Fit-for-purpose chromatographic method for the determination of amikacin in human plasma for the dosage control of patients.

PMID 26838437


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.