Xylazine: Pharmacology Review and Prevalence and Drug Combinations in Forensic Toxicology Casework.

Xylazine, an alpha-2 receptor agonist used in veterinary medicine for its sedative and muscle-relaxant effects, has been reported in forensic toxicology casework since the 1980s. It is not approved for human use, but it is used as an adulterant in heroin and illicit fentanyl. The prevalence and concentrations of xylazine in 2.5 years (January 2019-June 2021) of driving under the influence of drugs (DUID) and medico-legal death investigation (MDI) cases was investigated, including other drugs detected in combination with xylazine. Of over 170,000 cases screened for xylazine, 97% were classified as MDI. Over the course of the study period, the prevalence and geographical spread of xylazine increased. Overall, 2.8% of DUID and 2.1% of MDI cases screened positive for xylazine with concentrations of 5.1-450 ng/mL (mean = 36 ng/mL) and 5.0-11,000 ng/mL (mean = 41 ng/mL), respectively. Two MDI cases which had xylazine concentrations of 9,100 and 11,000 ng/mL were drug overdose suicides that did not involve any opioids. Opioids, primarily fentanyl and/or a fentanyl byproduct/metabolite were detected in 100% of DUID and all but two MDI cases. After opioids, stimulants, phyto-cannabinoids and benzodiazepines were the most common drug classes detected in conjunction with xylazine in both DUID and MDI casework. In summary, xylazine exposure continues to increase, mostly through the adulteration of illicit opioids. There is an extensive overlap in the concentrations between living and deceased individuals, making it difficult to interpret the role of the drug in MDI or DUID cases without other case information.

Reports of Adverse Events Associated with Use of Novel Psychoactive Substances, 2017-2020: A Review.

An important role of modern forensic and clinical toxicologists is to monitor the adverse events of novel psychoactive substances (NPS). Following a prior review from 2013 to 2016, this critical literature review analyzes and evaluates published case reports for NPS from January 2017 through December 2020. The primary objective of this study is to assist in the assessment and interpretation of these cases as well as provide references for confirmation methods. Chemistry, pharmacology, adverse events and user profiles (e.g., polypharmacy) for NPS are provided including case history, clinical symptoms, autopsy findings and analytical results. Literature reviews were performed in PubMed and Google Scholar for publications using search terms such as NPS specific names, general terms (e.g., 'designer drugs' and 'novel psychoactive substances'), drug classes (e.g., 'designer stimulants') and outcome-based terms (e.g., 'overdose' and 'death'). Government and website drug surveillance databases and abstracts published by professional forensic science organizations were also searched. Toxicological data and detailed case information were extracted, tabulated, analyzed and organized by drug category. Case reports included overdose fatalities (378 cases), clinical treatment and hospitalization (771 cases) and driving under the influence of drugs (170 cases) for a total of 1,319 cases providing details of adverse events associated with NPS. Confirmed adverse events with associated toxidromes of more than 60 NPS were reported including synthetic cannabinoid, NPS stimulant, NPS hallucinogen, NPS benzodiazepine and NPS opioid cases. Fifty of these NPS were reported for the first time in January 2017 through December 2020 as compared to the previous 4 years surveyed. This study provides insight and context of case findings described in the literature and in digital government surveillance databases and websites during a recent 4-year period. This review will increase the awareness of adverse events associated with NPS use to better characterize international emerging drug threats.

Brorphine-Investigation and quantitation of a new potent synthetic opioid in forensic toxicology casework using liquid chromatography-mass spectrometry.

New synthetic opioids continue to appear as novel psychoactive substances (NPS) on illicit drug markets. Isotonitazene emerged in mid-2019, becoming the most prevalent NPS opioid in the United States within a few months. Notification by the Drug Enforcement Administration of its intent to schedule isotonitazene in mid-2020 led to its decline in popularity and replacement with a new NPS opioid: brorphine. Brorphine is a potent synthetic opioid, but little information was previously available regarding its toxicity or involvement in impairment and death. Our laboratory developed an assay for the identification and quantitative confirmation of brorphine using standard addition. Quantitative analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vitro and in vivo metabolism studies were performed using pooled human liver microsomes and authentic biological specimens, respectively, with analysis by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Brorphine was confirmed in 20 authentic forensic cases, commonly found in combination with fentanyl (100%) and flualprazolam (80%). The average concentration of brorphine in blood was 2.5 ± 3.1 ng/mL (median: 1.1 ng/mL, range: 0.1-10 ng/mL). The average concentration of brorphine in urine was 4.6 ± 7.6 ng/mL (median: 1.6 ng/mL, range: 0.2-23 ng/mL). The majority of cases originated from Midwestern states. Metabolism was verified to included N-dealkylation and hydroxylation. Detailed case histories and autopsy findings are presented herein. The prevalence of brorphine continues to increase in the United States. Forensic scientists should remain aware of the ongoing emergence of new opioids, especially those outside a standard scope of toxicology testing.

