiomer inhibits to a 4-fold higher amount the neuronal serotonin reuptake than the other enantiomer. Additionally, racemic tramadol and (R,R)-tramadol also favor neuronal serotonin efflux; which is not the case for (S,S)-tramadol and O- desmethyltramadol [8, 108]. The total analgesic effect arises from a multimodal mechanism. Racemic trama- dol and O-desmethyltramadol exert a synergistic interaction in pain treatment and counterbalancing of adverse effects [109–112]. This multimodal mechanism may also explain the high varying inter-individual responses to tramadol. Absorption of tramadol occurs after oral administration fast and almost com- plete. Table 1.4 and table 1.5 summarize analytical and pharmacokinetic parameters of tramadol. With a LogP of 1.35 tramadol penetrates the BBB [113]. The absolute bioavailability (F ) is about 70% because of first-pass metabolism. Maxi-mal plasma concentrations are reached after about one hour (100 mg oral single dose: about 300 ng/mL). The volume of distribution (D) corresponds to 3 L/kg with a protein

17 Chapter 1. Introduction

Figure 1.8: Illustration of the metabolism of tramadol after oral administra- tion. 33 metabolites are identified, but most important human metabolites are M1 to M5. The metabolites M1 and M5 are known to have analgesic activity (adapted from Integrity database [9]).

18 1.5. Model Compounds Methylphenidate and Tramadol

Table 1.4: Dosage, formulations and analytical information about tramadol [87–89]. parameter tramadol CAS base 27203-92-5 CAS HCl 36282-47-0 Mw (base) 263.4 Mw (HCl) 299.8 pKa1 8.3 pKa2 9.42 LogP 1.35 UV maximum 272 nm daily dose 50 - 400 mg therapeutical plasma concentration 100 - 800 ng/mL MS molecular fragment [M+H]+ m/z 264 main fragment MS (m/z) m/z 58 formulations oral solution, suppository, sustained- release capsule/tablet, once-daily tablet, film-coated tablet, injection

Table 1.5: Pharmacokinetic core-parameters for tramadol enantiomers with oral administration in non-pregnant healthy individuals [8, 89, 113]. parameter (RS,RS)-tramadol t1/2 tramadol 6 h t1/2 O-desmethyltramadol 9 h V 3 L/kg F 70% fu 80% fe 30%

binding (1 - fu) of approximately 20%. Elimination from the body occurs via en- zymatic transformation and mainly renal excretion (90%) [114]. Main metabolizing enzymes are CYP2D6 and CYP3A4. CYP2D6 transforms tramadol stereoselectively to O-desmethyltramadol (M1) and CYP3A4 is responsible for enantioselective for- mation of N-desmethyltramadol (M2) [115]. Among today identified 33 tramadol metabolites [108], the metabolites M1, M2, M3 and M5 result as major metabolites (> 10% each) (figure 1.8). M4 is identified only in low amounts. 19 metabolites are known to be formed by functionalization enzymes and 14 metabolic products result from conjugation reactions. Elimination half-life (t1/2) of tramadol was found to be 6 hours and 9 hours for O-desmethyltramadol. About 30% of tramadol is eliminated unchanged (fe) via the kidneys [89, 113]. Under dry and light protected conditions solid tramadol hydrochloride and tra- madol base are stable for decades. Tramadol hydrochloride occurs in crystalline and amorphous form, where tramadol base is an oily liquid. It is stable in dissolved form, also in weak basic solution. Reduced stability is reported in acidic solution or

19 Chapter 1. Introduction

Introduction Page 33

(a) (b)

Figure 9: Graphical representation of a) peaks in a chromatogram as measure of response to time, Figureideal peaks 1.9: describe Graphical Gaussian representation peak form; b) limit of (a)of detection peaks in(LOD) a chromatogram or quantification (LOQ) as mea- is suredetermined of response as a minimal to time, ratio of ideal signal peaks to noise. describe Adapted Gaussianillustration referring peak form; to the (b)European limit Pharmacopoeia [86]. of detection (LOD) or quantification (LOQ) is determined as a minimal ra- tio of signal to noise (S/N). Adapted illustration referring to the European Pharmacopoeia [90]. Fundamental chromatographic parameters defined by the committee of Pharmacopoeia (eg. Ph.Eur.) are illustrated in Figure 9 and shortly discussed below [86] . oxidative conditions with epimerization and de-methylation respectively. Tramadol shows UV-absorption with a maximum at 272 nm [113]. Separated compounds are detected in form of peaks representing in optimal conditions a Considering pregnancy, tramadol is used for analgesia in labour. It turned out to haveGauss similar distribution. efficacy To asdetermine other opioids the effective (e.g. pethidine)symmetry, butthe factor with less(As) adverseis provided effects (nausea,from the vomiting width of and the peak respiration at one-twentieth depression) of peak [8, height116–118. A ].value differing from 1 is considered fronting (< 1) or tailing (> 1). Pharmacopoeias (eg. Ph.Eur.) define method The two model compounds methylphenidate and tramadol are used for the de- velopmentspecific acceptance of chromatographic ranges for individual methods compounds. described in the following section.

w0.05 A  1.6 Chromatography s 2d

ChromatographyPlate number (N is) describes a widely the used theoretical separation number technique. of distribution Compounds equilibriums are separated as a bymeasure different of distribution column efficiency, into immiscibledetermined from stationary the retention (liquid time or ( solid)tR) of a and defined mobile phasesanalyte (liquid and the or gas)width and of the a resultingpeak at half compound peak height specific (w0.5). migrationFor LC generally in the lower chromato- N graphic system. Frequently used chromatographic techniques are thin layer chro- matographyare obtained (TLC), than for gasGC. chromatography (GC) and liquid chromatography (LC). The isolated analytes are detected with adequate detection methods. 2  t  Fundamental chromatographicN parameters 5.54 R defined by the committee of Pharma- copoeia (e.g. Ph.Eur.) are illustrated in figure w0. 51.9 and shortly discussed below [90]. Separated compounds are detected in form of peaks representing in optimal conditionsResolution a Gauss(Rs) is used distribution. to measure the To separation determine between the effective two adjacent symmetry, peaks resulting the factor (A ) is provided from the width of the peak at one-twentieth of peak height. A sfrom the difference of retention time (tR) of the later eluting peak (tR2) and the earlier

20

1.6. Chromatography

value differing from 1 is considered fronting (< 1) or tailing (> 1). Pharmacopoeias (e.g. Ph.Eur.) define method specific acceptance ranges for individual compounds.

w0.05 A = (1.9) s 2d Plate number (N) describes the theoretical number of distribution equilibria as a measure of column efficiency, determined from the retention time (tR) of a defined analyte and the width of the peak at half peak height (w0.5). For LC generally lower N are obtained than for GC.

 t 2 N = 5.54 R (1.10) w0.05

Resolution (Rs) is used to measure the separation between two adjacent peaks resulting from the difference of retention time (tR) of the later eluting peak (tR2) and the earlier eluting peak (tR1) and the width of both peaks at half peak height (w0.5). Symmetrical peaks with similar height and a resolution ≥ 1.5 are considered to be baseline separated.

1.18(tR2 − tR1) Rs = 0.5 0.5 (1.11) w1 + w2 The signal-to-noise ratio (S/N) is especially important for peak identification and quantification. It corresponds to the quotient of the two-fold signal height (H) and the baseline noise. In general agreement, a signal with S/N = 3 is considered as a peak at the limit of detection (LOD) and a signal with S/N = 10 allows quantification of the peak, as it is considered as limit of quantification (LOQ). 2H S/N = (1.12) h 1.6.1 Gas Chromatography Gas chromatographic (GC) methods are very useful and ubiquitous for the sepa- ration of compounds being transferrable into gas phase without thermal degrada- tion [119]. Figure 1.10 describes schematically the instrumentation of a GC apparatus. A gas chromatograph consists basically of an injector, carrier gas of high purity, an oven with analytical column, a detection system and software to evaluate chromato- graphic results. Often an autosampler facilitates sample introduction and can even serve for sample preparation. Different injection techniques assist for sample injec- tion according to requested needs. The common and universal split/splitless injector (SSL) is used for on-column injection but also split injection, where the injected sam- ple is splitted by carrier gas. To prevent sample overload in the analytical column, the fraction entering the analytical column can be varied. A programmed temper- ature vaporization injector (PTV) may replace a SSL injector. Such an injector

21 Chapter 1. Introduction

Figure 1.10: Schematic illustration of a gas chromatograph, basically consisting of an injector, oven with column, a detection system and carrier gas.

Figure 1.11: Polysiloxane backbone of a stationary phase for capillary GC with methyl-, phenyl- or cyanopropyl side-chains. can be used similar to a SSL injector but allows rapid temperature changes during injection; and therefore a controlled vaporization of common volumes (0.5 - 1 µL) and large volumes (LV > 10 µL) is possible [120]. This is of particular interest, if solvent has to be eliminated prior to reaching the analytical column. The GC technology is characterized by high separation capacity with high plate numbers (N), depending on column length and compound. For analytical purposes nowadays exclusively glass capillary GC columns are employed. Capillary GC is a distribution chromatography with a stationary phase consisting of a viscous liquid and an inert gas (N2, He, H2) as mobile phase. Stationary phases for pharmaceutical applications mainly consist of bonded or non-bonded polysiloxane backbones with variable substituents like methyl-, phenyl- or cyanopropyl-rests (figure 1.11). A stan- dard column for drug compounds has a 5% phenyl- and 95% methyl-poly-siloxane stationary phase. Column efficiency is determined by the use of the van Deemter relation, where the height equivalent of a theoretical plate height (HETP ) is minimal. For opti- mal performance generally slightly higher operating velocity is chosen, resulting in shorter analysis time without loss of column efficiency (figure 1.12). In capillary GC term A (Eddy diffusion) can be disregarded. Only terms B and C, longitudinal diffusion and mass-transfer, contribute significantly with dependence to the velocity

22 1.6. Chromatography

HETP

Figure 1.12: Van Deemter relation for a specific capillary column (30 m x 0.32 mm I.D., 0.25 µm film thicknesses) and helium carrier gas (unpublished data). The minimal plate height (HETP ) is reached for a 25 - 30 cm/sec average velocity. of the carrier gas (u). This simplified relation is also known as Golay equation [121]. At a given gas velocity, specific for every column, a minimal HETP is obtained. HETP can also be determined as the quotient of column length (L) and plate number (N).

B HETP = A + + Cu (1.13) u

L HETP = (1.14) N Most common GC detectors are flame ionization detectors (FID), which are uni- versally used detectors for organic compounds, and mass selective detectors (MSD), having selective as well as universal properties. Both detectors have large dynamic areas with broad linear ranges (about 107 and 106). FID is a mass sensitive detector with detection limits in the low picograms-carbon per second. Trough combustion of organic compounds there is formation of methane and further formylium ion (CHO+), the primary FID signal-producing ion (figure 1.13). The ions are collected causing a current flow proportional to the collected ions, which is amplified and converted to a chromatogram [119]. MSD is a detector measuring the molecular mass of a compound by transfor- mation into charged ions. The resulting spectrum of the detected ions allows the identification of a molecule by spectral interpretation or consultation of a database. Mainly three steps are involved in GC-MS analysis with electron ionization mode

23 Chapter 1. Introduction

Figure 1.13: Shortly summarized ionization mechanism of a flame ionization detector (FID).

(EI). Firstly, there is the transformation of an analyte to characteristic ionized frag- ments through electron addition by an ion-source. Secondly, separation of the ion- fragments (quadrupole mass analyzer) and the molecular ion [M + H]+ on the basis of their mass-to-charge ratio (m/z) occurs, followed by thirdly measuring the ion- current of the separated ions with amplification and display in form of mass spectra. In EI mode, at low pressure (10−5 to 10−6 Torr), a broad range of compounds up to a molecular mass of 600 Da are detectable. Ionization occurs by application of kinetic electron energy exceeding the ionization energy of the sample molecule, generally 70 eV. The ionized molecular ion then dissociates into mass-fragments with characteristic pattern (fingerprint). The most abundant mass-fragment is considered as the base peak with a relative height of 100. All other mass-fragments are reported in relative percentage abundance to the base peak [122]. For maximal sensitivity and selectivity, selected-ion monitoring (SIM) can be performed. With the selection of only a few characteristic masses (e.g. base peak), the limit of detection (LOD) is steadily enhanced. SIM mode is only of interest when peak identity for a compound is complete or for a parallel record with SCAN mode at a chosen mass range. The choice of solvents in GC is a frequent problem. In many cases low boiling alcohols such as methanol or ethanol are used, but also apolar solvents like heptane. Preferentially, vapor pressure of solvent is far below that of the analyte. For the analysis of high boiling analytes, the choice of solvent is less crucial, but water content is avoided generally. Certain compounds are difficult to separate with GC because of impaired de- tectability, reduced thermal- and chemical stability or low volatility. Through deriva- tization by acylation, alkylation or silylation improved conditions are often achieved. Commonly used derivatization reagents for GC are summarized in table 1.6. Silyla- tion is the mostly used derivatization method for GC due to increased volatility and improved thermal stability of the derivatives. A disadvantage is the incompatibility of silylation reagents with protic solvents. Samples need to be dry and pure prior to silylation [123].

Chiral Gas Chromatography In gas chromatography direct approaches for chiral separation of enantiomer pairs have been employed successfully since 1966 [124]. Enantiomers are separated due to differently interacting with a chiral selector. Chiral recognition is based on si- multaneous interactions between the chiral selector and the enantiomer, forming a

24 1.6. Chromatography

Table 1.6: Examples of GC derivatization reagents [123]. derivatization target reagent acylation hydroxyl, amine TFAA (trifluoroacetic acid anhydride) alkylation amine, carboxylic acid TMSH (trimethylsulfonium) silylation amide, hydroxyl, carboxylic acid MSTFA (N-methyl-N-trimethylsilyl- trifluoro-acetamide)

diastereomeric relationship of the enantiomer intermediates. These reversible in- teractions enabling enantioseparation, consist of hydrogen bonds, π-π-interactions, van-der-Waals interactions and inclusion complexes [125, 126]. Today there is a common agreement that chiral recognition can simplified be explained with the ”three-point-interaction model”. This model states the need for at least three configuration dependent contact points between the chiral selector and the chiral substrate. The diastereomeric complex formation can consist of at- tractive or even repulsive properties. Also solvent molecules contribute to chiral recognition [127]. Most widely used chiral stationary phases (CSPs) in GC are based on cyclodex- trin derivatives [128]. Cyclodextrins (CD) are cyclic oligosaccharides being formed by enzymatic degradation of starch and show defined numbers of monomer units. Best known and characterized cyclodextrins are α-, β- and γ-CD with 6, 7 and 8 d-glucopyranose units respectively. Figure 1.14(a) illustrates a β-cyclodextrin with indicated monomer unit in red. The d-glucopyranose units are linked through α- 1,4-position and can be derivatized at the 2-O-, 3-O- or 6-O-position. Cyclodextrins have numerous chiral centers (35 for β-CD) and show a torus shape with an apo- lar cavity (figure 1.14(b)). Particular compound moieties may enter the cavity or interact at the outer side of the CD molecule trough the three-point interaction and therefore enable chiral separation. CDs have the advantage to lead to good enantioseparation without need of strongly interacting groups close to the chiral center [129–131]. The internal diameter of the rigid CD cavity varies with the number of monomer units. In table 1.7 particular information of native CDs is described [132]. The 2-O- and 3-O-positions point to the upper rim of the torus with wider diameter allowing interactions with amine and carboxyl groups. The 6-O-position of a CD unit is situated on the lower rim of the CD-cage [125]. The cylindrical CD configuration leads to a cavity with apolar character. The cavity of a β-CD revealed 6.5 A,˚ corresponding to a diameter of 0.78 nm [132, 133]. Cyclodextrins are soluble in water, with least solubility for β-CD. Enantioseparation is optimized by employing cyclodextrin derivatives. Table 1.8 describes examples of cyclodextrin derivatives used for CSP of chiral GC columns. Derivatization may even display new or different chemical properties and changed solubility [128, 134]. But cyclodextrin columns are easily harmed by use of polar solvents, water, buffer and high temperature, because CDs are mainly dissolved in

25 Chapter 1. Introduction

Table 1.7: Properties of native cyclodextrins [132].

cyclodextrin number of gluco- Mw cavity water solubility pyranose units (n) (g/mol) (A˚ [nm]) (g/100 mL) α 6 972 5.3 [0.57] 14.5 β 7 1135 6.5 [0.78] 1.85 γ 8 1297 8.3 [0.95] 23.2

Table 1.8: Selection of commercially available chiral CD-capillary columns. name cyclodextrin matrix of CSP provider BGB 173 50% 2,3-diacetyl-6-TBDMS- BGB-1701 (14% cyanopropyl- BGB Ana- α-cyclodextrin phenyl-, 86% methylpoly- lytics siloxane) BGB 174 50% 2,3-diacetyl-6-TBDMS- BGB-1701 (14% cyanopropyl- BGB Ana- β-cyclodextrin phenyl-, 86% methylpoly- lytics siloxane) BGB 175 50% 2,3-diacetyl-6-TBDMS- BGB-1701 14% cyanopropyl- BGB Ana- γ-cyclodextrin phenyl-, 86% methylpoly- lytics siloxane) BGB 176SE 20% 2,3-dimethyl-6-TBDMS- BGB-SE52 (5% phenyl-, 95% BGB Ana- β-cyclodextrin methyl-polysiloxane) lytics BGB 178 20% 2,3-diethyl-6-TBDMS- BGB-15 (15% phenyl-, 85% BGB Ana- β-cyclodextrin methylpolysiloxane) lytics Alpha DexTM 120 20% permethylated α-cyclo- SPB-35 poly (35% diphenyl/ Supleco dextrin, non bonded 65% dimethylsiloxane) Beta DexTM 120 20% permethylated β-cyclo- SPB-35 poly (35% diphenyl/ Supleco dextrin, non bonded 65% dimethylsiloxane) Gamma DexTM 120 20% permethylated γ-cyclo- SPB-35 poly (35% diphenyl/ Supleco dextrin, non bonded 65% dimethylsiloxane) Beta DexTM 325 25% 2,3-dimethyl-6-TBDMS- SPB-20 poly (20% phenyl/ Supelco β-cyclo-dextrin 80% dimethylsiloxane) Chiraldex A-DA 2,6-dipentyl-3-methoxy-α- not specified Astec cyclo-dextrin Chiraldex A-PH (S)-2-hydroxypropyl- not specified Astec methylether-α-cyclo-dextrin Chiraldex G-BP 2,6-dipentyl-3-butyryl-γ- not specified Astec cyclodextrin Chiraldex G-PN 2,6-dipentyl-3-propionyl-γ- not specified Astec cyclodextrin CP-Chirasil-Dex CB 2,3,6-tri-O-methyl-β-cyclo- SPB-35 poly (35% diphenyl/ Agilent dextrin chemically bonded to 65% dimethylsiloxane) Technolo- polysiloxane gies

26 1.6. Chromatography

Introduction Page 39

(a) (b)

FigureFigure 1.14:13: Native Native cyclodextrin cyclodextrin molecule molecule a) withβ-cyclodextrin variable with number 7 glucose (n = 6,7,8)units, ofin red a glycopyranose unit with variable number n = 6,7,8 is illustrated; b) schematic drawing of the cage- glycopyranoselike shape of a cyclodextrin units (red); molecule (a) illustration forming an inclusion of a β-cyclodextrin complex with methylphenidate. with 7 glucose units; (b) schematic drawing of the cage-like shape of a cyclodextrin molecule forming an inclusion complex with methylphenidate.

The internal diameter of the rigid CD cavity varies with the number of monomer units. polysiloxane stationary phases [135]. There are only few new phases available with chemicallyIn Table bonded 7 particular CDs informations (Chirasil-Dex) of andnative better CD are inertness. described [128]. The 2-O- and 3-O- positions point to the upper rim of the torus with wider diameter allowing (strong) Columninteractions Test Mixturewith amine and carboxyl groups. The 6-O-position of a CD unit is situated Theon state the andlower performance rim of the CD- of acage column [121]. need The to cylindrical be monitored CD configuration and results should leads to a be validated by the use of regular column performing checks. Commercially avail- ablecavity GC column with apolar test character. mixtures were developed by the Grob Laboratory [136, 137]. This column test is very useful for common capillary GC columns but is not ad- equateTable for 7: theProperties use with of nati chiralve cyclodextrins GC columns, [128]. in particular cyclodextrin containing 0 columns.Cyclodextrin Grob s test Number mixture of gluco- contains oneM racemicw compound.Cavity Ø AichholzWateret solubility al. designed a column test mixture for chiral columns, the CHIRAL-Test 1 mixture, pyranose units (g/mol) (Å [nm]) (g/100 mL) encasing certain column types [138]. The Schurig group developed a test mixture for β-cyclodextrin-polysiloxaneα 6 columns. All972 existing test mixtures5.3 [0.57] contain a range14.5 of apolarβ and polar compounds7 having different1135 functional6.5 groups [0.78] and being1.85 used for columnγ performance check8 [139]. 1297 8.3 [0.95] 23.2 GC analysis of high boiling compounds in pharmaceutical industry or foren- sic analyses is a commonly utilized method. But no high boiling compounds are included into the existing test mixtures, allowing the monitoring of separation per- The cavity of a β-CD has 6.5 Å, corresponding to a diameter of 0.78 nm [128], [129]. formance over time for such molecules. The utility of existing chiral test mixtures is reducedCyclodextrins to only are few soluble column in wate typesr, with and theyleast solubility demonstrate for β no-CD. broad applicability. Those aspects explain the need for further test mixtures.

27

Chapter 1. Introduction

Table 1.9: Provided information by characteristic compounds in a GC test mix- ture [136, 140]. compound characteristic informative value alcohol adsorption, peak shape amine adsorption, peak shape carbohydrate determination of plate number carboxylic acid adsorption, peak shape ester separation efficiency ketone adsorption, peak shape latone enantiomeric resolution additional marker information of proper interest

The development of a new chiral test mixture has the beneficial property to be ubiquitously used for non-chiral and chiral column check. Integrating a range of different functional groups (table 1.9), the mixture can be used for different kinds of column types and it facilitates the determination of an optimal column for a certain separation challenge.

1.6.2 Liquid Chromatography Liquid chromatography, in particular HPLC (high performance liquid chromato- graphy), is a widely used method for the separation of a broad range of molecules. In contrast to GC, a larger number of molecules can be analyzed, but generally broader peaks appear with LC and the limit of detection is significantly reduced compared to gas chromatography. Applications for pharmaceutical or forensic anal- yses are generally performed using reversed phase HPLC, where the mobile phase is hydrophilic and the column stationary phase consists of hydrophobic particles (diameter 3 - 5 µm). In most cases, modified silica (e.g. octylsilane: C8, octadecyl- silane: C18) build up the particles of the stationary phases. Different techniques for analyte detection are available. Economical and mostly used detectors are UV-VIS detectors, since many compounds have chromophores and are therefore suitable for UV-VIS detection. Besides UV-VIS detectors, there are MS detectors for universal use, but LC-MS has a limited range for mobile phases. Other more specific detectors are available, but are not discussed in more detail [141].

Chiral High Performance Liquid Chromatography Liquid chromatography offers for the separation of enantiomers a variety of dif- ferent chiral stationary phases (CSPs). Frequently used CSPs are cellulose and polysaccharide- based, but also protein and glycoprotein CSPs, macrocyclic anti- biotic CSPs, cyclodextrin CSPs or cyclodextrin mobile phase additives, as well as chiral ion- and ligand-exchange CSPs are available [128, 134, 142].

28 1.7. Sample Preparation

Chiral HPLC columns generally are more susceptible for damage and need a par- ticular handling. The prevalent α1-acid glycoprotein chiral stationary phase (AGP) is discussed in the following. AGP is a mammalian plasma glycoprotein of approximately 44 kDa. Its pI is very low with values of 2.8 - 3.8 and it has high carbohydrate content (45%). This unusual carbohydrate content makes this protein nicely soluble in water and also in polar organic solvents. AGP is synthesized by the liver and secreted by hepato- cytes. In vivo it is involved in various immune-modulatory or anti-inflammatory events [143, 144]. The ability of binding numerous compounds and net charge at physiologic pH is the reason for its application in chiral LC separation. AGP CSPs, although they consist of proteins, are quite robust, but need careful attendance. For better lifetime, an AGP column should not be employed without an appropriate pre-column. Equilibration time for AGP CSPs generally lasts 60 - 90 minutes and it is recommended to change the mobile phase not more than once per day. Generally used buffer systems are suitable (phosphate, citrate, acetate, formate) in typical concentrations of 10 - 20 mM. Organic content in the mobile phase is to be kept low (< 15%) and the pH working range is between pH 4 - 7. In this range the AGP molecule is negatively charged and leads to improved chiral separation of positively charged molecules. Maximal pressure should not exceed 140 bar and the flow rate for a 4 mm I.D. column (5 µm particle size) preferentially is in the range of 0.8 - 0.9 mL/min. If the column is not in use, the manufacturer recommends to store the column in a 2-propanol solution (15% V/V) to prevent bacterial growth.

1.7 Sample Preparation

Sample preparation is a very important, but often underestimated, part of an ana- lytical procedure. The removal of interfering sample matrix, for example biological fluids or buffers, can be accomplished using different approaches. Mainly three goals are envisaged with sample preparation

• Purification of analyte

• Enrichment of analyte

• Exchange of solvent

Standard sample preparation mainly is performed using liquid-liquid extraction (LLE), solid-phase extraction (SPE) or solid-phase microextraction (SPME). The appropriate method has to be chosen in accordance to the problem. But in com- mon, all approaches look for a minimal number of working steps, a selective and reproducible extraction with high recovery rate. LLE usually needs increased sol- vent volumes, is not effective for polar analytes and automation is hardly possible.

