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Pharmacokinetic studies and postmortem toxicological investigations require a validated analytical technique to quantify drugs on a large number of matrices. Three-step liquid/liquid extraction with online derivatization (silylation) ahead of analysis by gas chromatography-tandem mass spectrometry was developed and validated on rabbit specimens in order to quantify citalopram and 4 benzodiazepines (diazepam, nordazepam, oxazepam and temazepam) in 11 biological matrices (blood, urine, bile, vitreous humor, liver, kidney, skeletal muscle, brain, adipose tissue, bone marrow (BM) and lung). Since the 11 biological matrices came from the same animal species, full validation was performed on 1 matrix, bone marrow (considered the most complex), while the other 10 underwent partial validation. Due to non-negligible matrix effects, calibration curves were performed on each matrix. Within-day and between-day precision (less than 12.0% and 12.6%, respectively) and accuracy (from 88.9% to 106.4%) were acceptable on BM at both low and high concentrations. Assessment on the other matrices confirmed accuracy and within-day precision (less than 12%, and generally between 85.1% and 114.5%, respectively). The lower limit of quantification of the method was 1ng/g for nordazepam, 5ng/g for citalopram and 10ng/g for oxazepam, diazepam and temazepam. The combination of 3-step extraction and MS/MS detection provided good selectivity in all matrices, including the most lipid-rich. Application to real-case samples showed that the method was sensitive enough to describe distribution patterns in an animal experiment, and specific enough to detect molecules in highly putrefied samples from human postmortem cases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have developed a powerful and simple sensitive method for testing hair for anabolic steroids and their esters. A 100-mg amount of powdered hair was treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid-phase extraction on amino and silica cartridges. The residue was derivatized with N-methyl-N(trimethylsilyl)-trifluoracetamide (MSTFA) prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. The generally chosen parent ion was the molecular ion while two daughter ions were selected for each compound with collision energies ranging from -16 to -21 eV. Internal standards were nandrolone d3 for non-esterified drugs and testosterone phenyl propionate for esters. The limits of detection calculated from an analysis of the blanks (n=30) were 0.08 pg/mg for nandrolone, 6.20 pg/mg for boldenone, 0.07 pg/mg for methyl testosterone, 0.15 pg/mg for ethinyl estradiol, 2.10 pg/mg for metandienone, 0.86 pg/mg for testosterone propionate, 0.95 pg/mg for testosterone cypionate, 1.90 pg/mg for nandrolone decanoate, 3.10 pg/mg for testosterone decanoate and 4.80 pg/mg for testosterone undecanoate. Application to doping control has been demonstrated. In a series of 18 sportsmen, two tested positive for anabolic steroids in hair whereas urinalysis was negative for both of them. The first positive case was nandrolone and the second case concerned the identification of testosterone undecanoate. Measured in 10 white males aged between 22 and 31 years, the testosterone concentration was in the range 1.7-9.2 pg/mg (mean=5.0 pg/mg). The method was also applied in meat quality control. Of the 187 analyses realized based upon hair and urine sampling in slaughter houses, 23 were positive for anabolic steroids in hair: one case for boldenone, one case for metandienone, two cases for testosterone propionate, three cases for nandrolone, five cases for testosterone decanoate and 11 cases for methyl testosterone. In the meantime, urinalysis was always negative for these drugs or their metabolites.

After conversion of norpropoxyphene (NP) to its corresponding amide, dextropropoxyphene (DP) and NP are extracted from 1 ml of blood or 50 mg of powdered hair, on C18 cartridges and eluted using methanol containing 0.5% acetic acid. Automated extraction is conducted on-line with automated device, starting from buffered and centrifuged sample. After extraction, the dried residue is reconstituted with 40 microl of methanol, and then injected in a gas chromatograph at 250 degrees C. Quantitation is carried out by gas chromatography-mass spectrometry in the selected-ion monitoring mode, lidocaine being the internal standard. The method gave relative standard deviations lower than 6.2% in whole blood, and 6.0% in hair for the entire range of calibration from 0.5 to 10 microg/ml in blood and from 1 to 20 ng/mg in hair of both compounds. Limits of detection in blood and hair for DP are, respectively, 0.07 microg/ml and 0.05 ng/mg, whereas the respective limits of detection in whole blood and hair for NP are 0.09 microg/ml and 0.04 ng/mg. The present method was used for one year in our laboratory. Postmortem concentrations of DP in blood ranged from 1.6 to 44.0 microg/ml (mean=9.8microg/ml, n = 12) and are comparable to those found in the literature. Out of 30 hair samples from people who died from heroin overdose, 13 were positive both for DP and NP with concentrations ranging from 0.2 to 27.4 ng/mg (mean 8.7 ng/mg) for DP and 0.3 to 68.9 ng/mg (mean 24.1 ng/mg) for NP.

