Simultaneous determination of deoxyribonucleoside in the presence of ribonucleoside triphosphates in human carcinoma cells by high-performance liquidchromatography

Simultaneous determination of ribonucleoside and deoxyribonucleoside tripho sphates in cells by HPLC is an analytical challenge since the concentration of dNTP present in mammalian cells is several orders of magnitude lower th an the corresponding NTP. Hence, the quantitation of dNTP in cells is gener ally performed after selective oxidation or removal of the major NTP. The p rocedures reported so far are lengthy and cumbersome and do not enable the simultaneous determination of NTP. We report the development of a simple, d irect HPLC method for the simultaneous determination of dNTP and NTP in col on carcinoma WiDr cell extracts using a stepwise gradient elution ion-pairi ng HPLC with uv detection at 260 nm and with a minimal chemical manipulatio n of cells. Exponentially growing WiDr cells were harvested by centrifugati on, rinsed with phosphate-buffered saline, and carefully counted, The pelle ts were suspended in a known volume of ice-cold water and deproteinized wit h an equal volume of 6% trichloroacetic acid. The acid cell extracts (corre sponding to 2.5 x 10(6) cells/100 mu l) were centrifuged at 13,000g for 10 min at 4 degrees C. The resulting supernatants were stored at -80 degrees C prior to analysis. Aliquots (100 mu l) were neutralized with 4.3 mu l satu rated Na2CO3 solution prior the injection of 40 mu l onto the HPLC column ( injection speed 250 mu l/min). Chromatographic separations were performed u sing two Symmetry C18 3.5-mu m (2 x 3.9 x 150 mm) columns (Waters), connect ed in series equipped with a Sentry guard column (3.9 x 20 mm i.d.) filled with the same packing material. The HPLC columns were kept at 30 degrees C. The mobile phase was delivered at a flow rate of 0.5 ml/min, with the foll owing stepwise gradient elution program: % solvent A/solvent B, 100/0 at 0 min --> 100/0 at 1 min --> 36/64 at 5 min --> 31/69 at 90 min --> 31/69 at 105 min --> 0/100 at 106 min --> 0/100 at 120 min; 50/50 MeOH/solvent B fro m 121 to 130 min; 100% solvent A from 131 to 160 min. Solvent A contained 0 .01 M KH2PO4, 0.01 M tetrabutylammonium chloride, and 0.25% MeOH and was ad justed to pH 7.0 (550 mu l 10 N NaOH for 1 liter solvent A). Solvent B cons isted of 0.1 M KH2PO4, 0.028 M tetrabutylammonium chloride, and 30% MeOH an d was neutralized to pH 7.0 (1.4 ml 10 N NaOH for 1 liter solvent B). Even though dNTPs are minor components of cell extracts, satisfactory regression coefficients were obtained for their calibration curves (r(2) > 0.99) esta blished with the addition-calibration methods up to 120 pmol/40-mu l inject ion. The applicability of the method was demonstrated by in vitro studies o f the modulation of NTP and dNTP pools in WiDr colon carcinoma cell lines e xposed to various pharmacological concentrations of cytostatic drugs (i.e., FMdC, IUdR, gemcitabine). In conclusion, this optimized, simplified, analy tical method enables the simultaneous quantitation of NTP and dNTP and may represent a valuable tool for the detection of minute alterations of cellul ar cNTP/NTP pools induced by anticancer/antiviral drugs and diseases. (C) 1 999 Academic Press.