Simultaneous measurement of leucine and isoleucine by multivariate image analysis-thin layer chromatography (MIA-TLC)

This paper introduces a new application of multivariate image analysis-thin layer chromatography, as simple and inexpensive method, for semi-separation and simultaneous measurement of two isomeric amino acids leucine (Leu) and isoleucine (Ile). The images recorded by a digital camera were processed by home-made software and were converted to TLC chromatograms. Linear calibration graphs were obtained for individual analytes with detection limits of 0.60 and 0.48 mu g for Leu and Ile, respectively. The mobile phase composition was optimized for maximum separation. However, complete separation of the spots of the analytes was not obtained. So, the chromatograms were processed by partial least square regression to resolve the overlapped chromatogram. This offered simultaneous determination of Leu and Ile in their binary mixtures. Suitable models were obtained with root mean square error of prediction of 0.008 and 0.009 mu g for Leu and Ile, respectively. The prediction ability of the developed model was assessed by determination of the analytes in diluted serum plasma and the obtained percent of recoveries were in the range of 95-105 %.