Development and validation of a HPLC method for routine quantification of the decapeptide Cetrorelix in liposome dispersions.

The development and validation of an HPLC method for the quantification of the decapeptide Cetrorelix (acetyl-D-2-naphthylalanyl-D-4-chlorphenylalanyl-D-3-pyridylalanyl-seryl-tyrosyl-D-citrullyl-leucyl-arginyl-prolyl-d-alaninamide), a potent antagonist of the luteinising hormone-releasing hormone in liposome dispersions is described. An isocratic reversed phase method with UV-detection appeared most appropriate. Several detergents were tried to disrupt liposomes. Furthermore, detergents turned out to be useful, because they minimised unwanted loss of Cetrorelix due to adsorption to the vial surfaces. Triton X-100 was found most effective, while sodium cholate led to quantification problems. In the presence of 2.5% Triton X-100 calibration curves with a high degree of linearity were achieved in the desired range of 0.2-10 microg/ml. The limits of detection and quantification of Cetrorelix were calculated from the peak-to-noise ratio to be 11 and 37 ng/ml, respectively. The repeatability of the method in presence of phospholipid and Triton was good with relative standard deviations (R.S.D.) ranging from 0.8% (at 0.05 microg/ml) to 1.5% (at 0.2 microg/ml). The presence of liposomes at phospholipid contents of up to 0.25mg/ml did not significantly affect the slope or linearity of the calibration curve, nor the peak-to-noise ratio.