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.

Pharmacokinetics and metabolism of olerciamide A from Portulaca oleracea L. in rats by UHPLC-UV and UHPLC-ESI-Q-TOF/MS.

The aim of this study was to elucidate the pharmacokinetics of olerciamide A in rats after oral and intravenous administration of Portulaca oleracea L. extract by a simple and rapid ultra high-performance liquid chromatography method with bergapten as internal standard. The pharmacokinetic results indicated that olerciamide A was rapidly distributed with a time to peak concentration of 30 min after oral administration and presented a low oral absolute bioavailability of 4.57%. The metabolism of olerciamide A in rats was also investigated using ultra-high-performance liquid chromatography electrospray coupled with quadrupole-time of flight mass spectrometry to elucidate the reason for the low absolute bioavailability of olerciamide A and seven metabolites of oleraciamide A were found in rat plasma and urine.

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.

[Determination of dimemorfan in human plasma and urine with HPLC-MS/MS].

OBJECTIVE: To develop a sensitive and reproducible HPLC-MS/MS method for analyzing dimemorfan in human plasma and urine. METHODS: Dimemorfan was extracted from plasma and urine by redistilled ether, with lidocaine serving as the internal standard (IS). The analysis was performed on a column of ultimate C18 (50 mm x 4.6 mm, 5 microm) with the mobile phase consisting of methyl alcohol-water-formic acid = 75:25 : 0.05 at a flow rate of 0. 2 mL/min. Dimemorfan was detected by API 3000 mass spectrometer, with multiple reaction monitoring after protonated with ESI in positive electron ionization mode. The ion pairs being detected were (m/z) 256.4-->155. 3 (dimemorfan) and 235.4-->86.1 (lidocaine), respectively. RESULTS: The regression equation for dimemorfan showed excellent linearity (r = 0.995 7) from 0. 025 to 5.0 ng/mL of plasma with detecting limitation of 0.025 ng/mL and perfect linearity (r = 0.9983) from 0.1 to 20.0 ng/mL of urine with detecting limitation of 0.1 ng/mL. The method recoveries of dimemorfan in plasma and urine were ranging from 103.38% to 106.88% and 90.05% to 101.40%, respectively. The maximum intra-day and inter-day relative standard deviations (RSD) of concentration of dimemorfan were 5.92% and 5. 70% (for plasma), 10.35% and 8.80% (for urine), respectively. CONCLUSION: This new method was validated to be accurate and sensitive to determinate the concentration of dimemorfan in plasma and urine samples, and can be applied for pharmacokinetic studies of dimemorfan.

Naltrexone and Its Noroxymorphone Minor Metabolite - A Case Report.

As part of substance use maintenance programs, individuals are monitored for sobriety through urine drug screens. A positive screen, and its confirmation and interpretation, can have devastating consequences, sometimes even leading to termination from the program and relapse. Naltrexone metabolism involves several steps and metabolites - one minor metabolite with very little mention in medical literature being noroxymorphone. This is also the final intermediate in the metabolic pathway of oxycodone; hence, detection is naturally presumed by clinicians to be attributed to oxycodone use. Through this case report, we alert clinicians that, depending on individual pharmacogenomics, it is possible to obtain a positive confirmation of this component alone (without any oxycodone pathway intermediates) with naltrexone administration.

Separation and detection of isoquinoline alkaloids using MEEKC coupled with field-amplified sample injection induced by ACN.

New methods based on MEEKC coupling with field-amplified sample injection (FASI) induced by ACN were proposed for five isoquinoline alkaloids (berberine, palmatine, jatrorrhizine, sinomenine and homoharringtonine) in no salt and high salt sample solution (HS). For the separation of five isoquinoline alkaloids, a running buffer composed of 18 mM sodium cholate, 2.4% v/v butan-1-ol, 0.6% v/v ethyl acetate, 10% v/v (or 30% v/v) methanol and 87.0% v/v (or 67% v/v) 5 mM Na2B4O7~10 mM NaH2PO4 buffer (pH 7.5) was developed. In order to improve the sensitivity, FASI induced by ACN was applied to increase the detection sensitivity. The detection limit was found to be as low as 0.0002 microg/mL in no salt sample solution and 0.062 microg/mL in HS. The method has been applied for the analysis of human urine spiked with analytes, and the assay results were proved to be satisfactory, and also the determination of berberine in urine sample after oral administration berberine.

