Automated ion trap LC-MS screening for xenobiotics in vitreous humor

Abstract

Aim Screening for xenobiotics is a crucial part of post-mortem toxicological analysis. Vitreous humor is an easy-to-handle body fluid only little affected by putrefaction and therefore a smart alternative to urine or blood. Due to the blood-retinal barrier, interpretation of quantitative results in vitreous humor is quite difficult but is a suitable matrix for qualitative screening analysis. When analyzing body fluids, liquid chromatography - mass spectrometry has become the method of choice for a wide range of analytical questions. In this project, a previously developed automated LC-MSn approach was evaluated for the detection of drugs and drugs of abuse in vitreous humor to extend its application to post-mortem analysis. Methods Twenty-four samples (vitreous humor) autopsy cases were worked-up by a two-step SPE extraction (Isolute HCX5 ® ), and analyzed by LC-MSn. The LC-MSn system used is the Toxtyper™ (LC-IonTrap Amazon Speed Bruker and UHPLC RSLC 3000 Dionex-Thermo). The screening method is based on alternative polarity (Zero Delay), ultrascan mode (70–800 Da at 32,000 Da/s) and Data Dependend Acquisition (With Scheduled Precursor List). Acquired data (full scan MS, MS2 and MS3) were searched against the Toxtyper library (900 compounds). Results In total, 57 substances distributed to six mixtures were analyzed at three different concentrations (low, med, high). Medium concentration levels were adjusted to vitreous humor concentrations reported in the literature and cut-offs of other established screening methods. Approximately 94% of the compounds could be detected and identified correctly at each concentration level investigated. Mirtazepine could only be detected at the medium concentration level (10 ng/mL) while tramadol and olanzapine could only be identified correctly at high concentration levels (25 and 150 ng/mL). LSD was the only compound that could not be detected at all, probably due to its’ sensitivity to light. Limit of detection was set to the lowest concentration level that could be identified correctly in duplicate determination. The results from real cases were in good agreement with the findings from routine post-mortem analysis. In vitreous humor of 24 autopsy cases (c1–c24), 76% of the active agent consumed by the deceased could be identified in accordance to routine post-mortem analysis. For final method evaluation a confirmatory analysis to determine vitreous humor concentrations of at least the most commcon substances found in post-mortem analysis is mandatory. Conclusion The applied screening approach is a suitable tool for the detection of xenobiotics in vitreous humor. Besides the known physiological limitations of the matrix itself, the obtained limits of detection seem to be adequate for forensic casework. The easy handling of vitreous humor when compared to sample preparation of tissue samples combined with the LC-MSn analysis and automated data evaluation of the Toxtyper present a time- and cost-effective screening alternative for post-mortem cases if no urine and/or only a limited volume of blood is available.