HPLC DETERMINATION OF ANALGESICS IN HUMAN PLASMA AND SERUM BY DIRECT INJECTION ON 80-ANGSTROM PORE METHYL BONDED PHASE SILICA COLUMNS

The feasibility of using 5-mu-m particle, 80 Angstrom pore sized methyl bonded phase silica columns for direct plasma and serum injection was investigated. The approach was applied to the simultaneous determination of acetaminophen and salicylic acid in human plasma. The separation was achieved using a 15 cm analytical column coupled with a 1 cm guard column of the same material and a mobile phase of 1:100 isopropanol: 0.30% triethylamine in 0.05 M potassium dihydrogen phosphate (pH 6.1) with UV detection at 280 nm. The narrow pore packing gave good separation between the analytes and endogenous components. The very low organic content of the mobile phase did not induce protein precipitation. Analytical column lifetimes of approximately 140 plasma injections were obtained without loss of chromatographic resolution or excessive pressure buildup. Acetaminophen response was equivalent in plasma and serum and good correlation was shown between the direct inject HPLC approach and an enzymatic colorimetric method for acetaminophen determinations in human serum.