29 Chapter 1. Introduction

Recent advantages in SPE result in enhanced recovery, reproducibility and selectiv- ity for a broad range of analytes. Still, SPE contains a certain number of working steps. A wide range of compounds is specifically extracted using polar, non-polar and ion exchange packing material. The sorbent material is similar to the packing material for liquid chromatography, mainly modified silica with bound hydrocarbon chains (C2, C8, C18), or ionic side chains. Most commonly used are reversed-phase silica sorbents with C8 and C18 residues [145]. Trends to SPE miniaturization allow reduced solvent use and automation. A popular method is SPME, where no extraction solvent is needed and the analyte directly adsorbs to an extraction fiber. It is easily integrated into automated proce- dure, but recovery is reduced in comparison to general SPE due to the partitioning mechanism of the analytes between the sample and the coated fiber. With thermal desorption the analytes are transferred into GC mobile phase [146]. The fiber can be reused generally for more than 50 times. Disadvantages are the degradation risk of thermo-labile compounds during the injection process and the time to reach the equilibrium may take some time, in particular for trace analytes. Another approach for miniaturized SPE is microextraction by packed sorbent (MEPS) [147, 148]. This method also has advantages compared to conventional SPE. It is possible to au- tomate the procedure and combine to GC or HPLC using a standard autosampler system [149–154]. The MEPS instrument consists of a syringe with disposable needle (figure 1.15). The needle contains an integrated barrel, filled with SPE-sorbent. The mean particle size of MEPS packing is 45 µm with pore size 60 A[˚ 155]. By pulling up and down, analytes adsorb to the sorbent and are retained for later elution with an appropriate elution solvent. Low solvent volumes are required, generally less than one milliliter. Especially small sample volumes (< 1 mL) are nicely handled and analytes extracted. Trace analytes can be enriched on the miniaturized column packed with various stationary phases, as they are known from SPE or HPLC. Disadvantages are the resulting elution volume, which requests in case of GC analysis for a particular injector like PTV with large volume injection mode. An extraction cycle with a common C18 MEPS sorbent consists on the con- ditioning of the sorbent with organic solvent, followed by an activation step with water. This is simply done by aspiration and dispensation of the solvents. The number of analyte sampling depends on the enrichment wanted. After sampling, a washing step with water is performed to remove disturbing matrix residuals, fol- lowed by the aspiration of a small organic elution solvent volume to re-dissolve the analytes. This volume is then injected into the chromatographic system. During sample analysis, the MEPS barrel insert and needle (BIN) is cleaned by organic and water-based washing steps and carry over is minimized (figure 1.16). According to the manufacturer, MEPS needles can be reused for 40 - 100 times depending on the analyzed sample matrix [145]. Recovery is good with MEPS [146, 157], depending on the analyte and the sor- bent. Below saturation capacity of the sorbent, linear increase in enrichment is

30 1.7. Sample Preparation

Introduction Page 45

Figure 14: Microextraction by packed sorbent (MEPS) is effectuated by the use of a syringe with a Figuredisposable 1.15: needle, Microextraction containing a barrel by packed with a packed sorbent bed. (MEPS) This barrel is effectuated insert and needle by the (BIN) is useeasily of areplaced syringe and with several a disposable sorbents are needle, available, containing known from a barrel liquid with chromatography. a packed The bed.enlarged This viewing barrel of insert the MEPS and needle BIN shows (BIN) a barrel is easily containing replaced about and 1 several– 4 mg of sorbents sorbent [152], [153]. are available, known from liquid chromatography. The enlarged viewing of the MEPS BIN shows a barrel containing about 1 - 4 mg of sorbent [155, 156].

An extraction cycle with a common C18 MEPS sorbent consists on the conditioning of the sorbent with organic solvent, followed by an activation step with water. This is done by aspiration and dispensation of the solvents. The number of analyte sampling depends on the enrichment wanted. After sampling, a washing step with water is performed to remove disturbing matrix residuals, followed by the aspiration of a small organic elution solvent volume to re-dissolve the analytes. This volume is then injected into the chromatographic system. During sample analysis, the MEPS barrel insert and needle (BIN) is cleaned by organic and water based washing steps and carry over is minimized (Figure 15). According to the manufacturer, MEPS needles can be reused for 40 – 100 times depending on the analyzed sample matrix [142].

Figure 1.16: Standard procedure for a microextraction by packed sorbent (MEPS) with a C18 packing material. A cycle consists of condition- ing/activation of the sorbent, followed by the sampling, washing and analyte elution. After elution the sorbent is washed and ready for a next extraction.

31

Chapter 1. Introduction

reported. The loading capacity is described by 3 - 5% of the packing material (about 1 - 4 mg according to manufacturer) [155, 156]. Because the needle with the sorbent barrel contains a bed volume of about 6 µL, it is recommended to use an elution volume > 10 µL. Such volumes are commonly used with HPLC, but in GC a volume > 10 µL is considered as large volume requiring a technical adaptation to perform large volume injection.

32 Chapter 2

Aims and Scope

Nowadays more than 90% of pregnant women are exposed to medication, prescribed or self-medicated [29]. But a persisting limitation of appropriate models mimicking pregnancy status leads to a limited number of medical drugs to be used safely in pregnancy [16, 31]. Many, in particular newly designed compounds, provide no information about the use during gestation and are therefore not preferred for women at childbearing age. As earlier discussed in chapter1 a pregnant woman undergoes significant changes with regard to anatomy and physiology. Since protein level, blood and water amount change, a drug compound may lead to unusual pharmacological reactions. Therefore, single dose during pregnancy or permanent treatments enclose a potential risk for the health of mother and fetus. In addition, hormonally dependent alterations in en- zyme expression give potential rise to unknown or unwanted metabolites, increased or decreased blood drug-concentrations and accumulation of parent compounds or metabolites (weak bases) in the fetus. Also altered protein binding or modifications to the route of elimination are observed [17, 19]. In particular chiral drugs, used as single enantiomer or racemate, have high risk for unpredictable outcomes. Thus, synergistic or antagonistic metabolism of enan- tiomers or inversion into the other enantiomeric form might be observed in vivo [158]. This explains the need of considering a drug racemate as a mixture of two different compounds administered at the same time.

In this PhD project the main objective was the development of in vitro proto- cols to examine enzymatic transformation of chiral drugs for better understanding of drug metabolism and potential arising risks in pregnant women. With two chiral and racemic model compounds enantioseparation and chiral metabolism should be investigated. Enzymes with significantly altered expression in pregnancy were to be targeted for the investigation with the model compounds. Methylphenidate, the first compound, is the preferred drug for the treatment of attention/deficit hyperactivity disorder (ADHD) in children. Since it also has been approved for the treatment of adults for a few years, the need of pregnancy related security tests for methyl-

33 Chapter 2. Aims and Scope

phenidate appears to gain importance. Tramadol, the second model compound, is a weak opioid receptor agonist used for the treatment of moderate-severe pain. The investigation of both model compounds, being used in permanent or acute therapy, should be conducted using chromatographic methods like gas chromatography (GC) and high performance liquid chromatography (HPLC) with new application possi- bilities. The first basic method to investigate was chosen to be GC, because of its generally high sensitivity and broad applicability with same or just few parameters to adapt. Since chromatographic studies require a frequent monitoring of state and per- formance of chromatographic columns and no appropriate option was available for chiral GC columns, a new test mixture for cyclodextrin (CD) capillary columns had to be developed. Investigations over a series of different capillary columns with CD- derivatives as chiral stationary phases (CSP) should demonstrate the versatile use of that test mixture. Moreover, it should be utile for system performance checks during GC experiments.

The newly developed chromatographic method should allow the investigation of numerous compounds at low analyte concentrations with minimal working steps and possible automation of most fault-prone steps. In addition, it should meet the advantages to be useful for qualitative and quantitative analyses and also be helpful for isolation or separation as well as further identification of new metabolites. Metabolic studies of the selected pregnancy-relevant enzymes, including investi- gations on fundamental enzyme handling had to be conducted with a sample prepa- ration technique directly coupled to the analytical procedure. Furthermore, using a second chromatographic approach, outcomes from enzymatic experiments should be verified and contribute in the interpretation of critical or uncritical drug use during gestation. Concluding, the obtained in vitro results and published physiologically based pharmacokinetic models (PBPK) should facilitate prediction of safe drug use in pregnant women.

34 Chapter 3

Results and Discussion

3.1 Monitoring of GC Systems

To monitor the state and performance of chromatographic GC systems, in particular chiral cyclodextrin based columns, which risk to be easily harmed by inappropriate handling, a test mixture to detect early impairment of a CD-GC column or changed column behavior was developed.

3.1.1 Test Mixture for Chiral Cyclodextrin Capillary GC Columns

In chiral capillary GC with cyclodextrin columns, certain parameters appear to dif- fer compared to non-chiral GC conditions. Table 3.1 illustrates differences in use for non-chiral and chiral columns. Unexpected high flow rates need to be applied for suc- cessful enantiomeric separation with CD columns. Where 1.0 mL/min is commonly used for analytical GC analyses, flow rates of 2.5 mL/min are used for CD columns with same dimensions. The optimal flow rate was determined experimentally for each column. Highest column efficiency is defined as the minimum height equivalent to a theoretical plate HETP min (Golay equation). It was observed with flow rates of 3.0 ml/min and 2.5 ml/min for columns with an internal diameter of 0.32 mm and 0.25 mm respectively. Figure 3.1 illustrates two examples of CD column installation control and effi- ciency testing using methane gas. Both columns differ in their inner diameter of the capillary. Chiraldex A-PH (α-CD, A-PH) with broader inner diameter (0.32 mm) shows slightly lower column efficiency. It has an increased minimal HETP compared to Alpha DexTM 120 (α-CD, A-Dex 120) with 0.25 mm inner diameter. Symmetry was satisfying (0.9 < AS < 1.2) and all values were included. The results of each CD type (α-, β- or γ-CD) and diameter of every column are summarized in table 3.2.

35 Chapter 3. Results and Discussion

2.0 Chiraldex A-PH (0.32 mm) 1.5 ) AlphaDex 120

m (0.25 mm) m ( 1.0 P T E H 0.5

0.0 0 20 40 60 Velocity (cm/s)

Figure 3.1: CD columns with 0.32 mm inner diameter (Chiraldex A-PH, 0.32 mm I.D.) shows slightly higher minimal plate height and need higher car- rier velocity than 0.25 mm I.D. analytical CD columns (Alpha DexTM 120).

Table 3.1: Comparison of standard GC parameters using a non-chiral or a chiral capillary column. parameters common non-chiral capillary GC common chiral capillary GC column type 5%-phenyl- 95%-dimethyl-polysiloxane derivatized β-CD dissolved in 5%- phase phenyl- 95%-dimethyl-polysiloxane phase column length 30 m 30 m internal diameter 0.25 mm 0.25 mm film thickness 0.25 µm 0.25 µm rinsing of column recommended for impaired column not recommended baking out of column possible not recommended solvents to avoid water water, methanol, ethanol injector type SSL SSL injector temperature 250 ◦C 250 ◦C injection mode split split split ratio 1:25 1:25 injection volume 1 µL 1 µL carrier gas helium 5.6 helium 5.6 carrier mode constant flow constant flow flow rate 1-2 mL/min 2.5 mL/min detector type FID FID detector temperature 250 ◦C 250 ◦C FID parameters air / hydrogen 5.0 air / hydrogen 5.0 oven parameter: start temperature 50 ◦C 50 ◦C hold time 3-10 min 3 min heat rate 10 ◦C/min 2 ◦C/min final temperature 220 ◦C, or columns upper limit 200 ◦C hold time 10-15 min, or until last peak elutes 15 min

36 3.1. Monitoring of GC Systems

Table 3.2: Assembly of analyzed α-, β-, and γ-CD columns. As expected, higher flow rates were used for capillary columns with 0.32 mm I.D. than for columns with 0.25 mm inner diameter. α-cyclodextrins ADex 120 BGB-173 A-DA A-PH velocity at minimal HETP 24 cm/s 26 cm/s 25 cm/s corresponding flow rate 1.4 mL/min 2.0 mL/min 1.8 mL/min optimal velocity according to Golay relation 36.0 cm/s 39.0 cm/s 37.5 cm/s corresponding flow rate 2.5 mL/min 3.3 mL/min 3.0 mL/min optimal resolution resolution relation (max. Rs) 1.66 / 0.8 / 0.86 corresponding flow rate 2.5 mL/min / 2.0-3.5 mL/min flow rate interval for acceptable Rs 1.5-3.5 mL/min / 2.0-3. mL/min (mL/min) used flow rate 2.5 mL/min 2.5 mL/min 3.0 mL/min 3.0 mL/min β-cyclodextrins BDex 120 BDex 325 BGB- 174 BGB 176SE velocity at minimal HETP 60 cm/s 55 cm/s 25 cm/s 55 cm/s corresponding flow rate 4.5 mL/min 4.5 mL/min 1.3 mL/min 4.5 mL/min optimal velocity according to Golay relation 45-75 cm/s 45-65 cm/s 37.5 cm/s 40-65 cm/s corresponding flow rate 2.8 mL/min 3.0 mL/min 2.5 mL/min 2.8 mL/min optimal resolution resolution relation (max. Rs) 1.8 1.25 corresponding flow rate 2.5 mL/min 3.5 mL/min flow rate interval for acceptable Rs 2-4 ml/min 2-5 mL/min (mL/min) used flow rate 2.5 mL/min 2.5 mL/min 2.5 mL/min 2.5 mL/min γ-cyclodextrins BGB-175 G-BP G-PN GDex 120 velocity at minimal HETP 52 cm/s 50 cm/s 56 cm/s corresponding flow rate 4.5 mL/min 4.5 mL/min 4.5 mL/min optimal velocity according to Golay relation 40-65 cm/s 40-60 cm/s 40-70 cm/s corresponding flow rate 3.0 mL/min 3.5 mL/min 2.7 mL/min optimal resolution resolution relation (max. Rs) 1.55 0.92 corresponding flow rate 5.0 mL/min 3-4.5 mL/min flow rate interval for acceptable Rs 3.0-6.0 mL/min 3-6 mL/min (mL/min) used flow rate 2.5 mL/min 3.0 mL/min 3.0 mL/min 2.5 mL/min

Test Mixture

The final test mixture contains twelve enantiomer pairs (table 3.3)[159]. Distinction between enantiomer pairs is facilitated with higher compound amounts for levorotary forms, apart γ-valerolactone that is added in higher concentration by its dextrorotary form and the racemic compounds with equal enantiomer amounts. Every compound has no overlapping with other compounds of the test mixture, independent of the tested column. Elution orders, inversion of the enantiomer elution order or peak shape of the enantiomer pairs enable direct column comparison possibilities. Further stability checks of our test mixture did not show any impairments or quality deficiency over a period of a year.

37 Chapter 3. Results and Discussion

Table 3.3: Composition of the test mixture. The ingredients are dissolved in dichloromethane-hexane solvent (3:1). structural compound abbr. CAS number concentration characteristic (mg/mL) (+)-α-pinene A 7785-70-8 0.1 (-)-α-pinene A 7785-26-4 0.2 hydrocarbon (+)-β-pinene B 19902-08-0 0.1 (-)-β-pinene B 18172-67-3 0.2 (+)-limonene L 5989-27-5 0.1 (-)-limonene L 5989-54-8 0.2 (±)-propylene glycol P 57-55-6 1.0 alcohol (±)-linalool I 78-70-6 0.2 (-)-linalool I 126-91-0 0.1 ketone (+)-carvone C 2244-16-8 0.1 (-)-carvone C 6485-40-1 0.2 lactone (±)-γ-valerolactone G 108-29-2 0.2 (+)-γ-valerolactone G 58917-25-2 0.2 ester (+)-ethylmandelate E 13704-09-1 0.2 (-)-ethylmandelate E 10606-72-1 0.3 carboxylic acid (±)-α-methylhydrocinnamic acid M 1009-67-2 0.8 amine (±)-1-phenylethylamine H 618-36-0 0.5 drug (RR,SS)-methylphenidate Y 298-59-9 1.0 (R,S)-pentobarbital N 76-74-4 1.0

Separation Properties

In general, the chiral hydrocarbons α-pinene (A), β-pinene (B) and limonene (L) elute early and symmetrically on all columns. The pinenes are constitutional isomers and are separated by every column, but their two enantiomer pairs are not separated under every condition. The critical compounds 1-phenylethylamine, methylphenidate, pentobarbital, α- methylhydrocinnamic acid and propylene glycol show column selectivity and are challenging to analyze and separate. No co-elution is observed for any compound independent of the used column. Generally best enantiomeric separation is achieved with β-CD capillary columns. Thereof, on BGB-176SE ten enantiomers are baseline-separated. On the other co- lumns at least one compound is separated except the specific column Chiraldex A-DA, where no enantioseparation is obtained. In figure 3.2 the chromatograms with best enantiomeric separation are illustrated. BGB-176SE column shows sep- aration of all enantiomer pairs with minimal enantiomeric resolutions for carvone (C) (Rs = 1.3) and methylphenidate (Y) (Rs = 1.4). All other compounds are separated by their enantiomers with a resolution Rs more than 1.5 (100%) and are therefore baseline separated. Table 3.4 illustrates the resolution of the enantiomer pairs on all columns. The enantiomer pairs separated more than 85% (Rs ≥ 1.0)

38 3.1. Monitoring of GC Systems

are marked as X. Enantiomer separation with Rs ≥ 1.5 (100%) is shown with XX. Special mentioned are the enantiomers of γ-valerolactone and β-pinene. They even show a separation of their enantiomers with the inclusion of another compound. This particular behavior is marked with XXX. A compound not visible in the chro- matogram, is noted with a slash. If the peak is not significant, it is marked with a slash in brackets and if there is a peak but no chiral separation, there is an empty field in the table. The elution order of the enantiomer pairs varies on different columns. Only γ-valerolactone shows no inversion in enantiomer elution, independent to what col- umn was used. Table 3.5 shows the elution order of the compounds with known configuration. Enantiomer pairs revealing inversion of elution order, compared to the elution order of the column BGB-176SE, are accented in dark grey. As already mentioned, best separation is achieved with the β-CD column BGB- 176SE, separating every enantiomer pair of the test mixture (figure 3.2(c)). The β-CD column Beta DexTM 325 shows also powerful separation (figure 3.2(b)). Chiral separation with γ-CD columns is more compound-selective. The γ-CD column BGB- 175 showed best separation of its column type (figure 3.2(d)). Most compound- selective properties are found with α-CD columns. Best separation in this group demonstrates Alpha DexTM 120 (figure 3.2(a)).

BGB-176SE Column Column BGB-176SE was used to examine column stability and alterations over time. Good separation properties were still seen after a few hundred analyses, as figure 3.3(a) demonstrates. Extensive use and in particular studies of biological samples with water content, resulted in impaired column separation capacity. Figure 3.3(b) shows the chromatogram of a damaged column, where enantiomeric separation is not possible anymore. Pinenes, α-methylhydrocinnamic acid and pen- tobarbital are the only compounds still remaining partly separable. No enantiomeric separation reaches a resolution ratio Rs better than 1.2 (β-pinene peaks) and the symmetry of the peaks is impaired (table 3.6). Carvone and methylphenidate show only single peaks. Retention time also changes with the age of a column. The damaged column shows earlier elution compared to the other column.

Discussion Chiral capillary gas chromatography with CD-columns differs from common non- chiral GC. It has to be taken into account that in chiral GC not only the choice of right solvents is important, because of more fragile CSPs, but also upper tempera- ture limits are lower and baking out at high column temperature to regenerate the column may decrease chiral selectivity. Another important point is the significantly higher carrier flow in chiral GC. Earlier, Grob, Bicchi and Schurig [137, 140, 160] described the need of high velocities u (40 - 80 cm/s), that corresponds to a flow rate of about 2.5 - 7 ml/min (BGB-176SE column: 30 m length, 0.25 mm I.D. and

39 Chapter 3. Results and Discussion


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Figure 3.2: Chromatograms of the test mixture on α-, β- and γ-cyclodextrin columns from different providers; (a) Alpha DexTM 120, (b) Beta DexTM 325, (c) BGB-176SE, (d) BGB-175; levorotary enantiomers are added in higher con- centrations with α-pinene (A), β-pinene (B), limonene (L), linalool (I), carvone (C) and ethylmandelate (E); the dextrorotary enantiomer of γ-valerolactone (G) is added in higher concentration and 1-phenylethylamine (H), propylene glycol (P), α-methylhydrocinnamic acid (M), methylphenidate (Y) and pen- tobarbital (N) are used as racemates or as a mixture of enantiomers.

40 3.1. Monitoring of GC Systems TM : no 1.0); / Gamma Dex 120 < s R Chiraldex G-PN -CD columns γ Chiraldex G-BP (/) (X) / BGB- 175 XX XXX(X) X(X) XX XXX XXXX XX (X) XX (X) (X) / / //// XX (X) (/) / X (/) (X) BGB- 178 1.5); XX enantiomeric resolution is more > BGB- 176SE s R ≤ : indicates a peak but without chiral resolution; BGB- 174 XX  -CD columns TM β XXXXXX XX XX(X) XX XX XX XX XX XX X XX XXXX XX (X) (X) XX XX Beta Dex 325 TM Beta Dex 120 (X) X(/) X XX(/) (X) XX XX XX (X) XX/ XX /(X) XX XX XX / XX XX Chiraldex A-PH Chiraldex A-Da -CD columns α BGB- 173 TM Dex 120 X 1.5); XXX enantiomeric resolution is more than 100% with inclusion of another compound. ) no significant peak is observed; (X) enantiomeric resolution, but less than 85% separated ( / ≥ s R -pinene -methylhydrocinnamic -pinene -valerolactone XXX compound Alpha β limonene X (X) pentobarbital (/) carbohydrate α alcohol linalool propylene glycolketone carvonelactone Xγ ester ethylmandelate XX acid α (X)acid XXprimary amine phenylethylaminedrug methylphenidate (X) (X) / / / / Table 3.4: Comparison of the separation power of different columns; peak is visible, ( X enantiomeric resolution with more than 85% separated peaks (1.0 than 100% (

41 Chapter 3. Results and Discussion

Table 3.5: Elution order of the enantiomers with known conformation; column BGB- 176SE (β-CD column) figures as reference, where orange accented + or - symbols show inversion of the elution order; γ-valerolactone shows no inversion independent to the analyzed column. α-CD columns β-CD columns γ-CD columns compound BGB- Alpha BGB- Chiraldex Beta Beta BGB- BGB- BGB- Chiraldex Chiraldex 176SE DexTM 173 A-PH DexTM DexTM 174 178 175 G-BP G-PN 120 120 325 α-pinene - + - + - + + - β-pinene + - + - + - - + -+ -+ limonene - + - + - + - + - + + - linalool - + - + - + - + + - carvone + - - + -+ + - - + - + + - + - γ-valerolactone + - + - + - + - + - + - + - + - ethylmandelate + - + - + - + - - + -+ + - + - + - methylphenidate + - - + + - + -

15.00 (a) BGB-176SE column in use

(-)A (-)B (-)L (-)C N 10.00 (+)L H (-)I M N (+)A(+)B H (-)Y (+)C M (-)E (+)Y

(+)G PP (+)I (+)E

FID response (mVolt) 5.00 (-)G

0.00 5.00 21.00 3 7 . 0 0 5 3 . 0 0 6 9 . 0 0 T i m e ( m i n )

15.00

(b) Extensively used BGB-176SE column

10.00

(-)A 5.00 FID response (mVolt) Y (+)A (-)B M N (+)G C N (+)B H (-)E M P (+)L (-)L (-)G I (+)E

0.00 5.00 21. 00 37.00 53.00 69.00 Time (min) Figure 3.3: Chromatograms of the chiral test mixture (a) on a BGB-176SE col- umn after 250 analyses; (b) on an extensively used BGB-176SE column. Apart from the pinenes (A, B), α-methylhydrocinnamic acid (M) and pentobarbital (N), no satisfactory enantiomeric separation is achieved any more.

0.25 µm film thickness), for maximum CD column separation efficiency. These find- ings are consistent with the results of our experiments. High carrier flow is easily applied on GC equipped with flame ionization detectors. In contrast, when working with GC-MS, the tolerated carrier flow is inferior to optimal conditions and thus, a

42 3.1. Monitoring of GC Systems

Table 3.6: Comparison of BGB-176SE columns in use (250 analyses) and extensively used (damaged) as illustrated in figure 3.3. Retention times tR of the damaged column are shorter, symmetry As of the peaks is impaired and baseline separation of the enantiomers (Rs ≥ 1.5) is not reached anymore. compound abbr. retention time plates asymm. at 5% resolution (tR) NAs Rs column in use damaged in use damaged in use damaged in use damaged propylene glycol P 12.86 12.23 45616 1506 8.30 0.46 1.7 0.0 propylene glycol P 13.28 42442 8.05 (+)-α-pinene (+)A 14.41 12.64 165538 18288 1.78 1.70 2.9 0.7 (-)-α-pinene (-)A 14.80 12.97 206313 15534 1.71 9.00 (+)-β-pinene (+)B 16.64 14.73 313055 32735 2.00 3.18 4.2 1.2 (-)-β-pinene (-)B 17.17 15.23 266014 26141 2.19 6.49 (-)-limonene (-)L 19.03 17.37 356669 8137 1.97 1.63 6.4 0.9 (+)-limonene (+)L 19.83 18.13 405091 14730 2.07 0.97 (-)-γ-valerolactone (-)G 20.28 18.83 134755 5248 6.32 2.83 8.1 1.1 (+)-γ-valerolactone (+)G 21.80 20.23 313185 4807 3.88 3.30 1-phenylethylamine H 24.84 23.63 300438 2651 4.22 1.98 2.4 0.0 1-phenylethylamine H 25.31 253343 4.63 (-)-linalool (-)I 28.09 26.23 655426 19388 2.47 0.72 4.4 0.4 (+)-linalool (+)I 28.67 26.65 846469 8944 1.66 5.40 (+)-carvone (+)C 35.35 33.47 1081704 26379 2.06 2.28 1.2 0.0 (-)-carvone (-)C 35.53 832061 2.08 (+)-ethylmandelate (+)E 42.87 40.89 1526606 135306 2.22 0.51 2.3 0.5 (-)-ethylmandelate (-)E 43.22 41.24 1187696 52992 2.49 3.17 α-methylhydrocinnamic acid M 54.97 53.39 1265801 56925 3.59 1.13 3.9 0.7 α-methylhydrocinnamic acid M 55.74 54.16 1195312 60876 4.31 2.56 (R,R)-methylphenidate (+)Y 58.44 56.71 709219 43267 0.81 1.20 1.1 0.0 (S,S)-methylphenidate (-)Y 58.75 795771 0.88 pentobarbital P 68.42 67.17 4052247 106272 0.88 0.92 4.5 0.6 pentobarbital P 69.02 67.77 4490002 116016 0.89 2.59 compromise is needed. In general, it is recommended to check first a column0s behavior prior to routine use. Methane gas is examined isothermally and with short analysis time. These fast analyses give already lots of information about column installation, symmetry of non-interacting substances, column-tolerated compound amount, experimental dead time and optimal carrier flow. Subsequent analyses with the test mixture allow defining start conditions of a column and keeping it under control by repeated test mixture analyses in predefined intervals. The number of twelve compounds was chosen to get satisfying column compar- ison between column types, different providers and CSPs. As noticed in table 3.3, the different classes of compounds give some information about the column state and also about its enantiomeric separation capacity. Critical compounds are in some cases not eluted and were chosen to see early column impairment. The ester ethylmandelate appears to be an ideal compound for a multifunctional test mixture because it shows enantiomeric separation on most of the columns. Pentobarbital and methylphenidate are drugs, representing high boiling compounds with different separation properties. They elute at late retention times but are still enantiomeric separable with an appropriate column. The tailing properties of the compounds

43 Chapter 3. Results and Discussion

α-methylhydrocinnamic acid and 1-phenylethylamine exhibit alkaline column be- havior and acidic column behavior respectively. The latter is the most critical of all compounds in the test mixture, because it is not visible in every chromatogram. It is a challenge to separate its enantiomers and the symmetry As at 5% peak-height is in most cases not satisfying (As > 1.5). The hydrocarbons elute in general early and symmetric, even on affected columns. Contaminated and damaged columns become visible by the alcohols propylene glycol and linalool. Early damage is seen in particular with a tailing of propylene glycol because of its two hydroxyl groups and therefore the higher capacity to form hydrogen bonds. Ketone compounds are separable on almost all columns, also lactones. The lactone γ-valerolactone exhibits interesting properties. Where the elution order of other enantiomers is not pre- dictable, this small compound does always elute in the same order. It seems to interact almost entirely by inclusion and exclusion of the CD cavity. Consequently, other separating mechanisms are subordinate and they do not lead to a change of the elution order. For all other compounds prediction remains still impossible.

