Fast atom bombardment tandem mass spectrometric identification of diacyl, alkylacyl, and alk-1-enylacyl molecular species of glycerophosphoethanolamine in human polymorphonuclear leukocytes.

Fast atom bombardment ionization with tandem mass spectrometry of both positive and negative ions is a useful technique for the identification of intact glycerophosphoethanolamine (GPE) phospholipids, providing information as to polar head group and fatty acyl substituents. In the identification of GPE molecular species, positive ion neutral loss scanning for 141 units was attempted to confirm the presence of the phosphoethanolamine polar head group. This scan was found to discriminate against the abundant subclass of phospholipids having an 1-O-alk-1'-enyl linkage, termed plasmalogens, as well as 1-O-alkyl ether species. The neutral loss process is suggested to involve attack of a carbonyl oxygen from either sn-1 or sn-2 on the sn-3 methylene carbon with loss of neutral phosphoethanolamine. Using FAB/MS/MS alone, it is not possible to differentiate between plasmalogens and other 1-O-alkyl ether molecular species having the same molecular weight. The combination of mild acid hydrolysis, which selectively hydrolyzes the labile 1-O-alk-1'-enyl bond, with subsequent FAB/MS/MS distinguished species of these distinct subclasses. Using these techniques and precursor ion scans for the arachidonoyl carboxylate anion, m/z 303, the arachidonic acid containing glycerophosphoethanolamine molecular species were identified and the relative abundance of arachidonoyl plasmalogen, alkylacyl, and 1,2-diacyl GPE molecular species in the human polymorphonuclear leukocyte (neutrophil) was determined to be 75.4%, 12.1%, and 12.5%, respectively. These values were not significantly different from that reported in the literature using conventional methodology.

Incorporation and distribution of epoxyeicosatrienoic acids into cellular phospholipids.

The different regioisomers of epoxyeicosatrienoic acids derived from cytochrome P-450 monooxygenase are readily esterified into phospholipids of mastocytoma cells. Incorporation of 14,15-epoxyeicosatrienoic acid was concentration-dependent, with Km = 1.1 microM and Vmax = 36 pmol/min/10(7) cells. Half-maximal incorporation occurred in 30 min, reaching a steady-state concentration of 470 pmol/10(6) cells. This was slightly lower than the values for arachidonic acid (665 pmol/10(6) cells) or 5-hydroxyeicosatetraenoic acid (554 pmol/10(6) cells). The distribution of 14,15-epoxyeicosatrienoic acid was preferential in the order phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidyl serine much greater than neutral lipids plus fatty acids. This contrasted with 5(S)-hydroxyeicosatetraenoic acid, which was distributed primarily into phosphatidylcholine. Fast atom bombardment/tandem mass spectrometry facilitated identification of molecular species containing epoxyeicosatrienoic acids without relying on radioisotopes. Phosphatidylethanolamine plasmalogens with 16:1 or 18:2 at the sn-1 position, or an 18:0 acyl group, and phosphatidylcholine with 16:0 alkyl ether or an acyl group at the sn-1 position incorporated all possible epoxyeicosatrienoic acid regioisomers. Under basal conditions, cells eliminated 14,15-cis-epoxyeicosatrienoic acid slowly with a half-life of 34.9 +/- 7 h. Cells stimulated with calcium ionophore A23187 eliminated 14,15-epoxyeicosatrienoic acid rapidly. It was notable that its rate of release from phosphatidylcholine and phosphatidylinositol exceeded that for arachidonic acid. A coenzyme A-independent transacylase also catalyzed the transfer of epoxyeicosatrienoic acids from mastocytoma cell membranes into 1-palmitoyl-2-lysophosphatidylcholine. The cellular incorporation, release, and distribution of epoxyeicosatrienoic acids is distinctive and contrasts with most other eicosanoids, suggesting that these compounds may have both autocoid and nonautocoid functions.

