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HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY OF PORPHYRINS, CHLOROPHYLLS AND BILINS.
Lim, Chang Kee
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Últimas novedades química general química industrial
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The porphyrins, chlorophylls, bilins and related tetrapyrroles are vital for all living organisms. Natural and synthetic tetrapyrroles are used extensively in foods, cosmetics, biotechnology, pharmaceuticals, diagnostics and medicine. Methods for their separation and characterization therefore, have a very wide area of applications. Yet, there is a dearth of books dedicated to HPLC and HPLC/MS of tetrapyrroles. Lim addresses this problem admirably by providing practical HPLC and HPLC/MS protocols coupled with in-depth chromatographic and mass spectrometric reference data. These are invaluable in the analysis, identification and characterization of porphyrins, chlorophylls, bilins and other related compounds found in biological and clinical materials. HPLC method development and optimization for coupling to mass spectrometry are also described in rich detail. Sample preparation, and suggestions for avoiding procedural artifacts during extraction of clinical and biological samples are discussed. Clinical biochemists involved in biochemical diagnosis of human porphyrias will find this monograph assuredly helpful, as would analysts, biochemists and chemists involved in the separation, isolation and characterization of natural and synthetic tetrapyrroles. Undoubtedly, Lim has contributed a master-piece containing sufficient background material for beginners and up-to-date references for all researchers in the field. Contents: Structure, Distribution, Biosynthesis and Function High-Performance Liquid Chromatography of Porphyrins Mass Spectrometry of Porphyrins Porphyrin Profiles in Blood, Urine and Faeces by HPLC and HPLC/ESI-MS Isolation and Characterization of Protoporphyrin Glycoconjugates from Harderian Glands of Rodents by HPLC and HPLC/ESI-MS HPLC and HPLC/MS of Chlorophyll and Related Compounds HPLC and HPLC/MS of Bilins of Animal and Plant Origin Future Directions of HPLC and Mass spectrometry of Tetrapyrroles Readership: Analytical biochemists, clinical biochemists, researchers in tetrapyrolle chemistry and biochemistry, plant scientists, pharmaceutical chemists.
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vii Contents Preface ...................................................................................... v Chapter 1. Structure, Distribution, Biosynthesis and Function ........................................................ 1 1.1. Introduction..................................................................... 1 1.2. Nomenclature ................................................................. 2 1.3. Biosynthesis of Porphyrins, Haem and Chlorophyll ......... 3 1.4. Biosynthesis of Bilins in Animals and Plants .................... 9 1.4.1. Degradation of haem to bile pigments................ 9 1.4.2. Biosynthesis of bilins in plants, algae and cyanobacteria .............................................. 11 1.4.3. Degradation of chlorophyll in senescent higher plants....................................................... 13 1.5. Function of Porphyrins and Other Tetrapyrroles ............... 15 Chapter 2. High-Performance Liquid Chromatography of Porphyrins ........................................................ 25 2.1. Introduction..................................................................... 25 2.2. HPLC of Porphyrin Methyl Esters ..................................... 26 2.3. HPLC of Porphyrin Free Acids ......................................... 27 2.3.1. Stationary phases for reversed–phase HPLC of porphyrins ............................................ 28 2.3.2. Mobile phases for reversed–phase HPLC of porphyrins ...................................................... 29 2.3.3. Procedure for optimisation of porphyrin separation by reversed-phase HPLC.................... 32 2.3.4. Retention behaviour of porphyrins in reversed–phase HPLC..................................... 34 2.4 HPLC of Porphyrinogens................................................... 