44 3.2. Method Development for Automated Sample Preparation and Chromatography

Figure 3.4: Schematic drawing of the method development process for auto- mated MEPS extraction and GC large volume injection (LVI). Techniques for sample preparation (MEPS), LVI, using different approaches (solvent evap- oration, AT-line large volume injection, solvent gap large volume injection, backflushing (BKF)), and chiral GC were developed individually as far as possible. Most promising parameters were merged and adapted for success- ful combination. Resulting LVI method, MEPS-LVI-BKF method and chiral MEPS-LVI-BKF method were validated for linearity, limit of detection (LOD) and repeatability.

3.2 Method Development for Automated Sample Preparation and Chromatography

Figure 3.4 illustrates in an overview the method development for the automated sample preparation and GC analysis. Sample preparation as well as subsequent GC analysis were developed and adapted in a parallel way. The final method in sec- tion 3.2.4 (Chiral MEPS-LVI-BKF-GC) demonstrates a highly sensitive and broadly applicable method for chiral and non-chiral GC analyses.

45 Chapter 3. Results and Discussion

3.2.1 MEPS Sample Preparation

For the development of a general and most simple extraction method with broad applicability and coupling to GC, the first step was the assessment of starting pa- rameters. Because the investigated compounds evaporate at high temperature, nu- merous solvents apart from water were possible to consider. GC applications, par- ticularly chiral columns need absence of water, therefore the protocol was optimized to have minimal water content. Different solvents were investigated. Methanol (MeOH) and acetonitrile (ACN) turned out most suitable. Solvent mixtures did not show advantages compared to pure solvents. MeOH tuned out to be not the solvent of choice for methylphenidate experi- ments. Already low water content in the MeOH solvent led to hydrolysis of MPH and formation of ritalinic acid (RA) and MeOH. Because of noted MPH hydroly- sis and MeOH formation, this solvent has been discarded after a certain time as elution solvent and was replaced by ACN, where MPH shows good stability (see section 5.3.10: stability investigations of methylphenidate). The section 1.7 (sample preparation) explains the minimal reasonable elution volume of 10 µL as a result of the MEPS BIN bed volume with about 6 µL. An elution volume of 20 µL was chosen in early experiments. Later assays showed better analyte solubility and recovery with an elution volume of 30 µL. The selection of elution volume is crucial for the subsequent LVI steps. A preferable MEPS BIN sorbent is chosen according to the analyte properties or known HPLC compatible columns. Methylphenidate and tramadol have a LogP of 2.31 and 1.35 respectively and are therefore suitable for C18 sorbents. MEPS can be applied for various sample matrices like blood, plasma, saliva, buffer or water. In the experiments physiologic buffers (Tris and phosphate) as well as water-based extractions were exerted with success. Dilution steps before or after extraction were avoided, if possible. The investigated analytes were successfully extracted with the final method illustrated in figure 3.5. Lifetime of a MEPS BIN depends on sample matrix. In the present thesis, sorbents were used for 40 to 100 times, but were changed latest at first signs of decreasing performance, which was measured in reduced target peak areas and increased peak elution of MEPS BIN associated compounds. Conditioning and activation was performed with acetonitrile and water, followed by sampling steps for analyte enrichment. The number of samplings depends on the analyte concentration and the limit of detection for the chromatographic method. With increasing number of sampling, precision decreases (see section 3.2.4: method validation MEPS-LVI-BKF). In most cases MEPS was performed with three to five samplings per extraction cycle. For user simplification sampling was conducted by aspiration and direct dispensation of the aspirated volume into the waste vial. The advantage of that procedure minimized volume dependent mistakes in manipulation. Just the total needed volume needs to be taken into account, as the multiplied number of samplings (80 µL). The aspiration and dispensation speed is also valuable.

46 3.2. Method Development for Automated Sample Preparation and Chromatography

Figure 3.5: Final MEPS procedure for elaborated application. Conditioning is performed with organic solvent, followed by activation with aqueous phase. The number of sampling is dependent on sample volume and concentration. A washing step removes interfering matrix from the sorbent. If water absence is preferred a further washing step with 3x 50 µL air can be included. Subsequent aspiration of the elution solvent re-dissolves the analytes. This volume is consequently injected and analyzed. For re-use of the MEPS BIN sorbent, a post-washing step eliminates carry over and prepares the tool for a next extraction.

With 5 µL/s aspiration speed and 20 µL/s dispensation speed good extraction results were obtained. After sampling, the MEPS BIN is washed by a washing step (water) to remove remaining sample matrix. For water-sensitive processes a further drying step was introduced with three times aspiration of air or an inert gas (Argon). Elution of the concentrated analyte finally occurs by aspiration of 30 µL of acetonitrile and re- dissolution of the analytes. Dispensation of the dissolved analyte is performed either by direct injection into the GC injector with Large volume option at very low speed (3 µL/s for 1 s to 1 µL/s for 4 s with linear decrease; total time 26.4 s) and solvent evaporation (see section 3.2.2: large volume injection with PTV backflush) or by injection into a vial for further processes (derivatization, HPLC analysis), performed also using low injection speed (5 µL/s). After injection, the MEPS BIN is washed using an optimized washing procedure, starting with acetonitrile, followed by water, methanol and again water. After the cleaning procedure, the MEPS syringe is ready for a new extraction.

47 Chapter 3. Results and Discussion

3.2.2 Large Volume Injection with GC Large volume injection with GC is well established for different kinds of applications and various techniques are available [120, 161]. As it is mainly employed for small molecules with low boiling points, literature does not provide much information for high-temperature evaporating compounds. Since PTV injectors provide many modifiable parameters, different approaches were investigated. Most promising and robust methods were continued to pursue, keeping in mind the envisaged use with chiral columns. All approaches of method optimization were conducted with similar conditions (compound, sample concentra- tion) and compared with same criteria for qualification:

• Remaining solvent peak

• Analyte peak area, as an indication for method sensitivity

• Ratio of analyte degradation, as an indication for mild to harsh conditions

• Analyte elution time (tR), as a measure of throughput • Analyte peak shape, as an indication for good performance (double peak, broad peak, tailing, fronting)

Methylphenidate degrades at harsh conditions (high temperature or chemically ac- tive parts in the GC inlet). It is therefore a convenient compound for the establish- ment of mild conditions and provides information about the need of liner change. The inlet liner is to change at a degradation ratio exceeding 5% (figure 3.7(b)). As earlier mentioned, the selection of solvent is one of the primary steps in LVI method development. The inlet temperature is adapted to the vapor pressure of the solvent and the pressure in the injector. Therefore, operating conditions change by use of differing solvents. At early stages it was observed that methanol was not a solvent of choice because low water content in the solvent led to hydrolysis of methylphenidate to ritalinic acid with formation of methanol (figure 3.6). In addi- tion, MeOH is also formed as a product in enzymatic transformation (CES1A1) of methylphenidate to ritalinic acid. Those reasons led to the final discard of methanol as LVI solvent, although it showed promising properties and is widely used for LVI applications.

Solvent Evaporation with PTV Sample introduction is a very important part in GC analysis. The sample needs to be transferred into gas phase without contaminating the inlet. Too large volumes risk, due to volume expansion, leading to a back-flash into carrier tubing what has to be avoided. LVI was initiated by a general proposed approach for PTV. The sample is in- troduced at a controlled speed into the cold inlet. The analyte condenses to the

48 3.2. Method Development for Automated Sample Preparation and Chromatography

Figure 3.6: Transformation of methylphenidate: hydrolysis of methylpheni- date to ritalinic acid and methanol (i) and thermal degradation to methyl phenylacetate plus piperidine (ii).

(a) 1 µL injection

(b) 8 µL injection

Figure 3.7: (a) Standard injection (1 µL) of methylphenidate (10 µg/mL) and (b) starting method for LVI with same sample and 8 µL injection in solvent evaporation mode. MPH elutes at tR 8.0 min and 14.18 min respectively. In the LVI chromatogram a significant peak at tR 9.33 min corresponds to thermally degraded MPH: methyl phenylacetate (MPA). The ratio of the peak area of MPA to MPH gives the degradation ratio, a measure of operating conditions. See also figure 3.13.

49 Chapter 3. Results and Discussion

liner walls and the solvent evaporates via the split line. After solvent venting, the analyte is transferred in splitless mode to the column by rapidly increasing the inlet temperature. Preliminary tests in LVI were undertaken and compared to typical 1 µL split injections (figure 3.7).

Table 3.7: Starting conditions with LVI and improved solvent evaporation method. condition initial parameters improved LVI parameters sample concentration 10 µg/mL in MeOH 100 ng/mL in MeOH syringe maximal volume 10 µL 100 µL PTV large volume liner deactivated sintered glass for large volume initial temperature 65 ◦C 110 ◦C injection time 0.1 min 0.5 min vent flow 100 mL/min 100 mL/min evaporation temperature & time 110 ◦C for 3.0 min 110 ◦C for 2.0 min transfer temperature & time 210 ◦C for 1.0 min 180 ◦C for 1.0 min clean temperature & time 300 ◦C for 2.0 min 230 ◦C for 2.0 min carrier carrier mode constant flow constant flow carrier flow rate 1.5 mL/min 1.5 mL/min oven oven initial temperature & hold time 65 ◦C for 4.5 min 110 ◦C for 5.0 min oven heat rate 15 ◦C/min 17 ◦C/min oven final temperature & hold time 220 ◦C for 10.0 min 220 ◦C for 3.0 min detector mass scan range 50 - 400 30 - 400 mass SIM 56, 84, 91, 115, 172 56, 84, 91, 115, 172 autosampler sample volume 8 µL 20 µL injection speed 50 µL/s 1 µL/s external events None None total run time 16.85 min 13.48 min

The chromatogram of the LVI starting method (figure 3.7(b)) and table 3.7) shows successful analysis of methylphenidate. The peak area of LV injected analyte is significantly increased compared to the 1 µL split injection of the same sample. But the solvent peak is far too prominent and needs optimization in solvent evap- oration and elimination (solvent peak not shown). Additionally, methylphenidate is thermally decomposed to methyl phenylacetate (MPA). The peak appears at tR 9.33 min in the chromatogram of LV injected sample but does not arise for com- mon 1 µL injection. During LVI the analyte condenses to the liner in the inlet for a certain time, while the solvent is vented via the inlet split line. During this time par- ticularly labile compounds risk to decompose at contact of active inlet parts. This is hardly observed with split injection where residence time in the injector is short. In consequence, evaporation temperature and time are identified to be crucial points

50 3.2. Method Development for Automated Sample Preparation and Chromatography

in the optimization of a LVI procedure. But, evaporation time is also dependent on the injected volume. After having defined an injection volume, corresponding evap- oration time has to be chosen, considering elimination of the vast bulk of solvent and minimal analyte degradation. Transfer temperature and time, as well as cleaning temperature appeared to have significant impact on method improvement. This is also the case for injection speed; it is dependent on the investigated injection technique and solvent. Solvent evaporation with PTV usually needs slow injection rates (≤ 10 µL/s). Optimization of solvent evaporation with PTV led to improved results in regard to peak shape and degradation ratio and also led to enhanced sensitivity allowing continuation with lower sample concentrations (1 µg/mL or 100 ng/mL), but still, the solvent peak remained unfavorable (table 3.8). Further LVI techniques needed to be investigated, keeping found improvements in mind (table 3.7).

Table 3.8: Solvent evaporation LVI method optimization criteria qualification. compound solvent peak peak area degradation elution peak shape ratio time (n = 15) (tR = min) (As) methylphenidate to improve ok > 5% 10.3 double peak

Large Volume Injection with AT-Column Liner A second approach was investigated for LVI optimization. De Koning et al. pub- lished in 2004 a novel method for LVI, so called AT-column large volume injec- tion [162]. In this approach the sample volume is introduced into the PTV injector housing a particular PTV AT-column liner (figure 3.8). The gas chromatograph is equipped with a pre-column and an analytical column. The oven temperature is set a few degrees above the calculated solvent vapor temperature and the injector temperature below this temperature. After sample injection the solvent vaporizes in the pre-column and re-enters the injector, where it partly condenses. Remaining solvent vapor escapes via the split line. By formation of a temperature gradient between oven and injector a controlled vaporization of the solvent occurs as well as elimination via the PTV split line until just a small amount of solvent remains. A supplemental glass bead may assist in the vaporization process by restricting the liquid flow to the pre-column. There are only few parameters to be considered with this LVI procedure (injector temperature, vent flow, sample volume and oven tem- perature [162]) and it is proposed in particular for the analysis of labile compounds. Due to commercial lack of such a liner for the available PTV injector, a custom tailored liner was used. In fact, degradation ratio was reduced with this novel approach and repeated injection showed good reproducibility with nice peaks, but solvent elimination re- mained not successful (figure 3.9). Presence of glass beads (diameter 1 mm) or absence in the AT-liner did not lead to optimization.

51 Chapter 3. Results and Discussion

Figure 3.8: Schematic drawing of an AT-column liner for PTV. A side hole in the upper part of the liner allows vapor to exit via the solvent split. The glass bead assists in the control of the temperature gradient dependent vaporization process.

By varying the oven temperature a second peak appeared, eluting few seconds earlier to the methylphenidate peak. This second peak was assumed to be the inversed isomer erythro-methylphenidate as it shows same elution time and MS- spectra. For reasons not understood, in particular lower temperature favors the formation of this second peak. Conversion of isomers and newly arising peaks during analysis, need to be avoided. In contrast to methylphenidate, the analysis of other compounds showed tailing of late eluting peaks. But also low boiling compounds were not conveniently ana- lyzed and solvent elimination was not successful. Therefore, also this approach was discarded.

Table 3.9: AT-column LVI method optimization criteria qualification. compound solvent peak peak area degradation elution peak shape ratio time (n = 14) (tR = min) (As) methylphenidate unfavorable ok < 5% 16.2 < 1.2

52 3.2. Method Development for Automated Sample Preparation and Chromatography

.

.

.

. Figure 3.9: Chromatogram with adapted LVI method using an AT-column liner. Prominent solvent peak but no degradation of MPH was observed at m/z 91 (specific mass for MPH and MPA) in SCAN and SIM mode. Concen- tration was 1 µg/mL methylphenidate in methanol.

Large Volume Injection with Bypass Column

Another approach was performed by using a bypass column. It consisted of a pre- column and a press-fit 3-way splitter to the analytical column and a bypass column with possibility to open and close manually (for preliminary tests). For this approach a pre-column with 0.53 mm inner diameter and 15 m length was installed. The capillary is phenyl/methyl-deactivated (DPTMDS) and has a coating film at the last 3 meters with 5%-phenyl-dimethyl-polysiloxane. Pre-column, analytical column (0.25 mm ID) and the bypass column (0.53 mm ID) are connected through a press-fit 3-way splitter. During injection, inlet and oven generally have an initial temperature below the corrected solvent boiling point. The sample is injected at low speed. For solvent elimination the end of the bypass column is opened and solvent escapes from the system. After closing the bypass column again, concentrated analytes on the coated part of the pre-column and rests of solvent are transferred by oven heating to the

53 Chapter 3. Results and Discussion

analytical column. Solvent is successfully eliminated from the analytical system with this solvent gap method. But methylphenidate shows broad peaks and tends to be excessively degraded, and it would not result in highly sensitive and mild applications. Due to significant MPH degradation, seen as plateau formation in the chro- matogram preceding a MPH peak elution, this approach was also discarded.

Table 3.10: Bypass column LVI method optimization criteria qualification. compound solvent peak peak area degradation elution peak shape ratio time (n = 14) (tR = min) (As) methylphenidate ok to improve > 10% 16.5 broad peak

Large Volume Injection with PTV Backflush After all, solvent evaporation seemed to be the most convenient approach for the given challenge. But further technical adaption was indispensable to reduce the solvent burden on the analytical column. The implementation of a backflush tool (BKF) enabled advanced studies [163, 164]. A GC system equipped with backflush (figure 3.10) allows the change of carrier gas direction in the injector and pre-column at any given time during analysis, without disturbing the separation process. A pre-column (0.53 mm ID) is connected to the PTV injector and via a capillary-T-junction to the analytical column (0.25 mm ID). The BKF valve controls the direction of the gas flow and a restrictor (not shown) prevents opposite gas flow in the capillary-T-junction or carrier tubing in the inlet. With standard conditions the gas flow is directed from injector, to pre-column and analytical column with resulting MSD detection. When activating the BKF valve, direction of the carrier flow is changed. A predominant part of the carrier gas circulates from the capillary-T-junction through pre-column and PTV to the split line out of the system and only a minor part still assure continuous flow through the analytical column. The use of BKF is helpful in the elimination of large solvent amounts during injection procedure but it is also beneficial to protect the analytical column by elimination of heavy matrix prior to reach the column. Disadvantage of this additional tool are the limited applicability in LVI with low temperature boiling analytes, as well as an increasing number of parameters to adjust for satisfying results. Starting conditions for LVI-BKF were chosen from the solvent evaporation tech- nique with certain adaption for BKF use (table 3.11). Backflush is activated for the period of injection and evaporation to enhance solvent venting. During the transfer phase backflush is inactivated and only activated after end of transfer to remove residual solvent or heavy matrix in the inlet system. Generally, backflush is active until end of analysis.

54 3.2. Method Development for Automated Sample Preparation and Chromatography

- --- --

-

- - - -

Figure 3.10: Schematic drawing of a backflush (BKF) enabled GC system. During the injection and evaporation process the carrier gas direction in the injector and the pre-column is reversed. Green arrows indicate default carrier gas direction. With activated BKF valve, the carrier flows in opposite direction (red arrows), from capillary-T-junction to pre-column an injector and one part through the analytical column to keep the chromatographic conditions (black arrows). The gas flow in opposite direction facilitates the elimination of solvent through the split line. After the injection process, the direction is changed to default direction leading to the transfer of the analyte and remaining solvent to the analytical column.

At early LVI-BKF optimization, the selection of solvent was changed from MeOH to ACN. Acetonitrile had reduced impact on thermal degradation of methylpheni- date compared to methanol, as earlier mentioned. Again, criteria like remaining solvent peak, peak area of analyte, ratio of analyte degradation, elution time of analyte and analyte peak shape served as reference for the evaluation of the chromatograms. Solvent venting and also peak shape improved significantly with the use of LVI- BKF (figure 3.11), based on the method of solvent evaporation earlier described. Several statistical approaches (fractional factorial design, Plackett-Burman de- sign, surface design [165, 166]), were considered for optimization of LVI-BKF. Due to directly dependent parameters or impossible factor combinations without break down of the total system, systematic optimization was performed starting from the initial LVI-BKF method (table 3.11). In first attempts, dependent parameters were determined. Injection time and injection speed are closely related. The same is observed for transfer time and inlet purge time or splitless time, respectively. Also vent flow is a crucial parameter during injection phase and cleaning phase; and time of evaporation has to be selected according to the injection volume. A minimal amount of residual solvent is needed

55 Chapter 3. Results and Discussion

(a)

9

9 (b)

.

. Figure 3.11: Initial conditions for LVI (a) without and (b) with BKF. Same peak areas are obtained, but with BKF the solvent burden is tremendously reduced. Degradation product at tR 6.5 min is observed and a double peak at MPH elution time tR 10.74 min and 10.79 min. This method needs optimiza- tion. 56 3.2. Method Development for Automated Sample Preparation and Chromatography

Table 3.11: Initial and final parameters for LVI-BKF-GC-MSD application. condition initial LVI-BKF optimized parameters parameters sample concentration 100 ng/mL in MeOH 1 - 50 ng/mL in MeOH syringe maximal volume 100 µL 100 µL PTV large volume liner deactivated sintered glass for large volume initial temperature 65 ◦C 90 ◦C injection time 0.1 min 1.55 min vent flow 100 mL/min 120 mL/min evaporation temperature & time 70 ◦C for 1.0 min 90 ◦C for 1.55 min transfer temperature & time 180 ◦C for 5.0 min 180 ◦C for 2.0 min clean temperature & time 230 ◦C for 2 min 230 ◦C for 5.0 min oven oven initial temperature & hold time 69 ◦C for 2.0 min 90 ◦C for 3.1 min oven heat rate 17 ◦C/min 17 ◦C/min oven final temperature & hold time 220 ◦C for 3.0 min 220 ◦C for 3.25 min detector mass scan range 30 - 400 40 - 400 mass SIM 56, 84, 91, 115, 172 56, 58, 84, 86, 91, 120 autosampler sample volume 20 µL 30 µL injection speed 1 µL/s 3 µL/s to 1 µL/s; total time 26.4 s external events BKF active prep-run 0.8 min BKF inactive 1.10 min 3.15 min BKF active 6.28 min 5.25 min BKF inactive 10.0 min 14.0 min total run time 13.8 min 14.0 min for successful transfer of analyte to the analytical column, which also is described in literature [119]. Table 3.12 illustrates in an overview tested parameter variations. Each parameter was considered by its own and in combination with its dependent factors. Evaluation was done by reflecting the defined criteria. For assembling the findings to the final LVI-BKF method (table 3.11), values with best performance (bold values in table 3.12) were combined. Fine-tuning was done by variation of the parameters with most influencing behavior (figure 3.12). Final combination of optimized parameters led to a very sensitive and mild pro- cedure for solvent evaporation and elimination (figure 3.13). The optimized LVI-BKF method matched the defined criteria (table 3.13). Ther- mal degradation of methylphenidate is significantly reduced and can also be used for quality control in means of inlet liner change. Inlet liners need to be regularly

57 Chapter 3. Results and Discussion

(a) (b)

II SS

I S

(c) (d)

M

M

(e)

M

MM

Figure 3.12: (a) - (e) Final adjusting for LVI method. Standard deviation (n = 3 - 5) for resulting peak areas reflects the robustness of the method with chosen parameters.

58 3.2. Method Development for Automated Sample Preparation and Chromatography

Table 3.12: Initial and final parameters for LVI-BKF-GC-MSD application. parameter values 1 2 3 4 5 6 7 8 9 oven initial temperature (◦C) 76 78 82 84 86 88 90 92 95 initial hold time (min) 2.0 3.0 3.5 3.65 3.7 3.75 4.5 5.8 6.0 heating rate (◦C/min) 10 15 17 20 25 final temperature (◦C) 180 200 220 final hold time (min) 2 3 5 10 injector inlet start temperature (◦C) 70 80 90 95 100 102 104 106 108 stop septum purge (min) No 0.5 0.9 1.0 1.5 2.0 8.0 11.0 inlet inject time (min) 0.1 0.2 0.3 0.4 0.5 1.0 1.5 2.0 3.0 injection vent flow (mL/min) 100 120 140 200 evaporation temperature (◦C) 90 100 104 108 110 112 115 120 130 evaporation time (min) 0.8 1.0 1.5 1.75 2.0 2.25 3.0 3.5 4.0 transfer temperature (◦C) 150 180 190 200 210 220 250 transfer time (min) 0.5 0.6 0.7 0.8 0.9 1.0 1.5 2.0 2.5 cleaning temperature (◦C) 200 230 250 270 300 cleaning time (min) No 1.0 2.0 5.0 cleaning vent flow (mL/min) 100 120 140 150 200 carrier carrier flow (mL/min) 0.4 0.5 0.8 1.0 1.3 1.5 2.0 2.5 3.0 autosampler sample volume (µL) 8 20 30 Injection speed (µL/s) 50 20 10 5 4 3 2 1 < 1 sample pull-up speed (µL/s) 4 5 10 15 20 wash steps (n) 1 2 3 4 5 external events adjust in dependence on injection time, evaporation time, transfer time, oven run time replaced due to contamination. Contamination leads to higher degradation ratios of labile compounds. Degradation ratio of methylphenidate is therefore a sensitive and reproducible tool indicating a need of liner change. A peak area of methyl phenylacetate (MPA) with more than 5% compared to the methylphenidate peak, necessitates the installation of a new liner in the inlet.

Table 3.13: Criteria for LVI-BKF method optimization. compound solvent peak peak area degradation elution peak shape ratio time (n = 15) (tR = min) (As) methylphenidate ok ok < 2% 10.9 < 1.2 lidocaine ok ok no degradation 11.9 < 1.3

Advantages of this method are enhanced detection of low concentrated analytes

59 Chapter 3. Results and Discussion

I

I

Figure 3.13: Analysis of MPH (tR 10.91 min) and lidocaine (tR 11.92 min) at concentration 10 ng/mL for MPH and 50 ng/mL for lidocaine, with optimized LVI method. No thermal degradation at tR 7.01 min is detected for MPH. and successful solvent elimination. The procedure is therefore convenient for solvent susceptible applications. This leads to an increased column lifetime as a result of solvent venting and removal of heavy matrix prior to reach the analytical column. In addition, backflushing reduces the risk for carry over by inverting the carrier gas direction and removing potential analyte traces from the inlet system. This new method is further used as basis for the development of automated MEPS with large volume injection in non-chiral and chiral applications.

Method Validation Validation of the LVI-BKF method revealed good results for linearity at therapeutic concentrations. Also at higher concentrations linearity is still observed. The method shows very good repeatability (STDV% ≤ 2) for GC application (table 3.14). To check possible loss of analyte during backflushing, a standard injection of 1 µL (1 µg/mL, split 1:20) was compared to 30 µL LV injection (33.3 ng/mL). With both injections, same analyte amount was transferred on the column but different meth- ods applied. Therefore, same peak areas were expected. Resulting chromatograms

60 3.2. Method Development for Automated Sample Preparation and Chromatography

Table 3.14: Qualification of the LVI-BKF method. compound linearity R2 LOD repeatability n (ng/mL) (ng/mL) (STDV%) methylphenidate 1 - 60 0.9996 1 1.5 6 tramadol 60 - 370 0.9992 20 2.0 10

(a) 1 µL MPH solution (1 µg/mL)

M

M (b) 30 µL MPH solution (33.3 ng/mL)

.

