Application of deuterium labelling mass spectrometry in a study of the metabolism of the enantiomers of cyclophosphamide

The antitumour agent cyclophosphamide—{2‐[bis(2‐chloroethyl)amino]‐tetrahydro‐2H ‐1,3,2‐oxaza‐phosphorine 2‐oxide}—is chiral owing to asymmetry at phosphorus. The differential metabolism of the enantiomers [(+)‐cyclophosphamide and (−)‐cyclophosphamide] can be monitored by mass spectrometry if pseudoracemates consisting of either unlabelled [²H₀] (+)‐ and tetradeuterated [²H₄] (−)‐cyclophosphamide or of [²H₀](−) and [²H₄](+) enantiomers are administered. Using this principle, methodology has been developed for determining the enantiomer ratios of cyclophosphamide and two metabolites, 4‐ketocyclophosphamide {2‐[bis(2‐chloroethyl)amino] tetrahydro‐2H‐1,3,2‐oxazaphosphorin‐4‐one 2‐oxide} and carboxyphosphamide [2‐carboxyethyl N,N‐bis(2‐chloroethyl)phosphorodiamidate] recovered from the urine of mice. The drug and the two metabolites were quantified using [²H₁₀]cyclophosphamide, 4‐keto‐[²H₈]cyclophosphamide and [²H₆]carboxyphosphamide respectively, as internal standards. The amount of cyclophosphamide excreted was small and neither enantiomer preponderated markedly, but the minor metabolite, 4‐ketocyclophosphamide, was markedly depleted in the enantiomer derived from (−)‐cyclophosphamide, whereas the major metabolite, carboxyphosphamide, was slightly depleted in the enantiomer derived from (+)‐cyclophosphamide.