Negative Ion Chemical Ionization Mode Research Articles

Simultaneous identification of different classes of hydrocarbons and determination of nitro-polycyclic aromatic hydrocarbons by means of particle beam liquid chromatography-mass spectrometry

LC-MS with a particle beam interface was used to separate and identify aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs) and their nitrated derivatives (nitro-PAHs). In particular, the behaviour of nitro-PAHs and of two nitrated biphenyls was checked in terms of instrumental response. A normal-phase chromatographic system was used, with a silica column and heptane-tetrahydrofuran as mobile phase. Electron impact and positive- and negative-ion chemical ionization (NICI) modes were used; for nitro-PAHs, the best results were obtained under NICI conditions. In the flow-injection mode, detection limits as low as pg levels were obtained for 9-nitroanthracene, 3-nitro-9-fluorenone, 1-nitropyrene, 1,8-dinitronaphthalene, 2,7-dinitrofluorene and 2,7-dinitro-9-fluorenone, whereas the low-molecular-mass 1- and 2-nitronaphthalene were not detected even at μg levels; using the analytical column, a detection limit five times higher was determined for 1-nitropyrene. For most of the nitro-PAHs considered, the dependences of the intensities on the amounts injected were found to be linear in a range of more than two orders of magnitude.

The Fate of Leached Di(2-Ethylhexyl)Phthalate in Patients Undergoing Capd Treatment

To evaluate the degree of exposure to and the fate of di(2-ethylhexyl)phthalate (DEHP) and its major derivatives mono(2-ethylhexyl)phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA) in patients undergoing regular continuous ambulatory peritoneal dialysis (CAPD) during a 4-hour dwell period. Prospective, controlled. Teaching hospital, Department of Nephrology. Seven elderly patients on stable CAPD using Fresenius instruments and dialysate and 6 age-matched healthy controls. During a routinely performed peritoneal equilibration test (PET), blood and dialysate samples were drawn before and 120 and 240 min after the dwell was started. In addition, blood samples were taken from a group of volunteers participating in a pharmacological study. Quantitative analysis of DEHP and its hydrolysis products was performed by selected ion-monitoring gas chromatography/mass spectrometry, operating the mass spectrometer in a combined positive and negative ion chemical ionization mode. Serum concentrations of DEHP and PA were significantly higher in patients (median: 0.079 microgram/mL, range: 0.032-0.210 microgram/mL; and 0.167 microgram/mL, range: 0.097-0.231 microgram/mL, respectively) than in controls [0.0195 microgram/mL, range: 0.016-0.025 microgram/mL (p = 0.0027) and 0.0120 microgram/mL, range: 0.006-0.034 microgram/mL (p = 0.0026), respectively]. Concentration of MEHP in the fluid of CAPD bags prior to use was four times higher than that of the parent compound. During the first 4 hours of dwell time, the concentrations of MEHP and 2-EH in dialysate consistently decreased from 0.177 (range: 0.137-0.239 microgram/mL) to 0.022 microgram/mL (range: 0.005-0.058 microgram/mL) (p = 0.017), and from 0.087 (range: 0.075-0.097 microgram/mL) to 0.05 microgram/mL (range: 0.023-0.064 microgram/mL) (p = 0.017), respectively, while the concentration of DEHP remained stable. Remarkably high concentrations of PA (0.129 microgram/mL; range: 0.038-0.466 microgram/mL) were found in CAPD bags prior to use, and these concentrations tended to increase during dwell time, without statistical significance, however (0.135 microgram/mL; range: 0.073-0.659 microgram/mL, p = 0.062). Patients on CAPD are regularly exposed to considerable amounts of phthalic ester derivatives, mainly to MEHP and PA. MEHP seems to be well absorbed by the peritoneal membrane. The long-term effects of this exposure remain to be elucidated.

Gas chromatographic determination and negative-ion chemical ionization mass spectrometric confirmation of 4,4'-methylenebis(2-chloroaniline) in urine via thin-layer chromatographic separation.

