Current analytical methods for determination of glucosinolates in vegetables and human tissues.

Numerous epidemiological studies have indicated the potential effects of glucosinolates and their metabolites against cancer as well as other non-communicable diseases, such as cardiovascular disease and neurodegenerative disorders. However, information on the presence and quantity of glucosinolates in commonly consumed vegetables and in human fluids is sparse, largely because well-standardised methods for glucosinolate determination are not available, resulting in published data being inconsistent and conflicting. Thus, studies published since 2002 on the most recent developments of glucosinolate extraction and identification have been collected and reviewed with emphasis on determination of the intact glucosinolates by LC-MS and LC-MS/MS. This overview highlights the glucosinolate extraction methods used, the stability of glucosinolates during extraction, the availability of stable isotope labelled internal standards and the use of NMR for purity analysis, as well as the current analytical techniques that have been applied for glucosinolate analysis, e.g. liquid chromatography with mass spectrometric detection (LC-MS). It aims to interpret the findings with a focus on the development of a validated method, which will help to determine the glucosinolate content of vegetative plants and human tissues, and the identification and determination of selected glucosinolate metabolites.

Re-evaluation of phosphoric acid-phosphates - di-, tri- and polyphosphates (E 338-341, E 343, E 450-452) as food additives and the safety of proposed extension of use.

The Panel on Food Additives and Flavourings added to Food (FAF) provided a scientific opinion re-evaluating the safety of phosphates (E 338-341, E 343, E 450-452) as food additives. The Panel considered that adequate exposure and toxicity data were available. Phosphates are authorised food additives in the EU in accordance with Annex II and III to Regulation (EC) No 1333/2008. Exposure to phosphates from the whole diet was estimated using mainly analytical data. The values ranged from 251 mg P/person per day in infants to 1,625 mg P/person per day for adults, and the high exposure (95th percentile) from 331 mg P/person per day in infants to 2,728 mg P/person per day for adults. Phosphate is essential for all living organisms, is absorbed at 80-90% as free orthophosphate excreted via the kidney. The Panel considered phosphates to be of low acute oral toxicity and there is no concern with respect to genotoxicity and carcinogenicity. No effects were reported in developmental toxicity studies. The Panel derived a group acceptable daily intake (ADI) for phosphates expressed as phosphorus of 40 mg/kg body weight (bw) per day and concluded that this ADI is protective for the human population. The Panel noted that in the estimated exposure scenario based on analytical data exposure estimates exceeded the proposed ADI for infants, toddlers and other children at the mean level, and for infants, toddlers, children and adolescents at the 95th percentile. The Panel also noted that phosphates exposure by food supplements exceeds the proposed ADI. The Panel concluded that the available data did not give rise to safety concerns in infants below 16 weeks of age consuming formula and food for medical purposes.

A twenty-volunteer study using deuterium labelling to determine the kinetics and fractional excretion of primary and secondary urinary metabolites of di-2-ethylhexylphthalate and di-iso-nonylphthalate.

This study has obtained estimates of the kinetics and fractional excretion factors of metabolism of DEHP and DINP to their main primary and secondary metabolites. Samples were obtained from an open-label, fixed sequence, single oral dose study in 10 male and 10 female subjects. The dosed substances were deuterated di-2-ethylhexylphthalate (D(4)-DEHP) and di-isononylphthalate (D(4)-DINP) at two dose levels. Urine samples were collected at intervals up to 48 h post-dose. LC-MS/MS was used to measure metabolite concentrations. Excreted amounts were then calculated using urine volumes. Metabolite half-lives were estimated to be 4-8h with more than 90% of metabolites in the first 24h of urine collections and the remainder in the 24-48 h period. The four metabolites of DEHP amounted to 47.1 ± 8.5% fractional excretion on a molar basis. For DINP the identified metabolites totalled 32.9 ± 6.4%. For both DEHP and DINP the metabolites were in the abundance order -monoester<-oxo<-carboxy<-hydroxy. These robust fractional excretion values for the main primary and secondary phthalate metabolites along with estimates of their uncertainty can be used in future surveys of human exposure to DEHP and DINP.

Development and single-laboratory validation of an HPLC method for the determination of in foodstuffs using internal standardization and solid-phase extraction cleanup.

A selective, sensitive and rapid procedure based on liquid chromatography with photodiode array detection (HPLC-PDA) has been developed, single laboratory validated and cross-validated by an external laboratory for the determination of coumarin, a naturally occurring biologically active principle, in a range of foods and beverages. Reverse phase solid-phase extraction (SPE) was used to clean sample extracts when co-extractives were found to interfere with detection and the presence of coumarin was confirmed by comparison of chromatographic peak UV spectra with coumarin standards and quantified by the use of an internal standard. The method was applied to the analysis of flour products, breakfast cereal, gelatin confectionery, sugar confectionery, rice pudding, mixed spice, camomile infusion and soft drinks. Coumarin could not be confirmed in extracts of camomile infusions due to the presence of co-eluting peaks as confirmed by UV spectral analysis. Chromatographic separation was achieved on an octadecyl (C8) column using a mobile phase gradient of acetonitrile and 0.5% acetic acid with a total run time of 20 minutes. The limits of detection and quantification were sample dependent and ranged from 0.05 to 2.5 mg kg-1 and 0.05 to 8 mg kg-1 respectively. The method was successfully validated to show the standard range linearity, sensitivity, accuracy and precision in the matrices tested. Coumarin is regulated within the European Union so this method may be readily adapted for enforcement purposes.

