Quantification of Opioids in Urine Using an Aptamer-Based Free-Solution Assay.

The opioid epidemic continues in the United States. Many have been impacted by this epidemic, including neonates who exhibit Neonatal Abstinence Syndrome (NAS). Opioid diagnosis and NAS can be negatively impacted by limited testing options outside the hospital, due to poor assay performance, false-negatives, rapid drug clearance rates, and difficulty in obtaining enough specimen for testing. Here we report a small volume urine assay for oxycodone, hydrocodone, fentanyl, noroxycodone, norhydrocodone, and norfentanyl with excellent LODs and LOQs. The free-solution assay (FSA), coupled with high affinity DNA aptamer probes and a compensated interferometric reader (CIR), represents a potential solution for quantifying opioids rapidly, at high sensitivity, and noninvasively on small sample volumes. The mix-and-read test is 5- to 275-fold and 50- to 1250-fold more sensitive than LC-MS/MS and immunoassays, respectively. Using FSA, oxycodone, hydrocodone, fentanyl, and their urinary metabolites were quantified using 10 µL of urine at 28-81 pg/mL, with >95% specificity and excellent accuracy in ∼1 h. The assay sensitivity, small sample size requirement, and speed could enable opioid screening, particularly for neonates, and points to the potential for pharmacokinetic tracking.

Dilution of Urine Followed by Adulteration in an Attempt to Deceive the Laboratory.

Adulteration of samples submitted for toxicological analyses can present unique challenges to non-forensic clinical laboratories. With the number of overdose-related deaths expected to surpass 60,000 in 2018, it is incumbent on all members of the healthcare team to be active participants in curbing opioid dependence and identifying prescription drug misuse and diversion. Recently published guidelines have sought to provide guidance to laboratories overseeing prescription drug-monitoring programs. We present a case of sample adulteration in an attempt to conceal prescription non-compliance. The patient possessed only an active prescription for hydrocodone but on initial antibody-based screening the sample tested positive for benzodiazepines and oxycodone in addition to opiates. Active communication between the pain management clinic and the clinical laboratory alerted staff to conduct a more thorough investigation including sample validity testing, analyses of paired serum specimens by liquid chromatography tandem mass spectrometry. Analyses revealed the patient submitted a dilute urine specimen with a crushed hydrocodone pill inside in an attempt to hide prescription non-compliance. Previous screenings had been consistent with the medication list raising the question of whether this was an isolated incident or the patient had simply been more successful in manipulating specimens in the past. This case highlights the need for good communication among all members of the healthcare team and the widespread implementation of specimen validity testing for any laboratory that receives samples from pain clinics.

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.

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.

Urinary hydrocodone and metabolite distributions in pain patients.

Hydrocodone combined with acetaminophen is commonly used for moderate pain. Hydrocodone is metabolized by cytochrome P450 (CYP) 2D6 into hydromorphone and by CYP3A4 into norhydrocodone. This was a retrospective study evaluating hydrocodone, hydromorphone and norhydrocodone distributions in urine. Urine specimens (n = 76,924) were obtained from patients on chronic opioid therapy during their first or single visit and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The patients were at least 16 years of age and had documented hydrocodone use via a medication list. There were 48,710 specimens that were positive for all three analytes. Mean hydrocodone, hydromorphone and norhydrocodone mole fractions (95% confidence interval) were 0.39 (0.38-0.39), 0.12 (0.11-0.12) and 0.49 (0.48-0.49), respectively. Hydromorphone fractions were lower in women compared with men (0.11 versus 0.13; P < 0.0001). Hydrocodone mole fractions were higher in the 65-year and older age group compared with the 16- to 39-year age group (0.4 versus 0.36; P ≤ 0.005). Concurrent use of a CYP2D6 and/or CYP3A4 inhibitor altered hydromorphone and norhydrocodone mole fractions, compared with the control group. Patient factors affect hydrocodone and metabolite mole fractions and suggest increased awareness of their contribution when attempting to interpret urine drug testing results.

Observation of improved adherence with frequent urine drug testing in patients with pain.

