Characterization of Hg-phytochelatins complexes in vines (Vitis vinifera cv Malbec) as defense mechanism against metal stress

An approach to understand vines (Vitis vinifera) defense mechanism against heavy metal stress by isolation and determination of Hg-phytochelatins (PCs) complexes was performed. PCs are important molecules involved in the control of metal concentration in plants. PCs complex toxic metals through ?SH groups and stores them inside cells vacuole avoiding any toxic effect of free metals in the cytosol. The Hg-PCs identification was achieved by determination of Hg and S as hetero-tagged atoms. A method involving two-dimensional chromatographic analysis coupled to atomic spectrometry and confirmation by tandem mass spectrometry is proposed. An approach involving size exclusion chromatography coupled to inductively coupled plasma mass spectrometry on roots, stems, and leaves extracts describing Hg distribution according to molecular weight and sulfur associations is proposed for the first time. Medium–low molecular weight Hg–S associations of 29–100 kDa were found, suggesting PCs presence. A second approach employing reversed-phase chromatography coupled to atomic fluorescence spectrometry analysis allowed the determination of Hg-PCs complexes within the mentioned fractions. Chromatograms showed Hg-PC₂, Hg-PC₃ and Hg-PC₄ presence only in roots. Hg-PCs presence in roots was confirmed by ESI–MS/MS analysis.     

An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis

Phytochelatins (PCs) are peptides that function in heavy-metal chelation and detoxification in plants and fungi. A recent study showed that PCs have the ability to undergo long-distance transport in a root-to-shoot direction in transgenic Arabidopsis (Arabidopsis thaliana). To determine whether long-distance transport of PCs can occur in the opposite direction, from shoots to roots, the wheat (Triticum aestivum) PC synthase (TaPCS1) gene was expressed under the control of a shoot-specific promoter (CAB2) in an Arabidopsis PC-deficient mutant, cad1-3 (CAB2TaPCS1/cad1-3). Analyses demonstrated that TaPCS1 is expressed only in shoots and that CAB2TaPCS1/cad1-3 lines complement the cadmium (Cd) and arsenic metal sensitivity of cad1-3 shoots. CAB2TaPCS1/cad1-3 plants exhibited higher Cd accumulation in roots and lower Cd accumulation in shoots compared to wild type. Fluorescence HPLC coupled to mass spectrometry analyses directly detected PC2 in the roots of CAB2:TaPCS1/cad1-3 but not in cad1-3 controls, suggesting that PC2 is transported over long distances in the shoot-to-root direction. In addition, wild-type shoot tissues were grafted onto PC synthase cad1-3 atpcs2-1 double loss-of-function mutant root tissues. An Arabidopsis grafting technique for mature plants was modified to obtain an 84% success rate, significantly greater than a previous rate of approximately 11%. Fluorescence HPLC-mass spectrometry showed the presence of PC2, PC3, and PC4 in the root tissue of grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots, demonstrating that PCs are transported over long distances from shoots to roots in Arabidopsis.     

Complexation of Hg with phytochelatins is important for plant Hg tolerance

Three-week-old alfalfa (Medicago sativa), barley (Hordeum vulgare) and maize (Zea mays) were exposed for 7 d to 30 μm of mercury (HgCl(2) ) to characterize the Hg speciation in root, with no symptoms of being poisoned. The largest pool (99%) was associated with the particulate fraction, whereas the soluble fraction (SF) accounted for a minor proportion (<1%). Liquid chromatography coupled with electro-spray/time of flight mass spectrometry showed that Hg was bound to an array of phytochelatins (PCs) in root SF, which was particularly varied in alfalfa (eight ligands and five stoichiometries), a species that also accumulated homophytochelatins. Spatial localization of Hg in alfalfa roots by microprobe synchrotron X-ray fluorescence spectroscopy showed that most of the Hg co-localized with sulphur in the vascular cylinder. Extended X-ray Absorption Fine Structure (EXAFS) fingerprint fitting revealed that Hg was bound in vivo to organic-S compounds, i.e. biomolecules containing cysteine. Albeit a minor proportion of total Hg, Hg-PCs complexes in the SF might be important for tolerance to Hg, as was found with Arabidopsis thaliana mutants cad2-1 (with low glutathione content) and cad1-3 (unable to synthesize PCs) in comparison with wild type plants. Interestingly, high-performance liquid chromatography-electrospray ionization-time of flight analysis showed that none of these mutants accumulated Hg-biothiol complexes.     

