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False-positive MDA findings in HRMS-based screening of putrefied postmortem blood samples-Identification of the
Viviane C Stammer1, Dirk K Wissenbach1, Frank T Peters1
1Department Toxicology, Jena University Hospital, Institute for Forensic Medicine, Friedrich Schiller University Jena, Jena, Thuringia 00747, Germany.
Abstract:
An unidentified compound in putrefied postmortem blood samples showed identical accurate mass and chromatographic behavior as 3,4-methylenedioxyamphetamine (MDA) and led to false-positive preliminary screening results. The aim of the study was to identify this unknown interference. Postmortem blood samples were analyzed after protein precipitation on a QExactive Focus high-resolution mass spectrometer (Thermo Fisher, Germany) coupled to a RP C18 column (Macherey-Nagel, Germany). Based on the analysis of mass spectrometry (MS) adducts and isotope ratios using fullscan (m/z 134-330) information, the empiric formula of the protonated molecule [M + H]+ of the unknown compound was found to be C10H14O2N (+ 0.6 ppm). Product ion spectra recorded using normalized collision energy 22% showed a base peak of C8H9O1 (+ 1.5 ppm) and a low-abundant water loss to C7H9 (+ 1.9 ppm), neutral losses of C2H2O and NH3 were found. Based on fullscan and MS-MS information and under consideration of the observed order of neutral losses, the compound was presumptively identified as N-acetyltyramine. This assumption was supported by SIRIUS software showing a SIRIUS score of 99.43% for N-acetyltyramine. Finally, the putative structure annotation was confirmed by a reference compound. The described false-positive MDA findings could be attributed to the presence of N-acetyltyramine in putrefied blood samples. Being an isomer of MDA, N-acetyltyramine could not be distinguished by high-resolution data of the protonated molecules. The presented results once again highlight that false-positive findings may occur even in hyphenated high-resolution mass spectrometry (HRMS) when using full-scan information only.
Insights
An unidentified compound, N-acetyltyramine, in putrefied blood caused false-positive 3,4-methylenedioxyamphetamine (MDA) results. High-resolution mass spectrometry analysis confirmed its identity, highlighting potential interferences in forensic toxicology.
Area of Science:
- Forensic Toxicology
- Analytical Chemistry
- Mass Spectrometry
Background:
- Putrefied postmortem blood samples can contain unidentified compounds that interfere with drug screening.
- A specific unidentified compound mimicked 3,4-methylenedioxyamphetamine (MDA), leading to false-positive preliminary results.
Purpose of the Study:
- To identify the unknown interfering compound in putrefied postmortem blood samples.
- To investigate the cause of false-positive MDA screening results in forensic analysis.
Main Methods:
- Analysis of postmortem blood samples using high-resolution mass spectrometry (QExactive Focus) coupled with RP C18 chromatography.
- Utilized mass spectrometry (MS) adducts, isotope ratios, fullscan MS, and MS-MS data for compound characterization.
- Employed SIRIUS software for structural annotation and confirmed findings with a reference standard.
Main Results:
- The empirical formula of the unknown compound's protonated molecule was determined as C10H14O2N.
- Product ion spectra revealed characteristic fragments and neutral losses, leading to the presumptive identification of N-acetyltyramine.
- N-acetyltyramine was confirmed as the interfering compound, explaining the false-positive MDA findings due to its isomeric nature.
Conclusions:
- N-acetyltyramine in putrefied blood samples is responsible for false-positive MDA screening results.
- High-resolution mass spectrometry (HRMS) using only full-scan data can still yield false-positive findings.
- The study underscores the importance of comprehensive analysis beyond accurate mass and chromatographic behavior for definitive identification in forensic toxicology.
