A daptomycin assay by high-performance liquid chromatography

Abstract number: P1039

Tobin C.M., Lovering A.M., MacGowan A.P., Darville J.M.

Objectives: Daptomycin (DAPT) is a new lipopeptide antibiotic with a unique mode of action. Its spectrum of activity includes most clinically relevant Gram-positive pathogens. With this drug there are few published pharmacokinetic data and these have been limited to healthy volunteers. With all new agents, it is important to be able to measure the serum concentrations, for an understanding of the drug's pharmacokinetics and for the management of patients with multiple organ failure. Our aim was to develop a high performance liquid chromatography (HPLC) method for therapeutic drug monitoring (TDM).

Methods: The column was a C-18, 5-micron (100 x 4.6 mm) with a guard column of the same material. The mobile phase was 0.2 M phosphate buffer, pH 5.5 and acetonitrile (70:30) and the flow rate, 1.5 mL/min. The samples were prepared by mixing with equal volumes of acetonitrile, left for 5 min and centrifuged (13,000g) for 5 min. Ten micro-litres of the supernatant was injected. DAPT was detected by UV absorbance (223 nm). The reproducibility, linearity, accuracy, precision, and the lowest limit of quantification (LLOQ) were investigated.

Results: The retention time (RT) was 6 min. The assay was reproducible: The % CV was 2.8 for a spiked serum containing 2.5 mg/L DAPT (n = 3). For a serum 5 mg/L DAPT sample, the intra-day assay precision %CV was < 4 (n = 32). The inter-day %CV was < 8. The assay was linear from 0.5 to 100 mg/L (R^2 = 0.9993). Serum recovery was > 93% when aqueous and serum samples 2.5, 20.0 and 100.0 mg/L were compared. The accuracy was excellent: the observed and the target concentrations (20, 40 & 80 mg/L) were closely correlated, with all the % errors <2.5. The LLOQ was 0.5 mg/L (n = 4, %CV was 5.2).

Conclusion: The HPLC method was reproducible, linear and accurate. It was straightforward and rapid with a RT of 6 min for DAPT and a total assay time of 10 min. It will be suitable for both a busy TDM laboratory and for the rapid processing of large batches of samples for research purposes.