Amphetamines in Workplace Testing Hair Samples over the Years 2011-2020.

Amphetamines (AMPs) in hair were investigated with thousands of workplace testing head and body hair samples collected and analyzed over 10 years and tabulated by year. All samples were washed by a published extensive method prior to confirmation by liquid chromatography-mass spectrometry-mass spectrometry. Presented are concentrations of parent methamphetamine (METH), 3,4-methylenedioxymethamphetamine (MDMA) and methylenedioxyamphetamine as metabolite and AMP as metabolite and without the presence of parent drug. Some differences in METH concentrations from year to year were significant, and some ratios of metabolite to parent drug for both METH and MDMA also varied significantly. While rates of METH use may not have changed significantly, some aspects of the drugs ingested as demonstrated by hair analysis varied over the 10-year period.

Identifying contamination versus ingestion in hair testing, with cocaine as example.

When developing a procedure to identify external contamination of hair as opposed to drug that is in hair from ingestion, there are components of the process that must be considered in the final method. A method that does not achieve the objective may be missing one or more of these elements: choice of solvent, a drug-binding agent, ratio of solvent to hair, temperature, time, intactness of the hair, and establishing, for the chosen method, a criterion based on the drug contents of the wash and hair that indicates the hair may be contaminated.

Cannabinoids Tetrahydrocannabinol, Cannabinol, Cannabidiol, Tetrahydrocannabivarin and 11-nor-9-carboxy-∆9-THC in Hair.

The cannabinoids tetrahydrocannabinol (THC), tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabinol (CBN) and (-)-11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH) were determined in 4,773 hair samples. Confirmation of THC-COOH was by GC-MS-MS (gas chromatography--mass spectrometry-mass spectrometry). Confirmation of THC, THCV, CBN and CBD was by LC-MS-MS (liquid chromatoraphy--mass spectrometry-mass spectrometry) on an AB Sciex QTRAP 6500+ LC-MS-MS. The purpose of this work was not to utilize any analyte other than THC-COOH as indicative of ingestion, but to assess the absence or presence, and relative concentrations, of the other cannabinoid analytes in hair of marijuana users vs. primarily CBD users. In this regard, 10% of samples contained significantly higher concentrations of CBD relative to THC than the other 90%. A concentration of CBD that is five times greater than that of THC was proposed as good evidence of primarily CBD ingestion. THC concentrations in the samples ranged from below the limit of detection (5 pg/mg) to 47,808 pg/mg hair, varying widely in the relationship between parent THC and the metabolite THC-COOH. CBN was present in most samples, but concentrations relative to THC decreased with increasing THC concentrations. Only 26% of the samples contained THCV detectable by the method. When present, THCV concentrations averaged 1.77% of THC. A limitation of this study is the lack of subject histories to determine the types and amounts of products used and the mode of ingestion. Also, not all THC from external contamination may be removed. Nonetheless, the data provide a useful guide as to what cannabinoids may be found in hair, at what concentrations and under conditions of marijuana use vs. likely primarily CBD use.

Hydroxycocaines as Metabolic Indicators of Cocaine Ingestion.

Hydroxycocaines in hair were investigated with many hundreds of head and body hair samples. All samples were washed by a published extensive aqueous method prior to confirmation by LC-MS/MS. Concentrations, percent of cocaine, and ratios of para- and meta-hydroxycocaines to ortho-hydroxycocaine are presented. Hydroxycocaines as percent of cocaine did not appear to be affected by cocaine concentrations, but were shown to increase with cocaethylene concentrations. Stability of hydroxycocaines over a year of ambient storage was demonstrated. Ortho-hydroxycocaine was shown to be formed by exposure of cocaine-positive hair to peroxide, while para- and meta-hydroxycocaines were not. Presence of para- or meta-hydroxycocaine at > 0.05% of cocaine is proposed as indicating ingestion of cocaine. This indicator prevents black hair from being more likely interpreted as positive for ingestion than lighter colored hair.

A Retrospective Analysis of Selected Opioids in Hair of Workplace Drug Testing Subjects.

Opioids, both naturally occurring and semisynthetic, are effective pain management medications, but also possess the potential for abuse. Analyses of over 37,000 head and body hair samples containing codeine, morphine, hydrocodone, hydromorphone, oxycodone or oxymorphone provide a view of use habits of workplace-testing subjects that cannot be obtained from fluid matrices results. Testing was performed using FDA cleared immunoassays using either 2 ng morphine or oxycodone per 10 mg hair as calibrators. Non-negative screening samples were washed with an extended aqueous wash procedure followed by LC-MS-MS confirmation at a cutoff concentration of 2 ng opioid per 10 mg hair. The LC-MS-MS method measured codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone with an administratively established LOQ of 0.50 ng opioid per 10 mg hair. The linear range was 0.50-100 ng morphine per 10 mg hair, and 0.50-150 ng opioid per 10 mg hair for all other measured analytes. For all analytes, within run precision was ≤5.4%, and between-run precision was ≤6.4%. Analysis of samples containing metabolites found that, among codeine positive samples, 97% contained less than 10% morphine metabolite and 88% less than 20% hydrocodone metabolite, among hydrocodone positive samples, 97% contained less than 10% hydromorphone metabolite and 95% of oxycodone positive samples contained less than 10% oxymorphone metabolite. Our analysis of opioid-positive samples may provide guidelines for interpretation of hair opioid levels typically observed in workplace testing.

Nail Analysis for Drugs: A Role in Workplace Testing?

Analysis of nail clippings may be a useful back-up for hair analysis when hair is unavailable. One aspect of using nails or hair is the ability to analyze whether drug present is from ingestion or from contamination. A common method of three 15-s rinses in methanol failed to remove drug from nails that had been soaked in either 5 or 50 µg/mL cocaine, methamphetamine or morphine for 1 h. While methanol rinsing did not remove contaminating drug, washing the nails soaked with 5 and 50 µg/mL of these drugs with an extended wash, a method developed for hair analysis and consisting of a 15-min isopropanol wash, and three 30-min and two 60-min phosphate buffer-0.1% albumin washes, when applied to nails did remove most of the contaminating drug. The drug left in the nails after extended washing could be interpreted as contamination by applying a wash criterion that is routinely applied in hair analysis. Successful decontamination of the soaked contaminated nail model was followed by applying this extended wash method to presumptive positive nail samples identified in workplace testing. While the extended buffer wash and wash criterion distinguish contamination from ingestion with hair, we failed to demonstrate that the method effectively differentiates contamination from ingestion with nails.

Inhibitor-induced structural change in the HCV IRES domain IIa RNA.

Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90 degrees helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa construct in complex with a potent small-molecule inhibitor of HCV replication. Molecular dynamics refinement in explicit solvent and subsequent energetic analysis indicated that each inhibitor stereoisomer bound with comparable affinity and in an equivalent binding mode. The in silico analysis was substantiated by fluorescence-based assays showing that the relative binding free energies differed by only 0.7 kcal/mol. Binding of the inhibitor displaces key nucleotide residues within the bulge region, effecting a major conformational change that eliminates the bent RNA helical trajectory, providing a mechanism for the antiviral activity of this inhibitor class.