High-performance liquid chromatographic drug analysis by direct injection of whole blood samples. I. Determination of moderately hydrophobic drugs incorporated into blood corpuscles

Abstract

Amounts of a moderately hydrophobic drug incorporated into blood corpuscles were determined by column switching high-performance liquid chromatography with direct injection of whole blood samples. Some modifications were required in order to apply the conventional direct injection method to whole blood samples, as follows. Clogging of the pre-column by blood corpuscles or cytomembranes was avoided by using an end-fitting filter with pore size larger than 40 micron, and accordingly a packing with particle size larger than 40 micron was used. Haemoglobin was decomposed in contact with ODS silica gel and reduced the column efficiency. The protein-coated ODS pre-column (pore size less than 7 nm) was restored by washing with 0.1 M phosphate buffer (pH 3.0) containing 50% acetonitrile. Sodium dodecyl sulphate solution (0.5%, w/v) was preferable for rinsing and removes material remaining in the pre-column. A whole blood sample containing carbamazepine was directly injected into the pre-column, then blood cells were haemolysed promptly in contact with a mobile phase of low salt concentration. The total amount of carbamazepine could be determined with a coefficient of variation of 1.9% (within-run). Carbamazepine incorporated into rabbit blood cells (haematocrit value 33.6%) was determined to be 1.1-1.3 times higher than the concentration in plasma. Adsorption on the cytomembranes was not observed.