Impact of genetic variation in CYP2C19, CYP2D6, and CYP3A4 on oxycodone and its metabolites in a large database of clinical urine drug tests.

Urine drug testing (UDT) is a tool for monitoring drug use, including oxycodone. While variation in cytochrome P450 (CYP) genes is known to alter oxycodone metabolism, its impact on UDT results of oxycodone and its metabolites has not been well-studied. Here, multivariate analysis was performed on retrospective UDT results of 90,379 specimens collected from 14,684 genotyped patients prescribed oxycodone. Genetic variation in CYP2D6 and CYP2C19 had a significant impact on oxymorphone/oxycodone ratios, with a 6.9-fold difference between CYP2D6 ultrarapid metabolizers (UMs) and poor metabolizers (PMs; p < 10-300) and a 1.6-fold difference between CYP2C19 UMs and PMs (p = 1.50 × 10-4). CYP2D6 variation also significantly impacted noroxycodone/oxycodone ratios (p = 6.95 × 10-38). Oxycodone-positive specimens from CYP2D6 PMs were ~5-fold more likely to be oxymorphone-negative compared to normal metabolizers. These findings indicate that multivariate analysis of UDT data may be used to reveal the real-world impact of genetic and non-genetic factors on drug metabolism.

Electrochemical sensor based on glassy carbon electrode modified by polymelamine formaldehyde/graphene oxide nanocomposite for ultrasensitive detection of oxycodone.

Polymelamine formaldehyde/graphene oxide (PMF/GO) nanocomposite was used, for the first time, to study the ultrasensitive and selective electrochemical detection of oxycodone (OXC). The successful characterization of PMF/GO was verified based on scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), X-ray diffraction (XRD), energy-dispersive spectroscopy (EDS), and Raman spectroscopy. The modified GCE (PMF/GO-GCE) proved its electrocatalytic effect on OXC determination according to cyclic, linear sweep, and differential pulse voltammetry (CV, LSV, and DPV) and electrochemical impedance spectroscopy (EIS) studies. The developed sensor under optimal conditions offered a linear relationship in a limited range of  0.01 to 45 µmol L-1 with the limit of detection (LOD) of 2.0 nmol L-1. The proposed PMF/GO-GCE sensor was effectively employed for the OXC detection in human urine and serum samples. Graphical abstract.

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.

Determination of oxycodone and its major metabolites noroxycodone and oxymorphone by ultra-high-performance liquid chromatography tandem mass spectrometry in plasma and urine: application to real cases.

BACKGROUND: Oxycodone is a narcotic drug widely used to alleviate moderate and severe acute and chronic pain. Variability in analgesic efficacy could be explained by inter-subject variations in plasma concentrations of parent drug and its active metabolite, oxymorphone. To evaluate patient compliance and to set up therapeutic drug monitoring (TDM), an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay was developed and validated for the parent drug and its major metabolites noroxycodone and oxymorphone. METHODS: Extraction of analytes from plasma and urine samples was obtained by simple liquid-liquid extraction. The chromatographic separation was achieved with a reversed phase column using a linear gradient elution with two solvents: acetic acid 1% in water and methanol. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). RESULTS: Separation of analytes was obtained in less than 5 min. Linear calibration curves for all the analytes under investigation in urine and plasma samples showed determination coefficients (r2) equal or higher than 0.990. Mean absolute analytical recoveries were always above 86%. Intra- and inter-assay precision (measured as coefficient of variation, CV%) and accuracy (measured as % error) values were always better than 13%. Limit of detection at 0.06 and 0.15 ng/mL and limit of quantification at 0.2 and 0.5 ng/mL for plasma and urine samples, respectively, were adequate for the purpose of the present study. CONCLUSIONS: Rapid extraction, identification and quantification of oxycodone and its metabolites both in urine and plasma by UHPLC-MS/MS assay was tested for its feasibility in clinical samples and provided excellent results for rapid and effective drug testing in patients under oxycodone treatment.

Evaluation of 6 remote First Nations community-based buprenorphine programs in northwestern Ontario: Retrospective study.

