The invention discloses a method for simultaneously separation determination of the content of three
aminoglycoside antibiotics (AGs) (
tobramycin, etimicin and
netilmicin); the method successfully separates a mixture of the three
antibiotics, and also is applied in determination of the content of the AGs in
tobramycin eye drops, an
etimicin sulfate injection and a
netilmicin sulfate injection, and an HPLC-RRS combined technology is used for determination of the AGs. In an experiment, used chromatographic columns comprise Agilent Zorbox SB-C18 column (250 mm*4.6 mm i.d., 5 [mu]m) and a protective column (
Security Guard C18, 4*2 mm, Phenomenex). A mobile phase is
methanol:water (containing 0.2% TFA)=5:95 (v / v), the HPLC flow rate is 0.50 mL*min<-1>, [
lambda]ex=[
lambda]em=362 nm, the
column temperature is 30 DEG C, and the sample
injection volume is 20 [mu]L. The probe is a 2.0*10<-5> mol*L<-1> PSB solution, the pH is 9.5, the single-pump flow rate is 0.2 mL*min<-1>, the reaction
pipe length is 250 cm, and the internal
diameter is 0.5 mm. A brand-new HPLC detection mode established with RRS as a detection method is established, and is successfully applied to determination of the content of the AGs in different preparations. The application range of the RRS technology is widened, and the brand-new detection mode is provided for HPLC.