Confirmation of EMIT benzodiazepine assay with GLC/NPD.

A simple, rapid, and reliable gas-liquid chromatographic (GLC) confirmation procedure was developed for urine samples found positive by the EMIT-dau benzodiazepine metabolite assay. The procedure involves acid hydrolysis, organic extraction, and identification using GLC-nitrogen-phosphorus detection (NPD). The method was validated in three subjects who took 10 mg diazepam daily for five days. Urinary excretion of the diazepam related substances was monitored quantitatively for 12 days. Considerable differences in diazepam metabolism was observed, despite the small number of subjects used. The data further indicated that both the physical characteristics and the metabolic profile of each individual may determine whether or not their occasional benzodiazepine use would be detected by the EMIT procedure. In some individuals taking daily diazepam, 48 to 72 hours may be required before sufficient metabolites accumulate in the urine to give a positive EMIT reaction.

Evaluation of the TRI 'dipstick' test for the detection of drugs of abuse in urine.

An evaluation of Technology Resources Inc. (TRI) Amphetamine, Barbiturate, Narcotic (G) and Narcotic (S) "Dipsticks" for drugs of abuse in urine was made. The results obtained by six individuals reading the "Dipstick" papers was compared with the analysis of the same urine samples, by a combination of TLC, EMIT, RIA and GLC. The data obtained with "Dipstick" papers, regardless of the drug tested, were clearly unreliable (high percentage of false negatives, low percentage of true positives) and the assay was unsuitable as a technique for screening urines for drugs of abuse.

Quantitative determination of 2-hydroxy-3-methoxy-6 beta-naltrexol (HMN), naltrexone, and 6 beta-naltrexol in human plasma, red blood cells, saliva, and urine by gas liquid chromatography.

Two gas liquid chromatographic methods differing mainly in sensitivity are described for the quantitative determination of 2-hydroxy-3-methoxy-6 beta-naltrexol (HMN), a minor metabolite of naltrexone (NT), in human body-fluids. The methods also incorporate simultaneous determinations of naltrexone and its major human metabolite, 6 beta-naltrexol (beta-OL), in urine, serum (or plasma), red blood cells (RBC), and saliva. Flame ionization detection of the bis-(trimethylsylil) trifluoroacetamide (BSTFA) derivatives provided sufficient sensitivity for quantitation of the bases in urine. However, lower levels in serum, RBC and saliva necessitated the use of more sensitive electron capture detection of the pentafluoropropionate (PFPA) derivatives of the bases. Because HMN and 6 beta-naltrexol PFPA derivatives have nearly identical gas chromatographic retention times, their separation was achieved by differential extraction, based on their different partition characteristics between aqueous and organic solvents. In the plasma of 4 subjects, 16 and 24 hrs. after 2 X 200 mg NT doses, the relative percentages were 23.1% HMN, 3.4% NT and 73.5% beta-OL. In urine samples collected at the same time as the blood samples the relative percentages were 14.4% HMN, 9.0% NT and 76.6% beta-OL. The nonpolar nature of HMN and the greater polarity of beta-OL may have influenced their differential distribution into RBCs and saliva. In the RBCs, 96.1% HMN and no significant amount of beta-OL was found, in saliva, 92.3% of beta-OL and no HMN was found.

Radioimmunoassay of methaqualone in human urine compared with chromatographic methods.

The 125I-radioimmunoassay for methaqualone in human urine was evaluated by a comparison with newly modified gas-liquid chromatographic and thin-layer chromatographic methods. The statistically significant sensitivity value for the radioimmunoassay was at 2 microgram of methaqualone per liter of urine. The coefficient of variation was 2.88 +/- 0.39% interassay and 2.71 +/- 0.16% intraassay. There was cross-reactivity only with metabolites of methaqualone, 4'-hydroxymethaqualone being twice as sensitively measured as methaqualone. There was complete agreement between results by radioimmunoassay and by gas-liquid chromatography in 96.7% of the samples analyzed. Only 1.2% of the radioimmunoassay values were false positives, and 2.1% false negatives (phi = 0.8917, P less than 0.001). Comparisons between the thin-layer chromatographic data and the gas-liquid chromatographic or radioimmunoassay data showed less agreement because of the 50- to 200-fold higher sensitivity of the latter two techniques. Gas-liquid chromatography therefore appears to represent the best reference method for the evaluation of the radioimmunoassay, which appears to be a very sensitive and reliable technique for detecting methaqualone and its metabolites in human urine.

