A Dual-Column HPLC Method for the Simultaneous Determination of DHPG (9-[(1,3-Dihydroxy-2-Propoxy)Methyl]Guanine) and Its Mono and Diesters in Biological Samples

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Abstract
A convenient method for the simultaneous determination of various DHPG species present in biological samples is presented. This method utilizes a cation exchange column (25 cm × 4.6 mm i.d.) coupled in series to a short reversed-phase column (5 cm × 4.6 mm i.d.). The mobile phase consists of methanol:0.005M ammonium phosphate buffer, pH 2.5. There is a large polarity difference between DHPG and its esters due to the non-polar side chain of the ester moiety. The simultaneous determination of the diesters, monoesters, and DHPG in these samples using only the cation exchange or the reversed-phase column is not possible without time-consuming gradient elution. In the reversed-phase mode alone, the esters are highly retained relative to DHPG, whereas the esters are only slightly retained on a cation exchange column and are insensitive to changes in pH and ionic strength of the mobile phase. However, a combination of these two columns provides interesting selectivity for these compounds and offers a unique way of controlling the retention times of these species relative to each other. The retention time of esters can be selectively altered (with respect to DHPG) by changing the composition of methanol in the mobile phase. In contrast, the retention time of DHPG is controlled by changing the buffer strength and pH of the mobile phase.

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