Simultaneous determination of Δ9-tetrahydrocannabinol, cannabidiol, cannabinol and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair using liquid chromatography-tandem mass spectrometry.

For several years, hair analyses have become a powerful tool to investigate past exposure towards xenobiotics. In the case of illicit drugs and more precisely of cannabis exposure, four compounds are usually investigated: Δ(9)-tetrahydrocannabinol (THC), the main active compound of cannabis, one of its metabolites [11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] and two cannabinoids (cannabinol and cannabidiol). Up until now, the hair determination of the carboxylic metabolite of THC, which has been described as the only marker allowing distinguishing consumption and passive exposure, has been performed using a gas chromatography-tandem mass spectrometry method. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of the four markers. The sample preparation was based on an alkaline hydrolysis of hair samples followed by a liquid-liquid extraction of compounds in acidic conditions using a hexane/ethyl acetate mixture. The method was validated and the results were satisfactory: intra- and inter-assay accuracies below 9% and relative standard deviation below 15% for the four compounds. Moreover, the limit of quantification for THC-COOH, the most challenging compound, was validated at 0.2 pg/mg. This concentration is in accordance with the recommendations made by a scientific society which specializes in hair testing. It makes it possible to distinguish the kind of exposure to cannabis.

Development and validation of a single LC-MS/MS assay following SPE for simultaneous hair analysis of amphetamines, opiates, cocaine and metabolites.

The two major challenges in hair analysis are the limited amount of samples usually available and the low targeted concentrations. To overcome these limitations, a liquid chromatography-electrospray-tandem mass spectrometry method (LC-ESI-MS/MS) allowing the simultaneous analysis of 17 amphetamines (amphetamine, BDB, m-CPP, dexfenfluramine, DOB, DOM, ephedrine, MBDB, MDA, MDEA, MDMA, methamphetamine, methylphenidate, 4-MTA, norephedrine, norfenfluramine and PMA), 5 opiates (morphine, codeine, heroin, ethylmorphine, and 6AM), cocaine and 5 metabolites [ecgonine methyl ester (EME), benzoylecgonine (BZE), anhydroecgonine methyl ester (AME), cocaethylene, and norcocaine] has been developed. The validation procedure included linearity, intra-day and inter-day variability and accuracy for 5 days (5 replicates at 3 concentration levels). Proficiency studies were used to check the accuracy of the method. As a result, all amphetamines, opiates and cocaine derivatives were satisfactory identified by 2 MRM transitions in 15 min. Calibration curves were performed by a quadratic 1/X weighted regression. The calibration model fits from 0.05 to 10 ng/mg. The limits of detection (LODs) range between 0.005 and 0.030 ng/mg. Precision has been checked by intra-day and inter-day RSD, and associated relative bias, which were lower than 25% for the limits of quantifications (LOQs) and lower than 20% for the other levels tested. This method was routinely applied to hair samples: two positive results of adult drug addicts are presented.

Urinary determination of 2-isopropyl-4-methyl-6-hydroxypyrimidine in case of non fatal poisoning with diazinon.

We reported one non fatal case (42 month old boy) of intoxication with diazinon following accidental ingestion. Diazinon and three of its metabolites (2 common metabolites with other organophosphate pesticides: diethylphosphate and diethylthiophosphate; one specific metabolite: 2-isopropyl-4-methyl-6-hydroxypyrimidine) were determined in serum and in urine, respectively, using three liquid chromatography-tandem mass spectrometry methods. Diazinon was detected in serum while its metabolites were detected in urine. The concentrations of diazinon and its common metabolites were compared to concentrations previously described in literature in the same intoxication context and were discussed. The concentration of the specific metabolite was compared to concentrations highlighted in occupational exposure, because to the best of our knowledge, we reported here the first urinary concentration of this metabolite in an acute intoxication context.

Determination of glyphosate and AMPA in blood and urine from humans: about 13 cases of acute intoxication.

Acute intoxications after ingesting glyphosate are observed in suicidal or accidental cases. Despite low potential toxicity of this herbicide, a number of fatalities and severe outcomes are reported. Indeed, some authors have described the clinical features associated with blood and urine concentrations following intoxication. The purpose of this study is to describe the clinical feature and determinate the utility of the glyphosate concentration in blood and urine and the dose taken for predicting clinical outcomes. In 13 glyphosate poisoning cases treated in our laboratory within 7 years period from 2002 to 2009, we registered clinical observations and collected blood and urine samples to HPLC-MS-MS analysis. We classified our patients by the intoxication severity using simple clinical criteria. We obtained clinical observations from 10 patients and the others three patients were treated in forensic cases. Among the 10 patients, one was asymptomatic, 5 had mild to moderate poisoning and 2 had severe poisoning. There were 6 deaths whose 3 were forensic cases. The most common symptoms were oropharyngeal ulceration (5/10), nausea and vomiting (3/10). The main altered biological parameters were high lactate (3/10) and acidosis (7/10). We also noted respiratory distress (3/10), cardiac arrhythmia (4/10), hyperkaleamia, impaired renal function (2/10), hepatic toxicity (1/10) and altered consciousness (3/10). In fatalities, the common symptoms were cardiovascular shock, cardiorespiratory arrest, haemodynamic disturbance, intravascular disseminated coagulation and multiple organ failure. Blood glyphosate concentrations had a mean value of 61 mg/L (range 0.6-150 mg/L) and 4146 mg/L (range 690-7480 mg/L) respectively in mild-moderate intoxication and fatal cases. In the severe intoxication case for which blood has been sampled, the blood glyphosate concentration was found at 838 mg/L. Death was most of the time associated with larger taken dose (500 mL in one patient) and high blood glyphosate concentrations. To predict clinical outcomes and to guide treatment support in patients who ingested glyphosate, blood concentrations of this compound and the taken dose have been useful.

