Development and validation of ELISA and GC-MS procedures for the quantification of dextromethorphan and its main metabolite dextrorphan in urine and oral fluid.

The development of a highly sensitive enzyme-linked immunosorbent assay and gas chromatography-mass spectrometry confirmation method for the detection of dextromethorphan and its major metabolite dextrorphan in urine and oral fluid is described. For the screening assay, the intraday precision was less than 8% for urine and less than 5% for oral fluid. The interday precision was less than 10% for both drugs in urine and oral fluid. For the confirmatory procedure, both inter- and intraday precision was less than 5% for both matrices. The detection limit for both methods was 1 ng/mL. The quantifying ions chosen from the full scan mass spectra were m/z 271 for dextromethorphan, m/z 329 for dextrorphan, and m/z 332 for tri-deuterated dextrorphan-d(3). A high recovery yield (> 93%) from the Quantisal oral fluid collection device was achieved, and the drugs were stable in the collection device for at least 10 days at room temperature. The extracted drugs from both matrices were stable for at least 48 h while kept at room temperature. Both screening and confirmatory procedures were applied to authentic urine and oral fluid specimens obtained from volunteers following therapeutic ingestion of dextromethorphan.

Development and GC-MS validation of a highly sensitive recombinant G6PDH-based homogeneous immunoassay for the detection of buprenorphine and norbuprenorphine in urine.

Buprenorphine is now increasingly prescribed as an alternative to methadone for the treatment of heroin addiction. Because of its potency (dosage usages from 0.2 mg to 8 mg), the drug concentrations in body fluids are normally very low. Here, we report the first recombinant glucose-6-phosphate dehydrogenase (G6PDH)-based homogeneous immunoassay (EMIT-type assay) for free buprenorphine and free norbuprenorphine in urine. The antibody used in this assay cross-reacts nearly identically with buprenorphine and norbuprenorphine and, at the same time, has less than 1% cross-reactivity with a wide range of commonly prescribed opiates, particularly those structurally related compounds such as morphine, codeine, and dihydrocodeine. More importantly, this assay has a low detection limit of 1 ng/mL for buprenorphine or norbuprenorphine. Further evaluation of this technique using gas chromatography-mass spectrometry (GC-MS) of authentic urine samples demonstrated that the accuracy of the assay is greater than 95%. Because this assay is designed to measure the free drugs in urine, it resulted in simplification for GC-MS or liquid chromatography-MS confirmation methods that did not require urine hydrolysis before solid-phase or liquid-liquid extraction.

Determination of propoxyphene in oral fluid.

The determination of propoxyphene in oral fluid using solid-phase extraction and gas chromatography-mass spectrometry is described for the first time. The method employs collection of oral fluid with the Quantisal device, immunoassay screening of the specimen, confirmation of the positive screened samples after extraction using cation exchange/hydrophobic solid-phase extraction columns, optimized derivative formation, and gas chromatography-mass spectrometry in electron impact mode. Validated parameters including selectivity, linearity, accuracy, intra- and interday precision, extraction efficiency, and limit of quantitation were all within acceptable limits. The method was applied to authentic specimens taken from an individual prescribed propoxyphene following surgery.

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.

Identification and quantification of nicotine biomarkers in human oral fluid from individuals receiving low-dose transdermal nicotine: a preliminary study.

The objective of this preliminary study was to identify and quantify potential nicotine (NIC) biomarkers in post-exposure oral fluid samples collected from 10 NIC-abstinent human participants administered 7 mg transdermal NIC using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Oral fluid samples were collected prior to NIC patch application and at 0.5 and 0.75 h after patch removal using the Quantisal() oral fluid collection device. The validated LC-MS-MS analyte panel included nicotine-Nbeta-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1'-N-oxide, cotinine (COT), nornicotine, NIC, anatabine, anabasine, and cotinine-N-beta-D-glucuronide. Analytes and corresponding deuterated internal standards were extracted by solid-phase extraction. NIC and COT concentrations were quantifiable in oral fluid samples collected from 6 of the 10 participants 0.5 h after patch removal and in oral fluid samples collected from 7 of the 10 participants 0.75 h after patch removal. Based on the mean NIC and COT concentrations in oral fluid and plasma for the participants with both quantifiable NIC and COT at the 0.5 and 0.75 h collection times, the oral fluid-plasma ratio was 6.4 for NIC and 3.3 for COT. An ELISA procedure was also validated and successfully applied as a screening tool for these oral fluid samples in conjunction with LC-MS-MS confirmation. An ELISA cut-off concentration of 5.0 ng/mL provided excellent sensitivity for discrimination of COT-positive post-exposure oral fluid samples collected after low-level transdermal NIC exposure and oral fluid samples collected prior to patch application.