Development and Validation of a Sensitive UPLC-MS/MS Method for the Quantitation of [13C]Sucrose in Rat Plasma, Blood, and Brain: Its Application to the Measurement of Blood-Brain Barrier Permeability
Accurate and reproducible measurement of blood-brain barrier (BBB) integrity is critical in the assessment of the pathophysiology of the central nervous system disorders and in monitoring therapeutic effects. The widely-used low molecular weight marker [14C]sucrose is non-specific in the absence of chromatographic separation. The purpose of this study was to develop and validate a sensitive and reproducible LC-MS/MS method for the analysis of stable isotopemodified [13C12]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [13C6]sucrose), the marker and IS were recovered from diluted rat blood, plasma, and brain homogenate by protein precipitation using acetonitrile. The recovery of the marker and IS was almost quantitative (90-106%) for all three matrices. The recovered samples were directly injected into an isocratic UPLC system with a run time of 6 min. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r2 ≥ 0.99) in the concentration ranges tested for the diluted blood and plasma (10–1000 ng/mL) and brain homogenate (1-200 ng/mL). The lower limit of quantitation of the assay was 0.5 pg injected on column. The assay was validated (n = 5) based on acceptable intra- and inter-run accuracy and precision values. The method was successfully used for the measurement of serial blood and plasma and terminal brain concentrations of [13C12]sucrose after a single intravenous does (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [13C12]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models.
Miah MK, Bickel U, Mehvar R. Development and validation of a sensitive UPLC–MS/MS method for the quantitation of [13C]sucrose in rat plasma, blood, and brain: Its application to the measurement of blood-brain barrier permeability. J Chromatogr B. 2016;1015-1016:105-110. doi: 10.1016/j.jchromb.2016.02.017
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