Fentanyl as a marker of illicit drug use in morphine-positive urine specimens from workplace drug testing.

Total morphine is an important urinary marker of heroin use but can also be present from prescriptions or poppy seed ingestion. In specimens with morphine concentrations consistent with poppy seed ingestion (<4,000 ng/mL), 6-acetylmorphine has served as an important marker of illicit drug use. However, as illicit fentanyl has become increasingly prevalent as a contaminant in the drug supply, fentanyl might be an alternative marker of illicit opioid use instead of or in combination with 6-acetylmorphine. The aim of this study was to quantify opiates, 6-acetylmorphine, fentanyl and fentanyl analogs in 504 morphine-positive (immunoassay 2,000 ng/mL cutoff) urine specimens from workplace drug testing. Almost half (43%) of morphine-positive specimens had morphine concentrations below 4,000 ng/mL, illustrating the need for markers to differentiate illicit drug use. In these specimens, fentanyl (22% co-positivity) was more prevalent than 6-acetylmorphine (12%). Co-positivity of 6-acetylmorphine and semi-synthetic opioids increased with morphine concentration, while fentanyl prevalence did not. In 110 fentanyl-positive specimens, the median norfentanyl concentration (1,520 ng/mL) was 9.6× higher than the median fentanyl concentration (159 ng/mL), illustrating the possibility of using norfentanyl as a urinary marker of fentanyl use. The only fentanyl analog identified was para-fluorofentanyl (n = 50), with results from most specimens consistent with para-fluorofentanyl contamination in illicit fentanyl. The results confirm the use of fentanyl by employees subject to workplace drug testing and highlight the potential of fentanyl and/or norfentanyl as important markers of illicit drug use.

Prevalence of ∆8-tetrahydrocannabinol carboxylic acid in workplace drug testing.

∆8-Tetrahydrocannabinol (∆8-THC) recently became widely available as an alternative to cannabis. ∆8-THC is likely impairing and poses a threat to workplace and traffic safety. In the present study, the prevalence of ∆8-THC in workplace drug testing was investigated by analyzing 1,504 urine specimens with a positive immunoassay cannabinoid initial test using a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method quantifying 15 cannabinoid analytes after hydrolysis. ∆8-tetrahydrocannabinol-9-carboxylic acid (∆8-THC-COOH) was detected in 378 urine specimens (15 ng/mL cutoff), compared to 1,144 specimens containing ∆9-THC-COOH. The data could be divided into three general groups. There were 964 (76%) ∆9-THC-COOH-dominant (<10% ∆8-THC-COOH) and 139 (11%) ∆8-THC-COOH-dominant (>90% ∆8-THC-COOH) specimens, with the remaining 164 (13%) specimens showing a mixture of both analytes (>90% ∆8-THC-COOH). Similar concentrations of ∆9-THC-COOH (median 187 ng/mL) and ∆8-THC-COOH (150 ng/mL) as the dominant species support the use of similar cutoffs and decision rules for both analytes. Apart from the carboxylic acid metabolites, 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-∆9-THC, n = 1,282), ∆9-tetrahydrocannabivarin-9-carboxylic acid (∆9-THCV-COOH, n = 1,058), ∆9-THC (n = 746) and 7-hydroxy-cannabidiol (7-OH-CBD, n = 506) were the most prevalent analytes. Two specimens (0.13%) contained ≥140 ng/mL ∆9-THC without ∆9-THC-COOH, which could be due to genetic variability in the drug-metabolizing enzyme CYP2C9 or an adulterant targeting ∆9-THC-COOH. The cannabinoid immunoassay was repeated, and five specimens (0.33%) generated negative initial tests despite ∆9-THC-COOH concentrations of 54-1,000 ng/mL, potentially indicative of adulteration. The use of ∆8-THC is widespread in the US population, and all forensic laboratories should consider adding ∆8-THC and/or ∆8-THC-COOH to their scope of testing. Similar urinary concentrations were observed for both analytes, indicating that the decision rules used for ∆9-THC-COOH are also appropriate for ∆8-THC-COOH.

∆8-THC-COOH cross-reactivity with cannabinoid immunoassay kits and interference in chromatographic testing methods.

