Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 mul) was directly introduced onto a Capcell Pak MF Ph-1 column (20 x 4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35 x 2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100 x 2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the Valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD less than or equal to2.3%) and accuracy (bias: +/-2.0%) and speed (total analysis time 17 min). The response was linear (r(2) greater than or equal to 0.999) over the concentration range 10-1000 ng/ml. (C) 2001 Elsevier Science B.V. All rights reserved.