Determination of cocaine and metabolites in hair by column-switching LC-MS-MS analysis.

A method for rapid, selective, and robust determination of cocaine (CO) and metabolites in 5-mg hair samples was developed and fully validated using a column-switching liquid chromatography-tandem mass spectrometry system (LC-MS-MS). Hair samples were decontaminated, segmented, incubated overnight in diluted HCl, and centrifuged, and the diluted (1:10 with distilled water) extracts were analyzed in positive ionization mode monitoring two reactions per analyte. Quantifier transitions were: m/z 304.2→182.2 for CO, m/z 290.1→168.1 for benzoylecgonine (BE), and m/z 318.2→196.2 for cocaethylene (CE). The lower limit of quantification (LLOQ) was set at 0.05 ng/mg for CO and CE, and 0.012 ng/mg for BE. Imprecision and inaccuracy at LLOQ were lower than 20 % for all analytes. Linearity ranged between 0.05 and 50.0 ng/mg for CO and CE and 0.012 and 12.50 ng/mg for BE. Selectivity, matrix effect, process efficiency, recovery, carryover, cross talk, and autosampler stability were also evaluated during validation. Eighteen real hair samples and five samples from a commercial proficiency testing program were comparatively examined with the proposed multidimensional chromatography coupled with tandem mass spectrometry procedure and our reference gas chromatography coupled to mass spectrometry (GC-MS) method. Compared with our reference GC-MS method, column-switching technique and the high sensitivity of the tandem mass spectrometry detection system allowed to significantly reduce sample amount (×10) with increased sensitivity (×2) and sample throughput (×4), to simplify sample preparation, and to avoid that interfering compounds and ions impaired the ionization and detection of the analytes and deteriorate the performance of the ion source.

Rapid and robust confirmation and quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine by column switching LC-MS-MS analysis.

A method for the rapid and robust confirmation of 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (THCA) in urine involving basic hydrolysis with NaOH and direct injection of the hydrolysate in a column-switching LC-MS-MS system was developed and validated. THCA-d3 was used as internal standard. Detection was performed in negative-ion mode by monitoring the transitions from the [M-CO(2) ]- ion m/z 299.2→245.2 and and m/z 299.2→191.1 that were found to provide a better signal-to-noise ratio than the transition from the pseudomolecular ion at m/z 343. The high sensitivity of detection enabled the injection of a small volume (10 µl) of the NaOH hydrolysate which, together with the applied column switching system, proved to confer ruggedness to the method and to avoid the deterioration of the instrumental apparatus despite the large amount of inorganic ions in the hydrolysate. The LLOQ was established at 5 ng/ml, and the LLOD was calculated as 0.2 ng/ml (S/N =3). The method was submitted to thorough validation including evaluation of the calibration range (5-500 ng/ml), accuracy and precision, matrix effects, overall process efficiency, autosampler stability, carryover and cross-talk, and 10-times reduction of sample volume (0.1 ml). Proof of applicability was obtained by direct comparison with the reference GC-MS method in use in the lab (the R(2) between the two methods was 0.9951).

Prevalence of drug abuse among workers: strengths and pitfalls of the recent Italian Workplace Drug Testing (WDT) legislation.

BACKGROUND: In 2008 a Workplace Drug Testing (WDT) law became effective in Italy for workers involved in public/private transportation, oil/gas companies, and explosives/fireworks industry with the aim to ensure public safety for the community. AIMS: To examine and elaborate WDT data collected on a large group of workers (over 43,500) during March 2009-February 2010 in order to highlight pros and cons and to draw suggestions for policies in the field. SETTING: Northern Italy. METHODS: After ≤ 24 h notification, workers provided a urine sample screened for opiates, methadone, buprenorphine, cocaine, amphetamines, ecstasy, and cannabinoids (THC) by immunoassay. Positives were confirmed by GC-MS. RESULTS: The positive rate was 2.0%, THC being most frequent drug (1.3%; cocaine, 0.4%; opioids, 0.3%). 6.9% of the positive workers tested positive for ≥ 2 classes (most often THC+cocaine). Gender ratio and mean age were significantly lower in positives (F/M=0.007; 35.5 ± 8.3 years) than negatives (0.016 and 40.7 ± 9.5, respectively). No decline in rates of positives and an increase of diluted samples over time were observed. The highest rates of positives were detected when sampling was performed just before/after week-end and during morning hours. Possible correlation between job type and drugs used were observed (e.g. more cocaine positives among road vehicle-drivers than among lift truck-drivers). Declared use of medicine/illicit drugs during the preceding week showed that illicit drug use was likely not always detected in urine and that almost 4% workers declared use of medicine drugs possibly affecting performance. CONCLUSIONS: This survey enabled to evidence relevant pitfalls of the law and to define strategies to improve the outcomes of WDT policies.

Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures.

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.