Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro blood-brain barrier and intestinal drug permeability studies.
Sedative and anxiolytic-like properties of flavonoids such as kaempferol and quercetin, and of some of their intestinal metabolites, have been demonstrated in pharmacological studies. However, routes of administration were shown to be critical for observing in vivo activity. Therefore, the ability to cross intestinal and blood-brain barriers was assessed in cell-based models for kaempferol (KMF), and for the major intestinal metabolite of KMF, 4-hydroxyphenylacetic acid (4-HPAA). Intestinal transport studies were performed with Caco-2 cells, and blood-brain barrier transport studies with an immortalized monoculture human model and a primary triple-co-culture rat model. UHPLC-MS/MS methods for KMF and 4-HPAA in Ringer-HEPES buffer and in Hank's balanced salt solution were validated according to industry guidelines. For all methods, calibration curves were fitted by least-squares quadratic regression with 1/X(2) as weighing factor, and mean coefficients of determination (R(2)) were >0.99. Data obtained with all barrier models showed high intestinal and blood-brain barrier permeation of KMF, and no permeability of 4-HPAA, when compared to barrier integrity markers.
Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland; Department of Pharmacognosy, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.
Fahimeh
F
Moradi-Afrapoli
Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Mouhssin
M
Oufir
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt 62, 6726 Szeged, Hungary.
Fruzsina R
FR
Walter
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt 62, 6726 Szeged, Hungary.
Maria A
MA
Deli
Division of Molecular Modeling, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Martin
M
Smiesko
Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
Volha
V
Zabela
Department of Pharmaceutics, College of Pharmacy, University of Florida, 1345 Gainesville, FL, USA; School of Life Sciences Institute for Pharma Technology, Gründenstrasse 40,CH-4132 Muttenz, Switzerland.
Veronika
V
Butterweck
Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland. Electronic address: matthias.hamburger@unibas.ch.
Matthias
M
Hamburger
D000818:Animals; D001812:Blood-Brain Barrier / Q000737:chemistry* / Q000378:metabolism*; D018938:Caco-2 Cells; D002138:Calibration; D002478:Cells, Cultured; D018920:Coculture Techniques; D003198:Computer Simulation; D006801:Humans; D007408:Intestinal Absorption; D044949:Kaempferols / Q000032:analysis* / Q000493:pharmacokinetics*; D010648:Phenylacetates / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D044949:Kaempferols; D010648:Phenylacetates; C008070:4-hydroxyphenylacetic acid; C006552:kaempferol
4-HPAA; Blood-brain barrier; Caco-2 model; Kaempferol; LC–MS/MS; Method validation