Glutathione and Glutathione-Like Sequences of Opioid and Aminergic Receptors Bind Ascorbic Acid, Adrenergic and Opioid Drugs Mediating Antioxidant Function: Relevance for Anesthesia and Abuse.

Opioids and their antagonists alter vitamin C metabolism. Morphine binds to glutathione (l-γ-glutamyl-l-cysteinyl-glycine), an intracellular ascorbic acid recycling molecule with a wide range of additional activities. The morphine metabolite morphinone reacts with glutathione to form a covalent adduct that is then excreted in urine. Morphine also binds to adrenergic and histaminergic receptors in their extracellular loop regions, enhancing aminergic agonist activity. The first and second extracellular loops of adrenergic and histaminergic receptors are, like glutathione, characterized by the presence of cysteines and/or methionines, and recycle ascorbic acid with similar efficiency. Conversely, adrenergic drugs bind to extracellular loops of opioid receptors, enhancing their activity. These observations suggest functional interactions among opioids and amines, their receptors, and glutathione. We therefore explored the relative binding affinities of ascorbic acid, dehydroascorbic acid, opioid and adrenergic compounds, as well as various control compounds, to glutathione and glutathione-like peptides derived from the extracellular loop regions of the human beta 2-adrenergic, dopamine D1, histamine H1, and mu opioid receptors, as well as controls. Some cysteine-containing peptides derived from these receptors do bind ascorbic acid and/or dehydroascorbic acid and the same peptides generally bind opioid compounds. Glutathione binds not only morphine but also naloxone, methadone, and methionine enkephalin. Some adrenergic drugs also bind to glutathione and glutathione-like receptor regions. These sets of interactions provide a novel basis for understanding some ways that adrenergic, opioid and antioxidant systems interact during anesthesia and drug abuse and may have utility for understanding drug interactions.

Lower Cutoffs for LC-MS/MS Urine Drug Testing Indicates Better Patient Compliance.

BACKGROUND: Urine drug testing is used by health care providers to determine a patient's compliance to their prescribed regimen and to detect non-prescribed medications and illicit drugs. However, the cutoff levels used by clinical labs are often arbitrarily set and may not reflect the urine drug concentrations of compliant patients. OBJECTIVES: Our aim was to test the hypothesis that commonly used cutoffs for many prescribed and illicit drugs were set too high, and methods using these cutoffs may yield a considerable number of false-negative results. The goals of this study were to outline the way to analyze patient results and estimate a more appropriate cutoff, develop and validate a high sensitivity analytical method capable of quantitating drugs and metabolites at lower than the commonly used cutoffs, and determine the number of true positive results that would have been missed when using the common cutoffs. STUDY DESIGN: This was a retrospective study of urine specimens submitted for urine drug testing as part of the monitoring of prescription drug compliance described in chronic opioid therapy treatment guidelines. SETTING: The study was set in a clinical toxicology laboratory, using specimens submitted for routine analysis by health care providers in the normal course of business. METHODS: Lognormal distributions of test results were generated and fitted with a trendline to estimate the required cutoff level necessary to capture the normal distributions of each drug for the patient population study. A validated laboratory derived liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis capable of achieving the required cutoff levels was developed for each drug and/or metabolite. RESULTS: The study shows that a lognormal distribution of patient urine test results fitted with a trendline is appropriate for estimating the required cutoff levels needed to assess medication adherence. The study showed a wide variation in the false-negative rate, ranging from 1.5% to 94.3% across a range of prescribed and illicit drugs. LIMITATIONS: The patient specimens were largely sourced from patients in either a long-term pain management program or in treatment for substance use disorder in the US. These specimens may not be representative of patients in other types of treatment or in countries with different approaches to these issues. CONCLUSIONS: The high-sensitivity method reduces false-negative results which could negatively impact patient care. Clinicians using less sensitive methods for detecting and quantifying drugs and metabolites in urine should exercise caution in assessing patient adherence using and changing the treatment plan based on those results. KEY WORDS: Urine drug testing, patient adherence, clinical toxicology, immunoassay, LC-MS, definitive drug testing, REMS, negative test results, false negative.

Suitability of the DRI Hydrocodone/Hydromorphone Immunoassay in the Clinical Environment at a Lower Cutoff: Validation With LC-MS/MS Analysis.

