Confirmation of synthetic glucocorticoids with liquid chromatography/mass spectrometry: organization and results of an international interlaboratory comparison test

Within the framework of a European Union (EU) research project entitled "Food Safety Screening: Synthetic Glucocorticoids (QLK1-1999-00122)," an international interlaboratory ring test was organized to compare and evaluate different liquid chromatography/mass spectrometry (LC/MS) confirmatory methods that are applied in European monitoring programs for detecting the use of synthetic glucocorticoids. Liver and urine samples of bovines treated with synthetic glucocorticoids were collected and sent to the participants of the study for analysis. Participants received 3 liver and 3 urine samples and were free to use either their own LC/MS method or an LC/MS-based method developed during the EU research project. The residue concentrations in the samples were calculated as the mean of the concentrations reported by each laboratory. The mean dexamethasone concentration of liver sample L1 was calculated as 2.27 microg/kg [relative standard deviation (RSD) 43%, n = 9], which exceeds the maximum residue level (MRL) of 2 microg/kg. Three of the 9 laboratories (33%) reported concentration levels less than 2 microg/kg, resulting in obviously false compliant results. The overall mean concentration of flumethasone in liver sample L2 was calculated as 3.27 microg/kg (RSD 33%, n = 8). Applying a comparable limit for flumethasone of 2 microg/kg, 8 of the 9 laboratories would have obtained a correct noncompliant result. As for the blank liver sample, 1 participant found a false noncompliant result. The urine sample U1 contained prednisolone residues at a mean concentration of 1.58 microg/kg (RSD 43%, n = 9). Four out of 9 results were less than a theoretical minimum required performance level (MRPL) of 2 microg/kg. The calculated concentration of dexamethasone in urine sample U3 was 5.21 microg/kg (RSD 62%, n = 9). One of the 9 results was lower than 2 microg/kg. Urine sample U2 was correctly reported as blank by all participants.     

A confirmatory method for detection of a banned substance: the validation experience of a routine EU laboratory

The Commission Decision 2002/657/EC is a fundamental reference document for the UE laboratories involved in residue analysis although its implementation has caused some difficulties in the requirements interpretation. In this work a pragmatic validation approach of a quantitative confirmatory method for the detection of 17-alpha-(alpha-NT) and 17-beta-19-nortestosterone (beta-NT) in bovine urine by gas chromatography mass spectrometry is proposed. The 19-nortestosterone is a banned anabolic steroid for which no minimum required performance limit (MRPL) has been laid down, therefore the limit reported in Italian Residue Monitoring Plan (2 microg L(-1)) has been considered the reference level to evaluate the method performances. The decision limit (CCalpha) and the detection capability (CCbeta) were obtained by the calibration curve procedure. The minimum required performance level (mrpl), which represents the starting concentration of the calibration curves, was preliminary fixed estimating the results dispersion of blank urine samples fortified at 2 microg L(-1) for each isomer. The found CCalpha and CCbeta were 1.5 and 1.9 microg L(-1) for alpha-NT and 1.2 and 1.4 microg L(-1) for beta-NT. The precision (repeatability and within-laboratory reproducibility) and recoveries were suitable for the investigated concentration range (1-3 microg L(-1)). Finally, the method ruggedness (minor and major changes) has been also demonstrated.     

Quantitation of mercapturic acids from acrylamide and glycidamide in human urine using a column switching tool with two trap columns and electrospray tandem mass spectrometry

A sensitive and specific electrospray tandem mass spectrometry method using a column switching unit with two trap columns was established to quantify the mercapturates (MAs) of acrylamide (AA) and glycidamide (GA) in human urine. A specially endcapped material was applied for trapping the hydrophilic MAs and a pre-trap column was used to remove lipophilic compounds from the directly injected urine to protect the trap column. The limits of quantitation for AA-MA and GA-MA in urine were 0.5 microg/L and 1 microg/L, respectively. Urine was spiked with deuterated internal standards and injected directly into LC-MS/MS. Urine of smokers (n=13) revealed the highest concentrations of AA-MA and GA-MA in the range of 61-706 microg/L and 5-54 microg/L, respectively. Lower levels for AA-MA (14-102 microg/L) and GA-MA (1-11 microg/L) were detected in non-smokers (n=13).     