. Figure 3.14: Comparison of (a) standard GC injection and (b) LVI injection. Both chromatograms show similar peak shape, height and area for MPH at tR 8.00 min and 10.90 min respectively. showed similar signals in height and peak area (figure 3.14).

3.2.3 Automated Sample Preparation and Large Volume In- jection After successful development of LVI and MEPS as independent procedures, both protocols were combined for automated extraction and LVI with chromatographic separation and MS detection. The GC MSD equipment was adapted by implementation of an additional auto- sampler, a virtual auto-sampler, serving as second instrument. The main sampler was used for MEPS procedure and the virtual sampler for LVI-BKF. Automated extraction by MEPS usually takes one to five minutes, depending on the number of samplings. To circumvent time correction for the LVI-BKF method,

61 Chapter 3. Results and Discussion

A

Figure 3.15: Optimized method for MEPS-LVI-BKF-MSD analysis (sec- tion 5.3.3: GC-MSD method 6): Dots mark a valve change, temperature change or start/end of activity. Inlet: a - injection period, b - evaporation time, c - transfer rate to transfer temperature, d - transfer period, e - clean rate to clean temperature, f - clean temperature, g - re-establishment of initial conditions; Oven: A - initial temperature, B - heat rate to final temperature, C - final temperature period; DSQ: 1 - MS detector analyzing time period; Septum purge: 2 - period of active septum purge; BKF: 3 - period of active BKF, 4 - period of inactive BKF, 5 - period of active BKF.

extraction was programmed for performance in delayed mode. This allows the free selection of any number of samplings without further adaptations in regard to LVI- BKF chromatography. Generally no additional drying steps are needed before eluting the analytes with elution solvent into the GC inlet. Since water is to be avoided in the use of chiral columns, the MEPS syringe was dried by 3x aspiration of air or inert gas (argon) between the washing step and the analyte elution in chiral column application (Fig- ure 3.5). The final method is shown in figure 3.15, which can be employed also for other compounds than methylphenidate with few or even no changes of parameters. Methylphenidate was successfully extracted from physiologic phosphate buffer (100 mM, pH 7.4) at very low concentrations. Figure 3.16 illustrates the analysis of automated MEPS-LVI-BKF-GC-MSD of a methylphenidate sample with concen-

62 3.2. Method Development for Automated Sample Preparation and Chromatography

Figure 3.16: MES 2x with 1.0 ng/mL MPH in phosphate-buffer (P-buffer) is nicely detected in SCAN and SIM mode (tR = 10.90 min). tration 1.0 ng/mL and 2x MEPS sampling. The sample volume for MEPS depends on the expected number of samplings. 80 µL aspiration volume and 5x MEPS sampling requires 500 µL sample volumes. In case of lower sample volume, the MEPS protocol is simply adapted to the required volume or number of samplings. Also other compounds were tested. Figure 3.17 illustrates MEPS-LVI-BKF anal- ysis of methylphenidate, tramadol and lidocaine in concentration 10 ng/mL of each analyte.

Method Validation MEPS-LVI-BKF Extraction using MEPS turned out to be efficient and suitable for the given problem. After three cycles and 10x sampling only 30% of initial methylphenidate was left (figure 3.18(a)). Extraction is linear at low concentrations. MEPS of 1 ng/mL methylphenidate concentrations with variation in number of samplings shows linear increase in peak area (figure 3.18(b)). The same is true for a constant sampling number but changing concentrations. With 5x MEPS and concentrations 1 ng/mL to 20 ng/mL methylphenidate, a linear peak area increase (R2 = 0.9978) was observed (figure 3.18(c)). For tramadol a saturation of the MEPS BIN was observed at concentrations above 150 ng/mL with 5x MEPS sampling (appendix figure A.1). Preferentially, the number of samplings per MEPS cycle is below 10 for time reasons and improved precision. Moreover, the LVI-GC method is very sensitive,

63 Chapter 3. Results and Discussion

N

N

Figure 3.17: Analysis of MPH (tR 10.88 min), lidocaine (tR 11.90 min) and TMD (tR 12.65 min) in 10 ng/mL concentrations for every analyte with 5x MEPS (in SCAN and SIM mode). and already a concentration of 1.0 ng/mL (MPH) is easily detected. Therefore, the need for sample concentration in this work is only of limited impact. Linearity was tested with different numbers of MEPS sampling. The use of concentrations similar to physiologic plasma concentrations revealed for MPH (5x MEPS) and TMD (1x MEPS) good linearity with R2 0.9978 and 0.9731 respectively (table 3.15). Repeatability of MEPS shows in particular for TMD not satisfying values (48.1%). It is the result of imprecise volume aspiration during automated extraction. Aspiration of volumes > 20 µL out of a 1.5 mL crimp-vial can lead to increased risk for air-bubble formation in the syringe and to an impairment of pre- cision. With application of a minimal necessary number of MEPS cycles, precision stays in an acceptable range.

Table 3.15: Linearity and repeatability of MEPS-LVI-BKF with methylphenidate and tramadol at concentrations similar to physiologic plasma concentrations. compound MEPS cycles linearity R2 MEPS cycles repeatability n (n) (ng/mL) (n) (STDV%) methylphenidate 5 1 - 20 0.9979 5 7.4 6 tramadol 1 50 - 300 0.9731 5 48.1 6

64 3.2. Method Development for Automated Sample Preparation and Chromatography

(a) (b)

100 ) 6 ) % 3´10 ( a e e

80 r c a 6 (

n 2´10 e a c d 60 n

n 6 a 2´10 u d b n a

40 u 6 b e 1´10 a v i t e v a 20

i 5 l

t 5´10 e a l R e

0 R 0 x x x x x x x x 6 8 0 1 3 5 7 0 1 1 Number of MEPS samplings Number of MEPS samplings

(c)

R2

Figure 3.18: (a) Extraction with MEPS is efficient. A sample containing 1.0 mL of methylphenidate in a concentration of 10 ng/mL shows after three MEPS cycles and 10x sampling an analyte withdraw of over 70% from the sample; (b) at low concentrations a linear increase is observed with up-scaling number of MEPS sampling per cycle; methylphenidate at concentration 1.0 ng/mL; (c) changing concentrations but constant sampling number (5x) leads to linear increase in MPH peak area (n = 3 - 6).

65 Chapter 3. Results and Discussion

3.2.4 Chiral MEPS-LVI-BKF-GC A next challenge was the adaptation of MEPS-LVI-BKF to chiral GC conditions. Because chiral CD coated columns are sensitive to water, MEPS extraction needed to contain drying steps for minimizing water amount reaching the PTV inlet and subsequently the analytical chiral column. Preliminary tests were performed on a Focus GC-FID equipped with a chiral BGB 176SE column. Objective was the chiral separation of MPH enantiomers with minimal tR and initial apparatus conditions allowing the combination to LV injection. As chiral columns are generally used with increased flow rates, this has to be considered in particular for GC-MSD application. Certain carrier flow rates should not be exceeded due to MSD sensitivity loss with reduced vacuum in the MSD. Verification of the GC-FID method with GC-MSD showed good results. Ob- tained enantioseparation was still Rs > 1.5 for MPH, but a reduced flow rate was needed for good performance. On GC-FID, carrier flow of 2.5 mL/min led to good enantioseparation, but for GC-MSD it had to be reduced to 2.0 mL/min. Particular mention deserves the total carrier flow. Because injection of LVI turned out to be only possible with a flow rate of maximal 1.0 mL/min, the finally adapted carrier flow needed a programmed carrier flow rate. During injection 1.0 mL/min were ap- plied with an increase at the end of the injection period of 0.5 mL/min carrier rate to 2.0 mL/min until end of analysis. Also oven temperature rate was investigated. For BGB-176SE columns, a rate of 2 ◦C/min is proposed. Low oven temperature rate leads to long analysis time. With an initial rate of 16 ◦C/min and subsequent ◦ second rate of 3 C/min enantioseparation for MPH was still satisfying (Rs = 1.45) but analysis time significantly reduced from 45 minutes to 33 minutes. The final method is shown in table 3.16. Identification of the enantiomers was obtained with the injection of the single enantiomers Dex-MPH and Dex-TMD. Figure 3.19 illustrates schematically the final MEPS-LVI-BKF method for chiral application.

Method Validation Chiral MEPS-LVI-BKF-GC-MSD Not only the model compounds methylphenidate and tramadol were successfully extracted and analyzed (3x MEPS), but also other drug compounds like ibuprofen, pentobarbital, tolperisone, amphetamine and cocaine (figure 3.20). Using a C18 MEPS BIN and P-buffer at pH 7.4, basic compounds show better extraction than acidic compounds like ibuprofen. All analytes had similar concentra- tions (table 3.17). As earlier shown, precision exhibits an elevated STDV% (< 15), which can be significantly reduced by the use of an internal standard for quantitative analyses. As mentioned MEPS extraction procedure was not changed for use with chi- ral MEPS-LVI-BKF-GC-MS procedure besides the implementation of an additional drying step prior to sample elution. No influence on results was detected with this

66 3.2. Method Development for Automated Sample Preparation and Chromatography

g g

g g g g

gg g g g g

g g g

Figure 3.19: Graphical illustration of the chiral MEPS-LVI-BKF method (sec- tion 5.3.3: GC-MSD method 8): Dots mark a valve change, temperature change or start/end of activity. Inlet: a - injection period, b - evaporation time, c - transfer rate to transfer temperature, d - transfer period, e - clean rate to clean temperature, f - clean temperature, g - re-establishment of initial conditions; Oven: A - initial temperature, B - heat rate to intermediate tem- perature, C - heat rate to final temperature; DSQ: 1 - MS detector analyzing time period; Septum purge: 2 - period of active septum purge; BKF: 3 - period of active BKF, 4 - period of inactive BKF, 5 - period of active BKF. additional step. Interestingly, chiral LVI-BKF-GC-MSD analysis (no MEPS) of MPH shows good linearity (table 3.18) over the range of 0.25 - 10 ng/mL. But tramadol showed dif- ferent results. Peak areas did not increase linearly at used concentrations (50 - 300 ng/mL). With intermitted solvent wash steps, linear calibration for TMD was obtained. In the solvent runs no carry over was detected. Therefore, it is assumed that TMD interacts with the stationary phase leading to accumulation by ”sequence analysis”. With washing steps (”single run”) this accumulation is circumvented. Figure 3.21 illustrates the differing outcomes described in table 3.18 by TMD analysis in sequence or single runs. Both enantiomers behave similarly ((R,R)- enantiomer is shown). Single runs, considered as analyses with intermittent solvent runs, show repeatability with STDV% = 9.9, while sequence runs of identical samples lead to increasing peak areas (STDV% = 71.0). For further analyses all TMD samples were analyzed as single runs (with intermittent solvent step). The analysis of low concentrated TMD samples requires a pre-saturation of the chiral column with TMD for better sensitivity (section 3.3: enzyme experiments with Tramadol).

67 Chapter 3. Results and Discussion

Table 3.16: Final MEPS-LVI-BKF-GC-MS method used with chiral analytical col- umn (section 5.3.3: GC-MSD method 9 with drying step). condition optimized parameters sample concentration ng/mL range in ACN syringe maximal volume 100 µL pre-column 2.0 m, 0.53 mm I.D., DPTMDS deactivated PTV large volume initial temperature 90 ◦C injection time 4.2 min vent flow 120 mL/min transfer temperature & time 180 ◦C for 2.0 min clean temperature & time 230 ◦C for 5.0 min oven oven initial temperature & hold time 90 ◦C for 4.2 min oven heat rate 1 16 ◦C/min hold temperature 1 125 ◦C oven heat rate 2 3.0 ◦C/min oven final temperature & hold time 210 ◦C for 7.0 min carrier mode programmed flow initial value & time 1.0 mL/min for 6.35 min carrier rate 0.5 mL/min/min final value & time 2.0 mL/min for 32.0 min detector mass scan range 40 - 400 mass SIM 56, 58, 84, 91, 120, 249, 263 autosampler sample volume 30 µL injection speed 3 µL/s to 1 µL/s; total time 26.4 s external events BKF active 0.8 min BKF inactive 4.25 min BKF active 6.35 min BKF inactive 41.62 min total run time 41.72 min

68 3.2. Method Development for Automated Sample Preparation and Chromatography

N

N Figure 3.20: Chromatogram of automated MEPS 3x with LVI and sep- aration on a chiral BGB-176SE column of 7 chiral compounds: am- phetamine (tR 9.81 min), pentobarbital (tR 29.16 / 29.52 min), tramadol (tR 29.98 / 30.35 min), methylphenidate (tR 23.1 / 23.26 min), ibuprofen (tR 27.12 / 27.61 min), tolperisone (tR 28.22 min) and cocaine (tR 38.24 min), all in a concentration of 100 ng/mL. Ibuprofen, an acidic compound, is also extracted (would be increased with a different MEPS BIN sorbent), but in a much lower amount than all other compounds (weak bases). Simultaneous analysis in SCAN mode, considering masses m/z 40 - 400 and single ion mon- itoring mode (SIM) with selected masses, was used for increased sensitivity and peak identification.

Table 3.17: Automated MEPS of different compounds with subsequent chiral GC analysis. Amphetamine and tolperisone were not enantiomerically separated. Also enantiopure cocaine shows a single peak.

compound concentration tR (m/z) n STDV% (ng/mL) (min) amphetamine 144.8 9.81 44 5 8.6 methylphenidate 118.6 23.10 / 23.26 84 4 6.3 / 6.6 ibuprofen 97.8 27.12 / 27.61 161 3 16.7 / 14.7 tolperisone 112.6 28.22 98 3 5.8 pentobarbital 101.4 29.16 / 29.52 156 3 12.7 / 10.8 tramadol 111.4 29.98 / 30.35 58 3 12.2 / 12.8 cocaine 110.2 38.24 82 3 14.2

69 Chapter 3. Results and Discussion

Table 3.18: Qualification of the chiral LVI-BKF method. compound linearity R2 LOD enan- repeatability n enantiomer tiomer (ng/mL) (ng/mL) (STDV%) (R,R)-methylphenidate 0.25 - 10.0 0.992 0.25 4.2 7 (S,S)-methylphenidate 0.25 - 10.0 0.999 0.25 7 (R,R)-tramadol (sequence) 50 - 300 not linear 20.0 71.0 5 (S,S)-tramadol (sequence) 50 - 300 not linear 20.0 5 (R,R)-tramadol (single run) 50 - 300 0.993 20.0 9.9 5 (S,S)-tramadol (single run) 50 - 300 0.988 20.0 5

400000 )

M 300000 I S ( a

e 200000 r (R,R)-TMD (sequence) a

k (R,R)-TMD (single run) a

e 100000 P

0 1 2 3 4 5 Number of analysis (n)

Figure 3.21: Illustration of TMD analysis in a ”sequence” and in ”single runs”. Single runs are considered as TMD analyses with solvent runs in-between. Repetition of identical samples (50 ng/mL TMD racemate) led to different outcomes. Without intermittent solvent runs (= ”sequence”) a significant increase in peak area is observed for both TMD enantiomers ((S,S)-TMD not shown).

70 3.2. Method Development for Automated Sample Preparation and Chromatography

H

H Figure 3.22: Chromatogram of derivatized MPH and RA after 40 min reaction time. After a reaction time of 20 minutes, just carboxy-mono-silylation oc- curs (RA-tms). After 40 minutes of reaction also amine-silylation is observed leading to MPH-tms and RA-ditms.

3.2.5 Derivatization for GC Because ritalinic acid is not detectable by direct GC injection on the employed co- lumns, derivatization procedures were investigated. Derivatization should be simple and useful for numerous compounds with further possibility for implementation in automated procedure. Most promising results were obtained with silylation of a carboxylic acid group or amine-group using MSTFA (N-methyl-N-trimethylsilyl- trifluoro-acetamide). Direct derivatization as well as derivatization following the MEPS procedure was undertaken. MSTFA is water sensitive, so after MEPS procedure remaining water content had to be eliminated by evaporation. It turned out to be suitable for common GC-MSD applications but not useful in LVI-GC-MSD approaches (LVI and MEPS). Consequently, in this work it is used just for second confirmation of a result (figure 3.34(b)).

71 Chapter 3. Results and Discussion

Figure 3.23: Illustration of investigated LC processes for non-chiral and chi- ral LC methods with MEPS and stability tests. Non-chiral HPLC was per- formed for stability tests, chiral HPLC investigations with a α1-acid glycopro- tein (AGP) column were done with GC-MEPS extracted samples.

3.2.6 Liquid Chromatography

The chiral-GC results were verified with the second chromatographic method HPLC. Figure 3.23 summarizes the different steps and method combinations. LC and GC complement each other. GC allows the detection of very low sample concentrations, chiral separation of various compounds on a same column and single method. But certain compounds are undetectable in GC without derivatization and HPLC may be used as an alternative. As both model compounds have chromophores, HPLC- UV methods were developed for stability tests and verification of the enzyme assay results obtained with GC. But because of reduced sensitivity of this method, limited applicability of the same method for the analysis of more than one compound and increased number of needed working steps, the developed HPLC methods did not show advantages over the presented GC methods. Additional HPLC experiments were performed as part of work in master theses1 in the laboratory. Same MEPS procedure like in GC was employed for HPLC using TriPlus-auto- sampler of the GC-MSD. The autosampler was programmed to have a further in- jector. Instead of PTV-injection the extract was injected into a vial (position 300) and diluted in mobile phase for subsequent HPLC analysis. Automated MEPS with autosampler was preferred over manual MEPS because of better reproducibility and time reasons. Apart from this slight technical difference in procedure also the goal is different in MEPS-HPLC compared to MEPS-GC. The enrichment of analyte is focused in MEPS-LC, but not the analyte extraction with change of solvent.

1Master thesis Luca Castelnovo 2012, Hulda Brem 2013, Angela Tschabold 2014

72 3.2. Method Development for Automated Sample Preparation and Chromatography

R,R

S,S S,S R,R

Figure 3.24: MPH and RA enantioseparation on an AGP column. (R,R)-RA elutes at tR 2.90 min, its enantiomer (S,S)-RA at tR 5.94 min. The enantiomers (S,S)-MPH and (R,R)-MPH elute at tR 9.3 min and 11.53 min respectively.

Chiral Separation of Methylphenidate with HPLC-UV

Methylphenidate was successfully enantioseparated using ammonium acetate buffer and low content of organic modifier 2-propanol. The signals of MPH enantiomers (tR 9.30 min / 111.53 min) are baseline separated and show low tailing on an α1- acid glycoprotein (AGP) column. (R,R)-Dexmethylphenidate is the second eluting enantiomer (tR 11.53 min). Also ritalinic acid enantiomers (tR 2.90 min / 5.94 min) elute symmetrically and are nicely separated with the same method (Figure 3.24). Elution order of RA is inversed compared to MPH. The (R,R)-RA enantiomer elutes fist. Limit of quantification (LOQ) for each enantiomer was investigated with HPLC- UV. MPH and RA show high linearity (R2) in the chosen range. Repeatability is satisfying with low STDV% (table 3.19). Unfortunately, this HPLC-method turned out to be not suitable for combination with the elaborated MEPS protocol. Elution solvent ACN and residual P-buffer, even in traces, disturb the separation. Another chiral HPLC-method using another compatible buffer had to be de- veloped for combination with MEPS. The use of P-buffer with organic solvents (acetonitrile and ethanol) led to nicely separated enantiomers for both enantiomer pairs. Also combination with MEPS is possible (figure 3.25), but analysis time is

73 Chapter 3. Results and Discussion

Table 3.19: Qualification of chiral HPLC method. compound linearity R2 LOD enan- repeatability n enantiomer tiomer (µg/mL) (µg/mL) (STDV%) (R,R)-methylphenidate 0.05 - 6.75 0.9999 0.25 0.54 6 (S,S)-methylphenidate 0.05 - 6.75 0.9997 0.25 0.47 6 (R,R)-ritalinic acid 0.009 - 9.0 0.9999 0.1 0.35 6 (S,S)-ritalinic acid 0.009 - 9.0 0.9997 0.1 0.28 6

R,R

S,S

S,S R,R

Figure 3.25: Methylphenidate enantiomers, successfully MEPS enriched and separated with chiral HPLC-method (mobile phase: P-buffer, organic modifier ACN and EtOH). prolonged.

Chiral Separation of Tramadol with HPLC-UV Tramadol enantiomers and enantiomers of its most important metabolites (M1, M2) were investigated with HPLC-UV using P-buffer and a low amount of EtOH as organic modifier. In figure 3.26 chromatograms of baseline separated TMD and M1 enantiomers (figure 1.8) are presented (figure 3.26(a)). With same chromatographic conditions no enantioseparation of the M2 metabolite (figure 1.8) was achieved and its elution was at the same retention time (tR 17.0 min) as the parent enantiomer (S,S)-TMD (figure 3.26(b)).

74 3.2. Method Development for Automated Sample Preparation and Chromatography

R,R R,R

S,S

S,S

R,R

S,S

Figure 3.26: Chromatograms of TMD- and metabolite-enantiomers, analyzed with the same HPLC-method, compatible with MEPS; (a) TMD (tR 13 min and 17 min) and M1 (tR 10 min and 19 min) exhibit both base line separation of the enantiomers; (b) Metabolite M2-enantiomers are not separated and elute at the same tR as (S,S)-TMD (tR 17 min).

75 Chapter 3. Results and Discussion

Calibration for tramadol enantiomers exhibited good linearity in the range of 0.25 - 50 µg/mL and low standard deviation for repeatability.

Table 3.20: Qualification of chiral HPLC method. compound linearity R2 LOD enan- repeatability n enantiomer tiomer (µg/mL) (µg/mL) (STDV%) (R,R)-tramadol 0.5 - 50.0 1.0 0.5 0.64 3 (S,S)-tramadol 0.5 - 50.0 1.0 0.5 0.52 3

3.2.7 Stability Investigations

Methylphenidate

Methylphenidate stability testing in different solvents was performed using non- chiral HPLC. Since methylphenidate and ritalinic acid absorb light at 210 nm, sta- bility of MPH was measured as amount of RA formation over time. Dissolved in water, MPH quickly hydrolyzed to ritalinic acid at room temperature. This process was reduced at 4 ◦C but still remained significant. Apart from ACN, in all solvents formation of RA was observed (figure 3.27). Over time a further peak appeared, having same tR as erythro-MPH. Since the inversion of threo-MPH to erythro-MPH was also observed with GC experiments, it is assumed that MPH is slowly converted to the erythro-form in aqueous solution. Chiral separation with AGP columns allows low organic modifier content. There- fore, aqueous solutions are inevitable for the use of those columns, and stability of MPH was obtained with another approach. Acidification (pH 3.7) of the MPH stock solution results in enhanced MPH stability. No hydrolysis is observed for at least 1 hour (not shown). In all experiments, MPH, dissolved in aqueous solution, was used immediately or a stock solution in water pH 3.7 was prepared. The use of the MPH stock solution did not exceed 5 hours.

Tramadol

Tramadol is known to be stable in different solutions. Over a period of 1 week, chiral HPLC-UV analyses (271 nm) did not show TMD degradation in different solutions. But TMD, dissolved in ACN leads to highly deviating values, seen in figure 3.28. This is the result of base line affection of the column caused by ACN and therefore a reduced performance on the column. Although tramadol is stable in solution for at least 1 week, sample solutions were freshly prepared every day.

76 3.2. Method Development for Automated Sample Preparation and Chromatography

Figure 3.27: Stability of MPH, measured as the formation of RA in different solvents over a time period of 3 weeks at room temperature. MPH shows fast hydrolysis in water, and dissolution in mobile phase (solvent mix, section 5.3.10 also leads to rapid hydrolysis due to high water content. Dissolution in MeOH leads to formation of RA due to low water content in the solvent, but with- out further increase for RA after 2 days. MPH dissolved in ACN showed no formation of RA over this period.

5.5´10 5 TMD in P-buffer 10 mM

) TMD in solvent mix U

A TMD in P-buffer 100 mM m ( 5 TMD in ACN

a 5.0´10 e

r TMD in water a k a e P 4.5´10 5

0 2 4 6 8 Time (d)

Figure 3.28: Stability of TMD over a period of 1 week at room temperature. TMD appears to be stable in all tested solvents. Deviations in ACN dissolution are caused by reduced column compatibility to this solvent.

77 Chapter 3. Results and Discussion

Figure 3.29: Enzymatic hydrolysis of p-nitrophenyl acetate (NPA) to the yellow colored p-nitrophenolate (NP) in buffered solution [167].

3.3 Enzyme Experiments

Enzyme experiments with the model compounds MPH and TMD were analyzed using the developed chiral MEPS-LVI-BKF-GC-MSD method. For a second con- firmation of the results, also the developed HPLC protocols were used. But since HPLC methods were less versatile applicable and experiments were done with dif- ferent substrate concentrations than GC, because of reduced limit of detection and less obvious outcomes, HPLC results from enzyme experiments are shown in the appendices.

3.3.1 Stability and Performance Investigations on Carboxyl- esterase Since carboxylesterase 1A (CES1A) is known to be an important enzyme in drug metabolism and drug activation [54, 56], there is still relatively low information available about its stability and activity ranges. In preliminary experiments stability of recombinant CES was investigated in respect to parameters with possible influence in the laboratory (figure 3.30). All experiments were performed in triplicates. The stability tests were done with p-nitrophenyl acetate (NPA), a substrate for CES. Hydrolysis of the NPA (absorption 395 nm) leads to formation of p-nitro- phenolate (NP), yellow colored with absorption 400 nm (figure 3.29). The change of color allows immediate monitoring of the enzymatic reaction by UV-VIS detec- tion [167]. For experimental approaches, inter-batch variation of the recombinant enzyme was investigated as well as the behavior of the mostly similar CES enzymes CES1b and CES1c [59]. The volume of 1.0 mL substrate, dissolved in phosphate buffer (100 mM, pH 7.4) at concentration 0.35 mM, was transferred to a cuvette and an- alyzed after three minutes equilibration time at 37 ◦C. Immediately after addition of 3.2 µL CES enzyme and careful mixing with a pipette, UV-VIS measurements at 400 nm were started with sample reads in 0.3 - 0.5 min intervals. Two batches CES1b were used and showed different activity curves but same plateau for maximal

78 3.3. Enzyme Experiments

Figure 3.30: Overview of the carboxylesterase stability investigations with p- nitrophenyl acetate (NPA) as CES1 substrate.

