Nuclear magnetic resonance-based metabolomics for prediction of gastric damage induced by indomethacin in rats

Non-steroidal anti-inflammatory drugs (NSAIDs) have side effects including gastric erosions, ulceration and bleeding. In this study, pattern recognition analysis of the ¹H-nuclear magnetic resonance (NMR) spectra of urine was performed to develop surrogate biomarkers related to the gastrointestinal (GI) damage induced by indomethacin in rats. Urine was collected for 5 h after oral administration of indomethacin (25 mg kg⁻¹) or co-administration with cimetidine (100 mg kg⁻¹), which protects against GI damage. The ¹H-NMR urine spectra were divided into spectral bins (0.04 ppm) for global profiling, and 36 endogenous metabolites were assigned for targeted profiling. The level of gastric damage in each animal was also determined. Indomethacin caused severe gastric damage; however, indomethacin administered with cimetidine did not. Simultaneously, the patterns of changes in their endogenous metabolites were different. Multivariate data analyses were carried out to recognize the spectral pattern of endogenous metabolites related to indomethacin using partial least square-discrimination analysis. In targeted profiling, a few endogenous metabolites, 2-oxoglutarate, acetate, taurine and hippurate, were selected as putative biomarkers for the gastric damage induced by indomethacin. These metabolites changed depending on the degree of GI damage, although the same dose of indomethacin (10 mg kg⁻¹) was administered to rats. The results of global and targeted profiling suggest that the gastric damage induced by NSAIDs can be screened in the preclinical stage of drug development using a NMR based metabolomics approach.     

Sample cleanup-free determination of mycophenolic acid and its glucuronide in serum and plasma using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Mycophenolic acid (MPA) is an immunosuppressant drug which powerfully inhibits lymphocyte proliferation. Since the early 1990s it has been used to prevent rejection in organ transplantation. The requirement of therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and its major metabolite mycophenolic acid glucuronide (MPAG).The authors developed a sample cleanup-free, rapid, and highly specific method for simultaneous measurement of MPA and MPAG in human plasma and serum using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry. MPA- and MPAG-determinations were performed during a 2.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05-100 (r > 0.999) mg L⁻¹ and 4-4000 mg L⁻¹ (r > 0.999), respectively. Limits of Detection were 0.014 mg L⁻¹ for MPA and 1.85 mg L⁻¹ for MPAG. Lower Limits of Quantification were 0.05 mg L⁻¹ for MPA and 2.30 mg L⁻¹ for MPAG. Interassay imprecision was <10% for both substances. Mean recovery was 103.6% (range 78.1-129.7%) for MPA and 111.1% (range 73.0-139.6%) for MPAG. Agreement was good for MPA and MPAG between the presented method and a validated HPLC-MS/MS method. The Passing-Bablok regression line for MPA and MPAG was HPLC-MS/MS = 1.14 UPLC-MS/MS—0.14 [mg L⁻¹], r = 0.96, and HPLC-MS/MS = 0.77 UPLC-MS/MS + 0.50 [mg L⁻¹], r = 0.97, respectively. This sample cleanup-free and robust LC-MS/MS assay facilitates the rapid, accurate and simultaneous determination of MPA and MPAG in human body fluids.     

Determination of multi-class pesticides in wines by solid-phase extraction and liquid chromatography-tandem mass spectrometry

