Laboratory of Neuro-Oncology and Clinical & Cancer Proteomics. Dept of Neurology ErasmusMC P.O. Box 2040 3000CA Rotterdam, the Netherlands
Dr Molewaterplein 50, Room Ee-1981 (visiting address) 3015 GE Rotterdam, the Netherlands Telephone:+31-10-7043320 Fax:+31-10-7044365 Mobile:+31-6-24169522
Abstract: Kaletra (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)-ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.
Abstract: 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.
Abstract: Whole-body protein synthesis and breakdown are measured by a combined tracer infusion protocol with the stable isotope amino acids L-[ring-(2)H(5)]-phenylalanine, L-[ring-(2)H(2)]-tyrosine and L-[ring-(2)H(4)]-tyrosine that enable the measurement of the phenylalanine to tyrosine conversion rate. We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of very low tracer-tracee ratios (TTR) of the amino acids L-phenylalanine and L-tyrosine in human plasma. TTR calibration curves of the tracers L-[ring-(2)H(5)]-phenylalanine, L-[ring-(2)H(2)]-tyrosine and L-[ring-(2)H(4)]-tyrosine were linear (r(2)>0.99) in the range between 0.01% and 5.0% TTR and lowest measurable TTR for the tracers was 0.01% at a physiological concentration of 60 microM. The method was applied successfully to plasma samples from a clinical study reaching a steady state enrichment plateau (mean+/-SD) of 3.33+/-0.19% for L-[ring-(2)H(5)]-phenylalanine, 2.40+/-0.43% for L-[ring-(2)H(2)]-tyrosine and 0.29+/-0.07% for L-[ring-(2)H(4)]-tyrosine, respectively. The LC-MS/MS method can be applied for measurement of very low plasma enrichments of phenylalanine and tyrosine for the determination of whole-body protein synthesis and breakdown rates in humans.
Abstract: A method was developed for the determination of the monoterpene alcohols verbenol, myrtenol, perillyl alcohol, alpha-terpineol, Delta(3)-carene-10-ol, thymol and p-alpha,alpha-trimethylbenzylalcohol in urine samples. After an enzymatic cleavage of their glucuronide- and sulfate conjugates the monoterpene alcohols were converted in the urine matrix with 7-diethylaminocoumarin-3-carbonylazide into monoterpene-[7-(diethylamino)-coumarin-3-yl]-carbamate derivates prior to analyses. Enrichment of the monoterpene alcohols from the urine matrix was achieved by online-solid phase extraction (SPE) with restricted-access material (RAM). After removal of excess derivatization reagent and urine matrix components, the monoterpene derivatives were separated by high-performance liquid chromatography (HPLC) in combination with fluorescence (FLD) detection and simultaneous mass spectrometric (MS) identification. Detection limits (LOD) for studied monoterpene alcohols ranged between 22 and 197 ng/L. The method was validated and successfully applied to urine samples from human subjects orally exposed to monoterpenes trough an intake of cough medication containing monoterpenes as active medicinal ingredients.
Abstract: Sphingomonas sp. strain TTNP3 has been previously described as a bacterium that is capable of degrading the technical mixture of nonylphenol (NP) isomers and also the 4(3â²,5â²-dimethyl-3â²-heptyl)-phenol single isomer of NP. Until recently, 3,5-dimethyl-3-heptanol was the only reported metabolite of 4(3â²,5â²-dimethyl-3â²-heptyl)-phenol. A short time ago, the detection of an intracellular metabolite resulting from the oxidation of 4(3â²,5â²-dimethyl-3â²-heptyl)-phenol which was identified as 2(3,5-dimethyl-3-heptyl)-benzenediol has been reported. A decisive element for this identification was the occurrence of some slight differences with the two most probable metabolites i.e. 4(3â²,5â²-dimethyl-3â²-heptyl)-resorcinol and 4(3â²,5â²-dimethyl-3â²-heptyl)-catechol. These facts led us to hypothesise some NIH shift mechanisms explaining the formation of 2(3â²,5â²-dimethyl-3â²-heptyl)-benzenediol. In the present work, we describe the steps that led to the detection of these metabolites in the intracellular fraction of Sphingomonas sp. strain TTNP3. The formation of analogous intracellular metabolites resulting from the degradation of the technical mixture of NP is reported. To further elucidate these degradation products, studies were carried out with cells grown with 4(3â²,5â²-dimethyl-3â²-heptyl)-phenol as sole carbon source. The description of the syntheses of reference compounds, i.e. 4(3â²,5â²-dimethyl-3â²-heptyl)-resorcinol and 4(3â²,5â²-dimethyl-3â²-heptyl)-catechol and their comparative analyses with the intermediates of the degradation of 4(3â²,5â²-dimethyl-3â²-heptyl)-phenol are presented.
Abstract: 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.
Abstract: 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.
