title: Validation of a method for determination of ampicillin in human plasma using LC-DAD.
abstract: We describe the validation data of a simple but selective chromatographic method for determination of ampicillin in human plasma using liquid chromatography-diode array detector. Blank plasma free of drugs was transferred to eppendorf's tubes and spiked with ampicillin stock solution to obtain quality control samples at 1.00, 2.50, 5.00, and 10.00 microg/mL. Extraction of ampicillin and cephalexin (internal standard) from plasma samples (250 microL) was investigated using three different methods: precipitation with perchloric acid, ultra-filtration and solid-phase extraction. Chromatographic separation was achieved using a Shimpak C(18) column (300 mm x 4.6 mm i.d.; 5 microm), and detection was done at 215 nm with a diode array UV-Vis detector. The mobile phase consisted of dihydrogen phosphate (pH 3.5)-acetonitrile (87.5:12.5, v/v) delivered at a flow rate of 1.00 mL/min. Selectivity was evaluated with different pools of human plasma. Perchloric acid precipitation showed an excellent selectivity for normal plasma. The precipitation method presented recoveries above 84.0 +/- 3.3% and 82.0 +/- 1.6%, (n = 3) for ampicillin and cephalexin, respectively. The method has a limit of detection of 0.15 microg/mL and is linear in the range of 0.30 to 100.00 microg/mL. Standardized residue analysis demonstrated normality and homocedasticity. Inter-day precision was 4.5%, and accuracy was 11.1% (n = 9). Stability studies demonstrated instability of b-lactamics in human plasma at 20 and 2 degrees C after 6 and 360 h of storage, respectively.
authors.affiliation: Laboratório de Controle Qualidade de Fármacos e Medicamentos, Curso de Farmácia, Escola de Enfermagem e Farmácia, Universidade Federal de Alagoas, Campus A. C. Simões, BR 104 Norte-Km 97, Maceió-AL. CEP:57072-970. ticianogn@yahoo.com.br
authors.forename: Ticiano Gomes
authors.initials: TG
authors.lastname: do Nascimento
authors.forename: Cícero Flávio Soares
authors.initials: CF
authors.lastname: Aragão
authors.forename: Francinalva Dantas
authors.initials: FD
authors.lastname: de Medeiros
authors.forename: Eduardo de Jesus
authors.initials: Ede J
authors.lastname: Oliveira
authors.forename: Rui Oliveira
authors.initials: RO
authors.lastname: Macêdo
mesh_terms: D000667:Ampicillin / Q000097:blood*; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000667:Ampicillin