Identification of isobaric product ions in electrospray ionization mass spectra of fentanyl using multistage mass spectrometry and deuterium labeling

Abstract

Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS) conditions (m/z 188) was accounted for by a neutral loss of N-phenylpropanamide. 1-(2-Phenylethyl)-1,2,3,6-tetrahydropyridine (1) was proposed as the structure of the product ion. However, further fragmentation (MS(3)) of the fentanyl m/z 188 ion gave product ions that were different from the product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation of fentanyl in deuterium oxide moved one of the isobars to 1 Da higher mass, and left the other isobar unchanged in mass. Multistage mass spectral data from deuterium-labeled proposed isobaric structures provided support for two fragmentation pathways. The results illustrate the utility of multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product ions.

The characterization of isobaric product ions of fentanyl using multi‐stage mass spectrometry, high‐resolution mass spectrometry and isotopic labeling

“…These spectra contain the major structural fragments for each MS‐level. The distribution of product ions in Figure B is in agreement with the work of Wichitnithad et al…”

“…As described by Wichitnithad et al, the intermediate product ion at m/ z 188 forms through a 6‐centered rearrangement resulting in the loss of N ‐phenylpropanamide. However, based on our MS n studies, we have identified the intermediate product ion at m/ z 281 as an intermediate between the [M+H] + precursor and the fragment at m/ z 188.…”

“…However, fragmentation of this particular isomer at the MS 4 level only forms a product ion at m/ z 132 (Figure B). The product ion at m/ z 188 formed through the intermediate at m/ z 216 fragments differently from either of the two isomers at m/ z 188 identified by Wichitnithad et al Supplemental Figure 1 shows product ion spectra collected in pseudo‐MS 3 mode on the Q‐TOF instrument with proposed structures of the major fragments embedded. In these spectra, in‐source CID of the protonated molecular ion generated pseudo‐MS 2 primary fragments, which were then isolated at the MS 2 level and fragmented in the collision cell to generate pseudo‐MS 3 spectra.…”

“…The base peak in this spectrum is observed at m/ z 188, which has been identified as a characteristic ion for the identification of fentanyl derivatives . However, based on the competing mechanisms for the loss of the N ‐phenylpropanamide neutral, previous literature has demonstrated that this peak must contain at least two isobaric product ions with different constitutional structures …”

“…They recommended HRMS to help identify the elemental compositions of different fragment ions, including the fragment at m/ z 188.1439 in the case of fentanyl analogs . However, Wichitnithad et al observed at least two isobaric product ions with the same exact mass at m/ z 188.1439 . They were only able to distinguish the constitutional isomers using MS 3 and deuterium labeling .…”