Comparison of screening with the use of Biochip Array Technology to the direct analysis of 521 psychoactive compounds and their residues with the use of LC-MS/MS method – does it mean the end of an era for immunochemical research?

Abstract

The classical strategy for toxicology testing of road users involves preliminary immunochemical screening and in case of obtaining the positive result, the instrumental tests are necessary to confirm the result. Immunochemical tests are a cheap, quick and simple tool to confirm the presence of a particular substance in the biological material such as saliva, urine or blood. Their fundamental disadvantages are the limited specificity and the scope of analyses. The field tests allow to determine the presence of a few groups of compounds in one single test. Laboratory tests ELISA usually allow for detecting a few epitopes in one test. In the era of many NPS (novel psychoactive substances) and psychoactive drugs used for recreational purposes, the limited multiplexity factor related to immunochemical arrays is becoming a more serious problem. Therefore, some solution is the application of matrix screening. Biochip Array Technology matrix test allows to analyse 20 various groups of compounds on one chip. Another approach is to completely abandon screening for the sake of definite identification and quantitative determination. In order to satisfy the current situation, the multiplex method with the use of LC-MS/MS method to detect 521 psychoactive compounds and their metabolites was worked out. The experimental material consisted of 300 blood samples taken from road users in Poland, routinely tested in the laboratory of the Institute of Forensic Genetics from 2019 to 2021. The immunochemical tests were carried out with the use of Randox Evidence MultiSTAT analyser. The research with the use of LC-MS/MS method was carried out to detect the presence of 521 psychoactive compounds and their residues in blood taken from the drivers. Blood extraction was carried out using a one-step solid-liquid method. Validation was carried out for quantitative analysis of 521 analytes following the SWGTOX guidelines. Two MRM pairs were selected for each analyte according to the valid mass spectrometry standards and optimal analysis parameters were determined. The analyses were carried out using LC-MS/MS (Kinetex C18 100 × 3 mm, 2.6 μm chromatographic column, mobile phases (in formate buffer) water/methanol, QTRAP 5500 system operated in ESI mode). Comparison of results determined with the use of immunochemical and LC-MS/MS methods indicates that screening immunochemical tests may generate both false positive and false negative results. In spite of the application of the matrix immunochemical test, which allowed the detection of 20 groups of psychoactive substances and metabolites, the LC-MS/MS method made it possible to detect and determine the concentration in a greater number of cases. Taking into account the enormous number of compounds which detection is required while performing screening tests and the need to carry out the confirmation tests, it is justifiable to consider the change of strategy for the sake of the direct instrumental analysis in order to determine a great number of compounds with the use of multiplex analytical methods.