NP formation (figure 3.31(a)). Inter-batch comparison of results is only valid with standardized protocols and reference analyses of each batch. In addition, CES1b and CES1c have similar activity profiles for NPA transfor- mation as expected, because they just differ in one amino acid (figure 3.31(b)). CES enzymes show limited temperature sensitivity. This allows working with CES also at temperatures different from 37 ◦C. For other enzymes like cytochrome P450, the temperature range for performance is limited to a few degrees around 37 ◦C. CES showed optimal performance in the range of 35 ◦C to 40 ◦C. But at room temperature still good activity is observed in NPA to NP transformation (figure 3.32). Influence of carboxylesterase activity is obvious with different pH. A narrow range between pH 7 and 7.5 leads to good CES activity. In particular acidic pH decreases the activity of CES significantly (table 3.21). Investigation on influence of thawing cycles revealed a slight decrease in enzyme activity. After 13 freeze and thaw cycles an activity reduction of 12% is detected. Partitioning of the enzyme prevents this activity loss. The enzymes are not sensitive to increased temperature for thawing. Until 50 ◦C (for 5 min) no activity loss is

79 Chapter 3. Results and Discussion

(a)

150 )

M CES 1b batch 1 m ( CES 1b batch 2

n 100 o i t a r t n

e 50 c n o C

0 0 2 4 6 Time (min)

(b)

150 )

M CES 1b m ( CES 1c

n 100 o i t a r t n

e 50 c n o C

0 0 2 4 6 Time (min)

Figure 3.31: (a) Variations in batch activity for CES1b and (b) activity profiles for CES1b and CES1c. All tests were performed in triplicates. The volume of 1.0 mL substrate, dissolved in phosphate buffer (100 mM, pH 7.4) at con- centration 0.35 mM, was transferred to a cuvette and analyzed immediately after addition of CES enzyme (3.2 µL). UV-VIS measurements at 400 nm were started with sample reads in 0.3 - 0.5 min intervals (section 5.4.1).

80 3.3. Enzyme Experiments

observed, but at 70 ◦C no activity is measured anymore. The preliminary tests reveal no important influence on enzyme activity by thaw- ing time (1 - 15 min, data not shown). Generally, it is proposed to thaw enzymes rapidly at 37 ◦C and keep them on ice until further use. Considering literature and obtained results, all experiments with CES enzymes in this work were conducted with same thawing protocols, 5 minutes thawing at 37 ◦C and further holding on ice until use.

Table 3.21: Temperature and pH dependence for CES activity analyzed with NPA. Since the color of formed NP is pH dependent, no value could be obtained for pH 5. With increasing number of thawing the activity decreases, but thawing can be per- formed at elevated temperature without activity loss (n = 3 - 5 for all experiments). reaction activity pH activity thawing activity thawing activity temp. cycles temp. ◦C(µM/min) (µM/min) (n)(µM/min) ◦C(µM/min) 6 13.8 5 no value Control (2) 42.7 37 53.1 10 22.3 6 9.1 8 40.3 40 57.5 15 29.6 7 63.7 13 37.6 45 57.8 20 34.9 7.4 54.8 50 52.2 23 46.4 8 37.4 70 0.3 30 53.8 35 65.0 36 69.1 37 54.8 39 49.6 40 51.1 42 53.3 44 45.8

In summary, CES enzymes are robust and can be used in a simple assay. They are active without any cofactor or NADPH regenerating system at temperatures between 30 - 40 ◦C and P-buffer (100 mM, pH 7.4), but different batches need standard protocols for sample comparison. With enzyme partitioning an additional enzyme loss is prevented.

81 Chapter 3. Results and Discussion

(a) 150 )

M 6 °C m ( 10 °C

n 100 o

i 15 °C t a

r 20 °C t n

e 50 23 °C c n o C

0 0 2 4 6 8 Time (min) (b) 150 )

M 23 °C m ( 30 °C

n 100 o

i 35 °C t a

r 36 °C t n

e 50 c n o C

0 0 2 4 6 Time (min) (c) 150 )

M 37 °C m ( 39 °C

n 100 o

i 40 °C t a

r 42 °C t n

e 50 44 °C c n o C

0 0 1 2 3 4 5 Time (min) Figure 3.32: Overview for temperature dependence of CES as demonstrated in table 3.21; (a) low activity is observed at 6 ◦C, corrected to auto-hydrolysis. With increasing temperature, the activity increases and maximal transforma- tion is reached rapidly; (b) maximal activity is observed between 30 and 36 ◦C; (c) temperature above 40 ◦C leads to loss of activity. The experiments were done by transferring a volume of 1.0 mL substrate, in P-buffer (100 mM, pH 7.4) at concentration 0.35 mM to a cuvette and analyzing immediately after addi- tion of CES enzyme (3.2 µL) by UV-VIS measurements at 400 nm with sample reads in 0.5 min intervals (section 5.4.1).

82 3.3. Enzyme Experiments

3.3.2 Carboxylesterase Metabolism of Model Compounds Methylphenidate Metabolism with CES and GC-MSD Analysis

Proof of concept with MEPS-LVI-BKF-GC-MSD was performed on a standard non- chiral Rtx R column (30 m x 0.25 mm I.D., 0.25 µm film thicknesses). Apart from the oven parameters, same conditions were applied as for later chiral MEPS-LVI- BKF-GC-MSD experiments.

Sample concentrations were chosen to be as near as possible to physiologic plasma concentrations. Since in vivo enzymatic transformation (figure 3.33) occurs gener- ally under constant conditions with higher substrate concentration than enzyme concentration ([S] ≥ 104 x [E]), also for in vitro assays, the condition of higher substrate concentration is important [35]. Different enzyme and substrate concen- trations were investigated for the enzymatic assays. Minimal applicable enzyme concentration for CES experiments in a assay volume of 1.0 mL was 30 nM (corresponding to 1 µL supersome solution). Working concen- trations of 10 ng/mL for the substrate MPH (40 nM) therefore did not lead to reli- able results, but with substrate concentrations above 20 ng/mL (80 nM) successful enzyme reactions were obtained, even the rule of thumb with a substrate concen- tration >> enzyme concentration was not fulfilled. The preliminary experiment for proof of concept was investigated using the lowest substrate concentration for MPH. Chiral analyses with CES and MPH were conducted using a substrate concentra- tion of 100 ng/mL (420 nM). Besides transformation of MPH, also the detection of formed metabolites was of interest. Therefore this higher substrate concentration was chosen. In the case of TMD, plasma concentration is between 100 - 800 ng/mL and enzymatic investigations with CES were done using 100 ng/mL (380 nM) and 30 nM enzyme concentration. For enzymatic investigations with CYP enzymes, higher substrate concentrations were needed for reliable results and excess of substrate was essential. The working conditions for the substrates MPH and TMD were 1.0 µM. CYP2D6 and CYP3A4 were used in concentrations 10 nM and 40 nM, respectively.

Two different approaches were used for the investigation of proof of principle and method establishment. Both approaches contained a MPH sample concen- tration 20 ng/mL and same enzyme experiment conditions, but different analysis methods (figure 3.34). LVI-analysis showed significant enzymatic MPH hydrolysis (figure 3.34(a)). A control sample monitored hydrolysis occurring in aqueous so- lution. After 80 min incubation 87% of MPH is transformed to RA, but a little portion is hydrolyzed in the control sample after 80 minutes (section 5.4.2). Due to additional necessary steps, the second approach for enzymatic transformation just allowed a single result and no regard over time (figure 3.34(b)). The sample was analyzed after 80 min incubation and subsequent RA derivatization (section 5.3.9). Same results were obtained as in the first approach, with remaining MPH in a ratio

83 Chapter 3. Results and Discussion

Figure 3.33: Reaction equation for enzymatic biotransformation, where en- zyme (E) and substrate (S) form a complex for the transformation to the product (P). Rate constants k1 / k−1 and k2 / k−2 determine the velocity of association and dissociation of the enzyme-substrate complex (ES) or enzyme- product complex (PE). As the dissociation of the PE-complex is much more rapid than the transformation of the substrate to the product, it is considered of irrelevant for kinetics and is not particularly mentioned [35].

of 14% (MPH: tR 10.90 min, peak area 136443; RA-tms: tR 11.29 min, peak area 941640).

Methylphenidate Metabolism with CES and Chiral Chromatographic Analysis

Chiral separation of racemic MPH after CES1b metabolism shows stereoselective metabolism for the MPH enantiomers with MEPS-LVI-GC-MSD. (S,S)-MPH is faster metabolized than the (R,R)-enantiomer (figure 3.35). Since CES is a very ac- tive enzyme, already after 60 min stereoselectivity can be observed with GC analysis as it is known from in vivo experiments after more than 2 hours [102]. MEPS-HPLC control experiments were conducted at higher concentrations because of reduced limit of detection (GC: LOD MPH: 0.25 ng/mL, LOD TMD: 20 ng/mL; HPLC: LOD MPH 250 ng/mL, LOD TMD: 500 ng/mL). Metabolism of MPH to RA is confirmed but to a less significant proportion at same time points and therefore enantioselectivity is less obvious. The enzyme is also known to catalyze ester formation. In presence of ethanol, MPH is stereoselectively transformed to ethylphenidate, a drug compound with comparable drug action like MPH [62, 168]. For that purpose investigations were undertaken to check if also this back-reaction to MPH takes place (figure 3.36). In fact, and for the first time experimentally observed to our knowledge, incubation of RA (450 nM) with CES1b (30 nM) and methanol (100 µM) at 37 ◦C led to formation of MPH. In vivo this is of minor concern, because MeOH generally is not present and the formed MeOH rapidly eliminated. But in in vitro experiments it has to be considered in the interpretation of obtained results. The investigated racemic RA sample contained traces of MPH seen as bars in figure 3.37. The control analysis at 90 minutes without enzyme shows same results as for t0 (initial time). Comparing HPLC experiments showed only low MPH formation in presence of CES1b, but formation of RA was observed (appendices figure A.2).

84 3.3. Enzyme Experiments

(a)

5´10 6

6 ) 4´10 M I S ( 3´10 6 a e r a 2´10 6 k a e

P 1´10 6

0

in in in in in m m m m m 0 0 0 0 0 2 4 8 8 l o tr n o C

(b)

Figure 3.34: Metabolism of methylphenidate (20 ng/mL) by CES1b (1 µL) in P-buffer (100 mM, pH 7.4) using two different approaches; (a) MEPS- LVI-BKF-GC-MSD analysis of enzymatic methylphenidate hydrolysis. Af- ter 80 min only 13% of parent compound is left. Control 80 min shows non-enzymatic hydrolysis of methylphenidate. Only a minor part is hy- drolyzed, indicating the successful fast metabolism of methylphenidate by CES1b; (b) chromatogram of second approach with enzymatic methylpheni- date metabolism and ritalinic acid formation using a standard injection (1 µL) and metabolite derivatization (RA-tms). Methylphenidate (tR 10.90 min) and RA-tms (tR 11.29 min) show exclusive RA formation in MPH CES1b metabolism. Peak area of remaining MPH corresponds to 14% of RA-tms peak area, as it is seen in the main experiment figure 3.34(a).

85 Chapter 3. Results and Discussion

2.0´10 7 (R,R)-MPH

) (S,S)-MPH

M 7 I 1.5´10 S ( a e r

a 1.0´10 7 k a e P 5.0´10 6

in in in in m m m m 0 0 0 0 3 6 9 l o tr n o C

Figure 3.35: Chiral metabolism of racemic MPH (100 ng/mL) by CES1b (1 µL) in P-buffer (100 mM, pH 7.4) and chiral MEPS-LVI-GC-MSD analysis (n = 2). (S,S)-MPH is faster hydrolyzed than the (R,R)-enantiomer. According to the slope of the trend lines, already after 60 min tendency in enantiopreference is detected.

Figure 3.36: Enzymatic back-reaction of RA to MPH in presence of MeOH (excess).

Tramadol Metabolism with CES and Chiral Chromatographic Analysis

Tramadol is not known to be a substrate for CES1b, so lack of metabolism was expected with tramadol (100 ng/mL) and CES1b (30 nM) incubation. After 40 and 80 minutes incubation of racemic TMD a slight decrease compared to control t130 is observed but no tendency for enantiopreference (figure 3.38) with MEPS-LVI-GC- MSD. CES1b is a highly active, still insufficient investigated enzyme and its role in TMD metabolism is unclear. According to GC results, TMD is assumed to be a weak substrate for CES1b although it does not have any ester group for hydroly- sis. Unfortunately, there was no obvious new peak arising, so no proof of formed metabolite is available. With MEPS-HPLC, the obtained results could not be re- peated. The used substrate concentrations (10 µg/mL) were chosen according to

86 3.3. Enzyme Experiments

2.5´10 4 (R,R)-MPH

) (S,S)-MPH 2.0´10 4 M I S (

a 1.5´10 4 e r a

k 4 a 1.0´10 e P

5.0´10 3

in in in in m m m m 0 0 0 0 3 6 9 l o tr n o C

Figure 3.37: Formation of MPH in presence of racemic RA (450 nM), MeOH (100 µM) and CES1b (1 µL) dissolved in P-buffer (100 mM, pH 7.4). Already after 60 minutes MPH formation was detected with chiral MEPS-LVI-GC-MSD method (n = 2).

7´10 6 (R,R)-TMD

) (S,S)-TMD 6

M 6´10 I S ( a

e ´ 6

r 5 10 a k a

e 4´10 6 P

3´10 6

in in in in m m m m 0 0 0 0 4 8 3 1 l o tr n o C

Figure 3.38: CES1b metabolism (1 µL) of racemic TMD (100 ng/mL) in P- buffer (100 mM, pH 7.4). Only low decrease is observed but no enantioselective tendency (n = 2).

87 Chapter 3. Results and Discussion

2.0´10 7 (R,R)-MPH

) (S,S)-MPH

M 7 I 1.5´10 S ( a e r

a 1.0´10 7 k a e P 5.0´10 6

in in in in in m m m m m 0 5 0 0 0 4 0 4 0 1 1 2 l o tr n o C

Figure 3.39: Racemic methylphenidate incubation (1.0 µM) with CYP2D6 (5 µL and 30 µL NADPH regenerating system) in P-buffer (100 mM, pH 7.4) and chiral MEPS-LVI-GC-MSD analysis (n = 2). MPH appears to be a weak substrate for CYP2D6 but without clear tendency for enantioselectivity. After 140 min incubation, a significant decrease is observed in comparison to control t200 without any enzyme addition. Control t200 shows water caused hydrolysis. the limit of detection. They did not reflect the plasma concentration of TMD 100 - 800 ng/mL (appendices figure A.3).

3.3.3 Cytochrome P450 Metabolism of Model Compounds CYP2D6 Metabolism of Methylphenidate with Chiral Chromatographic Analysis MPH is mainly metabolized by CES1A1 to ritalinic acid and only to a minor amount by other enzymes to form 6-oxo-MPH and p-hydroxy-MPH (figure 1.6)[36]. CYP2D6 incubation and chiral MEPS-LVI-GC-MSD analysis shows MPH to be a substrate for this enzyme. After 140 min significant reduction of parent compound occurs, compared to control t200, where only hydrolysis takes place (figure 3.39). For successful and reliable enzyme reactions, elevated substrate concentrations were needed (1 µM). At therapeutic plasma concentrations (10 ng/mL or 40 nM) no satisfying results were obtained. Parallel MEPS-HPLC (in duplicate) could not repeat the MEPS-LVI-GC-MSD findings (appendices figure A.4).

88 3.3. Enzyme Experiments

4.0´10 7 (R,R)-TMD 3.0´10 7 ) (S,S)-TMD M I 7 (R,R)-ODT S 2.0´10 (

a (S,S)-ODT

e 7

r 1.0´10 a k

a 6 e 2´10 P 1´10 6 0

in in in in m m m m 0 0 0 0 0 5 0 1 1 2 l o tr n o C

Figure 3.40: Tramadol incubation (1.0 µM) with CYP2D6 (5 µL and 30 µL NADPH regenerating system) in P-buffer (100 mM, pH 7.4) and chiral analysis (n = 2). Enantioselective metabolism is observed with CYP2D6 preference for the (S,S)-enantiomer. Also formed O-desmethyltramadol is significantly increased for (S,S)-ODT.

CYP2D6 Metabolism of Tramadol with Chiral Chromatographic Analysis CYP2D6 stereoselectively metabolizes racemic tramadol according the MEPS-LVI- GC-MSD analysis. After 150 min incubation significant metabolism for both TMD- enantiomers is observed with stereopreference for the (S,S)-enantiomer. Tramadol is stable in aqueous solution, therefore control t200 shows similar results as for initial conditions (figure 3.40). In MEPS-HPLC analyses, same findings were confirmed. Enantioselective and fast metabolism of racemic TMD with CYP2D6 lead to formation of ODT-enantio- mers (M1) in stereopreference for the (S,S)-enantiomer (appendices figure A.5).

CYP3A4 Metabolism of Methylphenidate with Chiral Chromatographic Analysis Enzymatic investigation and analysis with MEPS-LVI-GC-MSD of methylphenidate (1.0 µM) with CYP3A4 does not result in any MPH degradation. Only water caused hydrolysis of MPH is observed (figure 3.41) with linear decrease (R2 = 0.9604). MEPS-HPLC experiments also demonstrate no metabolism of MPH in presence of CYP3A4 (appendices figure A.6).

89 Chapter 3. Results and Discussion

3.0´10 7 (R,R)-MPH

) (S,S)-MPH 7

M 2.5´10 I S ( a

e ´ 7

r 2.0 10 a k a

e 1.5´10 7 P

1.0´10 7

in in in in in m m m m m 0 0 0 0 0 3 6 9 0 2 l o tr n o C

Figure 3.41: Methylphenidate incubation (1.0 µM) with CYP3A4 (10 µL and 30 µL NADPH regenerating system) in P-buffer (100 mM, pH 7.4) and chiral MEPS-LVI-GC-MSD analysis (n = 2). No metabolism occurs. Methylpheni- date is no substrate for CYP3A4.

CYP3A4 Metabolism of Tramadol with Chiral Chromatographic Analysis CYP3A4 has reduced activity compared to CYP2D6 activity, so changes in parent compound are detectable, but enantioselectivity is not manifested over a time of a few hours. CYP3A4 is known to catalyze the formation of N-desmethyltramadol from the parent compound tramadol. Experiments demonstrate this fact. After 90 min in- cubation of racemic tramadol and CYP3A4, enzymatic transformation is observed, but with limited tendency for enantioselectivity (figure 3.42). Also formation of N-desmethyltramadol (NDT) is shown. In t0 samples already small NDT peaks are visible; this is due to needed pre-saturation steps for TMD injections. Observed interactions with the stationary phase lead to reduced sensitivity for tramadol and similar structures. Performing analyses at low concentrations, pre-saturation with the analyte was performed, followed by solvent washing steps indicating no carry- over of the analyte. Subsequent analysis of the sample allows detection of tramadol and the formed metabolite NDT. MEPS-HPLC experiments do not show metabolism of TMD with CYP3A4, also no formed metabolites are visible because of reduced limit of detection (appendices figure A.7).

90 3.3. Enzyme Experiments

2.0´10 7 (R,R)-TMD

) (S,S)-TMD M I 7 (R,R)-NDT

S 1.0´10 (

a (S,S)-NDT e r a k

a 5 e 2´10 P

0

in in in in m m m m 0 0 0 0 6 9 0 1 l o tr n o C

Figure 3.42: Tramadol incubation (1.0 µM) with CYP3A4 (5 µL and 30 µL NADPH regenerating system) in P-buffer (100 mM, pH 7.4) and chiral MEPS- LVI-GC-MSD analysis (n = 2). CYP3A4 catalyzes the N-demethylation of tramadol forming N-desmethyltramadol. A weak stereopreference for (S,S)- TMD is observed, as well as increased formation of the corresponding metabo- lite ((S,S)-NDT).

91 Chapter 4

Conclusions and Outlook

A useful tool for metabolic investigations of xenobiotics is presented. It includes a test mixture for chiral GC columns to check system performance, a method for automated sample preparation as well as chiral or non-chiral LVI-BKF-GC-MSD procedures and HPLC-UV methods. As no useful option for system performance check for chiral CD capillary GC columns was available, in particular for performance checks during analyses of high boiling compounds, a new chiral test mixture was designed, which is suitable for all types of cyclodextrin coated GC columns. It is a mixture of twelve different enantiomer pairs with different functional groups and two high boiling compounds, commercially available in racemic form or in single enantiomeric form. Preparation of the mixture is possible in every laboratory as well as individual addition of extra compounds of proper interest. This test mixture shows long-term stability and is able to fulfill needed purposes for column characterization like inertness, acidic or alkaline behavior, enantioselectivity, state or even identification of α-CD columns. With the development of the new LVI-BKF-GC-MSD methods, particularly high boiling compounds are successfully analyzed and the procedure is suitable for thermo-labile molecules. Chiral separations but also non-chiral analyses can be performed using this method and numerous compounds are analyzable with the same chromatographic parameters. Very low analyte concentrations are detectable (< 1 ng/mL), this is steadily enhanced by MEPS extraction. This also allows a fast and cheap analysis of compounds in water-based matrices, which generally need additional extraction steps prior to analyses with GC. The fact of GC-coupled au- tomated sample preparation makes this new procedure attractive also for further applications, for example environmental or forensic investigations. Thanks to auto- mated sample preparation (MEPS) reduced time consumption and reproducibility with low standard deviations is obtained. Furthermore, all steps can be handled with low volumes. A total consumption of less than 1.0 mL allows performing a complete extraction cycle. Moreover, the MEPS BIN can be re-used for up to 100-times, which has economic and environmental issues. To optimize and enlarge the spectrum of analyte candidates, the MEPS method

92 was expanded with new HPLC methods. Unfortunately, sensitivity was reduced in comparison to the GC approach and for both model compound different chro- matographic methods had to be developed. For the given problem, HPLC did not provide advantages compared to the GC method. However, enzymatic experiments were performed with HPLC, but the results were of limited usefulness. Carboxylesterase 1 enzymes manifest gaining importance with their role as pro- drug activator. For this reason, stability investigations and definition of optimal operating conditions for this enzyme were indispensable, because until today low in- formation is available about the accurate handling of those enzymes. CES1 turned out to be a robust enzyme with amazing working range. It is simply handled, as it does not need co-factors for enzyme activity and shows no particular susceptibility to temperature (23 - 40 ◦C) or number of thawing cycles. But the ability of CES for transesterification needs to be kept in mind for all kind of analyses, particu- larly in vitro experiments. CYP enzymes are more delicate and specific handling protocols must be respected. With the aid of the model compounds methylphenidate and tramadol, three dif- ferent enzyme types (CES1, CYP2D6, CYP3A4) in supersome presentation, were tested to investigate in vitro chiral drug metabolism. Conditions of pregnant women were mimicked with the selection of up-regulated enzymes for metabolic studies. Metabolites were formed, but no new compounds detected. Potential harm of those two drugs for pregnant women or the fetus is considered to be low. But further in- vestigations are needed to declare safe use. Methylphenidate is rapidly metabolized mainly to ritalinic acid, which is not known to have pharmacological effects. But since this compound is significantly lower eliminated than the parent compound, its potential residence time in the fetus is elevated. In addition, the lower fetal pH tends to capture ritalinic acid in the fetus because of its reduced lipophilicity. This results in fetal accumulation of ritalinic acid, what should be avoided. Consequently, only the minimal effective dose and particularly enantiopure drug formulations for methylphenidate are proposed to administrate during pregnancy. The parent com- pound methylphenidate is not assumed to have a direct or acute impact. Tramadol in contrast, appears to be of higher risk for the unborn. No malformations are known or acute administration does not seem to have any impact, but long-term use results in elevated risk for withdrawal symptoms in a newborn, as CYP2D6 expression is significantly elevated during pregnancy and therefore higher amounts of the µ-receptor agonist metabolite O-desmethyl-tramadol ((R,R)-M1) arise. This could be showed with CYP2D6 TMD experiments. Administration of tramadol during labour is not concerned, because of short exposure to the unborn. With the MEPS-LVI-BKF-GC-MSD method further applications are possible. Investigation of additional compounds, particularly racemic NMEs (new molecular entities), allows to obtain in vitro results for drug use in pregnancy. Since clinical trials are difficult to undertake with pregnant women, because of ethical reasons and animal models do not satisfyingly reflect the state of pregnancy in a woman, we present an alternative for drug testing. The MEPS-LVI-BKF-GC-MSD method can

93 Chapter 4. Conclusions and Outlook

be further expanded in the investigation of often prescribed, but not for pregnancy experienced chiral compounds, which are approved in enantiomeric or racemic for- mulations. Enantiomeric formulations are also to be considered because of possible inversion of the enantiomers (e.g. ibuprofen). Experiments with the up-regulated enzymes allow a directed investigation of racemic drug candidates for the formation of metabolites and will provide additional information of these drug compounds during pregnancy. Proposed are drugs for the treatment of psychological disorders (e.g. citalopram), analgesics (e.g. naproxen, tapentadol), gastrointestinal agents (e.g. pantoprazol) or muscle relaxants (e.g. tol- perisone), since these indications are of particular interest. The protocols for enzymatic investigations could be enlarged with further en- zymatic investigations using liver microsomes and further technical adaptations for GC or HPLC, allowing simple, rapid and reproducible analyses. In regard to preg- nant women, also the effective impact of placenta and fetus in the total metabolism could be explored in more detail.

94 Chapter 5

Experimental Section

5.1 General Information

All solvents were purchased in gradient grade or Ph.Eur. quality from Sigma Aldrich, J.T. Baker, Scharlau or Merck and were used without further purification. Com- mercial chemicals or drug compounds were not purified prior to use.

Infrared spectra (IR) were recorded on a Perkin Elmer R Spectrum 100 ATR FTIR instrument, with Perkin Elmer R Spectrum software (version 6.1.0.0038) in a range between 600 and 4000 cm-1. Resonance frequencies are given as wavenumbers in cm-1.

UV − VIS analyses were conducted with a Varian Cary 50 UV-Vis Spectropho- tometer (200 - 800 nm range) equipped with a 18 cells holder transporter or single holder and software Cary WinUV (version 3.00).

Optical rotations were analyzed on a Jasco R P-1020 polarimeter with Jasco R 24 cell 3.5 mm x 100 mm. Obtained angles of rotation are reported as [α]D with con- centration (g/100 mL) and solvent.

Derivatization of compounds and enzyme assays were performed on a VWR digital heatblock. A temperature of 70 ◦C was used for derivatization and 37 ◦C for enzyme assays, unless otherwise noted.

Enrichment and extraction of compounds from water-based samples were obtained with a 100 µL MEPS syringe and C18-BINs, using acetonitrile as elution solvent.

