Detection of Hydrocodone and Morphine as Metabolites in Oral Fluid by LC-MS/MS in Patients Prescribed Codeine.

A retrospective analysis of oral fluid drug testing results using LC-MS/MS was performed to determine the prevalence rates in oral fluid for codeine (COD) and 3 COD metabolites-morphine (MOR), norhydrocodone (NHC), and hydrocodone (HCOD). Oral fluid samples were collected using Quantisal oral fluid collection device (Immunalysis Inc.) and submitted to Millennium Health, LLC for the routine drug analysis by LC-MS/MS. Consistent with previously published literature, COD was the primary analyte detected in oral fluid after the use of COD. In COD-positive samples, HCOD, MOR, and NHC were detected at rates of 68.4%, 18.4%, and 6.3%, respectively. Concentration ranges of these analytes were 1.0 to >2000 ng/mL for COD, 1.0-20.2 ng/mL for MOR, 1.0-740.0 ng/mL for HCOD, and 2.1-47.5 ng/mL for NHC. In contrast to urine, where HCOD is typically detected as a minor metabolite of COD, HCOD was the most commonly detected metabolite in oral fluid in samples testing positive for COD with reported prescriptions for COD. This observation suggests that care should be taken when interpreting HCOD positives in oral fluid results, and that the use of COD should be considered as one possible explanation for HCOD positives.

Wastewater Effluent Hydrocodone Concentrations as an Indicator of Drug Disposal Program Success.

Drug disposal programs have been seen as a remedy to the concern of environmental contamination resulting from pharmaceutical disposal down the toilet or sink; however a thorough review of peer-reviewed literature and publicly available information on these programs indicates limited research has been conducted to validate the effectiveness of these programs at reducing pharmaceuticals in the environment. The purpose of this research was to determine if drug disposal programs could actually reduce pharmaceutical residues in the environment. The concentration of hydrocodone in wastewater effluent released from a wastewater treatment plant in Denton, Texas was monitored before and after a take back program called Denton Drug Disposal Day (D4). Data collected and analyzed suggests D4 events were successful in contributing to a reduction of pharmaceutical loading to the environment; however there was insufficient evidence to demonstrate that D4 events were exclusively responsible for these improvements.

A preliminary study of hydrocodone and hydromorphone to oxycodone ratios for distinguishing impurities from independent opioid use.

BACKGROUND: Urine drug testing (UDT) monitors prescription compliance and/or drug abuse. However, interpretation of UDT results obtained by liquid chromatography-tandem mass spectrometry (LC-MS-MS) can be complicated by the presence of drug impurities that are detected by highly sensitive methods. Hydrocodone is a drug impurity that can be found as high as 1% in oxycodone pills. OBJECTIVES: We evaluated the frequency and concentration of hydrocodone and its metabolite, hydromorphone, in patients taking oxycodone to check if the ratio of hydrocodone or hydromorphone to oxycodone could distinguish between oxycodone only use from those consuming additional opiates. DESIGN & METHODS: We correlated LC-MS/MS results with medication records of 319 patients with positive oxycodone results over 7 months (4/2021-11/2021). RESULTS: Fifteen of 319 patients with positive oxycodone results were taking oxycodone only. For these 15 patients, the mean ratio of hydrocodone to oxycodone was 0.57% (range 0.05%-3.35%), and the mean ratio of hydromorphone to oxycodone was 0.81% (range 0.18-3.51%). CONCLUSIONS: Hydrocodone and/or hydromorphone are detectable in patients taking only oxycodone and can likely be identified as an impurity if their calculated ratio to oxycodone is <1 %. Further validation of the ratios in a larger sample size is recommended.

