Thermospray liquid chromatography-mass spectrometry of corticosteroids.

A high-performance liquid chromatographic method was developed for thermospray mass spectrometric analysis of steroidal hormones. Using a Nova-Pak C18 reversed-phase column and isocratic elution with a solvent comprised of 25 mM ammonium formate in 30% acetonitrile, corticosteroids were separated within 10 min. This solvent also permitted ultraviolet absorbance detection down to 220 nm with low-nanogram sensitivity. The use of acetonitrile was favourable for thermospray mass spectrometric analysis because mass spectra were obtained with a pseudomolecular ion as the base peak. A combination of liquid chromatography, ultraviolet absorbance detection and thermospray mass spectrometry provided a sensitive and reliable method for unequivocal confirmation of the presence of steroidal drugs in equine urine.

Syntheses and comparison of three reagents for allyldimethylsilylation of prostaglandins and steroids: a gas chromatographic method.

Three reagents used for allyldimethylsilylation of prostaglandins and steroids have been prepared: N,O-bis(allyldimethylsilyl)trifluoroacetamide, N-methylallyl-dimethylsilyltrifluoroacetamide and allyldimethylsilyl-N,N-diphenylamine. The reaction kinetics of the reagents have been studied at room temperature in several solvents, and the optimal conditions for the derivatization of prostaglandins and steroids are discussed.

Analysis of leukotrienes by liquid chromatography/electrochemistry after conversion to dinitrobenzoate derivatives.

A sensitive liquid chromatography method has been developed using electrochemistry for the determination of leukotrienes in biological fluids. Biological specimens are treated with 3,5-dinitrobenzoyl chloride in acetonitrile which undergoes rapid reaction with hydroxyl groups of non-peptidic leukotrienes in the presence of pyridine and with amino groups of peptidic leukotrienes in the presence of potassium tetraborate buffer. The resulting dinitrobenzoate derivatives of leukotrienes are highly electroactive, suitable for reduction or oxidation at moderate potentials by an electrochemical detector. In reductive mode at -0.7 V or oxidative mode at +1.15 V potentials, the lower limits of detection for leukotriene derivatives were approximately 8 +/- 3 pg and 70 +/- 16 pg respectively, with a signal-to-noise ratio of 5 to 1. This method was applied to the detection of leukotrienes in plasma, nasal and bronchial fluids of patients with asthma.

Reversed-phase ion-interaction chromatography of leukotrienes, lipoxins and related compounds.

A novel mobile phase containing heptafluorobutyric acid has been used for ion-interaction high-performance liquid chromatography of leukotrienes, lipoxins and related compounds on octadecylsilane silica columns. The use of a hydrophobic perfluorinated carboxylic acid as an ion-interaction agent at optimized concentration and pH permitted rapid, isocratic separation of leukotrienes, lipoxins and monohydroxyeicosatetraenoic acids. A completely volatile mobile phase suitable for preparative chromatography was obtained by using triethylamine as the base for pH adjustment. With this novel mobile phase, leukotrienes and monohydroxyeicosatetraenoic acids were completely separated in less than 15 min.

Mass spectrometric evaluation of the tert.-butyldimethylsilyl derivatives of monohydroxyeicosatetraenoic acids and leukotrienes.

The tert.-butyldimethylsilyl (t-BDMS) derivatives of the hydroxyl and the carboxyl groups on the monohydroxyeicosatetraenoic acids (HETEs) 5-, 12- and 15-hydroxyeicosatetraenoic acid (5-HETE, 12-HETE and 15-HETE, respectively) as well as leukotriene B4 (LTB4), 20-carboxy-LTB4 (20-COOH-LTB4) and 20-hydroxy-LTB4 (20-OH-LTB4) have been prepared. The derivatization reagents N-methyl-N-tert.-butyldimethylsilyltrifluoroacetamide (MTBSTFA) and tert.-butyldimethylsilylimidazole (t-BDMSIM) were utilized and the derivatization yields evaluated by gas chromatographic analysis. While the derivatization of 12-HETE and 15-HETE was achieved with good yield using MTBSTFA, the 5-hydroxylated compounds could only be derivatized satisfactorily with t-BDMSIM. The t-BDMS ester derivatives of monohydroxy fatty acids and 20-COOH-LTB4 were found quite susceptible to hydrolysis upon attempted isolation. The mass spectrometric fragmentation patterns of the HETEs, LTB4, 20-COOH-LTB4 and 20-OH-LTB4 and their reduced analogues as the t-BDMS ester and ether derivatives were examined. The fragmentation of the unsaturated compounds was mainly influenced by the double bonds while, on the contrary, the saturated compounds showed intense high-mass ions derived from elimination of a tert.-butyl radical. Furthermore, it was observed that the t-BDMS ester moiety of the unsaturated compounds exerted certain influence upon the fragmentation pattern. With regard to earlier results obtained with the methyl ester t-BDMS ether derivative, it was demonstrated that the use of the t-BDMS ester derivative resulted in decreased intensities of high-mass ions with a few exceptions. Taking into account the mass spectrometric fragmentation and the poor hydrolytic stability of the t-BDMS ester derivatives of some of the studied compounds, the use of these derivatives was not found to constitute a major advantage over the use of the methyl ester derivative. However, a favourable fragmentation pattern with a very intense high-mass ion was obtained for 15-HETE and the levels of this compound were determined in human lung tissue samples.

