David L. Black

Prescription Opioids. I. Metabolism and Excretion Patterns of Oxycodone in Urine Following Controlled Single Dose Administration

Hydrocodone (HC) is a highly misused prescription drugs in the USA. Interpretation of urine tests for HC is complicated by its metabolism to two metabolites, hydromorphone (HM) and dihydrocodeine (DHC), which are also available commercially and are misused. Currently, there is interest in including HC and HM in the federal workplace drug-testing programs. This study characterized the disposition of HC in human urine. Twelve healthy, drug-free, adults were administered a single, oral 20 mg immediate-release dose of HC in a controlled clinical setting. Urine specimens were collected at timed intervals for up to 52 h and analyzed by LC-MS-MS (limit of quantitation = 50 ng/mL) with and without enzymatic hydrolysis. All specimens were also analyzed for creatinine and specific gravity (SG). HC and norhydrocodone (NHC) appeared within 2 h followed by HM and DHC. Peak concentrations of HC and metabolites occurred at 3-9 h. Peak hydrolyzed concentrations were in the order: NHC > HC > HM > DHC. Only HM was excreted extensively as a conjugated metabolite. At a cutoff concentration of 50 ng/mL, detection times were ∼28 h for HC, 40 h for NHC, 26 h for HM and 16 h for DHC. Some specimens did not contain HC, but most contained NHC, thereby facilitating interpretation that HC was the administered drug. Creatinine and SG measures were highly correlated. Creatinine corrections of HC urinary data had variable effects of lowering or raising concentrations. These data suggest that drug-testing requirements for HC should include a hydrolysis step and a test for HM.

Urine testing for norcodeine, norhydrocodone, and noroxycodone facilitates interpretation and reduces false negatives

Urine drug testing of pain patients provides objective information to health specialists regarding patient compliance, diversion, and concurrent illicit drug use. Interpretation of urine test results for semi-synthetic opiates can be difficult because of complex biotransformations of parent drug to metabolites that are also available commercially and may be abused. Normetabolites such as norcodeine, norhydrocodone and noroxycodone are unique metabolites that are not available commercially. Consequently, detection of normetabolite in specimens not containing parent drug, provides conclusive evidence that the parent drug was consumed. The goal of this study was to evaluate the prevalence and patterns of the three normetabolites, norcodeine, norhydrocodone and noroxycodone, in urine specimens of pain patients treated with opiates. Urine specimens were hydrolyzed with beta-glucuronidase and analyzed by a validated liquid chromatography tandem mass spectrometry (LC/MS/MS) assay for the presence of codeine, norcodeine, morphine, hydrocodone, norhydrocodone, hydromorphone, dihydrocodeine, oxycodone, noroxycodone, and oxymorphone. The limit of quantitation (LOQ) for these analytes was 50ng/mL. The study was approved by an Institutional Review Board. Of the total specimens (N=2654) tested, 71.4% (N=1895) were positive (>or=LOQ) for one or more of the analytes. The prevalence (%) of positive results for codeine, hydrocodone and oxycodone was 1.2%, 26.1%, and 36.2%, respectively, and the prevalence of norcodeine, norhydrocodone and noroxycodone was 0.5%, 22.1%, and 31.3%, respectively. For specimens containing normetabolite, the prevalence of norcodeine, norhydrocodone and noroxycodone in the absence of parent drug was 8.6%, 7.8% and 9.4%, respectively. From one-third to two-thirds of these specimens also did not contain other metabolites that could have originated from the parent drug. Consequently, the authors conclude that inclusion of norcodeine, norhydrocodone and noroxycodone is useful in interpretation of opiate drug source and reduces potential false negatives that would occur without tests for these unique metabolites.

