Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in human.

Two multiresidue methods based on different extraction procedures have been developed and compared for the liquid chromatography electrospray ionization tandem mass spectrometry analysis of 17 mycotoxins including ochratoxin A, aflatoxins (B(1), B(2), G(1), and G(2)), zearalenone, fumonisins (B(1) and B(2)), T-2 toxin, HT-2 toxin, nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, and neosolaniol in cereal-based commodities. The extraction procedures considered were a QuEChERS-like method and one using accelerated solvent extraction (ASE). Both extraction procedures gave similar performances in terms of linearity (r(2) > 0.98) and precision (both RSD(r) and RSD(iR) < 20%). Trueness was evaluated through participation in four proficiency tests and by the analysis of two certified reference materials and one quality control material. Satisfactory Z scores (|Z| < 2) and trueness values (73-130%) were obtained by the proposed procedures. Limits of quantification were similar by both methods and were within the 1.0-2.0 microg/kg range for aflatoxins, 0.5 microg/kg for ochratoxin A, and the 5-100 microg/kg range for all other mycotoxins tested. The QuEChERS-like method was found to be easier to handle and allowed a higher sample throughput as compared to the ASE method.

A multi-screening approach for monitoring potential chemical contaminants in honey by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed. A total of 42 veterinary drugs (5 tetracyclines, 7 macrolides, 3 aminoglycosides, 8 β-lactams, 2 amphenicols and 17 sulfonamides) were surveyed with the ultimate goal of unambiguously confirmed and quantified these analytes at a concentration level of 20μg/kg. A basic sample preparation including four subsequent liquid/liquid extraction steps was necessary to adequately extract the compounds of interest from the honey. The four extracts were injected into the LC-ESI-MS/MS using a stacking injection procedure. Validation of the entire procedure was carried out according to the European Union directive 2002/657/EC at three concentration levels, i.e., 10, 20 and 30μg/kg. Good performance data were obtained for 37 analytes, out of the 42 studied. Limit of compliance and detection limit were calculated based on an internal limit set at 20μg/kg for all the compounds and ranged between 24–30 and 27–80μg/kg, respectively. A limited survey on honeys of different geographical origins has demonstrated that positive honey samples were often contaminated by more than one class of drugs, thus highlighting the usefulness of such multi-screening approach to ensure and warrants the quality of honey.

A simple and fast selective extraction of the antibiotic chloramphenicol (CAP) from milk (raw milk, skimmed milk, and milk powder) using a molecularly imprinted polymer (MIP) sorbent is described. The method entails a single centrifugation step prior to loading the supernatant onto the MIP cartridge and subsequent elution with a mixture of solvents. CAP was further analyzed by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operating in negative ionization acquisition mode. The advantages of the MIP approach were assessed by comparing the data generated from a classical solid-phase and liquid-liquid extractions procedure, previously developed in our laboratory. A better recovery of CAP due to an enhanced selectivity and a faster turnaround time (18 samples processed within 3 h compared to 8 h with the classical approach) were evidenced when using the MIP cleanup. The analysis of CAP in raw milk was further validated according to the 2002/657/EC European Union criteria for the analysis of veterinary drug residues at the minimum required performance limit (MRPL) of 0.3 microg/kg, using CAP-d(5) as internal standard. Non-internal-standard corrected recovery values ranged between 50% and 87% over the range of concentrations considered. The decision limit (CCalpha) and detection capability (CCbeta) were calculated to be 0.06 and 0.10 microg/kg, respectively.

With the emergence of liquid chromatography coupled to tandem quadrupolar mass spectrometry (LC–QqQ) as a routine technique for quantitative analysis, analytical chemists claimed LC–QqQ to be the gold standard to reach the best compromise between versatility, high throughput, robustness, sensitivity and selectivity. In particular, a high selectivity is ensured when two or more transitions are monitored because not only the retention time and protonated molecule are controlled but also two or more product ions are. With the multiple-transition recording, the transition leading to the most intense signal is used for the quantification (quantifier), while the other one(s) is(are) aimed at confirming the detection of the analyte (qualifiers). The confirmation is based on the calculation of the relative intensity between the signal intensities of the quantifier and the qualifier(s). This useful approach raises the question of the validity of the limit of detection (LOD), initially employed for mono-channel detections such as HPLC combined with ultraviolet or fluorescence detection. Furthermore, it was shown that the multiple-transition recording leads to a confusing calculation of the decision limit (CCα) and detection capability (CCβ). In the present article, the LOD is split in three concepts defined as the limit of suspicion (LOS), recognition (LOR), and confirmation (LOC). For these three limits, applications and drawbacks are shown, while determination methods are proposed.

