Methadone and metabolite urine concentrations in patients maintained on methadone

As regulatory control over methadone maintenance relaxes, the need for methods of monitoring compliance will increase. In community clinics, monitoring would most likely involve immunoassays of outpatients' trough urine specimens. There are no published norms for such data. Therefore, we determined concentrations of methadone in 1093 urine specimens collected thrice weekly in 27 outpatients during up to 17 weeks of observed methadone ingestion (35 to 80 mg/day) using a semiquantitative homogeneous enzyme immunoassay (CEDIA). We used a separate CEDIA assay to measure methadone's main metabolite, 2-ethylidene-3,3-diphenylpyrrolidine (EDDP), which may help detect compliance in fast metabolizers or patients who adulterate samples to simulate compliance. Methadone concentrations were more variable than those of EDDP. Concentrations of methadone were < 100 ng/mL in one specimen, between 100 and 300 ng/mL in 27, and >or= 300 ng/mL in all others. EDPP concentrations were >or= 100 ng/mL in all specimens, suggesting that EDDP should be detectable in urine from compliant patients. Methadone and EDDP concentrations significantly increased with methadone dose and (in one participant with poor clinic attendance) significantly decreased following missed methadone doses. Nevertheless, variability was too great to permit estimation of methadone dose (or detect a single missed administration) from any single specimen.     

Analysis of methadone and its metabolites in meconium by enzyme immunoassay (EMIT) and GC-MS

An EMIT-ETS d.a.u. immunoassay screening method for methadone in meconium and a gas chromatography-mass spectrometry (GC-MS) method for methadone and its metabolites including 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) in meconium were described. The GC-MS method showed good linearity (r2 > or = 0.998) over a concentration range of 25-2000 ng/g with limits of detection of 10, 25, and 10 ng/g for methadone, EDDP, and EMDP, respectively, and a limit of quantitation of 25 ng/g for all three analytes. Fifty pooled meconium samples were screened using a cutoff of 200 ng/g, and all samples screened negative. GC-MS analysis of all samples showed four samples to contain methadone (35.2 to 79.9 ng/g), EDDP (28.5 to 557.2 ng/g), or both, with no detectable amount of EMDP. The negative results on the four specimens at the cutoff used may be explained by the fact that EMIT-ETS d.a.u. antibody for methadone was specific to the parent drug. The results point to the fact that immunoassays should be directed to EDDP for detection of prenatal exposure of methadone through analysis of meconium specimens.     

Methadone conversion to EDDP during GC-MS analysis of urine samples

During validation of a gas chromatography-mass spectrometry (GC-MS) method for the methadone metabolite 2-ethylidine-1,5dimethyl-3,3-diphenylpyrrolidine (EDDP), it was noted that detectable levels of EDDP were found during analysis of extracts from drug-free urine samples spiked with methadone. Different amounts of EDDP were detected by GC-MS during confirmation analysis; however, levels consistently exceeded 50 ng/mL at methadone concentrations > 10,000 ng/mL. Quantitation of EDDP was determined by the addition of EDDP-d3 to methadone-spiked urine samples. Subsequent analysis of methadone-spiked urine extracts by high-performance liquid chromatography (HPLC) indicated no EDDP as a result of contaminated standard or conversion during solid-phase extraction. Reducing the GC injector-port temperature from 260 degrees C to 180 degrees C reduced the observed EDDP concentration in one sample from 201 ng/mL to 53 ng/mL at the initial methadone concentration of 10,000 ng/mL. These results indicate GC injector-port temperature induces thermal conversion of methadone to EDDP as an artifact. When confirmation of methadone and EDDP is critical to determining individual compliance with maintenance programs, alternative chromatographic methods (e.g., capillary electrophoresis, HPLC, or liquid chromatography-mass spectrometry) should be considered.     

