title: Identification and fragmentation pathways of caffeine metabolites in urine samples via liquid chromatography with positive electrospray ionization coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry.
abstract: Liquid chromatography (LC) with positive ion electrospray ionization (ESI+) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was employed for the simultaneous determination of caffeine and its metabolites in human urine within a single chromatographic run. LC/ESI-FTICRMS led to the unambiguous determination of the molecular masses of the studied compounds without interference from other biomolecules. A systematic and comprehensive study of the mass spectral behaviour of caffeine and its fourteen metabolites by tandem mass spectrometry (MS/MS) was performed, through in-source ion trap collision-induced dissociation (CID) of the protonated molecules, [M+H](+). A retro-Diels-Alder (RDA) process along with ring-contraction reactions were the major fragmentation pathways observed during CID. The base peak of xanthine precursors originates from the loss of methyl isocyanate (CH(3)NCO, 57 Da) or isocyanic acid (HNCO, 43 Da), which in turn lose a CO unit. Also uric acid derivatives shared a RDA rearrangement as a common fragmentation process and a successive loss of CO(2) or CO. The uracil derivatives showed a loss of a ketene unit (CH(2)CO, 42 Da) from the protonated molecule along with the loss of H(2)O or CO. To assess the potential of the present method three established metabolite ratios to measure P450 CYP1A2, N-acetyltransferase and xanthine oxidase activities were evaluated by a number of identified metabolites from healthy human urine samples after caffeine intake.
authors.affiliation: Dipartimento di Chimica, Università degli Studi della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Bianco
authors.forename: Salvatore
authors.initials: S
authors.lastname: Abate
authors.forename: Cristiana
authors.initials: C
authors.lastname: Labella
authors.forename: Tommaso R I
authors.initials: TR
authors.lastname: Cataldi
mesh_terms: D001191:Arylamine N-Acetyltransferase / Q000378:metabolism; D002110:Caffeine / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D002110:Caffeine; D019363:Cytochrome P-450 CYP1A1; D014969:Xanthine Oxidase; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human