Development and evaluation of a generic HPLC-tandem MS-screening method for the detection of potential biomarkers for reactive intermediates [Elektronische Ressource] / vorgelegt von Karoline Simon, geb. Scholz

Martin Simon

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• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Development and Evaluation of
a Generic HPLC-Tandem MS Screening Method
for the Detection of Potential Biomarkers for Reactive Intermediates








Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg







vorgelegt von
Karoline Simon, geb. Scholz
aus Bad Königshofen


Würzburg 2006





Eingereicht am:............................................
bei der Fakultät für Chemie und Pharmazie



Gutachter:..............................................
der Dissertation



Prüfer:..............................................
des Öffentlichen Promotionskolloquiums



Tag des Öffentlichen Promotionskolloquiums: .....................................


Doktorurkunde ausgehändigt am: ........................................................






To my dear family Table of Contents - I -


TABLE OF CONTENTS
Table of Contents ...................................................................................................... I
Abbreviations............................................................................................................ V
1 Introduction and Background .............................................................................. 1
1.1 Significance of Reactive Intermediates ........................................................... 1
1.2 Formation of Reactive Intermediates............................................................... 3
1.3 Detoxification ................................................................................................... 10
1.4 Methods for the Generation and Detection of Reactive Intermediates ....... 12
1.4.1 In vitro Methods ......................................................................................... 12
1.4.2 In vivo Methods.......................................................................................... 13
1.4.3 Analytical Methods .................................................................................... 14
1.5 Applicability of a HPLC-MS/MS Screening Method for the Detection of
Mercapturic Acids and L-Lysine Conjugates in Drug Development............ 17
2 Objective .............................................................................................................. 20
3 Materials and Methods........................................................................................ 21
3.1 Instrumentation ................................................................................................ 21
3.2 Chemicals and Reagents ................................................................................. 21
3.3 Syntheses.......................................................................................................... 22
3.3.1 Syntheses of Sulfoxides of Mercapturic Acid Standards ....................... 22
3.3.2 Synthesis of Acetaminophen Mercapturic Acid ...................................... 22
3.3.3 Syntheses of L-Lysine and Acetyl-L-lysine Adduct Standards............... 23
3.4 Mercapturic Acids ............................................................................................ 23
3.4.1 Generation of HPLC-MS/MS Methods....................................................... 23
3.4.1.1 Generation of the IDA CNL/negative EPI Method (method 1)............... 24
3.4.1.2 Generation of the MRM Method for Quantitation of Acetaminophen
Mercapturic Acid (AAP-MA) in Rat Urine (method 2) ............................ 25
3.4.1.3 Generation of the IDA CNL/negative EPI Method after Optimization
(method 3)............................................................................................. 25
3.4.1.4 Generation of the IDA thMRM/negative EPI Method (method 4)........... 26
3.4.1.5 Generation of the IDA CNL/positive EPI Method (method 5) ................ 27
3.4.1.6 Method for Identification of the Isobaric Acetaminophen Metabolites
(method 6) 28 - II - Table of Contents


3.4.2 Sample Preparation.................................................................................... 28
3.4.2.1 Isolation and Preparation of Neutrophils from Human Whole Blood ..... 28
3.4.2.2 Sample Preparation of Microsomal Incubation Solutions and Urine
Samples ................................................................................................ 29
3.4.2.3 Sample Preparation for Determination of LOD with Method 3............... 29
3.4.2.4 eparation for Validation of Method 3 and 4........................... 30
3.4.2.5 Generation of Mercapturic Acids in vitro ............................................... 30
3.4.2.6 Generation of Mercapturic Acids by Rats.............................................. 31
3.4.2.7 rcapturic Acids of Acetaminophen by Humans .......... 32
3.4.3 Determination of DHN-MA and HNE-MA in Rat Urine.............................. 32
3.4.4 Quantitation of Acetaminophen Mercapturic Acid in Rat Urine............. 33
3.4.5 Quanrcapturic Acid in Human Urine....... 33
3.5 L-Lysine Adducts.............................................................................................. 33
3.5.1 Generation of the IDA CNL/negative EPI Method for the Determination
of Acyl Glucuronides................................................................................. 33
3.5.2 Development and Generation of the HPLC-MS/MS Method for the
Determination of L-Lysine Conjugates (method 7) ................................. 34
3.5.3 Generation of HPLC-MS/MS Method for the Detection of L-Lysine
Adducts on an API 3000 (method 8)......................................................... 35
3.5.4 Sample Preparation.................................................................................... 35
3.5.4.1 Sample Preparation for the Determination of Sensitivity of L-Lysine
Adducts ................................................................................................. 35
3.5.4.2 Generation of L-Lysine Adducts in Rat Liver Microsomes ..................... 36
3.5.4.3 Generation of L-Lysine Adducts in Human Urine Samples.................... 36
4 Results.................................................................................................................. 38
4.1 Mercapturic Acids ............................................................................................ 38
4.1.1 Analytical Method Development ............................................................... 38
4.1.1.1 Ionization and Fragmentation Pattern of Mercapturic Acid Standards and
their Sulfoxides obtained with MS/MS Techniques ............................... 38
4.1.1.2 Optimization of HPLC Conditions.......................................................... 43
4.1.1.3 Optimization of Source and Compound-Dependent Parameters .......... 45
4.1.1.4 Development of the IDA CNL/negative EPI Method (method 3)............ 46
4.1.1.5 Generation of the IDA thMRM/negative EPI Method (method 4)........... 49
4.1.1.6 Generation of the IDA CNL/positive EPI Method (method 5) ................ 50
4.1.2 Comparison of Sensitivity of the Methods............................................... 50
4.1.3 Validation of the Methods.......................................................................... 54