OBJECTIVE: To determine the relationship between urine drug testing (UDT) frequency and patient adherence for prescribed buprenorphine, carisoprodol, fentanyl, hydrocodone, methadone, morphine, and oxycodone. SETTING: Patients with pain routinely seen by private practitioners. DESIGN: A retrospective analysis was conducted on urinary excretion data analyzed by Millennium Laboratories between March 2008 and May 2011. PATIENT PARTICIPANTS: Patients in the United States with chronic pain who underwent routine UDT to confirm adherence for prescribed medications. INTERVENTIONS: Adherence for the urine drug test was defined as the presence of parent drug and/or metabolite(s) greater than or equal to the lower limit of quantitation. The percent of adherence for prescribed medications was compared to the average percent of the same in subjects with five or more visits. MAIN OUTCOMES: Correlation analyses were used to determine the relationship between adherence for prescribed medications and number of visits. RESULTS: There were 255,168 specimens submitted for testing from 166,755 individuals. When monitoring with more frequent visits (≥5 visits) adherence was higher by 1 percent for buprenorphine (89 percent vs 88 percent); 8 percent for carisoprodol (77 percent vs 69 percent); 5 percent for fentanyl (95 percent vs 90 percent); 7 percent for hydrocodone (83 percent vs 76 percent); 3 percent for methadone (96 percent vs 93 percent); 5 percent for morphine (92 percent vs 87 percent); and 8 percent for oxycodone (90 percent vs 82 percent). CONCLUSIONS: Adherence for prescribed medications is higher with frequent urine monitoring. UDT can be used as tool that may help improve this in patients with chronic pain.

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.

Update on hydrocodone metabolites in rats and dogs aided with a semi-automatic software for metabolite identification Mass-MetaSite.

1. There has been a lack of in vivo metabolite profiling update of hydrocodone since the original report on species differences was published in 1978. As such, the mechanism for its analgesic activity in different species has been ambiguous. To address safety concern from regulatory agencies, hydrocodone metabolite profiles in rats and dogs are updated herein aided by a newly developed software, Mass-MetaSite. 2. Samples collected from rats and dogs dosed orally with hydrocodone were analyzed with reversed phase liquid chromatography coupled with LTQ-Orbitrap. The exact mass measurement data collected with data-dependent acquisition methodology were analyzed both traditionally, using Xcalibur Qual Browser and MetWorks, and by Mass-MetaSite. 3. Profiling of hydrocodone metabolites in rat and dog plasma reflected previously reported species differences in circulating metabolites. While hydrocodone mainly underwent O-demethylation and ketone reduction in rats forming hydromorphone and reduced hydromorphone, which were then subsequently cleared via glucuronide conjugation, hydrocodone in dogs was cleared predominantly by N-demethylation and N-oxidation. 4. Given the success ratio of metabolite detection offered by Mass-MetaSite, the software will be able to aid chemists in early identification of drug metabolites from complex biomatrices.

Illicit drug use correlates with negative urine drug test results for prescribed hydrocodone, oxycodone, and morphine.

BACKGROUND: A number of studies indicate that 10.8%-34% of patients with chronic pain use illicit drugs. One hypothesis for this occurrence is that some patients may be supplementing their prescription medications with illicit drugs. OBJECTIVE: The primary purpose of this retrospective data analysis was to test the hypothesis that people whose urine specimens are positive for the medications that have been listed as being prescribed to them are positive for fewer illicit substances than those whose specimens were negative for their prescribed medications. The secondary purpose of the study was to correlate the use of illicit drugs and the amount of prescribed medications excreted in urine. STUDY DESIGN: A retrospective study of the incidence of patients using illicit drugs versus their consistency with reported medications. METHODS: Using urine specimens from a cohort of nearly 400,000 patients whose identities had been redacted, and who were being treated for chronic pain with opioid therapy, this study was performed to correlate the patients' positivity with their prescribed medication to the prevalence of illicit substance use. A secondary study was conducted to correlate the amount of prescribed medication excreted in urine (measured in ng/mL) with the incidence of illicit drug use. The specific prescription medications analyzed were hydrocodone, morphine, and oxycodone. RESULTS: Specimens defined as negative for prescribed hydrocodone (27.3%), morphine (11.5%) or oxycodone (19%) were more likely to contain illicit drugs than those found to be positive for the prescribed medication. The illicit drug prevalence among the inconsistent specimens was 15.3% for hydrocodone, 23.8% for morphine, and 24.4% for oxycodone. The secondary study showed no statistically significant difference in the excretion level of prescribed medication between those patients using and not using illicit drugs. LIMITATIONS: The study is limited in that no data was obtained to determine the causal relationships of illicit drug use. CONCLUSIONS: This work supports the hypothesis that people who are positive for their prescribed medications use fewer illicit drugs than those who do not take their medications. It may be beneficial for physicians to test more thoroughly for illicit drugs when patients' drug tests are negative for their prescribed medications.

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.