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.

Anticipation and consumption of food each increase the concentration of neuroactive steroids in rat brain and plasma.

Stressful stimuli and anxiogenic drugs increase the plasma and brain concentrations of neuroactive steroids. Moreover, in rats trained to consume their daily meal during a fixed period, the anticipation of food is associated with changes in the function of various neurotransmitter systems. We have now evaluated the effects of anticipation and consumption of food in such trained rats on the plasma and brain concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH DOC), two potent endogenous positive modulators of type A receptors for gamma-aminobutyric acid (GABA). The abundance of these neuroactive steroids was increased in both the cerebral cortex and plasma of the rats during both food anticipation and consumption. In contrast, the concentration of their precursor, progesterone, was increased in the brain only during food consumption, whereas it was increased in plasma only during food anticipation. Intraperitoneal administration of the selective agonist abecarnil (0.1 mg/kg) 40 min before food presentation prevented the increase in the brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC during food anticipation but not that associated with consumption. The change in emotional state associated with food anticipation may thus result in an increase in the plasma and brain levels of 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC in a manner sensitive to the activation of GABA(A) receptor-mediated neurotransmission. A different mechanism, insensitive to activation of such transmission, may underlie the changes in the concentrations of these neuroactive steroids during food consumption.

Social isolation-induced increase in alpha and delta subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABA receptor function.

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3alpha,5alpha-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the alpha(4) and delta subunits of the GABA(A) receptor in the hippocampus were increased in isolated rats as were GABA(A) receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABA(A) receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABA(A) receptor function and associated behaviour.

Structure-activity relationships and effects on neuroactive steroid synthesis in a series of 2-phenylimidazo[1,2-a]pyridineacetamide peripheral benzodiazepine receptors ligands.

A series of 36 imidazopyridineacetamides (2-37) were designed and synthesized to evaluate the effects of structural changes on the amide nitrogen at both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include variations in the length and number of the alkyl groups as well as introduction of different aromatic, heteroaromatic, and conformationally constrained groups. The affinities of these compounds for CBRs and PBRs were determined, and the results indicate that bulkiness of the substituents, their branching, and length beyond an optimal value may cause hindrance to the ligand in its interaction with the receptor. The presence of aromatic or conformationally constrained substituents on the carboxamide nitrogen can be conducive to high affinity and selectivity. Furthermore, the ability of a subset of the most active ligands to stimulate synthesis of neuroactive steroids in plasma and brain was evaluated in vivo and in vitro. Compound 3 exhibited very marked effects on the peripheral and central synthesis of neuroactive steroids, while 36 (potent at subnanomolar level) showed a slight ability to affect neuroactive steroid content in the cerebral cortex.

Increased expression of the gene for the Y1 receptor of neuropeptide Y in the amygdala and paraventricular nucleus of Y1R/LacZ transgenic mice in response to restraint stress.

A sustained increase in the brain concentrations of neuroactive steroids was previously shown to induce Y1 receptor gene expression in the amygdala of Y1R/LacZ transgenic mice which harbour a construct comprising the murine Y1 receptor gene promoter and the lacZ reporter gene. We now investigated the effects of restraint stress on both the cerebrocortical concentrations of neuroactive steroids and Y1 receptor gene expression in the amygdala and hypothalamic paraventricular nucleus (PVN) of Y1R/LacZ transgenic mice. The cerebrocortical concentrations of allopregnanolone and allotetrahydrodeoxycorticosterone were significantly increased immediately after a 1-h exposure to restraint stress and had returned to control values within 30 min. Expression of Y1R/LacZ was increased in the amygdala and PVN 6 h after restraint. The 5alpha-reductase inhibitor finasteride, that prevented the increase in neuroactive steroid concentrations, did not block that in transgene expression induced by 1-h restraint. Daily exposure to restraint for 10 days also increased the cerebrocortical concentrations of neuroactive steroids but failed to affect transgene expression. Acute but not repeated restraint thus increases Y1 receptor gene expression in the amygdala and PVN, suggesting that tolerance develops towards this stressor. The effect of acute restraint is not mediated by the increase in the brain concentrations of neuroactive steroids but may rather reflect a ligand-induced increase in Y1 receptor gene transcription. Data support a role of Y1 receptors in the behavioural and neuroendocrine responses to stress.

Social isolation increases the response of peripheral benzodiazepine receptors in the rat.

Social isolation of rats for 30 days immediately after weaning reduces the cerebrocortical and plasma concentrations of progesterone, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC). The percentage increases in the brain and plasma concentrations of these neuroactive steroids apparent 30 min after intraperitoneal injection of the peripheral benzodiazepine receptor (PBR) ligand CB 34 (25 mg/kg) have now been shown to be markedly greater in isolated rats than in group-housed controls. The CB 34-induced increase in the abundance of 3alpha,5alpha-TH PROG was more pronounced in the brain than in the plasma of isolated rats. Analysis of [3H]PK 11195 binding to membranes prepared from the cerebral cortex, adrenals, or testis revealed no significant difference in either the maximal number of binding sites for this PBR ligand or its dissociation constant between isolated and group-housed animals. Social isolation also induced a small but significant decrease in the plasma concentration of adrenocorticotropic hormone. Moreover, CB 34 increased the plasma concentration of this hormone to a greater extent in isolated rats than in group-housed animals. The persistent decrease in the concentrations of neuroactive steroids induced by social isolation might thus be due to an adaptive decrease in the activity either of the hypothalamic-pituitary-adrenal axis or of PBRs during the prolonged stress, reflecting a defense mechanism to limit glucocorticoid production. The larger increase in neuroactive steroid concentrations induced by CB 34 and the enhanced pituitary response to this compound in isolated rats indicate that this mild stressor increases the response of PBRs.

Social isolation-induced increase in the sensitivity of rats to the steroidogenic effect of ethanol.

Social isolation of rats for 30 days immediately after weaning results in marked decreases in the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC), as well as a moderate increase in the plasma concentration of corticosterone. This mildly stressful condition has now been shown to increase the sensitivity of rats to the effect of acute ethanol administration on the cerebrocortical and plasma concentrations of neuroactive steroids. The percentage increases in the brain and plasma concentrations of pregnenolone, progesterone, 3alpha,5alpha-TH PROG, and 3alpha,5alpha-TH DOC, apparent 20 min after a single intraperitoneal injection of ethanol (1 g/kg), were thus markedly greater in isolated rats than in group-housed animals. A subcutaneous injection of isoniazid (300 mg/kg) also induced greater percentage increases in the concentrations of these steroids in isolated rats than in group-housed animals. These results suggest that mild chronic stress, such as that induced by social isolation, enhances the steroidogenic effect of ethanol, a drug abused by humans under stress or affected by neuropsychiatric disorders. Social isolation also induced hyper-responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis, as was apparent after reduction of GABA-mediated inhibitory tone by isoniazid administration.