Simultaneous quantification of prostaglandins,isoprostane and thromboxane in cell-cultured medium using gas chromatography-mass spectrometry

We have developed a simultaneous quantification method for prostaglandin (PG) E(2), PGD(2), PGF(2 alpha), 8-epi-PGF(2 alpha), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2). Using [3,3,4,4-(2)H(4)]PGE(2), [3,3,4,4-(2)H(4)]PGD(2), [3,3,4,4-(2)H(4)]8-epi-PGF(2 alpha), [3,3,4,4-(2)H(4)]PGF(2 alpha), [3,3,4,4-(2)H(4)]6-keto-PGF(1 alpha) and [18,18,19,19-(2)H(4)]TXB(2) as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography-mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.     

Detection of leukotriene C4 and D4 in the exudate of rat carrageenin-induced pleurisy

Rat carrageenin-induced pleurisy was used as an acute exudative inflammatory model. The crude ethanol extract of the pleural fluid at 5 hr after carrageenin injection caused the very slow contraction of guinea-pig ileum, which was antagonized by FPL 55712 (1 μg/ml). The ethanol extract was cleaned by LH-20 and was rendered for separation of LTC₄ and LTD₄ by reversed-phase high-performance liquid chromatography (HPLC). Two peaks which showed the same retention time on HPLC as those of LTC₄ and LTD₄ had the contractile activity of guinea-pig ileum and the ratios of the contractile activity to the height on HPLC agreed with those of synthetic LTC₄ and LTD₄. Two peaks of Δ⁶-trans-LTB₄, 5S,12R-(E,E,E,Z)-diHETE and 5S, 12S-(E,E,E,Z)-diHETE, were detected, but the appreciable amount of LTB₄ was smaller than that of each Δ⁶-trans-LTB₄ in the pleural fluid at 5 hr.     

Amine metabolite profile of normal and uremic urine using gas chromatography—mass spectrometry

A method for the simultaneous analysis of phenolic amines and aliphatic amines in human urine is described. The amine metabolites in urine were extracted using Dowex 50W-X8 cationic resin, derivatized and analyzed by a gas chromatographic—mass spectrometric—computer system. The amine metabolites profile of 5 ml of urine was obtained with good gas chromatographic separation. The gas chromatographic method described here separates urinary phenolic amines, di- and polyamines and methylguanidine in a single chromatographic separation. The urinary levels of methylguanidine, putrescine, cadaverine, spermidine, p-tyramine, dopamine, and 3-methoxytyramine were quantitated by using a mass spectrometric technique. In uremic patients, only the urinary excretion of methylguanidine was increased in comparison with normal subjects, although the urinary excretion of other amines was decreased in uremic patients.     

Extraction of thromboxane B2 from urine using an immobilized antibody column for subsequent analysis by gas chromatography-mass spectrometry

This paper describes an antibody affinity (immunoaffinity) column which, in one step, extracts and sufficiently purifies urinary thromboxane B2 (TXB2) for quantitative analysis by high resolution gas chromatography-negative ion chemical ionization-selected ion monitoring-mass spectrometry (HRGC-NICI-SIM-MS). Polyclonal TXB2 antibody from rabbit was partially purified using immobilized Staphylococcus aureus Protein A. The purified IgG fraction was then immobilized using an N-hydroxysuccimidyl silica gel. The resulting matrix bound 570 ng TXB2 per ml of gel. TXB2 was quantitatively eluted with acetonitrile-water (19:1). Columns constructed from the gel could be used repeatedly since binding capacity was reconstituted using 0.01 M phosphate buffer (pH 7.4) with no apparent loss of activity. Using these columns, urinary TXB2 was sufficiently purified in one step such that in subsequent analysis by HRGC-NICI-SIM-MS interference free chromatograms were observed.     

