Cyclopentenyl cytosine 5'-triphosphate (
CPEC-TP) is the active metabolite of the
investigational drug cyclopentenyl cytosine (CPEC), a
nucleoside analogue which exhibits noteworthy
antineoplastic activity against several murine and human
tumors in tissue culture, and which is now undergoing Phase I clinical trials. This study describes a method to measure the intracellular
CPEC-TP levels in peripheral blood mononuclear cells (
PBM cells) from patients treated with CPEC, without using radiolabeled
drug. The method utilizes on-line multidimensional high-performance liquid chromatography (HPLC) with two columns of different retention mechanisms connected via an automated programmable switching valve. The elution fraction containing
CPEC-TP is initially separated from cellular components using a gel sizing column (TSK-G2000-SW) and then rechromatographed by means of a reversed-phase column operated in an ion-pairing mode (YMC-A-312-ODS). The limit of quantitation of
CPEC-TP by this method is 2.5 pmol per injection. When
CPEC-TP levels were measured in
PBM cells from 12
cancer patients after 20 h continuous infusion of CPEC at doses ranging from 3.5 to 5.9 mg/m2/h, the levels attained ranged from 1.4 to 13.4 microM (3.6 to 33.5 pmol/10(7) cells). However, wide variability in the concentrations of
CPEC-TP achieved were evident at each dose and did not appear to correlate either with the CPEC dose or with CPEC plasma levels. This method was validated by a comparison of the quantitation of
CPEC-TP in cultured
PBM cells and Molt-4 cells (a human T-cell line adapted for growth in tissue culture) after incubation with both unlabeled and radiolabeled CPEC.