Negative ion chemical ionization GC/MS-MS analysis of dialkylphosphate metabolites of organophosphate pesticides in urine of non-occupationally exposed subjects

Low level exposure to organophosphate (OP) pesticides can be determined by the measurement of dialkylphosphate (DAP) metabolites in urine. An analytical method is presented here which can measure the metabolites dimethyl phosphate (DMP), diethyl phosphate (DEP), dimethyl thiophosphate (DMTP), dimethyl dithiophosphate (DMDTP), diethyl thiophosphate (DETP), and diethyl dithiophosphate (DEDTP) at low levels. This was achieved by lyophilization of the urine, derivatization with pentafluorobenzyl bromide (PFBBr) and quantification by negative ion chemical ionization GC/MS-MS. The detection limits for the metabolites were 0.5 microg L(-1) DMP, 0.1 microg L(-1) DEP, 0.1 microg L(-1) DMTP, 0.04 microg L(-1) DMDTP, 0.04 microg L(-1) DETP and 0.02 microg L(-1) DEDTP. The RSD for the analytical method was 4-14% for the six metabolites. The method was used to monitor a group of non-occupationally exposed individuals in Sydney, Australia. The metabolites DMP, DEP, DMTP, DMDTP, DETP and DEDTP occurred in 73, 77, 96, 48, 100 and 2% of the samples with median values of 13, 3, 12, <1, 1 and 1 microg L(-1) respectively. The method is simple to use, sensitive and suitable for routine analysis of non-occupational exposure levels. These detection limits are between one and two orders of magnitude lower than those previously reported in the literature.     

Unambiguous identification of thiouracil residue in urine collected in non-treated bovine by tandem and high-resolution mass spectrometry

Thyrostats are banned compounds in Europe since 1981 (directive 81/602/EC) because of their carcinogenic and teratogenic properties. However, the occurrence of thiouracil (TU) in bovine urines from national monitoring plans with quantifications in the range 1-10 microg . L(-1) occasionally raises the question of its origin which might either be the consequence of an illegal administration or the result of 'endogenous' production. In order to definitively and unambiguously identify the so-called thiouracil signal in non-treated bovine urines, independent mass spectrometry (MS) approaches have been used. Different reagents (3-IBBr, 3-BrBBr and PFBBr) were used to derivatise and to extract TU from urine samples and characterisation of the residues was performed by means of different MS approaches [LC/(ESI-)MS/MS, GC/(EI+)MS/MS and HRMS (EI and NCI)]. These combined strategies allowed for an independent and confident identification of TU in bovine urine samples collected from animals never treated with any thyrostatic drugs. This result is of prime importance for laboratories and risk managers involved in the field of forbidden growth promoters control: detection of TU residue in bovine urine will have to be carefully considered as a non-systematic proof of illegal administration.     

Optimization of solid phase extraction clean up and validation of quantitative determination of corticosteroids in urine by liquid chromatography-tandem mass spectrometry

A solid phase extraction (SPE) method for extraction and clean up of 9 synthetic corticosteroids was optimized for quantification by reversed-phase high-performance liquid chromatography/negative electrospray ionisation mass spectrometry. Clean up was accomplished using a mixed mode polymeric strong anion exchange SPE column. The final method was validated according to EU regulations for determination of residues of veterinarian drugs in products of animal origin. Initial results showed a large difference in ion suppression between samples of porcine and bovine urine. The aim of optimisation was to design a procedure that minimised this difference while using a single SPE procedure and a fast HPLC method that enabled sufficient separation of the epimers beta- and dexamethasone. To include conjugated corticosteroids in the analysis, the sample was hydrolysed with Helix Pomatia beta-glucuronidase/aryl sulfatase. For the final method, which included fluocinolone acetonid, triamcinolone acetonid, beclomethasone, flumethasone, dexamethasone, betamethasone, 6alpha-methylprednisolone, prednisone and prednisolone, a quantification based on spiked samples carried through the entire analytical procedure was used. For quantification of triamcinolone acetonid an internal standard (triamcinolone acetonid-D6) was used. Relative average recoveries from 96 to 103% were found, except for beclomethasone (113%). Absolute average recoveries were 81-99%. Quantification limits (decision limits, CCalpha) were demonstrated to be not higher than 1 microg L(-1) (3 microg L(-1) for prednisone and prednisolone). The internal reproducibility, determined by triplicates from spiking at three different levels in six analytical series was 7-19% (at 2-4 microg L(-1)) except for prednisone and prednisolone (26-27% at 3-6 microg L(-1)).     