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.

Determination of naloxegol in human biological matrices.

AIM: Naloxegol is an oral peripherally acting µ-opioid receptor antagonist approved for the treatment of opioid-induced constipation. Sensitive, robust, bioanalytical methods were required to quantitate naloxegol in human biological matrices as part of the clinical development program. METHODOLOGY/RESULTS: Analytical plasma samples were prepared using Solid Phase Extraction (SPE) coupled with concentration. The method's linearity was established at 0.1-50 ng/ml with up to 100-fold dilution. Urine samples were analyzed directly postdilution; dialysate samples were extracted by supported liquid extraction. Sensitive liquid chromatography/mass spectrometry (LC-MS/MS) assays were developed and validated, and demonstrated acceptable precision, accuracy and selectivity for naloxegol in the appropriate matrices. CONCLUSION: Methods for quantifying naloxegol in human biological matrices have been successfully validated.

Prescription Opioids. V. Metabolism and Excretion of Oxymorphone in Urine Following Controlled Single Dose Administration.

Oxymorphone (OM), a prescription opioid and metabolite of oxycodone, was included in the recently published proposed revisions to the Mandatory Guidelines for Federal Workplace Drug Testing Programs. To facilitate toxicological interpretation, this study characterized the time course of OM and its metabolite, noroxymorphone (NOM), in hydrolyzed and non-hydrolyzed urine specimens. Twelve healthy subjects were administered a single 10 mg controlled-release OM dose, followed by a periodic collection of pooled urine specimens for 54 h following administration. Analysis for free and total OM and NOM was conducted by liquid chromatography tandem mass spectrometry (LC-MS-MS), at a 50 ng/mL limit of quantitation (LOQ). Following enzymatic hydrolysis, OM and NOM were detected in 89.9% and 13.5% specimens, respectively. Without hydrolysis, OM was detected in 8.1% specimens, and NOM was not detected. The mean ratio of hydrolyzed OM to NOM was 41.6. OM was frequently detected in the first pooled collection 0-2 h post-dose, appearing at a mean of 2.4 h. NOM appeared at a mean of 8.3 h. The period of detection at the 50 ng/mL threshold averaged 50.7 h for OM and 11.0 h for NOM. These data support that OM analysis conducted using a 50 ng/mL threshold should include hydrolysis or optimize sensitivity for conjugated OM.

Etrophine in man. II. Detectability in urine by common screening methods.

A single highly euphorogenic dose of etorphine, 100 mug, was administered subcutaneously to 7 nontolerant subjects, and all urine samples were collected for 1 day prior to and 3 days following drug administration. Samples were analyzed for the presence of opiates by radioimmunoassay (Abuscreen) and homogeneous enzyme immunoassay (EMIT), with cutoffs for "ositives" of 40 and 500 ng/ml, respectively. Samples were analyzed for etorphine by thin-layer chromatography (TLC) with iodoplatinate preceded by XAD-2 resin extraction (sensitivity = 0.2 mug etorphine/ml of urine) and by gas-liquid chromatography (GLC) preceded by organic solvent extraction and trimethylsilyl derivatization (sensitivity = 0.1 mug etorphine/ml of urine). The last pre-drug and first two post-drug samples were also analyzed after acid hydrolysis by TLC and after glucuronidase hydrolysis by TLC and GLC. No sample gave a "positive" opiate result in either immunoassay, and no etorphine was detected in the TLC and GLC analyses of any urine sample. Thus, it is unlikely that the abuse of etorphine could be diagnosed by urinalysis using the common screening methods of radioimmunoassay, EMIT, TLC preceded by XAD-2 resin extraction, or GLC preceded by organic solvent extraction and trimethylsilyl derivatization.

Polymorphic dextromethorphan metabolism: co-segregation of oxidative O-demethylation with debrisoquin hydroxylation.

