Gas chromatography-tandem mass spectrometry analysis of 52 monohydroxylated metabolites of polycyclic aromatic hydrocarbons in hairs of rats after controlled exposure.

A method based on gas chromatography-tandem mass spectrometry after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide was developed for the analysis of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in hair. The method focused on 52 target compounds corresponding to two- to six-ring monohydroxylated metabolites of polycyclic aromatic hydrocarbons (PAHs). The limits of quantification ranged from 0.2 to 50 pg mg(-1). The method was then applied to the analysis of hair samples collected from rats exposed to 12 PAHs at 0.01, 0.1, and 1 mg kg(-1), by intraperitoneal injection, for 28 days. The results of this study confirm that these metabolites can be incorporated in hair after intraperitoneal administration of the corresponding parent compound. Only 20 of the 52 metabolites were actually detected in hair samples and corresponded to nine parent PAHs. The mean concentrations of OH-PAHs in rat hair samples exposed to PAHs at 1 mg kg(-1) ranged from 0.6 ± 0.2 pg mg(-1) for 8-hydroxybenzo[b]fluoranthene to 6.7 ± 1.0 pg mg(-1) for 1-hydroxypyrene. The results also demonstrated that hair pigmentation has no influence on the concentration of most OH-PAHs. This animal experiment confirmed the incorporation of PAH metabolites in hair and demonstrated that the method was sufficiently sensitive to detect low levels of exposure to PAHs. These results confirmed the usefulness of hair analysis in the biomonitoring of human exposure to PAHs.

Determination of PAHs and OH-PAHs in rat brain by gas chromatography tandem (triple quadrupole) mass spectrometry.

An efficient and selective method for the quantitative determination of polycyclic aromatic hydrocarbons (PAHs) and their monohydroxylated metabolites (OH-PAHs) in rat brain tissue using gas chromatography tandem (triple quadrupole) mass spectrometry (GC-MS/MS) was developed and validated. The list of molecules investigated comprised the 16 PAHs from the US-EPA list and 53 of their OH-PAHs. Brain extract was submitted to enzymatic hydrolysis, followed by liquid-liquid extraction, and then purified by solid-phase extraction. Limits of quantification ranged from 0.6 to 29 pg/mg and from 0.5 to 30 pg/mg for PAHs and OH-PAHs respectively. The analysis of rat brain samples exposed to PAH mixture (0.01-1 mg/kg, 28 days, ip) demonstrated that this method allowed the detection of 16 PAHs and 28 OH-PAHs out of the 69 analytes investigated. Mean concentrations of PAHs in animal brain samples exposed to 1 mg/kg of PAH mixture ranged from 3.0 ± 2 pg/mg for benzo[b]fluoranthene to 146 ± 29 pg/mg for phenanthrene. Concomitantly, mean concentrations of OH-PAHs ranged from 0.49 ± 0.4 to 26.5 ± 23 pg/mg for 2-OH-chrysene and 1-OH-pyrene respectively. This study proves, for the first time, the bioavailability of most of the PAHs and OH-PAHs in mammalian brain tissue and should provide an important new tool for future neurotoxicological studies.

Clenbuterol determination in calf hair by UPLC-MS-MS: case report of a fraudulent use for cattle growth.

A method for clenbuterol determination in hair has been developed. Hair specimens collected from two calves were decontaminated using hot water followed by methylene chloride. Hair was cut into small pieces, and 100 mg was incubated in 1 mL 0.1M hydrochloric acid overnight at 45 degrees C in the presence of 1 ng acebutolol used as internal standard. After neutralization with 1 mL 0.1M NaOH, 2 mL of bicarbonate buffer (pH 8.6) were added and the preparation was then purified using solid-phase extraction with an Isolute C18 column. Methanolic eluent was evaporated to dryness and the residue was reconstituted with 50 microL methanol. A 5-microL portion was injected onto an ACQUITY UPLC BEH C18 column (2.1 x 50 mm, 1.7 microm) and separation was achieved using a gradient of acetonitrile and formate buffer delivered at a flow rate of 0.6 mL/min. Detection was done on a Waters Micromass Quattro Micro API triple-quadrupole mass spectrometer. Ionization was achieved using electrospray in positive mode. Clenbuterol was identified by two transitions (m/z 277.1 > 203.2 and m/z 277.1 > 132.1). Quantitation was performed with the most intensive transition (m/z 277.1 > 203.2) versus the internal standard monitored using the transition (m/z 337.3 > 116.1). When compared with gas chromatography methods that are generally used for the determination of beta-adrenergics, the major advantages of this method were the sensitivity, a shorter run time, and the absence of a derivatization step. The analysis of two hair samples from calves suspected of drug administration showed low clenbuterol concentrations at 3.6 and 4.8 pg/mg.

