Thermospray Liquid Chromatography-mass Spectrometry Research Articles

Naturally occurring monoepoxides of eicosapentaenoic acid and docosahexaenoic acid are bioactive antihyperalgesic lipids

Beneficial physiological effects of long-chain n-3 polyunsaturated fatty acids are widely accepted but the mechanism(s) by which these fatty acids act remains unclear. Herein, we report the presence, distribution, and regulation of the levels of n-3 epoxy-fatty acids by soluble epoxide hydrolase (sEH) and a direct antinociceptive role of n-3 epoxy-fatty acids, specifically those originating from docosahexaenoic acid (DHA). The monoepoxides of the C18:1 to C22:6 fatty acids in both the n-6 and n-3 series were prepared and the individual regioisomers purified. The kinetic constants of the hydrolysis of the pure regioisomers by sEH were measured. Surprisingly, the best substrates are the mid-chain DHA epoxides. We also demonstrate that the DHA epoxides are present in considerable amounts in the rat central nervous system. Furthermore, using an animal model of pain associated with inflammation, we show that DHA epoxides, but neither the parent fatty acid nor the corresponding diols, selectively modulate nociceptive pathophysiology. Our findings support an important function of epoxy-fatty acids in the n-3 series in modulating nociceptive signaling. Consequently, the DHA and eicosapentaenoic acid epoxides may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.

Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography–thermospray mass spectrometry

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC–thermospray mass spectrometry. The HPLC–thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa + and MH +–H 2O ions. As the other minor ions, MK +, MH + and m/ z 282 ions were detected. Although the preponderance of MNa + ions compared with the MH +–H 2O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH +–H 2O ions based on the elimination of the hydroxyl group introduced at the α-position of acyl-portion compared with the MNa + ions was detected in α-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor ( f) of each ceramide was calculated as follows; N-oleoyl- d-sphingosine ( f=0.45), N-palmitoyl- d-sphingosine ( f=0.40), N-stearoyl- d-sphingosine ( f=0.39), N-nervonoyl- d-sphingosine ( f=0.39) and N-lignoceroyl- d-sphingosine ( f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100 000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa +/MH +–H 2O ions corresponding to several ω-hydroxyacyl-ceramides were detected.

Metabolic fate of delmopinol in man after mouth rinsing and after oral administration

1. The metabolism of delmopinol, an inhibitor of plaque formation and gingivitis, has been studied after mouth rinsing or an oral dose of 14C-delmopinol to healthy male volunteers. The metabolite pattern was studied in urine and plasma samples (unhydrolysed or hydrolysed with conjugate cleaving enzymes) by liquid chromatography (LC) with radio detection. Metabolite identifications were carried out by gas chromatography-electron-impact mass spectrometry (GC-MS) and by liquid chromatography-thermospray mass spectrometry (LC-MS). 2. The metabolic clearance of delmopinol was high, and < 0.2% of the dose was excreted as intact delmopinol in the urine. The main metabolites were, for both administration routes, glucuronide conjugates of delmopinol and of (ω-1-hydroxy) delmopinol. These metabolites were predominant and accounted for nearly the entire urinary radioactivity and most of the plasma radioactivity. After mouth rinsing, parent delmopinol was also one of the main compounds in plasma. 3. Several other products of oxidation of the aliphatic side-chain were present in minor amounts in urine. These metabolites also appeared to be excreted as glucuronic acid conjugates. 4. Glucuronidated (ω-1-hydroxy) delmopinol separated into three peaks by the LC system used. This could be due to different chromatographic properties of conjugate isomers, positional or optical.

Glutathione oxidation in real time by thermospray liquid chromatography–mass spectrometry

The oxidation and reduction of glutathione and oxidized glutathione were studied in real time by liquid chromatography–mass spectrometry during exposure to hydrogen peroxide and mercaptoethanol. By mass spectrometry mixed disulfides and both reversible and irreversible oxidations of sulfur to higher states (sulfinic and sulfonic acids) were directly observed during exposure to hydrogen peroxide. The irreversible oxidation of glutathione to glutathione sulfonic acid could be detected after 30 min exposure of glutathione to 40 m M H 2O 2 at 20°C. A peak consistent with glutathione-sulfinic acid was transiently present, suggesting this compound behaved as an oxygen consuming antioxidant. Liquid chromatography–mass spectrometry appears to be an excellent method to study oxidation and reductions of sulfur containing peptides and amino acids.