Dextromethorphan hydrobromide, 25 mg po, was given to 268 unrelated Swiss subjects to study urinary drug and metabolite profiles. Rates of O-demethylation yielding the main metabolite dextrorphan were expressed by the urinary dextromethorphan/dextrorphan metabolic ratio. We found a bimodal distribution of this parameter in our population study, which indicates that there are two phenotypes for dextromethorphan O-demethylation. The antimode at a metabolic ratio of 0.3 separated the poor metabolizer (PM; n = 23; prevalence of 9%) from extensive metabolizer (EM) phenotypes. Urinary output of dextrorphan was less than 6% of the dose in all PMs and was 50% in the 245 EMs. Pedigree analysis of 14 family studies revealed an autosomal-recessive transmission of deficient dextromethorphan O-demethylation. In these families, 37 heterozygous genotypes could be identified; however, through use of the urinary drug and metabolite analysis it was not possible to identify the heterozygous genotypes within the EM phenotype group. Co-segregation of dextromethorphan O-demethylation with debrisoquin 4-hydroxylation was also studied. Complete concordance of the two phenotypic assignments was obtained, with a Spearman rank correlation coefficient of rs = 0.78 (n = 62; P less than 0.0001) for dextromethorphan and debrisoquin metabolic ratios. Presumably the two drug oxidation polymorphisms are under the same genetic control. Thus the innocuousness and ubiquitous availability of dextromethorphan render it attractive for worldwide pharmacogenetic investigations in man.

Quantitation of etorphine in urine by selective ion monitoring using tritiated etorphine as an internal standard.

Selective ion monitoring combined with GLC was used for the assay of etorphine in urine. Commercially available tritiated etorphine was added as an internal standard. The advantage of the methodology using this internal standard is higher sensitivity by a factor of about 20 when compared with ordinary GLC.

GLC determination of l-2-hydroxy-N-cyclopropylmethylmorphinan in plasma and urine.

A specific method was developed for the determination of l-2-hydroxy-N-cyclopropylmethylmorphinan in plasma and urine by GLC, using flame-ionization detection. The method involves the extraction of the compound into ether from plasma or urine at pH 7.4, followed by back-extraction into 1 N HCl. The acid phase is ether washed and made alkaline, and the compound is reextracted into ether. The ether is evaporated to dryness, the residue is dissolved in methanol, and an aliquot is analyzed by GLC. The same method is applicalble to plasma and urine samples following deconjugation of the compound with glucuronidase-sulfatase. The overall recovery is 93.1 +/- 9.4% SD) in the concentration range of 0.020-2.0 microgram/ml. The method was successfully applied to plasma and urine specimens obtained after administering single 25-mg oral doses to humans.

Tentative identification of novel oxycodone metabolites in human urine.

Oxycodone is a semisynthetic codeine derivative that has been used both as an analgesic and antitussive. In the mid 1990s, OxyContin was introduced as a slow-release formulation of oxycodone for use in patients with moderate to severe chronic pain from such ailments as arthritis, vertebral disc disease, and cancer. Doctors wrote 6.9 million prescriptions for OxyContin from May 2000 through May 2001. Thus, it is no surprise that hospitals and medical examiners' offices across the country have seen an increasing number of admissions and deaths resulting from oxycodone abuse and overdose. The laboratory identifies oxycodone as part of its routine abused and therapeutic drug-testing procedures. Routine gas chromatographic analysis of bile or urine in many of these cases revealed unidentified peaks in the region of oxycodone that appeared to be oxycodone metabolites. In humans, the only documented metabolites of oxycodone are oxymorphone and N-desmethyloxycodone (noroxycodone). This study attempts to characterize these compounds as "presumptive" metabolites based on circumstantial evidence from known metabolic pathways of oxycodone in other species, as well as of other opiates and narcotic analgesics.

GC/MS confirmatory method for etorphine in horse urine.

