title: Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry.
abstract: A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.
authors.affiliation: XenoBiotic Laboratories, Inc., 107 Morgan Lane, Plainsboro, New Jersey 08536, USA. pboner@xbl.com
authors.forename: Pamela L
authors.initials: PL
authors.lastname: Boner
authors.forename: Dave D W
authors.initials: DD
authors.lastname: Liu
authors.forename: William F
authors.initials: WF
authors.lastname: Feely
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Wisocky
authors.forename: Jinn
authors.initials: J
authors.lastname: Wu
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000032:analysis*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003002:Clonixin / Q000031:analogs & derivatives* / Q000032:analysis*; D004350:Drug Residues / Q000032:analysis*; D004355:Drug Stability; D005260:Female; D006900:Hydroxylation; D013058:Mass Spectrometry / Q000379:methods*; D008892:Milk / Q000737:chemistry*; D011786:Quality Control; D012680:Sensitivity and Specificity
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; C014557:flunixin; D003002:Clonixin