Degradation of procyanidins by Aspergillus fumigatus: identification of a novel aromatic ring cleavage product

Aspergillus fumigatus was able to grow on apple-purified procyanidins (PCs). PCs concentration decreased 30% over the first 60 h of liquid fermentation. The mean degree of polymerization (DPn) of apple-purified PCs increased from 8 to 15 during the fermentation. A fungal enzyme extract from the liquid fermentation was used to study procyanidin B2 [(-)-epicatechin-(4beta-8)-(-)-epicatechin] degradation. The major degradation product (PB2-X) had a retention time of 10.5 min and a molecular mass at m/z 609. High-performance liquid chromatography/multiple fragment mass spectrometry (HPLC/MS(n)) was used for the structural characterization of PB2-X as well as that of thiolysis-treated PB2-X. Twelve fragment ions at m/z 565, 547, 457, 439 (two fragment ions), 421, 413, 377, 395, 351, 287 and 277 were completely identified. It was therefore deduced that the terminal unit of procyanidin B2 dimer was modified by an oxygenase from A. fumigatus leaving the extension unit intact. In addition, FT-IR analysis confirmed a lactone formation in (-)-epicatechin moiety involved in oxidative degradation. Two reaction schemes were postulated for the interpretation of the results.     

Measurement of anti-cancer agent methoxyamine in plasma by tandem mass spectrometry with on-line sample extraction

In this work, we present the development and validation of a tandem mass spectrometry method for the quantitative determination of methoxyamine (CH3ONH2), a potential new chemotherapeutic agent, in human and mouse plasma. Methoxyamine together with the internal standard (I.S.) methoxyl-D3-amine was directly derivatized in plasma sample with a novel chemical agent 4-(N,N-diethylamino)benzaldehyde. The product solution was injected into an on-line Oasis HLB extraction column (2.1 mm x 20 mm) for analyte extraction. After the elution of extractives, the derivatized analytes were monitored by the positive-electrospray-ionization mass spectrometry (ESI-MS-MS). The structures of derivatized analytes were elucidated by fragmentation. Quantitation of plasma methoxyamine was carried out by the multiple reaction monitoring (MRM) mode. This method had a linear calibration range of 1.00-1000 ng/ml with a correlation coefficient of 0.9999 for methoxyamine in both human and mouse plasma. The limit of detection (LOD) and limit of quantification (LOQ) for methoxyamine in plasma were 0.150 and 0.500 ng/ml, respectively. It was demonstrated that the method had high recovery and accuracy (90.1-94.7 and 90.1-96.3%), as well as excellent intra- and inter-assay precision (2.2 and 3.7%), at three concentration levels (5.00, 50.0, 500 ng/ml). This method has been used to analyze the plasma levels of methoxyamine in samples obtained from male CD1 mice after bolus intraperitoneal injection of 2, 5 and 20mg methoxyamine hydrochloride (CH3ONH2.HCl) per kilogram mouse.     

Analysis of glycerophosphocholine molecular species as derivatives of 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin.