Simultaneous determination of five synthetic pyrethroid metabolites in urine by liquid chromatography-tandem mass spectrometry: application to 39 persons without known exposure to pyrethroids.

A sensitive and reliable method was developed and validated for the determination of five synthetic pyrethroid metabolites namely cis-Cl(2)CA, trans-Cl(2)CA, Br(2)CA, 3-PBA and 4-FPBA in human urine by liquid chromatography-tandem mass spectrometry. (2)D(6)-labelled trans-Cl(2)CA and (13)C(6)-labelled 3-PBA were used as internal standards. This method was based on a liquid-liquid extraction procedure in acidic conditions using hexane solvent with a basic purification, a chromatographic separation using a specific C18 column and mass spectrometric detection in the negative polarity. Suitable limits of detection (0.015µg/L for the five compounds) and quantification (from 0.020 to 0.030µg/L) were obtained for rendering the method usable for the biomonitoring of pyrethroids in the general population. The efficiency of the method was tested in 39 urine samples from French people without any known exposure to pyrethroids. At least three of the five metabolites were detected in each sample. The results of this study were compared to those obtained in previous ones and discussed.

Surprising hair analysis results following acute carbofuran intoxication.

We present two non fatal cases of intoxication with carbofuran (CBF) documented by hair analysis. Carbofuran and 3-hydroxycarbofuran (OHCBF, its main metabolite) hair concentrations were determined using a liquid chromatography-tandem mass spectrometry method. The obtained results were surprising if we consider several hair analyses previously published and based on a theory of the presence of xenobiotic in the only segment that comprised its intake. Among the two intoxication cases, we noticed the presence of CBF and OHCBF in hair segments corresponding to 45 days before, and more than 100 days after, the day of intoxication. Additionally, repeated hair samplings and subsequent analysis revealed a decrease of the carbofuran's concentration during the hair life.

Hair analysis to document non-fatal pesticide intoxication cases.

We reported two non-fatal cases of intoxication with pesticides namely alachlor and carbofuran. Hair stand samples were collected from two men approximately 1 year after alachlor intoxication for case 1, and 14 days after the last exposure for case 2. Hair analysis was performed using a liquid chromatography-tandem mass spectrometry method. In case 1, alachlor was detected in the 5 analysed hair segments (concentrations between 12 and 136 pg/mg) and its metabolites were not detected. In case 2, carbofuran and its main metabolite (3-hydroxycarbofuran) were detected in the hair strand (global analysis) at the concentrations of 207 and 164 pg/mg, respectively. However, additional data are required in order to interpret such results.

[Treatment of pesticides determination requests in biological samples: some problems frequently encountered ]. / Traitement des demandes de dosages de pesticides dans les milieux biologiques: quelques problèmes fréquemment rencontrés.

Currently, the determination of pesticides in human fluids is complex. Our scientific knowledge on pesticide intoxication is fragmentary. This leads to numerous difficulties regarding the diagnosis, the analysis request, the choice of the analytical technique, the interpretation of the results. Consequently the waiting time to provide a result is not compatible with the initiation of a treatment in the intoxicated patients. Very few information about Human intoxication with pesticides is available from the literature. In this context, we tried to acquire our own experience. We chose to deal with all pesticides intoxication suspicions received, in collaboration with clinicians. We are presenting the main problems we encounter.

Simultaneous determination of six dialkylphosphates in urine by liquid chromatography tandem mass spectrometry.

Dialkylphosphates (DAP) are urinary markers of the exposure to organophosphates pesticides. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of the following DAP: dimethylphosphate (DMP), dimethythiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP). Dibutylphosphate (DBP) was used as internal standard. This method was based on a liquid-liquid extraction procedure, a chromatographic separation using an Inertsil ODS3 C18 column and mass spectrometric detection in the negative ion, multiple reaction monitoring (MRM) mode, following two ion transitions per compound. It yielded a limit of quantification of 2 microg/L for the six compounds and intra-assay coefficients of variation (CV%) lower than 20%. This method was applied to the analysis of urines samples from a small cohort of non-exposed volunteers. At least one of the six DAP was detected in each sample. This result confirmed the feasibility of a LC-MS/MS procedure for monitoring the general population exposure to some frequently employed organophosphate pesticides.