This work reports a new sensitive multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection, confirmation and quantification of forty-six pesticides and transformation products belonging to different chemical classes in wines. The proposed method makes use of a solid-phase extraction (SPE) procedure with Oasis HLB cartridges that combines isolation of the pesticides and sample clean-up in a single step. Analysis is performed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-MS/MS) operated in the selected reaction monitoring (SRM) mode, acquiring two specific precursor-product ion transitions per target compound. An investigation of matrix effects has been performed during method validation showing medium to low effects for the majority of the compounds. Limits of detection (LODs) were in the range 0.0003–0.003 mg L⁻¹ and limits of quantification (LOQs) were in the range 0.001–0.01 mg L⁻¹. The average recoveries, measured at two concentration levels (0.010 and 0.050 mg L⁻¹), were in the range 70–110% for most of the compounds tested with % relative standard deviations below 20%, while a value of 0.010 mg L⁻¹ has been established as the method limit of quantification (MLOQ) for all target species. Expanded uncertainty values were in the range 10–40% while the Horrat ratios were below 1. The method has been successfully applied to the analysis of 60 wine samples in the course of an annual monitoring study with carbendazim-benomyl, thiophanate-methyl and carbaryl being the most frequently determined pesticides.     

Targeted analysis with benchtop quadrupole–orbitrap hybrid mass spectrometer: Application to determination of synthetic hormones in animal urine

Sensitive and unequivocal determination of analytes/contaminants in complex matrices is a challenge in the field of food safety control. In this study, various acquisition modes (Full MS/AIF, Full MS + tMS/MS, Full MS/dd MS/MS and tSIM/ddMS/MS) and parameters of a quadrupole–orbitrap hybrid mass spectrometer (Q Exactive) were studied in detail. One of the main conclusions has been that, reducing the scan range for Full MS (using the quadrupole) and targeted modes give higher signal-to-noise (S/N) ratios and thereby better detection limits for analytes in matrix. The use of Q Exactive in a complex case, for the confirmatory analysis of hormones in animal urine is presented. A targeted SIM data dependent MS/MS (tSIM/ddMS/MS) acquisition method for determination of eight synthetic hormones (trenbolone, 17α ethinylestradiol, zeranol, stanozolol, dienestrol, diethylstilbestrol, hexestrol, taleranol) and a naturally occurring hormone (zearalenone) in animal urine were optimized to have sensitive precursors from targeted SIM mode and trigger MS/MS scans over the entire chromatograph peak. The method was validated according to EC/657/2002. CCα (decision limit) for the analytes ranged between 0.11 μg L⁻¹ and 0.69 μg L⁻¹ and CCβ (detection capability) ranged between 0.29 μg L⁻¹ and 0.90 μg L⁻¹.     

Accelerated solvent-based extraction and enrichment of selected plasticisers and 4-nonylphenol, and extraction of tin from organotin sources in sediments, sludges and leachate soils

Enrichment techniques have become an important feature in the trace analysis of oestrogen mimicking chemicals in the environment. Recent developments such as accelerated solvent extraction (ASE) have improved extraction recoveries in a wide variety of solid matrices including sediments, sludges and leachate soils. Such samples taken from the Irish Midlands Shannon Catchment region during the winter of 2004/5 and suspected to contain certain xenooestrogens or hormonally active agents were extracted using this technique, which was then coupled with high performance liquid chromatography (HPLC) for quantification purposes. ASE was thus employed to both isolate and pre-concentrate targeted analytes using the minimum amount of solvent hence making extractions more conservational. Two simple, yet extremely sensitive liquid chromatographic methods were developed based on UV detection; one for phthalates and one for alkylphenols, with recoveries reaching up to 92.0%. Acid digestion was used for the extraction of the tin and organotin compounds with analysis by polarography. In river sediment, levels of up to 24.4 mg kg⁻¹ phthalate, 1.14 mg kg⁻¹ 4-nonylphenol and 118 mg kg⁻¹ tin were found. In leachate sediments, values up to 49.8 mg kg⁻¹ phthalate, 1.57 mg kg⁻¹ 4-nonylphenol, and 36.0 mg kg⁻¹ tin were determined. In sludge, values up to 174 mg kg⁻¹ phthalate and 22.8 mg kg⁻¹ 4-nonylphenol were quantified. The highest value of tin (118 mg kg⁻¹) was found present in an area of high leisure craft activity. Typical sediment levels of tin at other river locations ranged between 1.20 and 37.5 mg kg⁻¹.     

