Sara C. Cunha

Quantification of free and total bisphenol A and bisphenol B in human urine by dispersive liquid–liquid microextraction (DLLME) and heart-cutting multidimensional gas chromatography–mass spectrometry (MD–GC/MS)

A novel method combining dispersive liquid-liquid microextraction (DLLME) and heart-cutting multidimensional gas chromatography coupled to mass spectrometry was developed for the determination of free and total bisphenol A (BPA) and bisphenol B (BPB) in human urine samples. The DLLME procedure combines extraction, derivatization and concentration of the analytes into one step. Several important variables influencing the extraction efficiency and selectivity such as nature and volume of extractive and dispersive solvents as well as the amount of acetylating reagent were investigated. The temperature and time to hydrolyze BPA and BPB conjugates with a β-glucuronidase and sulfatase enzyme preparation were also studied. Under the optimized conditions good efficiency extraction (71-93%) and acceptable total DLLME yields (56-77%) were obtained for both analytes. Matrix-matched calibration curves were linear with correlation coefficients higher than 0.996 in the range level 0.1-5 μg/l, and the relative standard deviations (%RSD) were lower than 20% (n=6). The limits of detection were 0.03 and 0.05 μg/l for BPA and BPB, respectively. The applicability of the proposed method for determining urinary free and total BPA and BPB was assessed by analyzing the human urine of a group of 20 volunteers. Free BPA was detected in 45% of the sample whereas total BPA was detected in 85% of the samples at concentrations ranging between 0.39 and 4.99 μg/l. BPB was detected in conjugated form in two samples.

Development and validation of a method based on a QuEChERS procedure and heart-cutting GC-MS for determination of five mycotoxins in cereal products.

A new analytical method for the rapid and simultaneous determination of five mycotoxins (zearelenone, deoxynivalenol, Fusarenon X, 15-acetyldeoxynivalenol and nivalenol) in breakfast cereals and flours by heart-cutting GC-MS has been developed and validated. Extraction was performed with MeCN, applying a modified QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) procedure, and the extracts were analyzed after a silylation of the analytes under study. Careful optimization of the parameters of Deans Switch device and GC-MS was achieved in order to attain a fast separation in SIM mode, allowing a total run time of only 8 min. Acceptable recoveries for all mycotoxins at two different spiking levels (20 and 100 microg/kg) were achieved with good repeatability (from 9 to 21%). LOD ranged from 2 to 15 microg/kg and LOQ ranged from 5 to 50 microg/kg, which were lower than the maximum limit legal established by the European Union (EU). The method developed was applied to commercial breakfast cereals and flours; among the mycotoxins studied, deoxynivalenol and zearalenone were the most predominant.

Fast analysis of multiple pesticide residues in apple juice using dispersive liquid–liquid microextraction and multidimensional gas chromatography–mass spectrometry

A method for the rapid trace analysis of 24 residual pesticides in apple juice by multidimensional gas chromatography-mass spectrometry (MD-GC/MS) using dispersive liquid-liquid microextraction (DLLME) was developed and optimized. Several parameters of the extraction procedure such as type and volume of extraction solvent, type and volume of dispersive solvent and salt addition were evaluated to achieve the highest yield and to attain the lowest detection limits. The DLLME procedure optimized consists in the formation of a cloudy solution promoted by the fast addition to the sample (5 ml) of a mixture of carbon tetrachloride (extraction solvent, 100 microl) and acetone (dispersive solvent, 400 microl). The tiny droplets formed and dispersed among the aqueous sample solution are further joined and sedimented (85 microl) in the bottom of the conical test tube by centrifugation. Once extracted, all the 24 pesticides were directly injected and separated by a dual GC column system, comprising a short wide-bore DB-5 capillary column with low film thickness connected by a Deans switch system to a second chromatographic narrower column, with identical stationary phase. The instrumental setting used, in combination with carefully optimized operational fast GC and MS parameters, markedly decreased the retention times of the targeted analytes. The total chromatographic run was 8 min. Mean recoveries for apple juice spiked at three concentrations ranged from 60% to 105% and the intra-repeatability ranged from 1% to 21%. The limits of detection of the 24 pesticides ranged from 0.06 to 2.20 microg/L. In 2 of a total of 28 analysed samples were found residues of captan, although at levels below the maximum limit legal established.

