The enantiomer and the positional isomer of platelet-activating factor

We have compared for rabbit platelet aggregating and desensitizing activity two different preparations of platelet-activating factor (PAF-acether) (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and of its enantiomer (3-O-alkyl-2-O-acetyl-sn-glycero-1-phosphocholine). After phospholipase A2 treatment, the unnatural enantiomers appeared about 3000-fold less active than PAF-acether, a result which definitively establishes the stereospecificity of the mode of action of this mediator. A new structural analog of PAF-acether, 1-O-hexadecyl-3-O-acetyl-sn-glycero-2-phosphocholine, was isolated and characterized. It was a weak platelet agonist, stressing further the importance for PAF-acether activity of the acetyl group at position 2 of the glycerol.     

Systematic analysis of desorption chemical ionization (DCI) mass spectra of nucleic acids--7

The positive ion and negative ion pyrolysis mass spectra of the herring sperm DNA have been studied using desorption chemical ionization. The positive ion desorption chemical ionization spectra have been produced with CH4, i-C4H10, NH3, HCl and Cl2; the negative ones with N2O/CH4, N2O/i-C4H10, Cl2, CCl4, HCl and via electron capture. These spectra have been compared with the electron impact ionization spectra. We have observed an important increase of sensitivity when negative ionization has replaced the positive ionization mode. The series of diagnostic ions resulting from direct chemical ionization belong to the family of base + reagent ion X [BH + X] and base + X - HX ion [B]. Their abundance has increased considerably compared to the electron impact spectra. The application of these new diagnostic ions in nucleic acid studies is interesting especially for the much higher abundance of the usually weak dG fragment ion obtained in the negative ionization mode. The dG-base segment of the DNA is the most nucleophilic centre of the whole nucleic acid and is implicated in numerous important biochemical reactions involving, for example, proteins.     

Human neutrophil platelet-activating factor: molecular heterogeneity in unstimulated and ionophore-stimulated cells

The molecular heterogeneity of platelet-activating factor (PAF) in resting and ionophore (A23187) -stimulated human neutrophils was measured by a very sensitive gas chromatography-negative ion chemical ionization mass spectrometric method. The molecular species compositions of PAF, which are due to variations in the 1-O-alkyl chain length, were significantly different between resting and ionophore-stimulated polymorphonuclear leukocytes. The major species of PAF produced by unstimulated polymorphonuclear leukocytes were 16:0, 17:0, 18:1 and 18:0, representing 55, 14, 8 and 10%, respectively, of the total PAF; 16:0 was the predominant PAF (74%) in A23187-stimulated polymorphonuclear leukocytes. The PAF molecular species from unstimulated polymorphonuclear leukocytes was similar to compositions from those of the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, whereas those from the ionophore-stimulated polymorphonuclear leukocytes differed from the precursor 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, thus indicating a very high degree of substrate selectivity for PAF synthesis. Although the physiological implications of the variations in PAF composition are not known, these studies indicate that the PAF produced by resting polymorphonuclear leukocytes are significantly different from those produced in response to ionophore.     

Effects of hypochlorous acid on unsaturated phosphatidylcholines.

Effects of hypochlorous acid and of the myeloperoxidase-hydrogen peroxide-chloride system on mono- and polyunsaturated phosphatidylcholines were analyzed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Chlorohydrins and glycols were detected as main products according to the characteristic shift of molecular masses. Mainly mono-chlorohydrins result upon the incubation of HOCl/(-)OCl with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, whereas only traces of mono-glycols were detected. 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine yielded a complex mixture of products. Mono-chlorohydrins and glycols dominated only at short incubation, while bis-chlorohydrins as well as products containing one chlorohydrin and one glycol moiety appeared after longer incubation. Similarly, a complex product mixture resulted upon incubation of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine with hypochlorous acid. Additionally, tris-chlorohydrins, products with two chlorohydrin and one glycol moiety, as well as lysophosphatidylcholines and fragmentation products of the arachidonoyl side chain were detectable. Mono-chlorohydrins of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were detected after the incubation of the latter phospholipid with the myeloperoxidase-hydrogen peroxide-chloride system at pH 6.0. These chlorohydrins were not observed in the absence of chloride, hydrogen peroxide, or myeloperoxidase as well as in the presence of methionine, taurine, or sodium azide. Thus, mono-chlorohydrins in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine produced by hypochlorous acid from the myeloperoxidase-hydrogen peroxide-chloride system can also be detected by means of MALDI-TOF MS.     