Mineral content and botanical origin of Spanish honeys

Eleven elements (Zn, P, B, Mn, Mg, Cu, Ca, Ba, Sr, Na and K) were determined by inductively plasma coupled spectrometry in 40 honey samples from different places of Spain and four different botanical origins: Eucalyptus (Eucalyptus sp.), Heather (Erica sp.), Orange-blossom (Citrus sinensis) and Rosemary (Rosmarinus officinalis). K, Ca and P show the higher levels with average concentrations ranged between 434.1-1935 mg kg⁻¹ for K; 42.59-341.0 mg kg⁻¹ for Ca and 51.17-154.3 mg kg⁻¹ for P. Levels of Cu (0.531-2.117 mg kg⁻¹), Ba (0.106-1.264 mg kg⁻¹) and Sr (0.257-1.462 mg kg⁻¹) are the lowest in all honey samples. Zn (1.332-7.825 mg kg⁻¹), Mn (0.133-9.471 mg kg⁻¹), Mg (13.26-74.38 mg kg⁻¹) and Na (11.69-218.5 mg kg⁻¹) concentrations were found strongly dependent on the kind of botanical origin.Results were submitted to pattern recognition procedures, unsupervised methods such as cluster and principal components analysis and supervised learning methods like linear discriminant analysis in order to evaluate the existence of data patterns and the possibility of differentiation of Spanish honeys from different botanical origins according to their mineral content. Cluster analysis shows four clusters corresponding to the four botanical origins of honey and PCA explained 71% of the variance with the first two PC variables. The best-grouped honeys were those from heather; eucalyptus honeys formed a more dispersed group and finally orange-blossom and rosemary honeys formed a less distinguishable group.     

Determination of melamine in milk-based products and other food and beverage products by ion-pair liquid chromatography-tandem mass spectrometry

This paper describes a fast method for the sensitive and selective determination of melamine in a wide range of food matrices, including several milk-based products. The method involves an extraction with aqueous 1% trichloroacetic acid before the injection of the 10-fold diluted extract into the liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) system, using labelled melamine as the internal standard. As melamine is present in aqueous media in the cationic form, the chromatographic separation in reversed-phase LC requires the use of anionic ion-pair reagents, such as tridecafluoroheptanoic acid (THFA). This allows a satisfactory chromatographic retention and peak shape in all the types of food samples investigated. The method has been validated in six food matrices (biscuit, dry pasta and four milk-based products) by means of recovery experiments in samples spiked at 1 and 5 mg kg⁻¹. Average recoveries (n = 5) ranged from 77% to 100%, with excellent precision (RSDs lower than 5%) and limits of detection between 0.01 and 0.1 mg kg⁻¹. In addition, accuracy and robustness of the method was proven in different soya-based matrices by means of quality control (QC) sample analysis. QC recoveries, at 1 and 2.5 mg kg⁻¹, were satisfactory, ranging from 79% to 110%. The method developed in this work has been applied to the determination of melamine in different types of food samples. All detections were confirmed by acquiring two MS/MS transitions (127 > 85 for quantification; 127 > 68 for confirmation) and comparing their ion intensity ratio with that of reference standards. Accuracy of the method was also assessed by applying it to a milk-based product and a baking mix material as part of an EU proficiency test, in which highly satisfactory results were obtained.     