Collisionally induced dissociation of epoxyeicosatrienoic acids and epoxyeicosatrienoic acid-phospholipid molecular species.

Four isomers of epoxyeicosatrienoic acid (EET) can be formed by cytochrome P-450 oxidation of arachidonic acid: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. The collision-induced dissociation of the [M-H]- anion at m/z 319 from each of these isomers, using negative-ion fast atom bombardment ionization and a triple quadrupole mass spectrometer, resulted in a series of common ions as well as ions characteristic of each isomer. The common ions were m/z 301 [M-H2O]- and 257 [M-(H2O + CO2)]-. Unique ions resulted from cleavages alpha to the epoxide moiety to form either conjugated carbanions or aldehydes. Mechanisms involving charge site transfer are suggested for the origin of these ions. A distonic ion series that may involve a charge-remote fragmentation mechanism was also observed. The epoxyeicosatrienoic acids were also incorporated into cellular phospholipids following incubation of the free acid with murine mast cells in culture. Negative fast atom bombardment mass spectrometry of purified glycerophosphoethanolamine-EET species and glycerophosphocholine-EET species yielded abundant [M-H]- and [M-CH3]- ions, respectively. The collision-induced dissociation of these specific high-mass ions revealed fragment ions characteristic of the epoxyeicosatrienoic acids incorporated (m/z 319, 301, and 257) and the same unique ions as those seen with each isomeric epoxyeicosatrienoic acid. With this direct method of analysis, phospholipids containing the four positional isomers of EET, including the highly labile (5,6-EET), could be identified as unique molecular species in mast cells incubated with EET.

Molecular species analysis of arachidonate containing glycerophosphocholines by tandem mass spectrometry.

Carboxylate anions arising from collision-induced dissociation (CID) of the [M - 15](-) ion produced by fast atom bombardment (FAB) of glycerophosphocholine (GPCho) were previously shown to be produced in an abundance ratio of 1:3 for the carboxylic acids esterified at sn - 1 and sn - 2, respectively. This observation has been confirmed in a series of 13 synthetic GPCho molecular species. A good correlation was found between the isomeric purity of GPCho molecular species as determined by negative-ion FAB/CID analysis and the isomeric purity of the sn - 2 fatty acid using a phospholipase A2 assay. Negative-ion FAB mass spectra of several 1-0-alkyl-2-acyl-GPCho molecular species were found to be similar to those of diacyl GPCho. However, the cm spectra from the major high-mass ions are different from those of the diacyl species in that the [M - 15](-) ion yields only one carboxylate anion and the [M - 86](-) undergoes a neutral loss of the sn - 2 carboxylic acid as a major decomposition product. These results suggest several rules useful for structural characterization of GPCho molecular species by negative-ion tandem mass spectrometry (MS/MS): (1) For diacyl species, the mass of the two carboxyl anions plus the mass of the GPeho backbone (minus a methyl group) must correspond to the mass of the [M - 15] anion; (2) for diacyl species there is a carboxylate anion ratio approximately 1:3 for the substituents at sn - 1 and sn - 2; and (3) for alkylether species, only one fatty acyl group is present, and the difference between the [M - 15] ion and the GPCho backbone (minus methyl) plus the fatty acyl group at sn - 2 corresponds to an alkylether substituent. (4) Assignment of ether-linked molecular species can be made from the [M - 86](-) ion, which has a strong neutral loss of the sn - 2 fatty acid.Analysis of GPCho isolated from human neutrophils by total lipid extraction and normal-phase HPLC was carried out by negative-ion FABand MS/MS. The major arachidonate-eontaining molecular species, which comprise only 5% of total GPCho, were identified by using precursor ion scans for the arachidonate anion, m/ z 303. Decomposition of identified. precursor ions permitted the assignment of those molecular species of GPCho that contain arachidonate at sn - 2 and identification of the substituent at the sn - 1 position. These results were compared to previously identified molecular species from human neutrophils. Several minor arachidonate-containing molecular species were tentatively identified.

