Simultaneous determination of aceclofenac and diclofenac in human plasma by narrowbore HPLC using column-switching.

A fully automated narrowbore high performance liquid chromatography (HPLC) with column-switching was developed for the simultaneous determination of aceclofenac and diclofenac from human plasma samples. Plasma sample (100 microl) was directly introduced onto a Capcell Pak MF Ph-1 column (20 x 4 mm I.D.) where primary separation was occurred to remove proteins and concentrate target substances using acetonitrile potassium phosphate (pH 7, 0.1 M) (14:86, v/v). The drug molecules eluted from MF Ph-1 column were focused in an intermediate column (35 x 2 mm I.D.) by the valve switching step. The substances enriched in intermediate column were eluted and separated on the narrowbore phenyl hexyl column (100 x 2 mm I.D.) using acetonitrile-potassium phosphate (pH 7, 0.02M) (33:67, v/v) when the valve status was switched back to A position. The method showed excellent sensitivity (detection limit of 10 ng ml(-1)) with small volume of samples (100 microl), good precision and accuracy, and speed (total analysis time 17 min) without any loss in chromatographic efficiency. The response was linear (r2 > or = 0.999) over the concentration range of 50-10,000 ng ml(-1).