A method making use of turbulent flow chromatography automated online extraction with tandem mass spectrometry (MS/MS) was developed for the analysis of 4 quinolones and 12 fluoroquinolones in honey. The manual sample preparation was limited to a simple dilution of the honey test portion in water followed by a filtration. The extract was online purified on a large particle size extraction column where the sample matrix was washed away while the analytes were retained. Subsequently, the analytes were eluted from the extraction column onto an analytical column by means of an organic solvent prior to chromatographic separation and MS detection. Validation was performed at three fortification levels (i.e., 5, 20, and 50 microg/kg) in three different honeys (acacia, multiflower, and forest) using the single-point calibration procedure by means of either a 10 or 25 microg/kg calibrant. Good recovery (85-127%, median 101%) as well as within-day (2-18%, median 6%) and between-day (2-42%, median 9%) precision values was obtained whatever the level of fortification and the analyte surveyed. Due to the complexity of the honey matrix and the large variation of the MS/MS transition reaction signals, which were honey-dependent, the limit of quantification for all compounds was arbitrarily set at the lowest fortification level considered during the validation, e.g., 5 microg/kg. This method has been successfully applied in a minisurvey of 34 honeys, showing ciprofloxacin and norfloxacin as the main (fluoro)quinolone antibiotics administered to treat bacterial diseases of bees. Turbulent flow chromatography coupled to LC-MS/MS showed a strong potential as an alternative method compared to those making use of offline sample preparation, in terms of both increasing the analysis throughput and obtaining higher reproducibility linked to automation to ensure the absence of contaminants in honey samples.

Six ergot alkaloids belonging to the lysergic acid derivatives (ergonovine (EGN) and methysergide hydrogen maleinate (MHM)) and peptide-type derivatives (ergocristine (EGR), ergotamine (EGT), ergocornine (EGC) and alpha-ergokryptine (EGK)) were studied by positive electrospray tandem mass spectrometry. The fragmentation mechanisms of these compounds were studied by collision-induced dissociation (CID) using triple quadrupole and ion trap mass spectrometers, and the nature of the major product ions further confirmed by hydrogen/deuterium (H/D) exchange experiments. A common abundant product ion at m/z 223 was characteristic of the two classes of ergot alkaloids. Therefore, a precursor ion scan of m/z 223 that triggers information data acquisition (IDA) in combination with CID experiments was used to identify other potential ergot alkaloids. Using this approach, it was possible to confirm the presence of ergosine, another peptide-type ergot alkaloid, in a rye flour extract at trace levels. Copyright (c) 2006 John Wiley & Sons, Ltd.

An isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry method is presented for the simultaneous analysis of several 5-nitroimidazole-based veterinary drugs, which are dimetridazole (DMZ), ronidazole (RNZ), metronidazole (MNZ), ipronidazole (IPZ), and their hydroxylated metabolites (DMZOH, MNZOH, and IPZOH), in egg (fresh egg, whole egg powder, and egg yolk powder) and chicken meat. Data acquisition was achieved by applying multiple reaction monitoring, and quantitation was performed by means of five deuterated internal standards (ISs), namely, DMZ-d3, RNZ-d3, IPZ-d3, DMZOH-d3, and IPZOH-d3, whereas MNZ and MNZOH were quantitated using DMZOH-d3. At the lowest fortification levels (i.e., 0.5 microg/kg for fresh egg and chicken meat and 1.0 microg/kg for other egg-based matrices) and for compounds having their own corresponding deuterated analogue used as an IS, acceptable performance data were obtained (corrected recoveries, 88-111%; decision limits, 0.07-0.36 microg/kg; detection capabilities, 0.11-0.60 microg/kg; and within-lab precision, < or = 15%). The method failed to give acceptable quantitative results for MNZ and MNZOH due to the unavailability of the corresponding deuterated ISs. Nevertheless, a reliable identification of these two analytes at levels < or = 1 microg/kg was still feasible.