OBJECTIVE: To evaluate established opioid addiction treatment programs that use traditional healing in combination with buprenorphine-naloxone maintenance treatment in 6 First Nations communities in the Sioux Lookout region of northwestern Ontario. DESIGN: Retrospective cohort study. SETTING: Six First Nations communities in northwestern Ontario. PARTICIPANTS: A total of 526 First Nations participants in opioid-dependence treatment programs. INTERVENTION: Buprenorphine-naloxone substitution therapy and First Nations healing programming. MAIN OUTCOME MEASURES: Retention rates and urine drug screening (UDS) results. RESULTS: Treatment retention rates at 6, 12, and 18 months were 84%, 78%, and 72%, respectively. We estimate that the rate at 24 months will also be more than 70%. The UDS programming varied and was implemented in only 1 community. Initially urine testing was voluntary and it then became mandatory. Screening with either method found the proportion of urine samples with negative results for illicit opioids ranged between 84% and 95%. CONCLUSION: The program's treatment retention rates and negative UDS results were higher than those reported for most methadone and buprenorphine-naloxone programs, despite a patient population where severe posttraumatic stress disorder is endemic, and despite the programs' lack of resources and addiction expertise. Community-based programs like these overcome the initial challenge of cultural competence. First Nations communities in other provinces should establish their own buprenorphinenaloxone programs, using local primary care physicians as prescribers. Sustainable core funding is needed for programming, long-term aftercare, and trauma recovery for such initiatives.

Body pushing, prescription drugs and hospital admission.

A 39-year-old man died of multi-organ failure complicating mixed drug toxicity that included methadone, oxazepam, oxycodone and nitrazepam. His past medical history involved alcohol and poly-substance abuse with chronic self-harm and suicidal ideation. There had been multiple hospital admissions for drug overdoses. At autopsy the most unusual finding was of two packages of 10 tablets each, wrapped in thin plastic film within the rectum. The insertion of drugs into body orifices and cavities has been termed body pushing to distinguish it from body packing where illicit drugs are wrapped and swallowed for transport and smuggling, and body stuffing where small amounts of loosely wrapped or unwrapped drugs are swallowed to conceal evidence from police. This case demonstrates that body pushing may not always involve illicit drugs or attempted concealment from police or customs officials. It appears that the drugs had been hidden to ensure an additional supply during the time of residence in hospital. The extent to which body pushing is currently being used by patients to smuggle drugs into secure medical facilities is yet to be determined.

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.

Comparison of Response of DRI Oxycodone Semiquantitative Immunoassay With True Oxycodone Values Determined by Liquid Chromatography Combined With Tandem Mass Spectrometry: Sensitivity of the DRI Assay at 100 ng/ml Cut-Off and Validity of Semiquantitative Value.

OBJECTIVE: Oxycodone is a widely used opioid for pain management and patient's compliance with therapy is often monitored by using oxycodone immunoassay. The performance of the DRI oxycodone immunoassay was compared with liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) assay. MATERIALS AND METHODS: In 48 urine specimens collected from patients taking oxycodone, urinary oxycodone concentrations were determined using LC/MS/MS and the DRI oxycodone immunoassay for application on the Cobas c 501 analyzer (Roche Diagnostics, Indianapolis, IN). RESULTS: Out of 48 specimens, 14 specimens showed oxycodone value less than 100 ng/ml, seven specimens had low positive values (between 101 and 165 ng/ml) and all other specimens had values 165 to 1789 ng/ml using the LC/MS/MS assay. The DRI oxycodone assay successfully identified all oxycodone specimens with oxycodone concentrations over the 100 ng/ml. In addition, the DRI assay also showed positive response in 11 out of 14 specimens with oxycodone values less than 100 ng/ml. However, semiquantitative values obtained by the DRI assay did not match with true oxycodone and metabolite oxymorphone concentrations combined obtained by using LC/MS/MS. CONCLUSIONS: DRI oxycodone immunoassay at 100 ng/ml is a reliable immunoassay for analysis of oxycodone in urine.

Simultaneous quantification and confirmation of oxycodone and its metabolites in equine urine using ultra-high performance liquid chromatography-tandem mass spectrometry.

