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.

Prevalence of Cannabidiol, ∆9- and ∆8-Tetrahydrocannabinol and Metabolites in Workplace Drug Testing Urine Specimens.

Given the recent popularity of cannabidiol (CBD) use and the emergence of ∆8-tetrahydrocannabinol (∆8-THC), the prevalence and concentrations of these and other cannabinoids were investigated in 2,000 regulated and 4,000 non-regulated specimens from workplace drug testing. All specimens were screened using liquid chromatography coupled to mass spectrometry (LC-MS-MS) for the presence of 7-hydroxy-CBD (7-OH-CBD) and ∆9-tetrahydrocannabinol-9-carboxylic acid (∆9-THC-COOH), with a cutoff of 2 ng/mL. Specimens screening positive by LC-MS-MS were analyzed by immunoassay at 20, 50 and 100 ng/mL cutoffs and by an LC-MS-MS confirmation method for 11 cannabinoids and metabolites with a 1 ng/mL cutoff. Using a 1 ng/mL cutoff, 98 (4.9%) regulated and 331 (8.3%) non-regulated specimens were positive for ∆9-THC-COOH. Of these, 64% had concentrations below 15 ng/mL. Similarly, 59 (3.0%) regulated and 162 (4.2%) non-regulated specimens were positive for 7-OH-CBD (n = 210), CBD (n = 120) and/or 7-carboxy-cannabidiol (CBD-COOH, n = 120). The median concentrations of 7-OH-CBD, CBD and CBD-COOH in those 221 specimens were 6.3, 1.1 and 1.2 ng/mL, respectively. ∆8-Tetrahydrocannabinol-9-carboxylic acid (∆8-THC-COOH) was identified in 76 (1.3%) specimens. Parent ∆8-THC is a minor cannabinoid in marijuana, which appears to account for the typically low ∆8-THC-COOH concentrations (median 3.4 ng/mL) in most positive specimens. However, elevated concentrations suggested the use of ∆8-THC-containing products in some cases (range 1.0-415 ng/mL). Although 93% agreement was observed between confirmatory LC-MS-MS (15 ng/mL cutoff) and immunoassay (50 ng/mL cutoff), a false-negative specimen (66 ng/mL ∆9-THC-COOH) was identified.

Effects of fluid load on human urine characteristics related to workplace drug testing.

During workplace drug testing, urine is tested for dilution, substitution and adulteration. Donors argue that these findings are due to medical, health or working conditions or diet and genetic differences. There is a paucity of data correlating changes in urine characteristics after a fluid load to various body parameters. Therefore, five urine specimens (one in the morning, one prior to drinking 800 mL of a beverage, and three time intervals thereafter) from 12 males and 12 females were tested for four different beverages on separate occasions. Of the 480 samples, 376 were in sufficient amounts. Of these 376, 36 (10%) had creatinine <20 mg/dL but ≥2 mg/dL; 27 (75%) of 36 had specific gravity <1.0030 but >1.0010. Thus, these 27 samples can be considered to be dilute; 20 (74%) of 27 were from females. For males with at least one dilute sample, body fat was 11% less and resting metabolic rate (RMR) was 29% more than males with no dilute samples (p > 0.05); for females with at least one dilute sample, height was 8% less and weight 20% less than females with no dilute samples (p > 0.05). Individuals with a higher RMR appear to have a greater potential for producing dilute urine specimens than those with a lower RMR. Thus, a dilute sample does not necessarily indicate that it was intentionally diluted. Such samples must be carefully evaluated in consideration with recent consumption of liquid by donors to avoid false accusations.

Detection of THCA in oral fluid by GC-MS-MS.

A major marijuana metabolite, 11-nor-9-carboxy-tetrahydrocannabinol (THCA), has been identified in oral fluids from donors that previously tested positive for Delta(9)-tetrahydrocannabinol (THC). The method consisted of solid-phase extraction of the oral fluid samples followed by gas chromatography-tandem mass spectrometry analysis of the extracts. Testing for THCA was performed on 223 oral fluid samples previously analyzed for THC. The THCA assay was linear from 10 to 240 pg/mL. The mean recovery of spiked THCA in oral fluid was 104%, and precision was 4% at 20 pg/mL using fortified negative samples. This method was rugged and robust, providing detection and quantification of THCA in oral fluids at levels not previously reported. Results of this study showed that THCA was detectable in 21 of 26 oral fluid samples previously reported positive for THC. The range of concentrations from these samples was from 10 pg/mL up to 142 pg/mL THCA.