Detection of legal highs in the urine of methadone-treated patient by LC-MS.

Urine tests are the commonly accepted methods to control abstinence and adherence to treatment of patients who undergo methadone maintenance treatment (MMT). Depending on various national guidelines and accessibility of techniques, only selected psychoactive substances are routinely tested in urine of MMT patients. In general, they belong to the few groups of compounds: THC, cocaine, amphetamines, opiates, PCP and benzodiazepines. It is, however, well known that patients enrolled in such replacement programmes take psychoactive substances that are not routinely detected by the toxicology laboratories, to escape unexpected tests. Here, we report semiquantitative detection of legal highs taken by the MMT patient, using high-pressure liquid chromatography coupled to the flowing atmospheric pressure afterglow ion source (LC-FAPA-MS). To demonstrate effectivity of this technique, the data were confirmed by quantitative analysis using LC-ESI-MS/MS. In the analysed sample of MMT patient, a mixture of psychoactive compounds was found, namely 3-MMC (3-methylmethcathinone), pentedrone and methcathinone and determined at the concentrations of 670; 50 and 0.2 µg/mL, respectively. Such fast analytical technique may be useful for the efficient control of substances taken intentionally by MMT patients.

Monitoring changes in the healthy female metabolome across the menstrual cycle using GC × GC-TOFMS.

Urinary metabolomics offers a non-invasive means of obtaining information about the system-wide biological health of a patient. Untargeted metabolomics approaches using one-dimensional gas chromatography (GC) are limited due to the chemical complexity of urine, which poorly detects co-eluting low-abundance analytes. Metabolite detection and identification can be improved by applying comprehensive two-dimensional GC, allowing for the discovery of additional viable biomarkers of disease. In this work, we applied comprehensive two-dimensional GC coupled with time-of-flight mass spectrometry (GC × GC-TOFMS) to the analysis of urine samples collected daily across 28-days from 10 healthy female subjects for a personalized approach to female reproductive health monitoring. Through this analysis, we identified 935 unique volatile metabolites. Two statistical methods, a modified T-statistic and Wilcoxon Rank Sum, were applied to analyze differences in metabolome abundance on ovulation days as compared to non-ovulation days. Four metabolites (2-pentanone, 3-penten-2-one, carbon disulfide, acetone) were identified as statistically significant by the modified T-statistic but not the Rank Sum, after a false-discovery rate of 0.1 was set using a Benjamini-Hochberg correction. Subsequent analyses by boxplot indicated that the putative volatile metabolic biomarkers for fertility are expressed in increased or decreased abundance in urine on the day of ovulation. Individual analysis of metabolome expression across 28-days revealed some subject-specific features, which suggest a potential for long-term, personalized fertility monitoring using metabolomics.

Studies on the metabolism and detectability of the designer drug ß-naphyrone in rat urine using GC-MS and LC-HR-MS/MS.

Naphyrone (1-naphthalen-2-yl-2-pyrrolidin-1-yl-pentan-1-one; naphthylpyrovalerone, ß-naphyrone) is a cathinone designer drug and was marketed as replacement for the synthetic cathinone derivative mephedrone. Meanwhile, naphyrone is also classified as a controlled drug in several countries. Therefore, the aim of this study was to identify the metabolites of naphyrone in rat urine using gas chromatography-mass spectrometry techniques and to show its detectability in urine samples. The following metabolic steps could be detected in rat urine: oxidation of the pyrrolidine ring to the corresponding lactam, hydroxylation of the propyl side chain and the naphthyl ring, degradation to the primary amines after opening of the pyrrolidine ring, and combinations of these steps. Assuming similar kinetics, an intake of naphyrone should be detectable in human urine mainly via its metabolites.

Urinary volatile compounds as biomarkers for lung cancer.

Lung cancer is a leading cause of deaths in cancer. Hence, developing early-stage diagnostic tests that are non-invasive, highly sensitive, and specific is crucial. In this study, we investigated to determine whether biomarkers derived from urinary volatile organic compounds (VOCs) can be used to discriminate between lung cancer patients and normal control patients. The VOCs were extracted from the headspace by solid-phase microextraction and were analyzed by gas chromatography time-of-flight mass spectrometry. Nine putative volatile biomarkers were identified as elevated in the lung cancer group. Receiver operating characteristic curve analysis was also performed, and the markers were found to be highly sensitive and specific. Next we used principal component analysis (PCA) modeling to make comparisons compare within the lung cancer group, and found that 2-pentanone may have utility in differentiating between adenocarcinoma and squamous cell carcinomas.

Acetone, butanone, pentanone, hexanone and heptanone in the headspace of aqueous solution and urine studied by selected ion flow tube mass spectrometry.