An analytical method for monitoring 4,4'-methylenebis(2-chloroaniline) (MOCA) in urine was developed. The MOCA extract from a urine sample was derivatized with pentafluoropropionic anhydride and subsequently cleaned-up via TLC before being analysed by a gas chromatograph equipped with an electron capture detector. Heptafluorobutyric anhydride derivatized MOCA was used as the internal standard during quantification. The method showed that the analytical interferences are effectively minimized by using the TLC technique for sample clean-up. Recoveries of spiked MOCA in urine at levels of 7.5, 22 and 62.5 micrograms dm-3 were all over 90% with the limit of detection determined to be 1 microgram dm-3. Confirmation of MOCA in a urine sample was performed by using a gas chromatograph mass spectrometer in negative-ion chemical ionization mode.

Quantitative Analysis of THC, 11-OH-THC, and THCCOOH in Human Hair by Negative Ion Chemical Ionization Mass Spectrometry

A sensitive and specific method was developed for the quantitative analysis of delta9-tetrahydrocannabinol (THC), 11 -hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in human hair. Deuterated internal standards were added to hair samples, and samples were digested overnight in 1 N NaOH at 37 degrees C. Digest solutions were extracted with a liquid-liquid extraction procedure, which was previously developed in our laboratory for the analysis of plasma and whole blood. Derivatized extracts were analyzed on a Finnigan 4500" mass spectrometer in negative ion chemical ionization mode using methane as the reagent gas, hydrogen as the carrier gas, and a Restek Rtx 200-15M-0.25-microm capillary column. The assay was linear up to 50 ng/mg hair (r, 0.99) for all three compounds and was capable of detecting 10 pg THC and THCCOOH and 100 pg 11-OH-THC on column. The intra-assay precision ranged from 2.1 to 11.2% for the three analytes; the interassay precision ranged from 4.4 to 13.0%. The method was used to detect and quantitate the presence of THC, 11-OH-THC, and THCCOOH in human hair obtained from eight regular users of cannabis. THC, but not 11-OH-THC or THCCOOH, was detectable in the hair shaft above the assay limit of quantitation. Four laboratory wash procedures were also evaluated for their effect on the measured concentration of THC in hair. in seven of eight subjects, a methylene chloride wash procedure substantially reduced the measured THC concentration by up to 50%. The gas chromatographic-mass spectrometric assay is currently being used to support pharmacokinetic studies of drug disposition into the hair of humans and animals.

Regulation of Lysophospholipase Activity of the 85-kDa Phospholipase A2 and Activation in Mouse Peritoneal Macrophages

The regulation of the lysophospholipase activity of the 85-kDa cytosolic phospholipase A2 (PLA2) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA2 when assayed at a relatively low substrate concentration. Inhibition could be reversed if the substrate concentration was increased or if Ca2+ was present in the assay. Incubation of recombinant enzyme with macrophage membranes and lipid extracts from macrophage membranes resulted in the release of arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that with a bilayer substrate the PLA2 can sequentially deacylate the sn-2 then sn-1 acyl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glycerophosphocholine and glycerophosphoinositol by incubation with recombinant 85-kDa PLA2. The 85-kDa enzyme was identified as the main lysophospholipase activity in mouse peritoneal macrophage cytosols. Addition of Ca2+ to the assay enhanced activity, but this effect decreased as the substrate concentration was increased. Incubation of macrophages with zymosan increased the lysophospholipase activity of the 85-kDa PLA2 in cytosols. Phosphorylation of recombinant PLA2 with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA2 activity. In macrophages stimulated with zymosan release of stearic acid (18:0) and palmitic acid (16:0) was observed in addition to arachidonic acid (20:4). These results are consistent with a role of the 85-kDa PLA2 in regulating lysophospholipid levels in macrophages during zymosan stimulation.

