Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method.

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5-100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1-1,000 nM for all MTXPGn (R(2) > 0.99). The coefficient of variation ranged from 1-4% for intraday precision and 6-15% for interday precision. Samples were stable for at least 1 month at -80 °C. Recovery ranged from 98-100%, and the relative matrix-effect varied from 95-99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

Cytochrome P450-catalysed arene-epoxidation of the bioactive tea tree oil ingredient p-cymene: indication for the formation of a reactive allergenic intermediate?

1. The cytochrome P450-mediated metabolism of the tea tree oil ingredient p-cymene (p-isopropyltoluene) was studied by the application of in vitro enzymatic assays using different recombinant human cytochrome P450 enzymes. 2. In total, four enzymatic products were identified by gas chromatography-mass spectrometry. The enzymatic products identified were: thymol (2-isopropyl-5-methylphenol), p-isopropylbenzyl alcohol, p,alpha,alpha-trimethylbenzyl alcohol, and p-isopropylbenzaldehyde. 3. The enzymatic products of p-cymene resulted from catalysed enzymatic arene-epoxidation and hydroxylation reactions by the studied cytochrome P450 enzymes. 4. An in vivo study could only confirm the formation of one enzymatic product, namely thymol. Thymol was identified after enzymatic hydrolysis of glucuronide and sulphate conjugates in collected blood and urine samples. 5. The obtained results may help to increase the understanding of cases where skin sensitization and irritation by tea tree oil-containing products that are involved with allergic reactions of users of these products. The results also indicate that skin sensitization and irritation reactions not only can be explained by the frequently in literature reported auto-oxidation of tea tree resulting in bioactive oxidized products, but also now by the formation of epoxide intermediates resulting from catalysed arene-epoxidation reactions by selected human cytochrome P450 enzymes which are also located in different organs in humans.

Study on the cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool: indication of biological epoxidation.

The cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool was studied in vitro by enzymatic assays using recombinant human cytochrome P450 enzymes. Three different enzymatic products of allylic hydroxylation and epoxidation were identified by gas chromatography-mass spectrometry. Identified enzymatic products were 8-hydroxylinalool ((R/S)-3,7-dimethyl-1,6-octadiene-3,8-diol) and the cyclic ethers pyranoid-linalool oxide ((R/S)-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol) and furanoid-linalool oxide (R/S)-2-(1,1-dimethylethyl)-5-methyl-5-vinyltetrahydrofuran. The cyclic ethers result most likely from the epoxidation of the 6,7-carbon double carbon bond of (R/S)-linalool, followed by the intramolecular rearrangement of the 6,7-epoxy-linalool. Allylic-hydroxylation of the 8-methyl group of linalool was catalyzed by CYP2C19 and CYP2D6 while the enzymatic epoxidation of linalool was only observed with CYP2D6. The results indicate that the electrophilic oxidation products of linalool such as 6,7-epoxy-linalool which may cause sensitization and irritational skin reactions are not only produced by auto-oxidation reactions in the presence of air-oxygen as published in the past, but also by P450-mediated oxidative biological transformation.

Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions.

A new method involving zinc sulphate deproteinization was developed to study short chain fatty acids (SCFA) production in the colon and subsequent occurrence of SCFA in blood. SCFA were baseline separated in a 30 min cycle using ion-exclusion chromatography and detected by mass spectrometry. Concentrations could be measured down to 10 microM and isotopomeric distributions could be assessed, enabling the conduction of tracer studies to study changes in SCFA synthesis. The applicability of the method was tested in an extensively characterized pig model yielding portal SCFA concentrations ranging from 70 microM (butyric acid) to 150 microM (propionic acid) to 440 microM (acetic acid) prior to butyrate tracer infusion, reaching butyric acid isotopic steady state within 2 h.

Degradation of a nonylphenol single isomer by Sphingomonas sp. strain TTNP3 leads to a hydroxylation-induced migration product.

Sphingomonas sp. strain TTNP3 degrades 4(3',5'-dimethyl-3'-heptyl)-phenol and unidentified metabolites that were described previously. The chromatographic analyses of the synthesized reference compound and the metabolites led to their identification as 2(3',5'-dimethyl-3'-heptyl)-1,4-benzenediol. This finding indicates that the nonylphenol metabolism of this bacterium involves unconventional degradation pathways where an NIH shift mechanism occurs.

Mass spectrometric analytical methods for the determination of endocrine disrupting chemicals (EDCs).

This paper describes a method for the determination of endocrine disrupting chemicals (EDCs) in waste water. The method involves a SPE-C18 extraction followed by a SPE-silica gel column clean up and elution of the analytes with a mixture of acetone/pentane (2+1). Thereafter derivatisation of the analytes with heptafluorobutyric acid anhydride (HFBA) and analysis by gas chromatography/mass spectrometry (GC/MS) with positive electron impact ionisation (EI) or negative chemical ionisation (NCI) using methane as reactant gas was performed. The two different ionisation techniques were compared and the negative chemical ionisation technique proved significant lower limits of detection (LODs) and quantitation (LOQs) than positive electron impact ionisation. Recoveries of the analytes in various spiked waste water samples ranged from 75.4 to 96.9%. The concentrations of the EDCs in feed waste water from three different waste water treatment plants (WWTPs) ranged from non-detectable up to a concentration of 10,305 ng/L for 4-NP and in the corresponding effluent samples from non-detectable to 723 ng/L for BPA.

Perfluorooctane sulfonate--a quite mobile anionic anthropogenic surfactant, ubiquitously found in the environment.

The biochemical degradation of perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) under aerobic and anaerobic conditions in closed-loop systems was monitored in laboratory scale. Adsorptive effects of these compounds to glass and polypropylene were also examined. Liquid chromatography/mass spectrometry (LC-MS) under negative electrospray (ESI(-)) conditions was applied for determination. Elimination of PFOS was observed under anaerobic conditions whereas aerobic treatment was not effective.