Quantification of chlorinated paraffins by chromatography coupled to high-resolution mass spectrometry - Part B: Influence of liquid chromatography separation.

The analysis of chlorinated paraffins (CPs) is today an analytical challenge. Indeed, it is still impractical to describe their real composition in terms of polychlorinated alkanes (PCAs) homologue groups, which dominate technical mixtures. The co-elution of PCA congeners generates interferences due to the competition phenomena which occur during the ionisation process as well as to the dependence of the ionisation sources on the PCA chemistry. Therefore, the aim of this study was to investigate the influence of chromatographic separation, by LC-ESI-HRMS coupling, on the PCA homologue group pattern and, eventually, on their determination in food samples from interlaboratory studies. For this, three different mobile phases and six LC chromatographic columns were studied in order to optimise the analysis of CP mixtures. The first results showed that the use of a MeOH/H2O mobile phase reveals more appropriately the higher chlorinated PCAs. However, using ACN/H2O led to less ion species, with almost exclusively [M + Cl]- adducts, formed using post-column dichloromethane addition. Regarding the choice of the stationary phases, Hypercarb column provided a completely different homologue group pattern from the other chromatographic columns, in relation with the stronger retention of PCAs. Among the other columns, the C30 column better highlighted the short-chain PCAs compared to the C18 column conventionally used. Because the regulations now concern short-chain CPs, the quantification of food samples was then carried out on the C30 column. The optimised LC-ESI-HRMS conditions using C30 column and MeOH/H2O solvent mixture led to a quantification of PCAs in samples from interlaboratory studies with satisfactory accuracy (|Z-score| ≤ 2) and precision (<15%).

Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples.

This work evaluates the potential of ion mobility spectrometry (IMS) to improve the analytical performance of current liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determination of ergot alkaloids (EAs) in cereal samples. Collision cross section (CCS) values for EA epimers are reported for the first time to contribute to their unambiguous identification. Additionally, CCS values have been inter-laboratory cross-validated and compared with CCS values predicted by machine-learning models. Slight differences were observed in terms of CCS values for ergotamine, ergosine and ergocristine and their corresponding epimers (from 3.3 to 4%), being sufficient to achieve a satisfactory peak-to-peak resolution for their unequivocal identification. A LC-travelling wave ion mobility (TWIM)-MS method has been developed for the analysis of EAs in barley and wheat samples. Signal-to-noise ratio (S/N) was improved between 2.5 and 4-fold compared to the analog LC-TOF-MS method. The quality of the extracted ion chromatograms was also improved by using IMS.

Ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry for antidoping analyses: Assessment of the inter-laboratory reproducibility with urine samples.

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses. To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.0%. Only for two substances, namely fenbutrazate and niketamide, the retention was not repeatable between laboratories, with RSD% of approximately 15% in both cases. This behaviour was associated with (a) the low organic modifier percentage (around 2-4%) in the mobile phase at the corresponding retention times, and (b) the influence of the system volume on poorly retained analytes. An analysis on seven "blind" urines was subsequently carried out in the same four laboratories. In these blind samples, either one, two, or none of the 21 doping agents previously analyzed were present at an unknown concentration. Each laboratory had to perform the identification of the compounds in the samples and estimate their concentrations. All laboratories assigned all target analytes correctly in all blind urine samples and provide a comparable estimation of their concentrations.

Quantification of light polycyclic aromatic hydrocarbons in seafood samples using on-line dynamic headspace extraction, thermodesorption, gas chromatography tandem mass spectrometry, based on an isotope dilution approach.

The aim of our work was to develop an analytical strategy to quantify naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene and anthracene in fish products by on-line dynamic headspace extraction, followed by thermodesorption injection and gas chromatography analysis coupled with tandem mass spectrometry using electron ionization mode (DHS-TD-GC-EI-MS/MS). The developed protocol used 1 g of freeze-dried or oil sample supplemented with perdeuterated light PAHs. The sample was heated at [90 -100 °C], the headspace of the sample was swept by nitrogen and the trapping of the PAHs was carried out on a Tenax-type adsorbent placed at 25 °C. Analytes were thermodesorbed at 300 °C from the dried adsorbant and then cryofocused on a cooled injection system (CIS) at -25 °C before injection (12 °C s-1 up to 300 °C). The chromatographic separation of PAHs was carried out on a 5-MS type column (30 m × 0.25 mm, 0.25 µm) and the acquisition of the signals was performed in SRM following the transitions, involving the loss of one or two hydrogen atoms from the molecular ion. In view of the principle of extraction, the calibration curve was performed on a representative matrix or using the standard addition method. Quantification limits were determined between 0.01 and 0.6 ng g-1 of matrix from the method blank results. The method was validated by a series of multi-level supplemented matrix assays and by the analysis of a reference material from an inter-laboratory test (mussels, IAEA-432). The average of the expanded measurement uncertainty was from 9 to 44% for the four lightest PAHs, except for fluorene when the sample incubation was set at 90 °C. Occurrence measurements were performed on almost two hundred samples of molluscs, echinoderms and fish. The results have shown a quantification frequency greater than 66% for naphthalene and fluorene, at concentrations below a few ng g-1 of dry matter of fishery products. With this methodology, the light PAHs occurrence can now be measured in a wider range of foodstuffs in order to better characterize their contamination trends and the associated risk simultaneously.

