Metabolites of deadly NPS elucidated by LC-MS

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  • Published: Sep 16, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Metabolites of deadly NPS elucidated by LC-MS

Information is needed on metabolites of butyrfentanyl

Butyrfentanyl is an illegal new psychoactive substance (NPS) increasingly being used in many countries. It is analogous to the opioid painkiller fentanyl, being a butyramide derivative while fentanyl is a propionamide derivative. Butyrfentanyl has seven times the potency of morphine, but only 0.13 times that of fentanyl. It remains, however, a highly dangerous drug and has been linked to a number of deaths. To date, there has been little work carried out on the metabolism of butyrfentanyl, although such knowledge is needed by both medical and law enforcement analysts to determine if this particular compound has been taken. The Zurich researchers looked at the in vitro metabolism of the compound using human liver microsomes and also recombinant cytochrome enzymes. In addition, they carried out in vivo studies using blood and urine samples taken from a deceased victim of the drug.

Butyrfentanyl metabolites identified by UHPLC-QTOF

Blood and urine samples were treated with acetonitrile to precipitate proteins, which were removed by centrifugation. Rotary evaporation gave a metabolite mixture that was taken up in mobile phase (acetonitrile/aqueous ammonium formate). An alternative SPE (solid-phase extraction) method gave more intense peaks, but no additional metabolites were detected.

The metabolic mixture was separated by UHPLC using a Thermo Fisher UltiMate 3000 instrument fitted with a Phenomenex Synergy Polar-RP column. Gradient elution was carried out using 10mM ammonium formate solution (pH 3.5) and acetonitrile, both solvents containing 0.1% v/v formic acid. The amount of acetonitrile increased from 10 to 95 at 0.5 ml/min.

High-resolution mass spectrometry (HMRS) employed a Sciex 6600 QTOF instrument. Accurate masses for metabolite molecular ions were acquired, and the related daughter ions were detected in MS/MS mode and listed in a table. Detailed fragmentation patterns were obtained for the various metabolites, enabling the structures of many to be determined. In all, 36 metabolites had their structures assigned, although for some isomers the exact assignment was not certain (the precise position of OH groups was not clear). The metabolites included several glucuronides and one sulfate derivative. Clear metabolic pathways were mapped. Of the 36 metabolites, only eight were detected from the in vitro microsome experiment, while 14 were seen in the human blood sample and 34 in the human urine sample.

The metabolites from the human urine and blood samples differed from those typically seen in the case of fentanyl itself. For fentanyl and most of its analogues, the nor-compound (i.e. loss of ethylbenzene) is the main metabolite, but nor-butyrfentanyl was only a very minor metabolite in the current in-vivo study, despite being one of the main species present in the microsome in-vitro experiment. It is not clear whether this is due to the human subject having an atypical metabolism or not. The subject had been taking the antibiotic clarithromycin, which is known to inhibit the cytochrome enzyme CYP3A4. The recombinant enzyme studies showed that this enzyme, along with CYP2D6, was involved in the primary metabolic steps in butyrfentanyl degradation. In addition, post-mortem changes may also have occurred to the metabolites initially present.

Butyrfentanyl metabolites found, but are they typical?

The study clearly identified a range of butyrfentanyl metabolites using the comprehensive results from UHPLC and QTOF mass spectrometry. However, the results from human blood and urine were not in line with those seen with fentanyl, possibly because the samples came from a single subject, who may not have been typical. Moreover, the lack of samples meant that precise limits of detection (LODs) could not be established. Further work is needed using samples from a number of people.

Related Links

Drug Testing and Analysis, 2017, 9, 1085-1092. Steuer Jr. et al. Studies on the metabolism of the fentanyl-derived designer drug butyrfentanyl in human in vitro liver preparations and authentic human samples using liquid chromatography-high resolution mass spectrometry (LC-HRMS).

Forensic Science International, 2016, 266, 170-177. Staeheli et al. Time-dependent postmortem redistribution of butyrfentanyl and its metabolites in blood and alternative matrices in a case of butyrfentanyl intoxication.

Wikipedia, Tandem mass spectrometry

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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