BACKGROUND: We evaluated the analytical performance of the DRI hydrocodone/hydromorphone assay by comparing semiquantitative values obtained by this assay with values obtained by a liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method. We also evaluated the possibility of lowering the cutoff of the DRI assay from 300 to 100 ng/mL. METHODS: We compared semiquantitative values obtained by the DRI assay in 97 specimens with values obtained by the LC-MS/MS method including 10 specimens containing hydrocodone and/or hydromorphone concentrations between 105.0 and 145.0 ng/mL (determined by LC-MS/MS) to determine the sensitivity at 100 ng/mL. In addition, several opioids at a concentration of 5000 ng/mL were also analyzed by the DRI assay to determine its specificity. RESULTS: We observed no false-negative result using the DRI immunoassay in 96 specimens that showed semiquantitative values at 100 ng/mL or higher. However, one specimen containing 110 ng/mL of hydrocodone was false negative with the DRI assay (semiquantitative value 88 ng/mL, below 100 ng/mL cutoff). The semiquantitative values produced by DRI showed poor correlation with values determined by the LC-MS/MS method. The sensitivity of the DRI assay at 100 ng/mL was 90%, and the assay was very specific showing minimal cross-reactivity only with oxycodone and oxymorphone. CONCLUSIONS: DRI immunoassay for hydrocodone/hydromorphone is a cost-effective method of screening urine specimens in the clinical environment at a lower cutoff of 100 ng/mL.

Degradation of Opioids and Opiates During Acid Hydrolysis Leads to Reduced Recovery Compared to Enzymatic Hydrolysis.

Drug monitoring laboratories utilize a hydrolysis process to liberate the opiates from their glucuronide conjugates to facilitate their detection by tandem mass spectrometry (MS). Both acid and enzyme hydrolysis have been reported as viable methods, with the former as a more effective process for recovering codeine-6-glucuronide and morphine-6-glucuronide. Here, we report concerns with acid-catalyzed hydrolysis of opioids, including a significant loss of analytes and conversions of oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine. The acid-catalyzed reaction was monitored in neat water and patient urine samples by liquid chromatography-time-of-flight and tandem MS. These side reactions with acid hydrolysis may limit accurate quantitation due to loss of analytes, possibly lead to false positives, and poorly correlate with pharmacogenetic profiles, as cytochrome P450 enzyme (CYP2D6) is often involved with oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine conversions. Enzymatic hydrolysis process using the purified, genetically engineered ß-glucuronidase (IMCSzyme®) addresses many of these concerns and demonstrates accurate quantitation and high recoveries for oxycodone, hydrocodone, oxymorphone and hydromorphone.

Evaluation of a Newly Formulated Enzyme Immunoassay for the Detection of Hydrocodone and Hydromorphone in Pain Management Compliance Testing.

A new Hydrocodone Enzyme Immunoassay (HEIA; Lin-Zhi International, Inc.) was evaluated for the detection of hydrocodone and its main metabolite, hydromorphone. All specimens were tested with two different cutoff calibrators, 100 and 300 ng/mL, on an ARCHITECT Plus c4000 Clinical Chemistry Analyzer. Controls containing -25% (negative control) and +25% (positive control) of the cutoff calibrators and a drug-free control were analyzed with each batch. All 1,025 urine specimens were previously analyzed by ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS-MS) for opiates. Approximately, 33% (337/1,019) of the specimens yielded positive results by the HEIA assay at a cutoff concentration of 100 ng/mL and 19% (190/1,025) yielded positive results at the 300 ng/mL cutoff concentration. Of these presumptive positive specimens, UPLC-MS-MS confirmed the presence of hydrocodone and/or hydromorphone >100 ng/mL in 241 specimens and >300 ng/mL in 162 specimens, for each respective cutoff. With the 100 ng/mL cutoff, the HEIA demonstrated a sensitivity of 0.959, a specificity of 0.846 and an overall agreement with the UPLC-MS-MS of 87%. At 300 ng/mL cutoff, the HEIA demonstrated a sensitivity of 0.880, a specificity of 0.966 and an overall agreement of UPLC-MS-MS results of 95%. The Lin-Zhi HEIA 100 ng/mL cutoff assay demonstrated sensitivity for the detection of hydrocodone and hydromorphone in urine. The 300 ng/mL cutoff was less sensitive, but more selective, and should be part of an initial immunoassay screen, particularly in pain management compliance testing.