A confirmatory method for detecting five ergot alkaloids, ergocristine, ergotamine, ergonovine, ergocornine and alpha-ergokryptine, in rye flour is described using high performance liquid chromatography coupled to tandem mass spectrometry detection by monitoring two transition reactions per analyte. The procedure entails a liquid-liquid extraction followed by a clean-up step using a C18 solid-phase extraction (SPE) cartridge. An analogue compound, methysergide hydrogen maleinate, was used to assess both repeatability sample preparation and potential MS response fluctuations. The method was fully validated according to the European Union (EU) criteria. Detection and quantification limits of all analytes were calculated ranging from 7 to 11 microg/kg and from 23 to 37 microg/kg, respectively. Fifteen rye flour samples were investigated with the newly developed method, and none of them were above the current Swiss limits of 200mg/kg for total ergot alkaloids.

Ochratoxin A (OTA) is a renal carcinogen in rodents. Its human health significance is unclear. It likely depends upon the mechanism of carcinogenesis. In a previous microarray study a reduction in nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-dependent gene expression was observed in the kidney but not in the liver of rats fed OTA up to 12 months. Nrf2 regulates detoxification and antioxidant gene expression. The present report shows that OTA decreased the protein expression of several markers of the Nrf2-regulated gene battery in kidney in vivo indicating that the effects observed at mRNA level may be of biological significance. The OTA-mediated Nrf2 response could be reproduced in an NRK renal cell line and in primary hepatocyte cultures. In in vitro systems, an OTA-mediated inhibition of Nrf2 activity was demonstrated by electrophoretic mobility shift and Antioxidant Regulatory Element-driven luciferase reporter assays. The reduction of Nrf2-regulated gene expression resulted in oxidative DNA damage as evidenced by formation of abasic sites in vitro and confirmed in kidney in vivo. All OTA-mediated effects observed were prevented by pretreatment of cell cultures with inducers of Nrf2 activity. Our data suggest that reduction of cellular defense against oxidative stress by Nrf2 inhibition may be a plausible mechanism of OTA nephrotoxicity and carcinogenicity.

A confirmatory method based on isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the low-level determination of residues of four nitrofuran veterinary drugs in meat, e.g., furazolidone, furaltadone, nitrofurantoin, and nitrofurazone. The procedure entails an acid-catalysed release of protein-bound metabolites, followed by their in situ conversion into the 2-nitrobenzaldehyde (NBA) imine-type derivatives. Liquid–liquid extraction and clean-up on a polymeric solid phase extraction cartridge are then performed before LC–MS/MS analysis by positive electrospray ionisation (ESI) applying multiple reaction monitoring of three transition reactions for each compound. Reliable quantitation is obtained by using one deuterated analogue per analyte (d4-NBA derivative) as internal standard (IS). Validation of the method in chicken meat was conducted following the European Union (EU) criteria for the analysis of veterinary drug residues in foods. The decision limits (CCα) were 0.11–0.21μg/kg, and the detection capabilities (CCβ) 0.19–0.36μg/kg, thus below the minimum required performance limit (MRPL) set at 1μg/kg by the EU. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC–ESI-MS–MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid–liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS–MS in selecting m/z 321 → 257, 321 → 152 (35Cl2) and m/z 323 → 257, 323 → 152 (37Cl35Cl). Two calibration curves were constructed by plotting the area ratio of m/z 321 → 152 versus 326 → 157 and m/z 321 → 257 versus 326 → 262 against their corresponding amount ratio. Indeed, even if m/z 321 → 152 was found to give a higher MS–MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/z 321 → 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5μg/kg concentration levels using d5-CAP as internal standard. The decision limit (CCα) and detection capability (CCβ) of CAP in milk were calculated for m/z 321 → 152 at 0.02μg/kg and 0.03μg/kg, respectively, and for m/z 321 → 257 at 0.02μg/kg and 0.04μg/kg, respectively. At the lowest fortification level (i.e. 0.1μg/kg), repeatability and within-laboratory reproducibility were calculated for m/z 321 → 257 both at 0.02μg/kg and for m/z 321 → 152 at 0.03 and 0.05μg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.

