Simultaneous Determination of Deoxyribonucleoside in the Presence of Ribonucleoside Triphosphates in Human Carcinoma Cells by High-Performance Liquid Chromatography

Simultaneous determination of ribonucleoside and deoxyribonucleoside
triphosphates in cells by HPLC is an
analytical challenge since the concentration of dNTP
present in mammalian cells is several orders of magnitude
lower than the corresponding NTP. Hence, the
quantitation of dNTP in cells is generally performed
after selective oxidation or removal of the major NTP.
The procedures reported so far are lengthy and cumbersome
and do not enable the simultaneous determination
of NTP. We report the development of a simple, direct
HPLC method for the simultaneous determination of
dNTP and NTP in colon carcinoma WiDr cell extracts
using a stepwise gradient elution ion-pairingHPLCwith
uv detection at 260 nm and with a minimal chemical
manipulation of cells. Exponentially growing WiDr cells
were harvested by centrifugation, rinsed with phosphate-
buffered saline, and carefully counted. The pellets
were suspended in a known volume of ice-cold water
and deproteinized with an equal volume of 6% trichloroacetic
acid. The acid cell extracts (corresponding to 2.53
106 cells/100 ml) were centrifuged at 13,000g for 10 min at
4°C. The resulting supernatants were stored at 280°C
prior to analysis. Aliquots (100 ml) were neutralized with
4.3 ml saturated Na2CO3 solution prior the injection of 40
ml onto the HPLC column (injection speed 250 ml/min).
Chromatographic separations were performed using
two Symmetry C18 3.5-mm (2 3 3.9 3 150 mm) columns
(Waters), connected in series equipped with a Sentry
guard column (3.9 3 20 mm i.d.) filled with the same
packing material. The HPLC columns were kept at
30°C. The mobile phase was delivered at a flow rate of
0.5 ml/min, with the following stepwise gradient elution
program: % solvent A/solvent B, 100/0 at 0 min 3
100/0 at 1 min 3 36/64 at 5 min 3 31/69 at 90 min 3
31/69 at 105 min 3 0/100 at 106 min 3 0/100 at 120 min;
50/50 MeOH/solvent B from 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 adjusted to pH 7.0 (550 ml 10 N
NaOH for 1 liter solvent A). Solvent B consisted of 0.1
MKH2PO4, 0.028Mtetrabutylammonium chloride, and
30% MeOH and 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 (r2 > 0.99) established with the addition–calibration
methods up to 120 pmol/40-ml injection. The applicability
of the method was demonstrated by in vitro
studies of the modulation of NTP and dNTP pools in
WiDr colon carcinoma cell lines exposed to various
pharmacological concentrations of cytostatic drugs (i.e.,
FMdC, IUdR, gemcitabine). In conclusion, this optimized,
simplified, analytical method enables the simultaneous
quantitation of NTP and dNTP and may represent
a valuable tool for the detection of minute
alterations of cellular dNTP/NTP pools induced by anticancer/
antiviral drugs and diseases. © 1999 Academic Press