Oxycodone, an opioid commonly used to treat pain in humans, has the potential to be abused in racehorses to enhance their performance. To understand the pharmacokinetics of oxycodone and its metabolites in horses, as well as to detect the illegal use of oxycodone in racehorses, a method for quantification and confirmation of oxycodone and its metabolites is needed. In this study, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that can simultaneously quantify and confirm oxycodone and eight metabolites in equine urine. Samples were subjected to enzymatic hydrolysis and then liquid-liquid extraction using ethyl acetate. The analyte separation was achieved on a Hypersil Gold C18 sub-2 µm column and analytes were detected on a triple quadrupole mass spectrometer. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 25-50 pg/mL and 100 pg/mL, respectively. Excellent linearity of the calibration curves was observed over a range of 100-10000 pg/mL for all nine analytes. Retention time, signal-to-noise ratio, and product ion ratios were utilized as confirmation criteria, with the limits of confirmation (LOC) ranging from 100 to 250 pg/mL. The data from a pilot pharmacokinetic (PK) study suggested that oxycodone metabolites have longer detection periods in equine urine compared to oxycodone itself; thus, the detection of metabolites in equine urine extends the ability to detect oxycodone exposure in racehorses.

Urinary Pharmacokinetics of Immediate and Controlled Release Oxycodone and its Phase I and II Metabolites Using LC-MS-MS.

Oxycodone (OC) is a schedule II semisynthetic opioid in the USA that is prescribed for its analgesic effects and has a high potential for abuse. Prescriptions for OC vary based on the dosage and formulation, immediate release (IR) and controlled release (CR). Monitoring OC metabolites is beneficial for forensic casework. The limited studies that involve pharmacokinetics of the urinary excretion of OC metabolites leave a knowledge gap regarding the excretion of conjugated and minor metabolites, pharmacokinetic differences by formulation, and the impact of CYP2D6 activity on the metabolism and excretion of OC. The objectives of this study were to compare urinary excretion of phase I and II metabolites by formulation and investigate if ratio changes over time could be used to predict the time of intake. Subjects (n = 7) received a single 10 mg IR tablet of Oxycodone Actavis. A few weeks later the same subjects received a single 10 mg CR tablet of Oxycodone Actavis. During each setting, urine was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 9, 10, 12, 14, 24, 48 and 72 h. Urine samples (100 µL) were diluted with 900 µL internal standard mixture and analyzed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD using a previously validated method. The CYP2D6 phenotypes were categorized as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM). Comparisons between IR and CR were performed using two-tailed paired t-test at a significance level of P = 0.05. The metabolite ratios showed a general increase over time. Four metabolite to parent ratios were used to predict the time of intake showing that predictions were best at the early time points.

Real-time 2D separation by LC × differential ion mobility hyphenated to mass spectrometry.

The liquid chromatography-mass spectrometry (LC-MS) analysis of complex samples such as biological fluid extracts is widespread when searching for new biomarkers as in metabolomics. The success of this hyphenation resides in the orthogonality of both separation techniques. However, there are frequent cases where compounds are co-eluting and the resolving power of mass spectrometry (MS) is not sufficient (e.g., isobaric compounds and interfering isotopic clusters). Different strategies are discussed to solve these cases and a mixture of eight compounds (i.e., bromazepam, chlorprothixene, clonapzepam, fendiline, flusilazol, oxfendazole, oxycodone, and pamaquine) with identical nominal mass (i.e., m/z 316) is taken to illustrate them. Among the different approaches, high-resolution mass spectrometry or liquid chromatography (i.e., UHPLC) can easily separate these compounds. Another technique, mostly used with low resolving power MS analyzers, is differential ion mobility spectrometry (DMS), where analytes are gas-phase separated according to their size-to-charge ratio. Detailed investigations of the addition of different polar modifiers (i.e., methanol, ethanol, and isopropanol) into the transport gas (nitrogen) to enhance the peak capacity of the technique were carried out. Finally, a complex urine sample fortified with 36 compounds of various chemical properties was analyzed by real-time 2D separation LC×DMS-MS(/MS). The addition of this orthogonal gas-phase separation technique in the LC-MS(/MS) hyphenation greatly improved data quality by resolving composite MS/MS spectra, which is mandatory in metabolomics when performing database generation and search.

