Determination of dapsone in serum and saliva using reversed-phase high-performance liquid chromatography with ultraviolet or electrochemical detection

Abstract

A simple, extractionless method for the determination of dapsone in serum and saliva is described. Reversed-phase high-performance liquid chromatography is used with UV detection at 295 nm or electrochemical detection at 0.7 V. Diazoxide in buffer is the internal standard for UV detection and practolol for electrochemical detection. Sample preparation is minimal with protein precipitation of serum samples whilst saliva samples are simply diluted with addition of an internal standard. Low-level serum and saliva samples are front-cut on-line with a 3 cm laboratory-made precolumn in the loop position on a standard Valco injection valve. Isocratic separation is achieved on a 250 mm × 4.6 mm I.D. stainless-steel Spherisorb S5 ODS-1 column. The mobile phase for high levels of dapsone is acetonitrile—elution buffer (12:88, v/v) at 2 ml/min and a column temperature of 40°C for both serum and saliva separations. For the low-level assays using electrochemical detection and solid-phase clean-up, the mobile phase is acetonitrile—methanol—elution buffer (9:4:87, v/v/v). The UV and electrochemical detection limits are 25 ng/ml and 200 pg/ml, respectively, in both serum and saliva. This simple method is applicable to the routine monitoring of dapsone levels in serum from leprotic patients and electrochemical detection gives a simple, reliable method for the monitoring of trough values in subjects on anti-malarial prophylaxis.