Urine drug testing of chronic pain patients. III. Normetabolites as biomarkers of synthetic opioid use

Opioids are important therapeutic agents available to patients with moderate to severe pain. The synthetic opioids, buprenorphine, fentanyl, meperidine, methadone, and propoxyphene have been utilized for decades as analgesics. One of the major biotransformation pathways of these drugs occurs through N-demethylation leading to the formation and excretion of normetabolites. Normetabolites generally exhibit longer half-lives than the parent drug leading to accumulation with prolonged use. As part of continuing research efforts to improve monitoring programs of chronic pain patients undergoing opioid treatment, we evaluated the prevalence and relative abundance of normetabolites of buprenorphine, fentanyl, meperidine, methadone, and propoxyphene in patients? urine specimens. Selected sets of specimens were analyzed without prior immunoassay screening by liquid chromatography-tandem mass spectrometry for buprenorphine, fentanyl, meperidine, methadone, propoxyphene, and their respective normetabolites. Limits of quantitation (LOQ) were as follows: buprenorphine, 1 ng/mL; fentanyl, 0.5 ng/mL; meperidine, 50 ng/mL; methadone, 50 ng/mL; and propoxyphene, 50 ng/mL. LOQs for normetabolites were equal to the parent drug with the exception of norbuprenorphine (2.5 ng/mL). The percentage of positive specimens that contained normetabolite (only) ranged from 8.0% for EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) to 53.1% for norpropoxyphene. Inclusion of the five normetabolites in the test panel produced an increase in detection rates for parent drug use as follows: buprenorphine, 10.0%; fentanyl, 42.1%; meperidine, 98.7%; methadone, 8.7%; and propoxyphene, 113.2%. The authors conclude that testing for synthetic opioid normetabolites enhances the effectiveness of monitoring programs for pain patients.     

Urine concentrations of fentanyl and norfentanyl during application of Duragesic transdermal patches

A study of the urinary concentration of fentanyl (F) and its major metabolite norfentanyl (NF) in chronic pain patients treated with the Duragesic continuous release transdermal patches is presented. These patches are available in 10, 20, 30, and 40 cm(2) sizes releasing 25, 50, 75, and 100 microg/h F, respectively. F is rapidly and extensively metabolized, with NF as the major metabolite. Five hundred-forty six random urine specimens were collected from chronic pain patients wearing 25, 50, 75, or 100 ug F transdermal patches. Urine specimens were collected from hours after application to several days later after continuous F release. Each specimen was analyzed for F, NF, creatinine, and pH. Additionally, each was screened by enzyme immunoassay for the following: amphetamines, barbiturates, benzodiazepines, cocaine metabolite, methadone, phencyclidine, d-propoxyphene, opiates, and marijuana metabolites. All positive screening results were confirmed by gas chromatography-mass spectrometry (GC-MS). F and NF were isolated from urine by solid-phase extraction then identified and quantified by GC-MS in SIM mode. The LODs and LOQs for F and NF were 3 ng/mL, respectively. The results of F and NF analysis of urine form those wearing 25-microg patches (N = 142) was mean F, 47 ng/mL with a range of 0 to 983 ng/mL, and 97% of the specimens contained < 200 ng/mL and mean NF, 175 ng/mL with a range of 0-980 ng/mL, while 95% of the specimens contained < 400 ng/mL. The results of F and NF analysis of urine form those wearing 50 microg patches (N = 184) was: mean F, 74 ng/mL with a range of 0 to 589 ng/mL, and 92% of the specimens contained < 200 ng/mL and mean NF, 257 ng/mL with a range of 0-2200 ng/mL, and 98% of the specimens contained < 1000 ng/mL. The results of F and NF analysis of urine form those wearing 75 microg patches (N = 85) was mean F, 107 ng/mL with a range of 0 to 1280 ng/mL, and 98% of the specimens contained < 400 ng/mL and mean NF, 328 ng/mL with a range of 0-5630 ng/mL, and 99% of the specimens contained < 1000 ng/mL. The results of F and NF analysis of urine form those wearing 100 ug patches (N = 135) was mean F, 100 ng/mL with a range of 0 to 1080 ng/mL, while 96% of the specimens contained < 400 ng/mL and mean NF, 373 ng/mL with a range of 0-5730 ng/mL, and 95% of the specimens contained < 1000 ng/mL. The incidence of other drugs detected as a percentage the specimens was opiates, 48%, benzodiazepines, 43%; barbiturates, 3%; methadone, 4%; marijuana metabolite, 3%; and cocaine metabolite, 1%. With the exception of F and/or NF, no other drugs were detected in 25% of the specimens. These data demonstrate the wide variation in concentrations of F and NF in random urine specimens following application of Duragesic patches. However, these values obtained during therapeutic use far exceed concentrations previously reported in fatal poisoning. In general, one may expect to find urine NF concentrations 3-4 times higher than those of F.     