Direct determination of melamine in dairy products by gas chromatography/mass spectrometry with coupled column separation

A coupled capillary column system was developed for the qualitative and quantitative determination of melamine with isotope internal standard in dairy products by gas chromatography/mass spectrometry (GC/MS) without derivatization. A 30 m of DB-5ms ((5%-phenyl)-methylpolysiloxane, 0.25 mm i.d., 0.25 μm df) coupled with a 1.5 m of Innowax (polyethylene glycol, 0.32 mm i.d., 0.25 μm df) by a quartz capillary column connector was introduced as separation column. Three advantages were discussed for the coupled system. The sample was fortified with a ring-labeled ¹³C₃¹⁵N₃-melamine as an isotope internal standard and extracted by 1% of trichloroacetic acid aqueous solution. 2.2% of lead acetate solution was then added to deposit protein in the sample matrix. After purification by cation exchange cartridge, the sample solution was directly injected and detected by GC/MS. A six-point calibration curve ranging from 0.05 to 2 mg kg⁻¹ of melamine in sample was used to establish instrument response. The recovery was 93.9-102% with relative standard deviation from 3.1 to 8.7% when isotope internal standard used. The calculated method detection limit was 0.01 mg kg⁻¹.     

Assessment of cadmium and iron adsorption in sediment,employing a flow injection analysis system with on line filtration and detection by flame atomic absorption spectrometry and thermospray flame furnace atomic absorption spectrometry

This work presents an evaluation of iron and cadmium adsorption in sediment of the Furnas Hydroelectric Plant Reservatory located in Alfenas, Minas Gerais (Brazil). The metal determination was done employing a flow injection analysis (FIA) with an on-line filtering system. As detection techniques, flame atomic absorption spectrometry (FAAS) for iron and thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) for cadmium determinations were used. The developed methodology presented good limits of detection, being 190 μg L⁻¹ for iron and 1.36 μg L⁻¹ for cadmium, and high sampling frequency for both metals 144 and 60 readings h⁻¹ for iron and cadmium, respectively. Both metals obey the Langmuir model, with maximum adsorptive capacity of 0⋅169 mg g⁻¹ for iron and 7⋅991 mg g⁻¹ for cadmium. For iron, a pseudo-first-order kinetic model was obtained with a theoretical Q_e = 9⋅8355 mg g⁻¹ (experimental Q_e = 9⋅5432 mg  g⁻¹), while for cadmium, a pseudo-second-order kinetic model was obtained, with a theoretical Q_e = 0.3123 mg g⁻¹ (experimental Q_e = 0⋅3052 mg g⁻¹).     

Determination of the alkyl- and methoxy-phenolic content in wood extractives by micellar solid-phase microextraction and gas chromatography-mass spectrometry

This work combines the utilization of the micellar media during the extraction step and the focused microwave-assisted extraction followed by the solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) to determine the alkyl- and methoxy-phenolic content in wood extractives. The proposed environmental-friendly method is mainly characterized by short analysis times (5 min for the microwaves extraction step) and for avoiding the use of organic solvents. Different surfactants were tried for the extraction process: the cationic surfactant CTAB and the non-ionic surfactants Triton X-100 and POLE, with similar extraction efficiencies (85.5-99.7%). The overall method presents limits of detection in the ng g⁻¹ region for the alkylphenols (from 7 to 150 ng g⁻¹) and in the μg g⁻¹ region for the methoxyphenols (from 0.80 to 22.9 μg g⁻¹). The vanillin was the compound most abundant in the wood extractives studied, with concentrations up to 116.2 mg kg⁻¹.     

Quantitative metabolic profiling of 21 endogenous corticosteroids in urine by liquid chromatography-triple quadrupole-mass spectrometry