Isotonitazene Quantitation and Metabolite Discovery in Authentic Forensic Casework.

The synthetic opioid landscape continues to change as non-fentanyl-related substances appear in forensic toxicology casework. Among the newest synthetic opioids to emerge is isotonitazene, an analogue of a benzimidazole class of analgesic compounds. Isotonitazene is an active and potent synthetic opioid, but the extent to which this compound is causing toxicity among drug users was previously unknown. This report describes the confirmation and quantitation of isotonitazene in blood, urine and vitreous fluid through standard addition, as well as in vivo metabolic profile determination in drug users. Quantitative analysis was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS), and metabolite discovery was performed using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In total, 18 cases were confirmed positive for isotonitazene, nine of which were previously negative for any opioid. The average isotonitazene concentration in blood was 2.2 ± 2.1 ng/mL (median 1.75 ng/mL, range 0.4-9.5 ng/mL), and the average isotonitazene concentration in urine was 2.4 ± 1.4 ng/mL (median 2.7 ng/mL, range 0.6-4.0 ng/mL). The lowest concentration of isotonitazene in blood was 0.4 ng/mL (two cases) with no other opioids present; findings in death investigations. Four metabolites of isotonitazene were detected in vivo. N- and O-dealkylation products were determined to be the most prominent urinary biomarkers, while 5-amino-isotonitazene was identified in most blood samples. The prevalence and popularity of isotonitazene continue to increase in the United States in early 2020. Toxicologists, medical examiners and coroners should be aware of novel opioids outside the standard scope of testing, especially in medicolegal death investigations. Forensic scientists should add isotonitazene to testing procedures, and public health officials should counsel about potent new drugs and the dangers of opioid use.

4F-MDMB-BINACA: A New Synthetic Cannabinoid Widely Implicated in Forensic Casework.

This is the first report regarding the characterization of the new synthetic cannabinoid 4F-MDMB-BINACA. 4F-MDMB-BINACA was first analytically confirmed in seized drug material using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF), and nuclear magnetic resonance (NMR) spectroscopy. Subsequent to this characterization, 4F-MDMB-BINACA was detected in biological specimens collected as part of forensically relevant casework, including medicolegal death investigations and drug impaired driving investigations, from a variety of regions in the United States. Further analysis of biological specimens resulted in the identification of the metabolites 4F-MDMB-BINACA 3,3-dimethylbutanoic acid and 4-OH-MDMB-BINACA. 4F-MDMB-BINACA is appearing with increasing frequency as a contributory factor in deaths, creating morbidity and mortality risks for drug users. Laboratories must be aware of its presence and impact, incorporating 4F-MDMB-BINACA into workflows for detection and confirmation.

The Evolving Landscape of Designer Drugs.

Since 2008 there has been an onslaught of new drugs in the illicit marketplace. Often referred to as "research chemicals," "designer drugs," or "novel psychoactive substances" (NPS), these substances are used for their pharmacological effects which are often similar to more widely known drugs such as ecstasy or heroin. In some cases users specifically seek out these new chemicals, in other cases they are simply purchasing what they believe to be their normal drug of choice from a dealer, but the product is not what it is purported to be. Implementation of national and international systems to monitor the appearance of new compounds enables laboratories to be prepared with validated tests to detect them in biological specimens. The most common classes of NPS are synthetic cannabinoids, novel opioids, novel benzodiazepines, stimulants, and hallucinogens. Within these groups the compounds may be drugs that were originally synthesized for research purposes during the pursuit of new therapeutic agents such as the synthetic cannabinoid JWH-018 and the designer opioid U47700. Others like etizolam are compounds used in other countries but not commonly seen in the USA. Some are drugs synthesized specifically to circumvent legal controls. In all cases, these compounds present a unique challenge to forensic toxicology laboratories which must quickly develop and validate analytical methods for the identification and quantification in biological matrices.This chapter is a condensed and updated version of an article originally published in Clinical and Forensic Toxicology News.

