title: Fit-for-purpose chromatographic method for the determination of amikacin in human plasma for the dosage control of patients.
abstract: In this paper, a simple, rapid and sensitive method based on liquid chromatography with fluorimetric detection (HPLC-FLD) for the determination of amikacin (AMK) in human plasma is developed. Determination is performed by pre-column derivatization of AMK with ortho-phtalaldehyde (OPA) in presence of N-acetyl-L-cysteine (NAC) at pH 9.5 for 5 min at 80 °C. In our knowledge, this is the first time that NAC has been used in AMK derivatization. Derivatization conditions (pH, AMK/OPA/NAC molar ratios, temperature and reaction time) are optimized to obtain a single and stable, at room temperature, derivative. Separation of the derivative is achieved on a reversed phase LC column (Kromasil C18, 5 μm, 150 × 4.6 i.d. mm) with a mobile phase of 0.05 M phosphate buffer:acetonitrile (80:20, v/v) pumped at flow rate of 1.0 mL/min. Detection is performed using 337 and 439 nm for excitation and emission wavelengths, respectively. The method is fitted for the purpose of being a competitive alternative to the currently used method in many hospitals for AMK dosage control: fluorescence polarization immunoassay (FPIA). The method exhibits linearity in the 0.17-10 µg mL(-1) concentration range with a squared correlation coefficient higher than 0.995. Trueness and intermediate precision are estimated using spiked drug free plasma samples, which fulfill current UNE-EN ISO15189:2007 accreditation schemes. Finally, for the first time, statistical comparison against the FPIA method is demonstrated using plasma samples from 31 patients under treatment with AMK.
authors.affiliation: Departamento de Química Analítica, Universitat de València, Burjassot, Valencia, Spain; Instituto de Investigación Sanitaria INCLIVA, Hospital Clínico Universitario, Valencia, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Ezquer-Garin
authors.affiliation: Departamento de Química Analítica, Universitat de València, Burjassot, Valencia, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Escuder-Gilabert
authors.affiliation: Departamento de Química Analítica, Universitat de València, Burjassot, Valencia, Spain.
authors.forename: Y
authors.initials: Y
authors.lastname: Martín-Biosca
authors.affiliation: Instituto de Investigación Sanitaria INCLIVA, Hospital Clínico Universitario, Valencia, Spain.
authors.forename: R Ferriols
authors.initials: RF
authors.lastname: Lisart
authors.affiliation: Departamento de Química Analítica, Universitat de València, Burjassot, Valencia, Spain; Centro Interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Unidad Mixta Universidad Politécnica de Valencia-Universidad de València, Valencia, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Sagrado
authors.affiliation: Departamento de Química Analítica, Universitat de València, Burjassot, Valencia, Spain. Electronic address: maria.j.medina@uv.es.
authors.forename: M J
authors.initials: MJ
authors.lastname: Medina-Hernández
mesh_terms: D000583:Amikacin / Q000097:blood*; D000900:Anti-Bacterial Agents / Q000097:blood*; D001424:Bacterial Infections / Q000097:blood* / Q000175:diagnosis / Q000382:microbiology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D011786:Quality Control; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000900:Anti-Bacterial Agents; D000583:Amikacin
keywords: Amikacin; Fit-for-purpose; Fluorescence detection; Liquid chromatography; Ortho-phtalaldehyde/N-acetyl-L-cysteine derivatization; Plasma samples