Microfilter paper method for antipyrine determination in whole blood by high pressure liquid chromatography

A method for measuring antipyrine in whole blood collected on filter paper is described. A 6.2-mm diameter disc (1/4 in) is punched out, eluted with distilled water, and then extracted with 5 ml of dichloromethane/pentane (50:50, vol/vol). After reconstitution of the dried residue, reverse-phase high pressure liquid chromatography is used to quantitate antipyrine using an internal standard. A mixture of 15% acetonitrile in 0.006 M phosphate buffer pH 7.2 was used as the mobile phase. Chromatography was carried out under isocratic conditions for 15 min. The retention time of antipyrine was 3.6 min. Intra- and interassay coefficients of variation were 3.1% and 7.2%, respectively. The limit of sensitivity was 6 ng/injection. The amount of blood in a 6.2-mm filter paper disc was calculated to be 8.4 +/- 0.8 (SD) microliter. Antipyrine half-lives, apparent volumes of distribution, and metabolic clearance rates measured from the filter paper concentrations or directly from plasma were virtually identical. Antipyrine was stable on filter paper for less than or equal to 6 weeks at room temperature. The method reported is convenient; requires a small amount of blood, which can be easily obtained by fingerstick; and readily permits the measurement of antipyrine clearance in the pediatric as well as adult populations.