Determination of illicit drugs and their metabolites in human urine by liquid chromatography tandem mass spectrometry including relative ion intensity criterion

A method, using 0.5 mL of urine, was developed for the simultaneous determination of ecgonine methyl ester, benzoylecgonine, morphine, codeine, 6-acetylmorphine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and d-lysergic acid diethylamide (LSD). The analysis was performed by liquid chromatography with tandem mass spectrometry, after solid-phase extraction in the presence of their deuterated analogues. Reversed-phase separation on an Atlantis dC18 column was achieved in 12.5 min, under gradient conditions. The method was fully validated, including linearity (1-2000 microg/L for ecgonine methyl ester, benzoylecgonine, 6-acetylmorphine, methamphetamine, MDMA, and EDDP; 2-2000 microg/L for morphine, codeine, MDA, and methadone; 2-1000 microg/L for amphetamine, and 0.2-100 microg/L for LSD; r(2)>0.99); recovery (>65%), within-day and between-day precision, and accuracy (CV and MRE<15%); limit of detection (0.1 microg/L for LSD, 0.5 microg/L for ecgonine methyl ester, benzoylecgonine, methamphetamine, MDMA, 6-monoacetylmorphine, and EDDP, and 1 microg/L for amphetamine, MDA, morphine, and methadone); quantitation (lowest level of the calibration curve); relative ion intensities, freeze-and-thaw stability, and matrix effect. The procedure showed to be sensitive and specific, and was applied to real cases and quality control samples from a quality control program.     

Pharmacokinetics of morphine and its surrogates. VII: High-performance liquid chromatographic analyses and pharmacokinetics of methadone and its derived metabolites in dogs

