A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without beta-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with beta-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with beta-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (> 2 ng/mL) with peak concentrations at 12-36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15-20% of the total immunoassay crossreactive FNP metabolites.

In this paper are reported the synthesis and characterization of three LSD derivatives. On the basis of several analytical characterization studies, the most stable derivative has been selected and a procedure to covalently link the derivative to polystyrene microparticles through a carrier protein has been developed. In addition, two new LSD immunogens have been synthesized and characterized, and from these immunogens antibodies that recognize not only LSD but also several major LSD metabolites have been generated. Using the selected derivative and antibody, a homogeneous microparticle-based immunoassay has been developed for the detection of LSD in human urine with the required sensitivity and specificity for an effective screening assay. The performance of this LSD OnLine assay has been evaluated using the criteria of precision, cross-reactivity, correlation to the Abuscreen LSD RIA and GC/MS/MS, assay specificity, and limit of detection.

A homogenous microparticle-based immunoassay has been developed for the detection of d-lysergic acid diethylamide (LSD) in human urine using the Online technology. This immunoassay is based on the principle of the kinetic interaction of microparticles in a solution where the drug content of the urine is directly proportional to the inhibition of the microparticle aggregation. Antibodies to LSD were obtained by immunizing goats with an LSD analogue derivatized through the indole nitrogen and conjugated to bovine thyroglobulin. The assay cutoff is 0.5 ng/mL LSD, and the clinical sensitivity for the detection of LSD and its metabolites in human urine samples is equivalent to the LSD Abuscreen RIA. Thirty-one samples previously screened LSD positive by Abuscreen RIA and confirmed by gas chromatography-mass spectrometry were analyzed by the Abuscreen OnLine LSD and Abuscreen LSD RIA assays. All thirty-one samples screened positive in the Abuscreen OnLine and Abuscreen RIA. OnLine's cross-reactivity to nor-LSD is greater than thirty-five percent; other structurally related compounds have similar cross-reactivity to that of the Abuscreen RIA. One thousand presumed negative urine samples were also analyzed; 992 (99.2%) of these gave negative results. The eight OnLine positive samples from this set were found to be negative by Abuscreen RIA. Typical qualitative within-run precision on the Hitachi 717 (at x = cutoff of 0.5 ng/mL; 0.5x, 1.0x, and 1.5x) was found to be less than 2.5%. Between-run precision was less than 3.0%.