Optimisation of extraction procedures for analysis of benzoic and sorbic acids in foodstuffs

Benzoic and sorbic acids are the most commonly used preservatives in foodstuffs. They are usually analysed by RP-HPLC. However, in view of the complexity and diversity of foodstuffs composition, appropriate sample preparation procedures are required for reliable extraction of these preservatives from the matrices. Specific extraction procedures for analysis of jams, table olives, spreadable fats, sauces, fruit juices and wines were optimised. Thus, different types of food matrices were chosen, including those with high sugar content, with high fat content and beverages (with and without alcohol). A significant set of validation data was performed through recovery and precision studies. Chromatographic separation was achieved using a C18 column (S10 ODS2) and acetate buffer 0.005 M (pH=4.4)—methanol (65:35) as mobile phase, 1.4 ml/min flow rate and UV detection at 235 nm. The concentration of preservatives in the samples was calculated by external standard method. Benzoic and sorbic acids in jams, jellies and table olives were efficiently extracted with methanol after ground homogenization. Fortified samples, at 4 different concentration levels of benzoic and sorbic acids, presented average recoveries (after discarding outliers) for each preservative greater than 91% with a coefficient of variation (CV) less than 2.6%. Sorbic acid was extracted from spreadable fats and emulsified sauces with n-hexane and was back-extracted to an aqueous phase with acetate buffer 0.005 M (pH=4.4). Recoveries were higher than 98% for two levels of concentration and CV lower than 2.9%. The preservatives extraction from fruit juices (orange, apple and pineapple) and wines required purification using a Sep-Pak C18 cartridge, and its elution with methanol. Average recoveries of benzoic and sorbic acids at two levels of concentration were greater than 94% with CV less than 4.0%. Eighty-seven commercial brands were analysed including table olives (29), jams (24), jellies (2) spreadable fats (25), sauces (3), fruit juices (10) and table wines (3). All samples conformed to the legal prescriptions.

Multipesticide residue analysis in maize combining acetonitrile-based extraction with dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry.

A fast and simple gas chromatography-mass spectrometry (GC-MS) method for determination of forty-one pesticide residues in maize is introduced. The sample preparation involves liquid-liquid partitioning with acetonitrile in presence of anhydrous MgSO(4) and NaCl (QuEChERS) followed by dispersive liquid-liquid microextraction (DLLME) using carbon tetrachloride as extractive solvent and the extract obtained by QuEChERS as dispersive solvent. The main factors influencing DLLME efficiency including extractive solvent type and volume as well as the volume of dispersive solvent were evaluated in this study. The DLLME procedure effectively provides an enrichment of the extract and a cleanup of certain polar matrix components, which can maximize the sensitivity when a single quadrupole MS is used. For validation purposes, recoveries studies were carried out at two concentration levels, yielding recovery rates in the range 70-120% for 82% of the analytes. A good linearity and precision, with relative standard deviations generally below 20% were obtained for all forty-one pesticides. The limits of detection obtained were lower than 19 μg kg(-1) for more than 63% of the analytes. In two of a total of ten samples of maize, residues of lindane, tefluthrin, pirimicarb, folpet and bifenthrin were found, although at levels below the maximum limit established for this kind of samples.

Fast low-pressure gas chromatography-mass spectrometry method for the determination of multiple pesticides in grapes, musts and wines.

A fast method using low-pressure gas chromatography coupled to mass spectrometry (LP-GC/MS) was implemented and optimized to yield a complete separation of 27 representative pesticides in grapes, musts and wines. Extraction was performed with acetonitrile, applying quick, easy, cheap, effective, rugged and safe (QuEChERS) methodology. Several LP-GC/MS conditions such as column temperature, injection conditions, flow rate, MS conditions and matrix effects were evaluated to achieve the fastest separation with the highest sensitivity in MS detection (selected ion monitoring mode). After optimization, all 27 pesticides were extracted, chromatographically separated and detected in less than 20 min. Acceptable recoveries for nearly all pesticides at three different spiking levels (from 0.04 to 2.5 microg/g) were achieved with good repeatability (from 3 to 21%). Limits of quantification (from 0.02 to 5 microg/g) were lower than the maximum limit of residues, when established for pesticides.

Simultaneous determination of bisphenol A and bisphenol B in beverages and powdered infant formula by dispersive liquid–liquid micro-extraction and heart-cutting multidimensional gas chromatography-mass spectrometry