Arsenic metabolism in seaweed-eating sheep from Northern Scotland

Cation exchange and anion exchange liquid chromatography were coupled to an ICP-MS and optimised for the separation of 13 different arsenic species in body fluids (arsenite, arsenate, dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), trimethylarsine oxide (TMAO), tetramethylarsonium ion (TMA), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinoyl ethanol (DMAE) and four common dimethylarsinoylribosides (arsenosugars). The arsenic species were determined in seaweed extracts and in the urine and blood serum of seaweed-eating sheep from Northern Scotland. The sheep eat 2-4 kg of seaweed daily which is washed ashore on the most northern Island of Orkney. The urine, blood and wool of 20 North Ronaldsay sheep and kidney, liver and muscle from 11 sheep were sampled and analysed for their arsenic species. In addition five Dorset Finn sheep, which lived entirely on grass, were used as a control group. The sheep have a body burden of approximately 45-90 mg arsenic daily. Since the metabolism of arsenic species varies with the arsenite and arsenate being the most toxic, and organoarsenic compounds such as arsenobetaine the least toxic compounds, the determination of the arsenic species in the diet and their body fluids are important. The major arsenic species in their diet are arsenoribosides. The major metabolite excreted into urine and blood is DMAA (95 +/- 4.1%) with minor amounts of MMAA, riboside X, TMA and an unidentified species. The occurrence of MMAA is assumed to be a precursor of the exposure to inorganic arsenic, since demethylation of dimethylated or trimethylated organoarsenic compounds is not known (max. MMAA concentration 259 microg/L). The concentrations in the urine (3179 +/- 2667 microg/L) and blood (44 +/- 19 microg/kg) are at least two orders of magnitude higher than the level of arsenic in the urine of the control sheep or literature levels of blood for the unexposed sheep. The tissue samples (liver: 292 +/- 99 microg/kg, kidney: 565 +/- 193 microg/kg, muscle: 680 +/- 224 microg/kg) and wool samples (10470 +/- 5690 microg/kg) show elevated levels which are also 100 times higher than the levels for the unexposed sheep.     

Simultaneous determination of t,t-muconic, S-phenylmercapturic and S-benzylmercapturic acids in urine by a rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry method

We describe a rapid and sensitive high-performance liquid chromatography/electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for simultaneous determination of the most relevant metabolites of benzene and toluene, t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (S-PMA), and S-benzylmercapturic acid (S-BMA). Urine samples were purified before analysis by solid-phase microextraction (SPE) on SAX cartridges with 50 mg sorbent mass. The developed method fulfils all the standard requirements of precision and accuracy. Calibration curves were linear within the concentration range of the standards (0-80 microg/L(urine) for t,t-MA, and 0-25 microg/L(urine) for S-PMA and S-BMA), and had correlation coefficients > or =0.997. Limits of detection were 6.0 microg/L for t,t-MA, 0.3 microg/L for S-PMA, and 0.4 microg/L for S-BMA. The method was used to determine t,t-MA, S-PMA and S-BMA levels in urine of 31 gasoline-station workers, with personal monitoring data obtained from radial symmetry passive diffusive samplers. In the context of mean work-shift exposures of 75.9 microg/m(3) (range 9.4-220.2) for benzene and 331.9 microg/m(3) (78.2-932.1) for toluene, metabolite concentrations in end-of-shift urine samples ranged from 23.5-275.3 microg/g(creatinine) for t,t-MA, non-detectable to 0.9 microg/g(creatinine) for S-PMA, and 3.8-74.8 microg/g(creatinine) for S-BMA. No significant correlation was found between the environmental concentrations and urinary metabolites (p > 0.05 for all cases); the ratios of benzene metabolites could be influenced by exposure levels and co-exposure to xylenes and toluene. The high throughput of this procedure should facilitate exploration of the metabolic effects of benzene-related co-exposure to toluene and alkylbenzenes in large populations of subjects exposed to gasoline.     