Xanthones, triterpenes and a biphenyl from Kielmeyera coriacea

Analysis of the dichloromethane extracts of Kielmeyera coriacea leaves and stems by high performance liquid chromatography coupled with a photodiode array detector (LC-UV) and thermospray liquid chromatography-mass spectrometry (TSP/LC-MS) revealed the presence of several xanthones. Phytochemical investigation of these extracts resulted in the isolation and identification of ten xanthones, one biphenyl and two triterpenes. Their structures were established by chemical and spectroscopic methods (UV, EI-MS, D/CI-MS, 1H NMR, 13C NMR, HMQC and HMBC). One xanthone and two triterpenes are new compounds. Four xanthones and the biphenyl exhibited antifungal activity against the plant pathogenic fungus Cladosporium cucumerinum, while two prenylated xanthones inhibited the growth of Candida albicans.

Resolution and quantitative determination of coeluted pesticide mixtures in liquid chromatography–thermospray mass spectrometry by multivariate curve resolution

Resolution and quantitative determination of a mixture of coeluted pesticides (carbofuran, propoxur and pirimicarb) in liquid chromatography–thermospray mass spectrometry by multivariate curve resolution is shown. Carbofuran and pirimicarb both exhibit an ion with m/z 239 in their thermospray mass spectrum. For the first compound this ion is attributed to the ammonium adduct [M+NH4]+ ion, whereas the corresponding ion of pirimicarb is the protonated molecule, [M+H]+. A third carbamate, propoxur, was added because this compound partly coelutes with carbofuran under normal experimental conditions during multiresidue analysis of carbamates with C18 packed analytical columns. With this additional compound, the so-called suppression effects on the ion formation of these compounds in thermospray mass spectrometry and their quantitative resolution using chemometric methods were studied.

Identification of soyasaponins by liquid chromatography–thermospray mass spectrometry

Saponins are important bioactive molecules widespread in the plant kingdom. Soyasaponins, isolated from Glicine max (Leguminosae), have been shown to exhibit various biological activities, e.g., an inhibitory effect on lipid-oxidation and liver-lesion generation and an improving effect on hyper-cholesteremia. Mass spectral investigation of these metabolites requires soft ionization techniques such as desorption chemical ionization, fast atom bombardment and thermospray mass spectrometry. A high-performance liquid chromatographic method was developed for thermospray mass spectrometric analysis of saponins contained in soybean flour extracts. The analyses were performed using a ternary eluent (water–acetonitrile–methanol) in gradient conditions with post-column addition of aqueous ammonium acetate. Six saponin components could be separated and identified. The mass spectra obtained provided information concerning both molecular masses and aglycone composition.

Quantification of corticosteroids in human plasma by liquid chromatography–thermospray mass spectrometry using stable isotope dilution

Liquid chromatography–thermospray mass spectrometry (LC–TSP-MS) using isotope dilution was investigated for quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma. Complete separation attained by a LiChroCART Supersupher reversed-phase column and elution with 0.05 M ammonium formate–tetrahydrofuran–methanol (180:53:17, v/v/v) resulted in a significantly large isotope effect of the deuterium-labeled analogs on the HPLC behavior and caused difficulty in quantification. Reduction of the isotope effect on the retention times using 0.05 M ammonium formate–acetonitrile (65:35, v/v) permitted accurate quantification of cortisol and cortisone by the isotope dilution LC–TSP-MS, although separation between cortisol and prednisone was incomplete.

Determination of the furazolidone metabolite, 3-amino-2-oxazolidinone, in porcine tissues using liquid chromatography-thermospray mass spectrometry and the occurrence of residues in pigs produced in Northern Ireland

A method is presented for the detection of the furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ), in porcine tissue. Bound and extractable residues are detected following methanol-water homogenisation, repeated solvent washing and derivatisation with 2-nitrobenzaldehyde. Samples are analysed by using thermospray mass spectrometry-liquid chromatography, monitoring the positive ion m/z 253 with filament-assisted ionisation. There is no interference from tissue matrices or excess 2-nitrobenzaldehyde reagent. The limit of determination for liver and muscle is 10 ng/g. Recoveries are greater than 80%. The assay was used to investigate the occurrence of furazolidone residues in pigs from Northern Ireland. One hundred samples were analysed. Seventeen of these contained bound AOZ residues. The stability of tissue samples post mortem was investigated in order to achieve optimum storage conditions for samples. When liver was stored at 4 degrees C, the concentration of bound AOZ decreased by 22% within 48 h. However, there were no significant changes in AOZ concentrations in liver that was stored at -20 degrees C for six months.