Since corticosteroid metabolism may be affected by disease states, the accurate and precise measurement of endogenous corticosteroids in urine is necessary to understand their biochemical roles. An efficient quantitative profiling of 21 endogenous corticosteroids in urine has been validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After enzymatic hydrolysis with β-glucuronidase, samples were purified using a solid-phase extraction cartridge and then separated through a sub-2 μm particle C18 column (2.1 mm × 50 mm, 1.9 μm) and quantified within 12.1 min using a triple quadrupole MS with electrospray ionization in positive ion mode. All corticosteroids resulted in the base-line separation, which is even achieved for stereo-isomers, such as α-/β-cortol, α-/β-cortolone, and allo-tetrahydrocortisol/tetrahydrocortisol. Overall recoveries ranged from 85% to 106% with limit of quantification ranged from 0.5 to 2.0 ng mL⁻¹ for the corticosteroids examined. The precision (% CV) and accuracy (% bias) of the assay were 1.7-7.8% and 95.1-105.4%, respectively, in 0.5-200 ng mL⁻¹ calibration ranges (r² > 0.9903), for quality-control samples containing 21 endogenous corticosteroids at three different urinary concentrations. Clinical application included quantitative analysis from patients with both prostate cancer and benign prostatic hyperplasia with altered cortisol concentrations. The described LC-MS/MS method eliminates interference from other urine components, has excellent chromatographic resolution achieved by a small particle LC column with a sufficient sensitivity to allow the profiling of both gluco- and mineralo-corticosteroids at a time.     

A simple; efficient and environmentally friendly method for the extraction of pesticides from onion by matrix solid-phase dispersion with liquid chromatography-tandem mass spectrometric detection

An evaluation of the extraction of pesticides from onion by matrix solid-phase dispersion (MSPD) with the determination by liquid chromatography tandem mass spectrometry using electrospray as the ionization source (LC-ESI-MS/MS) was carried out. The performance of different sorbents, including reused C18 bonded silica, was evaluated. Different parameters affecting the extraction efficiency were evaluated, such as the type and amount of sorbent, the time of interaction after the fortification step, the time of sample dispersion and the elution solvent. The matrix effect regarding the recovery of the pesticides by MSPD was also investigated. The best results were obtained using 0.5 g of sample, 1.0 g reused C18, interaction time of 1 h, dispersion time of 5 min, and acetonitrile as the elution solvent. The method was validated by the fortification of the onion sample, free of pesticides, at different concentration levels (0.01, 0.1 and 1.0 mg kg⁻¹). Average recoveries ranged from 78.3 to 120.4% and relative standard deviation below 20% was obtained. Detection and quantification limits ranged from 0.003 to 0.03 mg kg⁻¹ and from 0.01 to 0.1 mg kg⁻¹, respectively.     

Phospholipidomic identification of potential plasma biomarkers associated with type 2 diabetes mellitus and diabetic nephropathy

Type 2 diabetes mellitus (T2DM) and its attendant complications, such as diabetic nephropathy (DN), impose a significant societal and economic burden. The investigation of discovering potential biomarkers for T2DM and DN will facilitate the prediction and prevention of diabetes. Phospholipids (PLs) and their metabolisms are closely allied to nosogenesis and aggravation of T2DM and DN. The aim of this study is to characterize the human plasma phospholipids in T2DM and DN to identify potential biomarkers of T2DM and DN. Normal phase liquid chromatography coupled with time of flight mass spectrometry (NPLC-TOF/MS) was applied to the plasma phospholipids metabolic profiling of T2DM and DN. The plasma samples from control (n = 30), T2DM subjects (n = 30), and DN subjects (n = 52) were collected and analyzed. The significant difference in metabolic profiling was observed between healthy control group and DM group as well as between control group and DN group by the help of partial least squares discriminant analysis (PLS-DA). PLS-DA and one-way analysis of variance (ANOVA) were successfully used to screen out potential biomarkers from complex mass spectrometry data. The identification of molecular components of potential biomarkers was performed on Ion trap-MS/MS. An external standard method was applied to quantitative analysis of potential biomarkers. As a result, 18 compounds in 7 PL classes with significant regulation in patients compared with healthy controls were regarded as potential biomarkers for T2DM or DN. Among them, 3 DM-specific biomarkers, 8 DN-specific biomarkers and 7 common biomarkers to DM and DN were identified. Ultimately, 2 novel biomarkers, i.e., PI C18:0/22:6 and SM dC18:0/20:2, can be used to discriminate healthy individuals, T2DM cases and DN cases from each other group.     