Prescription Opioids. III. Disposition of Oxycodone in Oral Fluid and Blood Following Controlled Single-Dose Administration

Oxycodone (OC) is recommended to be included as an analyte tested in the proposed Substance Abuse and Mental Health Services Administration (SAMHSAs) Mandatory Guidelines for Federal Workplace Drug Testing Programs using Oral Fluid (OF) Specimens. This study demonstrates the time course of OC and metabolites, noroxycodone (NOC), oxymorphone (OM) and noroxymorphone (NOM), in near-simultaneous paired OF and whole blood (BL) specimens by liquid chromatography-tandem mass spectrometry (LC-MS-MS) (limit of detection = 1 ng/mL OF, 5 ng/mL BL). A single dose of OC 20 mg controlled-release was administered to 12 healthy subjects followed by specimen collections for 52 h. Analyte prevalence was as follows: OF, OC > NOC > OM; and BL, OC > NOC > NOM. OC and NOC were frequently detected within 15-30 min in OF and 30 min to 2 h in BL. NOM and OM appeared between 1.5-5 h post-dose. The mean OF-to-BL (OF:BL) ratios and correlations were 5.4 for OC (r = 0.719) and 1.0 for NOC (r = 0.651). The period of detection for OF exceeded BL by ∼2-fold at similar cutoff concentrations. At a 1 ng/mL cutoff for OF, the mean detection time was 34 h for OC and NOC. These data provide new information that should facilitate interpretation of OC test results.

Urine Drug Testing of Chronic Pain Patients. V. Prevalence of Propoxyphene Following its Withdrawal from the United States Market

Propoxyphene is an opioid analgesic that was surrounded by controversy concerning its safety and efficacy during its lifespan in the US market. Propoxyphene was withdrawn in November of 2010 from the US market and is still being detected one year post-withdrawal in urine specimens from the pain management population. In this study, the prevalence of propoxyphene was determined in a total of 417,914 urine specimens collected from 630 clinics involved in pain management located in 24 states during the period of January 1, 2010, through December 31, 2011. Propoxyphene and norpropoxyphene were measured in urine by a validated liquid chromatography-tandem mass spectrometry procedure with a lower limit of quantitation of 50 ng/mL. The positivity rate for propoxyphene prevalence declined sharply between November and December of 2010 and further declined at a gradual rate, ending in a prevalence of 0.27% (one out of every 370 specimens, n = 25,658) for the month of December 2011. The presented data provide evidence of the dramatic decline in the use of propoxyphene products since their removal from the medical market, and may be beneficial to US urine drug testing programs determining the need for continual monitoring of propoxyphene levels.

Prevalence of heroin markers in urine for pain management patients.

Surveys of current trends indicate heroin abuse is associated with nonmedical use of pain relievers. Consequently, there is an interest in evaluating the presence of heroin-specific markers in chronic pain patients who are prescribed controlled substances. A total of 926,084 urine specimens from chronic pain patients were tested for heroin/diacetylmorphine (DAM), 6-acetylmorphine (6AM), 6-acetylcodeine (6AC), codeine (COD), and morphine (MOR). Heroin and markers were analyzed using liquid chromatography tandem mass spectrometry (LC-MS-MS). Opiates were analyzed following hydrolysis using LC-MS-MS. The prevalence of heroin use was 0.31%, as 2871 were positive for one or more heroin-specific markers including DAM, 6AM, or 6AC (a known contaminant of illicit heroin). Of these, 1884 were additionally tested for the following markers of illicit drug use: 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MAMP), 11-nor-9-carboxy-Δ(9)-tetracannabinol (THCCOOH), and benzoylecgonine (BZE); 654 (34.7%) had positive findings for one or more of these analytes. The overall prevalence of heroin markers were as follows: DAM 1203 (41.9%), 6AM 2570 (89.5%), 6AC 1082 (37.7%). MOR was present in 2194 (76.4%) and absent (<LOQ) in 677 (23.6%) of the heroin-positive specimens. COD was present in 1218 (42.4%) specimens. Prevalence of combinations for specimens containing MOR were as follows: DAM only 13 (0.59%), 6AM only 1140 (52.0%), 6AC only 24 (1.1%), DAM/6AM/6AC 710 (32.4%), 6AM/6AC 188 (8.6%), DAM/6AM 113 (5.2%), DAM/6AC 6 (0.27%). Importantly, the prevalence of combinations for specimens without MOR were as follows: DAM only 161 (23.8%), 6AM only 217 (32.1%), 6AC only 92 (13.6%), DAM/6AM/6AC 50 (7.4%), 6AM/6AC 7 (1.0%), DAM/6AM 145 (21.4%), DAM/6AC 5 (0.74%). Unexpected patterns of excretion were observed, such as the presence of DAM and 6AC in the absence of 6AM and MOR; therefore, multiple heroin markers may be useful to assess for heroin use.