Because of structural similarities, the presence of 11-Nor-9-carboxy-∆8-tetrahydrocannabinol (∆8-THC-COOH) in a urine specimen might interfere with testing for 11-Nor-9-carboxy-∆9-tetrahydrocannabinol (∆9-THC-COOH). A set of samples containing ∆8-THC-COOH with concentrations ranging from 10 to 120 ng/mL were tested at cut-offs of 20, 50 and 100 ng/mL using cannabinoid immunoassay reagents from three different manufacturers. Cross-reactivities ranged from 87% to 112% for ∆8-THC-COOH at the cut-off of 50 ng/mL for the three different platforms. Additionally, samples containing both ∆8-THC-COOH and ∆9-THC-COOH were fortified by the National Laboratory Certification Program (NLCP). U.S. Department of Health and Human Services (HHS)-Certified Laboratories tested the samples to determine the interference of ∆8-THC-COOH on confirmatory tests commonly used in workplace drug testing laboratories for the confirmation and quantification of ∆9-THC-COOH. When evaluating confirmation and quantification of ∆9-THC-COOH in the presence of ∆8-THC-COOH, unreportable results for ∆9-THC-COOH were observed because of chromatographic interference or mass ratio failures. However, there were no false-positive ∆9-THC-COOH reports from any HHS-certified laboratory.

Conversion of water-soluble CBD to ∆9-THC in synthetic gastric fluid-An unlikely cause of positive drug tests.

Cannabidiol (CBD) has been shown to convert to ∆9-tetrahydrocannabinol (∆9-THC) in acidic environments, raising a concern of conversion when exposed to gastric fluid after consumption. Using synthetic gastric fluid (SGF), it has been demonstrated that the conversion requires surfactants, such as sodium dodecyl sulfate (SDS), due to limited solubility of CBD. Recently, water-compatible nanoemulsions of CBD have been prepared as a means of fortifying beverages and water-based foods with CBD. Since these emulsions contain surfactants as part of their formulation, it is possible that these preparations might enhance the production of ∆9-THC even in the absence of added surfactants. Three THC-free CBD products, an oil, an anhydrous powder and a water-soluble formulation, were incubated for 3 h in SGF without SDS. The water-soluble CBD product produced a dispersion, while the powder and the oil did not mix with the SGF. No THC was detected with the CBD oil (<0.0006% conversion), and up to 0.063% and 0.0045% conversion to ∆9-THC was observed with the water-soluble CBD and the CBD powder, respectively. No formation of ∆8-THC was observed. In comparison, when the nano-formulated CBD was incubated in SGF with 1% SDS, 33-36% conversion to ∆9-THC was observed. Even though the rate of conversion with the water-soluble CBD was at least 100-fold higher compared to the CBD oil, it was still smaller than ∆9-THC levels reported in CBD products labeled "THC-free" or "<0.3% THC" based on the Agricultural Improvement Act of 2018 (the Farm Bill). Assuming a daily CBD dose of around 30 mg/day, it is unlikely that conversion of CBD to ∆9-THC could produce a positive urinary drug test for 11-Nor-9-carboxy-∆9-THC (15 ng/mL cut-off).

A pilot PT scheme for external assessment of laboratory performance in testing synthetic opioid compounds in urine, plasma, and whole blood.

A proficiency testing (PT) scheme was prepared for laboratories engaged in bioanalytical testing for synthetic opioid compounds in urine, plasma, and whole blood. Samples were prepared using compounds included in the Opioid Certified Reference Material Kit (Opioid CRM Kit) developed by the U.S. Centers for Disease Control and Prevention. Laboratories received samples during a 2-year project with each year consisting of two PT events 6 months apart. In the first year (pilot test), participants included 10 public health laboratories throughout the United States. In the second year, the group of laboratories expanded to include clinical and forensic drug testing laboratories, and 12 additional participating labs joined the program. In Year 1, overall detection percentages for the compounds present in the PT samples were 95.5% in Event 1% and 97.2% in Event 2. There were 31 apparent false positives reported in Event 1 and four apparent false positives reported in Event 2. Carryover or contamination in laboratory analytical systems were found to be the most significant causes of the false positive results, and none of the laboratories that reported false positives in Event 1 did so in Event 2. In Year 2, overall detection percentages for the compounds present in the PT samples were 89.5% in Event 3% and 94.8% in Event 4. There was one apparent false positive reported in Event 3 and three apparent false positives reported in Event 4. Improvements in drug detection between the two PT events in each year demonstrated the benefit of PT schemes in identifying and addressing potential deficiencies in laboratory systems.