Determination of metal content in some herbal drugs-Empirical and chemometric approach

The concentrations of Cu, Zn, Mn, Fe, K, Ca, Mg, Al, Ba and B in 26 herbal drugs of special importance in phytopharmacy were studied. Flame atomic absorption and emission spectrometry (FAAS, FAES), as well as inductively coupled plasma atomic emission spectrometry (ICP-AES), were applied in this work. The whole procedure, from sample preparation, via dissolution, to measurements, was validated by using CRM (NIST 1573a—tomato leaves), and the obtained recovery values are in the range from 91 to 102%. Drug samples originated from medicinal plants cultivated in Serbia contained Cu (4.47-14.08 mg kg⁻¹), Zn (8.4-54.5 mg kg⁻¹), Mn (9-155 mg kg⁻¹), Fe (47-546 mg kg⁻¹), K (0.20-6.24%), Ca (0.18-1.84%), Mg (0.13-1.09%), Al (16-416 mg kg⁻¹), Ba (11.70-84.83 mg kg⁻¹) and B (5.1-118.7 mg kg⁻¹). In order to get a better insight into the elemental patterns, a common chemometric approach to data evaluation was used. Four significant factors identified by principal component analysis (PCA) were attributed partly to the significant influential sources and high mobility of some elements thus referring to potential anthropogenic contamination as well.     

Conversion to isothiocyanates via dithiocarbamates for the determination of aromatic primary amines by headspace-solid phase microextraction and gas chromatography

A novel and highly selective method has been developed for the determination of aromatic primary amines by their conversion to dithiocarbamates by reaction with carbon disulphide, and then to isothiocyanates, which are volatile, by heating in the presence of a heavy metal ion. Zinc(II) was selected owing to its low toxicity and optimum yield of isothiocyanates. The latter were sampled by headspace-solid phase microextraction (HS-SPME) on divinylbenzene-carboxen-polydimethylsiloxane fibre, 50/30 μm. The HS-SPME procedure was optimized to provide adequate limits of detection in the analysis of aromatic amines in their real samples by gas chromatography with mass spectrometry (GC–MS) or flame ionization detection (GC–FID). The method gave rectilinear calibration graph, correlation coefficient and limit of detection, respectively, over the range 0.08–100 μg L⁻¹, 0.9950–0.9990 and 25–240 ng L⁻¹ in gas chromatography–mass spectrometry, and 0.01–10 mg L⁻¹, 0.9910–0.9991 and 0.8–3.0 μg L⁻¹ in gas chromatography–flame ionization detection. At two different levels, 10 and 40 μg L⁻¹, the range of intra-day RSD was 3.7–8.5% (GC–MS) and 3.3–9.2% (GC–FID), respectively. The proposed method is simple and rapid, and has been applied to determine aromatic primary amines in the environmental waters, food samples of ice cream powder and soft drinks concentrate, and food colours. The intra-day RSD in the analysis of real samples by GC–MS was in the range 3.6–6.2%. The food/colour samples were found to contain elevated levels of aniline and 2-toluidine.     

Determination of methylmercury and estimation of total mercury in seafood using high performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS): Method development and validation

