Validation of an HPLC-MS/MS method with QuEChERS extraction using isotopic dilution to analyze chlordecone in human serum.

Until 1993, chlordecone (CLD) was extensively used in banana fields in French West Indies. In a previous study, CLD was detected in 90 % of Martinican and Guadeloupean adult's serum. In order to simplify the analyses of CLD in the serum, a new QuEChERS-HPLC-MS/MS method was implemented and validated by the Pasteur Institute of Guadeloupe (IPG). This method was validated with accuracy profiles according to the French Standard NF V03-110 plus the ISO 15189 and European guidelines. Linearity, repeatability, accuracy, intermediate precision, specificity, limit of detection (LOD), limit of quantification (LOQ) and uncertainty were determined. The accuracy profile allowed the method to be validated between 0.06  µg L-1 and 1.00  µg L-1 of serum. The LOD was 0.02  µg L-1, the LOQ was 0.06  µg L-1 and the uncertainty of the method was 21 %. A comparison of 49 serum samples between the IPG (LC-MS/MS) and the LEAE-CART (GC-HRMS) laboratories demonstrated that this new method can reliably determine CLD in human serum. Stability tests were performed and duration of the storage of raw samples and extracts before analysis by HPLC-MS/MS. Raw samples were stable after collection for at least one week at 5 °C or 25 °C and for at least 3 months at -20 °C. Extracts in acetonitrile were stable for at least 1 month at -20 °C. These stability results facilitate the daily use of the method. This method should help the entire population of Guadeloupe and Martinique by allowing a routinely analyzed for CLD and will be useful for future projects aimed at improving population health monitoring.

Characterization and quantification of chlordecone elimination in ewes.

To reduce the exposure of the French West Indies population to the organochlorine insecticide chlordecone (Kepone; CLD), the contamination of currently consumed foodstuffs must be reduced. Depuration of contaminated animals before slaughter could be a strategy to obtain safe animal products. The aim of this study was to characterize and quantify CLD elimination in contaminated ewes during depuration process. Experiments A and B consisted in a single intravenous (i.v.) administration of CLD (n = 5) and CLDOH (chlordecol; n = 3) followed by a 84-d and 3-d depuration period respectively with collection of blood, faeces and urine samples. After CLD administration, CLD and conjugated-CLDOH (CLDOH-C) were quantified in serum and urine and CLD and CLDOH were quantified in faeces. Based on calculations of faecal, urinary and body clearances of CLD and CLDOH-C, faeces appeared as the major route of CLD excretion with 86 % of the CLD administered dose eliminated in faeces, either as CLD (51 %) or as CLDOH (35 %).

Linear toxicokinetic of chlordecone in ewe's serum.

Chlordecone (CLD) is an organochlorine pesticide used in banana fields of the French West Indies between 1972 and 1993. This use resulted in a long-term pollution of soils and the possible contamination of farm animals. Indeed, after involuntary ingestion of soil, CLD is absorbed and consequently leads to contaminated animals. The aim of this study was the determination of CLD half-life and the establishment of the linearity of CLD disappearance kinetics in non-lactating adult's ewes. Chlordecone diluted in cremophor was intravenously administrated to ewes at different doses: 0.04, 0.2, or 1 mg kg-1 body weight (n = 5 for each dose). Blood samples were collected from time t = 0 to time t = 84 days. Serum samples were extracted with a solid-phase extraction and analyzed by electron capture detection gas chromatography. A two-compartmental model was applied to the serum CLD kinetics. An additional statistical analysis was applied to the observed elimination parameters in serum according to the administrated dose, and no significant differences were detected. The linear elimination of CLD between 0.04 and 1 mg kg-1 body weight allowed the possibility of ewe's extrapolation half-life in this dose range. The estimated mean CLD half-life in ewes was 24 days. Overall, the results of this study will be useful to establish decontamination strategies in small ruminants reared in contaminated CLD areas. Graphical abstract Experimental design of the CLD toxicokinetic study in ewes.

Validation of analytical methods for chlordecone and its metabolites in the urine and feces of ewes.