A method has been developed for the analysis of derivatized diradylglycerols obtained from glycerophosphocholine (GPC) of transformed murine bone marrow-derived mast cells that provided high performance liquid chromatography (HPLC) separation of GPC subclasses and molecular species separation with on-line quantitation using UV detection. In addition, the derivatized diradylglycerol species were unequivocably identified by continuous flow fast-atom bombardment mass spectrometry. GPC was initially isolated by thin-layer chromatography (TLC), the phosphocholine group was hydrolyzed, and the resultant diradylglycerol was derivatized with 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin (CMMC). After separation of the derivatized subclasses by normal phase HPLC, the individual molecular species of the alkylacyl and diacyl subclasses were quantitated and collected during a subsequent reverse phase HPLC step. With an extinction coefficient of 14,700 l mol-1 cm-1 at a wavelength detection of 320 nm, the CMMC derivatives afforded sensitive UV detection (100 pmol) and quantitation of the molecular species. Continuous flow fast-atom bombardment mass spectrometry of the alkylacyl CMMC derivatives yielded abundant [MH]+ ions and a single fragment ion formed by loss of alkylketene from the sn-2 acyl group, [MH-(R = C = O)]+. No fragmentation of the sn-1 alkyl chain was observed. Diacyl derivatives also produced abundant [MH]+ ions plus two fragment ions arising from loss of RCOOH from each of the acyl substituents and two fragment ions from the loss of alkyketene from each acyl group. Individual molecular species substituents were assigned from these ions.     

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO₂)LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.     

The Composition of Metals Bound to Class III Metallothionein (Phytochelatin and Its Desglycyl Peptide) Induced by Various Metals in Root Cultures of Rubia tinctorum

The induction of phytochelatins (PCs) and their desglycyl peptides (both are referred to as class III metallothionein [CIIIMT]) by exposure to various metals (Ag+, As3+, As5+, Cd2+, Cu2+, Ga3+, Hg2+, In3+, Ni2+, Pb2+, Pd2+, Se4+, and Zn2+) and the metal composition in the CIIIMTs were investigated in root cultures of Rubia tinctorum L. All of these metal species induced PCs to various degrees when analyzed by the postcolumn derivatization high-performance liquid chromatography method. The desglycyl peptides of PCs often were also present. However, only Ag, Cd, and Cu were bound to the CIIIMTs that they induced when analyzed by the high-performance liquid chromatography-inductively coupled plasma-atomic emission spectrometry method. Cu was also bound to the CIIIMTs induced by Ag+, As3+, and Cd2+. After Ag+ exposure, an Fe peak that may be of Fe-CIIIMT was also observed. However, most of the metal species studied were not bound to the CIIIMTs that they induced.     

Synthesis of 5,9-hexacosadienoic acid phospholipids. 11. Phospholipid studies of marine organisms

The synthesis of phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS) containing two acyl chains of the naturally occurring sponge fatty acid (5Z,9Z)-5,9-hexacosadienoic acid as well as its hitherto unknown geometrical isomers is described. The PCs were prepared by deacylation of natural lecithins, followed by reacylation with fatty acid anhydrides. The synthesis of mixed-acid PCs is also reported: a diacyl product was converted to the lyso-PC by treatment with phospholipase A2 and subsequent acylation of the secondary hydroxyl group to give the desired mixed-acid PCs. The PEs and the PSs were prepared from the corresponding PCs by enzymatic transphosphatidylation catalyzed by phospholipase D. Structural assignments of the compounds were confirmed by spectroscopy (1H-NMR and MS). Ammonia chemical ionization mass spectrometry provided molecular ion and significant fragment peaks for PCs and PEs.     

Identification of plant hormones from cotton ovules

An extract from 8-day-old cotton ovules (Gossypium hirsutum L.) was partitioned into three fractions and each fraction was derivatized and analyzed separately. Gas-liquid chromatography and computer-controlled gas-liquid chromatography-mass spectrometry were used to separate, measure, and identify the naturally occurring plant hormones. A single extract contained abscisic acid, indoleacetic acid, and gibberellins A(1), A(3), A(4), A(7), A(9), and A(13) in the first fraction; ethyl indole-3-acetate and indole-3-aldehyde in the second fraction; and the cytokinins 6-(3-methyl-4-hydroxybutylamino)purine (dihydrozeatin), 6-(4-hydroxy-3-methyl-2-trans-butenylamino) purine (zeatin), 6-(3-methyl-2-butenylamino)purine(2iP), 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine(2iPA), and 6-(4-hydroxy-3-methyl-2-trans-butenylamino)-9-beta-d- ribofuranosylpurine (zeatin riboside) in the third fraction.     

