Positive-ion Electron Impact Mode Research Articles

Determination of Phthalic Acid Ester in Dunaliella by GC-MS

The aim of this study was to evaluate the levels of PAEs in Dunaliella Salina algae. A determination of PAEs in Dunaliella Salina algae was discussed. After concentration, the samples were directly injected into GC-MS in positive-ion electron impact mode. The method was applied to real sample and could potentially overcome the interference from large amounts of pigment. It was shown to be a reliable method for determination of PAEs in routine analysis.

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.

Determination of tricyclic antidepressants in human plasma using pipette tip solid‐phase extraction and gas chromatography–mass spectrometry

A method for the simultaneous extraction of four tricyclic antidepressants from human plasma samples using pipette tip SPE with MonoTip C(18) tips is presented. Human plasma (0.1 mL) containing four tricyclic antidepressants (amitriptyline, amoxapine, imipramine, and trimipramine) and an internal standard (IS), protriptyline, was mixed with 0.4 mL of distilled water and 100 microL 1 M NaOH solution. After centrifugation of the mixture, the supernatant was extracted to the C(18) phase of the tip by 20 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained in the tip were eluted with methanol by five repeated aspirating/dispensing cycles. Without evaporation and reconstitution, the eluate was directly injected into a gas chromatograph injector and detected by a mass spectrometer with SIM in the positive-ion electron impact mode. Recovery of the four antidepressants and IS spiked into human plasma was 80.2-92.1%. The regression equations for the four antidepressants showed excellent linearity in the range of 0.2-40 ng/0.1 mL. LODs and LOQs for the four drugs were 0.05-0.2 ng/0.1 mL and 0.2-0.5 ng/0.1 mL, respectively. Intra- and interday CVs for the four drugs in plasma were no greater than 9.5%.

Determination of fluoride in human whole blood and urine by gas chromatography-mass spectrometry

We developed a simple and sensitive method for determination of fluoride in human whole blood and urine using gas chromatography-mass spectrometry (GC-MS). Fluoride was alkylated with pentafluorobenzyl bromide in a mixture of acetone and phosphate buffer (pH 6.8). The derivative obtained was analyzed by GC-MS in the positive-ion electron-impact mode. The lower limit of detection for the compound was 0.5 mg/l for both matrices. The calibration curve for fluoride was linear over the concentration range of 1–100 mg/l. The precision and accuracy of the method were evaluated, and relative standard deviation was within 10%. Using this method, levels of fluoride in whole blood and urine were determined in a case of poisoning caused by hydrofluoric acid exposure.

Pipette tip solid-phase extraction and gas chromatography – mass spectrometry for the determination of methamphetamine and amphetamine in human whole blood

Methamphetamine and amphetamine were extracted from human whole blood samples using pipette tip solid-phase extraction (SPE) with MonoTip C(18) tips, on which C(18)-bonded monolithic silica gel was fixed. Human whole blood (0.1 mL) containing methamphetamine and amphetamine, with N-methylbenzylamine as an internal standard, was mixed with 0.4 mL of distilled water and 50 microL of 5 M sodium hydroxide solution. After centrifugation, the supernatant was extracted to the C(18) phase of the tip (pipette tip volume, 200 microL) by 25 repeated aspirating/dispensing cycles using a manual micropipettor. Analytes retained in the C(18) phase were eluted with methanol by five repeated aspirating/dispensing cycles. After derivatization with trifluoroacetic anhydride, analytes were measured by gas chromatography - mass spectrometry with selected ion monitoring in the positive-ion electron impact mode. Recoveries of methamphetamine and amphetamine spiked into whole blood were more than 87.6 and 81.7%, respectively. Regression equations for methamphetamine and amphetamine showed excellent linearity in the range of 0.5-100 ng/0.1 mL. The limits of detection for methamphetamine and amphetamine were 0.15 and 0.11 ng/0.1 mL, respectively. Intra- and interday coefficients of variation for both stimulants were not greater than 9.6 and 13.8%, respectively. The determination of methamphetamine and amphetamine in autopsy whole blood samples is presented, and was shown to validate the present methodology.

Simultaneous determination of methamphetamine and amphetamine in human urine using pipette tip solid-phase extraction and gas chromatography–mass spectrometry

Methamphetamine and amphetamine were extracted from human urine samples using pipette tip solid-phase extraction (SPE) with MonoTip C 18 tips (pipette tip volume, 200 μl), in which C 18-bonded monolithic silica gel was fixed. A sample of human urine (0.5 ml) containing methamphetamine, amphetamine, and N-methylbenzylamine as internal standard (IS), was mixed with 25 μl of 1 M sodium hydroxide solution. The mixture was extracted into the C 18 phase of the SPE tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. Analytes retained in the C 18 phase were then eluted with methanol by five repeated aspirating/dispensing cycles. After derivatization with trifluoroacetic anhydride, analytes were measured by gas chromatography/mass spectrometry with selected ion monitoring in the positive-ion electron impact mode. Recoveries of methamphetamine, amphetamine, and IS spiked into urine were more than 82.9, 82.2, and 78.2%, respectively. Regression equations for methamphetamine and amphetamine showed excellent linearity in the range of 0.25–200 ng/0.5 ml. Limit of detection was 0.04 ng/0.5 ml for methamphetamine and 0.05 ng/0.5 ml for amphetamine. Intra- and inter-day coefficients of variations for both stimulants were not greater than 10.8%. The data obtained from actual determination of methamphetamine and amphetamine in autopsy urine samples are also presented for validation of the method.