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

Hydromorphone (HM), a prescription opioid and metabolite of morphine and hydrocodone, has been included in proposed revisions to the Mandatory Guidelines for Federal Workplace Drug Testing Programs. This study characterized the time course of HM in hydrolyzed and non-hydrolyzed urine specimens. Twelve healthy subjects were administered a single 8 mg controlled-release HM dose, followed by periodic collection of pooled urine specimens for 54 h following administration. Analysis of total and free HM was conducted by liquid chromatography tandem mass spectrometry at a 50 ng/mL limit of quantitation. Detection periods were determined over a range of thresholds from 50 to 2,000 ng/mL. HM was detected in 85.3% and 47.6% of hydrolyzed and non-hydrolyzed post-dose specimens, respectively. Initial detection of total HM was frequently observed in the first 4-6 h following dosing. The period of detection at the 50 ng/mL threshold averaged 52.3 h for total HM and 38.0 h for free HM. These data support that HM detection is optimized by using low thresholds (50-100 ng/mL) and including conjugated HM in the analysis.

LC-MS-MS Method for Analysis of Opiates in Wastewater During Football Games II.

Continuing our previous studies analyzing drugs of abuse in municipal wastewater, a method was developed for the analysis of opiates in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Eight opiate drugs and metabolites were analyzed including codeine, hydrocodone, hydromorphone, 6-monoacetylmorphine (6-MAM, the primary urinary metabolite of heroin), morphine, norhydrocodone (the primary urinary metabolite of hydrocodone), oxycodone and oxymorphone. These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Waste Water Treatment Plant in Oxford, Mississippi (MS) and the University Wastewater Treatment Plant in University, MS. These wastewater samples were collected on weekends in which the Ole Miss Rebel football team held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain codeine, hydrocodone, hydromorphone, morphine, norhydrocodone, oxycodone and oxymorphone. None of the samples contained 6-MAM.

Sensitivity of an opiate immunoassay for detecting hydrocodone and hydromorphone in urine from a clinical population: analysis of subthreshold results.

Urine drug testing (UDT) is an emerging standard of care in the evaluation and treatment of chronic non-cancer pain patients with opioid analgesics. UDT may be used both to verify adherence with the opioid analgesic regimen and to monitor abstinence from non-prescribed or illicit controlled substances. In the former scenario, it is vital to determine whether the drug is present in the urine, even at low concentrations, because failure to detect the drug may lead to accusations of opioid abuse or diversion. Opiate immunoassays typically are developed to detect morphine and are most sensitive to morphine and codeine. Although many opiate immunoassays also detect hydrocodone (HC) and/or hydromorphone (HM), sensitivities for these analytes are often much lower, increasing the possibility of negative screening results when the drug is present in the urine. We selected 112 urine specimens from patients who had been prescribed HC or hydromorphone but were presumptive negative by the Roche Online DAT Opiate II™ urine drug screening assay, which is calibrated to 300 ng/mL morphine. Using a GC/MS confirmatory method with a detection limit of 50 ng/mL both for HC and for HM, one or both of these opiates were detected in 81 (72.3%) of the urine specimens. Examination of the raw data from these presumptive negative opiate screens revealed that, in many cases, the turbidity signal was greater than the signal obtained for the negative control, but less than the signal for the 300 ng/mL (morphine) threshold calibrator. A receiver operating characteristic curve generated for the reciprocal of the ratio of turbidity measurements in the patient specimens and negative (drug-free) controls, against the presence or absence of HC and/or HM by confirmatory analyses, produced an area under the curve of 0.910. We conclude that this opiate immunoassay has sufficient sensitivity to detect HC and/or HM in some urine specimens that screen presumptive negative for these commonly prescribed opiates at the established threshold.

Unauthorized drug use in the US Army based on medical review officer evaluations.