The Prevalence of Opioids in US Drinking Water Sources Detected Using Direct-Injection High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Active pharmaceutical ingredient (API) contamination of water sources, including opioid contamination, has become more common in recent years. Although drinking water-treatment plants help mitigate API infiltration, API contamination remains in some drinking water sources. Therefore, the ability to detect APIs at ultratrace concentrations is vital to ensure safe drinking water. A method for the ultratrace determination of fentanyl, hydrocodone, and codeine in drinking water via direct injection and high-performance liquid-chromatography tandem mass spectrometry (HPLC-MS/MS) was developed and validated. Drinking water samples (10 ml) are simply syringe-filtered and then analyzed by HPLC-MS/MS. A wide linear range (0.25-100 ng/L) and ultratrace limits of detection (80, 150, and 500 pg/L for fentanyl, hydrocodone, and codeine, respectively) were features of the method. The method produced excellent aggregate accuracies of 90%-115% and precisions of ≤11% for the three analytes tested. This method was used to test drinking water samples from 53 US locations, with hydrocodone and codeine detected in approximately 40% of the samples tested at concentrations between 0.3 and 20 ng/L. Codeine was detected at higher concentrations than hydrocodone (up to 7.3 times) for each sample containing these APIs. Fentanyl was not detected in any field drinking water sample. The detection of opioids in a large fraction of the US drinking water samples tested is cause for concern, and these levels should continue to be monitored to ensure that they do not become a threat to human health. Environ Toxicol Chem 2022;41:2658-2666. © 2022 SETAC.

[Simultaneous determination of 11 opiates in hair by liquid chromatography-tandem mass spectrometry].

The paper reports the establishment of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous analysis of 11 opiates in hair samples, and the study of presence of opiates in the hair of active heroin addicts. About 20 mg of decontaminated and pulverized hair sample was hydrolyzed with buffer solution for 30 min, in the presence of morphine-d3 and acetylmorphine-d6 used as internal standards, and then extracted with the mixture of dichlormethane and isopropanol, separated by the Allure PFP propyl column with a mobile phase consisting of acetonitrile and 20 mmol L(-1) ammonium acetate buffer, and then analyzed by LC-MS/MS. Multiple reaction monitoring (MRM) mode was used to analyze 11 opiates. Eleven opiates showed a fairly good linearity over the corresponding range (r > 0.996 0). The detection limits were less than 0.05 ng mg(-1). The recoveries were between 47.2% and 110%, and the deviations of intra- and inter-day precision were less than 14%. Heroin, acetylmorphine, morphine, codeine, acetylcodeine and hydrocodone were detected in hair samples of 21 herion addicts. The developed method shows high sensitivity and selectivity, and is suitable for the simultaneous analysis of 11 opiates in hair samples and identify legal and illegal use of opiates.

Opioid Deaths in Milwaukee County, Wisconsin 2013-2017: The Primacy of Heroin and Fentanyl.

Heroin and fentanyl are the overwhelming and increasing cause of opioid deaths in Milwaukee County, Wisconsin. We reviewed all drug and opioid deaths from 2013 to 2017 to delineate the specific opioid drugs involved and changes in their incidence. From 2013 to 2017, 980 deaths were due to opioids, rising from 184 in 2013 to 337 in 2017. In 2017, opioid deaths exceeded combined non-natural deaths from homicide and suicide. Illicit heroin and fentanyl/analogs caused 84% of opioid deaths and 80% of drug deaths, with no increase in deaths due to oral prescription drugs such as oxycodone and hydrocodone. Any approach to decreasing this dramatic increase in opioid deaths should first focus on interdicting the supply and cheap availability of these illicit opioids. Fentanyl and its analogs represent the most deadly opioids and the greatest threat to human life in our population.

Positivity rates of drugs in patients treated for opioid dependence with buprenorphine: A comparison of oral fluid and urine using paired collections and LC-MS/MS.

BACKGROUND: Drug testing is recommended as part of comprehensive monitoring for medication-assisted treatment. Alternative matrices including oral fluid offer a number of advantages when compared with conventional urine testing but are not as well characterized. This study aims to compare positivity rates of drugs and drug classes in oral fluid and urine as a measure of the clinical utility of oral fluid in the evaluation and treatment of patients with opioid use disorders. METHODS: A retrospective review of paired oral fluid and urine test results from Millennium Health's laboratory database was performed for 2746 patients with reported prescriptions for buprenorphine products used in the treatment of opioid dependence. Specimens were tested using quantitative LC-MS/MS for 34 medications, metabolites and illicit drugs. RESULTS: A number of medications and illicit drugs were detected at comparable or higher rates in oral fluid vs. urine such as cocaine (15.7% vs. 7.9%), opiates (13.4% vs. 10.0%), oxycodone (8.6% vs. 3.7%), hydrocodone (3.0% vs. 1.2%) and others. Lower detection rates were observed in oral fluid vs. urine for benzodiazepines (6.6% vs. 8.7%), cannabinoids (15.5% vs. 19.5%), oxymorphone (1.8% vs. 3.1%) and hydromorphone (0.8% vs. 4.5%). CONCLUSIONS: Clinicians may find oral fluid advantageous for detection of specific drugs and medications in certain clinical situations. Understanding the relative differences between urine and oral fluid can help clinicians carefully select tests best suited for detection in their respective matrix. To our knowledge, this is the largest inter-matrix patient comparison study using paired collections and direct to definitive testing.

