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.

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