New synthesis and characterization of (+)-lysergic acid diethylamide (LSD) derivatives and the development of a microparticle-based immunoassay for the detection of LSD and its 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.

Synthesis of new d-propoxyphene derivatives and the development of a microparticle-based immunoassay for the detection of propoxyphene and norpropoxyphene.

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).

Evaluation of the online immunoassay for propoxyphene: comparison to EMIT II and GC-MS.

A study was conducted to compare the clinical sensitivity of the OnLine and EMIT II assays for propoxyphene (PPX) use in human urine. A total of 5138 random clinical samples were evaluated by both OnLine and EMIT II. Samples that were positive for each immunoassay were confirmed for PPX and norpropoxyphene (NPPX) by gas chromatography-mass spectrometry (GC-MS). There were 14 samples that were identified positive by both immunoassays and confirmed positive by GC-MS. An additional six samples were positive by OnLine, negative by EMIT II, and confirmed positive by GC-MS. There was one unconfirmed positive sample identified by each immunoassay, and 5116 samples were identified as negative by both immunoassays. The increased sensitivity by OnLine can be attributed to the cross reactivity of the OnLine antibody, which is higher than the cross reactivity of the EMIT II antibody for NPPX (77% versus 7%, respectively). The high concentrations of NPPX, relative to those of PPX, found in all of the clinical samples suggest that laboratories that currently confirm for PPX should confirm for NPPX in order to obtain a better correlation between immunoassay results and GC-MS confirmations.

An online immunoassay for LSD: comparison with GC-MS and the Abuscreen RIA.

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%.