Profiling of bisenoic prostaglandins and thromboxane B2 in bronchoalveolar fluid from the lower respiratory tract of human subjects by combined capillary gas chromatography-mass spectrometry

Methods for the profiling of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 15(S),9 alpha,11 beta-trihydroxyprosta-5Z,13E-dien-1-oic acid (9 alpha,11 beta-PGF2), 6-keto-prostaglandin F1 alpha (6kPGF1 alpha), and thromboxane B2 (TxB2) in bronchoalveolar lavage (BAL) fluids from human subjects by combined capillary gas chromatography-mass spectrometry are described. Aliquots (5 ml) of BAL fluid obtained using a standardized lavage protocol were extracted on octadecylsilyl silica cartridges after addition of 0.8 to 2.0 nanograms of tetradeuterated analogs of PGE2, PGF2 alpha, and 6kPGF1 alpha as internal standards. Eluted analytes and internal standards were prepared for vapor phase analysis by sequential reactions resulting in the formation of methyloxime-pentafluorobenzyl ester-trimethylsilyl ether derivatives. The derivatized analytes were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The samples were of adequate purity for identification and quantitation of each of the prostanoids with detection limits of 0.1 to 0.2 picograms of each analyte per milliliter of BAL fluid. The time required for analysis of each sample was approximately 30 minutes. Standard curves of unlabeled species of the six prostanoids extracted after addition to BAL fluid were linear over a range from subpicogram to nanogram quantities. The differences between the amounts of prostanoid added and the amounts of prostanoid measured were typically less than 19%, and the intra-assay coefficients of variation for repeated measurements of a single sample were less than 20%. PGE2, PGD2, PGF2 alpha, and TxB2 were detectable in BAL fluids from normal subjects with levels of each of these compounds being less than 2.6 picograms/ml. BAL fluids from patients with lung disease presented qualitative and quantitative profiles of prostanoids markedly different than those from normal subjects. These analytical methods provide a basis for in vivo comparisons of prostanoid profiles in the lower respiratory tract of man and should be readily adaptable for use in a variety of clinical studies.     

Determination of plasma propofol levels using gas chromatography—mass spectrometry with selected-ion monitoring

Propofol (2,6-diisopropylphenol, I.C.I. 35 868) is a rapid-acting, intravenous anesthetic agent recently introduced for the induction and maintenance of general anesthesia. This paper describes a gas chromatographic—mass spectrometric procedure using selected-ion monitoring for the determination of plasma propofol levels. The drug and the internal standard (thymol) were extracted from plasma into diethyl ether—pentane, and derivatized to their trimethylsilyl derivatives before analysis. The reproducibility of the daily standard curves had coefficients of variation ranging from 2.7% to 10.2%. The precision of the assay yielded a coefficient of variation ranging from 4.5% to 5.6%, and the concentration means for the seeded control samples were found to be within −1.6% to +0.6% of the theoretical values for propofol. No interfering peaks have been observed in application of this procedure to either normal volunteer or patient samples. The minimum detectable level under the conditions described was 0.20 ng propofol/ml plasma. This assay and a high-performance liquid chromatographic assay with fluorescence detection were both used to measure plasma propofol concentrations in 89 human plasma samples, and the correlation between the two methods was excellent.     

Combined profile of androgen glucuro- and sulfoconjugates in post-competition urine of sportsmen: a simple screening procedure using gas chromatography-mass spectrometry

An analytical screening procedure has been developed for the estimation of total androgen conjugates in post-competition urine, using gas chromatography-mass spectrometry with computerized data acquisition and concentration calculation. Rapid acid-catalyzed methanolysis is a key feature of the method, which allows simulatenous cleavage of glucuronides and sulfates. Analytical data generated by this method for testosterone and epitestosterone are in accordance with our previous results obtained by more accurate isotope dilution mass spectrometry. The usefulness of the ratio of testosterone glucuronide-total epitestosterone as an aid for a better discrimination between physiologically high and pharmacologically high ratios of testosterone glucuronide-epitestosterone glucuronide, which was demonstrated previously, has been confirmed here.     

