Artefacts in the thin-layer chromatographic analysis of 99Tcm-tetrofosmin injections

In an evaluation of techniques for dispensing 99Tcm-tetrofosmin injections, variability in radiochemical purity measured by the recommended 'cut-and-count' thin-layer chromatographic (TLC) analytical technique was observed. This was due to inconsistency in the position of the 99Tcm-tetrofosmin on the chromatography plate. An investigation was therefore undertaken to identify the factors which influence the Rf value of 99Tcm-tetrofosmin and the subsequent effect on the values obtained in the measurement of radiochemical purity. TLC was performed on an ITLC/SG stationary phase with a mobile phase of dichloromethane:acetone (65:35). On a satisfactory chromatogram, the main peak had an Rf of approximately 0.5 and the radiochemical purity was measured at > 90%. The Rf of the main peak and the measured radiochemical purity were found to increase as the size of the sample applied to the plate was increased. Force-drying the sample spot after application to the TLC plate gave a single peak with an Rf of 0. The dimensions of the tank and the time allowed for mobile phase saturation did not affect the outcome of the analysis. Small variations in the composition of the mobile phase affected the Rf of the main peak but not the measured radiochemical purity. TLC plates that were dried at 11 degrees C before use were found to give distorted chromatograms and unreliable measurements of radiochemical purity. In conclusion, when using TLC to measure the radiochemical purity of 99Tcm-tetrofosmin, the following precautions should be observed: (1) the sample spot applied to the TLC plate should be in the range 10-20 microliters; (2) the spot should not be force-dried with air; and (3) ITLC/SG plates should not be dried before use.