Reversed-phase HPLC assays with on-column UV detection and post-column fluorescent detection of ion pair-extracted material were developed and used for the quantitative assay of methadone, its presumed metabolites, and acid- and alkali-hydrolyzable conjugates of these metabolites in biological fluids with assay sensitivities of 10-20 ng/mL. Plasma, urine, and bile were monitored in dogs after intravenous bolus administration of 0.8, 1.0, 2.0, and 2.2 mg/kg methadone hydrochloride. Plasma-time data showed two sequential half-lives of 8.3 +/- 3.4 (SEM) and 128 +/- 37 min, with apparent dose-independent pharmacokinetics in the studied dose range. Total body clearances were 899 +/- 103 (SEM) mL/min. Renal clearances (6-82 mL/min) of methadone were highly variable within and among studies but showed no significant variation with urinary pH or flow rate. The percentages of the dose excreted in the urine as methadone and (+)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrroline (2) were 3.6 +/- 0.5% (SEM) and 4.1 +/- 0.4% (SEM), respectively, but there were no significant concentrations of 2 in plasma. The presumed metabolites 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (3), 1,5-dimethyl-3,3-diphenyl-2-pyrrolidone (4), (-)-alpha-N-normethadol (7), 4-dimethylamino-2,2-diphenylvaleric acid (8), p-hydroxymethadone (9), and (-)-alpha-methadol (10) were not observed in the plasma of dogs given methadone. Quantities of presumed metabolites 3, 4, 7, 8, 9, and 10 were negligible in urine (less than 0.03% of dose). No acid-hydrolyzable conjugates, or generators on acidification, of 3, 4, 7, 8, or 10 were detectable in urine. No alkali-hydrolyzable conjugates, or generators on alkalinization, of 3, 4, 8, or 10 were detectable in urine. There was no significant biliary secretion of unchanged methadone; 2 in bile amounted to only 2% of the dose. In bile and urine, 50% and 17-27%, respectively, of the radiolabeled dose was not extractable into hexane. In a non-bile-cannulated dog, 35% of the total radiolabeled intravenous dose was present in the feces. As much as 88% of an intravenous radiolabeled dose could be accounted for, even though only small amount of methadone was disposed through the metabolic routes claimed in the literature. The intravenous administration of 2 resulted in two sequential half-lives of 3 and 270 min and no apparent pharmacokinetic dose dependency. Amounts of 2 excreted unchanged in urine and bile were 23% and 5-16% of the dose, respectively. Renal and total body clearances were 170 and 1150 mL/min.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monitoring urinary excretion of cannabinoids by fluorescence-polarization immunoassay: a cannabinoid-to-creatinine ratio study

Drug testing in substance abuse treatment programs is focused on urine analysis of parent drugs and major metabolites. Huestis reported that serial monitoring of the major urinary cannabinoid metabolite (delta9-THC-COOH)-to-creatinine ratios in paired urine specimens (collected at least 24 hours apart) could differentiate new marijuana or hashish use from residual cannabinoid metabolite excretion in urine after previous drug use. Subjects with a history of chronic marijuana use were screened for cannabinoids in urine over several months by an enzyme immunoassay (EMIT) with a cut-off value of 50 ng/mL. Presumptive positive specimens were confirmed by gas chromatography-mass spectrometry (GC-MS) for delta9-THC-COOH with a cut-off value of 15 ng/mL. The objective of this study was to determine whether a semiquantitative cannabinoids immunoassay (corrected for creatinine concentration) could differentiate new marijuana use from residual cannabinoid excretion in chronic users of marijuana or hashish compared with GC-MS. The criterion for new marijuana use was a cannabinoid-to-creatinine ratio > or =0.5 (dividing the immunoassay quantitative result to creatinine ratio of specimen 2 by the specimen 1 ratio, specimen 3 by the specimen 2 ratio, etc.). Urine specimens were analyzed by fluorescence-polarization immunoassay (FPIA) on an Abbott TDxFLx analyzer after analysis by GC-MS. In 90 urine specimens (group A) with delta9-THC-COOH values determined by GC-MS, the mean delta9-THC-COOH concentration was 44.4 ng/mL (range, 16-100), and the mean FPIA total cannabinoids value was 91.7 ng/mL (range, 21-204 ng/mL) with a correlation coefficient of 0.993 (group A). In 111 specimens (group B), the mean delta9-THC-COOH concentration was 361 ng/mL (range, 101-960 ng/mL). The mean FPIA value was 657 ng/mL (range, 211-1,270 ng/mL), and the correlation coefficient of the B series was 0.975. Percent cross-reactivity for delta9-THC-COOH standards prepared in drug-free urine by FPIA was 82% at 25 ng/mL, 45% at 50 ng/mL, and 50% at 100 ng/mL. Overall, there was 89% agreement (132 of 148 specimens) between FPIA and GC-MS. In 16 of 148 specimens, however, the FPIA and GC-MS paired urine data did not agree. The sensitivity of the FPIA assay was 95.3%, and the specificity was 44.4%. The authors conclude that FPIA cannabinoid analysis should be further evaluated as an alternative to GC-MS quantitation to help distinguish new marijuana use from residual marijuana metabolite excretion in clinical drug treatment programs.     

