title: Quantitation of brinzolamide in dried blood spots by a novel LC-QTOF-MS/MS method.
abstract: In the current study, a rapid and sensitive LC-QTOF-MS/MS method for the determination of brinzolamide in dried blood spots (DBS) was developed and validated. This novel sample collection, storage and transfer technique was suitable for analyzing a drug with high distribution into red blood cells and negligible plasma levels. The method included an isocratic mobile phase consisting of methanol and 10mM ammonium formate (90:10, v/v) and detection in positive electrospray mode (ESI+). The flow rate was adjusted to 0.350mL/min yielding retention times of 1.7min for both brinzolamide and internal standard (IS) rabeprazole on a Cyano analytical column, respectively. The validation of the proposed method over the concentration range 0.500-20.0μg/mL was performed in compliance with EMEA and FDA guidelines, assessing all major performance characteristics. Inter- and intra- assay precisions were less than 14%, while inter- and intra- assay accuracies varied from 92.2 to 111%. No matrix effect was observed and the mean brinzolamide extraction recovery was 93.5%. The method was successfully applied to real DBS samples from patients in steady state condition, receiving brinzolamide ophthalmic suspension 1% (w/v) for several months. Initial concentrations were corrected due to hematocrit effect, using image processing algorithm written in Matlab.
authors.affiliation: Laboratory of Pharmaceutical Analysis, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupoli Zografou GR-157 71, Athens, Greece.
authors.forename: Anargyros
authors.initials: A
authors.lastname: Foivas
authors.affiliation: Department of Drug Analysis, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia.
authors.forename: Anđelija
authors.initials: A
authors.lastname: Malenović
authors.affiliation: Department of Drug Analysis, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia.
authors.forename: Nađa
authors.initials: N
authors.lastname: Kostić
authors.affiliation: Faculty of Medicine, University of Belgrade, Dr Subotića 8, Belgrade, Serbia; Adnexal Department, Clinic of Eye Diseases, Clinical Center of Serbia, Pasterova 2, Belgrade, Serbia.
authors.forename: Marija
authors.initials: M
authors.lastname: Božić
authors.affiliation: Faculty of Medicine, University of Belgrade, Dr Subotića 8, Belgrade, Serbia; Adnexal Department, Clinic of Eye Diseases, Clinical Center of Serbia, Pasterova 2, Belgrade, Serbia.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Knežević
authors.affiliation: Laboratory of Pharmaceutical Analysis, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupoli Zografou GR-157 71, Athens, Greece.
authors.forename: Yannis L
authors.initials: YL
authors.lastname: Loukas
authors.affiliation: Laboratory of Pharmaceutical Analysis, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupoli Zografou GR-157 71, Athens, Greece. Electronic address: idotsik@pharm.uoa.gr.
authors.forename: Yannis
authors.initials: Y
authors.lastname: Dotsikas
mesh_terms: D060433:Administration, Ophthalmic; D000368:Aged; D002257:Carbonic Anhydrase Inhibitors / Q000008:administration & dosage / Q000097:blood* / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D059788:Dried Blood Spot Testing / Q000295:instrumentation / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D008297:Male; D009883:Ophthalmic Solutions; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013449:Sulfonamides / Q000008:administration & dosage / Q000097:blood* / Q000627:therapeutic use; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D013843:Thiazines / Q000008:administration & dosage / Q000097:blood* / Q000627:therapeutic use
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D009883:Ophthalmic Solutions; D013449:Sulfonamides; D013843:Thiazines; C111827:brinzolamide
keywords: Brinzolamide; Dried blood spots; Hematocrit; Mass spectrometry; RBC distribution