Pentafluorobenzyl derivatives for the gas chromatographic determination of hydroxy-polycyclic aromatic hydrocarbons in urban aerosols

A method for the analysis of hydroxy-substituted polynuclear aromatic hydrocarbons (hydroxy-PAHs), using gas chromatography with electron-capture (ECD) and mass spectrometric (MS) detectors, is described. Pentafluorobenzyl bromide (PFBBr) was used as derivatizing agent for the hydroxy-PAHs. Optimization of the derivatization conditions was performed, and electron impact and negative ion chemical ionization mass spectra were studied. Detection limits ranged 0.01 to 3.3 pg in ECD and negative ion chemical ionization mode (NICI)-SIM-MS values that allowed the determination of these compounds in environmental samples. Samples of air particulate matter were collected for the analysis of hydroxy-PAHs. The concentrations found for 5-hydroxyindol, 2-nitro-1-naphtho, 2-hydroxy-1,4-napthoquinone and 2-hydroxy-9-fluorenone ranged between 5 and 189 pg/m 3.

Identification of arachidonate epoxides/diols by capillary chromatography-mass spectrometry.

The identification of epoxide regioisomers of arachidonic acid (EETs) as methyl esters is difficult because they coelute during gas chromatography and possess similar mass spectra. In the present study, EETs and their hydrolysis products, dihydroxyeicosatrienoic acids (DHETs), were analyzed as pentafluorobenzyl ester derivatives and their properties were compared to other esters. The four EET regioisomers were not resolved by gas chromatography as pentafluorobenzyl, trimethylsilyl, t-butyldimethylsilyl, or methyl esters. However, after being hydrolyzed to DHETs, three of the four regioisomers were resolved as (bis)-t-butyldimethylsilyl ether, pentafluorobenzyl esters. The fourth regioisomer (5,6-DHET) was resolved after being converted to a delta-lactone. Thus, the EETs could be resolved by capillary gas chromatography once converted to DHETs. Pentafluorobenzyl esters of both EETs and DHETs (15-40 ng) provided diagnostic spectra when analyzed by electron ionization mass spectrometry. The mass spectral interpretations that indicated epoxide and diol positions were validated using synthesized EET/DHET [17,17,18,18-d4, 5,6,8,9,11,12,14,15 d8] standards. Lesser amounts of DHETs (5-150 fg) also indicated molecular weights when analyzed in the negative-ion chemical-ionization mode. In summary, EETs in nanogram quantities were identified as pentafluorobenzyl esters using electron ionization mass spectrometry. EETs in femtogram-to-picogram quantities were also identified after conversion to DHETs and analysis by gas chromatography-mass spectrometry in the negative ion-chemical ionization mode.

Characterization of anthraquinone-2-carbonyl chloride as an alcohol derivatization reagent for negative ion chemical ionization mass spectrometry

Anthraquinone-2-carbonyl chloride has been utilized as a derivatization reagent for alcohols to impart electron affinity and aid in transport via a particle beam liquid chromatography-mass spectrometry (LC/MS) interface. In addition, the gas chromatographic-mass spectrometry, UV, fluorescence, and electrochemical characteristics of the derivatives were determined. A series of model compounds, 2-phenylethanol (phenethyl alcohol), 1-phenyl-2-propanol, 2-methyl-l-phenyl-2-propanol, hexanol, and methyl 2-methylglycerate, were used as analytes.The particle beam LC/MS properties of the resultant anthraquinone carboxylate esters were determined in electron impact (EI) and negative ion chemical ionization (NCI) modes. The NCI responses of these anthraquinone carboxylate esters were compared with the corresponding 3,5-dinitrobenzoate esters. The anthraquinone carboxylate esters exhibited an NCI to EI sensitivity enhancement of 113 and were detected in NCI at a tenfold lower concentration than the corresponding 3,5-dinitrobenzoate esters. A detection limit of 26 pg injected on column was achieved for phenethyl anthraquinone carboxylate in NCI by using selected ion monitoring.