Development of a Quantitative LC-MS-MS Assay for Codeine, Morphine, 6-Acetylmorphine, Hydrocodone, Hydromorphone, Oxycodone and Oxymorphone in Neat Oral Fluid.

Recent advances in analytical capabilities allowing for the identification and quantification of drugs and metabolites in small volumes at low concentrations have made oral fluid a viable matrix for drug testing. Oral fluid is an attractive matrix option due to its relative ease of collection, reduced privacy concerns for observed collections and difficulty to adulterate. The work presented here details the development and validation of a liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone in neat oral fluid. The calibration range is 0.4-150 ng/mL for 6-acetylmorphine and 1.5-350 ng/mL for all other analytes. Within-run and between-run precision were <5% for all analytes except for hydrocodone, which had 6.2 %CV between runs. Matrix effects, while evident, could be controlled using matrix-matched controls and calibrators with deuterated internal standards. The assay was developed in accordance with the proposed mandatory guidelines for opioid confirmation in federally regulated workplace drug testing. The use of neat oral fluid, as opposed to a collection device, enables collection of a single sample that can be split into separate specimens.

Simultaneous determination of codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine in urine, serum, plasma, whole blood, and meconium by LC-MS-MS.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous analysis of six major opiates in urine, serum, plasma, whole blood, and meconium is described. The six opiates included are codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine (6-AM). The method was compared to an in-house gas chromatography (GC)-MS method and an LC-MS-MS method performed by another laboratory. The sample preparation time was decreased by eliminating the glucuronide hydrolysis and derivatization required for GC-MS analysis, as well as by adapting the solid-phase extraction to elute directly into autosampler vials. These improvements illustrate the advantages of an LC-MS-MS method over a GC-MS method for opiates. The structural similarity of these six opiates and others in the opiate class causes a high potential for interference and false-positive results. Twelve opiate analogues and metabolites were evaluated for interference. The potential for interference was reduced by altering the MRM transitions chosen for the six opiates. The increased specificity of LC-MS-MS decreased the interference rate in urine to 3.9% compared to 13.6% on the in-house GC-MS method. The rate of positivity for 6-AM in meconium is described for the first time. In urine, 11.0% of morphine positive specimens were also positive for 6-AM compared to 8.3% in serum/plasma and 0.9% in meconium. Although 6-AM is infrequent in meconium, it provides a definitive proof of illegal heroin abuse by the pregnant mother. This method has been routinely used in our laboratory over the last 6 months on more than 1500 patient specimens.

Disposition of hydrocodone in hair.

The use of prescription drugs, including synthetic opiates, is increasing in the U.S., with emergency room reports showing a dramatic rise in prescription opiate abuse. As part of an ongoing study, the hair of admitted opiate users was analyzed for hydrocodone and hydromorphone, as well as codeine, morphine, and 6-acetylmorphine in order to determine if there was any correlation between self-reported frequency of opiate intake and the concentration of drug detected in hair. The hairs were confirmed using gas chromatography-mass spectrometry following screening by enzyme linked immunosorbent assay (ELISA). Twenty-four hair specimens collected from volunteers showed the presence of hydrocodone (130-15,933 pg/mg); four of those also contained hydromorphone (59-504 pg/mg). The specimens were also analyzed for morphine, codeine, and 6-acetylmorphine. Hair specimens from five self-reported codeine users showed concentrations of hydrocodone between 592 and 15,933 pg/mg. In addition, codeine was present at concentrations of 575-20,543 pg/mg, but neither morphine nor hydromorphone were present in any of those hair specimens. Though the analysis of some opiates in hair has been previously published, this is the first study where the hydrocodone and hydromorphone concentrations have been measured following self-reported opiate intake.