A comparative study of methyl ester trimethylsilyl, allyldimethylsilyl and tert-butyldimethylsilyl ethers for electron impact mass spectrometry of leukotrienes.

The gas chromatographic and mass spectrometric properties of leukotriene B4, 20-hydroxy-leukotriene B4 and 20-carboxy-leukotriene-B4 were investigated as their methyl ester trimethylsilyl, methyl ester allyldimethylsilyl and methyl ester tert-butyldimethylsilyl ethers. The gas chromatographic properties of the trimethylsilyl and tert-butyldimethylsilyl ether derivatives were good with respect to peak shape and sensitivity, whereas the allyldimethylsilyl ether derivative gave a lower sensitivity. The sensitivity defined as the quantity that could be passed through the gas chromatographic column. The three derivatives showed a mass spectrometric fragmentation pattern with cleavage of the C12-C13 bond as an important feature. Particularly, the allyldimethylsilyl ether derivative of the three compounds studied exhibited a high tendency for C12-C13 bond cleavage resulting in a fairly intense ion at m/z 435. However, the mass spectra indicated multiple fragmentation pathways due to the presence of double bonds, leading to decreased intensities of the high mass ions. A quantitative analysis by selected ion monitoring of the most intense high mass ions in the respective mass spectrum demonstrated that neither derivative would allow measurements in the low picogram range. Catalytic hydrogenation of the double bonds was performed and the methyl ester trimethylsilyl, methyl ester allyldimethylsilyl and methyl ester tert-butyldimethylsilyl ether derivatives of the reduced compounds were prepared. Saturation of the double bonds increased the gas chromatographic sensitivity for the three derivatives as well as the intensities of the high mass ions in their mass spectra. The high sensitivity that can be obtained by measurement of such high mass ions was demonstrated by quantification of leukotriene B4 in lung tissue samples by selected ion monitoring.

Gas chromatography-mass spectrometry of monohydroxyeicosatetraenoic acids as their methyl esters trimethylsilyl, allyldimethylsilyl and tert.-butyldimethylsilyl ethers.

The gas chromatographic and mass spectrometric properties of the monohydroxy acids 5-hydroxyeicosatetraenoic acid (5-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) as their methyl ester trimethylsilyl, methyl ester allyldimethylsilyl and methyl ester tert.-butyldimethylsilyl ethers were investigated. The gas chromatographic properties of the trimethylsilyl and tert.-butyldimethylsilyl derivatives were found to be excellent while the allyldimethylsilyl derivative required a well deactivated column. The mass spectra of these silyl derivatives with the exception for 12-HETE did not exhibit particularly intense ions in the upper mass region. A quantitative analysis by selected-ion monitoring of the most intense ion in the upper mass region of respective mass spectrum demonstrated that a detection limit in the low picogram range could only be obtained for 12-HETE. Since the mass spectra indicated that the double bonds exerted a strong influence on the fragmentation pattern, the trimethylsilyl, allyldimethylsilyl and tert.-butyldimethylsilyl ethers of the methyl esters of the reduced analogues of the monohydroxy acids were prepared. The saturation of the double bonds completely altered the fragmentation patterns and very intense ions carrying a high percentage of the total ion abundance were found in all of the mass spectra. The developed technique was utilized for measurements of 5-HETE in lung tissue samples from patients with lung cancer.

Mass spectrometry of prostaglandins, leukotrienes and steroids as their allyldimethylsilyl ether derivatives.

Allyldimethylsilyl ethers of prostaglandin E2 and F2 alpha, leukotriene B4 and 5 beta-pregnane-3 alpha, 20 alpha-diol were prepared at room temperature in good yields with the novel reagent N,O-bis(allyldimethylsilyl)-trifluoroacetamide. The gas chromatographic properties of the derivatives of prostaglandins and the steroid were found to be excellent whereas those of leukotriene B4 were found to be less than satisfactory. The mass spectra of the allyldimethylsilyl ether derivatives of the compounds studied show intense ions in their upper mass region derived from the elimination of an allyl radical. In the mass spectrum of the derivative of leukotriene B4, the formation of a conjugated carbonium ion by alpha-cleavage is a major process. The detection limits for a quantitative assay were established by performing selected ion monitoring on the most intense ion in the upper mass region of respective mass spectrum. Detection limits in the low picogram range were obtained for the prostaglandins and the steroid but the allyldimethylsilyl ether derivative of leukotriene B4 exhibited a far higher detection limit. It is concluded for the latter compound that the detection limit depends primarily on the gas chromatographic properties.

Comparative study of solid phase extraction techniques for isolation of leukotrienes from plasma.