Species-specific variations in the molecular heterogeneity of the platelet-activating factor

The molecular heterogeneity of platelet-activating factor (PAF) synthesized by unstimulated and Ca2+ ionophore (A23187)-stimulated PMN from rat, mouse, and guinea pig and by rat basophilic leukemia (RBL) cells was investigated by gas chromatography-negative ion chemical ionization mass spectrometry. Several molecular species of PAF ranging from C14:0 to C19:0 were detected in all of the cells studied. PAF produced by each cell type exhibited a unique pattern of molecular species distribution. Although C16:0 was the major PAF molecular species of rat PMN and RBL cells representing 96% and 85% of the total PAF, respectively, PAF from mice PMN contained 81% of C16:0, 10% of C18:1, and 6% of C18:0. Alternatively, A23187-stimulated guinea pig PMN yielded PAF molecular species 35% in C16:0, 35% in C17:0, 8% in C18:1, and 3% in C18:0. Small but significant differences in the PAF molecular species distribution of resting and ionophore stimulated cells were also observed. In contrast to the PAF molecular species composition, the precursor 1-O-alkyl-2-acyl-glycero-3-phosphocholine of all the cell types was predominantly hexadecyl (C16:0) alkyl chain in the sn-1 position, representing 60 to 80% of the total 1-O-alkyl-2-acyl-glycero-3-phosphocholine. Thus, these results not only indicate a high degree of selectivity for utilization of precursor substrates for PAF biosynthesis, but also demonstrate that the selectivity is species specific.     

Analysis of the lipid composition of human and boar spermatozoa by MALDI-TOF mass spectrometry, thin layer chromatography and 31P NMR spectroscopy

Alterations in the phospholipid (PL) composition of spermatozoal membranes occur during the fertilization process. Furthermore, membrane lipid composition is of high interest with respect to cryopreservation. The PL and fatty acid compositions of human and boar spermatozoa are compared by using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) in combination with thin-layer chromatography and 31P NMR spectroscopy. The extreme sensitivity of alkenyl-linked PL against acid treatment was used to estimate the plasmalogen content of spermatozoa. Compared with humans, boar spermatozoa are characterized by a lower variability of their PL and fatty acid composition. Additionally, boar spermatozoa contain much higher moieties of alkyl-linked compounds, e.g. 1-palmityl-2-docosapentaenoyl-sn-glycero-3-phosphocholine and 1-palmityl-2-docosahexaenoyl-sn-glycero-3-phosphocholine as well as the corresponding phosphatidylethanolamine (PE), while human spermatozoa are characterized by high contents of diacyl-PL, e.g. 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine. A considerable plasmalogen moiety, for instance 1-palmitenyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine is a typical feature of both, human and boar spermatozoa. It will be shown that these differences in PL composition can be very rapidly and conveniently assessed by MALDI-TOF MS in combination with TLC and also by 31P NMR.     

Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production

To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.     

Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists

The platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and its analogs were examined to determine their effects on guinea pig peritoneal macrophages. PAF activated macrophages, but its effect on macrophages was much weaker than that observed on platelets: the concentration required for 50% maximum activation was 8.5 X 10(-6) M for macrophages and 2.9 X 10(-10) M for platelets. Three PAF agonists, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-glycero-3-phosphocholine (Compound I), 1-O-octadecyl-2-acetamido-2-deoxy-glycero-3-phosphocholine (Compound II), and 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Compound III), showed higher activity in stimulating macrophage function than PAF. The abilities of these non-metabolizable PAF agonists to activate macrophage paralleled their relative potency to induce platelet activation. The sn-3 enantiomers of PAF and Compound III exhibited activity, while the sn-1 did not. By comparing the activities of derivatives of Compound III, it was shown that the long-chain alkyl-ether group in the glycerol-1 position, a relatively small size of the substituent on the hydroxy group at the sn-2 position, and the choline moiety in the glycerol-3 position must play critical roles in the process of macrophage activation. A specific PAF antagonist, CV3988, which inhibits PAF-induced platelet activation and hypotension, inhibited the activation of macrophages caused by PAF and its agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment from Bacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.     

Ammonia desorption chemical ionization mass spectrometry of phospholipids

Ammonia desorption chemical ionization mass spectra (NH₃-DCIMS) of phosphatidyl-sulfocholines, a mixture of a homologous series of phosphatidylsulfocholines (PSC) and a mixture of a PSC phosphatidylcholine (PC), were measured by a flash heating method involving introduction of 1 μg of the samples on a tungsten wire, quickly heated, into the ion source of a Finnigan 4000/INCOS quadrupole instrument. It was possible to observe mass spectra during several seconds.The first spectra recorded of the PSCs gave essentially only the quasi-molecular [M + 18]⁺ peaks and the ‘A’ (see text) peaks. Spectra of a mixture of three homologous PSCs clearly showed three pairs of the above peaks. In contrast spectra of the PCs gave principally the [M + 1]⁺ and the ‘A’ peaks after 2s so that one can distinguish the diagnostic peaks in a mixture of a PC and a PSC.These results show that ammonia desorption chemical ionization by fast heating is a suitable technique for the charaterization of some head groups of phospholipids as well as a promising method for the analysis of phospholipid mixtures.     

Utility of mass spectrometry for proteome ana lysis: part I. Conceptual and experimental approaches

This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.     

High performance liquid chromatography of platelet-activating factors

Silica and C18 reverse phase high performance liquid chromatography (HPLC) were used to fractionate synthetic molecular species of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and semi-synthetic platelet-activating factor (PAF) synthesized from beef heart plasmalogens. A single coincident peak from silica HPLC was observed for either a mixture of synthetic AGEPC's with alkyl chain lengths from C12 to C18 or for beef heart-derived PAF. This peak was well separated from other classes of phospholipid standards including 2-lysophosphatidylcholine and 3H-labeled lyso-PAF. Subsequently, the synthetic AGEPC mixture or beef heart PAF was separated into individual species on a C18 reverse phase column. Beef heart-derived PAF was fractionated into at least four molecular species of PAF activity which had similar retention times as the radioactivity of 3H-labeled beef heart PAF. Approximately 56% of the radioactivity of 3H-labeled PAF was found in the fraction with a similar retention time as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 10% as 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, 11% as 1-O-pentadecyl-2-acetyl-sn-glycero-3-phosphocholine, and 13% in an unidentified fraction which eluted after C-16-AGEPC. The unidentified fraction did not correspond to any of the homologous series of synthetic AGEPCs with saturated alkyl chain lengths from C12 to C18. Recoveries of radioactive phospholipids from silica or reverse phase columns were greater than 95%.     

Specific receptor sites for 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) on rabbit platelet and guinea pig smooth muscle membranes

By using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF), we have directly identified its specific binding sites on rabbit platelet plasma membranes. The equilibrium dissociation constant for 3H-PAF is 1.36 (+/- 0.05) X 10(-9) M at 0 degrees C. The number of binding sites is 1.61 (+/- 0.34) X 10(12)/mg of membrane, which corresponds to approximately 150-300 receptors/platelet (depending on membrane vesicle orientation). Binding of 3H-PAF to rabbit platelet plasma membrane is rapid (t1/2 less than 5 min at 0 degrees C) and reversible. For a series of PAF analogues, their affinity for the receptor sites parallels with their relative potency to induce platelet aggregation. PAF can cause contraction of smooth muscle of heart, parenchymal strip, trachea, and ileum. Specific PAF receptor binding was demonstrated with purified plasma membrane from several smooth muscles and from polymorphonuclear leukocytes but not from presumably PAF nonresponsive cells such as erythrocytes and alveolar macrophages. It is likely that the interaction of PAF with these binding sites initiates the specific responses of platelets, polymorphonuclear leukocytes, and smooth muscles.     