This article examines the US Army's Medical Review Officer (MRO) drug positive urinalysis evaluations from 2009 through 2012. We retrospectively analyzed nearly 70,000 MRO results by year, drug and Army component. Of the MRO reviewable positive results, the Army's unauthorized drug positive rate was 22.21%. The component rates were 20.81, 24.17 and 26.09% for the Active Duty, Reserve and National Guard, respectively. By drug, the average unauthorized rates over these 4 years were 13.78% for oxycodone, 24.62% oxymorphone, 18.56% d-amphetamine, 98.04% d-methamphetamine, 21.97% codeine, 45.21% morphine and 100% steroids. In 2012 testing began for hydrocodone and hydromorphone and their unauthorized rates were 12.32 and 15.04%, respectively. The Army's unauthorized drug positive rate peaked in 2012 when it increased over 44% from the previous year. The 2012 rates in decreasing order were steroids > D-methamphetamine > morphine > oxymorphone > oxycodone > codeine > D-amphetamine > hydromorphone > hydrocodone. This comprehensive analysis showed that the majority of the Army's MRO reviews were associated with the use of authorized prescriptions; however, there appears to be significant abuse of oxycodone and D-amphetamine.

Observations on hydrocodone and its metabolites in oral fluid specimens of the pain population: comparison with urine.

OBJECTIVE: Hydrocodone undergoes metabolism via cytochrome P450 (CYP) 3A4 (N-demethylation) to norhydrocodone and via CYP2D6 (O-demethylation) to hydromorphone. Hydrocodone, hydromorphone, and norhydrocodone are excreted in urine and secreted in saliva. The goal was to characterize hydrocodone and its metabolites in oral fluid specimens of a pain population and compare to urine specimens. DESIGN: This retrospective analysis included more than 8,500 oral fluid specimens and more than 250,000 urine specimens collected between March and June 2012 that were sent to Millennium Laboratories (San Diego, CA) and analyzed for hydrocodone, hydromorphone, and norhydrocodone using liquid chromatography-tandem mass spectrometry. Statistical analyses and linear regressions were conducted using Microsoft Excel® 2010 and OriginPro v8.6. RESULTS: The median oral fluid concentrations of hydrocodone and norhydrocodone were 122 and 7.7 ng/mL, respectively. However, the oral fluid concentrations of hydromorphone were below detection in many specimens (<1 ng/mL). The positive detection rate of parent drug and metabolites in oral fluid (17-31 percent detection rates) was much lower than in urine (63-75 percent detection rates). The geometric median metabolic ratio (MR) of norhydrocodone to hydrocodone was 0.07 in oral fluid and 1.2 in urine. The observed hydrocodone oral fluid concentrations were approximately 10-fold greater than previously reported plasma concentrations. CONCLUSION: Oral fluid had a much lower norhydrocodone to hydrocodone MR compared to urine. Reference ranges for oral fluid drug concentrations should not be extrapolated from plasma ranges. The observed ranges of secreted hydrocodone and metabolite concentrations in oral fluid should help determine reference ranges for medication monitoring.

Observations on the urine metabolic profile of codeine in pain patients.

This retrospective data analysis explored the relationship between codeine and its metabolites morphine, hydrocodone and hydromorphone. The objectives were: (i) to determine urine concentrations and mole fractions of codeine and metabolites and (ii) to examine the effect of cytochrome P450 (CYP) 2D6 inhibition on metabolite mole fractions. De-identified urine specimens were collected between September 2010 and July 2011 and analyzed using LC-MS-MS to determine codeine, morphine, hydrocodone and hydromorphone concentrations. Geometric mean urine concentrations were 0.833, 0.085 and 0.055 for morphine, hydrocodone and hydromorphone, respectively. Mole fractions were 0.23, 0.025 and 0.014 for morphine, hydrocodone and hydromorphone, respectively. The fraction of excreted codeine in the urine increased (slope = 0.06 ± .01, R² = 0.02) with total moles. As the total amount of codeine and metabolites increased, the fraction of codeine increased, while the fraction of active metabolites decreased. CYP2D6 inhibition with paroxetine, fluoxetine, bupropion and methadone significantly decreased the fraction of morphine excreted. The prevalence of codeine metabolism to morphine was considerably higher than codeine to hydrocodone. The urine concentration of codeine excreted was the greatest, followed by morphine and hydrocodone. Subjects should be monitored during concomitant use of codeine and CYP2D6 inhibitors as this affects the amount of morphine metabolite formation.

Prescription opioids. II. Metabolism and excretion patterns of hydrocodone 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.

Excretion profile of hydrocodone, hydromorphone and norhydrocodone in urine following single dose administration of hydrocodone to healthy volunteers.