A method was developed for determination of methylmercury and estimation of total mercury in seafood. Mercury (Hg) compounds were extracted from 0.5 g edible seafood or 0.2 g lyophilized reference material by adding 50 ml aqueous 1% w/v l-cysteine·HCl·H₂O and heating 120 min at 60 °C in glass vials. Hg compounds in 50 μl of filtered extract were separated by reversed-phase high performance liquid chromatography using a C-18 column and aqueous 0.1% w/v l-cysteine·HCl·H₂O + 0.1% w/v l-cysteine mobile phase at room temperature and were detected by inductively coupled plasma-mass spectrometry at mass-to-charge ratio 202. Total Hg was calculated as the mathematical sum of methyl and inorganic Hg determined in extracts. For seafoods containing 0.055-2.78 mg kg⁻¹ methylmercury and 0.014-0.137 mg kg⁻¹ inorganic Hg, precision of analyses was ≤5% relative standard deviation (R.S.D.) for methylmercury and ≤9% R.S.D. for inorganic Hg. Recovery of added analyte was 94% for methylmercury and 98% for inorganic Hg. Methyl and total Hg results for reference materials agreed with certified values. Limits of quantitation were 0.007 mg kg⁻¹ methylmercury and 0.005 mg kg⁻¹ inorganic Hg in edible seafood and 0.017 mg kg⁻¹ methylmercury and 0.012 mg kg⁻¹ inorganic Hg in lyophilized reference materials. Evaluation of analyte stability demonstrated that l-cysteine both stabilized and de-alkylated methylmercury, depending on holding time and cysteine concentration. Polypropylene adversely affected methylmercury stability. Total Hg results determined by this method were equivalent to results determined independently by cold vapour-atomic absorption spectrometry. Methylmercury was the predominant form of Hg in finfish. Ratios of methylmercury/total Hg determined by this method were 93-98% for finfish and 38-48% for mollusks.     

Microcalorimetry as a possible tool for phenanthrene toxicity evaluation to eukaryotic cells

Microcalorimetry and measurement of culture turbidity using a Bioscreen C Analyzer System were applied to study the toxic effect of phenanthrene on Cunninghamella elegans IM 1785/21Gp spore germination. The results of C. elegans spore incubation in Bioscreen C microbiology reader showed the inhibition of spore germination by 70% (with 25 mg l⁻¹ of phenanthrene) and total inhibition of the fungus growth with a higher content of the xenobiotic (50-100 mg l⁻¹). The microcalorimetric technique showed to be useful for the estimation of metabolic activity of C. elegans spores in growth medium up to xenobiotic concentrations of 90 mg l⁻¹. These data corresponded with the microscopic observations. The obtained results showed that the microcalorimetry method could be a valuable supplement in the study on the mechanism of PAHs detoxification by fungi.     

Optimization of chemical and instrumental parameters in hydride generation laser-induced breakdown spectrometry for the determination of arsenic,antimony, lead and germanium in aqueous samples

A laser induced breakdown spectrometry hyphenated with on-line continuous flow hydride generation sample introduction system, HG-LIBS, has been used for the determination of arsenic, antimony, lead and germanium in aqueous environments. Optimum chemical and instrumental parameters governing chemical hydride generation, laser plasma formation and detection were investigated for each element under argon and nitrogen atmosphere. Arsenic, antimony and germanium have presented strong enhancement in signal strength under argon atmosphere while lead has shown no sensitivity to ambient gas type. Detection limits of 1.1 mg L⁻¹, 1.0 mg L⁻¹, 1.3 mg L⁻¹ and 0.2 mg L⁻¹ were obtained for As, Sb, Pb and Ge, respectively. Up to 77 times enhancement in detection limit of Pb were obtained, compared to the result obtained from the direct analysis of liquids by LIBS. Applicability of the technique to real water samples was tested through spiking experiments and recoveries higher than 80% were obtained. Results demonstrate that, HG-LIBS approach is suitable for quantitative analysis of toxic elements and sufficiently fast for real time continuous monitoring in aqueous environments.     

