Development of an LC-MS/MS method for the quantification of goserelin from Pheroid® formulation

Abstract

The oral route is for many reasons the most common route of drug administration, however, due to gastrointestinal (GI) tract enzymatic stability issues, peptide drugs are administered parenterally. Goserelin is an anticancer peptide drug which has been recently encapsulated in the Pheroid® delivery system to enhance its GI enzymatic stability. In vitro stability of Pheroid®-goserelin formulation was therefore evaluated in simulated intestinal fluids. An assay was required to extract and quantify goserelin from Pheroid® formulation in the presence of simulated intestinal fluids. However, a literature survey revealed no analytical method available for such task. Hence, in this study, novel LC-MS/MS assay was developed and validated for goserelin quantification in formulation and intestinal fluids. Pheroid®-goserelin formulation was incubated in simulated gastric fluid (pH 1.2), and simulated intestinal fluid (pH 6.8) for 120 min after which the enzymatic reaction was stopped with acetonitrile in the ratio of 1:3 (v/v). Goserelin was extracted from Pheroid®- goserelin formulation in the simulated intestinal fluids using a protein precipitation method prior to liquid-liquid extraction with water-saturated n-butanol and water. A gradient reverse-phase method with tandem mass spectrometry detection was optimized for the separation and quantification of the extracted goserelin. Several extraction methods and solvents investigated to extract goserelin from the lipids and proteins mixture led to either poor recovery or poor peak shape. A simple, reproducible and highrecovery extraction procedure for goserelin quantification was achieved using both protein precipitation method and liquid-liquid extraction with water-saturated n-butanol and water. Moreover, chromatographic separation and tandem mass spectrometry detection for goserelin and its internal standard were achieved within a total analysis time of 2 min. The challenge for this study was to develop a method to extract goserelin from a matrix comprising lipid and proteins. The stopping reaction reagent (acetonitrile) simultaneously functioned as the protein precipitation solvent, whilst the lipids were successfully extracted using liquid-liquid extraction with water-saturated n-butanol and water. This novel assay was found to be specific, rapid, precise and accurate and could be applied to the goserelin in vitro preclinical stability study