ABSTRACT
The separation and determination some Aminoglycosides Antibiotics [Amikacin, Tobramicin, Gentamicin] which is considerd non fluorescent compounds [lack of chromophore] after pre-column derivatization with ortho-phthaldehyde [OPA]were done by using reversed phase high performance liquid chromatography RP-HPLC and fluorescence detector [excitation 340 nm, emission 450nm]. Method was developed based on mixture [water: methanol: acetonitrile] ratio [60:35:5] as the mobile phase, flow rate 1ml/min and octadecyle column ODS c18 [250x4.6]. The mixture of three aminoghyccosides Antibiotics was separated and determined by the same mobile phase and same conditions, In addition, this method was applied for separation and determination of Amikacin in serum after liquid/ solid extraction technique. The extraction recoveries for standard sequence were between [95-104.3%] and regression coefficient=0.9997, limit of detection LOD=[0.01 micro g/ml] and limit of quantitation LOQ=[0.3 micro g/ml]. The validation for this method improved system suitability for qualitative studies through retention time, peak shape, selectivity and resolution and for quantitative studies through precision, accuracy, recovery, and linearity