A NICI-GC-MS Method to Analyze Endosulfan in Biological Samples

Abstract A mass spectrometric method in the negative ion chemical ionization mode was used to analyze α- and β-endosulfan in biological samples. The selected ion recording technique was employed, monitoring the ions at m/z 35 and 37. Biological samples of animals treated with endosulfan were analyzed. The method is selective and sensitive enough to analyze crude extracts of mouse brain and plasma directly without purification, since the chromatograms were clean

GC Analysis of Some Organochlorine Pesticides using a Brominated Internal Standard

Abstract The brominated compound 1,1 -bis(4-chloropheny 1)2-bromoethane was specifically synthesized for use as internal standard (IS) in the GC analysis of some organochlorine pesticides. The IS was prepared by reacting commercially available 2,2-bis(4-chlorophenyl)ethanol with triphenylphosphine and tetrabromomethane. Organochlorine pesticides were tested together with this IS for the linearity of the analytical method in the pg range of injected pesticides (HCB, α- and γ-HCH, heptachlor, aldrin, op'-and pp'-DDT and its metabolites: op'-and pp'-DDE, op'- and pp'-DDD), using MS detection in the negative ion chemical ionization mode (NICI). GC-ECD was also used to test the IS response and linearity of the method for some of the above pesticides in the pg range of injected analytes. The synthesized IS was added and analyzed in vegetable samples (spruce needle and branch) to prove the applicability of this compound in a GC-NICI-MS analytical method for organochlorine pesticides in such samples.

Stereospecific dehydrogenation of (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids by acyl-CoA oxidase in rat liver light mitochondrial fraction.

From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) has been studied with capillary gas chromatography (GC)/negative ion chemical ionization (NICI)-mass spectrometry. After incubation of (24R,25R)- or (24S,25S)-[24,25-2H2]THCA, synthesized from (24E)-delta 24-THCA by a deuterated diimide reduction, with a rat liver light mitochondrial fraction, 5 beta-cholestanoic acids were extracted and derivatized into a pentafluorobenzyl (PFB) ester-dimethylethylsilyl (DMES) ether. Subsequent resolution into THCA and delta 24-THCA was attained by GC on a cross-linked 5% phenylmethyl silicone fused-silica capillary column monitored with a corresponding characteristic carboxylate anion [M-PFB]- in the NICI mode. The stereospecific elimination of a pro-R hydrogen at C-24 in both (25R)- and (25S)-THCA indicated syn-elimination for the former, whereas anti-elimination for the latter was observed.

95/00600 Baseline performance of a 200 MWt pressurized fluidized bed combustor

We report on the quantitative determination of acetaminophen (paracetamol; NAPAP-d0) in human plasma and urine by GC–MS and GC–MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode after derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br). Commercially available tetradeuterated acetaminophen (NAPAP-d4) was used as the internal standard. NAPAP-d0 and NAPAP-d4 were extracted from 100-μL aliquots of plasma and urine with 300 μL ethyl acetate (EA) by vortexing (60 s). After centrifugation the EA phase was collected, the solvent was removed under a stream of nitrogen gas, and the residue was reconstituted in acetonitrile (MeCN, 100 μL). PFB-Br (10 μL, 30 vol% in MeCN) and N,N-diisopropylethylamine (10 μL) were added and the mixture was incubated for 60 min at 30 °C. Then, solvents and reagents were removed under nitrogen and the residue was taken up with 1000 μL of toluene, from which 1-μL aliquots were injected in the splitless mode. GC–MS quantification was performed by selected-ion monitoring ions due to [M−PFB]− and [M−PFB−H]−, m/z 150 and m/z 149 for NAPAP-d0 and m/z 154 and m/z 153 for NAPAP-d4, respectively. GC–MS/MS quantification was performed by selected-reaction monitoring the transition m/z 150 → m/z 107 and m/z 149 → m/z 134 for NAPAP-d0 and m/z 154 → m/z 111 and m/z 153 → m/z 138 for NAPAP-d4. The method was validated for human plasma (range, 0–130 μM NAPAP-d0) and urine (range, 0–1300 μM NAPAP-d0). Accuracy (recovery, %) ranged between 89 and 119%, and imprecision (RSD, %) was below 19% in these matrices and ranges. A close correlation (r > 0.999) was found between the concentrations measured by GC–MS and GC–MS/MS. By this method, acetaminophen can be reliably quantified in small plasma and urine sample volumes (e.g., 10 μL). The analytical performance of the method makes it especially useful in pediatrics.

