Measurement of Cyclopentenyl cytosine 5′-triphosphate In Vitro and In Vivo by multidimensional high-performance liquid chromatography

Riad Agbaria, Harry Ford, James A. Kelley, Fuming Xie, Pedro Politi, Jean L. Grem, David A. Cooney, Victor E. Marquez, Carmen J. Allegra, David G. Johns

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


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-3 12-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 μM (3.6 to 33.5 pmol/107 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.

Original languageEnglish
Pages (from-to)90-96
Number of pages7
JournalAnalytical Biochemistry
Issue number1
StatePublished - 15 Aug 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Measurement of Cyclopentenyl cytosine 5′-triphosphate In Vitro and In Vivo by multidimensional high-performance liquid chromatography'. Together they form a unique fingerprint.

Cite this