Abstract: The objective of the study was to monitor the occurrence of a wide range of OCPs and sediments of the Lake Volvi in northern Greece and to determine their temporal and spatial variations. The samples have been collected seasonally for a period of one year. Solid-phase extraction followed by gas-chromatographic techniques with electron-capture detection was used for the determination of the compounds. The compounds detected were HCB, lindane, β-HCH, heptachlor, methoxychlor, agr-endosulphan, and 2,4-DDT, while agr-HCH, aldrin, heptachlorperoxide, 4,4-DDE, dieldrin, endrin, 4,4-DDD, β-endosulphan, and 4,4-DDT could not be observed (detection limitsââ¥1ângâL-1). The frequencies of the compounds detected in the samples varied between 10 and 83% and between 6 and 88% in the water and sediment samples, respectively. Among the 16 OCPs pesticides monitored, only 2,4-DDT and methoxychlor were found in detectable concentrations in sediments throughout the whole sampling period. The water-sediment distribution pattern of the detected compounds reflects the great capacity of the lake sediments to adsorb and accumulate such compounds.
Abstract: The advanced oxidation process (AOP) reagents ozone (O3), O3/UV, O3/H2O2, and H2O2/Fe2+ (Fenton's reagent) were applied to the anionic and the non-ionic fluorinated surfactants perfluorooctanesulfonate (PFOS) and N-ethyl-N-(perfluoroalkyl)-sulfonyl-glycinic acid (HFOSA-glycinic acid) or N-ethyl-N-perfluoroalkyl sulfonylamido-2-ethanol polyethoxylates (NEtFASE-PEG), their methyl ethers (NEtFASE-PEG methyl ether) and partly fluorinated alkyl-ethoxylates (FAEO) dissolved in ultrapure water. To monitor the efficiencies of destruction samples were taken during the treatment period of 120 min. After sample concentration by C18-solid phase extraction (SPE) and desorption MS, coupled with atmospheric pressure chemical ionisation (APCI) or electrospray interface (ESI) was applied for detection. No elimination of PFOS was observed while HFOSA-glycinic acid and AOP treated non-ionic surfactants were eliminated by oxidation. Degradation products could be detected and identified. So PFOS was observed during HFOSA-glycinic acid oxidation. Polyethylene glycols (PEG) and PEG methyl ethers were generated from non-ionic fluorinated surfactants beside their oxidation products--aldehydes and acids--all identified by tandem (MS-MS) or multiple stage mass spectrometry (MSn). AOP treatment of FAEO blend resulted in a mixture of partly fluorinated alcohols, separated and identified using GC-MS.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: The endocrine disrupting chemical nonylphenol (NP) is a technical product which consists of a complex mixture of nonylphenols with different alkyl side-chain isomers. Since the bio-degradation of each NP isomer may lead to its own range of metabolites, the isolation and identification of transformation products is very difficult. In order to overcome this difficulty, the nonylphenol isomer 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) was synthesized, and its degradation by an axenic culture of Sphingomonas TTNP3 was investigated with [ring-U-14C]-labelled and non-labelled p353NP including a time-course study. Radioactive mass balancing resulted in different polar soluble fractions, in insoluble radioactivity associated with biomass, and volatile radioactivity in the form of the mineralization product 14CO2. In the extracellular media, the presence of nonanol corresponding to the nonyl chain of the NP isomer was confirmed and its concentration was determined during the course of fermentation. No other radioactive compounds were detected beside the parent isomer. Radioactive metabolites were only found in the intracellular fraction of S. TTNP3.
Abstract: An analytical method has been developed for simultaneous extraction and determination of two estrogenic active agents, 4-nonylphenol (4-NP) and bisphenol A (BPA), in activated sewage sludge and anaerobically stabilized sewage sludge. Both compounds were determined by GC/MS in freeze-dried sewage sludge samples that had been spiked with these compounds in order to indicate different contamination levels. Extractive steam distillation, Soxhlet extraction, supercritical fluid extraction (SFE), and accelerated solvent extraction (ASE) were applied, and the results were compared. ASE under use of ethyl acetate/formic acid and an extraction temperature of 170 degrees C provided the most efficient extraction procedure for simultaneous extraction and the only reliable extraction results. Analyses of the phenolic compounds were performed by capillary column gas chromatography/mass spectrometry (GC/MS), operating in selected ion monitoring (SIM) mode after an aluminum oxide column-cleanup step prior to acetylation. The observed recovery rates under optimized conditions-ASE with ethyl acetate/formic acid-for 4-NP in spiked activated sewage sludge samples (spiking levels: 51, 88, or 554 microg/g on dry weight basis (dwb)) were 90, 107, or 101%. BPA (spiking levels: 50, 87, or 474 microg/g dwb) was found with recovery rates of 70, 105, or 101%, respectively. For anaerobically stabilized sewage sludge, recoveries for 4-NP (spiking levels: 40, 66, or 196 microg/g dwb) were 97, 95, or 101% and 90, 95, or 101% for BPA (spiking levels: 41, 67, or 474 microg/g dwb), respectively.