40 Chapter 3. Mass Spectrometry of Porphyrins............................ 51 3.1. Introduction..................................................................... 51 3.2. Fast Atom Bombardment (FAB) Mass Spectrometry of Porphyrins ................................................................... 52 3.3. Laser Desorption/Ionisation Time-of-Flight (LDI-TOF) Mass Spectrometry of Porphyrins .................... 52 3.4 Electrospray Ionisation Mass Spectrometry (ESI-MS) and HPLC/ESI-MS of Porphyrins ....................................... 56 3.4.1. HPLC/ESI-MS of porphyrins ................................ 58 3.4.2. ESI-MS/MS fragmentation of porphyrins.............. 64 3.4.2.1. ESI-MS/MS product ion spectrum and fragmentation pathways of uroporphyrin................... 65 3.4.3. ESI-MS/MS fragmentation of hydroxyuroporphyrins .................................... 65 3.4.3.1. ESI-MS/MS product ion spectrum and fragmentation pathways of meso–hydroxyuroporphyrin............ 67 3.4.3.2. ESI-MS/MS product ion spectrum of hydroxyacetic acid uroporphyrin.... 69 3.4.3.3. ESI-MS/MS product ion spectrum of á-hydroxypropionic acid uroporphyrin ...................................... 71 3.4.3.4. ESI-MS/MS product ion spectrum and fragmentation pathways of â-hydroxypropionic acid uroporphyrin ...................................... 71 viii High-Performance LC and MS of Porphyrins, Chlorophylls and Bilins 3.4.3.5. ESI-MS/MS product ion spectra and fragmentation pathways of cis- and transhydroxyspirolactoneurochlorins........ 74 3.4.3.6. ESI-MS/MS product ion spectra and fragmentation pathways of cis- and transdihydroxyurochlorins........................ 75 3.4.3.7. Characteristic ESI-MS/MS product ions of hydroxyuroporphyrins........... 80 3.4.4. ESI-MS/MS product ion spectra of ketoacid uroporphyrins ................................................... 80 3.4.5. ESI-MS/MS of heptacarboxylic porphyrin.......... 83 3.4.6. ESI-MS/MS of hydroxyheptacarboxylic porphyrins ........................................................ 86 3.4.7. ESI-MS/MS of ketoacid heptacarboxylic porphyrins and formyl heptacarboxylic porphyrin.......................................................... 86 3.4.8. ESI-MS/MS product ion spectrum and fragmentation pattern of coproporphyrin .......... 89 3.4.9. ESI-MS/MS of hydroxymethylcoproporphyrin ..... 93 3.4.10. ESI-MS/MS of â-ketopropionic acid coproporphyrin and formylcoproporphyrin....... 93 3.4.11. ESI-MS/MS product ion spectrum and fragmentation pattern of protoporphyrin ............................................. 93 3.4.12. HPLC/ESI-MS and MS/MS of meso-tetraphenylporphyrin derivatives ............. 97 Chapter 4. Porphyrin Profiles in Blood, Urine and Faeces by HPLC and HPLC/ESI-MS................ 107 4.1. Introduction..................................................................... 107 4.2. Sample Collection and Preparation ................................. 108 4.2.1. Preparation of urine samples .............................. 109 4.2.2. Preparation of faecal sample .............................. 110 Contents ix 4.2.3. Extraction of porphyrins in plasma ..................... 111 4.2.4. Extraction of red blood cell porphyrins............... 112 4.3. Porphyrin Excretion Patterns and Enzyme Assays in the Porphyrias .................................................. 113 4.3.1. Aminolaevulinic acid dehydratase deficiency porphyria (ADP) ................................ 113 4.3.1.1. Determination of ALA and PBG ......... 113 4.3.1.2. Determination of erythrocyte Zn-protoporphyrin by HPLC............... 119 4.3.2. Acute intermittent porphyria (AIP) ...................... 119 4.3.3. Congenital erythropoietic porphyria (CEP)................................................... 123 4.3.4. Porphyria cutanea tarda (PCT) ............................ 127 4.3.4.1. Determination of uroporphyrinogen decarboxylase activity in erythrocytes by HPLC..................... 133 4.3.5. Hereditary coproporphyria (HCP) ....................... 135 4.3.5.1. HPLC assay of coproporphyrinogen oxidase activity in peripheral leucocytes .......................................... 140 4.3.6. Variegate porphyria (VP)..................................... 