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.

Hydromorphone and codeine concentrations in oral fluid specimens from patients receiving substitution therapy with Substitol™ (morphine sulfate).

This study aimed to determine whether hydromorphone and codeine can be detected in oral fluid specimens following administration of Substitol™, a slow-release formulation of morphine. This is of interest for those monitoring treatment compliance using drug testing. Oral fluid specimens collected for compliance assessment in routine clinical practice or as part of a clinical trial were subjected to quantitative analysis of hydromorphone, morphine, codeine, and 6-acetylmorphine using highly sensitive mass spectrometric methods. Oral fluid was collected using a Greiner Bio-One saliva collection system. Patients undergoing substitution treatment with Substitol™, methadone, or buprenorphine were included, together with patients undergoing pain treatment with hydromorphone. Hydromorphone was detected in 642 of the 663 (97%) samples from substitol-treated patients. Concentrations were not higher in methadone- and buprenorphine-treated patients who relapsed into heroin use, or in patients on hydromorphone therapy. Codeine was detected in 29% of the samples. These concentrations were lower than those in patients who had relapsed to heroin use. Clinical administration of morphine can lead to detectable concentrations of both hydromorphone and codeine in oral fluids. This should be taken into consideration when using drug testing in oral fluid samples for compliance assessment in this patient group.

Comparative Analysis of ELISA Immunoassay and LC-QTOF for Opiate Screening.

A comparative analysis of enzyme-linked immunosorbent assay (ELISA) and quadrupole time-of-flight mass spectrometry (LC-QTOF) for the detection of opioids in blood samples is presented. The Orange County Crime Lab (OCCL) was concerned that the opioid drug class was not accurately detected at low concentrations due to the use of LC-QTOF as a non-targeted screening method for multiple classes of drugs. In order to investigate this issue, 968 ante-mortem and postmortem blood samples were analyzed by ELISA for the presence of the following opioids: morphine, morphine-glucuronide, codeine, codeine-glucuronide, hydrocodone, hydromorphone, hydromorphone-glucuronide, oxycodone, oxymorphone and oxymorphone-glucuronide. All samples had been previously analyzed by LC-QTOF. Overall, 84 samples tested positive for opioids. Discrepant samples between ELISA and LC-QTOF were analyzed by a liquid chromatography tandem mass spectrometry confirmation method in order to determine the true composition of the sample. Upon review of the discrepant samples, no forensically relevant concentration of opioids was missed by LC-QTOF. Thus, the ability of the OCCL's LC-QTOF screening method was verified to detect opioids at low concentrations.

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.

Detection and quantification of codeine-6-glucuronide, hydromorphone-3-glucuronide, oxymorphone-3-glucuronide, morphine 3-glucuronide and morphine-6-glucuronide in human hair from opioid users by LC-MS-MS.

Current hair testing methods that rely solely on quantification of parent drug compounds are unable to definitively distinguish between drug use and external contamination. One possible solution to this problem is to confirm the presence of unique drug metabolites that cannot be present through contamination, such as phase II glucuronide conjugates. This work demonstrates for the first time that codeine-6-glucuronide, hydromorphone-3-glucuronide, oxymorphone-3-glucuronide, morphine-3-glucuronide and morphine-6-glucuronide are present at sufficient concentrations to be quantifiable in hair of opioid users and that their concentrations generally increase as the concentrations of the corresponding parent compounds increase. Here, we present a validated liquid chromatography tandem mass spectrometry method to quantify codeine-6-glucuronide, dihydrocodeine-6-glucuronide, hydromorphone-3-glucuronide, morphine-3-glucuronide, morphine-6-glucuronide, oxymorphone-3-glucuronide, codeine, dihydrocodeine, dihydromorphine, hydrocodone, hydromorphone, morphine, oxycodone, oxymorphone and 6-acetylmorphine in human hair. The method was used to analyze 46 human hair samples from known drug users that were confirmed positive for opioids by an independent laboratory. Glucuronide concentrations in samples positive for parent analytes ranged from ~1 to 25 pg/mg, and most samples had glucuronide concentrations in the range of ~1 to 5 pg/mg. Relative to the parent concentrations, the average concentrations of the four detected glucuronides were as follows: codeine-6-glucuronide, 2.33%; hydromorphone-3-glucuronide, 0.94%; oxymorphone-3-glucuronide, 0.77%; morphine 3-glucuronide, 0.59%; and morphine-6-glucuronide, 0.93%.