Synthesis and characterization of tyramine-derivatized (1 å 4)-linked α-d-oligogalacturonides

The reducing end C-1 of (1 → 4)-linked α-d-oligogalacturonides (oligogalacturonides), with degrees of polymerization (dp) 3 and 13, was coupled to tyramine via reductive amination in the presence of sodium cyanoborohydride. These derivatives were purified in milligram quantities and structurally characterized. Tyramination of trigalacturonic acid proceeded to completion. The yield of apparently homogeneous tyraminated trigalacturonic acid after desalting was 35%. Derivatization of tridecagalacturonide with tyramine was incomplete. The tyraminated tridecagalacturonide was purified to apparent homogeneity using semipreparative high-performance anion-exchange chromatography (HPAEC) with a yield of 30%. The structures of the derivatized oligogalacturonides were established by ¹H NMR spectroscopy and electrospray mass spectrometry.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) coupled to in vivo microdialysis sampling was used in order to monitor simultaneously a drug and several neurotransmitters in the brain extracellular fluid. Determination of the antiepileptic drug vigabatrin and the amino acid neurotransmitters glutamate (Glu), l-aspartate (l-Asp) and gamma-aminobutyric acid (GABA) was performed on low-concentration samples which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and separated using a pH 9.2 75 mM sodium borate running buffer containing 60 mM sodium dodecyl sulfate (SDS) and 5mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Glu, l-Asp and vigabatrin derivatized at a concentration of 1.0 x 10(-9) M, and GABA derivatized at a concentration of 5.0 x 10(-9) M, produced peaks with signal-to-noise ratios of 8:1, 8:1, 4:1 and 5:1, respectively. The nature of the neurotransmitter peaks found in rat brain microdialysates was confirmed by both electrophoretic and pharmacological validations. This method was used for monitoring vigabatrin and amino acid neurotransmitters in microdialysates from the rat striatum during intracerebral infusion of the drug and revealed rapid vigabatrin-induced changes in GABA and Glu levels. This original application of CE-LIFD coupled to microdialysis represents a powerful tool for pharmacokinetic/pharmacodynamic investigations.     

Utilization of different fatty acids for hepatic and biliary phosphatidylcholine formation and the effect of changes in phosphatidylcholine molecular species on biliary lipid secretion

Biliary cholesterol secretion is ordinarily tightly coupled to phosphatidylcholine (PC) secretion. Bile PCs are distinct in composition and predominantly composed of molecular species with 16:0 in the sn-1 position and 18:2 and 18:1 in the sn-2 position. In an attempt to acutely change the composition of biliary PCs and to assess the effect of a change in PCs on biliary cholesterol secretion, isolated livers were perfused with a variety of single free fatty acids. Rat livers with bile duct cannulas were perfused with a recirculating medium, taurocholate (40 mumol/h), and albumin-bound 16:1, 17:1, 18:1, 20:1, 18:2, 20:4, or 20:5 fatty acids (90 mumol/h) for 2 h. Biliary lipid secretion was measured and bile and liver PC compositions were compared at the start and end of perfusion. Results showed 1) greater utilization of shorter chain than longer chain fatty acids for bile PC formation (16:1 greater than 17:1 greater than 18:2 or 18:1 greater than 20:5, 20:4 or 20:1); 2) no similar pattern of FA utilization for liver PC formation; 3) preferentially greater incorporation of fatty acids into bile PCs compared to liver PCs when perfused fatty acids were used for esterification at both sn-1 and sn-2 positions of PC (to form diunsaturated PCs); and 4) increased biliary secretion of cholesterol relative to PC only when the population of PCs that was newly formed included more hydrophilic molecular species of PC than are present in native bile (that was observed only with perfusion of 16:1). Changes in biliary PC secretion or cholesterol/PC secretion occurred independently of any change in bile salt secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography.

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.     