On Alugram R SIL/G/UV 254 nm with 0.20 mm layer from Macherey-Nagel R thin layer chromatography was conducted.

95 Chapter 5. Experimental Section

Exact volumes were obtained with Mettler Toledo R pipettes (ranges 0.5 µL- 10.0 µL, 20.0 µL - 200.0 µL, 100.0 µL - 1000.0 µL)

pH measurements were conducted by a Schott Instruments R LAB 860 pH me- ter with BlueLine 14 electrode.

Water nanopur was obtained with an ELGA Labwater PURELAB Ultra An- alytik (UAL225563) instrument.

Exact weights above 20.0 mg were obtained with a Mettler Toledo R XP 205 DeltaRange balance (81 g / 220 g, d=0.01 mg / 0.1 mg), and masses below 20.0 mg with a Mettler Toledo R MX5 balance (5.1 g, d=1µg).

5.2 Identity of Methylphenidate and Tramadol

Figure 5.1: IR measurement for determination of methylphenidate hydrochlo- ride identity.

96 5.2. Identity of Methylphenidate and Tramadol

Figure 5.2: GC-MSD spectrum of methylphenidate. The five most abundant peaks in the spectrum are m/z 84 (999), 91 (276), 83 (93), 150 (80) and 55 (75).

Figure 5.3: IR measurement for determination of tramadol hydrochloride iden- tity.

97 Chapter 5. Experimental Section

Figure 5.4: GC-MSD spectrum of tramadol. The five most abundant peaks in the spectrum are m/z 58 (999), 263 (42), 57 (33), 59 (31) and 135 (27).

98 5.3. Chromatography

5.3 Chromatography

5.3.1 Gas Chromatography GC-FID Instrumentation

Oven Focus GC Thermo Electron Corporation with SSL inlet Detector FID with Hydrogen 5.0 and compressed air (80% N2, 20% O2) Autosampler AI/AS 3000 autosampler Thermo Fisher Scientific Carrier gas Helium 6.0 Software Chrom-Card Data System (version 2.3.3) Syringe 10 µL syringe from SGE company (cone tip) Solvent Dichloromethane or dichloromethane - hexane (1:1)

GC-MSD Instrumentation

Oven GC Ultra Thermo Fisher Scientific with PTV injector and backflush tool, transfer line 250 ◦C Detector DSQ II Thermo Fischer Scientific mass spectrometer Autosampler TriPlus Autosampler Thermo Fisher Scientific Carrier gas Helium 6.0 Software Xcalibur (version 2.2 SP1.48) MS databases FFNSC 1.3 (Flavors and Fragrances of Natural and Synthetic Com- pounds), Adams EO library, Mass Spectral Library of Drugs, Poi- sonos, Pesticides, Pollutants and Their Metabolites 2011 (Maurer, Hans H. / Pfleger, Karl / Weber, Armin A.); NIST 11 (NIST / EPA / NIH Mass Spectral Library, Scientific Instrument Services) Syringe 1 10 µL syringe from SGE company (cone tip) Syringe 2 100 µL syringe from Thermo Scientific (side hole) Syringe 3 100 µL MEPS syringe with disposable BIN-needle (cone tip) from SGE company

Analytical GC Columns

Non-chiral GC Column

name stationary phase dimension provider Rtx R -5Sil MS Crossbond R 1,4-bis(di- 30 m length, 0.25 mm I.D., Restek methylsiloxy)phenylene 0.25 µm df dimethyl polysiloxane

99 Chapter 5. Experimental Section

Chiral GC Columns name stationary phase dimension provider BGB 173 50% 2,3-diacetyl-6-TBDMS- 30 m length, BGB Analytics α-cyclodextrin in BGB-1701 0.25 mm I.D., matrix 0.25 µm df BGB 174 50% 2,3-diacetyl-6-TBDMS- 30 m length, BGB Analytics β-cyclodextrin in BGB-1701 0.25 mm I.D., matrix 0.25 µm df BGB 175 50% 2,3-diacetyl-6-TBDMS- 30 m length, BGB Analytics γ-cyclodextrin in BGB-1701 0.25 mm I.D., matrix 0.25 µm df BGB 176SE 20% 2,3-dimethyl-6-TBDMS- 30 m length, BGB Analytics β-cyclodextrin in BGB-SE52 0.25 mm I.D., matrix 0.25 µm df BGB 178 20% 2,3-diethyl-6-TBDMS- 30 m length, BGB Analytics β-cyclodextrin in BGB-15 0.25 mm I.D., matrix 0.25 µm df Alpha DexTM 120 20% permethylated α-cyclo- 30 m length, Supleco dextrin, non bonded in SPB- 0.25 mm I.D., 35 poly matrix 0.25 µm df Beta DexTM 120 20% permethylated β-cyclo- 30 m length, Supleco dextrin, non bonded in SPB- 0.25 mm I.D., 35 poly matrix 0.25 µm df Gamma DexTM 120 20% permethylated γ-cyclo- 30 m length, Supleco dextrin, non bonded in SPB- 0.25 mm I.D., 35 poly matrix 0.25 µm df Beta DexTM 325 25% 2,3-dimethyl-6-TBDMS- 30 m length, Supelco β-cyclo-dextrin in SPB-20 0.25 mm I.D., poly matrix 0.25 µm df Chiraldex A-DA 2,6-dipentyl-3-methoxy-α- 30 m length, Astec cyclo-dextrin in not specified 0.32 mm I.D., matrix df not reported Chiraldex A-PH (S)-2-hydroxypropyl- 30 m length, Astec methylether-α-cyclo-dextrin 0.32 mm I.D., in not specified matrix df not reported Chiraldex G-BP 2,6-dipentyl-3-butyryl-γ- 30 m length, Astec cyclodextrin in not specified 0.32 mm I.D., matrix df not reported Chiraldex G-PN 2,6-dipentyl-3-propionyl-γ- 30 m length, Astec cyclodextrin in not specified 0.32 mm I.D., matrix df not reported CP-Chirasil-Dex CB 2,3,6-tri-O-methyl-β-cyclo- 25 m length, Agilent Tech- dextrin chemically bonded to 0.25 mm I.D., nologies polysiloxane 0.25 µm df 100 5.3. Chromatography

5.3.2 HPLC-UV HPLC Instrumentation

Pump VWR Hitachi R L-2130 Autosampler VWR Hitachi R L-2200 UV-detector VWR Hitachi R L-2420 Oven VWR Hitachi R L-2300 Software Agilent Corp., EZChrom Elite R (version 3.2.1, build 3.2.1.31)

Analytical HPLC Columns

Non-chiral HPLC Column

name stationary phase dimension provider LiChrospher R 100 particles of silica with 250 mm length, 4.0 mm Merck RP-18e octadecyl derivative end- I.D., 5 µm particle size capped

Chiral HPLC Column

name stationary phase dimension provider R Chiralpak AGP α1-acid glycoprotein im- 100 mm length, 4.0 mm Supelco mobilized on silica parti- I.D., 5 µm particle size cles

101 Chapter 5. Experimental Section

5.3.3 Chromatographic Methods GC Methods

Chiral GC-FID Method 1 (0.25 mm I.D.)

Injector temperature 250 ◦C Injection mode Split Split ratio 1:25 Injection volume 1.0 µL Carrier mode Constant flow Carrier flow rate 2.5 mL/min Detector temperature 250 ◦C Oven initial temperature 50 ◦C Oven heat rate 2 ◦C/min Oven final temperature 200 ◦C Oven final temperature hold time 15 min

Total run time 90 min

Chiral GC-FID Method 2 (0.32 mm I.D.)

Injector temperature 250 ◦C Injection mode Split Split ratio 1:25 Injection volume 1.0 µL Carrier mode Constant flow Carrier flow rate 3.0 mL/min Detector temperature 250 ◦C Oven initial temperature 50 ◦C Oven heat rate 2 ◦C/min Oven final temperature 200 ◦C Oven final temperature hold time 15 min

Total run time 90 min

102 5.3. Chromatography

Chiral GC-FID Method 3 (test of LVI on GC-FID)

Injector temperature 250 ◦C Injection mode Split Split ratio 1:25 Injection volume 1.0 µL Carrier mode Constant flow Carrier flow rate 2.5 mL/min Detector temperature 250 ◦C Oven initial temperature 90 ◦C Oven heat rate #1 17 ◦C/min Oven intermediate temperature hold time 125 ◦C Oven heat rate #2 1 ◦C/min Oven final temperature 200 ◦C Oven final temperature hold time 5 min

Total run time 82 min

Non-Chiral GC-MSD Method 4 (standard chiral GC-MSD)

PTV Injection mode PTV Split Split flow 41 mL/min Initial temperature 150 ◦C

Carrier Carrier mode constant flow Carrier flow rate 1.5 mL/min

Oven Oven initial temperature 50 ◦C Oven initial temperature hold time 4.0 min Oven heat rate 15 ◦C/min Oven final temperature 220 ◦C Oven final temperature hold time 10.0 min

103 Chapter 5. Experimental Section

Non-Chiral GC-MSD Method 4 (Continuation)

Detector MS mode EI Polarity positive ions Electron energy 70 eV Source temperature 250 ◦C Start time 5.0 min Mass scan range 40 - 400

Autosampler Analysis type single injection mode basic Injector port Injector A (PTV) Synchronization mode standard Sample volume 1.0 µL Vial sampling depth bottom Injection speed 50 µL/s Sample pull up speed 80 µL/s Pre-injection wash solvents 3x 6 µL A (ACN) Post-injection wash solvents 3x 6 µL A (ACN))

Non-Chiral LVI-GC-MSD Method 5

PTV Injection mode Large volume Split flow 50 mL/min Initial temperature 90 ◦C Inject time 1.55 min Vent flow 120 mL/min Evaporation temperature 90 ◦C Evaporation time 1.5 min Transfer rate 10 ◦C/s Transfer temperature 180 ◦C Transfer time 2.0 min Clean rate 14.5 ◦C/s Clean temperature 230 ◦C Clean time 5.0 min

Carrier Carrier mode constant flow Carrier flow rate 1.0 mL/min

104 5.3. Chromatography

Non-Chiral LVI-GC-MSD Method 5 (Continuation)

Oven Oven initial temperature 90 ◦C Oven initial temperature hold time 3.1 min Oven heat rate 17 ◦C/min Oven final temperature 220 ◦C Oven final temperature hold time 3.25 min

Detector MS mode EI Polarity positive ions Electron energy 70 eV Source temperature 250 ◦C Start time 6.8 min Mass scan range 40 - 400 Mass SIM 56, 58 (TMD), 84 (MPH), 86, 91, 120 Autosampler Analysis type single injection mode basic Injector port Injector A (PTV) Synchronization mode standard Sample volume 30 µL Vial sampling depth 90% Injection speed 3 µL/s for (1 s) to 1 µL/s (4 s), total 26.4 s Sample pull up speed 5 µL/s Pre-injection wash solvents 3x 80 µL A (ACN) Post-injection wash solvents 3x 80 µL B (ACN))

Run table External event for virtual autosampler: 0.4 min event #1 on 3.0 min event #1 off Valve for backflushing contol: Prep-run valve #1 switch on 0.8 min valve #1 switch off 3.15 min valve #1 switch on 5.25 min valve #1 switch off 13.90 min valve #1 switch on

Total run time 14.0 min

105 Chapter 5. Experimental Section

Non-Chiral MEPS LVI-GC-MSD Method 6

PTV Injection mode Large volume Split flow 50 mL/min Initial temperature 90 ◦C Inject time 1.55 min Vent flow 120 mL/min Evaporation temperature 90 ◦C Evaporation time 1.5 min Transfer rate 10 ◦C/s Transfer temperature 180 ◦C Transfer time 2.0 min Clean rate 14.5 ◦C/s Clean temperature 230 ◦C Clean time 5.0 min

Carrier Carrier mode constant flow Carrier flow rate 1.0 mL/min

Oven Oven initial temperature 90 ◦C Oven initial temperature hold time 3.1 min Oven heat rate 17 ◦C/min Oven final temperature 220 ◦C Oven final temperature hold time 3.25 min

Detector MS mode EI Polarity positive ions Electron energy 70 eV Source temperature 250 ◦C Start time 6.75 min Mass scan range 40 - 400 Mass SIM 56, 58 (TMD), 84 (MPH), 86, 91, 120

106 5.3. Chromatography

Non-Chiral MEPS LVI-GC-MSD Method 6 (Continuation)

Autosampler Analysis type single injection mode enrichment Injector port Injector C (Waste bottle) Synchronization mode delayed Sample volume 80 µL Vial sampling depth 90% Injection speed 20 µL/s Sample pull up speed 5 µL/s Pre-injection wash solvents 80 µL B (ACN), 80 µL A (water) Post-injection wash solvents 80 µL A (water) Number of enrichments variable (1 - 10)

Virtual autosampler Analysis type single injection mode basic Injector port Injector A (PTV) Synchronization mode standard Sample volume 30 µL Vial sampling depth 90% Injection speed 3 µL/s for (1 s) to 1 µL/s (4 s), total 26.4 s Sample pull up speed 5 µL/s Pre-injection wash solvents none Post-injection wash solvents 2x 80 µL B (ACN), 2x 80 µL A (water), 2x 80 µL C (MeOH), 2x 80 µL A (water)

Run table External event for virtual autosampler: 0.4 min event #1 on 3.0 min event #1 off Valve for backflushing contol: Prep-run valve #1 switch on 0.8 min valve #1 switch off 3.15 min valve #1 switch on 5.25 min valve #1 switch off 13.90 min valve #1 switch on

Total run time 14.0 min

107 Chapter 5. Experimental Section

Chiral LVI-GC-MSD Method 7

PTV Injection mode Large volume Split flow 50 mL/min Initial temperature 90 ◦C Inject time 1.55 min Vent flow 120 mL/min Evaporation temperature 90 ◦C Evaporation time 1.5 min Transfer rate 10 ◦C/s Transfer temperature 180 ◦C Transfer time 2.0 min Clean rate 14.5 ◦C/s Clean temperature 230 ◦C Clean time 5.0 min

Carrier Carrier mode Programmed flow Initial flow value 1.0 mL/min Hold time initial flow value 5.25 min Carrier flow rate 0.5 mL/min/min Final flow value 2.0 mL/min Hold time final flow value 32.0 min

Oven Oven initial temperature 90 ◦C Oven initial temperature hold time 3.1 min Oven heat rate #1 16 ◦C/min Oven intermediate temperature hold time 125 ◦C Oven heat rate #2 3 ◦C/min Oven final temperature 210 ◦C Oven final temperature hold time 7 min

Detector MS mode EI Polarity positive ions Electron energy 70 eV Source temperature 250 ◦C Start time 6.75 min Mass scan range 40 - 400 Mass SIM 56, 58 (TMD), 84 (MPH), 91, 120, 249 (ODT), 263

108 5.3. Chromatography

Chiral LVI-GC-MSD Method 7 (Continuation)

Autosampler Analysis type single injection mode basic Injector port Injector A (PTV) Synchronization mode standard Sample volume 30 µL Vial sampling depth 90% Injection speed 3 µL/s for (1 s) to 1 µL/s (4 s), total 26.4 s Sample pull up speed 5 µL/s Pre-injection wash solvents 3x 80 µL B (ACN) Post-injection wash solvents 3x 80 µL B (ACN)

Run table Valve for backflushing contol: Prep-run valve #1 switch on 0.8 min valve #1 switch off 3.15 min valve #1 switch on 5.25 min valve #1 switch off 40.52 min valve #1 switch on

Total run time 40.62 min

109 Chapter 5. Experimental Section

Chiral MEPS LVI-GC-MSD Method 8

PTV Injection mode Large volume Split flow 50 mL/min Initial temperature 90 ◦C Inject time 1.55 min Vent flow 120 mL/min Evaporation temperature 90 ◦C Evaporation time 1.5 min Transfer rate 10 ◦C/s Transfer temperature 180 ◦C Transfer time 2.0 min Clean rate 14.5 ◦C/s Clean temperature 230 ◦C Clean time 5.0 min

Carrier Carrier mode Programmed flow Initial flow value 1.0 mL/min Hold time initial flow value 5.25 min Carrier flow rate 0.5 mL/min/min Final flow value 2.0 mL/min Hold time final flow value 32.0 min

Oven Oven initial temperature 90 ◦C Oven initial temperature hold time 3.1 min Oven heat rate #1 16 ◦C/min Oven intermediate temperature hold time 125 ◦C Oven heat rate #2 3 ◦C/min Oven final temperature 210 ◦C Oven final temperature hold time 7 min

Detector MS mode EI Polarity positive ions Electron energy 70 eV Source temperature 250 ◦C Start time 6.75 min Mass scan range 40 - 400 Mass SIM 56, 58 (TMD), 84 (MPH), 91, 120, 249 (ODT), 263

110 5.3. Chromatography

Chiral MEPS LVI-GC-MSD Method 8 (Continuation)

Autosampler Analysis type single injection mode enrichment Injector port Injector C (Waste bottle) Synchronization mode delayed Sample volume 80 µL Vial sampling depth 90% Injection speed 20 µL/s Sample pull up speed 5 µL/s Pre-injection wash solvents 80 µL B (ACN), 80 µL A (water) Post-injection wash solvents 80 µL A (water) Number of enrichments variable (1 - 10)

Virtual autosampler Analysis type single injection mode basic Injector port Injector A (PTV) Synchronization mode standard Sample volume 30 µL Vial sampling depth 90% Injection speed 3 µL/s for (1 s) to 1 µL/s (4 s), total 26.4 s Sample pull up speed 5 µL/s Pre-injection wash solvents none Post-injection wash solvents 2x 80 µL B (ACN), 2x 80 µL A (water), 2x 80 µL C (MeOH), 2x 80 µL A (water)

Run table External event for virtual autosampler: 0.4 min event #1 on 3.0 min event #1 off Valve for backflushing contol: Prep-run valve #1 switch on 0.8 min valve #1 switch off 3.15 min valve #1 switch on 5.25 min valve #1 switch off 40.52 min valve #1 switch on

Total run time 40.62 min

111 Chapter 5. Experimental Section

Chiral MEPS LVI-GC-MSD with Drying Step Method 9

PTV Injection mode Large volume Split flow 50 mL/min Initial temperature 90 ◦C Inject time 4.2 min Vent flow 120 mL/min Transfer rate 10 ◦C/s Transfer temperature 180 ◦C Transfer time 2.0 min Clean rate 14.5 ◦C/s Clean temperature 230 ◦C Clean time 5.0 min

Carrier Carrier mode Programmed flow Initial flow value 1.0 mL/min Hold time initial flow value 6.35 min Carrier flow rate 0.5 mL/min/min Final flow value 2.0 mL/min Hold time final flow value 32.0 min

Oven Oven initial temperature 90 ◦C Oven initial temperature hold time 4.2 min Oven heat rate #1 16 ◦C/min Oven intermediate temperature hold time 125 ◦C Oven heat rate #2 3 ◦C/min Oven final temperature 210 ◦C Oven final temperature hold time 7 min

Detector MS mode EI Polarity positive ions Electron energy 70 eV Source temperature 250 ◦C Start time 7.75 min Mass scan range 40 - 400 Mass SIM 56, 58 (TMD), 84 (MPH), 91, 120, 249 (ODT), 263

112 5.3. Chromatography

Chiral MEPS LVI-GC-MSD with Drying Step Method 9 (Continuation)

Autosampler Analysis type single injection mode enrichment Injector port Injector C (Waste bottle) Synchronization mode delayed Sample volume 80 µL Vial sampling depth 90% Injection speed 20 µL/s Sample pull up speed 5 µL/s Pre-injection wash solvents 80 µL B (ACN), 80 µL A (water) Post-injection wash solvents 80 µL A (water) Number of enrichments variable (1 - 10)

Virtual autosampler Analysis type single injection mode basic Injector port Injector A (PTV) Synchronization mode standard Sample volume 30 µL Vial sampling depth 90% Injection speed 3 µL/s for (1 s) to 1 µL/s (4 s), total 26.4 s Sample pull up speed 5 µL/s Pre-injection wash solvents 3x 50 µL D (air) Post-injection wash solvents 2x 80 µL B (ACN), 2x 80 µL A (water), 2x 80 µL C (MeOH), 2x 80 µL A (water)

Run table External event for virtual autosampler: 0.4 min event #1 on 3.0 min event #1 off Valve for backflushing contol: Prep-run valve #1 switch on 0.8 min valve #1 switch off 4.25 min valve #1 switch on 6.35 min valve #1 switch off 41.62 min valve #1 switch on

Total run time 41.72 min

113 Chapter 5. Experimental Section

Automated MEPS with External-Elution Method 10

PTV Injection mode Large volume Split flow 50 mL/min Initial temperature 90 ◦C Inject time 0.3 min Vent flow 50 mL/min Transfer temperature 90 ◦C Transfer time 0.0 min

Carrier Carrier mode constant flow Flow rate 1.0 mL/min

Oven Oven initial temperature 90 ◦C Oven temperature hold time 7.0 min

Detector MS mode EI Polarity positive ions Electron energy 70 eV Source temperature 250 ◦C Start time 6.5 min Mass scan range 40 - 400 Mass SIM 56, 84, 91, 115, 172

Autosampler Analysis type single injection mode enrichment Injector port Injector C (Waste bottle) Synchronization mode delayed Sample volume 80 µL Vial sampling depth 80% Injection speed 20 µL/s Sample pull up speed 5 µL/s Pre-injection wash solvents 80 µL B (ACN), 80 µL A (water) Post-injection wash solvents 80 µL A (water) Number of enrichments variable (1 - 10)

114 5.3. Chromatography

Automated MEPS with External-Elution Method 10 (Continuation)

Virtual autosampler Analysis type single injection mode basic Injector port Injector D (Vial position 300) Synchronization mode standard Sample volume 30 µL Vial sampling depth 90% Injection depth 25 mm Injection speed 5 µL/s Sample pull up speed 5 µL/s Pre-injection wash solvents none Post-injection wash solvents 2x 80 µL B (ACN), 2x 80 µL A (water), 2x 80 µL C (MeOH), 2x 80 µL A (water)

Run table External event for virtual autosampler: 0.4 min event #1 on 3.0 min event #1 off

Total run time 7.0 min

115 Chapter 5. Experimental Section

HPLC methods

Column Test HPLC-UV Method 1

Column LiChrospher R 100 RP-18e Mobile phase 40% (V/V) ACN, isocratic 60% (V/V) water nanopur Flow rate 1.0 mL/min Pressure 150 bar UV detection 254 nm Temperature 40 ◦C Injection volume 20 µL

Total run time 20 min

HPLC-UV Method 2

Column LiChrospher R 100 RP-18e Mobile phase ACN (A) Sodium octane sulfonate (1.36 g) and triethylamine (1.0 mL) in 1000.0 mL water nanopur, adjusted to pH 2.7 with phosphoric acid (B) Flow rate 1.0 mL/min Gradient 0-15 min 20% A : 80% B; 15-35 min 20-40% A : 80-60% B UV detection 209 nm Injection volume 20 µL

Total run time 35 min

Chiral Column Test HPLC-UV Method 3

Column Chiralpak R AGP Mobile phase 99% (V/V) P-buffer 10 mM, pH 7.0 with 1% (V/V) 2-propanol, isocratic Flow rate 0.9 mL/min Pressure 73 bar UV detection 225 nm Temperature 40 ◦C Injection volume 20 µL

Total run time 20 min

116 5.3. Chromatography

Chiral HPLC-UV Method 4

Column Chiralpak R AGP Mobile phase 99.6% (V/V) Amm. ac. buffer 10 mM, pH 5.8 with 0.4% (V/V) 2-propanol, isocratic Flow rate 0.8 mL/min Pressure 68 bar UV detection 210 nm Temperature 25 ◦C Injection volume 10 µL

Total run time 18 min

Chiral HPLC-UV Method 5

Column Chiralpak R AGP Mobile phase 98% (V/V) P-buffer 10 mM, pH 6.3 with 1% (V/V) ACN and 1% (V/V) ethanol, isocratic Flow rate 0.8 mL/min Pressure no data UV detection 210 nm Temperature 25 ◦C Injection volume 20 µL

Total run time 25 min

Chiral HPLC-UV Method 6

Column Chiralpak R AGP Mobile phase 99.25% (V/V) P-buffer 10 mM, pH 6.3 with 0.75% (V/V) ethanol, isocratic Flow rate 0.8 mL/min Pressure no data UV detection 271 nm Temperature 25 ◦C Injection volume 20 µL

Total run time 26 min

117 Chapter 5. Experimental Section

5.3.4 Chiral Test Mixture Determination of Optimal Carrier Flow

Determination of optimal carrier flow at minimal HETP (HETPmin) was performed using methane gas 4.5 purchased from PanGas AG (Switzerland) as analyte and He- lium 6.0 from PanGas AG (Switzerland) as carrier gas with each examined column. GC-crimp-vials (1.5 mL) were filled with methane and analyzed at 50 ◦C and isother- mal conditions for 5 minutes, employing carrier gas flow rates between 0.5 mL/min and 7.0 mL/min. The obtained retention time tR and known column parameters allowed the determination of plate number (N), velocity µ, effective flow rate (F ) and HETP with the following equations. The measured and calculated values for the column Alpha DexTM 120 are noted in table 5.1 and the overlaid chromatograms are illustrated in figure 5.5. The summary of all tested columns is listed in table 5.2.

 t 2 N = 5.54 R (5.1) w0.5 L µ = (5.2) tR

F = πr2µ (5.3)

L HETP = (5.4) N

Table 5.1: Experimental determination of HETP for the chiral column Al- pha DexTM 120.

length diameter flow rate tR N µ eff. flow rate HETP (cm) (cm) (mL/min) (min) (cm/s) (mL/min) (mm) 3000 0.025 0.5 4.207 13569 11.88 0.350 2.211 1.0 2.432 46066 20.56 0.605 0.651 1.5 1.823 46045 27.43 0.807 0.652 2.0 1.502 31232 33.29 0.980 0.961 2.5 1.305 28070 38.31 1.128 1.069 3.0 1.167 22435 42.84 1.261 1.337 3.5 1.067 15758 46.86 1.379 1.904 4.0 0.988 13529 50.61 1.490 2.217 4.5 0.925 11850 54.05 1.591 2.532

118 5.3. Chromatography

Figure 5.5: Chromatograms of methane peaks at flow rates 1.0 mL/min to 3.0 mL/min using chiral column Alpha DexTM 120 in overlaid view.