An electrospray ionization tandem mass spectrometry based system with an online dual-loop cleanup device for simultaneous quantitation of urinary benzene exposure biomarkers trans,trans-muconic acid and S-phenylmercapturic acid

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) system with an online dual-loop cleanup device was developed for simultaneous quantitation of the urinary benzene exposure biomarkers trans,trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA). The cleanup device was constructed from an autosampler, two electrically operated two-position switching valves, a reversed-phase C18 trap cartridge, a 200-microL loop, and two solvent-delivery pumps. The device was interfaced directly with a triple-quadrupole mass spectrometer and fully controlled by computer software and hardware. Because isotope dilution by introducing 13C-labeled ttMA and SPMA as internal standards was employed, the precision of the analytical system was high (for ttMA, intra- and inter-day CV values ranged from 3.82-4.53%; for SPMA, 2.13-7.06%). The calibration curves obtained using human urine spiked with ttMA were linear from 15.6-4000 microg/L (R = 0.9998) and SPMA at concentrations from 0.78-200 microg/L (R = 0.9993). The method detection limit (MDL) for SPMA was 0.23 microg/L. The MDL of ttMA could not be determined accurately because of unavailability of an appropriate blank urine matrix, but was estimated to be lower than 7.43 microg/L. Without tedious manual sample cleanup procedures the analytical system is fully automated and is therefore useful for high-throughput simultaneous determination of urinary ttMA and SPMA. The sample throughput is roughly 100 samples per day. With the selectivity and the sensitivity provided by MS/MS detection, the analytical system can be used for large-scale monitoring of environmental or occupational exposure of humans to benzene.     

Enantioselective analysis of pheniramine in urine by charged CD-mediated CZE provided with a fiber-based DAD and an on-line sample pretreatment by capillary ITP

Application potentialities of CZE on-line coupled with capillary ITP and DAD to the identification and determination of trace concentration levels (microg/L) of pheniramine (PHM) enantiomers and their metabolites present in complex ionic matrices of biological origin (urine) are shown. An enhanced (enantio)selectivity of the CZE separation system obtained by the addition of carboxyethyl-beta-CD (CE-beta-CD) to the carrier electrolyte provided CZE conditions for a reliable identification of similar/identical DAD spectra of structurally related compounds (PHM enantiomers and their metabolites) in clinical urine samples differing in qualitative and quantitative composition of sample matrix constituents. A high sample loadability (a 30 microL sample injection volume), partial sample clean-up (removing macroconstituents from the sample), and preconcentration of the analytes in ITP stage resulted in the decrease of concentration LOD for PHM enantiomers in urine to 5.2 and 6.8 microg/L (2.2 x 10(-8) and 2.8 x 10(-8) mol/L), without using any sample pretreatment technique. The background correction and smoothing procedure applied to the raw DAD spectra provided analytically relevant DAD spectra of PHM enantiomers and their metabolites also when they were present in urine sample (30 microL injection volumes of ten-times diluted urine sample) at a 9 x 10(-) (8) mol/L concentration. DAD spectra of PHM enantiomers present in urine samples matched their reference spectra with reasonable certainties. DAD spectra of PHM metabolites were compared with the reference spectra of PHM enantiomers and a good match was found which indicates the similarities in the structures of enantiomers and their metabolites detected in the urine samples. This fact allows performing the quantitative analyses of PHM metabolites in the urine samples by applying the calibration parameters of PHM enantiomers also for PHM metabolites and the results show the possibilities of using the ITP-CZE-DAD combination for the direct analysis of PHM enantiomers and/or their metabolites in urine without any sample pretreatment. ITP-CZE-DAD method with oppositely charged selector is suggested to use in clinical research as it provides favorable performance parameters including sensitivity, linearity, precision, recovery, and robustness with minimal demands on sample preparation.     

