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Free haem was isolated from the shell gland of the quail, Coturnix coturnix japonica, and of the fowl, Galinus domesticus, and characterized by HPLC-ESI-MS/MS. Quantification by HPLC gave values of 1.17-1.40 nmol/mg quail shell gland protein for haem, 1.66-2.17 nmol/mg protein for protoporphyrin and 0.25-0.40 nmol/mg protein for biliverdin. Possible implications of this previously unreported finding are discussed but they are not considered incompatible with the conclusion that all eggshell pigments are endogenously synthesized in the oviduct system., (Copyright © 2011 John Wiley & Sons, Ltd.)

The in vitro metabolism of toremifene has been studied in liver microsomal preparations from rat, mouse and human sources using high-performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESIMS). The metabolites detected were N-desmethyltoremifene (m/z 392), 4-hydroxytoremifene (m/z 422), 4'-hydroxytoremifene (m/z 422) and toremifene N-oxide m/z 422). In addition, a new polar metabolite with a protonated molecule at m/z 422 has been detected in all three species. The compound was identified by tandem MS-MS as alpha-hydroxytoremifene, an analogue of alpha-hydroxytamoxifen. The results showed that alpha-hydroxylation is a common feature of tamoxifen and toremifene metabolism and that alpha-hydroxytamoxifen is unlikely to be the reactive metabolite responsible for the hepatocarcinogenesis in rat, as widely believed., (Copyright 2002 John Wiley & Sons, Ltd.)

Congenital erythropoietic porphyria is a rare genetic disorder in which deficiency of uroporphyrinogen III synthase results in excessive production of Type I porphyrins. The main clinical features are severe photodestruction of the skin and haemolytic anaemia. Treatment consists of shielding from light, blood transfusions and splenectomy, but is generally unsatisfactory. Previous studies have suggested that oral charcoal may be of benefit by binding porphyrins in the gut. A trial was therefore undertaken to evaluate this possibility. Porphyrins in urine, plasma and erythrocytes were measured by HPLC in a 23-year-old male patient with congenital erythropoietic porphyria, during an 8 week "run-in" period, and for a further 3 weeks when oral charcoal was given. Total urinary porphyrin excretion was 79-283 mumol/24 h consisting of 75% uroporphyrin I, 15% coproporphyrin I and smaller amounts of hepta-, hexa-, and pentacarboxylic porphyrins. Similar proportions were found in plasma and erythrocytes. During the first 24 h of charcoal administration a minor decrease in plasma and erythrocyte porphyrins was detected but this was not maintained during the remainder of the trial. In bile and faeces coproporphyrin I constituted approximately 95% of the porphyrins, with 2-3% coproporphyrin III and smaller amounts of pentaporphyrins I and III, but only trace amounts of uroporphyrin I. Oral charcoal was of no value in this case. Reasons are discussed in the context of biochemical differences between this patient with classical Gunther's disease and the similar clinical syndrome due to deficiency of uroporphyrinogen decarboxylase.

The enantiomneric separation and the detection of 2-arylpropionic acids after derivatization with the fluorescent reagents with a benzofurazan structure, (S)-(+)-4-(N,N- dimethylaminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-ben zoxadiazole ((S)-DBD-Apy), (R)-(-)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3- benzoxadiazole ((R)-NBD-Apy), 4-N,N-dimethylaminosulphonyl-7-piperazino-2,1,3-benzoxadi zole (DBD-PZ) and N-hydrazinoformylmethyl-N-methylamino-4,4- N,N-dimethylaminosulphonyl-2,1,3-benzoxadiazole (DBD-CO-Hz) by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were examined. The diastereomeric derivatization with (S)-DBD-Apy or (R)-NBD-Apy and the separation on the reversed phase column afforded the high sensitivity. The separation on chiral stationary phase after non-chiral derivatization with DBD-PZ or DBD-CO-Hz provided less sensitivity. The signal-to-noise ratio of (S)-DBD-Apy-(S)-ketoprofen of 200:1 was observed for 12.5 picomole (pmol) injection and selected ion monitoring (SIM) of the quasi-molecular ion after splitting 1:7 before entering into the electrospray ion sources. As a result, the usefulness of these reagents for MS detection has been demonstrated.