GC-MS confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine.

A procedure for the simultaneous confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine specimens by gas chromatography-mass spectrometry (GC-MS) is described. After the addition of nalorphine and naltrexone as the two internal standards, the urine is hydrolyzed overnight with beta-glucuronidase from E. coli. The urine is adjusted to pH 9 and extracted with 8% trifluoroethanol in methylene dichloride. After evaporating the organic, the residue is sequentially derivatized with 2% methoxyamine in pyridine, then with propionic anhydride. The ketone groups on hydrocodone, hydromorphone, oxycodone, oxymorphone, and naltrexone are converted to their respective methoximes. Available hydroxyl groups on the O3 and O6 positions are converted to propionic esters. After a brief purification step, the extracts are analyzed by GC-MS using full scan electron impact ionization. Nalorphine is used as the internal standard for codeine, morphine, and 6-acetylmorphine; naltrexone is used as the internal standard for the 6-keto-opioids. The method is linear to 2000 ng/mL for the 6-keto-opioids and to 5000 ng/mL for the others. The limit of quantitation is 25 ng/mL in hydrolyzed urine. Day-to-day precision at 300 and 1500 ng/mL ranged between 6 and 10.9%. The coefficients of variation for 6-acetylmorphine were 12% at both 30 and 150 ng/mL. A list of 38 other basic drugs or metabolites detected by this method is tabulated.     

Validation of an ELISA method for screening methadone in postmortem blood

In this study, we evaluate Venture Labs' enzyme-linked immunosorbent assay (ELISA) for the detection of methadone in postmortem specimens. Sixty-one postmortem specimens that previously screened positive for methadone along with 59 specimens which screened negative for methadone were included. All specimens were screened using the Venture Labs methadone assay in conjunction with a liquid-liquid basic extraction and gas chromatographic-mass spectrometric (GC-MS) analysis. All cases screening positive by either method were confirmed for methadone and its metabolite 2-ethylidene-1,5-dimethyl- 3,3-diphenylpyrrolidine by a solid-phase extraction utilizing deuterated internal standards and GC-MS-SIM. Twenty-four postmortem samples that screened negative by both methods were also extracted and analyzed using the confirmation method to demonstrate the validity of both screening methods. The intra- and interassay precision for the ELISA method was evaluated at the cut-off concentration used for the analysis (50 ng/mL). True positives, true negatives, false positives, and false negatives were calculated for the ELISA results as compared to the GC-MS screening data. The Venture Labs methadone assay demonstrated a sensitivity of 96.7%+/-2.3% and a specificity of 98.3%+/-1.7% relative to the GC-MS method.     

An enantiomer-selective liquid chromatography-tandem mass spectrometry method for methadone and EDDP validated for use in human plasma, urine, and liver microsomes

A liquid chromatography-electrospray ionization-tandem mass spectrometry method has been developed and validated to detect (R)- and (S)-methadone and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human plasma with cross-validation to urine and liver microsomes. Use of deuterated internal standards and liquid-liquid extraction coupled with chiral separation provided baseline separation with a lower limit of quantitation (LLOQ) of 2.5 ng/mL. The LLOQ was established from comparison of signal in blanks from six different sources per matrix with the same sources fortified at the LLOQ (none exceeded 19% of LLOQ) and precision and accuracy at the LLOQ determined in the same six sources per matrix. The assay was precise (% coefficients of variation within 13.8%) and accurate (% targets within 15%) in all three matrices. No interference was seen from addition of other psychoactive drugs. Stability was determined in plasma (24 h at room temperature, 321 days at -20 degrees C, 3 freeze-thaw cycles); processed plasma samples (5 days at -20 degrees C, 12 days on autosampler); urine (24 h at room temperature); and stock solutions (20 h at room temperature, 61 days at -20 degrees C). Applications of varying degree are presented for each matrix. Plasma from five subjects maintained on 100 mg oral methadone per day permitted comparison of the pharmacokinetics of the enantiomers. The t(1/2) of (R)-methadone was significantly longer than for (S)-methadone, and (S)-methadone was more tightly protein bound. The C(max), AUC, C(min), and % protein bound of (S)-EDDP were significantly greater than (R)-EDDP, while the t(1/2) of (R)-EDDP was significantly greater than (S)-EDDP. In spot urines, (R)- was higher than (S)-methadone, and (S)- was generally higher than (R)-EDDP. (R)- and (S)-EDDP production was detected after incubation of therapeutic concentrations of racemic methadone with human liver microsomes, and (S)-EDDP production was twofold greater than (R)-EDDP in three human placental microsomes incubated with racemic methadone.     