A simplified approach to direct SPE-MS.

Microextraction by packed sorbent (MEPS) has been directly hyphenated with ESI-MS for the rapid screening of opiates and codeine metabolites in urine. This study introduces a novel format of MEPS that incorporates a two-way valve in the barrel of the syringe enabling the direction of liquid flow to be manipulated. Controlled directional flow (CDF) MEPS allows sharp, concentrated sample bands to be delivered directly to the MS in small volumes and effectively eliminates the need to optimize elution. The method optimization assessed the recovery, matrix effects, and the speed of infusion, all critical variables for optimum ESI performance. Matching extraction workflows demonstrated a reduction in carryover from 65% for conventional MEPS to only 1% for CDF MEPS. The recovery (<89% for 50 µL sample), matrix effects (<42%), linearity (r(2) > 0.99), and LODs (<5 ng/mL) were determined to demonstrate method performance. The optimized approach was employed for the screening of codeine metabolites in urine. The ion trace revealed sharp sample bands corresponding to the codeine metabolites. At-line MEPS-ESI-MS allowed both sample preparation and analysis to be completed in only 5 min facilitating high throughput and alleviating the burden of method development.

The analytical performance of six urine drug screens on cobas 6000 and ARCHITECT i2000 compared to LC-MS/MS gold standard.

BACKGROUND: Immunoassays provide a rapid tool for the screening of drugs-of-abuse (DOA). However, results are presumptive and confirmatory testing is warranted. To reduce associated cost and delay, laboratories should employ assays with high positive and negative predictive values (PPVs and NPVs). Here, we compared the results of urine drug screens on cobas 6000 (cobas) and ARCHITECTi2000 (ARCHITECT) platforms for six drugs against LC-MS/MS to assess the analytical performance of these assays. METHODS: Eighty nine residual urine specimens, which tested positive for amphetamine, THC-COOH, benzoylecgonine, EDDP, opiates and/or oxycodone during routine drug testing, were stored frozen until later confirmation by LC-MS/MS. Immunoassays were performed on cobas and ARCHITECT using a split sample. A third aliquot from these samples was tested by LC-MS/MS to assess the percentage of false positive, false negative, true positive and true negative results and calculate the PPVs and NPVs for each immunoassay. RESULTS: The PPVs of THC-COOH and EDDP assays were 100% on both platforms. Suboptimal PPVs were achieved for oxycodone (cobas, 57.1% vs ARCHITECT, 66.7%), amphetamine (77.8 vs. 100%), opiates (80.0 vs. 84.6%) and benzoylecgonine (88.9 vs. 84.2%) assays. The NPV was 100% for cobas and ARCHITECT oxycodone assays. Lower NPVs were achieved for THC-COOH (cobas, 28.6% vs ARCHITECT, 25.0%), EDDP (72.7% for both assays), benzoylecgonine (74.4% vs 73.8%), amphetamine (83.3% vs 82.8%) and opiates (100% vs 85.3%). CONCLUSION: Overall, cobas and ARCHITECT urine drug screens have comparable analytical performance. Confirmatory testing is warranted for positive test results especially for oxycodone, amphetamine, opiates and cocaine. Negative drug screen results must be interpreted with caution especially for THC-COOH, EDDP, benzoylecgonine, amphetamine and opiates.

Tunable detection sensitivity of opiates in urine via a label-free porous silicon competitive inhibition immunosensor.