Analysis of Nitrite in Adulterated Urine Samples by Capillary Electrophoresis

A simple method for analyzing nitrite in urine has been developed to confirm and quantify the amount of nitrite in potentially adulterated urine samples. The method involved separation of nitrite by capillary electrophoresis and direct UV detection at 214 nm. Separation was performed using a bare fused silica capillary and a 25 mM phosphate run buffer at a pH of 7.5. Sample preparation consisted of diluting the urine samples 1:20 with run buffer and internal standard, and centrifuging for 5 min at 2500 rpm. The sample was hydrodynamically injected, then separated using -25 kV with the column maintained at 35 degrees C. The method had upper and lower limits of linearity of 1500 and 80 microg/mL nitrite, respectively, and a limit of detection of 20 microg/mL. The method was evaluated using the National Committee for Clinical Laboratory Standards (NCCLS) protocol (Document EP10-A2), and validated using controls, standards, and authentic urine samples. Ten anions, ClO-, CrO4(-2), NO3-, HCO3-, I-, CH3COO-, F-, SO4-, S2O8(-2), and Cl-, were tested for potential interference with the assay. Interferences with quantitation were noted for only CrO4(-2) and S2O8(-2). High concentrations of Cl- interfered with the chromatography. The method had acceptable accuracy, precision, and specificity.

Determining Zolpidem Compliance: Urinary Metabolite Detection and Prevalence in Chronic Pain Patients

Zolpidem (Ambien(®)) is the most prescribed insomnia treatment in the USA; however, little is known about zolpidem metabolite excretion in chronic pain patients. As zolpidem is extensively metabolized in vivo to zolpidem 4-phenyl carboxylic acid (ZCA), metabolite detection may provide improved accuracy for compliance determinations, thereby improving clinical decisions. Zolpidem and ZCA were extracted from 1 mL human urine by mixed-mode solid-phase extraction. Samples were analyzed by LC-MS-MS using positive electrospray ionization with multiple reaction monitoring mode employed for detection and quantification. Gradient chromatographic separation was achieved with a reversed-phase column in a rapid 1.8 min analysis. The assay was linear from 4 to 1,000 µg/L for zolpidem and 4 to 10,000 µg/L for ZCA. Interday recovery (bias) and imprecision (n = 20) were 100-107% of target and 2.4-3.7% relative standard deviation, respectively. Extraction efficiencies were 78-90%. Pain compliance samples (n = 3,142) were de-identified and analyzed for zolpidem and ZCA. Zolpidem was detected greater than limit of quantification in 720 specimens (22.9%), while ZCA was detected in 1,579 specimens (50.3%). Only five specimens contained zolpidem alone. ZCA was observed without parent zolpidem in 864 specimens, thereby increasing population detection rates by 27.5%. Addition of a zolpidem metabolite to compliance determinations substantially improved detection for zolpidem intake and also should prove useful in clinical and forensic settings.

Urine drug testing results and paired oral fluid comparison from patients enrolled in long-term medication-assisted treatment in Tennessee.

Urine drug testing is recommended for individuals receiving medication-assisted treatment. It provides objective information for practitioners to consider and may serve as a protective factor against drug-related mortality. The primary objective of our study was to describe urine drug testing results for patients undergoing long-term medication-assisted treatment (≥6months). The secondary objective was to provide further evidence to establish oral fluid as a reliable alternative to urine. All subjects (n=639) included in the study were enrolled in one of five treatment centers in the state of Tennessee, and all urine specimens were positive for either methadone or buprenorphine. Nicotine (87%), caffeine (70%), marijuana (15%), alcohol (14%) and gabapentin (10%) were the most prevalent substances identified through urine drug testing. The presence of non-maintenance opioids (prescription and/or heroin) may represent relapse; these drugs were present in 10% of specimens tested. Evidence of illicit drug use (cocaine, heroin, marijuana and/or methamphetamine) was detected in 19% specimens. For 126 of the 639 subjects included in the study, paired oral fluid and urine test results were compared for agreement. Of the total paired urine and oral fluid tests, approximately 7% were positive for a drug in both specimen types and 91% were negative in both, resulting in an overall agreement of 98%. The study demonstrates continued use of illicit and commercially available medications in a medication-assisted treatment population undergoing long-term treatment. The results affirm the reliability of oral fluid as an alternative specimen type for compliance testing in this population.