Quantitation of benzodiazepines in urine, serum, plasma, and meconium by LC-MS-MS

A single method for confirmation and quantitation of a panel of commonly prescribed benzodiazepines and metabolites, alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam, and 7-aminoclonazepam, was developed for three specimen types, urine, serum/plasma, and meconium. Quantitation was by liquid chromatography tandem-mass spectrometry (LC-MS-MS) using a Waters Alliance-Quattro Micro system. The instrument was operated in multiple reaction monitoring mode with an electrospray ionization source in positive ionization mode. The method was evaluated for recovery, imprecision, linearity, analytical measurement range, specificity, and carryover. Average recovery and imprecision (within-run, between-run, and total % CV) were within +/- 15% of the target concentrations for urine (10 to 5000 ng/mL) and serum/plasma (10 to 2500 ng/mL) and within +/- 20% for meconium (10 to 5000 ng/g). In all, 205 patient specimens were analyzed, and the results compared to a previous in-house gas chromatography-MS method or LC-MS-MS results from an outside laboratory. Oxazepam glucuronide was evaluated as a hydrolysis control for the urine and meconium specimens.     

Experience with a urine opiate screening and confirmation cutoff of 2000 ng/mL.

Until recently, most laboratories used an opiate immunoassay screening and confirmation cutoff value of 300 ng/mL for codeine and morphine detection by gas chromatography-mass spectrometry (GC-MS). The cutoff value for opiates was increased to 2000 ng/mL or higher in various laboratories because of concerns that small doses of codeine and foods containing poppy seeds would give a positive opiate-screening result. Workplace drug-testing programs in the U.S. raised the opiate cutoff value to 2000 ng/mL on 30 November 1998. The objective of this study is to describe the results of opiate testing of 8600 urine specimens collected over 24 months with a 2000-ng/mL screening and confirmation (codeine and morphine) cutoff value. Specimens were screened by the EMITdau opiate assay using an in-house 2000-ng/mL morphine calibrator. Presumptive positive findings (N = 621) were analyzed quantitatively by GC-MS for codeine and morphine. One hundred and eighty six urine specimens were positive for codeine and morphine (> 2000 ng/mL), 298 specimens were positive for codeine only (> 2000 ng/mL) and 26 specimens were positive for morphine only (> 2000 ng/mL). All remaining specimens had codeine and morphine values < 2000 ng/mL. The codeine and morphine confirmation rate in this program reduced from 7.1% in 1994-1996 (300-ng/mL cutoff) to 2.1% in 1997-1998 with a 2000-ng/mL cutoff value. The codeine-only confirmation rate lowered from 6.6% (300-ng/mL cutoff) to 3.4% (2000-ng/mL cutoff). It was concluded that increasing opiate screening and codeine and morphine confirmation cutoff values led to > 300% reduction in the confirmed-positive rate for codeine and morphine and a 47% reduction in codeine-only confirmations in a urine drug-testing program where codeine was the major opiate used.     