Macrophage activation by PAF incorporated into dipalmitoylphosphatidylcholine-cholesterol liposomes

The phospholipid mediator, platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine), was recently reported to activate macrophage-monocyte cells as well as neutrophils or platelets. PAF was incorporated into dipalmitoylphosphatidylcholine-cholesterol liposomes, and their effect on guinea pig peritoneal macrophages was examined. PAF incorporated into liposomes was found to activate macrophages much more potently than PAF in the free form, whereas the effect of PAF in liposome on platelets was weaker than that of PAF in the free form. A large difference between PAF in liposomes and PAF in the free form was observed in the rate of degradation of PAF during incubation with macrophages. This rapid degradation of PAF in the free form may partially explain the poor activation by PAF of macrophages.     

Fast ATOM Bombardment Mass Spectra of Nucleosides. Comparison with Electron Impact and Chemical Ionization Mass Spectra

Abstract

The fast atom bombardment (FAB) mass spectra of the eight major nucleosides found in RNA and DNA, pseudouridine and 2′,3′-O-isopropylidene adenosine are described and compared to El, CI, and desorption chemical ionization (DCI) spectra reported in the literature or obtained in this laboratory. Bcty, cocltl nun FAB spectra are reported. The FAB spectra are simple and provide unambiguous molecular weight information along with structurally significant fragment ions. Mechanisms of ion formation are thought to closely parallel those suggested earlier to be operating in the CI mode. Advantages and disadvantages of FAB relative to the standard ionization modes are discussed.     

Binding of platelet activating factor to albumin

Binding of platelet activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) to albumin is an important facet of the biological activity of this phospholipid. Measurement of that binding has been hampered by the physical nature of the lipid, which made estimation of the free and bound concentrations difficult. With the use of ultracentrifugation to generate an albumin gradient and to produce a region free of protein, the successful measurement of free PAF and PAF bound to albumin was accomplished. This study has demonstrated that PAF binds to albumin at four binding sites and that the average equilibrium dissociation constant for this binding is 1.10(-7) M. Consideration of these data has led to the hypothesis that the receptor active form of PAF is the albumin-PAF complex, rather than free PAF.     

Hypochlorous acid-mediated generation of glycerophosphocholine from unsaturated plasmalogen glycerophosphocholine lipids

The myeloperoxidase-derived metabolite hypochlorous acid (HOCl) promotes the selective cleavage of plasmalogens into chloro fatty aldehydes and 1-lysophosphatidylcholine (LPC). The subsequent conversion of the initially generated LPC was investigated in plasmalogen samples in dependence on the fatty acid residue in the sn-2 position by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and (31)P NMR spectroscopy. Plasmalogens containing an oleic acid residue in the sn-2 position are converted by moderate amounts of HOCl primarily to 1-lyso-2-oleoyl-sn-glycero-3-phosphocholine and at increased HOCl concentrations to the corresponding chlorohydrin species. In contrast, plasmalogens containing highly unsaturated docosahexaenoic acid yield upon HOCl treatment 1-lyso-2-docosahexaenoyl-glycerophosphocholine and glycerophosphocholine. The formation of the latter product denotes a novel pathway for the action of HOCl on plasmalogens.     