Pipette tip solid-phase extraction and gas chromatography–mass spectrometry for the determination of mequitazine in human plasma

Mequitazine has been found to be extractable from human plasma samples using MonoTip C 18 tips, inside which C 18-bonded monolithic silica gel was fixed. Human plasma (0.1 mL) containing mequitazine and cyproheptadine as an internal standard (IS) was mixed with 0.4 mL of distilled water and 25 μL of 1 M potassium phosphate buffer (pH 8.0). After centrifugation of the mixture, the supernatant fraction was extracted to the C 18 phase of the tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C 18 phase were then eluted with methanol by five repeated aspirating/dispensing cycles. Without evaporation and reconstitution, the eluate was injected into a gas chromatograph injector and detected by a mass spectrometer with selected ion monitoring in the positive-ion electron impact mode. The separation of mequitazine and the IS from each other and from impurities was generally satisfactory using a DB-1MS capillary column (30 m × 0.32 mm i.d., film thickness 0.25 μm). The recoveries of mequitazine and the IS spiked into plasma were more than 90.0%. The regression equation for mequitazine showed excellent linearity in the range of 0.2–200 ng 0.1 mL −1, and the detection limit was 0.05 ng 0.1 mL −1of plasma. The intra-day and inter-day coefficients of variation for mequitazine in human plasma were not greater than 8.16 and 9.24%, respectively. Accuracy for the drug was in the range of 90.0–97.4%. The data obtained from determination of mequitazine in human plasma after oral administration of the drug are also presented.

Simultaneous determination of ten antihistamine drugs in human plasma using pipette tip solid‐phase extraction and gas chromatography/mass spectrometry

Ten antihistamine drugs, diphenhydramine, orphenadrine, chlorpheniramine, diphenylpyraline, triprolidine, promethazine, homochlorcyclizine, cyproheptadine, cloperastine and clemastine, have been found to be extractable from human plasma samples using MonoTip C18 tips, inside which C18- bonded monolithic silica gel was fixed. Human plasma (0.1 mL) containing the ten antihistamines was mixed with 0.4 mL of distilled water and 25 microL of a 1 M potassium phosphate buffer (pH 8.0). After centrifugation of the mixture, the supernatant fraction was extracted to the C18 phase of the tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C18 phase were then eluted with methanol by five repeated aspirating/dispensing cycles. The eluate was injected into a gas chromatography (GC) injector without evaporation and reconstitution steps, and was detected by a mass spectrometer with selected ion monitoring in the positive-ion electron impact mode. The separation of the ten drugs from each other and from impurities was generally satisfactory using a DB-1MS column (30 m x 0.32 mm i.d., film thickness 0.25 microm). The recoveries of the ten antihistamines spiked into plasma were 73.8-105%. The regression equations for the ten antihistamines showed excellent linearity with detection limits of 0.02-5.0 ng/0.1 mL. The within-day and day-to-day coefficients of variation for plasma were not greater than 9.9%. The data obtained from determination of diphenhydramine and chlorpheniramine in human plasma after oral administration of the drugs are also presented.

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

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.

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.

Positive and negative ion mass spectrometry and rapid isolation with Sep-Pak C 18 cartridges of ten local anaesthetics

The positive ion electron impact (PIEI), positive ion chemical ionization (PICI) and negative ion chemical ionization (NICI) mass spectra and a rapid isolation procedure using Sep-Pak C 18 cartridges are presented for ten local anaesthetics. In the PIEI mode, molecular peaks were very small or missing for most compounds. Peaks at m z 86 due to the diethylaminomethyl or propylaminoethyl group constituted base peaks in six compounds. In the PICI mode, peaks due to M + H and M + C 2H 5 appeared. The cation at m z 86 was also observed for the six compounds. This ion seems useful for the screening of local anaesthetics. In the NICI mode, anions at m z M − H constituted base peaks for all compounds, peaks at m z M + 12 appeared in many compounds. The total ion current in the PIEI and PICI modes generally gave higher sensitivity than in the NICI mode. Local anaesthetics present in whole blood or cerebrospinal fluid (CSF) could be rapidly isolated by use of Sep-Pak C 18 cartridges with chloroform/methanol as an elution solvent. Their detection was possible using wide-bore capillary gas chromatography with SPB-1 and HP-17 wide-bore capillary columns with satisfactory separation from impurities.