Quantification of Designer Opioids by Liquid Chromatography-Tandem Mass Spectrometry.

Opioids including heroin and commonly prescribed drugs such as oxycodone and fentanyl are among the most commonly abused drugs. In recent years, the abuse of opioids has spread beyond these commonly encountered analytes and now includes novel psychoactive drugs such as AH-7921 and U47700 and a variety of fentanyl-related compounds such as acetyl fentanyl and furanyl fentanyl. The assay described is for the quantitative determination of 19 designer opioids in serum, plasma, and whole blood. Also included is a discussion on the challenges of keeping an analytical method current as new analytes appear on the illicit drug market.

Reports of Adverse Events Associated with Use of Novel Psychoactive Substances, 2013-2016: A Review.

Novel psychoactive substances (NPS) represent significant analytical and interpretive challenges to forensic and clinical toxicologists. Timely access to case reports and reports of adverse incidents of impairment or toxicity is imperative to clinical diagnosis and treatment, as well as to interpretation of forensic results. Delays in identifying the presence of a novel intoxicating agent have significant consequences for public health and public safety. Adverse effects of intoxications with novel cannabinoids, stimulants, hallucinogens, benzodiazepines and opioids spanning January 2013 through December 2016 as reported in emergency departments, death investigations, impaired driving cases and other forensic contexts are the subject of this review. Discussion of the chemistry, pharmacology and adverse events associated with novel drug classes is summarized and described within. Adverse effects or symptoms associated with ingestion of more than 45 NPS have been abstracted and summarized in tables, including demographics, case history, clinical or behavioral symptoms, autopsy findings and drug confirmations with quantitative results when provided. Based on these findings and gaps in the available data, we provide recommendations for future toxicological testing of these evolving substances. These include development and management of a national monitoring program to provide real-time clinical and toxicological data, confirmed analytically, on emerging drugs and their known toxidromes and side effect profiles. Increased efforts should be made to analytically confirm the agents responsible for clinical intoxications involving adverse events in emergency department admissions or hospitalizations. Evidence-based community preparedness among analytical laboratories gained through active communication and sharing of toxicological findings and trends in NPS is imperative to assist in enabling early detection of new drugs in forensic and clinical populations.

Analysis of Novel Synthetic Opioids U-47700, U-50488 and Furanyl Fentanyl by LC-MS/MS in Postmortem Casework.

Following series of synthetic cannabinoid and synthetic cathinone derivatives, the illicit drug market has begun to see increased incidence of synthetic opioids including fentanyl and its derivatives, and other chemically unrelated opioid agonists including AH-7921 and MT-45. Among the most frequently encountered compounds in postmortem casework have been furanyl fentanyl (N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylfuran-2-carboxamide, Fu-F) and U-47700 (trans-3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide). Both drugs have been reported to be present in the heroin supply and to be gaining popularity among recreational opioid users, but were initially developed by pharmaceutical companies in the 1970s as candidates for development as potential analgesic therapeutic agents. A method was developed and validated for the analysis of U-47700, U-50488 and furanyl fentanyl in blood specimens. A total of 20 postmortem cases, initially believed to be heroin or other opioid-related drug overdoses, were submitted for quantitative analysis. The analytical range for U-47770 and U-50488 was 1-500 and 1-100 ng/mL for furanyl fentanyl. The limit of detection was 0.5 ng/mL for all compounds. Within the scope of the method, U-47700 was the only confirmed drug in 11 of the cases, 5 cases were confirmed for both U-47700 and furanyl fentanyl, and 3 cases were confirmed only for furanyl fentanyl. The mean and median blood concentrations for U-47700 were 253 ng/mL (±150) and 247 ng/mL, respectively, range 17-490 ng/mL. The mean and median blood concentrations for furanyl fentanyl were 26 ng/mL (±28) and 12.9 ng/mL, respectively, range 2.5-76 ng/mL. Given the widespread geographical distribution and increase in prevalence in postmortem casework, toxicology testing should be expanded to include testing for "designer opioids" in cases with histories consistent with opioid overdose but with no traditional opioids present or insufficient quantities to account for death.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the identification and quantification of JWH-018, JWH-073, JWH-019, and JWH-250 in human whole blood.