Isolation of Polyphenols fromPolygala gazensis and Liquid Chromatography-Mass Spectrometry of Related AfricanPolygala Species

The chromonocoumarins frutinones A and B and lignans eudesmin, magnolin, yangambin and kobusin were isolated from lipophilic extract of P. gazensis. This species and two other related Polygala species were subsequently analysed by thermospray liquid chromatography–mass spectrometry and high pressure liquid chromatography coupled with photodiode-array detection. Frutinone A was also found in the roots and aerial parts of P. teretifolia and P. fruticosa, while the presence of frutinone B was limited principally to the roots. Lignans were present in the aerial parts of P. teretifolia. Frutinone A showed antifungal activity against Cladosporium cucumerinum and Candida albicans while eudesmin and kobusin were antifungal against Cladosporium cucumerinum only. © 1997 by John Wiley & Sons, Ltd.

Determination of trimethoprim in tissues using liquid chromatography-thermospray mass spectrometry.

A method is described for the determination of the antibacterial drug trimethoprim in tissues. Minced tissue is homogenised with chloroform-acetone (1 + 1 v/v), filtered, and the filtrate evaporated to an oily residue using a rotary evaporator. The residue is redissolved in methanol-water-acetic acid (50 + 48.7 + 1.3 v/v) and any fats present are partitioned into hexane. The aqueous phase is analysed by liquid chromatography-thermospray mass spectrometry in positive mode with the protonated molecular ion at m/z 291 being monitored. Recoveries ranged between 60% in liver and 79% in muscle. The limit of determination was 25 micrograms kg-1 and the limit of detection was approximately 4 micrograms kg-1. The method is suitable for monitoring tissues taken under national surveillance schemes for veterinary drug residues.

Determination of dimetridazole in poultry tissues and eggs using liquid chromatography-thermospray mass spectrometry.

A method is presented for the determination of the nitroimidazole drug dimetridazole (DMZ) in poultry tissues and eggs by liquid chromatography (LC)-thermospray mass spectrometry (MS). Deuteriated DMZ was employed as an internal standard. Samples were extracted with dichloromethane (muscle) or toluene (liver, egg) and applied to silica gel cartridges. Dimetridazole was eluted with acetone and the eluate evaporated to dryness at 40 degrees C under nitrogen. The residue was redissolved in methanol-water (1 + 1, v/v) and washed with hexane before LC-MS analysis. Quantification was by the ratios of the positive [M + H]+ ions at m/z 142 and 145 for DMZ and the internal standard, respectively. Internal standard corrected recoveries were between 93 and 102% with RSDs between 1.2 and 7.7% for liver spiked at 5, 10 and 20 ng g-1 and muscle and eggs spiked at 5 ng g-1. Absolute recoveries were approximately 80%. The method is suitable for statutory residue testing and was used to measure DMZ residues in eggs from chickens fed a diet containing DMZ at 10 mg kg-1.

Analysis of mesocarb analogues in urine and plasma of rats by high-performance liquid chromatography and thermospray liquid chromatography-mass spectrometry.

The pharmacokinetics and metabolism of synthetic 2-hydroxymesocarb and 4-methyl-2-hydroxymesocarb analyzed by HPLC-DAD and thermospray LC-MS were studied in rats. Multistep liquid-liquid extraction (LLE) was used with diethyl ether at pH 7.0. The major metabolites of 2-hydroxymesocarb in both urine and plasma of the rat were the p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug. The metabolites of 4-methyl-2-hydroxymesocarb in urine of rats may be the oxidized forms at the phenylcarbamoyl group of the parent drug. Absorption (ka) and elimination (ke) rate constants in plasma of 2-hydroxy-mesocarb were 0.0300 and 0.00485 min-1, respectively; those of 4-methyl-2-hydroxymesocarb were 0.0546 and 0.00797 min-1, respectively. The half-lives (t1/2) of 2-hydroxymesocarb and 4-methyl-2-hydroxymesocarb in plasma were 144 and 86 min, respectively.