Analytical method for the quantitative determination of urinary ethylenethiourea by liquid chromatography/electrospray ionization tandem mass spectrometry

A direct, rapid and selective method for the quantitative determination of the ethylenethiourea (ETU) in human urine has been validated and is reported in the present study. It allows the accurate quantification of ETU in this complex matrix without the use of any internal standard as the sample cleanup is effective enough for the removal of interferences that could lead to ion suppression in the electrospray ionization (ESI) source. This simple and rapid purification system, based on the use of a Fluorosil phase of a BondElut column followed by a liquid-liquid extraction procedure, achieves mean extracted recoveries, assessed at three different concentrations (2.5, 10.0, and 25.0 microg/L), always more than 85%. High-performance liquid chromatography (HPLC) with positive ion tandem mass spectrometry, operating in selected multiple reaction monitoring (MRM) mode, is used to quantify ETU in human urine. The assay is linear over the range 0-50 microg/L, with a lower limit of quantification (LOQ) of 1.5 microg/L and a coefficient of variation (CV) of 8.9%. The lower limit of detection (LOD) is assessed at 0.5 microg/L. The overall precision and accuracy were determined on three different days. The values for within- and between-day precision are < or = 8.3 and 10.1%, respectively, and the accuracy is in the range 97-118%. The relative uncertainties for the LOQ and QC concentrations have been estimated to be 18 and 8%, respectively. The assay was applied to quantify ETU in human urine from growers that regularly handle ethylenebisdithiocarbamate pesticides in large crop plantations. The biological samples were collected at the start and end of the working day, and the ETU urine levels were found to vary between 1.9 and 8.2 microg/L.     

Simultaneous determination of cyclophosphamide, ifosfamide, doxorubicin, epirubicin and daunorubicin in human urine using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry: bioanalytical method validation

A reversed-phase high-performance liquid chromatography (rp-HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed-phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 microg.L(-1) for CP, IF, DXR, EPI and 0.15-2.0 microg.L(-1) for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 microg.L(-1) for CP, IF, EPI and was set at 0.3 and 0.15 microg.L(-1) for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter- and intra-day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp-HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI-MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug-manufacturing plants.     

Determination of acephate in human urine

Acephate is a commonly used organophosphate insecticide applied on agricultural crops and in residential communities. Because very little acephate is metabolized prior to excretion, the parent pesticide compound can be measured in human urine. The residue method must be sensitive enough to determine human exposure and potential health risk for both agricultural workers and their families who may be exposed by pesticide drift or by inadvertent carry-home residues. A reliable and sensitive method was developed to measure acephate concentrations in human urine. Urine was diluted with water and acetone, adjusted to a neutral pH, and partitioned twice in acetone-methylene chloride (1 + 1, v/v), with NaCl added to aid separation. The solvent-reduced organic phase extracts were clarified by activated charcoal solid-phase extraction and then adjusted to a final volume with the addition of a D-xylose analyte protectant solution to reduce matrix enhancement effects. Acephate concentrations in urine were determined by gas chromatography using pulsed flame photometric detection. The method limit of detection was established at 2 microg/L, with a method limit of quantitation of 10 microg/L. The average recovery from urine fortified with 10-500 microg/L was 102 +/- 12% (n = 32).     

Development of a high-performance liquid chromatography method for the simultaneous quantification of quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal tissues

A method of high-performance liquid chromatography with UV detection has been established for simultaneous quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the marker residues for carbadox (CBX) and olaquindox (OLA), respectively, in the muscles and livers of porcine and chicken and in the muscle of fish. Tissue samples were subject to acid hydrolysis followed by liquid-liquid extraction and Oasis MAX solid-phase extraction clean-up. The method was validated according to the EU Commission Decision 2002/657/EC. The decision limits (CCalpha) were 0.7-2.6microg/kg and the detection capabilities (CCbeta) were 1.3-5.6microg/kg for QCA and MQCA in tissues. The recoveries of QCA and MQCA, spiked at levels of 2-100microg/kg, were from 70 to 110%; the relative standard deviation values were <20%. This simple, fast and economic method could be applied to the monitoring for the possible misuse of CBX and OLA in animal edible tissue samples.     