Pesticide detection in air samples from contrasted houses and in their inhabitants' hair.

In order to identify associations between indoor air contamination and human exposure to pesticides, hair samples from 14 persons (9 adults and 5 children below 12 years) were collected simultaneously with the air of their 5 contrasted houses. Three houses were situated in Alsace (France), one in Lorraine (France) and one in Luxembourg (Luxembourg). Houses were located in urban (n=3), semi-urban (n=1) and rural areas (n=1). Twenty five (25) pesticides were detected at least once in indoor air samples and 20 pesticides were detected at least once in hair samples. The comparison between hair and air samples for the same sampling periods shows that pesticides detected in the two matrices were not necessarily associated. Exposure profiles varied from one home to another but also between inhabitants of the same home, suggesting that exposure can be different between inhabitants of the same home. This study demonstrated the usefulness and the complementarity of hair analysis, for the personalized biomonitoring of people exposure to pesticides, and air analysis, for the identification of airborne exposure and house contamination.

Behavioral toxicity and physiological changes from repeated exposure to fluorene administered orally or intraperitoneally to adult male Wistar rats: A dose-response study.

Fluorene is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in the environment by reason of its high volatility. Demonstrated to be a neurotoxicant through inhalation, it was also identified as a contributive PAH to food contamination. Since no data are available on its oral neurotoxicity, the purpose of the present study was to assess the behavioral and physiological toxicity of repeated oral administration of fluorene to adult Wistar male rats. Animals were daily treated with fluorene at 1, 10 or 100mg/kg/day for 28 consecutive days. Administration was intraperitoneal (i.p.) or oral (p.o.) to evaluate the influence of the route of exposure on fluorene toxicity. Following this period of treatment, animals in both groups were subjected to similar cognitive evaluations, namely anxiety (elevated-plus maze), locomotor activity (open-field) and learning and memory abilities (eight-arm maze and avoidance test of an aversive light stimulus), as well as physiological measurements. The behavioral testing occurred from the 28th to the 60th day of the experiment during which fluorene treatment continued uninterrupted. At the end of this period, the concentration levels of fluorene and of three of its monohydroxylated metabolites in blood and brain were determined using a GC-MS/MS method. The results demonstrated a reduction in rat anxiety level at the lowest doses administered (1 and 10mg/kg/day) regardless of the treatment route, whereas locomotor activity and learning abilities remained unchanged. Moreover, a less significant weight gain was noticed in animals i.p.- and p.o.-treated with 100mg/kg/day during the 28-day period of treatment, which, upon comparison with the three other groups, induced a body weight gap that was maintained throughout the experiment. Significant increases in relative liver weight were also observed in a dose-dependent manner in orally treated rats and only in animal treated i.p. with 100mg/kg/day. According to the dose, higher concentration levels of fluorene and its monohydroxylated metabolites were measured in blood and brain compartments of i.p.-treated rats compared to p.o.-treated animals. In conclusion, fluorene reduced the anxiety level of rats related to dose, treatment route, duration of exposure and blood concentration levels of metabolites.

Multi-residue analysis of organic pollutants in hair and urine for matrices comparison.

Urine being currently the most classically used matrix for the assessment of human exposure to pesticides, a growing interest is yet observed in hair analysis for the detection of organic pollutants. The aim of the present work was to develop and to validate multi-residue analytical methods, as similar as possible, in order to determine pesticides and their metabolites in these two biological matrices despite their different nature. The list of parent compounds and their metabolites investigated here consisted of 56 compounds, including organochlorines, organophosphates, pyrethroids, carbamates, other pesticides and polychlorinated biphenyls (PCBs). Two different approaches were necessary for the analysis of non-polar compounds (mainly parents) on one hand and polar analytes (mainly metabolites) on the other hand. In the final procedure, extraction from hair was carried out with acetonitrile/water after sample decontamination and pulverization. Extract was split into two fractions, which were analyzed directly with solid phase microextraction (SPME) injection for non-polar compounds and after derivatization with liquid injection for polar compounds. In urine, non-polar compounds were analyzed directly using SPME. Polar compounds were analyzed after acidic hydrolysis, liquid-liquid extraction with acetonitrile-cyclohexane-ethyl acetate, derivatization and liquid injection. Analysis was performed with gas chromatography tandem mass spectrometry operating in negative chemical ionization (GC-MS/MS-NCI) for all the compounds (non-polar and polar) in the two matrices. In hair, limits of quantification (LOQ) ranged from 0.02 pg/mg for trifluralin to 5.5 pg/mg for diethylphosphate. In urine, LOQ ranged from 0.4 pg/mL for α-endosulfan to 4 ng/mL for dimethyldithiophosphate. The analysis of samples supplemented with standards and samples collected from an animal previously submitted to chronic exposure to pesticides confirmed that all the compounds were analyzable in both hair and urine. In addition, the levels of sensitivity reached with these methods were quite satisfactory with regard to previously published studies, and also considering the number of compounds investigated.

