Enantiospecific gas chromatographic-mass spectrometric analysis of urinary methylphenidate: implications for phenotyping

Abstract

A chiral derivatization gas chromatographic-mass spectrometric (GC-MS) method for urine methylphenidate (MPH) analysis was developed and validated to investigate preliminary findings regarding a novel MPH poor metabolizer (PM). Detection was by electron impact (EI) ionization-selected ion monitoring of the N-trifluoroacetylprolylpiperidinium fragments from MPH and the piperidine-deuterated MPH internal standard. The PM eliminated approximately 70 times more l-MPH in urine (9% of the dose over 0-10h), and approximately 5 times more of the d-isomer (10% of the dose), than the mean values determined from 10 normal metabolizers of MPH. Only minor amounts of the metabolite p-hydroxy-MPH were found in the urine of both the PM and normal metabolizers, while the concentration of MPH lactam was not high enough to be detectable. The described method indirectly gauges the functional carboxylesterase-1 status of patients receiving MPH based on the evaluation of relative urine concentrations of d-MPH:l-MPH. Clinical implications concerning rational drug selection for an identified or suspected MPH PM are discussed.

Fig. 1

Structures of MPH enantiomers and corresponding human metabolites . Figure adapted from Patrick et al. [16].

Fig. 2

l-MPH plasma concentration over time for the poor MPH metabolizer (●) compared to the mean values for 19 normal metabolizers (o). Subjects received MPH·HCl (0.3 mg/kg).

Fig. 3

GC-MS-electron impact ionization of urine extracts from a MPH normal metabolizer (left) and from the MPH PM (right). Detection was by selected ion monitoring of the TFP-piperidinium fragment m/z 277. Chiral derivatization generated separable diasteriomers and the piperidine-deuterated MPH provided analytical control (D₅-chromatograms). The urinary concentration of both l-MPH and d-MPH was greatly elevated in the PM.

Fig. 4

GC-MS-EI total ion monitoring of a D₂-D₈ deuterated MPH isotopolog mixture. Selected monitoring of D₅-MPH (TFP-piperidinium fragment m/z 282) served as the internal standard.

Fig. 5

GC-MS-NICI ion chromatogram of reference standard N,O-bis-TFP-dl-HO-MPH. The assignment of d-(R:R’)-HO-MPH (t_R = 20.88 min) and l-(S:S’)-HO-MPH (t_R = 22.18 min) configurations were tentatively assigned-see Results. p-Hydroxylation is a minor MPH metabolic pathway in humans but was investigated as an alternative metabolic pathway for the MPH esterase PM.

Fig. 6

GC-MS-EI total ion monitoring of the MPH lactam metabolite reference standard. This metabolite was not detectable in urine. This 6′-position oxidative metabolite is a minor metabolic pathway in humans but was investigated as an alternative metabolic pathway for the MPH esterase PM.