Development and Validation of a High-Performance Liquid Chromatography Method for the Evaluation of Niflumic Acid Cross-Reactivity of Two Commercial Immunoassays for Cannabinoids in Urine

Abstract

Niflumic acid is a nonsteroidal, anti-inflammatory drug widely prescribed in Greece. We recently noticed that this drug cross-reacts for cannabinoids in a kinetic interaction of microparticles in a solution (KIMS) immunoassay method but does not in an enzyme multiplied immunoassay technique (EMIT) immunoassay method. The objective of the study was to develop and validate a high-performance liquid chromatographic method in order to evaluate niflumic acid cross-reactivity in two commercial immunoassays for cannabinoids in urine, both in niflumic acid standards as well as in urine specimens obtained from subjects receiving niflumic acid. Urine niflumic acid standards were prepared in drug-free urine at 13 concentrations ranging from 1.25 to 1000 microg/mL. The standards gave presumptive positive cannabinoids results when analyzed by the KIMS immunoassay method when the concentration was above 2.5 microg/mL. None of the prepared standards gave a false-positive cannabinoid result when analyzed by the EMIT immunoassay method. By applying a 50 ng/mL cutoff for cannabinoids in these assays, all 55 urine specimens collected from the 5 subjects who participated gave negative results by the EMIT and false-positive results by the KIMS immunoassay method. It is concluded that KIMS is more prone to cross-reactions by niflumic acid compared to EMIT. Therefore, all positive screening tests for cannabinoids obtained by KIMS should be confirmed by another technique.

Forensic drug analysis and microfluidics

“…Current on‐site screening devices, such as Drugwipe and the Cozart DDSV , are based on immunoassay technologies . Immunoassays are indispensable tools for initial rapid screening to eliminate negative samples , although the general drawback is that they suffer from false positives and cross‐reactivity . In addition, positive samples are followed by confirmatory moderate‐cost tests, such as GC‐MS and HPLC‐MS .…”