Rapid direct analysis of retinyl palmitate (vitamin A) in fortified vegetable oils by HPLC-FLD.

Large-scale food fortification of vegetable oils with vitamin A has been implemented successfully for decades in numerous African and Asian countries, contributing demonstrably to reductions in vitamin A deficiency. For these programmes, reliable and validated analytical data are essential to demonstrate compliance with legal standards and fortification levels. Commonly, many analytical laboratories use a saponification method for the quantitative analysis of retinyl palmitate (the mostly used form of vitamin A for fortification) in fortified oils, which implies a multiple-step procedure with long analysis times and the potential risk of analyte loss. The aim of the present study was to develop and validate a direct High-performance Liquid Chromatography (HPLC) method that reduces these sample preparation steps, leading to the cost- and time-efficient quantification of retinyl palmitate in fortified oils. Oil samples are dissolved into the HPLC solvents, then injected directly into a common C18 column, and subsequently detected by a fluorescence detector. The limit of quantification (1.0 mg retinyl palmitate kg-1) and the working range of 1.0-100 mg retinyl palmitate kg-1 with a linearity of R2 = 0.9989 are appropriate to analyse fortified oil samples. The method also showed adequate precision (RSD between 1.1% and 3.1%) and recoveries (86-103%) at two different concentration levels. The accuracy of the direct HPLC method was additionally proven by the comparison of spiked samples with two external laboratories that used the saponification method. The robustness of the method was confirmed by the analysis of various spiked edible oils. The HPLC column is not deteriorated by the lipid matrix and shows excellent stability and long lifetime. Also, 9-cis-retinyl palmitate formed mainly by light exposure could be detected by this method. The direct HPLC method is a well-suited alternative to the saponification method for the rapid and reliable routine analysis of fortified oil samples.

Screening of stereoisomeric chloramphenicol residues in honey by ELISA and CHARM ® II test - the potential risk of systematically false-compliant (false negative) results.

Chloramphenicol (CAP) is a broad-spectrum antibiotic used widely both in human and in veterinary medication but due to adverse health effects is not authorised anymore for use in food-producing animals in many countries. CAP molecule contains two asymmetric centers resulting in four para-CAP stereoisomers, but only the RR-CAP enantiomer is bioactive with significant antimicrobial activity. In this study the detection of the four CAP stereoisomers was tested by five commercial ELISA kits and the Charm® II Chloramphenicol Test. These immunoassay tests are commonly used and widely accepted for screening of CAP residues in foods of animal origin, including honey. The test results definitely show that SS-CAP residues are not detected; even high SS-CAP concentrations are missed due to the lack of any cross reactivity and the high specificity of the CAP antibodies to RR-CAP. In former studies chiral LC-MS/MS analysis indicated clearly that honey samples with raised CAP concentrations often contain the SS-CAP enantiomer in addition to the bioactive RR-CAP. According to this study, the investigated screening tests carry the risk of systematically false-compliant (false negative) results for CAP and a discrepancy between LC-MS/MS and ELISA/Charm® test results. As a consequence of this study, it is recommended that immunoassay manufacturers develop and use CAP antibodies which also bind SS-CAP. The origin of SS-CAP residues in honey samples is discussed and general toxicological and regulatory aspects of CAP stereoisomers are raised.

Enantioselective analysis of chloramphenicol residues in honey samples by chiral LC-MS/MS and results of a honey survey.

Chloramphenicol (CAP) is a broad-spectrum antibiotic used widely in both human and veterinary medication. Since 1994, CAP has not been authorised for use in food-producing animals in the European Union due to several adverse effects. A minimum required performance level (MRPL) of 0.3 µg kg-1 was established in 2003. The CAP molecule contains two asymmetric centres, thus in total four para-CAP stereoisomers exist. Only the RR-CAP enantiomer is bioactive, having significant antimicrobial activity. For the first time a chiral LC-MS/MS method is reported to identify and quantify the four CAP enantiomers at residue levels in honey samples. The method was validated at two concentration levels. The decision limits (CCα) and detection capabilities (CCß) were well below 0.3 µg kg-1, with limits of quantification (LOQs) between 0.08 and 0.12 µg kg-1 for all four enantiomers. The method provides a sensitive and reliable analysis of CAP enantiomers in honey, and proved its robustness during the daily routine analyses of numerous honey samples. In an internal honey survey, in total 40 honey samples from different geographical regions with identified CAP residues at or above the MRPL were reanalysed by chiral LC-MS/MS. In nine honey samples only the bioactive RR-CAP was detected as anticipated. However, in all other 31 honey samples the non-bioactive SS-CAP was also identified and quantified unambiguously. In 10 of these samples, mixtures of RR- and SS-CAP were analysed, and in 21 samples only the SS-CAP enantiomer, with concentrations up to 2.2 µg kg-1. Most of these samples are honeys from Ukraine and Eastern Europe. This is the first report of SS-CAP residues in food samples. The potential sources for these findings are discussed and the need of further systematic studies emphasised. It is recommended to examine in more depth the toxicological profile of the individual CAP stereoisomers.