Comparison of solid phase- and liquid/liquid-extraction for the purification of hair extract prior to multi-class pesticides analysis.

The present study focuses on the influence of a purification step - after extraction of pesticides from hair and before analysis of the extract - on the sensitivity of analytical methods including compounds from different chemical classes (both parent and metabolites). Sixty-seven pesticides and metabolites from different chemical classes were tested here: organochlorines, organophosphates, carbamates, pyrethroids, ureas, azoles, phenylpyrazoles and neonicotinoids. Two gas chromatography-negative chemical ionization-tandem mass spectrometry methods and one based on ultra-performance liquid chromatography-electrospray tandem mass spectrometry were used. Seven solid-phase extraction cartridges: C18, S-DVB, PS-DVB, GCB, GCB/PSA, SAX/PSA and Florisil/PSA were tested and compared to more classical liquid-liquid extraction procedures using ethyl acetate, hexane and dichloromethane. Although LLE allowed obtaining good results for some compounds, on the whole, SPE clearly provided better recovery for the majority of the pesticide residues tested in the present study. GCB/PSA was clearly the best suited to non-polar compounds such as organochlorines, pyrethroids and organophosphates, with recovery ranging from 45.9% (diflufenican) to 117.1% (parathion methyl). For hydrophilic metabolites (e.g. dialkyl phosphates and other organophosphate metabolites, pyrethroid metabolites, phenols and carbamate metabolites), the best results were obtained with PS-DVB, with recovery ranged from 10.3% (malathion monocarboxylic acid) to 93.1% (para-nitrophenol). For hydrophilic parent pesticides (e.g. neonicotinoids, carbamates, azoles) and metabolites without nucleophilic functions, the best recovery was obtained with SAX/PSA, with recovery ranging from 52.1% (3-hydroxycarbofuran) to 100.9% (3,4-dichloroaniline). Solid phase extraction was found to be more suitable than the liquid-liquid extraction for pesticides and their metabolites determination in terms of number of extracted compounds and their recovery. Moreover, the use of solid phase extraction cartridges has enabled the reduction of the analytical background noise, resulting in better chromatographic separations.

Hair decontamination procedure prior to multi-class pesticide analysis.

Although increasing interest is being observed in hair analysis for the biomonitoring of human exposure to pesticides, some limitations still have to be addressed for optimum use of this matrix in that specific context. One main possible issue concerns the need to differentiate chemicals biologically incorporated into hair from those externally deposited on hair surface from contaminated air or dust. The present study focuses on the development of a washing procedure for the decontamination of hair before analysis of pesticides from different chemical classes. For this purpose, three different procedures of artificial contamination (with silica, cellulose, and aqueous solution) were used to simulate pesticides deposition on hair surface. Several washing solvents (four organic: acetone, dichloromethane, methanol, acetonitrile; and four aqueous: water, phosphate buffer, shampoo, sodium dodecylsulfate) were evaluated for their capacity to remove artificially deposited pesticides from hair surface. The most effective washing solvents were sodium dodecylsulfate and methanol for aqueous and organic solvents, respectively. Moreover, after a first washing with sodium dodecylsulfate or methanol, the majority of externally deposited pesticides was removed and a steady-state was reached since significantly lower amounts were removed by additional second and third washings. Finally, the effectiveness of a decontamination procedure comprising washing with sodium dodecylsulfate and methanol was successively demonstrated. In parallel, it was determined that the final procedure did not affect the chemicals biologically incorporated, as hair strands naturally containing pesticides were used. Such a procedure appears to remove in one-shot the fraction of chemicals located on hair surface and does not require repeated washing steps.

Tetrahydroxylated-benzo[a]pyrene isomer analysis after hydrolysis of DNA-adducts isolated from rat and human white blood cells.