Currently, there is need for laboratory-based high-throughput and reliable point-of-care drug screening methodologies. We demonstrate here a chip-based label-free porous silicon (PSi) photonic sensor for detecting opiates in urine. This technique provides a cost-effective alternative to conventional labeled drug screening immunoassays with potential for translation to multiplexed analysis. Important effects of surface chemistry and competitive binding assay protocol on the sensitivity of opiate detection are revealed. Capability to tune sensitivity and detection range over approximately 3 orders of magnitude (18.0 nM to 10.8 muM) was achieved by varying the applied urine specimen volume (100-5 muL), which results in systematic shifts in the competitive binding response curve. A detection range (0.36-4.02 muM) of morphine in urine (15 muL) was designed to span the current positive cutoff value (1.05 muM morphine) in medical opiate urine screening. Desirable high cross-reactivity to oxycodone, in addition to other common opiates, morphine, morphine-3-glucuronide, 6-acetyl morphine, demonstrates an advantage over current commercial screening assays, while low interference with cocaine metabolite was maintained. This study uniquely displays PSi sensor technology as an inexpensive, rapid, and reliable drug screening technology. Furthermore, the versatile surface chemistry developed can be implemented on a range of solid-supported sensors to conduct competitive inhibition assays.

Label-free porous silicon immunosensor for broad detection of opiates in a blind clinical study and results comparison to commercial analytical chemistry techniques.

In this work, we evaluate for the first time the performance of a label-free porous silicon (PSi) immunosensor assay in a blind clinical study designed to screen authentic patient urine specimens for a broad range of opiates. The PSi opiate immunosensor achieved 96% concordance with liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS) results on samples that underwent standard opiate testing (n = 50). In addition, successful detection of a commonly abused opiate, oxycodone, resulted in 100% qualitative agreement between the PSi opiate sensor and LC-MS/MS. In contrast, a commercial broad opiate immunoassay technique (CEDIA) achieved 65% qualitative concordance with LC-MS/MS. Evaluation of important performance attributes including precision, accuracy, and recovery was completed on blank urine specimens spiked with test analytes. Variability of morphine detection as a model opiate target was <9% both within-run and between-day at and above the cutoff limit of 300 ng mL(-1). This study validates the analytical screening capability of label-free PSi opiate immunosensors in authentic patient samples and is the first semiquantitative demonstration of the technology's successful clinical use. These results motivate future development of label-free PSi technology to reduce complexity and cost of diagnostic testing particularly in a point-of-care setting.

Measurement of multiple drugs in urine, water, and on surfaces using fluorescence covalent microbead immunosorbent assay.

There are a range of applications that require the measurement of multiple drugs such as urine analysis, drug determination in water, and screening for drug contamination on surfaces. Some of the procedures used such as enzyme-linked immunosorbent assay (ELISA) are simple but can only determine one drug at a time, and others such as GC-MS or LC-MS are complex, time-consuming, and expensive. In this study, fluorescence covalent microbead immunosorbent assay (FCMIA) was investigated as a simple method for the measurement of multiple drugs simultaneously in three matrices: diluted urine, water, and on surfaces. Five different drugs of abuse or their metabolites (methamphetamine, caffeine, benzoylecgonine (a metabolite of cocaine), tetrahydrocannabinol (THC), the active ingredient in marijuana, and oxycodone) were studied over the range 0-15 ng/ml. There was no measureable cross-reactivity among the drugs at the concentrations studied. Urine dilutions from 1/50 to 1/2.5 were studied and dilutions less than 1/20 had a significant effect on the methamphetamine assay but limited effects on the benzoylecgonine and oxycodone assays and almost no effect on the THC assay. For assays performed in 1/20 urine dilution, water, and diluted surface sampling buffer, least detectable doses (LDD) were 1 ng/ml or less for the drugs. Surfaces spiked with drugs were sampled with swabs wetted with surface sampling buffer and recoveries were linear over the range 0-100 ng/100 cm(2) surface loading for all drugs. FCMIA has potential to be used for the measurement of multiple drugs in the matrices studied.

Criteria for opiate identification using liquid chromatography linked to tandem mass spectrometry: problems in routine practice.

