A new software tool for confident identification and quantitation of drugs in urine and serum

Abstract

Aim High-resolution time-of-flight mass spectrometry has become an excellent tool in forensic toxicology. The accurate mass based inherent characteristics like sensitive wide scope screening together with retrospective and general unknown analysis capabilities make it an ideal tool for this work. We describe the development and evaluation of a new software tool allowing screening and quantification using ultra-high-resolution LC-Q-ToF, accurate mass analysis. The new software allows applying the diagnostic ion concept, using enhanced confirmation criteria such as isotopes, adducts, fragments and qualifier ratios for reducing false positive detection rates. Methods Sixty-one compounds covering several compound classes based on their relevance in post-mortem and routine drug screening were used in this study. After acetonitrile precipitation, urine and serum samples were spiked with the toxicological compound mixes at four levels (between 10–500 ng/mL) and analyzed in ESI(+) mode by LC-Q-ToF-MS (Impact II, Bruker Daltonics, USA), in full scan and bbCID modes using a 14 minute gradient, reverse phase UHPLC separation. The data acquired in bbCID mode were processed with the new software package including a database with more than 2000 compounds of forensic relevance. Results In the full scan ToF-MS channel, all compounds were detected at all concentration levels, no false negatives were encountered. In urine, the high matrix load produced a high number of false positives (547 compared to the 276 expected positives). Only seven compounds were causing ∼ 75% of all -false positives. These false positives typically arose due to a very low detection threshold. In total 35 different compounds appeared as false positives. After applying the enhanced diagnostic ion detection criteria the false positives were completely removed. A challenge was tramadol in the presence of o-desmethylvenlafaxine as these compounds have the same retention time, same sum-formula and the same main fragment ion. However, two smaller characteristic fragments allowed the unambiguous identification of o-desmethylvenlafaxine. Conclusion The screening method with the new software is robust and has been shown to work reproducibly for forensic toxicological screening of blood and urine samples. The application of the ‘diagnostic ion’ concept has proven to be a very powerful tool to eliminate false positive findings.