Closely related oxidized phospholipids differentially modulate the physicochemical properties of lipid particles

Oxidation of glycerophospholipids results in the formation of large variety of oxidized phospholipid products that differs significantly in their chemical compositions and molecular structures. Biological activities of these oxidized products also differ considerably. Here we report the comparisons of the physicochemical properties of non-oxidized phospholipid particle containing two closely related tOx-PLs: 1-palmitoyl-2-(5-keto-6-octendioyl)-sn-glycero-3-phosphocholine (KOdiA-PC) and 1-palmitoyl-2-(9-keto-10-dodecendioyl)-sn-glycero-3-phosphocholine (KDdiA-PC). DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was used as a model membrane non-oxidized phospholipid. Physicochemical properties of the lipid particles were characterized by using fluorescence spectroscopy, native polyacrylamide gel and agarose gel electrophoresis. Our result shows that the presence of closely related tOx-PLs, which differ only in the chemical composition of the oxidized fatty acyl chains at the sn-2 position, exerts considerably different effect on the physicochemical properties of non-oxidized phospholipid particles containing them.     

Detection of Biosignatures by Geomatrix-Assisted Laser Desorption/Ionization (GALDI) Mass Spectrometry

Identification of mineral-associated biosignatures is of significance for retrieving biochemical information from geological records here on Earth and for detecting signs of life on other planets, such as Mars. An investigation using laser desorption Fourier transform mass spectrometry was conducted to determine whether geomatrix-assisted laser desorption/ionization (GALDI) can be used to detect amino acids (e.g., histidine, threonine, and cysteine) and small proteins (e.g., gramicidin S) associated with mineral phases and whether the geomatrix impacts detection. Iron oxide (Fe₂ O ₃ ) and sodium chloride (NaCl) were investigated as clean chemical analogues of hematite and halite, respectively, which have both been detected on the surface of Mars. Samples were prepared by 2 methods: (1) application of analyte solution to the geomatrix surface with subsequent drying; and (2) physical mixing of analyte and geomatrix. Amino acids incorporated within NaCl by physical mixing yielded a better signal-to-noise ratio than those that were applied to the surface of a NaCl pellet. The composition of the geomatrix had an influence on the detection of biomolecules. Peaks corresponding to the cation-attached biomolecular ions were observed for the NaCl prepared samples. However, no biomolecular ion species were observed in samples using Fe ₂ O ₃ as geomatrix. Instead, only minor peaks that may correspond to ions derived from fragments of the biomolecules were obtained.     

Quantitative measurement of delta 9-tetrahydrocannabinol and two major metabolites in physiological specimens using capillary column gas chromatography negative ion chemical ionization mass spectrometry

delta 9-Tetrahydrocannabinol and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, can be measured in a single 1-ml sample of blood, plasma, or urine by a new assay which combines a relatively rapid extraction procedure with capillary column gas chromatography and negative ion chemical ionization mass spectrometry. Deuterium-labeled analogs of each cannabinoid are added to the physiological specimen as internal standards. Two extracts are obtained from each sample: a neutral fraction containing delta 9-tetrahydrocannabinol and 11-hydroxy-delta 9-tetrahydrocannabinol, and an acid fraction containing 11-nor-9-carboxy-delta 9-tetrahydrocannabinol. The neutral fraction is derivatized by treatment with trifluoroacetic anhydride; the acid fraction is first treated with BF3-methanol followed by reaction with trifluoroacetic anhydride. Under electron-capture chemical ionization conditions the derivatized delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol give abundant molecular anions ideally suited for selected ion monitoring. The negative ion chemical ionization spectrum of the HO-THC-trifluoroacetate shows no molecular anion. Consequently, quantitation of the hydroxy metabolite is achieved by monitoring a fragment ion formed by loss of CF3CO2 from its molecular anion. The limits of reliable measurement are judged to be 0.1 ng ml-1 for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, 0.2 ng ml-1 for delta 9-tetrahydrocannabinol and 0.5 ng ml-1 for 11-hydroxy-delta 9-tetrahydrocannabinol. Four examples are given of the application of the assay to the analysis of specimens of medico-legal importance.