[Determination of opiates in biological human samples by liquid chromatography-tandem mass spectrometry].

OBJECTIVE: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of opiates in biological samples according to the emerging problem in drugs abuse. METHODS: Opiates such as heroin, 6-acetylmorphine, morphine, codeine, acetylcodeine, hydrocodone and hydromorphone were isolated from human blood, urine, oral fluid and hair using a simple extraction and consequently analyzed using LC-MS/MS. The method was evaluated by real cases. RESULTS: The mobile phase give the optimum separation for opiates. The detection limit of morphine in urine with dilution and liquid-liquid extraction and in hair is 10ng/mL, 0.01 ng/mL and 0.01 ng/mg, respectively. CONCLUSION: The method is simple and rapid, offering superior sensitivity and selectivity for opiates. The target compounds comprising hydrocodone and hydromorphone enlarge the applied area.

Hair analysis for opiates: hydromorphone and hydrocodone as indicators of heroin use.

BACKGROUND: Identification of external contamination is a challenge in hair analysis. This study investigates metabolite ratios of hydromorphone to morphine and hydrocodone to codeine as indicators to distinguish contamination from heroin use provided that hydromorphone/hydrocodone intake is excluded. RESULTS: Hair samples after external contamination with street heroin proved to be negative for hydromorphone/hydrocodone. Hair samples from individuals with suspected street heroin use/contamination or opiate medication were analyzed for 6-monoacetylmorphine, morphine, acetylcodeine, codeine, hydromorphone and hydrocodone, and metabolite ratios of hydromorphone to morphine and hydrocodone to codeine were assessed. Hair samples from individuals with medicinal heroin/morphine/codeine use displayed significantly higher metabolite ratios than those with suspected street heroin use/contamination. CONCLUSION: Hydromorphone/hydrocodone are solely formed during body passage. Thus, metabolite ratios can be used to distinguish morphine/heroin use from external contamination.

Evaluation and comparison of postmortem hydrocodone concentrations in peripheral blood, central blood and liver specimens: a minimal potential for redistribution.

Postmortem changes can alter the concentration of drugs in the vascular compartment as compared with concentrations originally present at the time of death. Numerous drugs have been reported to increase due to postmortem redistribution (PMR). The potential for PMR of hydrocodone, a therapeutic opioid analgesic used to manage pain, is of particular interest due to its wide use. Hydrocodone concentrations in 39 peripheral blood, central blood, and liver specimens were compared. Dihydrocodeine (DHC), a commonly encountered hydrocodone metabolite, was present in 61% of the cases with an average concentration that was 29% of the hydrocodone value. Central blood to peripheral blood hydrocodone ratios were well correlated (R(2)=0.965) with an average (±S.D.) of 1.3 (±0.35) and a median of 1.2. The liver to peripheral blood (L/P) hydrocodone ratio was also well correlated (R(2)=0.915) with an average (±S.D.) of 3.4 (±1.7) L/kg and a median of 3.0 L/kg. This low L/P ratio suggests that hydrocodone is unlikely to undergo substantial PMR changes.

Simultaneous assay of hydrocodone bitartrate and acetaminophen in a tablet formulation.

A reversed-phase pressurized liquid chromatographic procedure is presented for the simultaneous quantitation of hydrocodone bitartrate and acetaminophen in a tablet formulation. The separation method was based on an octadecylsilane column with a buffered (pH 4.5) methanol-water mobile phase. Measurement was with a UV spectrophotometer set at 283 nm, compared to external standards. Assays for the active ingredients in tablet samples averaged 99.7% of the label claim for hydrocodone bitartrate and 100.3% for acetaminophen. The respective relative standard deviations of the retention time and precision were 2.2 and 1.75% for hydrocodone and 3.3 and 0.95% for acetaminophen. The range of interest studied was 0.035 to 0.065 mg/ml for hydrocodone bitartrate and 3.50 to 6.50 mg/ml for acetaminophen. The assay method was also compared to colorimetric and USP procedures for the active ingredients. The method was suitable for control, content uniformity, and stability-indicating use.