BACKGROUND: Liquid chromatography linked to tandem mass spectrometry (LC/MS/MS) is being increasingly used for drug confirmation. At present, no official criteria exist for drug identification using this technique although the European Union (EU) criteria for compound identification have been adopted. These criteria are evaluated with respect to opiate confirmation by LC/MS/MS and problems highlighted. METHODS: Urine samples screened positive for opiates by immunoassay were subjected to confirmation by LC/MS/MS using multiple reaction monitoring (MRM) and two separate buffer systems of pH 6.8 and 8.0, respectively. The EU criteria for compound identification were applied for confirmation of morphine, 6-monoacetylmorphine (6MAM), codeine and dihydrocodeine (DHC). RESULTS: Using the pH 6.8 buffer, confirmation could be achieved for 84%, 94%, 96% and 95%, respectively, for samples demonstrating MRM chromatographic peaks at retention times for morphine, 6MAM, codeine and DHC. Failure to meet the EU criteria was mainly attributed to low signal-to-noise (S:N) ratios or excessively high drug concentrations. Isobaric interferences and poor chromatography were also contributing factors. The identification of morphine was considerably improved with chromatography at pH 8.0 owing to resolution of interferences. Oxycodone metabolites were a potential problem for the identification of DHC. CONCLUSION: Isobaric interferences can pose a problem with drug identification using LC/MS/MS. Optimizing chromatographic conditions is important to overcome these interferences. Consideration needs to be given to investigating drug metabolites as well as parent drugs in method development.

Determination of oxycodone and its major metabolites in haematic and urinary matrices: Comparison of traditional and miniaturised sampling approaches.

Oxycodone is a widely prescribed, full agonist opioid analgesic. As such, it is used clinically to treat different kinds of painful conditions, with a relatively high potential for doping practices in athletes. In this paper, different classic and innovative miniaturised matrices from blood and urine have been studied and compared, to evaluate their relative merits and drawbacks within therapeutic drug monitoring (TDM) and to implement new protocols for anti-doping analysis. Plasma, dried blood spots (DBS) and dried plasma spots (DPS) have been studied for TDM purposes, while urine, dried urine spots (DUS) and volumetric absorptive microsamples (VAMS) from urine for anti-doping. These sampling techniques were coupled to an original bioanalytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the evaluation and monitoring of the levels of oxycodone and its major metabolites (noroxycodone and oxymorphone) in patients under pain management and in athletes. The method was validated according to international guidelines, with good results in terms of precision, extraction yield and accuracy for all considered micromatrices. Thus, the proposed sampling, pre-treatment and analysis are attractive strategies for oxycodone determination in human blood and urine, with advanced options for application to derived micromatrices. Microsampling procedures have significant advantages over classic biological matrices like simplified sampling, storage and processing, but also in terms of precision (<9.0% for DBS, <7.7% for DPS, <7.1% for DUS, <5.3% for VAMS) and accuracy (>73% for DBS, >78% for DPS, >74% for DUS, >78% for VAMS). As regards extraction yield, traditional and miniaturised sampling approaches are comparable (>67% for DBS, >74% for DPS, >75% for DUS, >75% for VAMS). All dried matrices have very low volumes, leading to a significant advantage in terms of analysis feasibility. On the other hand, this also leads to a corresponding decrease in the overall sensitivity.

Rapid quantification of urinary oxycodone and oxymorphone using fast gas chromatography-mass spectrometry.

Human urine specimens that were determined to be presumptively positive for oxycodone and its metabolite, oxymorphone, by immunoassay screening were assayed using fast gas chromatography-mass spectrometry to positively identify and quantify the oxycodone and oxymorphone present. Urine specimens were first spiked with deuterated internal standards, oxycodone-d(3) and oxymorphone-d(3), subjected to acid hydrolysis, and then extracted using a positive-pressure manifold and mixed-bed solid-phase cartridge extraction methodology. Extracts were derivatized using methoxylamine and acetic anhydride. The acetylated-oxime derivatives of oxycodone and oxymorphone were identified and quantified using a selective ion monitoring (SIM). The method was found to be linear for both analytes to 1600 ng/mL, and limits of detection for oxycodone and oxymorphone were found to be 40 ng/mL and 20 ng/mL, respectively. Interlaboratory data comparisons (n = 40) showed correlation coefficients of 0.9999 and 0.9997 for oxycodone and oxymorphone, respectively. Twelve semisynthetic, structurally similar compounds at concentrations of 5000 ng/mL were assayed in the presence of oxycodone and oxymorphone and found not to interfere with identification and quantitation by this method. Finally, exact mass and tandem mass spectrometry techniques were employed to elucidate the structures of the SIM ions.