Gas chromatograph-mass spectrometric method for the determination of carvedilol and its metabolites in human urine

A sensitive and efficient method was developed for the determination of carvedilol and its metabolites in human urine by gas chromatography-mass spectrometry (GC-MS). Urine samples were hydrolyzed with beta-glucuronidase/arylsulfatase (from Helix pomatia) and the target compounds were extracted with liquid-liquid extraction. The extracts were completely derivatized with MSTFA and MBTFA and analyzed by GC-MS using an Ultra-2 column. The linearity of the assay ranges were 0.75-75 ngmL(-1) for carvedilol and o-desmethyl carvedilol (o-DMC), and 3.0-75 ngmL(-1) for 4-hydroxyphenyl carvedilol (4-HPC) and 5-hydroxyphenyl carvedilol (5-HPC). The absolute recovery of carvedilol and its metabolites added to a blank urine sample was 80.1-97.8%. The limits of detection (LOD) and quantitation (LOQ) of carvedilol and o-DMC were 0.30 and 0.75 ngmL(-1), and its of 4-HPC and 5-HPC were 0.75 and 3.0 ngmL(-1), respectively. The reproducibilities were 1.86-11.5% for the intra-day assay, and 0.70-1.71% for the inter-day assay precision and the degree of inaccuracy was -3.0 to 3.9% at the concentration of 75 ngmL(-1). The proposed GC-MS method was effective for the determination of carvedilol and its three metabolites in human urine.     

Hydroxymethyl-phytochelatins [(gamma-glutamylcysteine)n-serine] are metal-induced peptides of the Poaceae.

Exposure of several species of the family Poaceae to cadmium results in the formation of metal-induced peptides of the general structure (gamma-Glu-Cys)n-Ser (n=2-4). They are assumed to be formed from hydroxymethyl-glutathione (gamma-Glu-Cys-Ser) and are termed hydroxymethyl-phytochelatins (hm-PCs) in analogy to the homo-phytochelatins [(gamma-Glu-Cys)n-beta-Ala], discovered in legumes, and the phytochelatins [PCs, (gamma-Glu-Cys)n-Gly] found in most other plants and many fungi. The hm-PCs were isolated from the roots of cadmium-exposed rice (Oryza sativa L. cv Strella), and their structure was confirmed by amino acid analysis after total and enzymic hydrolysis and by tandem mass spectrometry. The hm-PCs probably play a significant role in heavy metal detoxication in rice. In addition to this new form of gamma-Glu-Cys (gamma EC) peptide, PCs and gamma EC peptides without C-terminal Ser or Gly are found. All gamma EC peptides are synthesized without delay after incubation of rice plants in 100 microM CdCl2 in the roots as well as in the shoots. Incubation times exceeding 24 h or higher concentrations of cadmium result in a selective enrichment of gamma EC peptides with higher chain length and an increased ratio of PCs to hm-PCs. gamma EC peptide synthesis is accompanied by a decrease of the glutathione content and an increase of the hydroxymethyl-glutathione content in roots and shoots of rice plants.     

Highly sensitive quantification of 7alpha-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.     

Profiling of prostaglandin biosynthesis in biopsy fragments of human lung carcinomas and normal human lung by capillary gas chromatography-negative ion chemical ionization mass spectrometry

Methods for the profiling of prostaglandin F2 alpha (PGF2 alpha), prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) biosynthesis in tissue samples of clinical origin by capillary gas chromatography-negative ion chemical ionization mass spectrometry (CGC-NICIMS) are detailed. Aliquots (25 microliter 1) of incubates (1 ml volume) of human lung carcinoma and normal human lung tissue fragments (total protein content = 0.2 to 2.0 mg) were derivatized for vapor phase analysis in the presence of 0.75 to 1.60 ng of tetradeuterated analogs of PGE2, PGF2 alpha and 6KPGF1 alpha without prior extraction and/or chromatography. The derivatized analytes and internal standards were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The inter-sample and intra-sample coefficients of variation for the assay method were typically less than 12%. The analysis of biological samples was completed with less than 2.5% of each derivatized sample per injection. The samples were of adequate purity for the identification and quantitation of each of the eicosanoids. The methods described in this report are highly selective and highly sensitive with detection limits of 0.1 to 0.2 picograms per injection. The analytical procedures provide the basis for comparisons of the qualitative and quantitative profiles of prostaglandin biosynthesis and should be adaptable for use in a variety of biological and clinical studies.