Importance of Pre-Analytical Factors Contributing to Measurement Uncertainty, When Determining Sulfadoxine and Sulfamethoxazole from Capillary Blood Dried on Sampling Paper

A bioanalytical method is developed and validated for determination of sulfadoxine (SD) and sulfamethoxazole (SM) in 100 microL capillary blood dried on sampling paper (Whatman 31ET Chr). SD and SM are extracted with 2000 microL perchloric acid and the liquid phase is loaded onto ENV+ solid-phase extraction columns. SD, SM, and the internal standard are separated on a Purospher STAR RP-18 liquid chromatography column (150 x 4.6 mm) with a mobile phase consisting of acetonitrile-sodium acetate buffer pH 5.2, I = 0.1 (33:67, v/v). Analytes are detected with UV at 256 nm. Lower limit of quantitation is 5 micromol/L, where precisions are 4.2% and 3.9% for SD and SM, respectively. Three brands of sampling papers have been compared with respect to absorption properties, extraction recoveries, and variations. Punching out dried blood spots (DBS) instead of cutting spots into strips prior to extraction has been evaluated by examining precision and accuracy of SD and SM determinations. Importance of uniformity of types of sampling paper, sampling volume and biological matrix, benefit of punching out discs from DBS, and impact on absorption properties of different brands of sampling papers are discussed. Avoiding pre-analytical errors whenever possible results in concentrations determined being more accurate and precise.