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection

Pyrovalerone and its hydrolated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.     

Urinary metabolites of cannabidiol in dog, rat and man and their identification by gas chromatography—mass spectrometry

Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography—mass spectrometry as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS and methyloxime—TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4″- and 5″-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of β-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2″-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the Δ-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.     

Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10⁶ DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10⁶ DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Metabolism and toxicological detection of the new designer drug 4'-methoxy-alpha-pyrrolidinopropiophenone studied in rat urine using gas chromatography-mass spectrometry

R,S-4'-Methoxy-alpha-pyrrolidinopropiophenone (MOPPP) is a new designer drug with assumed amphetamine-like effects, which has appeared on the illicit drug market. The aim of this study was to identify the MOPPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and GC-MS. Analysis of urine samples of rats treated with MOPPP revealed that MOPPP [limit of detection (S/N 3) was 100 ng/ml] was completely metabolized by demethylation of the methoxy group, hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam and/or oxidative desamination to the corresponding diketo compounds. To some extent, the demethylated MOPPP metabolites were hydroxylated with partial subsequent methylation in position 3'. The hydroxy groups were found to be partly conjugated. Based on these data, MOPPP could be detected in urine via its metabolites by full-scan GC-MS using MS for screening and library search for identification by comparison of the spectra with reference spectra.     

Rapid and sensitive procedure for the separation and quantitation of - and -serine in rat brain using gas chromatography-mass spectrometry

A very simple and rapid GC-MS procedure for the separation and quantitation of - and -serine has been developed utilizing a conventional bonded-phase capillary column. The procedure involves initial esterification with isobutanol followed by acylation with the chiral derivatizing reagent S-(−)-N-(heptafluorobutyryl)prolyl chloride (HPC). This procedure requires neither extraction nor clean-up steps and is sensitive to 50 pg on-column. Total time of the procedure is under 3 h and derivatives are stable at room temperature for at least 5 days, making this procedure ideal for automated injections. A simple, one-day synthesis of HPC is described which yields >99.9% optical purity.     

Determination of the terfenadine metabolite azacyclonol in human serum using gas chromatography-mass spectrometry

In this work, a method for the determination of azacyclonol, a primary metabolite of terfenadine, in human serum is described. Sample preparation is carried out by liquid-liquid extraction under basic conditions. For an efficient clean up, the analytes are back-extracted into diluted hydrochloric acid and, after alkalinization, are once again extracted into the organic phase. No derivatisation step is performed. The samples are measured by gas chromatography-mass spectrometry with good selectivity. The limit of detection is 2 ng/ml. The coefficients of variation are 12.6% at 10 ng/ml and 6.44% at 200 ng/ml in the day-to-day control measurements.     

Metabolic network analysis on Phaffia rhodozyma yeast using C–labeled glucose and gas chromatography-mass spectrometry

Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for ‘all natural’ products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-¹³C]glucose were conducted with the wildtype strain (CBS5905 T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes.Amino acid labeling patterns, as determined by GC–MS, were in accordance with a metabolic network consisting of the Embden–Meyerhof–Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway (65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower.     

Screening and confirmatory analysis of β-agonists, β-antagonists and their metabolites in horse urine by capillary gas chromatography—mass spectrometry

A method for the screening and confirmatory analysis of β-agonists and -antagonists in equine urine is described. Following initial enzymic hydrolysis, the basic drugs and metabolites are extracted using Clean Screen^® DAU or Bond Elut Certify™ cartridges, and analysed as their trimethylsilyl ether or 2-(dimethyl) silamorpholine derivatives by capillary gas chromatography—mass spectrometry. The method proved to be very sensitive and selective for basic drugs. After administration of therapeutic doses of propranolol, metoprolol, timolol, isoxsuprine and clenbuterol to thoroughbred horses, the parent compound/metabolites could be detected in urine for upto 14–120 h depending on the drug.     

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.