The detection of hydrocodone in meconium: two case studies.

The detection of hydrocodone in meconium samples is reported for the first time. Hydrocodone, a semisynthetic antitussive and analgesic, is often prescribed as a painkiller following minor surgery or dental work. It also cross-reacts in enzyme and fluorescence polarization immunoassay opiate systems. In two cases recently received by our laboratory, hydrocodone was detected following a positive opiate immunoassay screening result. The meconium samples were not hydrolyzed because previous work in our laboratory showed that greater than 70% of morphine in meconium is not glucuronide bound. The confirmatory procedures showed that, in case No. 1, codeine was also detected but at a much lower concentration than the hydrocodone. In case No. 2, morphine was detected but, again, at a much lower concentration than the hydrocodone. Even though in both samples another opiate was also detected, the possibility still remains that a positive opiate test result may be reported by screen-only laboratories based solely on the presence of hydrocodone. We present a novel extraction method and a gas chromatographic-mass spectrometric confirmatory procedure for the determination of hydrocodone and hydromorphone in meconium.

A closer look at acetyl and pentafluoropropionyl derivatives for quantitative analysis of morphine and codeine by gas chromatography/mass spectrometry.

PFPA and acetic anhydride derivatives of morphine and codeine were evaluated with respect to stability, chromatography, potential for analytical interferences by other opiates, and suitability of major fragment ions for analysis by GC/MS with deuterated internal standards and selected ion monitoring (SIM). The PFPA derivatives showed acceptable stability and could be analyzed without interference from other opiates, but the codeine derivative had relatively poor chromatography and its mass spectrum had only two ions suitable for SIM. The acetic anhydride derivatives were stable and chromatographed well, but diacetyl hydromorphone enol, a minor product of derivatization of hydromorphone, interfered with analysis of morphine. 3-Monoacetylmorphine, a minor product of derivatization of morphine, prevented use of the abundant m/z 285 ion of derivatized D3-codeine as a qualifying ion in quantitative assays. The acetic anhydride derivative of morphine cannot be distinguished from the corresponding derivative of the heroin metabolite 6-monoacetylmorphine.

The simultaneous determination of codeine, morphine, hydrocodone, hydromorphone, 6-acetylmorphine, and oxycodone in hair and oral fluid.

Recently, the abuse of prescription opiates as alternatives to heroin has become a national concern. The determination of a six-drug opiate panel, codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, and oxycodone, in hair and oral fluid using solid-phase extraction and capillary gas chromatography-mass spectrometry (GC-MS) is described. Oral fluid was obtained from the donor by insertion of absorptive collectors into the mouth. Hair was collected from the patient and powdered using stainless steel ball bearings in a mini bead-beater apparatus. Opiates present in the samples were extracted from a buffered, aqueous matrix using a solid-phase cartridge. The extracts were concentrated and the methoxime/BSTFA derivatives prepared in order to eliminate interference from the keto-opiates. The extracts were separated by GC-MS in electron impact mode. By utilizing methoxyamine, we were able to produce the methoxime derivatives required for single derivative production and chromatographically separate all six opiates. The routine analysis of these opiates in hair and oral fluid using GC-MS is described for the first time.

[Evaluation of methods of trace analysis of various opiates including hydrocodone and hydromorphone in the blood and urine using gas chromatography-mass spectrometry]. / Zhodnocení metod stopové analýzy ruzných opiátu vcetne hydrokodonu a hydromorfonu v krvi a v moci pomocí GC-MS.

A GC-MS method suitable to conduct practical trace toxicological analyses of various opiates including hydrocodone or hydromorphone is presented both in scan and selected ion monitoring modes. Silylation can be a convenient reaction to derivatize unknown analytes in toxicological samples. However, silylation of drugs with tautomeric keto/enol groups in molecular structure can cause analytical problems due to poor reproducibility of conversion degree. After using N-Methyl-N-trimethyl-silyltrifluoracetamide (MSTFA) acceptable results were achieved.

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.

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.