Evaluation of alternate lines of Fe for sequential multi-element determination of Cu, Fe, Mn and Zn in soil extracts by high-resolution continuum source flame atomic absorption spectrometry

The usefulness of the secondary line at 252.744 nm and the approach of side pixel registration were evaluated for the development of a method for sequential multi-element determination of Cu, Fe, Mn and Zn in soil extracts by high-resolution continuum source flame atomic absorption spectrometry (HR-CS FAAS). The influence of side pixel registration on the sensitivity and linearity was investigated by measuring at wings (248.325, 248.323, 248.321, 248.329, and 248.332 nm) of the main line for Fe at 248.327 nm. For the secondary line at 252.744 nm or side pixel registration at 248.325 nm, main lines for Cu (324.754 nm), Mn (279.482 nm) and Zn (213.875 nm), sample flow-rate of 5.0 mL min⁻¹ and calibration by matrix matching, analytical curves in the 0.2-1.0 mg L⁻¹ Cu, 1.0-20.0 mg L⁻¹ Fe, 0.2-2.0 mg L⁻¹ Mn, 0.1-1.0 mg L⁻¹ Zn ranges were obtained with linear correlations better than 0.998. The proposed method was applied to seven soil samples and two soil reference materials (IAC 277; IAC 280). Results were in agreement at a 95% confidence level (paired t-test) with reference values. Recoveries of analytes added to soil extracts containing 0.15 and 0.30 mg L⁻¹ Cu, 7.0 and 14 mg L⁻¹ Fe, 0.60 and 1.20 mg L⁻¹ Mn, 0.07 and 0.15 mg L⁻¹ Zn, varied within the 94-99, 92-98, 93-101, and 93-103% intervals, respectively. The relative standard deviations (n = 12) were 2.7% (Cu), 1.4% (Fe - 252.744 nm), 5.7% (Fe - 248.325 nm), 3.2% (Mn) and 2.8% (Zn) for an extract containing 0.35 mg L⁻¹ Cu, 14 mg L⁻¹ Fe, 1.1 mg L⁻¹ Mn and 0.12 mg L⁻¹ Zn. Detection limits were 5.4 μg L⁻¹ Cu, 55 μg L⁻¹ Fe (252.744 nm), 147 μg L⁻¹ Fe (248.325 nm), 3.0 μg L⁻¹ Mn and 4.2 μg L⁻¹ Zn.     

A new reliable sample preparation for high throughput focused steroid profiling by gas chromatography–mass spectrometry

The use of steroid hormones as growth promoters in cattle has been banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. If an efficient monitoring of synthetic compounds (screening and confirmation) is ensured today by many laboratories, pointing out suspicious samples from a natural steroids abuse remains a tricky challenge due to the difficulty to set relevant threshold levels for these endogenous compounds. The development of focused profiling or untargeted metabolomic approaches is then emerging in this context, with the objective to reveal potential biomarkers signing an exogenous administration of such natural steroids. This study aimed to assess sample preparation procedures based on microextraction and adapt them to high throughput urinary profiling or metabolomic analyses based on gas chromatography–mass spectrometry measurement. Two techniques have been tested and optimised, namely solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS), using five model steroid metabolites (16α-hydroxyandrosterone, 2α-hydroxytestosterone, 11-keto,5β-androstanedione, 6α-hydroxyestradiol and 7β-hydroxypregnenolone). The considered performance criteria included not only the absolute response of the targeted compounds but also the robustness of the materials, and the global aspect of the diagnostic ion chromatograms obtained. After only five successive urinary extractions, a clear degradation of the SPME fiber was observed which led to discard this method as a relevant technique for profiling, whereas no degradation was observed on MEPS sorbent. Repeatability and recovery yields were calculated from urine samples fortified at 500 μg L⁻¹ and extracted by MEPS. They were found respectively below 11% and above 60% for all model compounds. Detection limits were in the 5–15 μg L⁻¹ range depending on the compounds, and a good linearity was observed on the 10–75 μg L⁻¹ range (R² > 0.99). This methodology was applied on urine samples collected from control versus androstenedione-treated bovines, revealing a significant concentration increase for several well-known metabolites such as etiocholanolone, 5α-androstane-3β,17α-diol, 5β-androstane-3α,17α-diol and 5-androstene-3β,17α-diol. Finally, these results allowed to confirm the suitability of the developed strategy and give to this new MEPS application a promising interest in the field of GC–MS based steroid profiling and metabolomic.