We have compared five commercially available absorbent materials (i.e. C18 Sep-Pak, C18 J.T. Baker, Amberlites XAD-7, XAD-2 and XAD-4) for their applicability as effective tools for extraction of leukotrienes from plasma samples. Leukotriene C4 (LTC4) and B4 (LTB4) were selected as representatives of peptidic and non-peptidic leukotrienes, respectively. These leukotrienes were added to 1 ml of plasma and passed through columns containing the above described adsorbent materials. The recovery was determined for each material using different combinations of solvents. XAD-4 gave the highest recovery for LTB4 (90%), whereas XAD-4 and XAD-2 gave identical recoveries for LTC4 (90%) when an eluting solvent mixture of pyridine-water--dimethylformamide (50:45:5) was used. The efficiency of the other three solid adsorbent materials for leukotriene extraction were in order of decreasing magnitude, C18 J.T. Baker greater than XAD-7 greater than C18 Sep-Pak. XAD-7 was shown to be more efficient for LTB4 than for LTC4, whereas the octadecylsilane C18 materials gave approximately similar recoveries for both of the leukotrienes. In addition to very good extraction properties of XAD-4 and XAD-2 as compared to octadecylsilane silica, these solid adsorbent materials retained less plasma impurities than the C18 materials, giving cleaner chromatograms for leukotrienes extracted from plasma. Therefore, XAD-4 or XAD-2 are the best overall choice for extraction of leukotrienes from plasma for reversed-phase high-performance liquid chromatographic analysis.

Immunological and non-immunological release of leukotrienes and histamine from human nasal polyps.

Nasal polyps were obtained from 22 patients undergoing polyp surgery. They were chopped into fragments of approximately 2 mm2, washed free of blood, and passively sensitized with serum from timothy allergen sensitive patients (RAST 30-40%), then, challenged with timothy allergen. Analysis of the incubation medium after a 30 min challenge as assessed by reversed phase high performance liquid chromatography and bioassay revealed the presence of leukotriene (LT) LTB4, LTC4, LTD4 and LTE4 (29 +/- 9, 6.6 +/- 3, 62 +/- 13 & 86 +/- 23 pmol/g tissue wet weight, respectively). The human nasal polyps also released histamine which reached a maximum after approximately 5 min (2.8 +/- 1.28 nmol/g tissue), as measured by radioenzymatic assay. A similar profile of leukotrienes and histamine release was observed when the nasal polyps were stimulated with ionophore A23187. However, the ionophore stimulated release was greater than that observed for the antigen challenge. When human polyps were stimulated with a mixture of the ionophore and arachidonic acid, all the above products as well as 5-hydroxy-eicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE were detected in the medium. These data, taken together, demonstrate that human nasal polyps are able to release significant amounts of leukotrienes and histamine upon both immunological and non-immunological stimulations and that these responses might significantly contribute to the symptoms of allergic rhinitis.

Metabolism of prostaglandin E analogs in guinea pig and rat liver microsomes.

Tritium-labelled 16,16-dimethyl-PGE2, 9-methylene-PGE2 (9-deoxo-16,16-dimethyl-9-methylene-prostaglandin E2) and tetranor-9-methylene-PGE2 were incubated with guinea pig liver microsomes. All three compounds were converted to omega-oxidized products in yields of a few per cent. In addition, from incubations with 9-methylene-PGE2 and tetranor-9-methylene-PGE2 were also obtained metabolites with the methylene group transformed into a dihydrodiol. In a comparative study with rat liver microsomes, it was found that these converted tetranor-9-methylene-PGE2 in a 50 per cent yield to omega-oxidized products. Finally, 20.000 X G supernatants from guinea pig and rat liver were compared with respect to omega-oxidation. The rat liver 20.000 X G supernatant was found to convert the substrate to the same extent as washed microsomes. By contrast, the guinea pig liver 20.000 X G supernatant was considerably more efficient than washed microsomes.

Metabolism of prostaglandin E analogs in the guinea pig liver mitochondrial fraction.

Tritium-labeled 16,16-dimethyl-PGE2 and 9-methylene-PGE2 were incubated with the guinea pig liver mitochondrial fraction. The tetranor and dinor metabolites were obtained, a large portion of the latter contained a saturated carboxyl side chain. Also the beta-hydroxydinor metabolites, analogous to the beta oxidation of fatty acids, were identified. Such metabolites have not been reported earlier in connection with beta oxidation of prostaglandins or prostaglandin analogs. Furthermore, omega-hydroxylated analogs were incubated with the guinea pig liver mitochondrial fraction and were found to pass through beta oxidation to a certain extent.

Metabolism of 9-deoxo-16,16-dimethyl-9-methylene prostaglandin E2 in humans.

Tritium-labeled 9-deoxo-16,16-dimethyl-9-methylene prostaglandin E2 was administered by intravenous injection into humans. The initial half-life in plasma was determined to be about 3 min. About 35% of the injected radioactivity was excreted in the urine and about 55% in the feces. Twenty-three urinary metabolites were identified. The metabolites included beta-oxidized and omega-oxidized products as well as metabolites in which the 9-methylene group had been biotransformed.