A sensitive and specific method for the quantification of JWH-018, JWH-073, and JWH-250 and the qualitative identification of JWH-019 in whole blood was developed and validated. Samples fortified with JWH-018-d9 and JWH-073-d9 underwent liquid-liquid extraction and were analyzed by liquid chromatography-positive ion electrospray ionization-tandem mass spectrometry. Two transitions were monitored for all analytes except JWH-250, for which there was only one available transition. JWH-019 did not meet the stringent requirements for quantitative analysis, and thus this method is only appropriate for the qualitative identification of this compound in whole blood. The linear range was 0.1-20 µg/L for all quantitative analytes. The maximum average within and between-run imprecision was 7.9% and 10.2%, respectively, and all controls quantified within 8.2% of target concentrations. Process efficiency, a measurement that takes into effect extraction efficiency and matrix effect, was ≥ 32.0% for all quantitative analytes; similar results were obtained for the deuterated internal standards. All analytes were stable at room, refrigerated, and frozen temperatures for at least 30 days. The method was used to quantify JWH-018 and JWH-073 in a blood specimen collected from a person known to have used an herbal incense blend containing these substances.

Urinary excretion of buprenorphine, norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide in pregnant women receiving buprenorphine maintenance treatment.

BACKGROUND: Buprenorphine (BUP) is under investigation as a medication therapy for opioid-dependent pregnant women. We investigated BUP and metabolite disposition in urine from women maintained on BUP during the second and third trimesters of pregnancy and postpartum. METHODS: We measured BUP, norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and NBUP-Gluc concentrations in 515 urine specimens collected thrice weekly from 9 women during pregnancy and postpartum. Specimens were analyzed using a fully validated liquid chromatography-mass spectrometry method with limits of quantification of 5 microg/L for BUP and BUP-Gluc and 25 microg/L for NBUP and its conjugated metabolite. We examined ratios of metabolites across trimesters and postpartum to identify possible changes in metabolism during pregnancy. RESULTS: NBUP-Gluc was the primary metabolite identified in urine and exceeded BUP-Gluc concentrations in 99% of specimens. Whereas BUP-Gluc was identified in more specimens than NBUP, NBUP exceeded BUP-Gluc concentrations in 77.9% of specimens that contained both analytes. Among all participants, the mean BUP-Gluc:NBUP-Gluc ratio was significantly higher in the second trimester compared to the third trimester, and there were significant intrasubject differences between trimesters in 71% of participants. In 3 women, the percent daily dose excreted was higher during pregnancy than postpregnancy, consistent with other data indicating increased renal elimination of drugs during pregnancy. CONCLUSIONS: These data are the first to evaluate urinary disposition of BUP and metabolites in a cohort of pregnant women. Variable BUP excretion during pregnancy may indicate metabolic changes requiring dose adjustment during later stages of gestation.

Development and validation of a liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of buprenorphine, norbuprenorphine, and metabolites in human urine.

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and norbuprenorphine glucuronide (NBUP-Gluc) in human urine was developed and fully validated. Extensive endogenous and exogenous interferences were evaluated and limits of quantification were identified empirically. Analytical ranges were 5-1,000 ng/mL for BUP and BUP-Gluc and 25-1,000 ng/mL for NBUP and NBUP-Gluc. Intra-assay and interassay imprecision were less than 17% and recovery was 93-116%. Analytes were stable at room temperature, at 4 degrees C, and for three freeze-thaw cycles. This accurate and precise assay has sufficient sensitivity and specificity for urine analysis of specimens collected from individuals treated with BUP for opioid dependence.

Validation and application of a method for the determination of buprenorphine, norbuprenorphine, and their glucuronide conjugates in human meconium.

A novel liquid chromatography tandem mass spectrometry method for quantification of buprenorphine, norbuprenorphine, and glucuronidated conjugates was developed and validated. Analytes were extracted from meconium using buffer, concentrated by solid-phase extraction and quantified within 13.5 min. In order to determine free and total concentrations, specimens were analyzed with and without enzyme hydrolysis. Calibration was achieved by linear regression with a 1/x weighting factor and deuterated internal standards. All analytes were linear from 20 to 2000 ng/g with a correlation of determination of >0.98. Accuracy was >or=85.7% with intra-assay and interassay imprecisionor=85.0%. There was suppression of ionization by the polar matrix; however, this did not interfere with sensitivity or analyte quantification due to inclusion of deuterated internal standards. Analytes were stable on the autosampler, at room temperature, at 4 degrees C, and when exposed to three freeze/thaw cycles. This sensitive and specific method can be used to monitor in utero buprenorphine exposure and to evaluate correlations, if any, between buprenorphine exposure and neonatal outcomes.

