Validation of two methods for the quantitative analysis of cocaine and opioids in biological matrices using LCMSMS.

The proliferation of misuse of prescription and non-prescription opioids, in recent years, has caused an opioid epidemic in the United States. Forensic toxicology laboratories often encounter implications of abuse in both driving under the influence of drugs and death investigation cases. The Virginia Department of Forensic Science has seen over a 190% increase in the number of reported opioids in death investigation cases over the past several years. Traditionally, analyses are completed by individual drug class, which subsequently requires an individual case to be evaluated using multiple analytical techniques for comprehensive analysis. To ease the impact of increasing case submissions and case complexity, two liquid chromatography-tandem mass spectrometry (LCMSMS) methods for the quantitative analysis of opioids, cocaine, and cocaine metabolites in biological matrices have undergone a fit-for-purpose validation. The methods were compared to determine the advantages and disadvantages of each analytical technique. Two sample preparation techniques, protein precipitation and solid-phase extraction, were employed for quantitative validation. Validation aspects evaluated included accuracy and precision, sensitivity, linearity, matrix effects, recovery, carryover, interferences, dilution integrity, and post-extraction stability. The accuracy of the protein precipitation method ranged from 80 ± 3% to 101 ± 10%. The accuracy of the solid-phase extraction method ranged from 88 ± 9% to 119 ± 3%. Additionally, within-run and intermediate precision was within ±20% for both extraction techniques. Although ionization suppression was noted, it was determined to have minimal effect on the methods. The newly developed methods require less sample volume and combine four analytical techniques into one method, which significantly impacts laboratory productivity.

PCB congener specific oxidative stress response by microarray analysis using human liver cell line.

In this study we have examined the effect of exposure to different congeners of PCBs and their role in oxidative stress response. A metabolically competent human liver cell line (HepG2) was exposed with two prototype congeners of PCBs: coplanar PCB-77 and non-coplanar PCB-153. After the predetermined times of exposure (0-24h) at 70 µM concentration, the HepG2 cells showed significant apoptotic changes by fluorescent microscopy after 12h of exposure. Gene set enrichment analysis (GSEA) identified oxidative stress as the predominant enrichment. Further, paraquat assay showed that PCB congeners lead to oxidative stress to different extents, PCB-77 being more toxic. This study, with emphasis on all recommended microarray quality control steps, showed that apoptosis was one of the most significant cellular processes as a result of oxidative stress, but each of these congeners had a unique signature gene expression, which was further validated by Taqman real time PCR and immunoblotting. The pathways involved leading to the common apoptotic effect were completely different. Further in-silico analysis showed that PCB-153 most likely acted through the TNF receptor, leading to oxidative stress involving metallothionein gene families, and causing apoptosis mainly by the Fas receptor signaling pathway. In contrast, PCB-77 acted through the aryl hydrocarbon receptor. It induced oxidative stress through the involvement of cytochrome P450 (CYP1A1) leading to apoptosis through AHR/ARNT pathway.

Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Jordan.

Molecular screening for glucose-6-phosphate (G6PD) mutations in two Jordanian populations revealed six different mutations and higher incidences of G6PD deficiency and G6PD A- (376A-->G + 202G-->A) mutation in Jordan Valley than in the Amman area. These observations may be explained by historically higher rates of malaria and African ancestral origins, respectively.

Olfactory biopsies demonstrate a defect in neuronal development in Rett's syndrome.

Rett's Syndrome (RTT) is a neurodevelopmental disorder resulting from mutation in the mecp2 gene that encodes methyl CpG binding protein 2, a transcriptional repressor. Because this disease primarily affects neurons, tissue is not available during active disease. We used the olfactory system as a model to investigate abnormalities in neuronal development in RTT, because olfactory receptor neurons (ORNs) are replaced throughout life by ongoing postnatal neurogenesis. Thus, even in the adult, the olfactory epithelium contains neurons at various developmental stages. We obtained biopsies of nasal epithelium containing ORNs from RTT patients and age-matched controls to study the status of the neuronal population using antibodies to stage-specific developmental markers. There were no postprocedure complications. Compared with age-matched controls, there were far fewer mature ORNs, as defined by olfactory marker protein expression, and significantly greater numbers of immature neuron-specific tubulin-positive ORNs present. In RTT biopsies, olfactory marker protein-positive neurons displayed abnormal structure. These results suggest that dysfunction of MeCP2 results in decreased survival of mature ORNs with a compensatory increase in neurogenesis, or a failure of immature neurons to mature. Our study indicates that olfactory biopsies provide a method to study neuronal developmental diseases in adults and children.

Rapid genotyping of common MeCP2 mutations with an electronic DNA microchip using serial differential hybridization.

Rett syndrome is a neurodevelopmental disorder that affects females almost exclusively, and in which eight common point mutations on the X-linked MeCP2 gene are knows to cause over 70% of mutation-positive cases. We explored the use of a novel platform to detect the eight common mutations in Rett syndrome patients to expedite and simplify the process of identification of known genotypes. The Nanogen workstation consists of a two-color assay based on electric hybridization and thermal discrimination, all performed on an electronically active NanoChip. This genotyping platform was tested on 362 samples of a pre-determined genotype, which had been previously identified by a combination of DHPLC (denaturing high performance liquid chromatography) and direct sequencing. This genotyping technique proved to be rapid, facile, and displayed a specificity of 100% with 3% ambiguity. In addition, we present consecutive testing of seven mutations on a single pad of the NanoChip. This was accomplished by tagging down two amplimers together and serially hybridizing for seven different loci, allowing us to genotype samples for seven of the eight common Rett mutations on a single pad. This novel method displayed the same level of specificity and accuracy as the single amplimer reactions, and proved to be faster and more economical.