Positive- and negative-ion mass spectrometry and rapid clean-up of some carbamate pesticides

Positive-ion electron impact (PIEI), positive-ion chemical ionization (PICI) and negative-ion chemical ionization (NICI) mass spectra of 9 carbamate pesticides are presented. In the PIEI mode, the spectra showed small molecular peaks, intense or base peaks due to M − CH 3NHCO + H and peaks at m z 58 due to CH 3NHCO. In the PICI mode, peaks due to M + H, M + C 2H 5, M − CH 3NHCO + 2H, CH 3NHCO( m z 58) and M-28 appeared. The cations at m z 58 found in both PIEI and PICI modes seem very useful for screening of a carbamate. In the NICI mode, the spectra showed peaks due to M − CH 3NHCO and characteristic anions appearing at mass numbers higher than molecular ones, which were probably due to dimerization of [M − CH 3NHCO] − followed by hydrogen attachment. Carbamates, which had been added to urine, plasma, whole blood, the liver, kidney and brain, could be rapidly isolated by use of Sep-Pak C 18 cartridges with chloroform as an elution solvent. They could be detected by wide-bore capillary gas chromatography with a SPB-5 column, with satisfactory separation from impurities in their underivatized forms.

Positive- and negative-ion mass spectrometry and rapid clean-up of 19 phenothiazines

Positive-ion electron impact (PIEI), positive-ion chemical ionization (PICI) and negative-ion chemical ionization (NICI) mass spectra of 19 phenothiazines are presented. In the PIEI mode, peaks due to M, M minus side chain (M − R 1), M − R 1 + H, and side chain itself (R 1) appeared for most compounds. The M − R 1 and R 1 ions were very useful for drug screening. In the PICI mode, most spectra showed base or intense peaks due to M + H, and small peaks due to M + C 2H 5; peaks due to M − R 1 + 2H and R 1 also appeared in many compounds. In the NICI mode, fragmentation modes were different in different compound groups; molecular or [M − H] − quasi-molecular anions appeared in many compounds with aliphatic side chains. Anions at m z 98 and 115 were characteristic for compounds with ( N-methylpiperazinyl)propyl side chains. Selected ion monitoring in the PIEI mode generally gave much higher sensitivity than in the PICI and NICI modes. Phenothiazines present in urine or plasma could be rapidly isolated by use of Sep-Pak C 18 cartridges. Thirteen of 19 phenothiazines could be detected by HP-17 wide-bore capillary gas chromatography with satisfactory separation from impurities in their underivatized forms.

Positive and negative ion mass spectrometry of antiepileptic hydantoins and their analogs

Positive ion electron impact (PIEI), positive ion chemical ionization (PICI), and negative ion chemical ionization (NICI) mass spectra of seven compounds of hydantoins and their analogs are presented; their probable fragmentation modes are also presented. In the PIEI mode, intensities of molecular ions differed according to different compounds. Cleavage at outsides of both carbonyl groups was commonly observed for all compounds. In the PICI mode, all compounds showed [M + 1]+ quasi-molecular ions constituting the base peaks. In the NICI mode, [M - 1]- quasi-molecular anions were the base peaks except for trimethadione and paramethadione. All negative spectra showed anions at m/z 42 due to [NCO]-; these peaks seem useful for screening of antiepileptics. An extraction procedure for the anti-epileptics from human urine or plasma, and their separation by gas chromatography (GC), are also presented to serve for their actual identification by GC/mass spectrometry.

Positive- and negative-ion mass spectrometry of 24 benzodiazepines

Positive-ion electron impact (PIEI), positive-ion chemical ionization (PICI) and negative-ion chemical ionization (NICI) mass spectra of 24 benzodiazepines are presented. In the PIEI mode, peaks due to M, M − H, M − CO and a liberated R 3-phenyl ring appeared in many compounds. The peaks at m z M − 47 and m z 56 or 70 were characteristic for nitro- and oxazolo-compounds, respectively. In the PICI mode, most spectra showed the base peaks due to M + H and small peaks due to M + C 2H 5; small peaks due to M − R 1 or M − R 1 + 2H also appeared for many compounds. For the nitro-compounds, peaks at m z M − 29 commonly appeared. For the oxazolo-compounds, peaks due to the loss of the R 3-phenyl ring appeared. In the NICI mode, either molecular or [M − H] − quasi-molecular anions were the base peaks in 10 compounds. Small anions at m z M + 15 (M − H + O) also appeared in many compounds. Anions due to liberated halogens were observed for most compounds; bromine gave its base peaks. An procedure for benzodiazepines from human urine and plasma, and their separation by gas chromatography (GC) with a wide-bore capillary column are also presented to serve for their actual identification by GC/mass spectrometry (MS). The wide-bore capillary column was useful to prevent the drugs from decomposition.