Identification of Drugs in Parenteral Pharmaceutical Preparations from a Quality Assurance and a Diversion Program by Direct Analysis in Real-Time AccuTOFTM-Mass Spectrometry (DART-MS).

In healthcare settings drug diversion and impairment of physicians are major concerns requiring a rapid and efficient method for surveillance and detection. A Direct Analysis in Real Time ion source coupled to a JEOL AccuTOFTM time-of-flight mass spectrometer (DART-MS) method was developed to screen parenteral pharmaceutical formulations for potential drug diversion. Parenteral pharmaceutical formulations are also known as injectable formulations and are used with intravenous, subcutaneous, intramuscular and intra-articular administration. A library was created using the mass spectra data collected by a DART-MS operated in switching mode at 20, 60 and 90 V settings. This library contained 17 commonly encountered drugs in parenteral pharmaceutical formulations that included the surgical analgesic: fentanyl, hydromorphone and morphine; anesthetic: baclofen, bupivacaine, ketamine, midazolam, ropivacaine and succinylcholine; and a mixture of other drug classes: caffeine, clonidine, dexamethasone, ephedrine, heparin, methadone, oxytocin and phenylephrine. Randomly selected 200 de-identified parenteral pharmaceutical formulations containing one or more drugs were submitted for analysis to the FIRM Toxicology Laboratory at Virginia Commonwealth University Health and were screened using the DART-MS. The drug contents of the de-identified formulations were previously confirmed by a published high performance liquid chromatography (HPLC) method. The drugs in the formulations were rapidly and successfully identified using the generated library. The DART-MS and HPLC results were in complete agreement for all 200 parenteral pharmaceutical formulations.

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.

Separation of opiate isomers using electrospray ionization and paper spray coupled to high-field asymmetric waveform ion mobility spectrometry.

One limitation in the growing field of ambient or direct analysis methods is reduced selectivity caused by the elimination of chromatographic separations prior to mass spectrometric analysis. We explored the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS), an ambient pressure ion mobility technique, to separate the closely related opiate isomers of morphine, hydromorphone, and norcodeine. These isomers cannot be distinguished by tandem mass spectrometry. Separation prior to MS analysis is, therefore, required to distinguish these compounds, which are important in clinical chemistry and toxicology. FAIMS was coupled to a triple quadrupole mass spectrometer, and ionization was performed using either a pneumatically assisted heated electrospray ionization source (H-ESI) or paper spray, a direct analysis method that has been applied to the direct analysis of dried blood spots and other complex samples. We found that FAIMS was capable of separating the three opiate structural isomers using both H-ESI and paper spray as the ionization source.

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.

Breast milk hydrocodone and hydromorphone levels in mothers using hydrocodone for postpartum pain.

OBJECTIVE: To estimate the extent of passage of hydrocodone and its active metabolite, hydromorphone, into breast milk. METHODS: This is a pharmacokinetic study of 30 postpartum women receiving hydrocodone bitartrate for postpartum pain in the inpatient setting. Mothers donated timed breast milk samples for the analysis of hydrocodone and hydromorphone. RESULTS: Fully breastfed neonates received 1.6% (range 0.2%-9%) of the maternal weight-adjusted hydrocodone bitartrate dosage. When combined with hydromorphone, the total median opiate dosage from breast milk is 0.7% of a therapeutic dosage for older infants. Most mothers excreted little to no hydromorphone into breast milk. CONCLUSION: Standard postpartum dosages of hydrocodone bitartrate appear to be acceptable to use in women nursing newborns. Prolonged use of high dosages is not advisable.

Accidental death from hydromorphone ingestion.