Urine is commonly analysed in clinical practice by a variety of liquid-phase techniques to check for excessive ketone bodies, proteins and salts to name just a few compounds. However, little work has been carried out to measure the volatile compounds emitted by urine since these do not yet have an established role in clinical diagnosis. There is, however, a growing body of evidence that these volatile compounds can be indicators of adverse physiological conditions and disease and with the advent of sensitive gas-phase analytical methods they can be quickly quantified in urine headspace and potentially provide valuable support for clinical diagnosis. Thus, we are developing selected ion flow tube mass spectrometry, SIFT-MS, for the real-time analysis of urine headspace, ultimately to support rapid diagnosis in the clinical environment. In this paper we focus on volatile ketones in the headspace of aqueous solutions and urine donated by three healthy volunteers. Using SIFT-MS, we have unambiguously quantified in urine headspace acetone, by far the most abundant ketone, butanone, pentanone, hexanone and heptanone using NO(+) precursor ions. Further to this, we have determined the Henry's Law coefficients, HLC, for these ketones in aqueous solution to allow the liquid-phase concentrations in urine to be estimated from headspace levels of their vapours. In addition, the influence of the addition of physiological amounts of dissolved urea, sodium chloride and hydrochloric acid on the partitioning of these ketones between the aqueous phase and gas phase has been investigated and found to be small, which gives greater credence to the use of the HLC obtained using aqueous solutions for the estimation of ketone concentrations in urine. Finally, parallel measurements of the levels of acetone in exhaled breath and urine headspace have been obtained and shown to be very similar, which gives support to the previous deduction from breath analysis that acetone is a truly systemic compound.

Metabolite profiles of two [14C]-labelled catechol O-methyltransferase inhibitors, nitecapone and entacapone, in rat and mouse urine and rat bile.

The metabolites of two inhibitors of catechol O-methyltransferase, nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)2,4-pentanedione] and entacapone [(E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide++ +], excreted in urine and bile by rats and in urine by mice, were compared and quantified by using HPLC with radiochemical detection after administration of [14C]-labelled compounds. With the exception of 3-O-methylated nitecapone, no major metabolites were found in rat bile that were not found in rat urine. For both compounds the major biotransformations were the same in the mouse and the rat. However, a bisulfite adduct of nitecapone was found in rat urine only, and reduction of the C = C and C = O groups of the nitecapone side chain was more extensive in the mouse. After entacapone administration, the products of amide N-dealkylation were more abundant in rat urine than in mouse urine. Most of the dose was excreted in urine and bile as O-conjugates. Most abundant were the O-glucuronides, while smaller amounts of O-sulfates and O-methylated metabolites were found in both species. One non-glucuronide glycoside of entacapone was found in urine of both rats and mice.

Identification of major metabolites of the catechol-O-methyltransferase inhibitor nitecapone in the rat and dog.

Metabolites of nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione], a potent catechol-O-methyltransferase inhibitor with gastroprotective and antiulcerogenic effects, were isolated by extraction and HPLC from dog and rat urine after enzymatic hydrolysis and as glucuronic acid and sulfate conjugates. Eight and 10 nonconjugated metabolites and unchanged nitecapone were found in hydrolyzed dog and rat urine, respectively, and identified by HPLC with diode-array UV detection, electron ionization mass spectrometry, and IR spectroscopy. In both species the main phase I metabolic pathways were: 1) reduction of the side chain carbon-carbon double bond and carbonyl groups and 2) cleavage of the side-chain double bond, giving an aromatic aldehyde that was partly oxidized to the corresponding carboxylic acid. These phase I metabolites and unchanged nitecapone were excreted in urine mainly as their glucuronides and sulfates in both species. Additionally, the 3-O-methylated metabolite, not found in urine, was identified in rat plasma.

Determination of a catechol-O-methyltransferase inhibitor, nitecapone, in human plasma and urine by liquid chromatography.

Methods based on reversed-phase liquid chromatography with amperometric detection have been developed for determination of nitecapone, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione, a COMT inhibitor, in human plasma and urine. Nitecapone was extracted with ethyl acetate-hexane mixtures from plasma after acidification with hydrochloric acid and from urine as the tetrabutylammonium ion-pair of its diphenylborate derivative. The recoveries of both methods exceeded 70% and the relative standard deviations for within-day precision were less than 4% and 8% at 50 ng ml-1 and at the quantitation limits, respectively. The methods are selective, sensitive and precise enough for determination of 4-5 ng ml-1 of nitecapone in plasma and urine and are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.

Identification of major metabolites of the catechol-O-methyltransferase-inhibitor nitecapone in human urine.

Metabolites of nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione], a potent new catechol-O-methytransferase-inhibitor, were isolated from human urine both after hydrolysis with beta-glucuronidase and as intact conjugates. Seven phase-I metabolites and corresponding glucuronides were identified using electron ionization and fast atom bombardment mass spectrometry, IR spectroscopy, and proton NMR spectrometry. The most abundant metabolite in urine was the glucuronide of unchanged nitecapone, representing 60-65% of the metabolites found. The main phase-I metabolic reaction was reduction of the side chain double bond and carbonyl groups. One of the major metabolites was formed by cleavage of the side chain by retro aldol condensation. All phase-I metabolites were present mainly as their glucuronic acid conjugates. The 3-nitrocatechol-structure of nitecapone seems to hinder nitro-reduction, catechol-O-methylation, and sulfation reactions.