A sensitive and selective isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) method is presented for the simultaneous analysis of the metabolites of four nitrofuran veterinary drugs, that is, furazolidone, furaltadone, nitrofurantoin, and nitrofurazone, in honey samples. The method entails a combined hydrolysis of protein-bound drug metabolites and derivatization of the resulting metabolites with 2-nitrobenzaldehyde (NBA) during an overnight incubation, followed by a liquid-liquid extraction and a cleanup on a polymeric solid-phase extraction cartridge. Mass spectral acquisition is carried out in the positive ion mode by applying multiple reaction monitoring (MRM) of three diagnostic transition reactions for each analyte under survey. A reliable quantification is obtained by the use of one deuterated analogue per analyte (NBA-d(4) derivative). The method has been validated in honey according to the European Union criteria for the analysis of veterinary drug residues in food. Expressed in underivatized nitrofuran metabolite concentrations, the decision limits (CCalpha) ranged within 0.07-0.46 microg/kg, and the detection capabilities (CCbeta) were within 0.12-0.56 microg/kg. The method has been successfully applied in a survey of honeys of various geographical origins, showing that furazolidone is the main nitrofuran antibiotic administered to treat bacterial diseases of bees.

Evidence is presented for the first time showing that semicarbazide (SEM) is a minor thermal decomposition product of the blowing agent azodicarbonamide (ADC). A novel direct analytical method based on liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) has been developed to determine SEM in foamed polyvinyl chloride (PVC) seals of metal lids, as well as in commercially available ADC. The direct LC-MS/MS method for gaskets entails extraction of the gaskets in hot water, addition of ((15)N(2)(13)C)-SEM as internal standard, and injection of an aliquot directly into the LC-MS system, achieving good sensitivity (S/N = 348 for 2 ng injected on-column) and monitoring three characteristic mass transitions (m/z 76-->31; 76 -->44; 76-->59). Semicarbazide can be detected in thermally treated ADC, reaching up to 0.93 mmol mol(-1) at 220 degrees C, as determined by the direct LC-MS/MS method. This new method is also compared to the classical derivatization method using 2-nitrobenzaldehyde (2-NBA) that is routinely employed to determine SEM as an indicator of the usage of the antimicrobial drug nitrofurazone, the use of which is not authorized in the European Union (EU). Both methods revealed proportional results, with approx. 3-fold higher levels recorded by the direct SEM approach, probably due to differences in the extraction procedures used. A limited survey of plastic seals from used press twist and twist-off metal lids on food jars (non-foamed and foamed) revealed levels of SEM ranging from 2 to 8689 microg kg(-1)(average = 1593 microg kg(-1), n= 57 determinations).

A confirmatory method based on isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry is described for the determination of the antibiotic chloramphenicol (CAP) in foods. The method is quantitative and entails liquid-liquid extraction followed by a clean-up step on a silica gel solid-phase extraction cartridge. Mass spectral acquisition is done in the negative ion mode applying multiple reaction monitoring of two diagnostic transition reactions for CAP (m/z 321 --> 257 and m/z 321--> 152). In addition, the presence of two chlorine atoms in the CAP molecule provides further analyte certainty by assessing the 37Cl/35Cl ratio using the transition reactions m/z 323 --> 257 and m/z 323 --> 152. Validation of the method in chicken meat is conducted according to the latest European Union criteria for the analysis of veterinary drug residues at levels of 0.05, 0.10, and 0.20 microg/kg, employing [2H5]-chloramphenicol as internal standard. The decision limit and the detection capability were calculated at 0.01 microg/kg and 0.02 microg/kg, respectively. At the lowest fortification level (i.e. 0.05 microg/kg), precision values below 14 and 17% were achieved under repeatability and within-laboratory reproducibility conditions, respectively. The accuracy of the method was within 20, 15, and 5% of the target values at the 0.05, 0.10, and 0.20 microg/kg fortification levels, respectively. The applicability of this procedure was demonstrated by the analysis of other meat (turkey, pork, beef) and seafood (fish, shrimps) products. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.

