Unusual mass spectrometric dissociation pathway of protonated isoquinoline-3-carboxamides due to multiple reversible water adduct formation in the gas phase.
The study of the collision-induced dissociation behavior of various substituted isoquinoline-3-carboxamides, which are amongst a group of drug candidates for the treatment of anemic disorders (e.g., FG-2216), allowed for the formulation of the general mechanisms underlying the unusual fragmentation behavior of this class of compounds. Characterization was achieved with high-resolution/high accuracy LTQ-Orbitrap tandem mass spectrometry of the protonated precursor ions. Presented data were substantiated by the synthesis and analysis of proposed gas-phase intermediate structures and stable isotope labeled analogues, as well as by density functional theory calculations. Exemplary, CID of protonated N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-yl)carbonyl]glycine gives rise to the isoquinoline-3-carboxy-methyleneamide product ion which nominally eliminates a fragment of 11 u. This was attributed to the loss of methyleneamine (-29 u) and a concomitant spontaneous and reversible water addition (+18 u) to the resulting acylium ion to yield the protonated isoquinoline-3-carboxylic acid. The same water addition pattern is observed after loss of propylene (-42 u). A further nominal loss of 10 u is explained by the elimination of carbon monoxide (-28 u) followed by another water adduct formation (+18 u) to yield the protonated 1-chloro-3,4,7-trihydroxy-isoquinoline. The source of the multiple gas-phase water addition remained unclear. This atypical fragmentation pattern proved to be highly characteristic for all studied and differentially substituted isoquinoline-3-carboxamides, and offers powerful analytical tools for the establishment of a LC/MS(/MS) based screening procedure for model HIF-stabilizers and their potential metabolites in clinical, forensic and sports drug testing.
Beuck
Simon
S
Center for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.
Schwabe
Tobias
T
Grimme
Stefan
S
Schlörer
Nils
N
Kamber
Matthias
M
Schänzer
Wilhelm
W
Thevis
Mario
M
D002853:Chromatography, Liquid / Q000379:methods; D004300:Doping in Sports / Q000517:prevention & control*; D005740:Gases / Q000737:chemistry*; D006801:Humans; D007477:Ions; D007546:Isoquinolines / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D004364:Pharmaceutical Preparations / Q000737:chemistry; D044367:Phase Transition; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D015813:Substance Abuse Detection / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014867:Water / Q000737:chemistry*
D005740:Gases; D007477:Ions; D007546:Isoquinolines; D004364:Pharmaceutical Preparations; D011522:Protons; D014867:Water; C039109:isoquinoline