Performance of Hair Testing for Cocaine Use-Comparison of Five Laboratories Using Blind Reference Specimens.

The purpose of this study was to compare results from five commercial hair testing laboratories conducting workplace drug testing with regard to bias, precision, selectivity and decontamination efficiency. Nine blind hair specimens, including cocaine-positive drug user specimens (some contaminated with methamphetamine) and negative specimens contaminated with cocaine, were submitted in up to five replicates to five different laboratories. All laboratories correctly identified cocaine in all specimens from drug users. For an undamaged hair specimen from a cocaine user, within-laboratory Coefficients of Variation (CVs) of 5-22% (median 8%) were reported, showing that it is possible to produce a homogenous proficiency testing sample from drug user hair. Larger CVs were reported for specimens composed of blended hair (up to 29%) and curly/damaged hair (19-67%). Quantitative results appeared to be method-dependent, and the reported cocaine concentrations varied up to 5-fold between the laboratories, making interlaboratory comparisons difficult. All laboratories reported at least one positive result in specimens contaminated with cocaine powder, followed by sweat and shampoo treatments. Benzoylecgonine, norcocaine, cocaethylene and hydroxylated cocaine metabolites were all detected in cocaine powder-contaminated specimens. This indicates that current industry standards for analyzing and reporting positive cocaine results are not completely effective at identifying external contamination. Metabolite ratios between meta- or para-hydroxy-cocaine and cocaine were 6- and 10-fold lower in contaminated specimens compared to those observed in cocaine user specimens, supporting their potential use in distinguishing samples positive due to contamination and drug use.

Conversion of 7-Carboxy-Cannabidiol (7-COOH-CBD) to 11-Nor-9-Carboxy-Tetrahydrocannabinol (THC-COOH) during Sample Preparation for GC-MS Analysis.

The growing use of cannabidiol (CBD) products by the general public is expected to result in an increase in the prevalence of CBD and the CBD metabolites in drug testing laboratories. CBD converts into tetrahydrocannabinol (THC) under acid conditions which could produce false-positive results, but little is known about how the presence of the urinary metabolite of CBD, 7-carboxy-cannabidiol (7-COOH-CBD), would affect urine drug testing for 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). As the operators of the National Laboratory Certification Program (NLCP), we prepared a set of performance testing samples containing 7-COOH-CBD for cannabinoid testing at the laboratories accredited by the NLCP to investigate if 7-COOH-CBD can produce false-positive results for THC-COOH during immunological screening analysis and if 7-COOH-CBD can be converted to THC-COOH. At concentrations up to 2,500 ng/mL, 7-COOH-CBD was not reactive by immunoassay in any of the four different immunoassay kits used. Additionally, we did not observe any significant conversion of 7-COOH-CBD to THC-COOH in assays used by NLCP-certified laboratories. However, we did see conversion when we requested that selected laboratories retest their samples using derivatization with perfluorinated anhydrides in combination with perfluorinated alcohols or when samples containing 7-COOH-CBD were exposed to acid for an extended time.

Assessment of the stability of DNA in specimens collected under conditions for drug testing-A pilot study.

For forensic biological sample collections, the specimen donor is linked solidly to his or her specimen through a chain of custody (CoC) sometimes referenced as a chain of evidence. Rarely, a donor may deny that a urine or oral fluid (OF) specimen is his or her specimen even with a patent CoC. The goal of this pilot study was to determine the potential effects of short-term storage on the quality and quantity of DNA in both types of specimen under conditions that may be encountered with employment-related drug testing specimens. Fresh urine and freshly collected oral fluid all produced complete STR profiles. For the "pad" type OF collectors, acceptable DNA was extractable both from the buffer/preservative and the pad. Although fresh urine and OF produced complete STR profiles, partial profiles were obtained after storage for most samples. An exception was the DNA in the Quantisal OF collector, from which a complete profile was obtained for both freshly collected OF and stored OF.