Identification of hydrocodone in human urine following controlled codeine administration

Allegations of illicit hydrocodone use have been made against individuals who were taking physician-prescribed oral codeine but denied hydrocodone use. Drug detection was based on positive urine opiate immunoassay results with subsequent confirmation of hydrocodone by gas chromatography-mass spectrometry (GC-MS). In these cases, low concentrations of hydrocodone (approximately 100 ng/mL) were detected in urine specimens containing high concentrations of codeine (> 5000 ng/mL). Although hydrocodone has been reported to be a minor metabolite of codeine in humans, there has been little study of this unusual metabolic pathway. We investigated the occurrence of hydrocodone excretion in urine specimens of subjects who were administered codeine. In a controlled study, two African-American and three Caucasian male subjects were orally administered 60 mg/70 kg/day and 120 mg/70 kg/day of codeine sulfate on separate days. Urine specimens were collected prior to and for approximately 30-40 h following drug administration. In a second case study, a postoperative patient self-administered 960 mg/day (240 mg four times per day) of physician-prescribed oral codeine phosphate, and urine specimens were collected on the third day of the dosing regimen. Samples from both studies were extracted on copolymeric solid-phase columns and analyzed by GC-MS. In the controlled study, codeine was detected in the first post-drug-administration specimen from all subjects. Peak concentrations appeared at 2-5 h and ranged from 1475 to 61,695 ng/mL. Codeine was detected at concentrations above the 10-ng/mL limit of quantitation for the assay throughout the 40-h collection period. Hydrocodone was initially detected at 6-11 h following codeine administration and peaked at 10-18 h (32-135 ng/mL). Detection times for hydrocodone following oral codeine administration ranged from 6 h to the end of the collection period. Confirmation of hydrocodone in a urine specimen was always accompanied by codeine detection. Codeine and hydrocodone were detected in all specimens collected from the postoperative patient, and concentrations ranged from 2099 to 4020 and 47 to 129 ng/mL, respectively. Analyses of the codeine formulations administered to subjects revealed no hydrocodone present at the limit of detection of the assay (10 ng/mL). These data confirm that hydrocodone can be produced as a minor metabolite of codeine in humans and may be excreted in urine at concentrations as high as 11% of parent drug concentration. Consequently, the detection of minor amounts of hydrocodone in urine containing high concentrations of codeine should not be interpreted as evidence of hydrocodone abuse.     

Semiautomated preparation of 3,5,6-trichloro-2-pyridinol in human urine using a Zymate XP laboratory robot with quantitative determination by gas chromatography-negative-ion chemical ionization mass spectrometry

A rapid and sensitive semiautomated method was developed for quantitation of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCP) in human urine. A Zymark Zymate XP laboratory robotics system was used to mix urine samples, transfer aliquots, add the stable-isotope-labeled TCP internal standard (13C2- or 13C2,15N-), and liberate conjugates of TCP from urine via acid hydrolysis. Samples were manually extracted into toluene, derivatized, and analyzed by gas chromatography-negative-ion chemical ionization mass spectrometry. Determination of the metabolic TCP was performed by selected ion monitoring of the dichloropyridinol fragment ions: m/z 161 for TCP and m/z 165 for 13C2-TCP or m/z 168 for 13C2,15N-TCP. Interday precision and accuracy were demonstrated over 3 years of analyses using the 13C2-TCP internal standard, with an average recovery from fortified urine samples of 93+/-12% (N = 54, concentration range 1-140 ng/mL). The method was found to be linear over the range of 0.5 to 200 ng/mL, and the limit of detection for TCP in urine was estimated to be 0.2 ng/mL with a limit of quantitation of 1 ng/mL. The effect of solids distribution on the concentration of TCP in the thawed urine samples was examined, and the results indicated that homogeneous distribution is critical for quantitation. The precision and accuracy of the automated method with respect to the transfer of homgeneous urine aliquots and delivery of internal standard yielded equivalent or improved results over the manual techniques. Overall, this method is more simple than existing methodologies, and it yields results with improved precision, accuracy, and sensitivity over previously developed methods.     