A highly sensitive procedure for GC/MS determination of etorphine in horse urine is described. This assay provides both specificity and reliability and is particularly well suited for the confirmation of radioimmunoassay screening procedures usually used for etorphine. After solvent extraction and purifications, the etorphine is characterized as a pentafluoroacetic derivative (PFAA) by using mass fragmentography. The detection limit is 0.1 ng/mL in urine; the coefficient of variation of the estimations is 10.9%. The procedure has been validated after on-field administration of 5 to 90 micrograms of etorphine to five thoroughbred horses (10 to 180 ng/kg).

The pharmacokinetics of oxycodone in uremic patients undergoing renal transplantation.

STUDY OBJECTIVE: To determine the pharmacokinetics of oxycodone and the excretion of oxycodone and its metabolites noroxycodone and oxymorphone in uremic patients undergoing renal transplantation. DESIGN: Open study of the pharmacokinetics and excretion of oxycodone. SETTING: IV Department of Surgery, Helsinki University Central Hospital. PATIENTS: 10 uremic patients undergoing renal transplantation and 10 ASA status I patients undergoing general surgery. INTERVENTIONS: Intravenous (IV) oxycodone chloride 0.07 mg/kg was administered 30 minutes before induction of standardized anesthesia. Sampling of blood and urine was conducted for 24 hours. MEASUREMENTS AND MAIN RESULTS: The concentrations of oxycodone and noroxycodone in plasma and the 24 hour urine recoveries of the conjugated and unconjugated forms of oxycodone, noroxycodone, and oxymorphone were measured. Mean elimination half-life was prolonged in uremic patients due to increased volume of distribution and reduced clearance. Interindividual variation was very great. Plasma concentrations of noroxycodone were higher in uremic patients. Significantly smaller quantities of free oxycodone and noroxycodone and both free and conjugated oxymorphone were excreted in the urine in the uremic than in the control patients. CONCLUSIONS: Elimination of oxycodone is impaired in end-stage renal failure.

[Determination of sinomenine HCl in serum and urine by HPLC and its pharmacokinetics in normal volunteers].

A RP-HPLC method was developed to determine the concentrations of sinomenine HCl in serum and urine and its pharmacokinetics was studied in healthy volunteers. C18H37 column was eluted with the mobile phase of acetonitrile--0.01 mol.L-1 sodium phosphate monobasic--N, N, N', N'-tetramethylenediamine (46:54:0.22 v/v, pH 6.9) and the ultraviolet absorbance was monitored at 263 nm. Triazolan was used as internal standard. The calibration curves were linear in the range of 6-480 ng.ml-1 in serum and 0.06-3 micrograms.ml-1 in urine, with mean recoveries of 75.46% and 91.38% respectively. The lowest detectable limits were 4 ng.ml-1 in serum and 40 ng.ml-1 in urine and the RSD for the intra-day and inter-day were less than 5%. A single oral dose of 80 mg sinomenine HCl tablet was given to 8 healthy male volunteers. The concentrations of sinomenine HCl in serum and urine were determined. The serum concentration--time curve was found to fit a two-compartment open model with first order elimination. The pharmacokinetic parameters were: T1/2 alpha 0.791 +/- 0.491 h, T1/2 beta 9.397 +/- 2.425 h, Tmax 1.040 +/- 0.274 h, Cmax 246.604 +/- 71.165 ng.ml-1, AUC 2651.158 +/- 1039.050 ng.h.ml-1, CL 0.033 +/- 0.010 ng.ml-1.

Monitoring oxycodone use in patients with chronic pain: analysis of oxycodone and metabolite excretion in saliva and urine.