Evaluation of the radioimmunoassay for benzoylecgonine (a cocaine metabolite) in human urine.

The 125I-radioimmunoassay (RIA) for benzylecgonine (cocaine metabolite) in urine was evaluated by comparison with gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) and the Enzyme-Multiplied Immunoassay Technique (EMIT). By RIA, a statistically significant concentration, 2 microng/liter, was observed for urinary benzoylecgonine. The coefficient of variation for the RIA was 2.58+/-0.38% inter-assay and 2.20+/-0.14% intraassay. There was cross-reactivity with cocaine (more reactive than benzoylecgonine) and other members of the tropane family of alkaloids. There was agreement between results by RIA and GLC in 95.5% of the samples, between RIA and TLC in 87.0%, and between RIA and EMIT in 84.5%. The percentage of true false-positives was 3.5% for the RIA in comparison to GLC, 8.8% in comparison to TLC, and 9.1% in comparison to EMIT. True false-negatives were insignificant (0 to 1.0%). GLC and RIA results correlated highly (phi=0.908). GLC, therefore, was the best comparison method for this evaluation study. RIA for benzoylecgonine is sensitive, reproducible, and reliable for the detection of cocaine in urine.

A gas-liquid chromatographic method for the determination of naltrexone and beta-naltrexol in human urine.

A rapid quantitative method was developed to assay in urine naltrexone and its major urinary metabolite, beta-naltrexol. Following solvent extraction and elimination of interfering materials, the weakly basic drug, its metabolite and the internal standard (etorphine) were silylated and analyzed by gas-liquid chromatography. As little as 0.02 mug/ml of naltrexone and beta-naltrexol was detectable using a hydrogen flame ionization detector. Twenty-four-hour urine samples were analyzed from three subjects taking 180 mg naltrexone daily. The major urinary excretion product was beta-naltrexol which accounted for 48.6% of the administered dose. Only 5% of the administered dose excreted in the urine was naltrexone. Beta-naltrexol was excreted 70% as free drug and 30% conjugated while naltrexone was 90% conjugated.

Radioimmunoassay of drugs subject to abuse: critical evaluation of urinary morphine-barbiturate, morphine, barbiturate, and amphetamine assays.

Radioimmunoassays for morphine-barbiturate (MOR-BARB), morphine, barbiturate, and amphetamine were evaluated by a direct comparison with differential elution extraction thin-layer chromatography, the "enzyme multiplied immunoassay technique," and XAD-2 resin extraction thin-layer chromatography for the detection in urine of drugs subject to abuse. Statistically significant (Psmaller than 0.01) concentrations for detection were: 50-100 mug/liter for MOR-BARB; 5 Mug/liter for morphine, 10 mug/liter for barbiturate, and 100 mug/liter for amphetamine. Unconfirmed and unaccounted-for radioimmunoassay positives (false) were: 0% for morphine in the radioimmunoassay for MOR-BARB and that for morphine alone; 2.8% for barbiturates in the MOR-BARB assay and that for barbiturates alone; 0-6% when a combination of these drugs was present in the MOR-BARB, morphine, or barbiturate assay; and 2.4% in the amphetamine radioimmunoassay. Less than 1% of all radioimmunoassay-negative samples were unconfirmed (false). Cross-reactivity was observed with drugs of a similar chemical structure in each of the radioimmunoassays tested. All the radio-immunoassays were easy to use, highly sensitive, and extremely reliable for detecting drug use or abuse.