Determination of tapentadol and its metabolite N-desmethyltapentadol in urine and oral fluid using liquid chromatography with tandem mass spectral detection

An analytical procedure for the determination of the new pain medication tapentadol and its main metabolite N-desmethyltapentadol (DMT), in urine and oral fluid has been developed and validated using liquid chromatography with tandem mass spectral detection (LC-MS-MS). Oral fluid was collected using Quantisal? devices, and drugs present were quantified using solid-phase extraction followed by LC-MS-MS. For confirmation, two transitions were monitored and one ratio determined which had to be within 20% of that of the known calibration standard. For tapentadol, 222.1 > 107 was used as the quantifying transition; 222.1 > 121 for the qualifier. For DMT, 208.1 > 107 was used for quantification; 208.1 > 121 as the qualifier. For saliva, the linear range was 10-100 ng/mL; the lower limit of quantitation (LLOQ) was 10 ng/mL; the intraday precision was 3.6% (n = 6) and interday precision was 13.6% (n = 24). The recovery of tapentadol and DMT from the oral fluid collection pad was > 99%. For urine, the specimens were diluted and injected directly into the LC-MS-MS. The LLOQ was 50 ng/mL; the intraday and interday precisions were 2.1% and 4.4%, respectively, for tapentadol and 2.9% and 5.7%, respectively, for DMT. This is the first analytical procedure for tapentadol and DMT in urine and oral fluid.     

Evaluating the relationship of methadone concentrations and EDDP formation in chronic pain patients

Methadone is used to treat moderate to severe pain in patients not responsive to non-narcotic analgesics and for maintenance treatment of opioid addiction. Methadone is primarily metabolized by N-demethylation to an inactive metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidene (EDDP) by CYP3A4 and CYP2B6. Establishing expected concentrations for metabolism of methadone to EDDP using urine excretion data may be useful for monitoring "medications" and toxicity. Urine specimens from chronic pain patients were collected during routine clinic visits. Methadone and EDDP were quantified by liquid chromatography-tandem mass spectrometry. Approximately 8,000 subjects who reported taking methadone had creatinine concentrations ≥20 mg/dL, and excreted concentrations of methadone and EDDP above ≥100 ng/mL were selected. The median methadone urine concentration was 3.03 mg/g cr. Ninety-five percent of the population had concentrations between 0.175 and 20.9 mg/g cr. EDDP was, on average, twice the methadone concentration. The wide variance in relationship of methadone to its metabolite was not concentration-dependent. Variability between subjects was larger than variability within subjects. As the urinary pH increased, the proportion of excreted EDDP increased, implying a preferred excretion of EDDP.     

The effect of the use of mouthwash on ethylglucuronide concentrations in urine

Two studies were performed to evaluate the effect of alcohol containing mouthwash on the appearance of ethyl glucuronide (EtG) in urine. In the first study, 9 volunteers were given a 4-oz bottle of mouthwash, which contained 12% ethanol. They gargled with all 4 oz. of the mouthwash at intervals over a 15-min period. All urine samples were collected over the next 24 h. Of 39 provided urine samples, there were 20 > 50 ng/mL, 12 > 100 ng/mL, 5 > 200 ng/mL, 3 > 250 ng/mL, and 1 > 300 ng/mL. The peak concentrations were all within 12 h after the exposure. In the second study, 11 participants gargled 3 times daily for 5 days. The first morning void was collected. Sixteen of the 55 submitted samples contained EtG concentrations of greater than 50 ng/mL. All of them were less than 120 ng/mL. These studies show that incidental exposure to mouthwash containing 12% ethanol, when gargling according to the manufacturer's instructions, can result in urinary EtG values greater than 50 ng/mL. All specimens were negative for ethanol. The limits of detection and quantitation for the EtG testing were 50 ng/mL.