OBJECTIVE: Saliva is purported to have a close correspondence to plasma concentrations due to a passive diffusion process from plasma to saliva. However, limited data are available characterizing oxycodone and its metabolites in saliva. The purpose of this analysis was to evaluate the use of saliva monitoring in patients prescribed oxycodone and to compare the disposition of oxycodone in saliva and urine. DESIGN: This retrospective analysis examined deidentified urine and saliva specimens collected from patients with chronic pain. These specimens were received at Millennium Laboratories between March and June 2012 and analyzed using LCMS/MS to quantitate oxycodone, noroxycodone, and oxymorphone concentrations. RESULTS: The geometric mean metabolic ratio (MR) of noroxycodone to oxycodone in saliva was 0.11, whereas the geometric mean MR in urine was 1.7. The geometric mean oxycodone concentration in saliva was 860 ng/mL (range, 1.5-8,600,000 ng/mL; 95% CI, 770-950 ng/mL), whereas the geometric mean noroxycodone concentration was 98 ng/mL (range, 2.3-8,800 ng/mL; 95% CI, 90-107 ng/mL). Fifty-four of the saliva specimens (6 percent) had oxycodone concentrations between 10,000 and 9,000,000 ng/mL. CONCLUSIONS: Oxycodone is predominant over noroxycodone in saliva (similar to plasma), while the reverse relationship exists in urine. Much greater oxycodone concentrations were found in saliva than are expected in plasma (up to a 1,000-fold difference). Saliva concentrations are lower than urine concentrations but still may not reflect plasma disposition. Possible explanations include medication residue in the mouth (recent medication use or misuse) or active secretion into saliva. Saliva analysis may be used for qualitative drug monitoring of oxycodone, with detection rates similar to urine; however, further characterization is needed for appropriate interpretation.

The effects of renal impairment on the pharmacokinetics, safety, and tolerability of naloxegol.

The impact of renal impairment on the pharmacokinetics of a 25-mg oral dose of naloxegol was examined in patients with renal impairment classified as moderate, severe, or end-stage renal disease (ESRD) and compared with healthy subjects (n = 8/group). Geometric mean area under the plasma concentration-time curve (AUC) was increased in patients with moderate (1.7-fold) or severe (2.2-fold) impairment, and maximum plasma concentrations (Cmax ) were elevated in patients with moderate (1.1-fold) or severe (1.8-fold) impairment. These findings were driven by higher exposures in two patients in each of the moderate and severe impairment groups; exposures in all other patients were similar to the control group. Overall exposures in ESRD patients were similar and Cmax was 29% lower versus normal subjects. Renal impairment minimally affected other plasma pharmacokinetic parameters. As renal clearance was a minor component of total clearance, exposure to naloxegol was unaffected by the degree of renal impairment, with no correlation between either AUC or Cmax and estimated glomerular filtration rate (eGFR). Hemodialysis was an ineffective means to remove naloxegol. Naloxegol was generally well tolerated in all groups. Renal impairment could adversely affect clearance by hepatic and gut metabolism, resulting in the increased exposures observed in outliers of the moderate and severe renal impairment groups.

Observations of urinary oxycodone and metabolite distributions in pain patients.

Oxycodone is an opioid analgesic metabolized to oxymorphone and noroxycodone by cytochrome P450 (CYP) 2D6 and 3A4/5, respectively. This was a retrospective study to evaluate sex, age, urinary pH and concurrent medication use on oxycodone, oxymorphone and noroxycodone distributions. Urine specimens obtained from patients on chronic opioid therapy were analyzed by LC-MS-MS. There were 108,923 specimens from a subject's first or single visit, who were at least 18 years of age, and had documented physician-reported oxycodone use. The majority of specimens had detectable oxycodone urine concentrations (n = 106,852) resulting in oxycodone mole fractions (arithmetic mean ± SD) of 0.44 ± 0.27. Ninety-eight percent (n = 106,229) and 49% (n = 53,394) had detectable oxymorphone and noroxycodone, respectively. Oxycodone and oxymorphone mole fractions were lower in women compared with men (P < 0.0001). Mean ± SD age was 49.1 ± 12.9 years. Noroxycodone mole fractions were highest in the 65 years and older age group. Concurrent use of a CYP2D6 inhibitor, but not a CYP3A4/5 inhibitor, altered oxycodone and oxymorphone mole fractions. Dual inhibition of CYP2D6 and CYP3A4/5 did not result in a statistical difference upon comparison with CYP2D6 inhibitor or CYP3A4/5 inhibitor use. Patient factors affect oxycodone and metabolite mole fractions and suggest increased awareness of each contribution when attempting to monitor therapy with urine drug testing.