A novel and rapid LC/MS/MS assay for bioanalysis of Azurin p28 in serum and its pharmacokinetics in mice

Azurin p28 (NSC745104) is a 28 amino acid peptide fragment that inhibits proliferation of human solid and hematological malignancies in vitro and in vivo by reducing proteasomal degradation of oncogene p53. The present study aimed at developing a novel and fast liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the bioanalysis of p28 in mouse serum, and determining Azurin p28 stability and pharmacokinetics in mice after full method validation. Both Azurin p28 and its internal standard MP-1 were separated and extracted from serum by using perchloric acid (7%, v/v) without time-consuming reconstitution. Chromatographic separation of Azurin p28 and MP-1 from the serum matrix was achieved using a C18 column with a gradient elution profile consisting of 5 mM ammonium acetate and acetonitrile, both containing formic acid. Mass analysis was conducted using positive ion electrospray ionization (ESI) and multiple reaction monitoring (MRM). It took 7.5 min to analyze one sample. The validated concentration range of the method extended from 100 to 10,000 ng/ml with accuracies of 85–115% and inter-day precision (CV) of