Targeted toxicological screening for acidic, neutral and basic substances in postmortem and antemortem whole blood using simple protein precipitation and UPLC-HR-TOF-MS

A broad targeted screening method based on broadband collision-induced dissociation (bbCID) ultra-performance liquid chromatography high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS) was developed and evaluated for toxicological screening of whole blood samples. The acidic, neutral and basic substances covered by the method were identified in postmortem and antemortem whole blood samples from forensic autopsy cases, clinical forensic cases and driving under the influence of drugs (DUID) cases by a reverse target database search. The screening method covered 467 substances. Validation was performed on spiked whole blood samples and authentic postmortem and antemortem whole blood samples. For most of the basic drugs, the established cut-off limits were very low, ranging from 0.25 ng/g to 50 ng/g. The established cut-off limits for most neutral and acidic drugs, were in the range from 50 ng/g to 500 ng/g. Sample preparation was performed using simple protein precipitation of 300 μL of whole blood with acetonitrile and methanol. Ten microliters of the reconstituted extract were injected and separated within a 13.5 min UPLC gradient reverse-phase run. Positive electrospray ionization (ESI) was used to generate the ions in the m/z range of 50⿿1000. Fragment ions were generated by bbCID. Identification was based on retention time, accurate mass, fragment ion(s) and isotopic pattern. A very sensitive broad toxicological screening method using positive electrospray ionization UPLC-HR-TOF-MS was achieved in one injection. This method covered basic substances, substances traditionally analyzed in negative ESI (e.g., salicylic acid), small highly polar substances such as beta- and gamma-hydroxybutyric acid (BHB and GHB, respectively) and highly non-polar substances such as amiodarone. The new method was shown to combine high sensitivity with a very broad scope that has not previously been reported in toxicological whole blood screening when using only one injection.