The reconstitution of fractionated Cardiolite kits with sodium 99Tcm-pertechnetate eluted from a wet-column 99Mo/99Tcm generator results in a preparation that contains elevated amounts of a polar radiochemical impurity, which is normally present in only trace amounts when such kits are reconstituted with eluate from a dry-column generator. A consequence of this impurity, which appears to be an organotechnetium compound, is that it causes a positive bias in several quality control methods for 99Tcm-sestamibi, including that recommended by the manufacturer. This impurity was isolated, and its biodistribution determined in Wistar rats. Two alternative methods for the preparation of 99Tcm-sestamibi from fractionated Cardiolite kits using eluate from a wet-column generator were investigated. A rapid procedure, based on a C18 solid phase extraction (SPE) cartridge, for measuring the radiochemical purity of 99Tcm-sestamibi samples, which had been prepared using fractionated Cardiolite kits, was developed and compared with two other methods including high-performance liquid chromatography (HPLC). Over a radiochemical purity range of 49-98%, the values obtained using our quality control method deviated from those obtained using HPLC by 1.1 +/- 1.3% (n = 26). In contrast to some of the rapid quality control methods assessed in our investigation, the SPE technique was not biased by the presence of the organotechnetium impurity.