Negative ion tandem mass spectrometry of leukotriene E4, and LTE 4, metabolites: Identification of LTE 4, in human urine.

The sulfidopeptide leukotrienes, leukotriene E4, (LTE4,) and its N-acetyl derivative and several ω- and ß-oxidized metabolites of LTE4, have been analyzed by tandem mass spectrometry. [M-H](-) ions were produced by continuous flow fast atom bombardment, and collision-induced dissociation of these ions was studied by using a triple quadrupole instrument. The product ion spectra obtained were characteristic of the structure of LTE4, and mechanisms of ion formation were investigated by using deuterated compounds. ß-Elimination of the peptide portion of LTE4, by loss of CO2, and ethylene amine leaves the C-l carboxyl group ionized in the most abundant fragment ion for LTE4, and all metabolites. Tandem mass spectrometry of fast atom bombardment-generated anions from ω- and ß-oxidized metabolites of LTE4, produced similar ions with only a minor influence of the third carboxyl group at the omega terminus evident. Tandem mass spectrometry was used to identify unequivocally the presence of unmodified LTE4, in a high performance liquid chromatography-purified fraction of urine from a normal healthy volunteer after infusion with LTE4.

Determination of plasma dexamethasone by chemical oxidation and electron capture negative ionization mass spectrometry.

A quantitative assay for dexamethasone in human plasma is described as based on methodology employing electron capture negative ionization mass spectrometry (ECNI/MS). The unique feature of this assay is the sample preparation involving chemical oxidation which transforms dexamethasone to a highly electrophilic species while not significantly affecting the electrophilic character of the biochemical matrix, thereby permitting selective and sensitive detection of the analyte. Optimized chemical procedures and instrumental parameters are described for the detection of oxidized dexamethasone. Finally, the newly developed methodology, based on gas chromatography/mass spectrometry (GC/MS) with ECNI, is evaluated by comparison with a conventional GC/MS assay using electron impact of an 11,17,21-tris-trimethylsilyl ether 20-enol-trimethylsilyl ether derivative of dexamethasone and with a radioimmunoassay in the analysis of pooled samples of human plasma containing widely varying concentrations of the drug as obtained from patients at different stages of the dexamethasone suppression test.

Comparison of tandem and conventional mass spectrometry using electron capture negative ionization in the detection of chemically oxidized dexamethasone in human plasma.

Chemistry and Biochemistry, Michigan State University, East Lansing, Michigan, USA Dexamethasone, a synthetic steroid, can be oxidized chemically to a ketonic steroid structure that is readily detected by electron capture negative ionization mass spectrometry (ECNI/MS). Previous work from this laboratory has demonstrated that the chemical oxidation procedure provides advantages of low detection limits and high selectivity for detection of oxidized dexamethasone against chemical background that would otherwise interfere with detection of this steroidal drug in biological samples using more conventional methodology. This report describes the extent to which tandem mass spectrometry (MS/MS) can further enhance the selectivity of the oxidation/ECNI methodology for the detection of dexamethasone during the analysis of human plasma and presents evidence that sample introduction by direct inlet probe (DIP) can be used successfully under ECNI conditions. For purposes of comparing the methodologies, the same human plasma samples are analyzed by ECNI, first with detection by conventional mass spectrometry using selected ion monitoring (SIM) and then by MSIMS using selected reaction monitoring (SRM) with sample introduction by the gas chromatographic (GC) inlet and by the DIP. The results indicate that use of the DIP is a viable means of sample introduction for ECNI when sample processing involves the specialized oxidation procedure described herein because the sample matrix does not compete significantly for the thermal electrons in the ion source. Whereas SIM and SRM provide comparable results when sample introduction is achieved by the GC inlet, the MS/MS approach offers the possibility for sample introduction using the DIP, which significantly simplifies and shortens the analysis.