A new, simple method for quantifying gemcitabine triphosphate in cancer cells using isocratic high‐performance liquid chromatography

Abstract

A deoxycytidine analog, gemcitabine (dFdC), is effective for treating solid tumors and hematological malignancies. After being transported into cancer cells, dFdC is phosphorylated to dFdC triphosphate (dFdCTP), which is subsequently incorporated into the DNA strand, thereby inhibiting DNA synthesis. Intracellular dFdCTP is the critical determinant for dFdC cytotoxicity, so therapeutic drug monitoring or in vitro testing of the capability of cancer cells to accumulate dFdCTP may be informative for optimizing dFdC administration. We have developed a new isocratic-elution high-performance liquid chromatography method for quantifying dFdCTP in cancer cells. Samples (500 µ µ µ µL) were eluted isocratically using 0.06 M Na 2 HPO 4 (pH 6.9) containing 20% acetonitrile, at a constant flow rate of 0.7 mL/min and at ambient temperature. Separation was carried out using an anion-exchange column (TSK gel DEAE-2SW; 250 mm × × × × 4.6 mm inside diameter, particle size 5 µ µ µ µL) and monitored at 254 nm. The standard curve was linear with low within-day and interday variability. The lower detection limit (20 pmol) was as sensitive as that of the previous gradient-elution method. dFdCTP was well separated from other nucleoside triphosphates. The method could measure dFdCTP in cultured or primary leukemic cells treated in vitro with dFdC. The method was also applicable to simultaneous determination of dFdCTP and cytarabine triphosphate, the results of which demonstrated ara-CTP production augmented by dFdC pretreatment. Thus, our isocratic high-performance liquid chromatography assay method will be of great use because of its sensitivity and simplicity as well as its applicability to biological materials. (Cancer Sci 2006; 97: 1274-1278) G emcitabine (2′,2-difluorodeoxycytidine, dFdC) is the most important deoxycytidine analog to be developed since cytarabine: it has geminal fluorine atoms inserted at the 2′-carbon of the deoxyribofuranosyl ring.(1,2) The drug is active against not only leukemic cells in vitro but also several experimental solid tumors such as non-small-cell lung cancer, small-cell lung cancer and pancreatic cancer.(3-6) Clinically, dFdC has become the standard first-line therapy for patients with advanced pancreatic cancer, and it is rapidly becoming incorporated into first-line regimens in non-small-cell lung cancer and transitional-cell carcinoma of the bladder.(7-9) The efficacy of dFdC has also been evaluated for leukemic patients. (10,11) Thus, dFdC is now used widely for treating both solid tumors and hematological malignancies. (7)(8)(9)(10)(11) 2′,2-Difluorodeoxycytidine is a prodrug that requires intracellular activation.(1,2) After being transported into cancer cells, it is phosphorylated via dFdC monophosphate and diphosphate to dFdC triphosphate (dFdCTP), the process of which is catalyzed by several kinases including the rate-limiting enzyme deoxycytidine kinase. dFdCTP is incorporated into DNA at the penultimate position and, after the incorporation of one or more nucleotides, blocks further elon...

Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: A review

“…They achieved a satisfactory separation between araCTP, CTP, UTP, ATP, GTP and dFdCTP, however the run time of the analysis was very long (∼2 h). They showed that deoxyribonucleotide triphosphates were eluted at retention times almost identical to those of the corresponding ribonucleotide triphosphates [96].…”

“…Several chromatographic modes were employed for the determination of the nucleoside gemcitabine monophosphate (dFdCMP), diphosphate (dFdCDP) and triphosphate: either the IE-HPLC [96][97][98], or IP-HPLC [2,58] or RP-HPLC on a PGC column [99]. Veltkamp et al [98] developed an assay based on a weak anion exchange liquid chromatography coupled to a detection with MS/MS detection.…”

“…Nishi et al [96] proposed an HPLC method for simultaneous determination of araCTP and gemcitabine triphosphate (dFdCTP) in cancer cells using an anion exchange column coupled with UV detection at 254 nm. Samples were eluted with isocratic mode using a mixture of phosphate buffer (60 mM, pH 6.9) and acetonitrile (80/20, v/v).…”

“…Finally, before the storage, analytical column was back-flushed with 20 volumes of tetrahydrofuran and then stored in ammonium acetate buffer (1 mM)/acetonitrile (85/15, v/v) [100]. Three studies used UV detection for the quantification of diand triphosphate derivatives of gemcitabine [58,96,97]. According to study, the wavelengths were setting to 254, 271 and 275 nm.…”

“…dFdCTP was extracted from biological matrix using a protein precipitation with PCA and neutralization with KOH [96,97]. The protein precipitation could be performed using TCA and then the supernatant was processed with a mixture trioctylamine-freon to allow the cleaning and neutralization of the sample (step repeated three times) [58].…”