Validation of a reversed-phase high-performance liquid chromatography method with fluorescence detection for the bioequivalence study of norfloxacin in plasma samples.
A robust method for the determination of norfloxacin in human plasma, using reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detection, has been developed. The method involves precipitation of plasma protein with acetonitrile and the use of ciprofloxacin as internal standard (IS). Chromatographic separations were performed on a Synergi MAX-RP 150 x 4.6-mm, 4-micro column with an elution system consisting of a mixture of phosphate buffer-acetonitrile (85:15, v/v). The calibration curve was linear, in the range of 30 to 3500 ng/mL. The recoveries at concentrations of 90, 1400, and 2800 ng/mL were 103.5%, 100.2%, and 100.2%, respectively. The quantification limit for norfloxacin was 30 ng/mL per 10-microL injection employing fluorescence detection with excitation and emission set at 300 and 450 nm, respectively. The method validation included examining the within-run and between-run precision and accuracy and ensuring that these were within accepted limits; in summary, the precision was <8.6% and accuracy ranged from 95.8% to 104.1% for concentration from 90 to 2800 ng/mL. The precision and accuracy for the lowest calibration standard (30 ng/mL) was well within accepted limits for lower limit of quantification. The method was then applied in a bioequivalence study in healthy volunteers given 400-mg doses of reference and test formulations of norfloxacin in random order and including a 7-day washout phase.
Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco, Cidade Universitária, Recife-PE, Brazil.
Maria Bernadete Sousa
MB
Maia
Ismael Leite
IL
Martins
Demétrius Fernandes
DF
do Nascimento
Adriano Nunes
AN
Cunha
Francisco Evanir Gonçalves
FE
de Lima
Fernando Antônio Frota
FA
Bezerra
Manoel Odorico
MO
Moraes
Maria Elisabete Amaral
ME
Moraes
D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000493:pharmacokinetics*; D001798:Blood Proteins / Q000378:metabolism; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D002939:Ciprofloxacin / Q000008:administration & dosage / Q000493:pharmacokinetics; D018592:Cross-Over Studies; D004311:Double-Blind Method; D016568:Drugs, Generic; D006801:Humans; D007202:Indicators and Reagents; D009643:Norfloxacin / Q000008:administration & dosage / Q000493:pharmacokinetics*; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013810:Therapeutic Equivalency
D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D016568:Drugs, Generic; D007202:Indicators and Reagents; D002939:Ciprofloxacin; D009643:Norfloxacin