Comprehensive analysis of synthetic cannabinoids and metabolites in oral fluid by online solid-phase extraction coupled to liquid chromatography-triple quadrupole-mass spectrometry.

The class of novel psychoactive substances known as synthetic cannabinoids (SC) includes illicit compounds that are sprayed on plant material and smoked or sold as liquids to be vaporized in e-cigarettes. In toxicological analysis of SC, fast analytical methods are needed for the detection and confirmation of parent drugs and metabolites at very low levels. While various analytical methods have been developed for SC in blood and urine, few are available for alternative matrices such as oral fluid (OF). There are numerous advantages to using OF as a sample matrix for SC analysis, including non-invasive collection, lesser risk of adulteration, and presence of both parent drug and metabolites. Here we report a validated online solid-phase extraction (online SPE) method coupled to LC-QqQ-MS for rapid confirmation and quantitation of 72 structurally diverse SC parent drugs and metabolites in OF with 2.5 min of preconcentration time and a total elution time of < 10 min. The use of online SPE for sample pretreatment facilitates rapid and consistent processing and greatly increases sample throughput. The method was fully validated according to relevant guidelines (ANSI/ASB Standard 036). Bias and precision values were within ± 20% for all compounds in human OF matrix. Method detection and quantitation limits ranged from 0.4 to 3.8 ng/mL and from 1.1 to 11.6 ng/mL, respectively. Recovery, matrix effects, process efficiency, carryover, and stability were also within acceptable limits for the majority of compounds. Successful application of the method was demonstrated using blank human OF fortified with SC in addition to a set of authentic OF specimens previously tested by another laboratory. Graphical abstract.

Rapid quantitative analysis of methylphenidate and ritalinic acid in oral fluid by liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS).

Methylphenidate (MPH), which is metabolized into ritalinic acid (RA), is an amphetamine derivative largely used in the treatment of attention-deficit hyperactivity disorder, a neurological condition commonly diagnosed in early childhood. Ensuring that patients comply with clinical treatment is crucial and compliance is generally monitored in blood or urine specimens which, especially in the case of children, can be challenging to obtain on a repetitive basis. Here we report validation of a specific, non-invasive, and rapid dilute-and-shoot analytical method for the detection and quantitation of MPH and RA in oral fluid (OF). The method is based on liquid chromatography coupled to triple quadrupole MS with electrospray ionization utilizing dynamic MRM mode. Subject OF specimens were collected using a Quantisal™ device, processed, and diluted for analysis with seven-point quadratic calibration curves (weighting of 1/x) using MPH-d9 and (±)-threo-RA-d10 as internal standards. QC samples and diluted specimens showed intra- and inter-day bias and imprecision values no greater than ±12%. The LOD and LOQ for MPH were 0.1 and 0.5 ng/mL, respectively, and 0.2 ng/mL and 0.5 ng/mL for RA, respectively, indicating the validity of the method for identification and confirmation at low concentrations. Selectivity was specific for the analytes of interest and matrix effects were minimized through the use of internal standard based quantitation.

Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters.

AIMS: Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction. RESULTS: In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGE2) in plasma when compared with either HIV or alcohol alone. INNOVATION: This article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users. CONCLUSION: These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to immune toxicity, and further exacerbation with alcohol use. Antioxid. Redox Signal. 28, 324-337.

HIV-1 subtypes B and C Tat differentially impact synaptic plasticity expression and implicates HIV-associated neurocognitive disorders.

Earlier studies have established that infection with HIV-1 subtypes (clades) might differentially influence the neuropathogenesis of HIV-1-associated neurocognitive dysfunction (HAND). HIV-1 Trans activator of transcription protein (Tat) is of considerable significance and plays a major role in the central nervous system (CNS) dysfunction. However, these HIV-1 clades exert diverse cellular effects that leads to neuropathogenic dysfunction has not been well established. We hypothesized that the HIV-1 clade B and clade C Tat proteins effect synaptic plasticity expression in neuroblastoma cells (SK-N-MC) by diverse methods, and accordingly modulates the development of HAND. In the present study, we have analyzed important and highly expressed 84 key human synaptic plasticity genes expression which differentially impact in clade B and clade C Tat treated SK-N-MC cells using RT(2) Profile PCR Array human Synaptic Plasticity kit. Observed results demonstrate that out of 84 key synaptic plasticity genes, 36 and 25 synaptic genes were substantially (≥3 fold) up-regulated and 5 and 5 genes considerably (≥3 fold) down-regulated in clade B and clade C Tat treated cells, respectively, compared to the control SK-N-MC. We have also estimated the levels of glutamine and glutamate in HIV-1 clade B and C Tat exposed SK-N-MC cells compared to untreated cells. Our results indicate that levels of glutamate, glutamine and expression of synaptic plasticity genes were highly dysregulated by HIV-1 clade B Tat compared to clade C Tat in SK-N-MC cells. In summary, this study suggests that clade B Tat substantially potentiates neuronal toxicity and further dysregulated synaptic plasticity genes in SK-N-MC may contribute to the severe neuropathogenesis linked with HAND.