title: Development and validation of a fast and sensitive bioanalytical method for the quantitative determination of glucocorticoids--quantitative measurement of dexamethasone in rabbit ocular matrices by liquid chromatography tandem mass spectrometry.
abstract: A sensitive, selective, accurate and robust LC-MS/MS method was developed and validated for the quantitative determination of glucocorticoids in rabbit ocular tissues. Samples were processed by a simple liquid-liquid extraction procedure. Chromatographic separation was performed on Phenomenex reversed phase C18 gemini column (50mmx4.6mm i.d.,) with an isocratic mobile phase composed of 30% of acetonitrile in water containing 0.1% of formic acid, at a flow rate 0.2mL/min. Dexamethasone (DEX), prednisolone (PD) and hydrocortisone (HD) were detected with proton adducts at m/z 393.20-->355.30, 361.30-->147.20 and 363.20-->121.0 in multiple reaction monitoring (MRM) positive mode respectively. Finally, 50microL of 0.1% novel DEX mixed micellar formulation was topically administered to a rabbit eye and concentrations were measured. The method was validated over a linear concentration range of 2.7-617.6ng/mL. Lower limit of quantitation (LLOQ) of DEX and PD was measured in the concentration range of 2.7 and 11.0ng/mL respectively. The resulting method demonstrated intra and inter-day precision within 13.3% and 11.1% and accuracy within 19.3% and 12.5% for DEX and PD, respectively. Both analytes were found to be stable throughout freeze-thaw cycles and during bench top and postoperative stability studies (r(2)>0.999). DEX concentrations in various ocular tissue samples i.e., aqueous humor, cornea, iris ciliary body, sclera and retina choroid were found to be 344.0, 1050.07, 529.6, 103.9 and 48.5ng/mg protein respectively. Absorption of DEX after topical administration from a novel aqueous mixed micellar formulation achieved therapeutic concentration levels in posterior segment of the rabbit eye.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, 5258 Health Science Building, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA.
authors.forename: Ravinder
authors.initials: R
authors.lastname: Earla
authors.forename: Sai H S
authors.initials: SH
authors.lastname: Boddu
authors.forename: Kishore
authors.initials: K
authors.lastname: Cholkar
authors.forename: Sudharshan
authors.initials: S
authors.lastname: Hariharan
authors.forename: Jwala
authors.initials: J
authors.lastname: Jwala
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D003907:Dexamethasone / Q000008:administration & dosage / Q000032:analysis* / Q000493:pharmacokinetics; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005123:Eye / Q000187:drug effects / Q000378:metabolism*; D005938:Glucocorticoids / Q000008:administration & dosage / Q000032:analysis* / Q000493:pharmacokinetics; D008297:Male; D011817:Rabbits; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005938:Glucocorticoids; D003907:Dexamethasone