Rapid alcohol determination in plasma and urine by column liquid chromatography with biosensor detection

An enzyme based amperometric biosensor used as a selective and sensitive de tection unit in column liquid chromatography for the determination of ethan ol and methanol in biological fluids such as plasma and urine is described. The reagentless enzyme electrode is based on the co-immobilisation of alco hol oxidase and horseradish peroxidase in carbon paste. The selectivity of the biosensor was found to vary when four various alcohol oxidase enzyme pr eparations from Candida boidinii, Pichia pastoris, and Hansenula polymorpha were used in the biosensors described. High sensitivity could be obtained for a number of alcohols, organic acids, and aldehydes. Optimisation regard ing the sensitivity and selectivity of the four alcohol oxidase co-immobili sed biosensors are outlined. A fast and reliable liquid chromatographic sep aration system with a PLRP-S polymer based separation column used with a ph osphate buffer as the mobile phase was optimised using the best biosensor w hich was based on alcohol oxidase from P. pastoris and which showed the hig hest turnover rate for alcohols, as the detector for the determination of e thanol and methanol in human urine and plasma samples. The selectivity and stability of the biosensor were retained by working at an applied potential of -50 mV versus Ag/AgCl, the optimal operational potential, and by the ca sting of a protective membrane on the electrode surface. High selectivity o f the enzyme electrode was also found towards other easily oxidisable inter fering species normally present in biological fluids. It was found that sta ble and reliable determinations of ethanol and methanol in plasma and urine could be performed with only a simple dilution and centrifugation step pri or to injection into the liquid chromatographic system. An analysis time of 4 min was required for the assay, with a sample throughput of 13 samples h (-1). (C) 1998 Published by Elsevier Science B.V. All rights reserved.