Applications and implications of nanotechnologies for the food sector.

A review of current and projected nanotechnology-derived food ingredients, food additives and food contact materials is presented in relation to potential implications for consumer safety and regulatory controls. Nanotechnology applications are expected to bring a range of benefits to the food sector, including new tastes, textures and sensations, less use of fat, enhanced absorption of nutrients, improved packaging, traceability and security of food products. The review has shown that nanotechnology-derived food and health food products are set to grow worldwide and, moreover, a variety of food ingredients, additives, carriers for nutrients/supplements and food contact materials is already available in some countries. The current level of applications in the European food sector is at an elementary stage; however, it is widely expected that more and more products will be available in the EU over the coming years. The toxicological nature of hazard, likelihood of exposure and risk to consumers from nanotechnology-derived food/food packaging are largely unknown and this review highlights major gaps in knowledge that require further research. A number of uncertainties and gaps in relevant regulatory frameworks have also been identified and ways of addressing them proposed.

Development and validation of a rapid headspace gas chromatography-mass spectrometry method for the determination of diethyl ether and acetone residues in Tween extracts of shellfish intended for mouse bioassay for diarrhoetic toxins.

A validated analytical method using headspace capillary gas chromatography with mass spectrometric detection, which utilises deuterated analogues of the target analytes as internal standards has been developed and applied to the determination of acetone and diethyl ether in Tween extracts of cockles (Cerastoderma edule) and mussels (Mytilus edulis) destined for mouse bioassay for lipophilic toxins. The optimal conditions for headspace incubation were 50 degrees C for 6 min. The limits of detection and quantitation for both DEE and acetone were 2 and 7 microg/mL, respectively, based on signal to noise ratios of 3 and 10, respectively. The linear dynamic range of the instrument was 0 to ca. 4000 microg/mL for both acetone (r(2)=0.995) and DEE (r(2)=0.999). Tween extracts of cockle spiked with acetone and DEE at 3925 and 3570 microg/mL, respectively, gave mean (n=3) recovery figures of 101% (RSD=13.1%) for acetone and 90% (RSD=7.3%) for DEE in cockle matrix. The corresponding figures obtained from spiked mussel matrix were 114% (RSD=5.7%) for acetone and 95% (RSD=6.7%) for DEE, respectively, which were within acceptable range.

Method development and HPLC analysis of retail foods and beverages for copper chlorophyll (E141[i]) and chlorophyllin (E141[ii]) food colouring materials.

An analytical method using high performance liquid chromatography with photodiode array and fluorescence detection has been developed and applied to the determination of the food colour additives copper chlorophylls and copper chlorophyllins (E141[i] and [ii]) in foods and beverages. The analytical procedures from previously reported methods have been refined to cover a range of food colour formulations and retail foods. The method was single-laboratory validated. Recoveries of the polar copper chlorophyllins from spiked samples (at 14.5 mg/kg in all but one case) were in the range 79-109%, except for jelly sweets (49%). Recoveries of relatively non-polar copper chlorophylls were in the range 77-107% (except for 'made' jelly at 50%). The %RSD for recoveries was generally below 12%. Quantitative estimates of the total copper chlorophyll/chlorophyllin content of a small range of food commodities are reported, based on the use of trisodium copper chlorophyllin as a surrogate standard. The majority of E141-containing foods and colour formulations analysed exhibited a multiplicity of components due to the various extraction and purification processes that are used to obtain these colour additives. This was confounded by the presence of overwhelming amounts of native chlorophylls in certain samples (e.g. mint sauce). Food commodities containing significant amounts of emulsifiers (i.e. ice cream), gelatine or fats were problematic during extraction hence further development of extraction regimes is desirable for such products. All of the samples analysed with added E141, had estimated total copper chlorophyllin contents of below 15 mg/kg (range 0.7-13.0).

Determination of isotopically labelled monoesterphthalates in urine by high performance liquid chromatography-mass spectrometry.

A method of analysis for monoesters of phthalic acid ('monoesterphthalates') in human urine has been developed. The method was needed to determine the hydrolysis and excretion efficiency of isotopically-labelled phthalate diesters ('phthalates') when they were fed to volunteers as part of a biomarker study to estimate total exposure to phthalates. The targeted substances were 13C-monobutylphthalate (MBP), 2H4-monobutylphthalate (MBP), 2H4-monobenzylphthalate (MBeP), 13C-monocyclohexylphthalate (MCHP), 13C-monoethylhexylphthalate (MEHP), and 13C-monoisodecylphthalate (MIDP). The monoesters in urine were deconjugated enzymatically, extracted into solvent, and then determined by high performance liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure chemical ionisation in the negative ion mode. The limits of determination were 10 ng ml(-1) for MBP, MCHP, MBeP and MEHP, and 40 ng ml(-1) for MIDP. The recovery from urine spiked at 100 ng ml(-1) was in the range from 70 to 85% except for MIDP which was lower at 55%. The between-batch reproducibility of the analysis was in the range 8 to 17% (n = 6 batches on separate days).