title: Development and validation of a sensitive UPLC-MS/MS method for the quantitation of [(13)C]sucrose in rat plasma, blood, and brain: Its application to the measurement of blood-brain barrier permeability.
abstract: 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 [(14)C]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 isotope-modified [(13)C12]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [(13)C6]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 (r(2)≥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 [(13)C12]sucrose after a single intravenous dose (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [(13)C12]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA.
authors.forename: Mohammad K
authors.initials: MK
authors.lastname: Miah
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA; Center for Blood-Brain Barrier Research, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA. Electronic address: Ulrich.Bickel@ttuhsc.edu.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Bickel
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA; Center for Blood-Brain Barrier Research, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA; Department of Biomedical and Pharmaceutical Sciences, Chapman University, School of Pharmacy, Irvine, CA, USA. Electronic address: mehvar@chapman.edu.
authors.forename: Reza
authors.initials: R
authors.lastname: Mehvar
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000502:physiology*; D001923:Brain Chemistry; D002199:Capillary Permeability / Q000502:physiology*; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D008954:Models, Biological; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013395:Sucrose / Q000097:blood / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D013395:Sucrose
keywords: Blood; Blood-brain barrier permeability; Brain; Plasma; Sucrose; UPLC–MS/MS