Positive and negative ion mass spectrometry of ten xanthine derivatives and their rapid clean-up with Sep-Pak C 18 cartridges from biological samples

Positive ion electron impact (PIEI), positive ion chemical ionization (PICI) and negative ion chemical ionization (NICI) mass spectra are presented for ten xanthine derivatives; and each fragmentation pathway has been analyzed. In the PIEI mode, molecular ions were generall intense and constituted base peaks in five compounds. Peaks at m z 42 (NCO) appeared in all compounds; fragment cations at m z 55, 68 (or 67) and 82 were observed in many compounds. In the PICI mode, all drugs showed intense [M+H] + quasi-molecular peaks together with small peaks at m z M+C 2H 5 and M+C 3H 5; fragment peaks at m z 44, 58, 72 and/or 84 (or 86) were also observed. In the NICI mode, [M−H] − quasi-molecular peaks appeared in nine compounds and constituted base peaks in five compounds; adduct anions at m z M+32 and/or M+C 3H 7, and fragment anions at m z 42, 165 and/or 179 were also observed in many compounds. Detection limits for total ion monitoring of the drugs in the PIEI, PICI and NICI modes were 2.2–10 ng, 11–28 ng and 7.8–14 ng on column, respectively. Xanthine derivatives present in human plasma or urine could be rapidly extracted by use of Sep-Pak C 18 cartridges with chloroform/methanol as elution solvent; recovery of the drugs from the plasma and urine was > 75%. They were also detected by capillary gas chromatography (GC) with both flame ionization detection (FID); their limits were 1.6–8.3 ng on column.

Determination of prostaglandin E1 and its main plasma metabolites 15-keto-prostaglandin E0 and prostaglandin E0 by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry.

Prostaglandin E1 (PGE1), 15-keto-PGE0 and PGE0 in plasma were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of deuterated internal standards, the prostaglandins were extracted by a solid-phase cartridge and derivatized to the pentafluorobenzyl ester methoxime. The samples were purified by thin-layer chromatography, converted to the trimethylsilyl ethers and quantified by gas chromatography/triple-stage quadrupole mass spectrometry. The parent ions in the negative ion chemical ionization mode were [M-pentafluorobenzyl]- ([P]-), the daughter ions used for quantification were [P-(CH3)3SiOH]- (PGE0 and 15-keto-PGE0) and [P-2(CH3)3SiOH]- (PGE1), respectively. Plasma concentrations in healthy subjects were at about 1-3 pg ml-1 for PGE1 and PGE0 and 2-15 pg ml-1 for 15-keto-PGE0. After infusion of 60 micrograms PGE1 in 2 h, the concentrations in plasma were 3-10 pg ml-1 for PGE1, 8-17 pg ml-1 for PGE0 and 115-205 pg ml-1 for 15-keto-PGE0.

Formation, Metabolism, and Action of Hepoxilin A3in the Rat Pineal Gland

The present study was undertaken to investigate the possible formation of hepoxilin A3 in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 microM) led to the appearance of hepoxilin A3 (HxA3) analyzed as its stable trihydroxy derivative, trioxilin A3 by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA3 is estimated to be 1.43 +/- 0.66 ng/micrograms of protein. This amount is not modified when the tissue is boiled (2.07 +/- 0.66 ng/micrograms of protein). However, the formation of this compound was stimulated to 21.26 +/- 5.82 ng/micrograms of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 micrograms) resulted in a partial blockade of hepoxilin formation. Using [1-14C]HxA3, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA3 into trioxilin A3. This hydrolysis was inhibited by 1 mumol/L of 3,3,3-trichloropropene-1,2-oxide. In a separate study, HxA3 in the presence of 3,3,3-trichloropropene-1,2-oxide to block the hydrolysis of HxA3 decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5'-N-ethylcarboxamidoadenosine, an A1/A2 adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (-88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA3 in the rat pineal gland.