Excretion of methamphetamine and amphetamine in human sweat following controlled oral methamphetamine administration.

BACKGROUND: Understanding methamphetamine (MAMP) and amphetamine (AMP) excretion in sweat is important for interpreting sweat and hair testing results in judicial, workplace, and drug treatment settings. METHODS: Participants (n = 8) received 4 10-mg (low) oral doses of sustained-release S-(+)-MAMP HCl (d-MAMP HCl) within 1 week in a double-blind, institutional review board-approved study. Five participants also received 4 20-mg (high) doses 3 weeks later. PharmChek sweat patches (n = 682) were worn for periods of 2 h to 1 week during and up to 3 weeks after dosing. The mass of MAMP and AMP in each patch was measured by GC-MS, with a limit of quantification of 2.5 ng/patch. RESULTS: MAMP was measurable in sweat within 2 h of dosing. After low and high doses, 92.9% and 62.5% of weekly sweat patches were positive, with a median (range) MAMP of 63.0 (16.8-175) and 307 (199-607) ng MAMP/patch, respectively; AMP values were 15.5 (6.5-40.5) and 53.8 (34.0-83.4) ng AMP/patch. Patches applied 2 weeks after the drug administration week had no measurable MAMP following the low doses, and only 1 positive result following the high doses. Using criteria proposed by the Substance Abuse Mental Health Services Administration, 85.7% (low) and 62.5% (high) weekly sweat patches from the dosing week were positive for MAMP, and all patches applied after the dosing week were negative. CONCLUSIONS: These data characterize the excretion of MAMP and AMP after controlled MAMP administration and provide a framework for interpretation of MAMP sweat test results in clinical and forensic settings.

Opioid disposition in human sweat after controlled oral codeine administration.

BACKGROUND: Characterization of opioid excretion in sweat is important for accurate interpretation of sweat tests in drug treatment, criminal justice, and workplace drug testing programs. METHODS: Participants (n=20) received placebo, 3 low (60 mg/70 kg) or 3 high (120 mg/70 kg) codeine sulfate doses (used as a model for opioid excretion) within 1 week. Codeine and metabolites in sweat were collected with PharmChek Sweat Patches; hourly patches were applied for 1 to 15 h (n=775) and weekly patches for 7 days (n=118). Patches were analyzed by solid-phase extraction and gas chromatography-mass spectrometry for codeine, norcodeine, morphine, normorphine, and 6-acetylmorphine. Limits of quantification were 2.5 ng/patch (codeine and morphine) and 5 ng/patch (other analytes). RESULTS: Codeine was the only analyte identified in 12.6% of hourly patches and 83.3% of weekly sweat patches worn during dosing. Weekly patch concentrations (SD) were 38.6 (59.9) ng/patch [median (range), 15.9 (0-225.1) ng/patch] for low and 34.1 (32.7) ng/patch [24.0 (0-96.2) ng/patch] for high codeine doses. Codeine detected 1 week after dosing was 4.6 (5.3) ng/patch [median (range), 4.0 (0-17.1) ng/patch; n=11] after low and 7.7 (7.1) ng/patch [6.9 (0-20.5) ng/patch; n=10] after high doses. In total, 2.6% of hourly, 38.5% of low-dose, and 45.5% of high-dose weekly patches contained codeine at the proposed Substance Abuse and Mental Health Services Administration cutoff. CONCLUSIONS: Codeine was the only analyte detected, at highly variable concentrations, up to 2 weeks after dosing. These results are consistent, considering the complex processes of codeine deposition in sweat. Sweat testing is a useful alternative technique for qualitative monitoring of opioid use.

Disposition of cocaine and its metabolites in human sweat after controlled cocaine administration.

