Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability
CONTENTS
LIST OF ORIGINAL PUBLICATIONS.................................................................................... 4
ABBREVIATIONS....................................................................................................................... 5
ABSTRACT................................................................................................................................... 7
1 INTRODUCTION...................................................................................................................... 8
2 ADME (absorption, distribution, metabolism and excretion) ............................................... 9
2.1 Intestinal absorption of drugs ........................................................................................... 10
2.1.1 Caco-2 cells for predicting intestinal absorption .................................................... 11
2.2 Metabolism of drugs........................................................................................................... 12
2.2.1 In vitro models in drug metabolism studies ............................................................. 13
2.2.2 In vitro determination of drug interaction and metabolic stability....................... 14
2.3 Increasing throughput in ADME studies by cocktail dosing.......................................... 15
3 LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC/MS)........................ 16
3.1 Sample preparation ............................................................................................................ 17
3.2 Liquid chromatography..................................................................................................... 17
3.3 Mass spectrometry.............................................................................................................. 20
3.3.1 Ionization .................................................................................................................... 20
3.3.2 Mass analysers............................................................................................................ 21
3.4 Liquid chromatography-mass spectrometry in metabolite profiling ............................ 22
4 AIMS OF THE STUDY.......................................................................................................... 24
5 EXPERIMENTAL................................................................................................................... 25
5.1 Chemicals and reagents...................................................................................................... 25
5.2 Recombinant enzymes, hepatocytes and human urine ................................................... 28
5.3 Caco-2 cell culture and permeability experiments .......................................................... 28
5.4 In vitro incubations ............................................................................................................ 29
5.5 Sample pretreatment.......................................................................................................... 29
5.6 LC/MS/MS Instruments .................................................................................................... 30
6 RESULTS AND DISCUSSION .............................................................................................. 31
6.1 Cocktail dosing and n-in-one analysis in permeation and metabolic stability studies. 31
6.1.1 Development of n-in-one LC/MS/MS....................................................................... 31
6.1.2 Transport of compounds across Caco-2 cell monolayer (I,IV).............................. 37
6.1.3 Determination of metabolic clearance (Clint) (II).................................................... 39
6.2 Metabolite profiling of biological matrices (III, V) ......................................................... 42
7 SUMMARY AND CONCLUSIONS...................................................................................... 51
ACKNOWLEDGEMENTS ....................................................................................................... 54
REFERENCES............................................................................................................................ 56 |
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