Determination and Confirmation of Pirlimycin Residue in Bovine Milk and Liver by Liquid Chromatography/Thermospray Mass Spectrometry: Interlaboratory Study

Three laboratories participated in trials of methods to determine and confirm pirlimycin residue in bovine milk and liver. The methods used liquid chromatography/thermospray mass spectrometry (LC/MS) with an internal standard to measure residue concentration. The internal standard was isopirlimycin, a stereoisomer of pirlimycin, which was resolved chromatographically. Determinative procedures were validated by replicate analyses of negative control, fortified control, and residue-incurred milk. For the milk method, average corrected recoveries (and coefficients of variation, CVs) were 83-113% (CV, 7.5-15.4%) at 0.2 ppm, 91-98% (CV, 3.4-18.5%) at 0.4 ppm, and 89-102% (CV, 8.8-22.9%) at 0.8 ppm. For the liver method, average corrected recoveries were 94-103% (CV, 2.2-7.1%) at 0.25 ppm, 87-94% (CV, 4.8-10.3%) at 0.5 ppm, and 96-101% (CV, 5.5-6.9%) at 1.0 ppm. There were no interferences in control samples of either matrix. Pirlimycin was confirmed by matching the retention time and relative abundances of 4 ions from sample extracts to corresponding values obtained for pirlimycin standard. Pirlimycin was confirmed in all residue-incurred samples and all samples fortified at regulatory tolerances (0.4 ppm in milk and 0.5 ppm in liver) by 2 of the 3 laboratories and in most samples by the third laboratory.

Simultaneous Determination of Illicit Drugs in Human Urine by Liquid Chromatography-Mass Spectrometry

The method for simultaneous determination and confirmation of illicit drugs (e.g., methamphetamine, amphetamine, ephedrine, methylephedrine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, 6-acetylmorphine, cocaine, and benzoylecgonine) in human urine by thermospray liquid chromatography-mass spectrometry (LC-MS) was studied. The LC-MS separation was performed on a reversed phase column (L-column ODS; 150 mm x 4.6-mm i.d.) using a gradient mobile phase system of 100mM ammonium acetate for 1 min then linear ramps to 100mM ammonium acetate including 40% acetonitrile at 20 min. Extraction was conducted by solid-phase extraction using a Sep-pak C18 cartridge. The drugs were eluted with 2 mliters of 40% acetonitrile in 100 mM ammonium acetate, pH3 (adjusted with acetic acid), from the cartridge. A 50-microL volume of the eluate was injected into the LC-MS. The recoveries by this extraction were 88 to 99%. The mass spectra of methamphetamine, amphetamine, ephedrine, methylephedrine, morphine, 6-acetylmorphine, cocaine, and benzoylecgonine showed the quasi-molecular [M + H]+ ion as the base peak, whereas morphine-3-glucuronide and morphine-6-glucuronide showed [MH-glucuronide]+ ion as the base peak. The calibration graphs were linear and reproducible. Detection limits of these drugs ranged from 2 to 40 ng/mliters by selected ion monitoring (SIM) mode and from 50 to 400 ng/mliters by scan mode. The coefficients of variation for the analysis of these drugs ranged from 4.5 to 9.5% at a concentration of 0.4 micrograms/mliters (n = 10).

Analysis of quaternary ammonium drugs by thermospray liquid chromatography-mass spectrometry using a resin-based stationary phase

The analysis of quaternary ammonium drugs by thermospray liquid chromatography-mass spectrometry (LC-TSP-MS), using a resin-based stationary phase, is described. The use of such a stationary phase eliminates the need of using an ion-pairing agent, which often is incompatible with mass spectrometric detection. Gradient elution has been used to improve the separation and to reduce the analysis time. Detection limits in selective ion monitoring mode as well as in selective reaction monitoring, are in the order of 50 pg for each compound. The usefulness of the method has been demonstrated with the bioanalysis of antrenyl in plasma, using mepenzolate as an internal standard, showing a limit of determination of 1 ng ml −1.

Subcellular localization of docosahexaenoic acid and arachidonic acid ω-hydroxylation activity in the brain, liver and colonic adenocarcinoma

A homogenate of rat brain, rat liver or human colonic well differentiated adenocarcinoma was prepared in 250 m M sucrose isoosmolaric buffer (pH 7.6) and fractionated by differential centrifugation at 10 3, 10 4 and 10 5 g. Each precipitate or supernatant was incubated with NADPH and docosahexaenoic acid or arachidonic acid as a substrate for 30 min at 37°C under aerobic conditions. ω-Hydroxydocosahexaenoic acid or ω-hydroxyeicosatetraenoic acid from an incubation mixture was detected by reversed-phase high-performance liquid chromatography-thermospray mass spectrometry with selected-ion monitoring. ω-Hydroxy polyunsaturated fatty acids were characterized by high intensity of the molecular ion (MH +) although common hydroxy polyunsaturated fatty acids were characterized by high intensity of the MH + - H 2O ion. For the rat brain, ω-hydroxylation activity (the amount of ω-hydroxy product produced in 30 min) was concentrated to a 103 g precipitate although the specific activity (the activity per 1 mg of protein) in the 10 3 g precipitate did not indicate superiority over other fractions. However, the specific activity of the rat brain increased on addition of a 10 4 or 10 5 g precipitate. For the rat liver, although ω-hydroxylation activity was concentrated to a 10 3 g precipitate, the specific activity was concentrated to a 10 5 g precipitate and the Subcellular localization differed from that of rat brain. In the human colonic well differentiated adenocarcinoma, although ω-hydroxylation activity was relatively high in the 10 3 g supernatant, the specific activity was relatively high in the 10 3 and 10 5 g precipitates. These results suggest that there is a difference regarding subcellular localization of the ω-hydroxylation activity depending on the species of the organs.