Since exposure to benzo[a]pyrene is suspected to be associated with several health issues, significant efforts have been made to develop efficient strategies for the assessment of human exposure to this ubiquitous compound. In this context, a method was developed for the analysis of four tetrahydroxylated-benzo[a]pyrene isomers resulting from the hydrolysis of their respective diol-epoxide precursors which are involved in DNA-adduct formation. The analytical sensitivity necessary to reach environmental levels of concentration was obtained by using gas chromatography-tandem mass spectrometry. The recovery determined at the four concentration levels were estimated in average at 83% for benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol(±), 29% for benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol(±), and 82% for benzo[a]pyrene-r-7,t-8,C-9,c-10-tetrahydrotetrol(±). The coefficient of determination of the calibration curve was above 0.997 for all the analytes investigated and the limit of quantification ranged from 0.5 to 2 adduct/10(8) nucleotides. The precision was between 5.3% and 22.3%. The suitability of the method was firstly evaluated by the analysis of DNA isolated from white blood cells of rats submitted after controlled exposure to benzo[a]pyrene. The four targeted tetra-OH-benzo[a]pyrenes as well as two unknown isomers were detected in all the treated animals. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol(±) appeared as the most abundant isomer in both treated and control animals followed by benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol(±). The method was afterwards applied to the analysis of DNA isolated from white blood cells of human volunteers. The results confirmed that this method was sufficiently sensitive to monitor environmental levels of exposure since all the specimens analyzed were above the limit of quantification for benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol(±) and two of them were positive for benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol(±), thereby highlighting interspecies differences in the nature of the tetrahydroxylated-benzo[a]pyrene isomers formed. This study confirms the necessity to focus on all the tetrahydroxylated-benzo[a]pyrene isomers, which could be indicators of benzo[a]pyrene-associated toxicity related to an individual's own metabolism, rather than limit to a single form.

Short-term effects of a perinatal exposure to a 16 polycyclic aromatic hydrocarbon mixture in rats: assessment of early motor and sensorial development and cerebral cytochrome oxidase activity in pups.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs), a family of ubiquitous neurotoxic pollutants, mainly through ingestion of contaminated food. Developing organisms can be exposed also to PAHs due to the ability of these compounds to pass through the placental barrier as well as through the breast milk. Previous animal studies have reported that the exposure of rats to a 16 PAH mixture at environmental doses strictly limited to gestation did not induce any long-lasting consequences, whereas gestational and lactational PAH exposure induced long-term behavioral and cerebral metabolic effects. In the present study, short-term effects of exposures to the same PAH mixture during gestation, or during gestation and lactation, were assessed by evaluating motor and sensory development of rat pups, and by measuring cerebral cytochrome oxidase activity (a marker of energetic metabolism) in different brain areas. Brain levels of PAHs and some monohydroxylated metabolites were also evaluated in pups at birth and at 21 days of postnatal life. No significant short-term modifications of behavioral development and of cerebral metabolism were observed following an early PAH exposure whatever the dose and the period of exposure. Surprisingly, the same brain levels of concentration of PAHs and metabolites were observed in control and exposed pups in both studies. These analytical results raise the difficulty in overcoming environmental contamination of control animals and the choice of such controls in experimental studies which focus on neurotoxicity of exposure to low levels of pollutants.

Neurobehavioral toxicity of a repeated exposure (14 days) to the airborne polycyclic aromatic hydrocarbon fluorene in adult Wistar male rats.

Fluorene is one of the most abundant polycyclic aromatic hydrocarbons in air and may contribute to the neurobehavioral alterations induced by the environmental exposure of humans to PAHs. Since no data are available on fluorene neurotoxicity, this study was conducted in adult rats to assess the behavioral toxicity of repeated fluorene inhalation exposure. Male rats (n = 18/group) were exposed nose-only to 1.5 or 150 ppb of fluorene 6 hours/day for 14 consecutive days, whereas the control animals were exposed to non-contaminated air. At the end of the exposure, animals were tested for activity and anxiety in an open-field and in an elevated-plus maze, for short-term memory in a Y-maze, and for spatial learning in an eight-arm maze. The results showed that the locomotor activity and the learning performances of the animals were unaffected by fluorene. In parallel, the fluorene-exposed rats showed a lower level of anxiety than controls in the open-field, but not in the elevated-plus maze, which is probably due to a possible difference in the aversive feature of the two mazes. In the same animals, increasing blood and brain levels of fluorene monohydroxylated metabolites (especially the 2-OH fluorene) were detected at both concentrations (1.5 and 150 ppb), demonstrating the exposure of the animals to the pollutant and showing the ability of this compound to be metabolized and to reach the cerebral compartment. The present study highlights the possibility for a 14-day fluorene exposure to induce some specific anxiety-related behavioral disturbances, and argues in favor of the susceptibility of the adult brain when exposed to volatile fluorene.

Multi-class pesticide analysis in human hair by gas chromatography tandem (triple quadrupole) mass spectrometry with solid phase microextraction and liquid injection.