Simultaneous determination of low levels of methotrexate and cyclophosphamide in human urine by micro liquid chromatography/electrospray ionization tandem mass spectrometry

The aim of this study was to develop and validate a novel solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of two antineoplastic drugs, cyclophosphamide (CP) and methotrexate (MTX), in human urine using trophosphamide as internal standard. The method showed good precision and accuracy (mean RSD 2.8% and 0.9%; bias 2.7% and 2.4% for MTX and CP, respectively). The lower limits of detection obtained, 0.2 microg/L(urine) for MTX and 0.04 microg/L(urine) for CP, were lower than the best previously reported values. The use of a 96-well SPE plate for matrix purification ensures a high throughput (50 samples/day), allowing the routine biological monitoring of CP and MTX as measures of occupational exposure at very low levels.     

Application of positive ion chemical ionisation and tandem mass spectrometry combined with gas chromatography to the trace level analysis of ethyl carbamate in bread

A rapid, sensitive and selective method has been developed and validated for the analysis of the contaminant ethyl carbamate (EC) in bread products at the part-per-billion level. The new procedure uses positive ion chemical ionisation (PICI) and tandem mass spectrometry (MS/MS), combined with gas chromatography (GC), on a 'bench-top' triple-quadrupole mass spectrometer. Ammonia was the PICI reagent gas of choice because of its ability to produce abundant [M+H]+ and [M+NH4]+ ions from EC and deuterium-labelled EC (LEC) used as an internal standard. For identification and quantification, selected reaction monitoring (SRM) was used to follow the precursor-to-product ion transitions of m/z 107 --> 90, m/z 107 --> 62 and m/z 90 --> 62 for EC, as well as m/z 112 --> 63 for the LEC internal standard. The limits of detection and quantification were 0.6 and 1.2 microg kg(-1), respectively, and the recovery of the method was 101 +/- 10% at 10 microg kg(-1) and 98 +/- 5% at 100 microg kg(-1). The precision of the method, established under conditions of intermediate reproducibility, did not exceed a relative standard deviation of 7%. The quantitative performance of the new GC/PICI-SRM procedure compared favourably with that of a reference method based on GC/MS and selected ion monitoring (correlation coefficient, r = 0.997). However, the new method had the advantages of reduced sample preparation time, improved sensitivity and unambiguous identification of EC at all concentrations. Application of the new method to the analysis of 50 UK breads showed that levels of EC ranged from 0.6 to 2.3 microg kg(-1) in retail products and from 3.1 to 12.2 microg kg(-1) for breads prepared using domestic breadmaking machines (dry weight basis). Toasting bread in a domestic toaster led to increases of between two- and three-fold in mean EC concentrations.     

Phenolic compounds in field horsetail (Equisetum arvense L.) as natural antioxidants

In this paper, the study of antioxidant activity and phenolic composition of three different extracts (EtOAc, n-BuOH and H(2)O) of field horsetail (Equisetum arvense L.) is presented. The antioxidant activity has been evaluated measuring the total reducing power (expressed by Ascorbate Equivalent Antioxidant Capacity - AEAC), inhibition of lipid peroxidation, and free radical scavenging capacity (RSC) towards 2,2-diphenyl-1-picrylhydrazyl (DPPH radical) and nitric oxide (NO), respectively. In addition, the total flavonoid content (TFC) and phenolic constituents of each extract have been determined. The results obtained show that the highest RSC regarding both DPPH and NO radicals is expressed by EtOAc extract (EC(50)=2.37 microg/mL and EC(50)=90.07 microg/mL, respectively), and the lowest by H(2)O extract (EC(50)=37.2 microg/mL and EC(50)>333.33 microg/mL, respectively). n-BuOH extract showed the highest total reducing power (AEAC=13.40 microg/mL). Differences in the phenolic composition of examined extracts are found comparing the HPLC chemical profiles. Although, isoquercitrin is the main flavonoid in both EtOAc and n-BuOH extracts, a considerable amount of di-E-caffeoyl-meso-tartaric acid was presented in the n-BuOH extract. In H(2)O extract high content of phenolic acids and low percentage of flavonoids were detected.     

Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry

Determination of 16 priority pesticides and transformation products specified in the EU Baby Food Directive 2003/13/EC has been compared using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) coupled to tandem quadrupole mass spectrometry (MS/MS). Prior to analysis, co-extractives were removed from acetonitrile extracts using dispersive solid-phase extraction (SPE) with primary secondary amine (50 mg). Extracts spiked with pesticides at 1 microg kg(-1) yielded average recoveries in the range 85-119%, with relative standard deviations less than 17%. The HPLC-MS/MS and UPLC-MS/MS multi-residue methods developed are simple, rapid and suitable for the quantification and confirmation of the 16 priority pesticides in fruit-, potato- and cereal-based baby food at 1 microg kg(-1). The major advantages of UPLC, using 1.7 microm particles, over HPLC are the speed of analysis, the narrower peaks (giving increased signal-to-noise ratio) and improved confirmation for the targeted pesticides in the analyses of baby foods.     

Determination of total urinary mercury by on-line sample microwave digestion followed by flow injection cold vapour inductively coupled plasma mass spectrometry or atomic absorption spectrometry

The total mercury content in urine was determined by inductively coupled plasma mass spectrometry with the so-called cold vapour method after on-line oxidative treatment of the sample in a microwave oven (FI-MW-CV-ICPMS). Use of a KBr/KBrO(3) mixture, microwave digestion, and the final oxidation with KMnO(4), assure the complete recovery of the organic forms of Hg which would be difficult to determine otherwise if using only the CV-ICPMS apparatus. Quantitative recoveries were obtained for phenyl Hg chloride (PMC), dimethyl Hg (DMM), Hg acetate (MA) and methyl Hg chloride (MMC). Use of automatic flow injection microwave systems (FI-MW) for sample treatment reduces environmental contamination and allows detection limits suitable for the determination of reference values. Since no certified reference materials were commercially available in the concentration ranges of interest, the accuracy of the proposed procedure has been assessed by analysing a series of urine samples with two independent techniques, ICP-MS and AAS. When using the FI-MW-CV-ICP-MS technique, the detection limit was assessed at 0.03microg/L Hg, while with FI-MW-CV-AAS it was 0.2microg/L Hg. The precision of the method was less than 2-3% for FI-MW-CV-ICP-MS and about 3-5% for FI-MV-CV-AAS at concentrations below 1microg/L Hg. These results show that ICP-MS can be considered as a "reference technique" for the determination of total urinary Hg at very low concentrations, such as are present in non-exposed subjects.     

Multi-detection of corticosteroids in sports doping and veterinary control using high-resolution liquid chromatography/time-of-flight mass spectrometry

A liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) method was developed using the latest high-resolution LC column technology, the ultra performance liquid chromatography (UPLC), and electrospray ionization (ESI) in the positive ion mode. Gradient UPLC separation conditions were optimized for a group of 22 analytes comprising 17 glucocorticosteroids, specific designer steroids such as tetrahydrogestrinone (THG) and specific beta2-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5 min only for all the substances tested. Even the critical pair of dexamethasone and betamethasone isomers was almost completely resolved. Thanks to the over 10,000 full-width at half maximum (FWHM) mass resolution and high mass accuracy features of TOFMS 50 mDa window accurate mass chromatograms could be reconstructed for the individual analytes. Sensitive screening in human and calf urine samples fortified at the glucocorticosteroids minimum required performance limit (MRPL) of 30 microg L(-1) (human urine, sports doping) and 2 microg L(-1) (calf urine, veterinary control) could be obtained. The potential of UPLC/TOFMS for confirmatory analysis was shown by determining the accurate mass of all compounds and fragment ions upon in-source collision-induced dissociation (CID) at different energies. The exact mass measurement errors for all glucocorticosteroids were found to be within 6 ppm. Considering veterinary control, limits of detection (LOD) and limits of quantification (LOQ) were determined for most of the analytes in calf urine and found to range from 0.1 to 3.3 and from 0.4 to 4.4 microg L(-1), respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta2-agonists to the original analyte mixture in urine, without causing any interferences.