A convenient method is presented for the preparation of the carbon-13-labeled 2-nitrobenzaldehyde derivatives of the nitrofuran metabolites 3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 1-aminohydantoin (AH), and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), with the purpose of using them as internal standards for the quantification of trace levels of nitrofuran residues by liquid chromatography-tandem mass spectrometry in foods of animal origin. The synthesis encompasses the nitration of [1,2,3,4,5,6-(13)C(6)]toluene prior to chromyl compound-mediated oxidation of the methyl group into the corresponding aldehyde. The four metabolites of nitrofuran antibiotics were derivatized independently with the resulting ring-labeled 2-nitrobenzaldehyde (NBA) to obtain the target compounds. Both the isotopically enriched and native substances were used to perform a comprehensive fragmentation study by electrospray ionization (ESI) collision-induced dissociation (CID) mass spectrometry (MS). Full characterization of the nitrofuran derivatives was accomplished with ultraviolet (UV) and exhaustive nuclear magnetic resonance (NMR) analysis. A major advantage of the described procedure is that it can be extended to the preparation of other carbon-13-labeled derivatives of metabolites of nitrofuran antibiotics.

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve problems in the measurement of DNA oxidation that have resulted in varying estimates of the extent of this damage in humans. HeLa cells, sent to members for analysis, were either untreated, or treated with light in the presence of a photosensitizer to induce different amounts of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA. Laboratories employing HPLC with electrochemical detection were able to measure the induced damage with similar efficiency; dose response gradients for seven of the eight sets of results were almost identical. GC-MS and HPLC-MS/MS, employed in three laboratories, did not convincingly detect the dose response. An alternative approach to measuring base oxidation employs the enzyme formamidopyrimidine DNA N-glycosylase (FPG) to convert 8-oxoGua to strand breaks, which are then measured by alkaline unwinding, alkaline elution, or the comet assay. Ten laboratories used this approach; five were able to detect the dose response in cells treated with photosensitizer plus light (at lower doses than for chromatographic methods, because the enzymic methods are more sensitive and less prone to spurious oxidation). Median values for 8-oxoGua (or FPG-sensitive sites) in untreated cells were 4.01 per 10(6) guanines for chromatographic methods, and 0.53 per 10(6) guanines for techniques based on FPG. (C) 2003 Elsevier Inc.

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CV of less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10(6) Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve the problems associated with the measurement of background levels of oxidative DNA damage (in particular 8-oxo-7,8-dihydroguanine, or 8-oxoGua) in human cells. A tendency for DNA oxidation to occur during sample preparation prior to chromatography has been recognized as the source of a very substantial artefact. To assess the success of attempts to eliminate the artefact, ESCODD has distributed to its members standard samples of pig liver and HeLa cells for analysis. Estimates of 8-oxoGua in pig liver, using chromatographic techniques, ranged from 2.23 to 441 per 10(6) guanines, with a median of 10.47 per 10(6) guanines. Chromatographic analysis of HeLa cell DNA gave a range of 1.84 to 214 per 10(6) guanines with a median of 5.23 per 10(6) guanines. HeLa cell DNA was also analysed by an enzymic approach, in which whole cell DNA was treated with formamidopyrimidine DNA glycosylase, which nicks DNA at sites of 8-oxoGua, and the breaks measured with the comet assay, alkaline unwinding or alkaline elution. Values with these methods ranged from 0.06 to 4.98 8-oxoGua per 10(6) guanines, with a median of 0.79 per 10(6) guanines. Although there are clearly still serious discrepancies between methods and laboratories, the lowest estimates by chromatography (arguably those in which the artefact was best controlled) are only 2.5 times higher than the median value obtained with the enzymic approach.