HPLC-thermospray Mass Spectrometry Research Articles

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.

Characterization of proanthocyanidins from grape seeds

Leucoselect™ (grape seed selected proanthocyanidins) was analyzed. HPLC thermospray mass spectrometry (TSP-MS) allowed the detection of monomeric flavan-3-ols and dimeric proanthocyanidins. Fractionation over Sephadex® LH-20 resin and analysis of the isolated fractions by gel permeation chromatography (GPC) and electrospray mass spectrometry (ESI-MS) led to the complete characterization of the proanthocyanidin constituents of Leucoselect™. The analysis revealed the presence of approximately 15% of (+)-catechin (1) and (−)-epicatechin (2), 80% of (−)-epicatechin 3- O-gallate (3), dimers, trimers, tetramers and their gallates and 5% of pentamers, hexamers, heptamers and their gallates.

Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins: studies utilizing amino acid and peptide models

Quinone methides (QMs) are electrophiles formed in several biological processes including direct oxidations of 4-alkylphenols by cytochromes P450. These species may be responsible for the adverse effects of certain phenolic compounds through protein alkylation, but little information is available concerning specific targets or the resulting mechanisms of cell injury. The present goal was to determine the most likely sites of adduct formation among competing protein nucleophiles utilizing QMs of varying electrophilicity. Reactions of poorly reactive, moderately reactive, and highly reactive QMs, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM), 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4-methylene-2,5-cyclohexadienone (BHTOH-QM), and 2-tert-butyl-6-methyl-4-methylene-2,5-cyclohexadienone (BDMP-QM), respectively, were investigated in aqueous solutions with nucleophilic amino acids. Each QM rapidly formed a thioether derivative of cysteine with little or no competition from the addition of water (hydration). The α-amino groups were the primary sites of alkylation for all other amino acids examined including lysine, histidine, tyrosine, and serine, and the pseudo-first order rates were 5 to 8-fold greater than the rates of hydration. Alkylation of the side chain nitrogens of lysine and histidine occurred at about one-fourth the rate of hydration for BDMP-QM, but no reaction was detectable for BHT-QM and no reactions occurred between QMs and amino acid hydroxyl groups. The results indicate that, based on chemical reactivity, peptide alkylation should occur in the order cysteine thiol>N-terminal amino>N ε-lysine=NIm-histidine, with side chain modifications occurring only with the more electrophilic QMs. Reactions of QMs with the tripeptide Gly-His-Lys confirmed the results with amino acids as N α-glycine alkylation predominated, but side chain adducts also formed with BHTOH-QM and BDMP-QM. Human hemoglobin was treated with QMs, hydrolyzed, and assayed by HPLC-thermospray mass spectrometry. This work revealed that N ε-lysine was the main alkylation site, emphasizing the importance of factors, in addition to chemical reactivity, which influence protein modification by electrophiles.

Three complementary liquid chromatographic methods for determination of the peptoid cholecystokinindashB antagonist, CI-988, in rat plasma

Three different liquid chromatographic methods (two quantitative methods which employ fluorescence detection and one qualitative method which employs selected iondashmonitoring detection) were developed and validated to provide complementary specificity for determination of CI-988, a cholecystokinindashB antagonist, in rat plasma. The first quantitative method involves isocratic separation of “nondashionized” CI-988 and internal standard on a C-18 column, whereas the alternative quantitative method involves isocratic separation of the “anionic” analytes. These two quantitative HPLC methods rely on the intrinsic fluorescence of CI-988 and internal standard for detection, and both methods are equally sensitive (linear range of 2.0–1000 ng ml −1). accurate (±15%, relative error), and precise (⩽15% relative standard deviation). Plasma CI-988 concentrations for samples ( N = 69) assayed with the “nondashionized” separation are linearly correlated with concentrations for the same samples assayed with the “anionic” separation ( y = 1.08 x − 0.57, R = 0.999). In addition, a third qualitative method, HPLC-thermospray mass spectrometry, was developed to provide complementary evaluation of assay specificity through the use of selected CI-988 fragment ion monitoring. When investigating an anomalous chromatographic result that calls into question the specificity of a method, the availability and use of alternative validated chromatographic separations and orthogonal detection schemes are beneficial.

Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine ( p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 m M potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 m M ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.

Trace analysis of impurities in 3′-azido-3′-deoxythymidine by reversed-phase high-performance liquid chromatography and thermospray mass spectrometry

An analytical method has been developed for the detection of trace amounts of impurities in 3′-azido-3′-deoxythymidine referred to herein as AZT (Zidovudine). A sample extract was preconcentrated by normal-phase high-performance liquid chromatography (HPLC) with subsequent on-line reversed-phase HPLC-thermospray mass spectrometry (TSP-MS). During the sample extraction and concentration step, carried out by semipreparative normal-phase chromatography, the preliminary separation of the impurities from the AZT takes place. The organic solvent (dichloroethane-acetonitrile, 40:60) is evaporated from the collected fractions and the compounds are redissolved in a smaller volume of the reversed-phase mobile phases for a further degree of concentration. The collected fractions are then subjected to reversed-phase HPLC-TSP-MS. The influence of acetonitrile concentration and pH on the reversed-phase separation together with the sensitivity of the TSP-MS detection have been examined to maximise detection levels. The 3′-azido-3′-deoxy-5′-O-tritylthymidine, triphenyl methanol and 3′-chloro-3′-deoxythymidine, which are route-indicative impurities formed during the synthesis can be detected in the 50–100 ppb (w/w) range.

Characterization of three rifamycins via electrospray mass spectrometry and HPLC-thermospray mass spectrometry.

Rifamycins are a class of antibiotic compounds of which rifampicin is the most commonly prescribed. Conventional electron-impact mass spectrometry of rifampicin has not been found to provide useful data. Thermospray and electrospray mass spectrometry are studied as potential tools for the analysis of rifampicin, rifamycin SV, and rifamycin B. Using thermospray and electrospray ionization, all three compounds provide significant ion intensity for either the [MH]+ or [MNa]+ ions. In addition, combined high-performance liquid chromatography-thermospray mass spectrometry provides useful analytical data for a mixture of the three rifamycins.

Identification of RP-HPLC peaks of bisphenol F and of bisphenol F diglycidyl ether and its hydrolysis products by thermospray mass spectrometry and gas chromatography/mass spectrometry

The need to determine the migration of toxic unreacted compounds in bisphenol diglycidyl ether epoxy resins prompted us to investigate the HPLC properties of bisphenol F diglycidyl ether and its hydrolysis products in the water-based food simulants 3% (w/v) acetic acid, distilled water and 15% (v/v) ethanol. Peaks were identified by reversed-phase HPLC thermospray mass spectrometry and gas chromatography/mass spectrometry.

N-hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [13C,15N2]MeIQx and [14C]MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M+H]+) from the parent compound, MeIQx and at 230/233 ([M+H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M+H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.

Determination of Rafoxanide in Plasma Using High Performance Liquid Chromatography (HPLC) and in Tissue Using HPLC-Thermospray Mass Spectrometry

Abstract Methods are described for the determination in plasma and tissue samples, of the drug rafoxanide which is used to control liver fluke in sheep and cattle. HPLC with UV detection is used to measure levels of the drug in plasma and combined HPLC-thermospray mass spectrometry is used to measure residue levels in tissue. The methods are both specific and sensitive with detection limits of 0.1 ug ml−1 for plasma and 0.02 ug g−1 for tissue.

Metabolism of the mutagen MeIQx in vivo: Metabolite screening by liquid chromatography-thermospray mass spectrometry

2-Amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx) is a potent mutagen found in cooked food. MeIQx and its isotopically labelled (13C, 15N2 and 14C) analogues were synthesised and used for metabolic studies in vivo. An equimolar mixture of MeIQx and its 13C, 15N2 stable isotope labelled analogue (containing tracer amounts of 14C-MeIQx) was given intraperitoneally to mice. Some 67% of the radioactivity was eliminated in urine and faeces within 24h. Four radiolabelled species were observed when urine was analysed by HPLC, corresponding to unchanged MeIQx and three more polar metabolites. Urine was analysed directly by HPLC-thermospray mass spectrometry. Four signals were observed containing the characteristic 1:1 isotopic doublet, corresponding to unchanged MeIQx, an MeIQx glucuronide, and two uncharacterized metabolites.