Analysis of urine specimens collected from individuals ingesting 2 and 4 mg flunitrazepam (FN) showed positive results by OnLine and OnTrak immunoassays for up to 60 h. Gas chromatographic-mass spectrometric (GC-MS) analysis of these specimens for FN, 3-OH-FN, 7-amino-FN, 7-amino-3-OH-FN, desmethyl-FN, and 3-OH-desmethyl-FN after glucuronidase treatment showed only low levels of 7-amino-FN with almost none of the other metabolites. These levels were far below the expected results based on the immunoassay data. This study reports on a GC-MS procedure for FN and the previously listed metabolites. The method is based on acid hydrolysis of the urine specimens, which converts FN and all its metabolites described previously to one of four amino-benzophenone derivatives (1-4) with oxazepam-d5 as the internal standard. Under the experimental conditions, the internal standard is converted to 2-amino-5-chloro-benzophenone-d5. The limit of detection for 7-amino-FN and 7-amino-desmethyl-FN and their 3-OH derivatives was less than 1 ng/mL. Analysis of urine specimens collected for 72-h postingestion of 1, 2, or 4 mg FN showed appreciable levels of benzophenone 3 (product of 7-amino-FN and 7-amino-3-OH-FN) and lower levels of benzophenone 4 (product of 7-amino-desmethyl-FN and 7-amino-3-OH-desmethyl-FN) with no detectable levels of benzophenones 1 and 2. The method makes it possible to confirm the presence of FN metabolites in urine at least 72-h postingestion of small doses of the drug.

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A study was conducted to determine the conditions needed to achieve the equilibrium concentration for the epimerization of d-lysergic acid diethylamide (LSD) to iso-LSD. The reaction was followed by integration of the C-9 resonance of LSD and iso-LSD by proton nuclear magnetic resonance (NMR). The C-9 resonance of LSD and iso-LSD appear as singlets at 6.35 and 6.27 ppm respectively. Starting with pure LSD, the conversion to iso-LSD is attained at temperatures above 37 degrees C and pH levels over 7.0. At a pH of 7.0 or higher, the LSD/iso-LSD ratio of 9:1 is achieved after one week at 45 degrees C or two weeks at 37 degrees C. Starting with iso-LSD, the conversion to LSD requires more vigorous conditions. The 9:1 LSD/iso-LSD ratio is attained only after 6 weeks at a temperature of 45 degrees C and a pH of 9.7. At lower pH levels, the reaction proceeds more slowly. The 9:1 LSD/iso-LSD ratio is achieved whether the starting material is LSD or iso-LSD and therefore represents an equilibrium concentration (K = 9). In addition, the more vigorous conditions needed to achieve equilibrium of iso-LSD to LSD demonstrate the difficulty in extraction of the epimerizable proton of iso-LSD. This study is the first to quantitate the epimerization of LSD by NMR techniques and establishes the conditions needed to induce epimerization in solution.

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.

The synthesis of [S-(R,S)]-4-[[methyl[2-methyl-3-(1-oxopropoxy)-3, 4-diphenylbutyl]amino]-1-oxobutoxy]-2,5-pyrrolidinedione+ ++ (propoxyphene active ester, 2) is described. This was used as an intermediate to prepare a propoxyphene immunogen, [S-(R,S)]-4-[methyl][2-methyl-3-(1-oxopropoxy)-3,4-diphenylbuty l]-amino]- 1-oxobutyl-Bovine Thyroglobulin (3). This immunogen was then used to generate antibodies which demonstrate good cross-reactivity to d-propoxyphene, d-norpropoxyphene, and other propoxyphene metabolites. In addition, these antibodies were shown to have very low cross-reactivity to methadone, a structurally related compound. The introduction of an aminomethyl benzoate spacer into the propoxyphene active ester (2), followed by the activation of the carboxylic acid, provided for a more stable active ester (5). This stable active ester, together with the antibodies generated from the propoxyphene immunogen, has led to the development of an immunoassay based on the Kinetic Interaction of Microparticles in Solution (KIMS).