Abuse of prescription opioids for non-medical use has been on the rise over the past decade. The most commonly abused opioid is hydrocodone, a frequently prescribed pain medication metabolized by the body to hydromorphone, norhydrocodone and other minor metabolites. This study describes the excretion profile of hydrocodone, hydromorphone and norhydrocodone in urine following a single dose (10 mg) administration of hydrocodone to human subjects (n = 7) and presents a validated liquid chromatography-tandem mass spectrometry method for analysis of the drug and its metabolites. Limit of quantitation was 5 ng/mL for all analytes; limit of detection was 2.5 ng/mL for hydrocodone and norhydrocodone and 5 ng/mL for hydromorphone. Peak concentrations of hydrocodone were found at 3:30-7:00 hours post-dose and were in the range of 612-2,190 ng/mL. Hydromorphone peak concentrations were found at 6:15-26:45 hours post-dose and ranged from 102 to 342 ng/mL. For norhydrocodone, peak concentrations were found at 4:20-13:00 hours post-dose and ranged from 811 to 3,460 ng/mL. Although hydromorphone was found at lower levels than hydrocodone, in six of seven subjects, it persisted for as long as hydrocodone was detected. Norhydrocodone was found at higher levels and lasted for a longer period of time than hydrocodone, thus making the nor-metabolite a valuable tool in evaluating hydrocodone use and/or misuse.

Relationship between the concentration of hydrocodone and its conversion to hydromorphone in chronic pain patients using urinary excretion data.

Hydrocodone in combination with acetaminophen is commonly used to control moderate pain and is metabolized by cytochrome P4502D6 to form the active metabolite, hydromorphone. The purpose of this study was to determine the metabolic relationship and variability between hydrocodone and its conversion to hydromorphone using urinary excretion data from chronic pain patients. Liquid chromatography-tandem mass spectrometry was used to quantitate hydrocodone and hydromorphone concentrations in urine specimens. The first visits of 25,200 subjects who took hydrocodone and not hydromorphone and had measurable concentrations were included in this study. The geometric mean (95% confidence index) of hydrocodone and hydromorphone urine concentrations were 1.39 (1.37-1.41) mg per gram of creatinine and 0.224 (0.221-0.227) mg per gram of creatinine, respectively. The log of creatinine-corrected hydromorphone versus the log of creatinine-corrected hydrocodone showed a positive relationship (R(2) = 0.20), with 60-fold variability between subjects. The plot of the log of the metabolic ratio ([hydromorphone] divided by [hydrocodone]) versus the log of creatinine-corrected hydrocodone had a coefficient of determination of R(2) = 0.42, with 125-fold variability between subjects. Ultra-rapid metabolizers represented 0.6% of the population, whereas 4% were poor metabolizers. Within-subject variability for the excretion of hydrocodone in urine was 23-fold, whereas between-subject variability was 134-fold. Hydrocodone and hydromorphone urine concentrations showed great variability within and between subjects.

Dilute and shoot: analysis of drugs of abuse using selected reaction monitoring for quantification and full scan product ion spectra for identification.

Our objective was to develop a "dilute and shoot" liquid chromatography-tandem mass spectrometry confirmatory procedure that uses full scan product ion spectra to identify drugs that are present above cutoff values as determined by isotope dilution relative to a deuterium-labeled internal standard. Deuterium-labeled internal standards are added to urine which is then diluted prior to analysis. Full scan product ion spectra were obtained in the data-dependent mode using a linear ion trap (ABI 4000 Qtrap). Identification was based on a purity fit of greater than 70. Ninety-seven urine specimens were analyzed by the method described, and results were compared to values obtained from a reference laboratory using selected reaction monitoring (SRM). The ion trap provided about 30-fold increase in signal-to-noise ratio as compared with the same instrument operated in a traditional full scan product ion mode. The assays were linear to at least 10 times the cutoff. Selecting appropriate triggers for obtaining full scan product ion spectra minimized space charging for specimens that contained high concentrations of drugs. There was 100% concordance between the full scan identification and the SRM results for identification of amphetamine, methamphetamine, benzoylecgonine, morphine, codeine, hydrocodone, and hydromorphone. The ability to "dilute and shoot" reduces the turnaround time for results. The data acquired with SRM and full scan product ion spectra provide accurate quantification and a high degree of specificity.