Toward a new European threshold to discriminate illegally administered from naturally occurring thiouracil in livestock.

Thiouracil is a thyrostat inhibiting the thyroid function, resulting in fraudulent weight gain if applied in the fattening of livestock. The latter abuse is strictly forbidden and monitored in the European Union. Recently, endogenous sources of thiouracil were identified after frequently monitoring low-level thiouracil positive urine samples and a "recommend concentration" (RC) of 10 µg/L was suggested by the EURL to facilitate decision-making. However, the systematic occurrence of urine samples exceeding the RC led to demands for international surveys defining an epidemiologic threshold. Therefore, six European member states (France, Poland, The Netherlands, United Kingdom, Norway, and Belgium) have shared their official thiouracil data (2010-2012) collected from bovines, porcines, and small livestock with 95 and 99% percentiles of 8.1 and 18.2 µg/L for bovines (n = 3894); 7.4 and 13.5 µg/L for porcines (n = 654); and 7.4 µg/L (95% only) for small livestock (n = 85), respectively. Bovine percentiles decreased with the animal age (nonadults had significantly higher levels for bovines), and higher levels were observed in male bovines compared to female bovines.

Assessment of dietary exposure to bisphenol A in the French population with a special focus on risk characterisation for pregnant French women.

Bisphenol A (BPA) is used in a wide variety of products and objects for consumers use (digital media such as CD's and DVD's, sport equipment, food and beverage containers, medical equipment). Here, we demonstrate the ubiquitous presence of this contaminant in foods with a background level of contamination of less than 5 µg/kg in 85% of the 1498 analysed samples. High levels of contamination (up to 400 µg/kg) were found in some foods of animal origin. We used a probabilistic approach to calculate dietary exposure from French individual consumption data for infants under 36 months, children and adolescents from 3 to 17 years, adults over 18 years and pregnant women. The estimated average dietary exposure ranged from 0.12 to 0.14 µg/kg body weight per day (bw/d) for infants, from 0.05 to 0.06 µg/kg bw/d for children and adolescents, from 0.038 to 0.040 µg/kg bw/d for adults and from 0.05 to 0.06 µg/kg bw/d for pregnant women. The main sources of exposure were canned foods (50% of the total exposure), products of animal origin (20%) and 30% as a background level. Based on the toxicological values set by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) for pregnant women, the risk was non negligible. Thus, we simulated scenarios to study the influence of cans and/or food of animal origin on the BPA-related risk for this specific population.

Relative bioavailability to laying hens of indicator polychlorobiphenyls present in soil.

Transfer of indicator polychlorobiphenyls (PCBs) from soil into hen eggs may occur in hens reared outdoor, which ingest significant amounts of soil. This transfer depends on the bioavailability of the ingested compounds. The impact of soil on the bioavailability of indicator PCBs was assessed by means by a relative bioavailability (RBA) trial, in which their deposition in egg yolk and in abdominal fat, in response to their ingestion through contaminated-soil and through spiked-oil were compared. A sandy soil (709 µg indicator PCBs kg(-1) dry matter) was collected in the vicinity of a former fire involving treated wood. Twenty-eight laying hens were individually housed and fed one of the seven experimental diets during 14 d. The seven experimental diets were an uncontaminated control diet, three diets in which contaminated soil was introduced at levels of 3%, 6% and 9% and three diets in which spiked oil was introduced to achieve similar levels and profile of contaminants. Yolk, abdominal fat and liver were collected at the end of exposure. Indicator PCBs were extracted by ASE (Accelerated Solvent Extraction) and analyzed by GC-HMRS. Within each ingested matrix, the concentration of indicator PCBs in yolk and in abdominal fat linearly increased with the amount of indicator PCB ingested (P<0.001). Except for PCB 28, the slopes of the responses to soil and to oil could not be differentiated (P>0.1). RBA estimates did not differ from 1 for all indicator PCBs except for PCB 28, for which it was 0.58-0.59. Measurements performed on liver confirm these conclusions.

Polychlorinated biphenyl and low polybrominated diphenyl ether transfer to milk in lactating goats chronically exposed to contaminated soil.