BACKGROUND: Sweat testing is a noninvasive technique for monitoring drug exposure in treatment, criminal justice, and employment settings. METHODS: We evaluated cocaine excretion in 9 participants' sweat after they received 3 low doses (75 mg/70 kg) of cocaine HCl subcutaneously within 1 week and, 3 weeks later, 3 high doses (150 mg/70 kg). Six additional participants completed portions of the study. PharmChek sweat patches (n = 1390) were collected throughout a 3-week washout period, reflecting previously self-administered drugs, and during and after controlled dosing. RESULTS: Cocaine was the primary analyte detected with 24% of patches positive at the gas chromatography-mass spectrometry limit of quantification of 2.5 ng/patch and 7% of patches at the proposed Substance Abuse and Mental Health Services Administration cutoff of 25 ng/patch. Ecgonine methyl ester (EME) was detected more often and at generally higher concentrations than benzoylecgonine. In patches containing both metabolites, there was no statistically significant difference in the benzoylecgonine/EME ratio based on length of patch wear. During washout, 2 participants' weekly patches tested positive (> or =25 ng/patch) during the first week; one remained positive during week 2; and none were positive during week 3. Cocaine and EME were detectable within 2 h; benzoylecgonine was not detected until 4-8 h after low doses and slightly sooner after high doses. The majority of drug was excreted within 24 h. Over 70% of weekly patches worn during low doses were positive for cocaine (> or =25 ng/patch), increasing to 100% during high doses. CONCLUSION: Sweat testing is an effective and reliable method of monitoring cocaine exposure.

Performance characteristics of the Cozart RapiScan Oral Fluid Drug Testing System for opiates in comparison to ELISA and GC/MS following controlled codeine administration.

Oral fluid is an interesting alternative matrix for drug testing in many environments, including law enforcement, workplace drug testing, and drug treatment facilities. Performance characteristics of the FDA-cleared, qualitative, Cozart RapiScan Opiate Oral Fluid Drug Testing System (Opiate Cozart RapiScan System or Opiate CRS) were compared to the semi-quantitative Cozart Microplate EIA Opiate Oral Fluid Kit (Opiate ELISA) and to gas chromatography/mass spectrometry (GC/MS). The following oral fluid opiate cutoffs were evaluated: the GC/MS limit of quantification (LOQ) of 2.5 mg/l; 15 microg/l currently used for oral fluid testing in the United Kingdom (UK); 30 microg/l (Opiate CRS cutoff); and 40 microg/l, the proposed Substance Abuse and Mental Health Services Administration (SAMHSA) cutoff. Subjects provided informed consent to participate in this IRB-approved research and resided on the closed research ward throughout the study. Three oral codeine doses of 60 mg/70 kg were administered over a 7-day period. After a 3-week break, subjects received three doses of 120 mg/70 kg within 7 days. Oral fluid specimens (N = 1273) were analyzed for codeine (COD), norcodeine (NCOD), morphine (MOR) and normorphine (NMOR) by GC/MS with an LOQ of 2.5 microg/l for all analytes. MOR and NMOR were not detected in any sample; 26.5% of the specimens were positive for COD and 13.7% for NCOD. Opiate CRS uses a preset, qualitative cutoff of 10 microg/l; this is equivalent to 30 microg/l in undiluted oral fluid as the oral fluid collection process involves a 1:3 dilution with buffer. Sensitivity, specificity, and efficiency of Opiate CRS compared to Opiate ELISA were 98.6, 98.1, and 98.2% at a 30 microg/l cutoff and 99.0, 96.2, and 96.6% at a 40 microg/l cutoff. Compared to the much lower GC/MS LOQ of 2.5 microg/l, sensitivity, specificity and efficiency were 66.8, 99.3 and 90.7%. Increasing the GC/MS cutoff to the current UK level yielded performance characteristics of 81.5% (sensitivity), 99.3% (specificity), and 95.4% (efficiency). Using a GC/MS cutoff identical to the preset Opiate CRS cutoff yielded sensitivity, specificity, and efficiency of 88.5, 99.2, and 97.5%, respectively. At the proposed SAMSHA confirmation cutoff of 40 microg/l, sensitivity increased with little change in specificity and efficiency (91.3% sensitivity, 98.9% specificity, and 97.5% efficiency). Oral fluid is a suitable matrix for detecting drugs of abuse. Opiate CRS, with a 30 microg/l cutoff, is sufficiently sensitive, specific and efficient for oral fluid opiate analysis, performing similarly to Opiate ELISA at the same cutoff, and having performance characteristics >91% when compared to GC/MS at the proposed SAMHSA cutoff.