The purpose of this study was to establish a reliable, cost-effective, fast and simple method to quantify simultaneously both bisphenol A (BPA) and bisphenol B (BPB) in liquid food matrixes such as canned beverages (soft drinks and beers) and powdered infant formula using dispersive liquid–liquid micro-extraction (DLLME) with in-situ derivatisation coupled with heart-cutting gas chromatography-mass spectrometry (GC-MS). For the optimisation of the DLLME procedure different amounts of various extractive and dispersive solvents as well as different amounts of the derivative reagent were compared for their effects on extraction efficiency and yields. The optimised procedure consisted of the injection of a mixture containing tetrachloroethylene (extractant), acetonitrile (dispersant) and acetic anhydride (derivatising reagent) directly into an aliquot of beverage samples or into an aqueous extract of powdered milk samples obtained after a pretreatment of the samples. Given the compatibility of the solvents used, and the low volumes involved, the procedure was easily associated with GC-MS end-point determination, which was accomplished by means of an accurate GC dual column (heart-cutting) technique. Careful optimisation of heart-cutting GC-MS conditions, namely pressure of front and auxiliary inlets, have resulted in a good analytical performance. The linearity of the matrix-matched calibration curves was acceptable, with coefficients of determination (r2) always higher than 0.99. Average recoveries of the BPA and BPB spiked at two concentration levels into beverages and powdered infant formula ranged from 68% to 114% and the relative standard deviation (RSD) was <15%. The limits of detection (LOD) in canned beverages were 5.0 and 2.0 ng l–1 for BPA and BPB, respectively, whereas LOD in powdered infant formula were 60.0 and 30.0 ng l–1, respectively. The limits of quantification (LOQ) in canned beverages were 10.0 and 7.0 ng l–1 for BPA and BPB, respectively, whereas LOQ in powdered infant formula were 200.0 and 100.0 ng l–1, respectively. BPA was detected in 21 of 30 canned beverages (ranging from 0.03 to 4.70 µg l–1) and in two of seven powdered infant formula samples (0.23 and 0.40 µg l–1) collected in Portugal. BPB was only detected in canned beverages being positive in 15 of 30 samples analysed (ranging from 0.06 to 0.17 µg l–1). This is the first report about the presence of BPA and BPB in canned beverages and powdered infant formula in the Portuguese market.

Polybrominated diphenyl ethers (PBDEs) contents in house and car dust of Portugal by pressurized liquid extraction (PLE) and gas chromatography-mass spectrometry (GC-MS).

Dust is the repository of various compounds including flame retardants. In this study an analytical method based on PLE extraction and gas chromatography-mass spectrometry was selected for the analysis of 16 PBDEs congeners in house and car dust samples collected in Portugal. The analytical performance of the method was validated using standard reference material (SRM); values from 90% to 109% and from 2% to 11% were obtained for recovery and precision, respectively. The PBDE congeners distribution in whole and sieved fractions of the dust samples, as well as influence of the source on the levels of these contaminants, were obtained. The wide range of PBDEs contents found in the dust samples indicates heterogeneous levels of contamination in these matrices. The clearest feature of the results obtained was that Deca-BDE was the main PBDE in both house and car dust samples. The total PBDEs measured in house dust (ranging from 34 to 1928 ng g(-1)) was lower than those found in car dust (ranging from 193 to 22955 ng g(-1)). However, house dust provides a major contribution to human exposure due to the time spent there, much higher than in cars.

Monitoring pesticide residues in greenhouse tomato by combining acetonitrile-based extraction with dispersive liquid-liquid microextraction followed by gas-chromatography-mass spectrometry.

A multiclass and multiresidue method for pesticide analysis in tomato was validated. Extraction and pre-concentration of the pesticide residues from acetonitrile extracts was performed by using dispersive liquid-liquid microextraction (DLLME) technique, followed by gas chromatography-mass detection. DLLME was performed using carbon tetrachloride as extractive solvent and acetonitrile extract as dispersive solvent, in order to increase enrichment factor of the extraction procedure. Validation parameters indicated the suitability of the method for routine analyses of thirty pesticides in a large number of samples. In general, pesticide recoveries ranged between 70% and 110% and repeatability ranged between 1% and 20%. The proposed method was applied to the monitoring of pesticides in tomatoes grown during winter in greenhouses. Among the compounds considered in this work, cyprodinil was found in tomato at concentrations of 0.33mg/kg, other pesticides like azoxystrobin, fenhexanid, tolyfluanid, λ-cyhalothrin and trifloxystrobin were also detected, but, not quantified.

Brominated flame retardants and seafood safety: A review

Brominated flame retardants (BFRs), frequently applied to industrial and household products to make them less flammable, are highly persistent in the environment and cause multi-organ toxicity in human and wildlife. Based on the review of BFRs presence in seafood published from 2004 to 2014, it is clear that such pollutants are not ideally controlled as the surveys are too restricted, legislation inexistent for some classes, the analytical methodologies diversified, and several factors as food processing and eating habits are generally overlooked. Indeed, while a seafood rich diet presents plenty of nutritional benefits, it can also represent a potential source of these environmental contaminants. Since recent studies have shown that dietary intake constitutes a main route of human exposure to BFRs, it is of major importance to review and enhance these features, since seafood constitutes a chief pathway for human exposure and biomagnification of priority environmental contaminants. In particular, more objective studies focused on the variability factors behind contamination levels, and subsequent human exposure, are necessary to support the necessity for more restricted legislation worldwide.