Table 5.2: Experimental determination of optimal carrier flow rate for different chiral GC columns. column name length diameter optimal flow rate (m) (mm) (mL/min) BGB 173 30 0.25 2.5 BGB 174 30 0.25 2.5 - 3.0 BGB 175 30 0.25 2.5 BGB 176SE 30 0.25 2.0 - 5.0 BGB 178 30 0.25 2.5 - 3.0 Alpha DexTM 120 30 0.25 1.5 - 3.5 Beta DexTM 120 30 0.25 2.5 - 3.0 Gamma DexTM 120 30 0.25 2.5 - 3.0 Beta DexTM 325 30 0.25 2.0 - 4.0 Chiraldex A-DA 30 0.32 2.5 - 3.5 Chiraldex A-PH 30 0.32 2.0 - 3.5 Chiraldex G-BP 30 0.32 3.0 - 6.0 Chiraldex G-PN 30 0.32 3.0 - 6.0 CP-Chirasil-Dex CB 25 0.25 2.5 - 3.5

119 Chapter 5. Experimental Section

Preparation of Chiral Test Mixture Dichloromethane (0.02% water, preserved with about 20 ppm amylene) was pur- chased from J.T. Baker and n-hexane 96% (analytical grade ACS) from Scharlau, (+)-α-pinene, (-)-α-pinene, (+)-β-pinene, (-)-β-pinene, (-)-limonene, (+)-limonene, (-)-linalool, (+)-carvone, (-)-carvone from Fluka (Sigma Aldrich, Switzerland) and (±)-linalool, (±)-γ-valerolactone, (+)-γ-valerolactone, (+)-ethylmandelate, (-)-ethyl- mandelate, (±)-1-phenylethylamine and (±)-α-methylhydrocinnamic acid were pro- vided by Sigma Aldrich (Germany). Racemic methylphenidate hydrochloride and (R,S)-pentobarbital were purchased from Siegfried (Switzerland) and (±)-propylene glycol from Haenseler AG (Switzerland). The chemical structures are illustrated in figure 5.6 Two stock solutions were prepared according to table 5.3 and 5.4. The levorotary- and the dextrorotary-enantiomers were added in different amounts to differentiate them in the chromatogram. The final test mixture used for analysis was prepared by dilution of 1.0 mL stock solution 1 and 5.0 mL stock solution 2 in a 10.0 mL graduated flask and filled to the mark with dichloromethane - n-hexane (1:1). All solutions were stored in common glassware at 4 ◦C.

Table 5.3: Stock solution 1 for column test mixture. compound amount concentration (mg) (mg/mL) (±)-propylene glycol 198.54 9.9 (+)-α-pinene 25.67 1.3 (-)-α-pinene 33.64 1.7 (+)-β-pinene 24.55 1.2 (-)-β-pinene 33.8 1.7 (+)-limonene 23.26 1.2 (-)-limonene 32.97 1.6 (±)-γ-valerolactone 37.00 1.9 (+)-γ-valerolactone 32.22 1.6 (±)-1-phenylethylamine 96.12 4.8 (±)-linalool 30.13 1.5 (-)-linalool 19.59 1.0 (+)-carvone 28.12 1.4 (-)-carvone 42.62 2.1 (+)-ethylmandelate 30.35 1.5 (-)- ethylmandelate 52.04 2.6 (±)-α-methylhydrocinnamic acid 161.09 8.1 (R,S)-pentobarbital 200.31 10.0

The test mixture (table 5.5) was analyzed on all mentioned GC-columns with GC-FID using chiral GC-FID method 1 (columns with I.D. 0.25 mm) or chiral GC-

120 5.3. Chromatography

Table 5.4: Stock solution 2 for column test mixture. compound amount concentration (mg) (mg/mL) (R,S)-methylphenidate HCl 20.06 2.0

FID method 2 (columns with I.D. 0.32 mm) as described in section 5.3.3 and was stored at 4 ◦C.

Table 5.5: Test mixture for chiral GC columns. compound concentration (mg/mL) (±)-propylene glycol 1.0 (+)-α-pinene 0.1 (-)-α-pinene 0.2 (+)-β-pinene 0.1 (-)-β-pinene 0.2 (+)-limonene 0.1 (-)-limonene 0.2 (±)-γ-valerolactone 0.2 (+)-γ-valerolactone 0.2 (±)-1-phenylethylamine 0.5 (±)-linalool 0.2 (-)-linalool 0.1 (+)-carvone 0.1 (-)-carvone 0.2 (+)-ethylmandelate 0.2 (-)- ethylmandelate 0.3 (±)-α-methylhydrocinnamic acid 0.8 (R,S)-pentobarbital 1.0 (R,S)-methylphenidate HCl 1.0

Peak Identification Determination of the test mixture elution order, elution time of each component and control of possible co-elution was performed with the analysis of single-enantiomer- pair solutions in comparable concentrations to the test mixture. All solutions were prepared in a volume of 10.0 mL and further diluted before analysis. The volume of 1.0 mL stock solution was transferred into a 10.0 mL flask and filled to the mark with same solvent (dilution 1:10). In table 5.6 the composition of each solution and the subsequent dilution for analysis is described. All solutions were stored in common glassware at 4 ◦C. After a first analysis of the test mixture, all single enantiomer solutions were analyzed with chiral GC-FID method 1 (columns with I.D. 0.25 mm) or chiral GC-

121 Chapter 5. Experimental Section

Figure 5.6: Structures of test mixture ingredients.

FID method 2 (columns with I.D. 0.32 mm) for peak identification (section 5.3.3). In figure 5.7, the example of enantiomeric separation of methylphenidate and peak identification of its enantiomers is illustrated for the chiral column BGB 176SE, using chiral GC-FID method 1 (section 5.3.3). Separation properties (symmetry, resolution) are calculated according to mentioned equations and elution order was determined for each compound. Same procedure was used for all other examined columns.

Extraction of Dexmethylphenidate The content of 2 capsules Focalin R XR (10 mg, Novartis, Switzerland) was extracted at room temperature with dichloromethane, filtered with Spartan 13/0.2 RC filters from Schleicher & Schuell (Germany) and was investigated with all chiral columns.

122 5.3. Chromatography

Table 5.6: Single 12 solutions as single components for GC peak identification on all columns. sol. compound solvent amount conc. dil. conc. (mg) (mg/mL) (mg/mL) 1 (±)-propylene glycol DCM-hexane 108.11 10.8 1.1 (R,S)-pentobarbital DCM-hexane 101.21 10.1 1.0 2 (+)-α-pinene DCM-hexane 11.37 1.1 0.1 (-)-α-pinene DCM-hexane 16.90 1.7 0.2 3 (+)-β-pinene DCM-hexane 11.08 1.1 0.1 (-)-β-pinene DCM-hexane 17.01 1.7 0.2 4 (+)-limonene DCM-hexane 12.03 1.2 0.1 (-)-limonene DCM-hexane 15.23 1.5 0.2 5 (±)-γ-valerolactone DCM-hexane 20.31 2.0 0.2 (+)-γ-valerolactone DCM-hexane 16.35 1.6 0.2 6 (±)-1-phenylethylamine DCM-hexane 57.55 5.8 0.6 7 (±)-linalool DCM-hexane 17.03 1.7 0.2 (-)-linalool DCM-hexane 15.34 1.5 0.2 8 (+)-carvone DCM-hexane 20.33 2.0 0.2 (-)-carvone DCM-hexane 21.61 2.2 0.2 9 (+)-ethylmandelate DCM-hexane 15.26 1.5 0.2 (-)- ethylmandelate DCM-hexane 22.44 2.2 0.2 10 (±)-α-methylhydrocinnamic acid DCM-hexane 80.54 8.1 0.8 11 (RR,SS)-methylphenidate HCl DCM 20.14 2.0 1.0 12 (R,R)-dexmethylphenidate DCM 10.0 1.0 -

Also the adjuvant E 1505 (triethyl citrate) [169] was extracted (figure 5.7). On several columns its elution time is similar to tR of (±)-α-methylhydrocinnamic acid. The solution was stored at 4 ◦C without further dilution.

123 Chapter 5. Experimental Section

Figure 5.7: (a) Chromatogram of solution 11 (table 5.6), containing racemic methylphenidate, on BGB 176SE with tR for the enantiomers 57.3 min and 57.6 min respectively; (b) Chromatogram of solution 12 (table 5.6) on the same column; dexmethylphenidate, the (R,R)-enantiomer elutes at tR 57.3 min, the adjuvant triethyl citrate, identified with GC-MSD databases, at tR 54.0 min ([169].

124 5.3. Chromatography

5.3.5 LVI-BKF-GC-MSD Methylphenidate hydrochloride (1.0 mg) from Siegfried (Switzerland) was dissolved in 100.0 mL ACN (10.0 µg/mL) and lidocaine hydrochloride (1.0 mg) from H¨anseler AG (Switzerland) was dissolved in 50.0 mL ACN (20.0 µg/mL). Test solutions con- taining 50.0 ng/mL of both analytes or 10.0 ng/mL and 50 ng/mL of analyte in ACN, were analyzed during method development with numerous methods and were finally analyzed with LVI-BKF-GC-MSD method 5 (section 5.3.3).

Methylphenidate hydrochloride (1.0 mg) from Siegfried (Switzerland) was dis- solved in 100.0 mL ACN (10.0 µg/mL). Six dilutions in ACN of this stock solution in the range of 1.0 ng/mL to 60.0 ng/mL were analyzed in triplicates with LVI-BKF- GC-MSD method 5 (section 5.3.3) for calibration and determination of limit of de- tection. Repeatability was tested with a MPH dilution at concentration 40.0 ng/mL with six repetitions. Tramadol hydrochloride (1.00 mg) from Siegfried (Switzerland) was dissolved in 50.0 mL ACN (20.0 µg/mL). Seven dilutions in ACN of this stock solution in the range of 60.0 ng/mL to 370.0 ng/mL were analyzed in triplicates with LVI-BKF- GC-MSD method 5 (section 5.3.3) for calibration and determination of limit of de- tection. Repeatability was tested with a TMD dilution at concentration 60.0 ng/mL with ten repetitions.

For comparison of LVI versus common GC injection two MPH solutions with concentration 1.0 µg/mL and 33.3 ng/mL, dissolved in ACN, were prepared and analyzed with non-chiral GC-MSD method 4 and LVI-BKF-GC-MSD method 5 respectively (section 5.3.3).

5.3.6 MEPS-LVI-BKF-GC-MSD Phosphate buffer (P-buffer, 100 mM, pH 7.4) and water nanopur pH 3.7 were pre- pared for MEPS sample preparation with coupled LVI-BKF-GC-MSD analysis. Exact amounts of 2.68 g potassium phosphate monobasic (KH2PO4) and 11.40 g sodium phosphate dibasic (Na2HPO4) were weighed in a 1000 mL volumetric flask and filled with water nanopur to 950 mL. If necessary, the pH was adjusted with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland) to pH 7.4. The volumetric flask was filled to 1000 mL and stored at 4 ◦C. The P-buffer was used within three days. To a volume of approximately 500 mL H2O nanopur, hydochloric acid (1.0 M) from Sigma-Aldrich (Switzerland) was added drop by drop until pH 3.7. The acidic water was freshly prepared every two days.

Methylphenidate hydrochloride (1.0 mg) from Siegfried (Switzerland) was dis- solved in 50.0 mL water nanopur (pH 3.7) and tramadol hydrochloride (1.0 mg) from

125 Chapter 5. Experimental Section

Siegfried (Switzerland) was dissolved in P-buffer. These stock solutions (20.0 µg/mL) were used for further dilution. The MPH stock solution in acidic water was not used for more than 6 hours.

Extraction efficiency MPH was diluted to 10.0 ng/mL in P-buffer and immediately analyzed with MEPS and the analysis method non-chiral MEPS LVI-GC-MSD method 6 (variation: no dispensation of aspired volume into the waste, but dispensation back into the sam- ple vial) (section 5.3.3). MEPS was performed in triplicates with sampling from the same vial with a sample volume of 1.0 mL. The chosen samplings were 6x, 8x and 10x per MEPS cycle.

Extraction linearity First approach: MPH was diluted to 1.0 ng/mL in P-buffer and immediately ana- lyzed with MEPS and the analysis method non-chiral MEPS LVI-GC-MSD method 6 (section 5.3.3). A calibration for 1 - 10 MEPS cycles was performed in triplicates. Second approach: MPH was diluted to four concentrations: 1.0 ng/mL, 5.0 ng/mL, 10.0 ng/mL and 20.0 ng/mL in P-buffer and immediately analyzed with MEPS 5x sampling and the analysis method non-chiral MEPS LVI-GC-MSD method 6 (sec- tion 5.3.3). The calibration was performed in triplicates.

Repeatability and Calibration MPH was diluted to 10.0 ng/mL in P-buffer and immediately analyzed with 5x MEPS sampling and the analysis method non-chiral MEPS LVI-GC-MSD method 6 (section 5.3.3). With six times repetition, check of repeatability was performed. Calibration with 5x MEPS sampling was performed with five MPH dilutions (P- buffer) in the range of 1.0 ng/mL to 20.0 ng/mL with freshly prepared solutions immediately before analysis. All samples were analyzed in triplicates. Tramadol was diluted to 50.0 ng/mL in P-buffer and analyzed with 5x MEPS sampling and the analysis method non-chiral MEPS LVI-GC-MSD method 6 (sec- tion 5.3.3). With six times repetition, check of repeatability was performed. Cali- bration with 1x MEPS sampling was performed with four TMD dilutions (P-buffer) in the range of 50.0 ng/mL to 300.0 ng/mL and the same chromatographic method. All samples were analyzed in triplicates.

5.3.7 Chiral LVI-BKF-GC-MSD Racemic methylphenidate hydrochloride (0.5 mg) from Siegfried (Switzerland) was dissolved in 50.0 mL ACN (10.0 µg/mL). Twelve dilutions in ACN of this stock solution in the range of 0.5 ng/mL to 20.0 ng/mL were analyzed in triplicates with chiral LVI-BKF-GC-MSD method 7 (section 5.3.3) for calibration and determination of limit of detection. Repeatability was tested with a MPH dilution at concentration 10.0 ng/mL with seven repetitions.

126 5.3. Chromatography

Racemic tramadol hydrochloride (1.0 mg) from Siegfried (Switzerland) was dis- solved in 50.0 mL ACN (20.0 µg/mL). Seven dilutions in ACN of this stock solution in the range of 50.0 ng/mL to 300.0 ng/mL were analyzed in triplicates with chiral LVI-BKF-GC-MSD method 7 (section 5.3.3) for calibration and determination of limit of detection. Repeatability was tested with a TMD dilution at concentration 50.0 ng/mL with five repetitions.

5.3.8 Chiral MEPS-LVI-BKF-GC-MSD

Phosphate buffer (P-buffer, 100 mM, pH 7.4) and water nanopur pH 3.7 were pre- pared for MEPS sample preparation with coupled chiral LVI-BKF-GC-MSD analy- sis. Exact amounts of 2.68 g potassium phosphate monobasic (KH2PO4) and 11.40 g sodium phosphate dibasic (Na2HPO4) were weighed in a 1000 mL volumetric flask and filled with water nanopur to 950 mL. If necessary, the pH was adjusted with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland) to pH 7.4. The volumetric flask was filled to 1000 mL and stored at 4 ◦C. The buffer was used within three days. To a volume of approximately 500 mL H2O nanopur, hydochloric acid (1.0 M) from Sigma-Aldrich (Switzerland) was added drop by drop until pH 3.7. The acidic water was freshly prepared every two days.

Methylphenidate hydrochloride (0.5 mg) from Siegfried (Switzerland) was dis- solved in 50.0 mL water nanopur pH 3.7 (10.0 µg/mL), ibuprofen (0.1 mg) from Soparchim SA (Switzerland) was dissolved in 50.0 mL P-buffer (2.0 µg/mL), pen- tobarbital (1.0 mg) from Siegfried (Switzerland) was dissolved in 50.0 mL P-buffer (20.0 µg/mL), tolperisone (1.0 mg) from TCI Chemicals Europe was dissolved in 50.0 mL P-buffer (20.0 µg/mL), amphetamine sulfate (1.0 mg) from Siegfried (Switzer- land) was dissolved in 50.0 mL P-buffer (20.0 µg/mL), cocaine hydrochloride (1.0 mg) from Siegfried (Switzerland) was dissolved in 50.0 mL P-buffer (20.0 µg/mL) and tra- madol hydrochloride (1.0 mg) from Siegfried (Switzerland) was dissolved in P-buffer (20.0 µg/mL). These stock solutions were used for further dilution. The MPH stock solution in acidic water was not used for more than 6 hours.

A mixture was prepared containing all analytes in a concentration 100 ng/mL, dissolved in P-buffer. The freshly prepared mixture was analyzed with chiral MEPS3x- LVI-GC-MSD method 9 (with drying step) and five repetitions (section 5.3.3). For analyte identification specific masses (m/z) were selected as described in table 5.7.

127 Chapter 5. Experimental Section

Table 5.7: Selected masses (m/z) for identification of the analytes compound specific mass (m/z) amphetamine 44 / 91 tramadol 58 cocaine 82 methylphenidate 84 tolperisone 98 pentobarbital 156 ibuprofen 161

5.3.9 Derivatization for GC

Derivatization and GC-MSD analysis To methylphenidate hydrochloride (0.1 mg) from Siegfried (Switzerland) and rital- inic acid (0.1 mg) from Novartis (Switzerland) a volume of 10.0 µL MSTFA was added. After 40 min at 70 ◦C, the reaction mixture was diluted with 30 µL acetoni- trile and analyzed with non-chiral GC-MSD method 4 (variation: instead of oven heat rate 15 ◦C/min, a oven heat rate of 10 ◦C/min was used (section 5.3.3).

Derivatization after MEPS sample preparation Phosphate buffer (P-buffer, 100 mM, pH 7.4) and water nanopur pH 3.7 were pre- pared for MEPS sample preparation with coupled LVI-BKF-GC-MSD analysis. Exact amounts of 2.68 g potassium phosphate monobasic (KH2PO4) and 11.40 g sodium phosphate dibasic (Na2HPO4) were weighed in a 1000 mL volumetric flask and filled with water nanopur to 950 mL. If necessary, the pH was adjusted with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland) to pH 7.4. The volumetric flask was filled to 1000 mL and stored at 4 ◦C. The buffer was used within three days. To a volume of approximately 500 mL H2O nanopur, hydochloric acid (1.0 M) from Sigma-Aldrich (Switzerland) was added drop by drop until pH 3.7. The acidic water was freshly prepared every two days.

Methylphenidate hydrochloride (1.0 mg) from Siegfried (Switzerland) was dis- solved in 50.0 mL water nanopur (pH 3.7) and ritalinic acid (1.0 mg) from Novartis (Switzerland) was dissolved in P-buffer. These stock solutions (20.0 µg/mL) were used for further dilution. The MPH stock solution in acidic water was not used for more than 6 hours. A dilution containing 100.0 ng/mL of both analytes in P-buffer was analyzed with automated MEPS and external-elution method 10 (section 5.3.3). The MEPS ex- tracted MPH and RA in acetonitrile were then brought to dryness under nitrogen and further dried at 70 ◦C for 5 min. A volume of 10.0 µL MSTFA was added. After

128 5.3. Chromatography

20 min at 70 ◦C the reaction mixture was diluted with 30 µL acetonitrile and ana- lyzed with non-chiral GC-MSD method 4 (section 5.3.3).

Derivatization after metabolism and MEPS sample preparation A 50.0 mL stock solution of MPH hydrochloride (20.0 µg/mL), dissolved in freshly prepared H2O pH 3.7 (section 5.4), was prepared and further diluted to a solution of 1.0 µg/mL in H2O pH 3.7 (Dil-1, 20.0 mL). The sample solution (20 ng/mL) was freshly prepared immediately before enzyme addition by dilution of 200 µL Dil-1 to 10.0 mL with P-buffer. An aliquot of CES1 (25 µL, enzyme content 5 mg/mL, activity 2400 nmol/min/mg) purchased by BD Biosciences - Discovery Labware (Massachusetts, USA) was thawed at 37 ◦C for 5 min and kept on ice until use. Sample solution was prepared and 1.0 mL transferred to a 1.5 mL GC vial (control A). Control A is instantly MEPS extracted (3x sampling) with automated MEPS and external-elution method 10 (section 5.3.3). The MEPS extracted analytes in acetonitrile were then brought to dryness under nitrogen and further dried at 70 ◦C for 5 min. A volume of 10.0 µL MSTFA was added. After 20 min at 70 ◦C, the reaction mixture was diluted with 30 µL acetonitrile and analyzed with non-chiral GC-MSD method 4 (section 5.3.3).

A volume of 1.0 mL sample solution was added to two eppendorff tubes (1,5 mL, polypropylene). The samples in eppendorff tubes were incubated at 37 ◦C for 20 min. After incubation, 3.2 µL of CES1 enzyme were added to one eppendorff tube, gen- tly mixed with a 200 µL pipette and again incubated at 37 ◦C. After incubation of 80 min, the content of the eppendorff tube with enzyme, was transferred to a 1.5 mL GC vial and processed with same conditions as control A. The second control (eppen- dorff tube incubated for 80 min without enzyme) was subsequently processed with automated MEPS and external-elution method 10 (section 5.3.3) and derivatized according to the described procedure.

5.3.10 Stability of Methylphenidate with Non-Chiral HPLC Analysis A column performance check was conducted before sample analysis and after analysis of the last sample. The parabene-mixture was prepared firstly. Methyl parabene (20.71 mg) from Sigma Aldrich (Switzerland) were dissolved in 100 mL methanol-water solution (50:50), ethyl parabene (29.49 mg) from Sigma-Aldrich (Swit-zerland) were dissolved in 100 mL methanol-water solution (50:50), propyl parabene (24.99 mg) from Sigma Aldrich (Switzerland) were dissolved in 100 mL methanol-water solution (50:50) and butyl parabene (23.51 mg) from Sigma Aldrich (Switzerland) were dissolved in 100 mL methanol-water solution (50:50). The volume 10.0 mL of each solution was trans- ferred into a 100.0 mL graduated flask and filled to the mark with methanol-water (50:50). This solution was used for performance check of the column with the HPLC-

129 Chapter 5. Experimental Section

UV method 1 (section 5.3.3).

Stability of methylphenidate hydrochloride from Siegfried (Switzerland) was per- formed by the HPLC analysis of methylphenidate solutions (200 mg/200 mL) dis- solved in methanol, solvent mixture, acetonitrile and water over a time period of three weeks. Solvent mixture was prepared by the mixture of 20 % (V/V) acetonitrile and 80 % (V/V) sodium octane sulfonate (1.36 g, Sigma-Aldrich, Switzerland) and 1.0 mL triethylamine (Sigma-Aldrich, Switzerland) in 1000.0 mL water nanopur, ad- justed to pH 2.7 with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland). HPLC analyses were performed after defined time intervals (section 5.3.10) with HPLC-UV method 2.

5.3.11 Chiral Column Performance Test A column performance check was conducted before sample analysis and after analysis of the last sample. Mobile phase for isocratic LC was prepared by addition of 1% 2-propanol from Sigma-Aldrich (Switzerland) to phosphate buffer (10 mM, pH 7.0). The preparation of 1000.0 mL phosphate buffer 10 mM, pH 7.0 was done by dissolu- tion of 1.550 g Na2HPO4 7H2O (Sigma-Aldrich, Switzerland) and 0.658 g NaH2PO4 2H2O (Sigma-Aldrich, Switzerland) in 950 mL water nanopur, pH adjustment to pH 7.0 with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland) and addition of water nanopur to 1000.0 mL. The buffer was filtered with a PES filter (250 mL, pore size 0.22 µm) from TPP (Switzerland). Pentobarbital (2.055 mg) from Siegfried (Switzerland) was dissolved in 100.0 mL mobile phase and used for column performance check. The analysis of the test solution was performed with chiral column test HPLC-UV method 3 (section 5.3.3).

5.3.12 Chiral HPLC of Methylphenidate System 1: Mobile phase for isocratic LC was prepared by addition of 0.4% 2- propanol from Sigma-Aldrich (Switzerland) to ammonium acetate buffer (10.0 mM, pH 5.8). Water nanopur pH 3.7, for enhanced stability of methylphenidae, was prepared by dropwise addition of hydochloric acid (1.0 M) from Sigma-Aldrich (Switzerland) until pH 3.7. The acidic water was freshly prepared every two days. Preparation of 2000 mL ammonium acetate buffer 10 mM, pH 5.8 was done by dissolution of 1.540 g CH3COONH4 from Sigma-Aldrich (Switzerland) in 1950 mL water nanopur, pH adjustment to pH 5.8 with acetic acid ≥ 99.8 % (Sigma-Aldrich, Switzerland) and addition of water nanopur to 2000 mL. The buffer was filtered with a PES filter (250 mL, pore size 0.22 µm) from TPP (Switzerland).

System 2: The mobile phase for MEPS-HPLC experiments was prepared by ad-

130 5.3. Chromatography

dition of 1% ACN and 1% ethanol from Sigma-Aldrich (Switzerland) to phosphate buffer (10.0 mM, pH 6.3). Preparation of 1000 mL phosphate buffer 10 mM, pH 6.3 was done by dissolution of 1.23 g Na2HPO4 7H2O (Sigma-Aldrich, Switzerland) and 0.574 g NaH2PO4 2H2O (Sigma-Aldrich, Switzerland) in 950 mL water nanopur, pH adjustment to pH 6.3 with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland) and addition of wa- ter nanopur to 1000 mL. The buffer was filtered with a PES filter (250 mL, pore size 0.22 µm) from TPP (Switzerland).

Methylphenidate hydrochloride (1.0 mg) from Siegfried (Switzerland) was dis- solved in 100.0 mL water nanopur pH 3.7 (10.0 µg/mL) and ritalinic acid (1.0 mg) from Novartis (Switzerland) was dissolved in 100.0 mL water nanopur (10.0 µg/mL).

For calibration and determination of the limit of quantification, three MPH stock solutions with concentrations 10 µg/mL each, were prepared for analyses with system 1 and 2. Five dilutions with water pH 3.7, in the range of 0.1 µg/mL to 13.5 µg/mL were prepared and analyzed with the chiral HPLC-UV method 4 and chiral HPLC- UV method 5 (section 5.3.3). Also for ritalinic acid, three stock solutions containing 10.0 µg/mL were prepared and analyzed for calibration and determination of the limit of quantification in the range of 0.02 µg/mL to 18.0 µg/mL with the same mentioned method. All analyses were performed in triplicate.