Toxicological risks to humans of toxaphene residues in fish.

A revised risk assessment for toxaphene was developed, based on the assumption that fish consumers are only exposed to toxaphene residues that differ substantially from technical toxaphene due to environmental degradation and metabolism. In vitro studies confirmed that both technical toxaphene and degraded toxaphene inhibit gap junctional intercellular communication that correlates with the mechanistic potential to cause tumor promotion. In vivo rat studies established the NOAEL for degraded and technical toxaphene at the highest dose tested in the bioassay. Toxaphene residue intakes from European fishery products were estimated and compared to the provisional tolerable daily intakes (TDIs) from various regulatory agencies including Canada, the United States, and Germany. The estimated intake was also compared to a new calculated provisional MATT pTDI. The MATT pTDI is based on new toxicological information (in vivo rat studies) developed on a model for environmental toxaphene residues rather than technical toxaphene. A MATT pTDI (1.08 mg total toxaphene for a person of 60 kg) for tumor promotion potency was adopted for use in Europe and is referred to here as the MATT pTDI. These new data result in a better estimate of safety and a higher TDI than previously used. Based on realistic fish consumption data and recent baseline concentration data of toxaphene in European fishery products, the toxaphene intake for the consumers of Germany, Ireland, Norway, and the Netherlands was estimated. For an average adult fish consumer, the average daily intake of toxaphene was estimated to be 1.2, 0.4, 0.5, and 0.2 µg for the consumers of Norway, Germany, Ireland, and the Netherlands, respectively. The toxaphene intake of these average fish consumers was far below the MATT pTDI of 1.08 mg/60 kg bw. In conclusion, based on the most relevant toxicological studies and the most realistic estimates of fish consumption and recent concentrations of toxaphene in European fishery products, adverse health effects are unlikely for the average European consumer of fishery products. In no case is the MATT pTDI exceeded.

Baseline survey of concentrations of toxaphene congeners in fish from European waters.

The European Union project "Investigation into the monitoring, analysis and toxicity of toxaphene"(MATT) began in 1997 involving participants from the Netherlands, Ireland, Norway and Germany. Concentration information, analytical methodology and statistical interpretation of 207 samples covering 23 different fish species from European waters are presented for three toxaphene indicator congeners: CHBs 26, 50 and 62 (CHB = chlorobornane). Concentrations for the Sigma3CHBs were more elevated in fish from more northern latitudes, such as the Barents and Norwegian Sea, compared to fish from Irish, Dutch and German waters. Concentrations were lowest in shellfish and in fish species having low lipid content and were highest in medium/high lipid species. Females from a number of fish species were shown to contain significantly higher concentrations than those observed in male fish. Overall no samples were shown to exceed existing German MRL legislation, with only one Greenland halibut sample shown to exceed Canadian TDI recommendations.

Comparative tests to improve the gas chromatographic analysis of chlorobornanes in fish samples.

A comparison was made between electron capture negative ionization quadrupole mass spectrometry (ECNI-MS) and electron capture detection (ECD) with regard to repeatability and reproducibility for the gas chromatographic (GC) analysis of toxaphene congeners [chlorobornanes (CHBs)]. The tests, including standard solutions and several cleaned fish extracts, showed larger relative standard deviations (RSDs) for the repeatability of ECNI-MS but no differences in the reproducibility of the 2 techniques. The sensitivity of the GC-ECNI-MS was considerably better than that of GC/ECD. Four stepwise-designed comparative tests were also conducted on GC analysis, cleanup, and the complete method. The results showed that, according to the current state-of-the-art, coefficients of variation for the between-laboratory performance were not < 20% and were usually between 20 and 30%. In spite of separation problems, e.g., for CHB 26, which cannot be separated into a single-component peak, a 95% methyl 5% phenyl polysiloxane (CP Sil 8) column was preferred to more polar columns for the analysis of CHBs 26, 40, 41, 44, 50, and 62. CHB 62 was more difficult to determine than CHB 26 and 50. Addition of the CHBs 40, 41, and 44 to the standard set of 3 chlorobornanes (26, 50, and 62) resulted in more separation problems. A 3-step cleanup method was recommended.