Analysis of medicinal plant phenoloids by coupled tandem mass spectrometry Ph.D. Thesis Balázs Blazics Semmelweis University, Department of Pharmacognosy Semmelweis University Doctoral School of Pharmaceutical Sciences Supervisor: Dr. Ágnes Kéry, Ph.D. Referees: Dr. Mária Báthori, Ph.D., D.Sc. Dr. László Drahos, Ph.D. Chair of exam comittee: Dr. Imre Klebovich, Ph.D., D.Sc. Exam comittee: Dr. Éva Lemberkovics, Ph.D. Dr. Károly Vékey, Ph.D., D.Sc. Budapest 2010 Contents Contents CONTENTS ...................................................................................................................................2 LIST OF ABBREVIATIONS ......................................................................................................4 1. INTRODUCTION ....................................................................................................................6 1.1. PRELIMINARIES ................................................................................................................................6 2. AIMS OF STUDIES ................................................................................................................8 3. BIBLIOGRAPHY REVIEW ..................................................................................................10 3.1. MASS SPECTROMETRY IN PHENOLOID ANALYTICS ..................................................................................10 3.1.1. Phenoloids .........................................................................................................................10 3.1.2. Extraction and sample preparation...................................................................................14 3.1.3. Conditions and instrumentation........................................................................................15 3.1.4. Mass spectrometry of phenolic acids, salicylates and derivatives ....................................17 3.1.5. Mass spectrometry of flavonoid aglycons.........................................................................20 3.1.6. Mass spectrometry of flavonoid glycosides.......................................................................25 3.2. OTHER METHODS COMMONLY USED FOR THE INVESTIGATION OF PHENOLOIDS (HPLC, NMR, IN VITRO ASSAYS ) .........................................................................................................................................................28 3.3. MODEL PLANTS ..............................................................................................................................30 3.3.1. Euphrasia rostkoviana HAYNE (Scrophulariaceae)............................................................31 3.3.2. Satureja hortensis L. (Lamiaceae).....................................................................................32 3.3.3. Filipendula ulmaria L. MAXIM (Rosaceae)........................................................................33 3.3.4. Sempervivum tectorum L. (Crassulaceae) ........................................................................34 3.3.5. Epilobium parviflorum Schreb. (Onagraceae) ..................................................................35 4. MATERIALS AND METHODS ..........................................................................................37 4.1. GENERAL ......................................................................................................................................37 4.1.1. Solvents and chemicals......................................................................................................37 4.1.2. Solid-phase extraction.......................................................................................................37 4.1.3. Liquid chromatography-mass spectrometry apparatus ....................................................38 4.2. EUPHRASIA ROSTKOVIANA HAYNE ......................................................................................................38 4.2.1. Plant material....................................................................................................................38 4.2.2. Extraction, fractionation and isolation..............................................................................38 4.2.3. Content of main groups of compounds .............................................................................39 4.2.4. Antioxidant activity assay .................................................................................................39 4.2.5. High performance liquid chromatographic conditions......................................................40 4.2.6. Triple quadrupole mass spectrometric conditions.............................................................41 4.2.7. Time-of-flight mass spectrometric conditions...................................................................41 4.2.8. NMR spectroscopy, apparatus and conditions..................................................................42 4.2.9. Method validation.............................................................................................................42 4.3. SATUREJA HORTENSIS L. ..................................................................................................................44 4.3.1. Plant material....................................................................................................................44 4.3.2. Extraction and sample preparation...................................................................................44 4.3.3. High performance liquid chromatographic conditions......................................................44 4.3.4. Triple quadrupole mass spectrometric conditions.............................................................45 4.3.5. Testing of method performance........................................................................................45 4.4. FILIPENDULA ULMARIA L. MAXIM ....................................................................................................45 4.4.1. Plant material....................................................................................................................45 4.4.2. Extraction and sample preparation...................................................................................46 4.4.3. High performance liquid chromatographic conditions......................................................46 2 Contents 4.4.4. Triple quadrupole mass spectrometric conditions.............................................................46 4.4.5. Method validation.............................................................................................................47 4.5. SEMPERVIVUM TECTORUM L. ...........................................................................................................48 4.5.1. Plant material....................................................................................................................48 4.5.2. Extraction and sample preparation...................................................................................48 4.5.3. High performance liquid chromatographic conditions......................................................49 4.5.4. Triple quadrupole mass spectrometric conditions.............................................................49 4.6. EPILOBIUM PARVIFLORUM SCHREB . ...................................................................................................49 4.6.1. Plant material....................................................................................................................49 4.6.2. Extraction and sample preparation...................................................................................50 4.6.3. High performance liquid chromatographic conditions......................................................50 4.6.4. Triple quadrupole mass spectrometric conditions.............................................................50 5. RESULTS AND DISCUSSION ...........................................................................................52 5.1. EUPHRASIA ROSTKOVIANA HAYNE ......................................................................................................52 5.1.1. Polyamide fractionation and isolation ..............................................................................52 5.1.2. Antioxidant effect..............................................................................................................53 5.1.3. Qualitative analysis ...........................................................................................................55 5.1.4. Quantitation of acteoside..................................................................................................64 5.1.5. NMR spectroscopy.............................................................................................................65 5.1.6. Quantitative result and method validation .......................................................................66 5.1.7. Summary ...........................................................................................................................68 5.2. SATUREJA HORTENSIS L. ..................................................................................................................68 5.3. FILIPENDULA ULMARIA L. MAXIM ....................................................................................................72 5.3.1. Qualitative analysis ...........................................................................................................72 5.3.2. Quantitative analysis.........................................................................................................77