A method for the simultaneous detection and quantification of 22 pesticides from different chemical classes was developed using solid-phase microextraction (SPME) and gas chromatography tandem (triple quadrupole) mass spectrometry. Pesticides were extracted from 50mg of pulverized hair with acetonitrile. The extract was submitted to two successive steps of direct immersion-SPME at 30°C and 90°C or to a liquid injection without SPME in order to obtain optimized conditions for each of the 22 analytes investigated. Validation parameters were significantly influenced by both the injection mode (SPME vs liquid injection) and the temperature of SPME. Limits of quantification ranged from 0.05 pg mg(-1) for trifluralin to 10 pg mg(-1) for pentachlorophenol. The application of the validated method to the analysis of samples collected from non-occupationally exposed volunteers demonstrated the presence of pesticides in all the samples tested. Altogether, 13 different analytes were detected at concentration above the limit of quantification.

Determination of farm workers' exposure to pesticides by hair analysis.

In the present work, a highly sensitive method based on solid phase microextraction and gas chromatography tandem (triple quadrupole) mass spectrometry was used to test hair samples for 50 pesticides including 39 molecules from different chemical families currently used in agriculture and 11 organochlorines. The population investigated was composed of 18 farm workers who provided hair samples repeatedly collected during the entire treatment period (from March to November 2009). Among the 62 hair samples that were collected, 33 different target molecules were detected. The most frequently detected agricultural pesticides were Diflufenican and Pyrimethanil, two herbicides which were detected in 13 subjects. The concentration in volunteers' hair matched with agricultural activity and the highest concentration was observed for Cyprodinil (1161pg/mg), an anilinopyrimidine used as a fungicide. For organochlorines, p,p'-DDE and γ-HCH were the most frequently detected molecules as they were present in at least one of the hair samples provided by each of the 18 volunteers. The highest concentrations detected for these chemicals reached 21.0pg/mg for p,p'-DDE and 23.5pg/mg for γ-HCH, but the highest concentration of organochlorine was observed for ß-endosulfan (105pg/mg). The results suggest that farm workers have a weak, though constant exposure to organochlorine pesticides, especially to p,p'-DDE and γ-HCH, while exposure to currently used pesticides is strongly associated with occupation. Observations also suggest that spraying work would not necessarily be the only source of exposure to agricultural pesticides and that worker not directly involved in spraying can also be submitted to significant level of exposure.

Ethyl glucuronide: unusual distribution between head hair and pubic hair.

Ethyl glucuronide (EtG) is a minor metabolite of ethanol that can be detected in hair. In some specific situations, head hair can be missing, and therefore, alternative anatomical locations of hair are of interest. In this study, paired hair specimens (head hair and pubic hair) from eight social drinkers were analyzed for EtG. Each sample was decontaminated by two dichloromethane bathes (5 ml) for 2 min. After cutting into small pieces, about 50 mg of hair was incubated in 2 ml water in the presence of 10 ng of EtG-d5, used as internal standard and submitted to ultra-sonication for 2 h. The aqueous phase was extracted by SPE using Oasis MAX columns. The hair extract was separated on an ACQUITY BEH HILIC column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 221-85 and 75 and m/z 226-75 for EtG and the IS, respectively. This laboratory is using a positive cut-off at 50 pg/mg. All eight head hair specimens were negative for EtG at a limit of quantitation fixed at 10 pg/mg. Surprisingly, EtG was identified at high concentrations in pubic hair, in the range 12-1370 pg/mg. It appears, therefore, that it is not possible to document the drinking status of a subject by simply switching from head hair to pubic hair.

Hair analysis for diphenhydramine after surreptitious administration to a child.

Diphenhydramine is one of the first effective antihistamine agents to have been discovered. The compound is also used for its sedative and antiemetic effects. The first case involving repetitive sedation linked to the use of diphenhydramine as a drug-facilitated crime and subsequent impairment of a 9-year-old female victim is reported. Due to the long delay between the alleged crime and clinical examination, collection of blood or urine was of little value. Hence, the laboratory developed an original approach based on hair testing by LC-MS/MS. A single strand of hair from the victim was sampled about 7 weeks after the last suspected administration and was cut into small segments. After cutting into small pieces, about 20 mg of hair per segment was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted with 5 ml of a mixture of methylene chloride/diethyl ether (80/20), in presence of diazepam-d5, used as internal standard (IS). The hair extract was separated on an XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 256.2-152.1 and 167.1 and m/z 289.9-154.0 for diphenhydramine and the IS, respectively. In the hair of the child, diphenhydramine was detected at concentrations in the range 33-39 pg/mg, depending on the segment.