This study investigated milk excretion kinetics of PCBs, tetra-BDE (BDE 47), and penta-BDE (BDE 99) in goats exposed to contaminated soil under controlled conditions. The animals were fed (80 days) with feed containing 5% of contaminated soil. During this exposure period, milk was analyzed weekly. At the end of the experiment the PCBs and PBDEs retained in hepatic and adipose tissues were also determined. The soil-milk carry over rates (CORs) of PCBs ranged from 6 to 62%. This result suggests that a large part of ingested soil-bound PCBs was recovered in milk. Significantly different levels between the congeners were reported in the tissues (fat, liver). BDE 47 and 99 excretions in milk achieved a plateau after 2 weeks of exposure, and their corresponding CORs were about 30%. These two congeners showed a significantly (P < 0.05) lower accumulation in the adipose tissue than the major PCB congeners. The concentrations of BDE 47 and 99 in the liver were the same as PCB concentrations. This result suggests that the low brominated congeners are submitted to the metabolism more extensively than the major PCBs.

Assessment of circulating sex steroid levels in prepubertal and pubertal boys and girls by a novel ultrasensitive gas chromatography-tandem mass spectrometry method.

CONTEXT: Estrogens and androgens play key roles for pubertal onset and sexual maturation. Most currently used immunoassays are not sensitive enough to accurately measure the low circulating levels of sex steroids in children without any signs of puberty. However, this does not exclude that sex steroids have important biological roles in prepubertal children. OBJECTIVES: To accurately determine levels of sex steroid hormones and their metabolites in serum of healthy children before any physical signs of puberty and to evaluate possible sex differences. MAIN OUTCOME MEASURES: Total (unconjugated plus conjugated) serum levels of 17beta-testosterone, 17alpha-testosterone, 5alpha-dihydrotestosterone, 5beta-dihydrotestosterone, androsterone, etiocholanolone, estradiol, and estrone measured by an ultrasensitive method based on gas chromatography-tandem mass spectrometry in samples from 81 healthy schoolchildren (42 boys) without any signs of puberty. For comparison, 48 pubertal children were studied. RESULTS: 17beta-Estradiol levels in prepubertal boys were undetectable or extremely low (median < 3.7 pmol/liter), whereas levels in prepubertal girls were significantly higher (median 9.6 pmol/liter, P < 0.001). Among the older prepubertal children (>8 yr), girls had significantly higher androsterone (4.07 vs. 1.45 nmol/liter, P < 0.05), etiocholanolone (5.45 vs. 1.95 nmol/liter, P < 0.0001), 5alpha-dihydrotestosterone (0.11 vs. <0.10 nmol/liter, P < 0.01), and 17beta-testosterone concentrations (0.69 vs. 0.47 nmol/liter, P < 0.05) compared with similarly aged prepubertal boys. CONCLUSION: Using an accurate and sensitive method, we found significantly higher levels of estrogens as well as androgen metabolites in prepubertal girls compared with age-matched boys. The higher prepubertal sex steroid levels in girls may contribute to their earlier onset of puberty including pubic hair development.

Analytical strategies for the direct mass spectrometric analysis of steroid and corticosteroid phase II metabolites.

The use of steroid hormones as growth promoters remains illegal in Europe. A classical approach used to control their utilization consists to measure the parent drug in target biological matrices. However, this strategy may fail when the parent drug is submitted to extensive metabolism reactions. For urine and tissue samples, chemical or enzymatic hydrolysis is usually applied in order to deconjugate glucuronide and sulfate phase II metabolites. But this treatment lead to the loss of information such as nature and relative proportions of the different conjugated forms, which can be useful, for example, to discriminate an endogenous production from an exogenous administration for natural hormones, or for other clinical or biochemical specific applications. For these purposes, direct measurement of conjugated metabolites using liquid chromatography-tandem mass spectrometry may represent a solution of choice. In this context, the mass spectrometric behavior of 14 steroid and corticosteroid phase II metabolites after electrospray ionization was investigated. Their fragmentation pathways in tandem mass spectrometry revealed some specificities within the different group of conjugates. A specific acquisition program (MRM mode) was developed for the unambiguous identification of the studied reference compounds. A more generic method (Parent Scan mode) was also developed for fishing approaches consisting to monitor several fragment ions typical of each conjugate class. A reverse phase HPLC procedure was also proposed for efficient retention and separation of the studied compounds. Finally, a protocol based on quaternary amine SPE was developed, permitting the separation of free, glucuronide, and sulfate fractions. Preliminary results on biological samples demonstrated the suitability of this analytical strategy for direct measurement of dexamethasone glucuronide and sulfate residues in bovine urine.