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Ampicillin analysis by fully validated HPLC assay in human plasma

Reem Saleh Alswayeh, Syed N.Alvi, Muhammad M.Hammami

Asimple and rapidHPLCassay for ampicillinmeasurement in human plasma was developed and validated. 0.25 ml plasma sample was precipitated with 50 µl of perchloric acid, and 100 µl of the supernatant was directly injected into 4.6ï‚´150mm, SymmetryShield,RP8, 4-µm steel column at room temperature (RT). The mobile phase, 0.05Mdibasic sodiumphosphate buffer (pH = 7.3) and acetonitrile (87:13, v:v), was delivered at 1.0ml/minwith a run time of 9min. Ciprofloxacin (internal standard, IS) and ampicillinwere detected at 3.9 and 8.1 min, respectively, usingWaters 2998 photodiode array detector set at 210 nm. The response was linear over the range of 0.3–15 µg/ml, and the intra-and inter-run coefficient of variations were ï‚£ 10.7% and 10.4%, respectively. Extraction recovery and intra- and inter-run bias were  86% (mean 91%), ï‚£ 11%, and ï‚£ 7%, respectively.Ampicillinwas stable in plasma for 24 hours at RT ( 87%), 5 weeks at –20 0C( 93%), and after 3 cycles of freeze at –20C and thaw at RT ( 89%). In precipitated plasma samples, ampicillin was stable for 24 hours at RT ( 93%) and 48 hours at –20ï‚°C ( 85%). Stock solution of ampicillin (1mg/ml inwater) was stable for 48 hours at RT (93%) and 5 weeks at -20ï‚°C(97%).