A 15-year-old male orally consumed an unknown but fatal amount of sustained release hydromorphone. He was naïve to opioid use. No other drugs or alcohol were involved. The cause of death was acute aspiration-related bronchopneumonia, secondary to hydromorphone ingestion; the manner of death was accidental. Hydromorphone and hydromorphone-3-glucuronide were quantified in postmortem fluids by tandem liquid chromatography-mass spectrometry. The hydromorphone concentrations in the peripheral blood, urine, and vitreous humor were 57, 4460, and 31 ng/mL, respectively. The hydromorphone-3-glucuronide concentrations in the corresponding three fluids were 459, 36,400, and 40 ng/mL. Hydromorphone-3-glucuronide accumulation probably did not contribute significantly to the opiate toxicity. The proposed minimum lethal hydromorphone blood concentration in the nontolerant user is in the vicinity of 60 ng/mL.

Prevalence of dihydrocodeine in hydrocodone positive postmortem specimens.

Hydrocodone (HC) has received renewed interest in the US due to reported increases in opiate related deaths involving psychotherapeutic drugs. The relative contribution of dihydrocodeine (DHC) in these deaths is unknown since little testing of this compound is performed. The objective of the study was to determine the prevalence of DHC in HC positive decedents and report the range of concentrations detected in these cases in order to evaluate the potential role of DHC in the deaths and determine the usefulness of including this analyte in opioid testing protocols. Specimens were assayed by liquid-liquid or solid phase extraction followed by gas chromatography/mass spectrometry operated in the selected ion monitoring mode. A multipoint calibration was utilized in the linear range 2-600ng/mL. Accuracy for HC, DHC and hydromorphone (HM) was 101-106% and between day precision at 160ng/mL between 7% and 11%. One hundred and thirty six cases were identified with the majority male (62%) and white (83%). A search of HC positive cases identified 64 with DHC (47%). The range of HC concentrations was 9-3039ng/mL heart blood (n=43) and 42-12353ng/mL urine (n=21). DHC concentrations in these cases ranged 3-243ng/mL in heart blood and 5-1842ng/mL in urine. DHC/HC ratios ranged 0.00(7)-2.90 in blood (n=43), and 0.01-5.04 in urine (n=21) with 16% and 24% of these cases with ratios >0.50, respectively. HM was detected in only 9 HC cases with the majority positive in urine.

A novel approach to evaluate the extent and the effect of cross-contribution to the intensity of ions designating the analyte and the internal standard in quantitative GC-MS analysis.

In gas chromatography-mass spectrometry methods of analysis adopting the analyte's isotopic analog as the internal standard (IS), the cross-contribution (CC) phenomenon -- contribution of IS to the intensities of the ions designating the analyte, and vice versa -- has been demonstrated to affect the quantitation data. A novel approach based on the deviations of the empirically observed concentrations of a set of standards was developed to assess the accuracy of the empirically derived CC data. This approach demonstrated that normalization of ion intensities derived from the analyte and the IS generates reliable CC data. It further demonstrated that an ion-pair (designating the analyte and the IS) with approximately 5% or higher CC will result in a very limited linear calibration range.

An estimate of the proportion of drug-facilitation of sexual assault in four U.S. localities.

In recent years, drugs including flunitrazepam, gamma-hydroxybutyrate, ketamine, and ethanol, have become popularly associated with drug-facilitated sexual assault. Other drugs are also candidates as factors in "drug facilitated sexual assault" (DFSA). The true extent of DFSA is not known, and is difficult to estimate. We recruited sexual assault complainants at four clinics in different parts of the U.S. to anonymously provide urine and hair specimens, and to answer questions about suspected drugging, drug use, and the sexual assault incident. Urine and hair specimens were tested for 45 drugs, including ethanol, and those pharmacologically capable of inducing sedation, amnesia, or impairment of judgment. Analytical test results were used to estimate the proportion of subjects, and the proportion of all complainants to the clinic in the same time period, who were victims of DFSA. Overall, cases of 43% of 144 subjects, and 7% of 859 complainants, were characterized as DFSA. Subjects underreported their use of drugs. The role of toxicological results and history in characterizing DFSA cases is discussed.

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.