In previous work certain hydroxylated and peroxylated derivatives of uroporphyrin (URO) have been isolated from the urine of patients suffering from porphyria. We have now investigated the mechanism of production of these oxygenated derivatives of URO, using both enzymic and chemical model systems and also the effect of exposure to light during reoxidation of uroporphyrinogen (URO'gen). When URO'gen was incubated with haemolysates, peaks with the same retention times as peroxyacetic acid URO, meso-hydroxy URO and beta-hydroxypropionic acid URO were all detected. The first of these was formed in sufficient amounts to allow its characterization by mass spectrometry. Under these conditions, peroxyacetic acid derivatives of heptacarboxylate and pentacarboxylate porphyrins could also be produced from the corresponding porphyrinogens, but no peroxylated product could be obtained from coproporphyrinogen (COPRO'gen, where no acetic acid side chains are present) or from the fully oxidized URO. Similar results were obtained on re-oxidation of URO'gen in the xanthine oxidase-xanthine system and in the presence of hydrogen peroxide/Fe-EDTA (ethylenediamine-tetraacetic acid) and here again no peroxylated product could be detected from either COPRO'gen or URO. Finally, formation of peroxyacetic acid URO could be demonstrated during photo-oxidation of URO'gen and this was followed by light-induced loss of both URO and its peroxylated derivative. It is concluded that the oxygenated derivatives arise from the action of reactive oxygen species on the porphyrinogens (rather than the porphyrins), with one of the acetic acid side chain serving as the preferential (or exclusive target) for peroxylation.

Porphyrin metabolisms in human porphyria cutanea tarda (PCT) and in rats treated with hexachlorobenzene (HCB) have been studied in detail by high performance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC) and liquid secondary ion mass spectrometry (LSIMS). The analyses of porphyrin metabolites in the urine, faeces and liver biopsies of patients with PCT have shown that apart from uroporphyrin I and III and their expected decarboxylation intermediates and products, a complex mixture of many other porphyrins are present. The new porphyrins identified are: meso-hydroxyuroporphyrin III, beta-hydroxypropionic acid uroporphyrin III, hydroxyacetic acid uroporphyrin III, peroxyacetic acid uroporphyrin III, beta-hydroxyproionic acid heptacarboxylic acid porphyrin III, hydroxyacetic acid hepatocarboxylic porphyrin III and peroxyacetic acid pentacarboxylic porphyrin III.

A procedure for the extraction and high performance liquid chromatographic (HPLC) determination of the photodynamic therapeutic agent 5,10,15,20-tetra(m-hydroxyphenyl)chlorin in human, rat and mouse tissues following intravenous administration of the drug is described. The tissue (tumour, skin, muscle and liver) was homogenized and extracted into a mixture of methanol:dimethyl sulphoxide:water (32:8:1 by vol.) containing, 5,10,15,20-tetra(p-hydroxyphenyl)chlorin as the internal standard. The precipitated proteins were removed by centrifugation and the supernatant was separated by reversed phase HPLC on a Hypersil-ODS column with 77% (v/v) acetonitrile in 0.1% trifluoroacetic acid as the mobile phase. The solute was detected with high sensitivity and specificity by a UV-VIS detector set at 423 nm.

A high performance liquid chromatographic method for the determination of the photodynamic chemotherapeutic agent 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) in human plasma following intravenous infusion is described. The procedure involves extraction of the drug in plasma with methanol/dimethyl sulphoxide (4:1 v/v) containing 5,10,15,20-tetra(p-hydroxyphenyl)chlorin as the internal standard and separation on a C18 reversed phase column with acetonitrile:0.1% trifluoroacetic acid (77:23 v/v) as the mobile phase. The drug was detected specifically and sensitively at its absorption maximum of 423 nm with a detection limit of 15 ng/mL (signal-to-noise ratio of 5). The intra- and inter-assay coefficients of variation (CV) on analysis of a plasma spiked with m-THPC (1 micrograms/mL) were 2.3 and 3.4% (n = 6), respectively.

beta-Hydroxypropionic acid- and hydroxyacetic acid-uroporphyrin I have been isolated from the urine of a patient with congenital erythropoietic porphyria by reversed phase high performance liquid chromatography. The compounds were characterized by their chemical behaviour and confirmed by liquid secondary ion mass spectrometry.

A rapid, reliable, sensitive and reproducible HPLC method was developed for the assay of ferrochelatase activity in rat liver. The assay was carried out aerobically with Zn2+ and mesoporphyrin or protoporphyrin IX as substrates. Zn-porphyrins formed were extracted with dimethyl sulphoxide/methanol (30:70, v/v) containing Zn-deuteroporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for mesoporphyrin was 5.9 microM, for protoporphyrin IX 8.8 microM and for zinc 6.0 microM. The specific activities were 33.1 +/- 5.0 nmol Zn-mesoporphyrin or 13.4 +/- 2.0 nmol Zn-protoporphyrin formed per hour per mg of protein for mitochondria and 12.3 +/- 2.2 nmol Zn-mesoporphyrin or 4.6 +/- 0.9 nmol Zn-protoporphyrin per hour per mg of protein for liver homogenate.