Positive- and negative-ion mass spectrometry of diphenylmethane antihistaminics and their analogues and rapid clean-up of them from biological samples

Positive-ion electron impact (PIEI), positive-ion chemical ionization (PICI) and negativeion chemical ionization (NICI) mass spectra are presented for 15 compounds of diphenylmethane antihistaminics and their analogues, and each fragmentation pathway was analyzed. In the PIEI mode, molecular peaks were very small or missing for most compounds. Peaks at m z 58, due to a dimethylaminomethyl group liberated, constituted base peaks in five compounds. Peaks at m z 165 and/or 167, due to diaromatic rings plus a methyl group, appeared in most compounds. In the PICI mode, peaks due to M + H and M + C 2H 5 appeared in all compounds. Peaks due to diaromatic rings plus a methyl or ethyl group constituted base peaks in five compounds, which had an ether bond in their structures. In the NICI mode, anions at m z M-H appeared in most compounds. Peaks at m z 35 were observed for compounds having a chlorine group in their structures. Detection limits for total ion monitoring of these compounds were 20–50 ng on column in the PIEI mode, 100–200 ng in the PICI mode and 500–1000 ng in the NICI mode. A rapid and simple clean-up procedure of these drugs with use of Sep-Pak C 18 cartridges is also presented. The drugs could be detected by gas chromatography with DB-1 and DB-17 capillary columns with satisfactory separation from impurities in their underivatized forms. The recovery of the drugs, which had been added to whole blood and urine, was more than 60% except for meclizine.

Quantification of the carcinogens 2-amino-3,8-dimethyl- and 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine in food using a combined assay based on gas chromatography—negative ion mass spectrometry

A gas chromatographic—mass spectrometric assay has been developed for the measurement of 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) in food. Stable isotope-labelled analogues of MeIQx and PhIP are used as internal standards and the synthesis of deuterated PhIP is described. The mass spectrometer is operated in the electron-capture negative ion chemical ionisation mode and the amines are chromatographed as their di-3,5-bistrifluoromethylbenzyl derivatives. All three compounds can be measured in a single chromatographic run and detection limits of 0.05, 0.1 and 0.2 ng/g for MeIQx, DiMeIQx and PhIP, respectively, in food are obtained. Various home-cooked and commercially prepared foodstuffs were analysed with this assay and several were found to contain measurable amounts of one or more of the three amines. These results are presented and discussed.

Determination of an acetylcholinesterase inhibitor, MDL 73745, in dog plasma and urine by combined gas chromatography/negative ion chemical ionization mass spectrometry

A highly sensitive and specific assay has been developed for the determination of MDL 73745 [2,2,2-trifluoro-1-(3-trimethylsilyl-phenyl) ethanone] (I) and the internal standard (MDL 74398) at the nanomolar level in dog plasma and urine by gas chromatography/mass spectrometry. After a single-step extraction process, an aliquot was directly injected onto the gas chromatograph column. The mass spectrometer was run in the negative ion chemical ionization mode with ammonia as reagent gas, and was set to monitor the abundant M-. ion at m/z 246 of both compounds. The method yielded a linear response over the concentration range 0.1-10 pmol 100 microliters -1 plasma or urine. Within-day reproducibility at a concentration of 0.25, 1 and 5 pmol 100 microliters -1 plasma was 8.6%, 1.0% and 1.0%, respectively. The method was applied to the determination of I in plasma and urine after administration of 1 mg kg-1 i.v. and 10 mg kg-1 p.o. to dogs.

Stereoselective disposition: enantioselective quantitation of 3,4-(methylenedioxy) methamphetamine and three of its metabolites by gas chromatography/electron capture negative ion chemical ionization mass spectrometry.