Hydrophilic interaction LC-MS/MS analysis of opioids in urine: significance of glucuronide metabolites.

BACKGROUND: In clinical laboratories, a large proportion of the toxicology workload is drug confirmations. Our GC-MS method for opioid confirmation detects total codeine, morphine, hydrocodone, hydromorphone, oxycodone and oxymorphone. The objective of this study was to develop a LC-MS/MS assay measuring the above drugs and glucuronide metabolites. In addition, to determine if measuring free drug only would lead to false negative results. RESULTS: In 85 patient urines, 43% were positive for morphine glucuronide, but not morphine, 48% were positive for hydromorphone glucuronide, but not hydromorphone, 33% were positive for codeine glucuronide, but not codeine, and 44% were positive for oxymorphone glucuronide, but not oxymorphone. CONCLUSION: We developed an LC-MS/MS assay capable of detecting codeine, morphine, hydrocodone, hydromorphone, oxycodone, oxymorphone and glucuronide metabolites. Detection of free drug only led to false negative confirmations.

Evaluation of the usefulness of an oxycodone immunoassay in combination with a traditional opiate immunoassay for the screening of opiates in urine.

Oxycodone is a semisynthetic opioid analgesic largely prescribed for post-operative and chronic pain management. The introduction of a slow release formulation of oxycodone has led to its frequent abuse and to an increase in emergency cases related to oxycodone overdose. Until recently, oxycodone testing has been confined to gas chromatography-mass spectrometry (GC-MS) analysis because the widely used automated opiate immunoassays poorly react to this compound. We investigated the utility of a new oxycodone immunoassay as a screening procedure to eliminate inappropriate GC-MS testing of negative urine specimens. We analyzed 96 urine specimens using GC-MS and two immunoassays, CEDIA((R)) opiates and DRI((R)) oxycodone assays from Microgenics, on a Hitachi 917 analyzer. The GC-MS allowed us to detect codeine, hydrocodone, hydromorphone, morphine, oxycodone, and oxymorphone following enzymatic hydrolysis and derivation by acetylation. The combination of the two immunoassays gave the best performance (98% sensitivity and specificity) when considering a positive result from GC-MS for any of the opiates. Considering positive GC-MS results for oxycodone or oxymorphone only, the oxycodone immunoassay resulted in two false-positives and one false-negative (50 ng/mL cutoff). Using these immunoassays for screening before GC-MS analysis provides a reduced opiate GC-MS workload without compromising quality.

Quantitation of opioids in blood and urine using gas chromatography-mass spectrometry (GC-MS).

The opioid and 6-acetylmorphine assays utilize gas chromatography-mass spectrometry (GC-MS) for the analysis of morphine, codeine, hydromorphone, hydrocodone, and 6-acetylmorphine in blood and urine. The specimens are fortified with deuterated internal standard and a five-point calibration curve is constructed. Specimens are extracted by mixed-mode solid phase extraction. The morphine, codeine, hydromorphone, hydrocodone, and 6-acetylmorphine extracts are derivatized with N-methyl-bis(trifluoroacetamide) (MBTFA) producing trifluoroacetyl derivatives. The final extracts are then analyzed using selected ion monitoring GC-MS.

Quantitation of morphine, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, and 6-monoacetylmorphine (6-MAM) in urine, blood, serum, or plasma using liquid chromatography with tandem mass spectrometry detection.

Opiates and opioids currently rank among the most commonly prescribed pain medications. We describe two liquid chromatography tandem mass spectrometry (LC-MS-MS) methods for the quantification of morphine, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, and 6-monoacetylmorphine (6-MAM). In the first, urine samples are pretreated by acidifying with sodium acetate containing appropriate deuterated internal standards and hydrolyzed with beta-glucuronidase. Samples are cooled, diluted with water, vortexed, centrifuged, and a portion is transferred to an autosampler vial for analysis. The second method allows for the measurement of the compounds in blood, serum, or plasma specimens. Analysis of these samples involves pretreatment with acetonitrile containing deuterated internal standards to deproteinize the sample, which is subsequently vortexed and centrifuged. A portion of the organic layer is transferred to a clean test tube, dried under nitrogen, and reconstituted with water for analysis. Quantitation of analytes is accomplished using a commercially available single-point calibrator (urine samples) or an in-house prepared six-point standard curve (blood samples).