Chlordecone (CLD) is a Persistent Organic Pollutant used between 1972 until 1993 in the French West Indies (FWI). Due to its persistence and extensive application, a quarter of the total local agricultural acreage is still moderate to heavily polluted. In consequence, livestock may be contaminated at various levels. This is a major public health concern, particularly for local consumers. In order to better understand the fate of CLD in livestock organisms, in vivo studies are required. There is no information available about its metabolism and elimination in ruminants, common livestock in the FWI. To be able to monitor the fate of chlordecone and its metabolites in livestock and to assess if the compounds could be released in the environment, urinary and fecal samples were logically targeted. In order to reach this goal, robust and validated analytical methods are required. For this purpose, Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction methods were validated to analyze CLD and its metabolites in the urine and feces. The analysis was carried using liquid phase chromatography with tandem mass spectrometry and validated according to French standard NF V03-110 and SANTE guidelines. Matrix effect, Accuracy, within-laboratory repeatability, specificity, Q/q relative ion intensities and uncertainty were reported. Recoveries between 70% and 120% were obtained from urine and feces. The limits of quantification (LOQ) in urine samples were 0.1 µg CLD L-1, 0.1 µg total CLD (CLD and its conjugates)·L-1, 1.3 µg CLDOH L-1 and 2.4 µg total CLD (chlordecol and its conjugates) L-1 of urine. LOQ in fresh feces were 3.2 µg CLD kg-1 and 5.8 µg CLDOH kg-1. Contaminated urinary and fecal samples from ewes were analyzed to confirm the relevance of the methods. In urine, CLD and conjugated CLDOH could be quantified whereas only free CLD and free CLDOH were found in feces. These methods are essential for future toxicokinetic studies and also to estimate the environmental contamination.

Classification of trace elements in tissues from organic and conventional French pig production.

This study assesses the impact of the farming system on the levels of copper, zinc, arsenic, cadmium, lead and mercury in pig tissues from three types of production (Organic (n = 28), Label Rouge (n = 12) and Conventional (n = 30)) randomly sampled in different slaughterhouses. All the concentrations were below regulatory limits. In muscles, Cu, Zn and As were measured at slightly higher levels in organic samples but no differences between organic and Label Rouge was observed. Livers from conventional and Label Rouge pig farms exhibited higher Zn and Cd contents than the organic ones, probably due to different practice in zinc or phytase supplementation of fattening diets. Principal component analysis indicated a correlation between Cu and As concentrations in liver and carcass weight, and between Zn and Cd liver levels and lean meat percentage. The linear discriminant analysis succeeded in predicting the farming process on the basis of the lean meat percentage and the liver Cd level.

Development and validation of an HPLC-MS/MS method with QuEChERS extraction using isotopic dilution to simultaneously analyze chlordecone and chlordecol in animal livers.

A QuEChERS extraction method followed by HPLC-MS/MS analysis was developed to simultaneously analyze chlordecone and its metabolite chlordecol in animal livers. The overall method was validated with accuracy profiles according to the French Standard NF V03-110 and European Union guidelines. The validation was performed on bovine, ovine and porcine liver samples. Linearity, matrix effect, accuracy, within-laboratory repeatability, specificity, LOQ, Q/q relative ion intensities, and uncertainty were reported. Recoveries were between 70% and 120%. LOQs of 1.36 µg chlordecone kg-1 and 2.50 µg chlordecol kg-1 of fresh liver were found. Twelve contaminated livers of bovine, ovine and porcine origin from the French West Indies or samples from in vivo studies were analyzed. In these liver samples from contaminated animals, chlordecone was quantified at concentrations higher than the maximum residue limit and chlordecol in very low amounts in all the samples. In addition, these results confirm that chlordecone can be metabolized in ruminant species.

Micropollutants and chemical residues in organic and conventional meat.

The chemical contamination levels of both conventional and organic meats were assessed. The objective was to provide occurrence data in a context of chronic exposure. Environmental contaminants (17 polychlorinated dibenzodioxins/dibenzofurans, 18 polychlorinated biphenyls (PCBs), 3 hexabromocyclododecane (HBCD) isomers, 6 mycotoxins, 6 inorganic compounds) together with chemical residues arising from production inputs (75 antimicrobials, 10 coccidiostats and 121 pesticides) have been selected as relevant compounds. A dedicated sampling strategy, representative of the French production allowed quantification of a large sample set (n=266) including both conventional (n=139) and organic (n=127) raw meat from three animal species (bovine, porcine, poultry). While contamination levels below regulatory limits were measured in all the samples, significant differences were observed between both species and types of farming. Several environmental contaminants (Dioxins, PCBs, HBCD, Zn, Cu, Cd, Pb, As) were measured at significantly higher levels in organic samples.