This study describes a real-life situation involving nine calves, 106 days old, which received oral doses of clenbuterol administered through their milk. Powdered skim milk containing 6.7 mg of clenbuterol was given daily for fifteen days under supervision (i.e. 100 mg per calf for the whole study) to seven calves, and two calves did not receive the drug. Hair samples and urine were taken and subjected to analysis by gas chromatography-mass spectrometry. Hairs were pulverized in a ball mill and 100 mg were incubated in a mildly acidic medium. The sample clean-up procedure involved solid-phase extraction on C18 cartridges. Metoprolol was used as the internal standard for quantitation, after formation of methylboronate derivatives. The calibration curve for clenbuterol in hair was linear in the range 20-5000 pg/mg. The limit of detection of clenbuterol was 16 pg/mg in hair and 0.14 ng/ml in urine. Hair testing was effective after 7-10 days of treatment, and concentrations were in the range of 20 to 4372 pg/mg. Urinalysis can detect clenbuterol for up to two weeks after discontinuation of the drug. Conveniently, this is around the time when the hair samples attain greatest sensitivity. Therefore, the combination of the two matrices appears to be the method of choice for testing for the illegal use of drugs in meat-producing animals.

We have described a rapid and simple solid-phase extraction on C18 cartridges of moclobemide suitable for the analysis of post-mortem whole blood and urine. The methods used for identification were GC-MS and HPLC-PDA. Quantification was performed by the HPLC-PDA technique with detection at 238 nm. The limit of detection was 0.012 microgram/ml in blood. A between-day precision study gave relative standard deviations which were always less than 4.7% over the entire range of calibration (0.2 to 20.0 micrograms/ml). The method was applied in a case of moclobemide overdose due to a deliberate ingestion of 4.5 g of the drug. A second case concerned a polyintoxication including moclobemide as one of the main toxins. The post-mortem whole blood concentrations were 15.5 and 13.8 micrograms/ml respectively. Determination of the drug in other biological specimens is also reported.

We describe a simple method for the urinary identification and blood quantitation of meprobamate suitable for any toxicological laboratory. After urinary screening using GC-MS technology, quantitation is performed by GC-MS in the selected-ion monitoring mode. Isolation of the drug is achieved by solid phase extraction on a C-18 cartridge. A specific elution is obtained by three volumes of acetone:triethylamine (99:1 v/v). Lidocaine is used as internal standard. RSDs (%) of the within-day and between-day precision studies are always less than 7.2 on the entire range of calibration. Linearity is inspected using an analysis of variance ANOVA. Homogeneity of the variances is tested using Hartley's test. Weighted linear regression is then computed. Limits of detection and quantification are given by an analysis of the blanks. The present method was applied in our laboratory over a period of 1 year. Meprobamate appeared as a drug which still has a significant frequency (5.5%) and is the most frequently involved in fatal pharmaceutical overdoses (15.3%). Post mortem concentrations ranged from 41 to 397 mg/l (mean = 182) and are compared to those of the literature.

We have developed a new solid-phase extraction (SPE) on C18 cartridges which allows a very simple protocol of manipulation and a single elution of opiates and cocainics from human hair samples. The method involved decontamination in a phosphate buffer and dichloromethane, pulverization in a ball mill, addition of deuterated internal standards, heated acid hydrolysis and SPE. Quantitation utilized gas chromatography and mass spectrometry. Between days precise study gave relative standard deviations always inferior to 8.9% for each compound at 4 ng/mg (except methylecgonine ester = 15.7%). Accuracy was tested using a t-statistic versus a reference material from the NIST. Limits of detection were calculated from an analysis of the blanks which contained between 0.12 and 0.28 ng/mg for each drug. The method was applied in forensic cases for 1 year of toxicological activity. Among the 108 analyses performed, 30 were positive for cocaine and 33 for opiates. Concentrations were in the range 0.9-242.0 ng/mg (cocaine), 0.3-71.3 ng/mg (benzoylecgonine), 0.0-9.8 ng/mg (methylecgonine ester), 0.0-2.9 ng/mg (cocaethylene), 0.1-11.5 ng/mg (codeine), 0.4-44.6 ng/mg (morphine) and 0.7-131.2 ng/mg (6-acetylmorphine). Ratios of the metabolites to parent drugs were proposed to avoid risk of external contamination.

A method is described to screen for a wide range of pharmaceuticals in human hair. 75 mg of powdered hair are incubated (12 h at +56 degrees C) in 2 ml of distilled water (acidic compounds) or 0.1 M hydrochloric acid (neutral and basic compounds). A twin solid-phase extraction on C18 cartridges is used for the sample clean-up procedure. Acidic drugs are fixed at pH 2 and eluted with 1% ammoniacal methanol while neutral and basic drugs are retained on the column at pH 8.6 and eluted with methanol containing 0.5% acetic acid. The internal standard (I.S.) for the acidic extraction was bupivacaine while the I.S. for the basic extraction was prazepam. The separation of the drugs was performed using both the liquid and the gas chromatographic techniques whereas identification was achieved using photodiode array and mass spectrometric detection, respectively. The liquid chromatographic system gives an elution of the drugs following a multi step gradient from a Symmetry C8 (Waters) 5 microns column (250 x 4.6 mm I.D.) at +30 degrees C with acetonitrile-phosphate buffer (pH 3.8). Identification is achieved using the reference data (retention times and spectra) of 675 pharmaceuticals, toxicants and drugs of abuse stored in a personal library. The present method has been applied during 6 months in our laboratory. By establishing a victim's drug use history, it is a very powerful tool in forensic medicine. We illustrate the method with some real cases of police crime investigation.