Multiple response optimization of a liquid chromatographic method for determination of fluoroquinolone and nitroimidazole antimicrobials in serum and urine

• A rapid RP-HPLC method for analysis of four antimicrobial drugs was optimized and validated using design of experiments.
• Chromatographic separation was achieved on a phenyl hexyl column and analytes were resolved in less than 5 min.
• Solid phase extraction with polymeric cartridge offered good and higher recoveries (≥ 94%).
• A detection limit of 0.06–0.15 µg/mL suggests its potential use in routine clinical studies.

ObjectivesDevelopment and validation of a sensitive, selective and robust SPE assisted HPLC method for the quantification of fluoroquinolones and nitroimidazoles in human serum and urine using design of experiments methodology.Design and methodsDesign of experiments was employed for method optimization (Box–Behnken design) and robustness testing (Plackett–Burman design). Sample preparation involved a simple solid phase extraction, which offered a satisfactory recovery (≥ 94%). Analytes were separated on a phenyl hexyl column with mobile phase comprising water, acetonitrile and triethyl amine in ratio of 74:26:0.15 v/v, with a flow rate of 1.1 mL/min.ResultsCalibration curves were linear over selected range (≥ 0.995) for all the analytes. The method was sensitive with detection limits of 0.06–0.16 μg/mL in serum and urine samples. Inter and intra-day precision data (in terms of %RSD) was found to be less than 7%. Stability studies were carried out to assess freeze thaw, short term and long term stability and all analytes were found to be stable. The method was successfully applied for determination of antimicrobial drugs in spiked serum and urine.ConclusionThe obtained results corroborated the potential of the proposed method for determination of all the four antimicrobial drugs in therapeutic drug monitoring, bioequivalence and drug–drug interaction studies.

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