A new chiral assay for 3,4-(methylenedioxy)methamphetamine (MDMA) and three of its metabolites in biological specimens is based on direct aqueous derivatization with N-heptafluorobutyryl-S-prolyl chloride, followed by capillary chromatographic separation of the diastereomeric derivatives and detection by a mass spectrometer operated in the electron capture negative ion chemical ionization mode. The assay is linear from 5 to 1000 ng ml-1 for each enantiomer and allows simultaneous quantitation of MDMA and three of its metabolites in biological specimens. Investigation of the disposition of racemic MDMA in rats and mice revealed quantitative differences in the disposition of the enantiomers of MDMA in these species; the most noteworthy result was a two-fold greater urinary excretion of the neurotoxic S-(+)-MDMA by mice. Only MDMA and 3,4-(methylenedioxy)amphetamine (MDA) enantiomers were detected at measurable concentrations in the frontal cortices and hippocampis from rats dosed with 10 mg kg-1 of racemic MDMA; in this species the enantiomeric profiles of these two compounds were similar in brain and urine.

Renal vasodilator activity of 5,6-epoxyeicosatrienoic acid depends upon conversion by cyclooxygenase and release of prostaglandins

The 5,6-epoxyeicosatrienoic acid (5,6-EET), a renal vasodilator metabolite of arachidonic acid via cytochrome P450 (P450) requires cyclooxygenase for expression of its vasoactivity as the responses are inhibited by indomethacin and other aspirin-like drugs. We now report on the metabolism of 5,6-EET by rabbit kidneys in order to characterize those metabolites that may account for its vasoactivity. The 5,6-EET was injected close-arterially into the rabbit isolated Krebs-Henseleit perfused kidney, preconstricted with phenylephrine, and the effluent collected throughout the response period. Basal collections, following injection of 100 microliters of vehicle, were made at 20-min intervals before each 5,6-EET injection. Prior to acidic extraction, deuterated 6-keto-prostaglandin (PG) F1 alpha and PGE2 were added as internal standards. The extracts were separated by TLC and prostaglandins were derivatized for gas chromatography-mass spectrometry analysis using a negative ion chemical ionization mode. Injection of 0.5, 1, 5, 10, and 20 micrograms of 5,6-EET (n = 4) resulted in dose-related decreases in perfusion pressure of 6 +/- 2, 12 +/- 4, 21 +/- 4, 26 +/- 4, and 27 +/- 7 mm Hg, respectively. Basal perfusates contained 6-keto-PGF1 alpha and PGE2, levels of which were increased by 2-fold or more by 5,6-EET. Perfusates, collected during 5,6-EET administration, also contained 5-hydroxy-PGI1 and 5,6-epoxy-PGE1, cyclooxygenase metabolites of 5,6-EET. This is the first report of the recovery and identification of these 5,6-EET metabolites from an intact organ. Since the responses to 5,6-EET are endothelial-dependent, we also studied the profile of eicosanoids formed following incubation of 5,6-EET with cultured bovine pulmonary endothelial cells. Endothelial cells metabolized 5,6-EET to products with a similar radioactive profile on reverse-phase high pressure liquid chromatography compared to kidney perfusates. We compared the vasodilator activity of 5,6-epoxy-PGE1 and 5-hydroxy-PGI1, chemically synthesized by us from PGE2 and PGF2 alpha, respectively, with PGE2 and PGI2 in the rabbit kidney. The 5,6-epoxy-PGE1 was equipotent to PGE2 as a vasodilator. The ED50 values for 5,6-EET, 5,6-epoxy-PGE1, and PGE2 were 4.69, 0.43, and 0.42 nmol, respectively. Although PGI2 was a potent vasodilator (ED50, 0.24 nmol), 5-hydroxy-PGI1 was devoid of activity. Thus, the cyclooxygenase-dependent vasoactivity of 5,6-EET in the rabbit kidney has two components: release of vasodilator prostaglandins, PGE2 and PGI2, and metabolism of 5,6-EET to a prostaglandin analog, 5,6-epoxy-PGE1.