143 4.3.6.1. Determination of protoporphyrinogen oxidase by HPLC............................................. 144 4.3.7. Erythropoietic protoporphyria (EPP) .................... 146 4.3.7.1. HPLC assay for ferrochelatase in leucocytes or lymphocytes ............. 147 4.3.8. Mixed porphyria ................................................. 149 Chapter 5. Isolation and Characterisation of Protoporphyrin Glycoconjugates from Harderian Glands of Rodents by HPLC and HPLC/ESI-MS.................................. 155 5.1. Introduction..................................................................... 155 5.2. Extraction of Porphyrins from Harderian Glands.............. 159 x High-Performance LC and MS of Porphyrins, Chlorophylls and Bilins 5.3. HPLC Separation of Porphyrins from Rat Harderian Gland Extract .................................................. 160 5.4. Identification of Protoporphyrin Glycoconjugates from Rat Harderian Glands by Capillary Electrophoresis and HPLC/ESI-MS ................................... 162 5.4.1. Characterisation of protoporphyrin xyloside isolated from Harderian glands........................... 166 5.4.2. Characterisation of protoporphyrin monoglucoside isolated from Harderian glands of the rat.................................................. 172 5.5. Origin and Function of Protoporphyrin Glycoconjugates in Harderian Glands of Rodents ........... 173 Chapter 6. HPLC and HPLC/MS of Chlorophyll and Related Compounds ...................................... 177 6.1. Introduction..................................................................... 177 6.2. Reversed-Phase HPLC of Chlorophyll and Related Compounds in Plant Extracts ........................................... 177 6.3. Normal-Phase HPLC of Chlorophyll and Related Compounds..................................................................... 181 6.4. HPLC Separation of Fluorescent and Non–Fluorescent Chlorophyll Catabolites ................................................... 183 6.5. HPLC/MS and MS/MS of Chlorophyll and Related Compounds..................................................................... 185 Chapter 7. HPLC and HPLC/MS of Bilins of Animal and Plant Origin................................................... 191 7.1. Introduction................................................................... 191 7.2. HPLC Separation of Bilirubin IIIá, IXá and XIIIá Isomers .......................................................... 191 7.3. HPLC Separation of Bilirubin Conjugates ...................... 194 7.4. HPLC of Biliverdins ....................................................... 195 7.5. HPLC of Biliviolins ........................................................ 198 7.6. HPLC of Urobilinoids .................................................... 198 Contents xi 7.7. Analysis and Determination of Bile Pigments in Biological and Clinical Samples by HPLC ................. 201 7.7.1. HPLC methods for the determination of conjugated and unconjugated bilirubin in serum............................................. 202 7.7.1.1. Extraction and determination of conjugated and unconjugated bilirubin in serum following trans-esterification ............................ 203 7.7.1.2. Solid-phase extraction (SPE) and determination of conjugated and unconjugated bilirubin in serum ........................................... 204 7.7.1.3. Simultaneous determination of conjugated bilirubin, unconjugated bilirubin and biliprotein or ä-bilirubin by direct injection HPLC................................. 207 7.8. HPLC/ESI-MS and MS/MS of Bile Pigments.................... 208 7.9. Characterisation of Tetrapyrrole Pigments in Avian Eggshells.......................................................... 212 7.9.1. Extraction of pigments from eggshells............... 213 7.10. HPLC and HPLC/MS of Phycobilins............................... 217 7.11. HPLC separation of Phycobiliproteins............................ 217 Chapter 8. Future Directions of HPLC and Mass Spectrometry of Tetrapyrroles............................... 223 Index......................................................................................... 227 xii High-Performance LC and MS of Porphyrins, Chlorophylls and Bilins |
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