Determination of aciclovir and ganciclovir in human plasma by liquid chromatography-spectrofluorimetric detection and stability studies in blood samples

A sensitive HPLC method has been developed for the assay of aciclovir and ganciclovir in human plasma, by HPLC coupled with spectrofluorimetric detection. Plasma (1000 microl), with 9-ethyl-guanine added as internal standard, is submitted to protein precipitation with trichloroacetic acid solution 20%. The supernatant, evaporated to dryness at 37 degrees C, is reconstituted in 100 microl of a solution of sodium heptanosulfonate 0.4% adjusted with acetic acid to pH 2.60 and a 30 microl volume is then injected onto a Nucleosil 100-5 microm C18 column. Aciclovir and ganciclovir are analysed by spectrofluorimetric detection set at 260 nm (excitation) and 380 nm (emission) using a gradient elution program with solvents constituted of acetonitrile and a solution of sodium heptanosulfonate 0.4% adjusted to pH 2.60. The calibration curves are linear between 0.1 and 10 microg/ml. The mean absolute recovery of aciclovir and ganciclovir are 99.2+/-2.5 and 100.3+/-2.5%, respectively. The method is precise (with mean inter-day C.V.s within 1.0-1.6% for aciclovir and 1.2-3.5% for ganciclovir), and accurate (range of inter-day deviations -1.6 to +1.6% for aciclovir and -0.4 to -1.4% for ganciclovir). The method has been applied in stability studies of ganciclovir in patients' blood samples, demonstrating its good stability in plasma at -20 degrees C and at room temperature. The distribution of ganciclovir and aciclovir in plasma and red blood cells was also investigated in vitro in spiking experiments with whole blood, which showed an initial drop of ganciclovir and aciclovir levels in plasma (about -25%) due to the cellular uptake of aciclovir and ganciclovir by red blood cells. The method has been validated and is currently applied in a clinical study assessing the ganciclovir plasma concentration variability after administration of valganciclovir in a population of solid organ transplant patients.