Prescription Opioids. VI. Metabolism and Excretion of Hydromorphone in Urine Following Controlled Single-Dose Administration

Oxymorphone (OM), a prescription opioid and metabolite of oxycodone, was included in the recently published proposed revisions to the Mandatory Guidelines for Federal Workplace Drug Testing Programs. To facilitate toxicological interpretation, this study characterized the time course of OM and its metabolite, noroxymorphone (NOM), in hydrolyzed and non-hydrolyzed urine specimens. Twelve healthy subjects were administered a single 10 mg controlled-release OM dose, followed by a periodic collection of pooled urine specimens for 54 h following administration. Analysis for free and total OM and NOM was conducted by liquid chromatography tandem mass spectrometry (LC-MS-MS), at a 50 ng/mL limit of quantitation (LOQ). Following enzymatic hydrolysis, OM and NOM were detected in 89.9% and 13.5% specimens, respectively. Without hydrolysis, OM was detected in 8.1% specimens, and NOM was not detected. The mean ratio of hydrolyzed OM to NOM was 41.6. OM was frequently detected in the first pooled collection 0-2 h post-dose, appearing at a mean of 2.4 h. NOM appeared at a mean of 8.3 h. The period of detection at the 50 ng/mL threshold averaged 50.7 h for OM and 11.0 h for NOM. These data support that OM analysis conducted using a 50 ng/mL threshold should include hydrolysis or optimize sensitivity for conjugated OM.

Pseudoephedrine and False-Positive Immunoassay Urine Drug Tests for Amphetamine

We would like to address an erroneous assertion in the review article entitled “Drug Testing in the Workplace” by Dr. Phan and colleagues regarding urinary excretion of pseudoephedrine and its propensity for causing false-positive immunoassay screening results. The authors have underestimated the risk of false-positives from pseudoephedrine ingestion with the Microgenics (Fremont, CA) cloned enzyme donor immunoassay (CEDIA) for amphetamine and Ecstasy due to a high cross-reactive threshold (pseudoephedrine concentration of 160,000 ng/ml), citing a mean pseudoephedrine concentration in fatal overdoses of 105,000 ng/ml. The authors concluded that employers may “confidently make the correct decision to deny employment based only on the CEDIA urine drug test results.” However, the reference they cited for these data published the concentration not as a mean, but as a single case. That same reference cites urine concentrations up to 291,000 ng/ml after single doses used commonly in clinical practice. Pseudoephedrine concentrations of 2,500,000 ng/ml have been reported in the Department of Defense drug testing program. In our laboratory testing of 1855 student and professional athletes, urinary pseudoephedrine concentrations ranged from 813–6,670,097 ng/ml (median 56,200 ng/ml). The risk of false-positive amphetamine immunoassay results from pseudoephedrine has been well established. 6 A false-positive rate of 75% was reported with the Microgenics DRI amphetamine-methamphetamine immunoassay due to presence of pseudoephedrine in 1104 urine samples. Many of these false-positives occurred at concentrations far below the published pseudoephedrine threshold, indicating that cross-reactivity in human specimens is sometimes greater than the limits reported by the manufacturer. Interpreting a “fatal” range of concentrations in urine is problematic. Given the estimated time course of excretion, urine concentrations may not reflect the current clinical state and may be quite low or absent in cases of acute overdose. Thus, we advise any employer or medical practitioner to exercise caution when interpreting nonnegative immunoassay results, as falsepositive results can and do occur.