5.3.13 Chiral HPLC of Tramadol The mobile phase for MEPS-HPLC experiments was prepared by addition of 0.75% ethanol from Sigma-Aldrich (Switzerland) to phosphate buffer (10.0 mM, pH 6.3). Preparation of 1000.0 mL phosphate buffer 10 mM, pH 6.3 was done by dis- solution of 1.23 mg Na2HPO4 7H2O (Sigma-Aldrich, Switzerland) and 0.574 mg NaH2PO4 2H2O (Sigma-Aldrich, Switzerland) in 950 mL water nanopur, pH ad- justment to pH 6.3 with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland) and addition of water nanopur to 1000 mL. The buffer was filtered with a PES filter (250 mL, pore size 0.22 µm) from TPP (Switzerland). Tramadol hydrochloride (5.0 mg) from Cilag (Switzerland) was dissolved in 100.0 mL P-buffer pH 6.3 (50.0 µg/mL) and ritalinic acid (1.0 mg) from Novartis (Switzerland) was dissolved in 100.0 mL water nanopur (10.0 µg/mL). For calibration and determination of the limit of detection, three TMD stock so- lutions with concentrations 10 µg/mL were prepared. Four dilutions with P-buffer pH 6.3, in the range of 1.0 µg/mL to 100.0 µg/mL were prepared and analyzed with the chiral HPLC-UV method 6 (section 5.3.3). All analyses were performed in trip- licate.

Stability of tramadol hydrochloride from Cilag (Switzerland) was performed by

131 Chapter 5. Experimental Section

the HPLC analysis of TMD solutions (200 mg/200 mL) dissolved in P-buffer (10 mM, PH 6.3) and P-buffer (100 mM, pH 7.4), solvent mixture (P-buffer, 10.0 mM, pH 6.3 and 0.75% ethanol), acetonitrile and water over a time period of one week. HPLC analyses were performed after defined time intervals (section 5.3.10) with HPLC-UV method 6.

5.4 Enzyme Experiments

Preparation of Phosphate Buffer 100 mM, pH 7.4, 1000 mL

Exact amounts of 2.68 g potassium phosphate monobasic (KH2PO4) and 11.40 g sodium phosphate dibasic (Na2HPO4) were weighed in a 1000 mL volumetric flask and filled with water nanopur to 950 mL. If necessary, the pH was adjusted with ortho-phosphoric acid 85% (H¨anselerAG, Switzerland) to pH 7.4. The volumetric flask was filled to 1000 mL and stored at 4 ◦C. The buffer was used within three days.

Preparation of water nanopur pH 3.7

To a volume of approximately 500 mL H2O nanopur, hydochloric acid (1.0 M) from Sigma-Aldrich (Switzerland) was added drop by drop until pH 3.7. The acidic water was freshly prepared every two days.

5.4.1 Stability of CES Preparation of 4-Nitrophenyl Acetate Solution 4-Nitrophenyl acetate (4-NPA, 63.08 mg) was dissolved to 10.0 mL methanol for stock solution (35.0 mM). Because of 4-NPA instability, the stock solution was stored at 4 ◦C and freshly prepared every week. The stock solution was diluted immedi- ately before the experiments: 1.0 mL 4-NPA stock solution was diluted to 100.0 mL phosphate buffer (100 mM, pH 7.4, section 5.4). The concentration of 4-NPA - buffer solution for experiments was 0.35 mM.

Preparation of 4-Nitrophenolate Solutions 4-Nitrophenol (4-NP) solutions were prepared for calibration of 4-NP concentrations after enzyme kinetic measurements with UV-VIS spectroscopy. Two stock solutions were prepared: 22.14 mg (stock solution 1, 15.91 mM) and 34.08 mg (stock solution 2, 24.50 mM) of 4-nitrophenol were dissolved separately to 10.0 mL methanol (conc.: 14 mM, 24 mM). Seven further dilutions with phosphate buffer (100 mM, pH 7.4, section 5.4) were prepared in the concentration range 0.03 mM to 7.95 mM. UV-VIS measurements were performed at the maximum absorption of 4-NP at 400 nm (pH 7.4) for calibration with 4-NP and calculated with a linear equation.

132 5.4. Enzyme Experiments

UV-VIS Spectroscopy with Human Carboxylesterase 1

The volume of 1.0 mL substrate, dissolved in phosphate buffer (100 mM, pH 7.4, section 5.4) at concentration 0.35 mM, was transferred to a cuvette and analyzed after three minutes equilibration time at 37 ◦C. Immediately after addition of 3.2 µL CES enzyme (enzyme content 5 mg/mL, activity 2400 nmol/min/mg, kept on ice after thawing at 37 ◦C for 5 min) purchased by BD Biosciences - Discovery Labware (Massachusetts, USA) and careful mixing with a 1000 µL-pipette, UV-VIS measure- ments at 400 nm were started with sample reads in 0.5 min intervals. Temperature dependent activity of human CES1 (3.2 µL) was analyzed at dif- ferent temperatures by adaption of cell holder water bath temperature (6 ◦C, 10 ◦C, 15 ◦C, 20 ◦C, 23 ◦C, 30 ◦C, 35 ◦C, 36 ◦C, 37 ◦C, 39 ◦C, 40 ◦C, 41 ◦C, 42 ◦C, 44 ◦C). At each temperature, the substrate solution (0.35 mM) in the cuvette was equilibrated for three minutes. The analysis at 0 ◦C was performed by immediate analysis of 1.0 mL ice-cold substrate solution and enzyme (3.2 µL) addition. A control cuvette with only substrate was also put on ice. After 160 minutes absorption of 4-NP was measured and compared to the control (freshly prepared substrate solution, 0.35 mM). For the investigation of enzyme activity dependence on pH, phosphate buffers 100 mM with different pH’s were prepared (pH 5.0, 6.0, 7.0, 7.4, 8.0). According to ”Geigy Tabellen” [170] phosphat-buffer stock solutions (100 mM) were prepared and combined for corresponding pH. The resulting pH was checked with the pH meter, no adjustments with acid or base were necessary. 4-NP has pH dependent UV-VIS absorption. Before conducting pH analyses with substrate solution and enzyme, 4-NP analyses were performed with same pH’s. Finally, the 4-NPA stock solution (35 mM) was diluted to 0.35 mM with each buffer, enzyme was added and the reac- tion analyzed in the same manner as described earlier. The obtained absorption was then corrected with the correction factor, from pH-dependent 4-NP absorbance. Examination of enzyme thawing conditions and resulting enzyme activity was done by variable thawing conditions. Enzymes were kept at -80 ◦C (dry ice). CES1 aliquot were thawed at 37 ◦C for 5 min, 10 min and 15 min and subsequently analyzed with the standard protocol for activity analysis mentioned above. Also thawing tem- perature was investigated. The standard protocol was adapted by enzyme thawing of 10 min and changing thawing temperatures (40 ◦C, 45 ◦C, 50 ◦C, 70 ◦C,). The influence of freezing-thawing-cycles on enzyme activity was investigated by repeated thawing (37 ◦C for 5 min) and freezing (-80 ◦C for 2 min) of CES1 aliquot followed by UV-VIS analysis. Analyses were performed after 2, 8 and 13 freeze- thaw-cycles. Enzyme activity of CES1b and CES1c were compared by analysis of both en- zymes with the standard protocol for UV-VIS absorption.

133 Chapter 5. Experimental Section

5.4.2 Methylphenidate Metabolism with CES and GC-MSD Analysis A 50.0 mL stock solution of MPH HCl (20.0 µg/mL), dissolved in freshly prepared H2O pH 3.7 (section 5.4), was prepared and further diluted to a solution of 1.0 µg/mL in H2O pH 3.7 (Dil-1, 20.0 mL). Sample solution (20 ng/mL) was freshly prepared immediately before enzyme addition by dilution of 200 µL Dil-1 to 10.0 mL with phosphate buffer (100 mM, pH 7.4, section 5.4). An aliquot of CES1 (25 µL, enzyme content 5 mg/mL, activity 2400 nmol/min/mg) purchased by BD Biosciences - Discovery Labware (Massachusetts, USA) was thawed at 37 ◦C for 5 min and kept on ice until use. Sample solution was prepared and 1.0 mL transferred to a 1.5 mL GC vial (control A). Control A is instantly MEPS extracted (3x sampling) and analyzed with non-chiral MEPS-LVI-BKF-GC-MSD method 6 (section 5.3.3). A volume of 1.0 mL sample solution each is then added to 5 eppendorff tubes (1,5 mL, polypropylene). The samples in eppendorff tubes are incubated at 37 ◦C for 20 min. After incubation, 3.2 µL of CES1 enzyme are added to 4 eppendorff tubes, gently mixed with a 200 µL pipette and again incubated at 37 ◦C. The content of eppendorff tube 5 is transferred to a 1.5 mL GC vial (control B) and analyzed with same conditions as control A. The tubes containing CES1 are analyzed with non-chiral MEPS-LVI-BKF-GC-MSD method 6 (section 5.3.3) after 20 min, 40 min, 60 min and 80 min incubation time. After 20 min, a further eppendorff tube (1,5 mL, polypropylene) is filled with 1.0 mL sample solution and incubated for 80 min at 37 ◦C (control C). Control C is similarly analyzed with non- chiral MEPS-LVI-BKF-GC-MSD method 6 (section 5.3.3) to monitor water caused hydrolysis of methylphenidate.

5.4.3 Methylphenidate Metabolism with CES and Chiral Chromatographic Analysis A 50.0 mL stock solution of MPH HCl (10.0 µg/mL), dissolved in in freshly prepared H2O pH 3.7 (section 5.4), was prepared and further diluted to a solution of 0.1 µg/mL in H2O pH 3.7 (Dil-1, 10.0 mL). Sample solution (100 ng/mL) was freshly prepared immediately before enzyme addition, by dilution of 100 µL Dil-1 to 10.0 mL with phosphate buffer (100 mM, pH 7.4, section 5.4). An aliquot of CES1 (25 µL, enzyme content 5 mg/mL, activity 2400 nmol/min/mg) purchased by BD Biosciences - Discovery Labware (Massachusetts, USA) was thawed at 37 ◦C for 5 min and kept on ice until use. Sample solution was prepared and 1.0 mL transferred to a 1.5 mL GC vial (control A). Control A is instantly MEPS extracted (1x sampling) and analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 (sec- tion 5.3.3). A volume of 1.0 mL sample solution each is then added to 3 eppendorff tubes (1,5 mL, polypropylene). The samples in eppendorff tubes are incubated at 37 ◦C for 20 min. After incubation, 1.0 µL of CES1 enzyme is added to 2 eppendorff tubes, gently mixed with a 200 µL pipette and again incubated at 37 ◦C. The content

134 5.4. Enzyme Experiments

of eppendorff tube 3 is transferred to a 1.5 mL GC vial (control B) and analyzed with same conditions as control A. The tubes containing CES1 are analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 (section 5.3.3) after 30 min, and 60 min incubation time. After 30 min, a further eppendorff tube (1,5 mL, polypropylene) is filled with 1.0 mL sample solution and incubated for 60 min at 37 ◦C (control C). Then, control C is analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 (section 5.3.3) to monitor water caused hydrolysis of methylphenidate.

5.4.4 Tramadol Metabolism with CES and Chiral Chro- matographic Analysis A 50.0 mL stock solution of TMD HCl (10.0 µg/mL), dissolved in in freshly prepared H2O pH 3.7 (section 5.4), was prepared and further diluted to a solution of 0.1 µg/mL in H2O pH 3.7 (Dil-1, 10.0 mL). Sample solution (100 ng/mL) was freshly prepared immediately before enzyme addition, by dilution of 100 µL Dil-1 to 10.0 mL with phosphate buffer (100 mM, pH 7.4, section 5.4). An aliquot of CES1 (25 µL, enzyme content 5 mg/mL, activity 2400 nmol/min/mg) was thawed at 37 ◦C for 5 min and kept on ice until use. Sample solution was pre- pared and 1.0 mL transferred to a 1.5 mL GC vial (control A). Control A is instantly MEPS extracted (1x sampling) and analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 (section 5.3.3). A volume of 1.0 mL sample solution each is then added to 3 eppendorff tubes (1,5 mL, polypropylene). The samples in eppendorff tubes are incubated at 37 ◦C for 20 min. After incubation, 1.0 µL of CES1 enzyme is added to 2 eppendorff tubes, gently mixed with a 200 µL pipette and again incubated at 37 ◦C. The content of eppendorff tube 3 is transferred to a 1.5 mL GC vial (control B) and analyzed with same conditions as control A. The tubes containing CES1 are analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 (section 5.3.3) after 40 min, and 80 min incubation time. After 40 min, a further eppendorff tube (1,5 mL, polypropylene) is filled with 1.0 mL sample solution and incubated for 80 min at 37 ◦C (control C). Then, control C is analyzed with chiral MEPS-LVI-BKF-GC- MSD method 9 (section 5.3.3) to monitor tramadol stability at 37 ◦C.

5.4.5 CYP2D6 Metabolism of Methylphenidate with Chiral Chromatographic Analysis Recombinant cytochrom P450 enzyme 2D6 supersomes and NADPH regenerating system solutions A and B were purchased by BD Biosciences - Discovery Labware (Massachusetts, USA). Phosphate buffer (100 mM, pH 7.4) was prepared according to the protocol in section 5.4.

Enzyme (component E), NADPH regenerating system solutions A and B (com- ponents B and C) were rapidly thawed at 37 ◦C for 5 min and kept on ice until use. According to table 5.8 components B (25.0 µL) and C (5.0 µL) were pipetted into

135 Chapter 5. Experimental Section

the polypropylene reaction eppendorff tube (1.5 mL) and chilled on ice for 5 min. In a next step, 453.5 µL phosphate buffer was added and the reaction mixture warmed to 37 ◦C for further 5 min. A 50.0 mL stock solution (44 µM, component D) of MPH HCl (0.598 mg), dissolved in phosphate buffer (100 mM, pH 7.4), was prepared im- mediately before adding 11.5 µL to the reaction mixture. The reaction was started by additions of 5.0 µL CYP2D6 (component E) to the eppendorff tube at 37 ◦C. Also a control sample was prepared containing 977 µL phosphate buffer (100 mM, pH 7.4) and 23 µL substrate solution (component D) and was incubated at 37 ◦C for 200 min. Immediately after enzyme addition, 130 µL were withdrawn from the reaction mixture and pipetted to 260.0 µL ice cold water nanopur in a Mini-UniPrepTM RC filter vial (WhatmanTM GE Healthcare Life Sciences, Buckinghamshire, UK) to stop enzyme reaction (sample t0). The filtrate (approximately 270 µL) was transferred to a conical glass insert (300 µL) from BGB Analytik (Switzerland) and analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 performing 3 MEPS cycles (sec- tion 5.3.3). Same procedure was performed after 45 min, 100 min, 140 min and after 200 min for the control sample. Before the analysis of the sample t0 and after the analysis of the control sample, a solvent run (acetonitrile solvent with MEPS 3x) and the chiral MEPS-LVI-BKF- GC-MSD method 9 was conducted.

Table 5.8: Reaction mixture for CYP2D6 metabolism of methylphenidate. component assay reagent final concentration volume (µL) A phosphate buffer (100 mM, pH 7.4) 100 mM 453.5 1.3 mM NADP+ B NADPH regenerating system solution A 3.3 mM G6P 25.0 3.3 mM MgCl2 C NADPH regenerating system solution B 0.4 U/mL G6PDH 5.0 D substrate in phosphate buffer (44 µM) 1.0 µM 11.5 E CYP2D6 (1 pmol/µL) 10.0 pmol/mL 5.0

5.4.6 CYP2D6 Metabolism of Tramadol with Chiral Chro- matographic Analysis Recombinant cytochrom P450 enzyme 2D6 supersomes and NADPH regenerating system solutions A and B were purchased by BD Biosciences - Discovery Labware (Massachusetts, USA). Phosphate buffer (100 mM, pH 7.4) was prepared according to the protocol in section 5.4.

A 50.0 mL stock solution (104.0 µM, component D) of TMD HCl (1.523 mg), dissolved in phosphate buffer (100 mM, pH 7.4), was prepared. Enzyme (component

136 5.4. Enzyme Experiments

E), NADPH regenerating system solutions A and B (components B and C) were rapidly thawed at 37 ◦C for 5 min and kept on ice until use. According to table 5.8 components B (25.0 µL) and C (5.0 µL) were pipetted into the polypropylene reac- tion eppendorff tube (1.5 mL) and chilled on ice for 5 min. In a next step, 460.0 µL phosphate buffer was added and the reaction mixture warmed to 37 ◦C for further 5 min. Then, 11.5 µL of substrate solution (component D) were added to the reac- tion mixture. The reaction was started by additions of 5.0 µL CYP2D6 (component E) to the eppendorff tube at 37 ◦C. Also a control sample was prepared containing 990.0 µL phosphate buffer (100 mM, pH 7.4) and 10.0 µL substrate solution (compo- nent D) and was incubated at 37 ◦C for 200 min. Immediately after enzyme addition, 130 µL were withdrawn from the reaction mixture and pipetted to 260 µL ice cold water nanopur in a Mini-UniPrepTM RC fil- ter vial (WhatmanTM GE Healthcare Life Sciences, Buckinghamshire, UK) to stop enzyme reaction (sample t0). The filtrate (approximately 270 µL) was transferred to a conical glass insert (300 µL) from BGB Analytik (Switzerland) and analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 performing 3 MEPS cycles (sec- tion 5.3.3). Same procedure was performed after 45 min, 100 min, 150 min and after 200 min for the control sample. Before the analysis of the sample t0, between every sample analysis and after the analysis of the control sample, a solvent run (acetonitrile solvent with MEPS 3x) and the chiral MEPS-LVI-BKF-GC-MSD method 9 was conducted.

Table 5.9: Reaction mixture for CYP2D6 metabolism of tramadol. component assay reagent final concentration volume (µL) A phosphate buffer (100 mM, pH 7.4) 100 mM 460.0 1.3 mM NADP+ B NADPH regenerating system solution A 3.3 mM G6P 25.0 3.3 mM MgCl2 C NADPH regenerating system solution B 0.4 U/mL G6PDH 5.0 D substrate in phosphate buffer (100.0 µM) 1.0 µM 5.0 E CYP2D6 (1 pmol/µL) 10.0 pmol/mL 5.0

5.4.7 CYP3A4 Metabolism of Methylphenidate with Chiral Chromatographic Analysis Recombinant cytochrom P450 enzyme 3A4 supersomes and NADPH regenerating system solutions A and B were purchased by BD Biosciences - Discovery Labware (Massachusetts, USA). Phosphate buffer (100 mM, pH 7.4) was prepared according to the protocol in section 5.4.

Enzyme (component E), NADPH regenerating system solutions A and B (com- ponents B and C) were rapidly thawed at 37 ◦C for 5 min and kept on ice until use.

137 Chapter 5. Experimental Section

According to table 5.8 components B (25.0 µL) and C (5.0 µL) were pipetted into the polypropylene reaction eppendorff tube (1.5 mL) and chilled on ice for 5 min. In a next step, 455.0 µL phosphate buffer was added and the reaction mixture warmed to 37 ◦C for further 5 min. A 50.0 mL stock solution (101.0 µM, component D) of MPH HCl (1.368 mg), dissolved in phosphate buffer (100 mM, pH 7.4), was prepared immediately before adding 5.0 µL to the reaction mixture. The reaction was started by additions of 10.0 µL CYP3A4 (component E) to the eppendorff tube at 37 ◦C. Also a control sample was prepared containing 990.0 µL phosphate buffer (100 mM, pH 7.4) and 10.0 µL substrate solution (component D) and was incubated at 37 ◦C for 200 min. Immediately after enzyme addition, 120 µL were withdrawn from the reaction mixture and pipetted to 260.0 µL ice cold water nanopur in a Mini-UniPrepTM RC filter vial (WhatmanTM GE Healthcare Life Sciences, Buckinghamshire, UK) to stop enzyme reaction (sample t0). The filtrate (approximately 260 µL) was transferred to a conical glass insert (300 µL) from BGB Analytik (Switzerland) and analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 performing 3 MEPS cycles (sec- tion 5.3.3). Same procedure was performed after 30 min, 60 min, 90 min and after 200 min for the control sample. Before the analysis of the sample t0 and after the analysis of the control sample, a solvent run (acetonitrile solvent with MEPS 3x) and the chiral MEPS-LVI-BKF- GC-MSD method 9 was conducted.

Table 5.10: Reaction mixture for CYP3A4 metabolism of methylphenidate. component assay reagent final concentration volume (µL) A phosphate buffer (100 mM, pH 7.4) 100 mM 455.0 1.3 mM NADP+ B NADPH regenerating system solution A 3.3 mM G6P 25.0 3.3 mM MgCl2 C NADPH regenerating system solution B 0.4 U/mL G6PDH 5.0 D substrate in phosphate buffer (100 µM) 1.0 µM 5.0 E CYP3A4 (2 pmol/µL) 40.0 pmol/mL 10

5.4.8 CYP3A4 Metabolism of Tramadol with Chiral Chro- matographic Analysis Recombinant cytochrom P450 enzyme 3A4 supersomes and NADPH regenerating system solutions A and B were purchased by BD Biosciences - Discovery Labware (Massachusetts, USA). Phosphate buffer (100 mM, pH 7.4) was prepared according to the protocol in section 5.4.

A 50.0 mL stock solution (102.0 µM, component D) of TMD HCl (1.530 mg), dissolved in phosphate buffer (100 mM, pH 7.4), was prepared. Enzyme (component

138 5.4. Enzyme Experiments

E), NADPH regenerating system solutions A and B (components B and C) were rapidly thawed at 37 ◦C for 5 min and kept on ice until use. According to table 5.8 components B (25.0 µL) and C (5.0 µL) were pipetted into the polypropylene reac- tion eppendorff tube (1.5 mL) and chilled on ice for 5 min. In a next step, 455.0 µL phosphate buffer was added and the reaction mixture warmed to 37 ◦C for further 5 min. Then, 5.0 µL of substrate solution (component D) were added to the reaction mixture. The reaction was started by additions of 5.0 µL CYP3A4 (component E) to the eppendorff tube at 37 ◦C. Also a control sample was prepared containing 990.0 µL phosphate buffer (100 mM, pH 7.4) and 10.0 µL substrate solution (component D) and was incubated at 37 ◦C for 200 min. Immediately after enzyme addition, 120 µL were withdrawn from the reaction mixture and pipetted to 260 µL ice cold water nanopur in a Mini-UniPrepTM RC filter vial (WhatmanTM GE Healthcare Life Sciences, Buckinghamshire, UK) to stop enzyme reaction (sample t0). The filtrate (approximately 260 µL) was transferred to a conical glass insert (300 µL) from BGB Analytik (Switzerland) and analyzed with chiral MEPS-LVI-BKF-GC-MSD method 9 performing 1 MEPS cycle (section 5.3.3). Same procedure was performed after 45 min, 90 min, 140 min and after 150 min for the control sample. Before the analysis of the first sample, between every sample analysis and after the analysis of the control sample, a solvent run (acetonitrile solvent with MEPS 1x) and the chiral MEPS-LVI-BKF-GC-MSD method 9 was conducted.

Table 5.11: Reaction mixture for CYP3A4 metabolism of tramadol. component assay reagent final concentration volume (µL) A phosphate buffer (100 mM, pH 7.4) 100 mM 455.0 1.3 mM NADP+ B NADPH regenerating system solution A 3.3 mM G6P 25.0 3.3 mM MgCl2 C NADPH regenerating system solution B 0.4 U/mL G6PDH 5.0 D substrate in phosphate buffer (100.0 µM) 1.0 µM 5.0 E CYP3A4 (2 pmol/µL) 40.0 pmol/mL 10.0

139 Appendices

A HPLC Experiments

A.1 Saturation of MEPS BIN with 5x MEPS Sampling of TMD

R²

Figure A.1: A saturation of the MEPS-BIN was observed with 5x MEPS sampling of a TMD concentration 150 ng/mL.

140 A.2 CES Metabolism of MPH and Chiral HPLC Analysis

(a)

S R

R

(b)

S R

R

Figure A.2: CES-MPH metabolism analyzed with chiral HPLC-UV; (a) low decrease of MPH after 90 min incubation with CES and (b) observed formation of RA.

141 A.3 CES Metabolism of TMD and Chiral HPLC Analysis

S R

Figure A.3: CES-TMD metabolism is not observed after analysis with chiral HPLC-UV.

A.4 CYP2D6 Metabolism of MPH and Chiral HPLC Anal- ysis

S

R

Figure A.4: No CYP2D6-MPH metabolism is seen after analysis with chiral HPLC-UV.

142 A.5 CYP2D6 Metabolism of TMD and Chiral HPLC Anal- ysis

(a)

S R

R

(b)

S

R

R

Figure A.5: CYP2D6-TMD metabolism analyzed with chiral HPLC-UV; (a) enantioselective metabolism of TMD is observed and (b) formation of M1 metabolite.

143 A.6 CYP3A4 Metabolism of MPH and Chiral HPLC Anal- ysis

S R

Figure A.6: CYP3A4-MPH metabolism could not be demonstrated after anal- ysis with chiral HPLC-UV.

A.7 CYP3A4 Metabolism of TMD and Chiral HPLC Anal- ysis

S R

Figure A.7: CYP3A4-TMD metabolism could not be demonstrated after anal- ysis with chiral HPLC-UV.

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159 Publications and Presentations

Publications

A Multifunctional Test Mixture for Chiral Cyclodextrin GC Columns S. Wyss, I. A. Werner Pharmeuropa Bio & Scientific Notes. 5, 72-86 (2012).

Preparation and Structural Analysis of (plus or minus)-T hreo-Ritalinic Acid S. Wyss, I. A. Werner, S. M. Ametamey, S. Milicevic Sephton Acta Crystallographica Section C Crystal Structure Communications, International Union of Crystallography. 69, 1225-1228 (2013).

Modification of Different IgG1 Antibodies via Glutamine and Lysine Us- ing Bacterial and Human Tissue Transglutaminase T. L. Mindt, V. Jungi, S. Wyss, A. Friedli, G. Pla, I. Novak-Hofer, J¨u.Gr¨unberg, R. Schibli Bioconjugate chemistry, ACS Publications. 19, 271-278 (2007)

Oral and Poster Presentations

Development of Chromatographic Methods for the In V itro Investigation of Chi- ral Drug Metabolism in Pregnant Women, Doktorandentag, Autumn Session 2013, ETH Zurich (CH), September 2013.

Automated Micro Extraction and Large Volume Injection with Gas Chromatography, Pharma Posterday 2013, Zurich (CH), August 2013.

Automated Micro Extraction and Large Volume Injection with Gas Chromatography, Swiss Pharma Science Day 2013, Bern (CH), August 2013.

Chiral and Non-Chiral Separation of Drugs with Automated MEPS-LVI-GC-MSD Tech- nique,

160 38th International Symposium of Capillary Chromatography and 11th GCxGC Sym- posium 2014, Riva del Garda (IT), May 2014.

161