High-performance liquid chromatography-thermospray mass spectrometry of 5,6-dihydroxyeicosatrienoate-1,5-lactone from tissue homogenates

We have developed a method for the analysis of 5,6-dihydroxyeicosatrienoate-1,5-lactone (5,6-DiHETriE-δ-lactone) in tissue homogenates, supplemented with NADPH and arachidonic acid [20:4( n−6)] as a substrate. During the incubation and the extraction, most of the 5,6-epoxyeicosatrienoic acid (5,6-EpETriE) was converted to 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETriE), and most of the 5,6-DiHETriE was converted to 5,6-DiHETriE-δ-lactone. Consequently, the chief degradation product of 5,6-EpETriE and 5,6-DiHETriE in the incubation mixture was 5,6-DiHETriE-δ-lactone. 5,6-DiHETriE-δ-lactone, corresponding to [20:4( n−6)], was shown to be characterized by a high intensity of quasimolecular ions (MH + and MNH 4 +), using ion analysis obtained by reversed-phase HPLC-thermospray MS. On selected-ion monitoring (SIM) chromatograms of 5,6-DiHETriE-δ-lactone and with deuterium-labeled 15( s)-hydroxyeicosatetraenoic acid as the internal standard, the regression equation of the peak-area ratio and the amount of 5,6-DiHETriE-δ-lactone was y = 12.2 x + 0.7 ( r = 0.9996). 5,6-Epoxygenase activity was represented as the sum of the amount of 5,6-DiHETriE-δ-lactone, 5,6-EpETriE and 5,6-DiHETriE per mg protein, after 30 min in an incubation mixture. The activity from rat brain homogenate decreased considerably with growth of the rat.

Liquid chromatography-thermospray mass spectrometry of toremifene and its derivatives

A high-performance liquid chromatographic-thermospray mass spectrometric method was developed for analysis of toremifene and eight of its derivatives, including some main metabolites. The compounds were chromatographed with an acetonitrile-ammonium acetate buffer eluent. Thermospray mass spectra of the compounds, showing abundant protonatede molecular ions, were obtained with the exception of deaminohydroxy- and deaminocarboxy-toremifene, which apparently did not get protonated under these conditions. Selected-ion monitoring (SIM) of the protonated molecular ion of toremifene, the molecule of main interest in this series, was accomplished. According to the results, toremifene can be analyzed in the range from 0.01 μg/ml to 10 μg/ml, the limit of detection being 500 pg per injection. This method provides an effective means for purity assignation of forthcoming synthetic triphenylethylene derivatives. Liquid chromatography-thermospray mass spectrometry with selected-ion monitoring seems to be a sensitive and a selective quantitation method for toremifene.

Thermospray and Atomospheric Pressure Chemical Ionization Liquid Chromatographic-Mass Spectrometric Analysis of Benzodiazepines and Their Metabolites.

To assess the LC/MS methods for the determination of these compounds in biological materials positive ion mass spectra were measured for benzodiazepines and their metabolites by thermospray-liquid chromatography/mass spectrometry (TSP-LC/MS) and atomospheric pressure chemical ionization-liquid chromatography/mass spectrometry (APCI-LC/MS). In both TSP-LC/MS and APCI-LC/MS, LC analyses were performed on a TSKgel octyl-80Ts in the solvent system of methanol-ammonium acetate at a flow rate of 1.0 ml/min. TSP mass spectra showed only a [M+H]+ ion for most of compounds, while APCI mass spectra showed [M+H]+ and some fragment ions. The analysis of these compounds with TSP gave better detection limits than with APCI by scan mode, and gave the same results for detection limits as with APCI by selected ion monitoring (SIM). The data, described in this paper, should serve as a basis for the qualitative and quantitative analyses of benzodiazepines and their metabolites in biological samples.