Application of high-performance liquid chromatography-electrospray ionization-mass spectrometry to measure microsomal membrane transport of glucuronides.
A primary reason for poor characterization of microsomal transport to date is the limitations of the measurement techniques used. Radiodetection provides sufficient sensitivity, but it can be applied only when labeled analogue is available. In this article, we report the novel application of high-performance liquid chromatography and electrospray tandem mass spectrometry (LC-MS/MS) in "rapid filtration" transport assays. The method was developed using glucuronides, but it is adaptable to any compound that can be measured with LC-MS/MS. Because of the high sensitivity and accuracy of this detection technique, the substrates can be used at their physiological concentration in the experiments. The new methodology does not require radiolabeling, so it remarkably widens the range of possible substrates to investigate and allows simultaneous detection as well as monitoring of substrate stability during the experiments.
Staines
Adam G
AG
Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 4HN, Scotland, UK.
Burchell
Brian
B
Bánhegyi
Gábor
G
Mandl
József
J
Csala
Miklós
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004958:Estradiol / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D020719:Glucuronides / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism / Q000648:ultrastructure; D010635:Phenolphthaleins / Q000032:analysis / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D020719:Glucuronides; D010635:Phenolphthaleins; C016651:phenolphthalein glucuronide; C025483:estradiol-17 beta-glucuronide; D004958:Estradiol