Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometry

ABSTRACT: Neuropeptide Y (NPY) is a peptide widely distributed throughout the body that is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis and, as recently reported, the regulation of bone homeostasis. The Y1 receptors have been identified as major contributors to this latter biological effect. 5 - Carbamimidamido- 2 - (2,2 - diphenylacetamido) – N - [(4- hydroxyphenyl)methyl] pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized potential as therapeutic agent in bone regeneration and its quantification in cellular internalization assays was aimed in the present work. Using high-performance liquid chromatography coupled to triple quadrupole-tandem mass spectrometry, BIBP 3226 determination was attained with a reversed phase column, isocratic elution, detection in positive ionization mode and data acquisition in selected reaction monitoring mode. Calibration curves were linear for concentrations ranging from 0.25 to 30 ng mL-1 with LOD and LOQ values as low as 0.1 and 0.3 pg in cell extracts and 16 and 48 pg in supernatant culture media, respectively. BIBP 3226 was successfully quantified in cell extracts and supernatants obtained from internalization assays using bone marrow cells (Wt C57/Bl6 mice) and also breast cancer cells (MCF7), which overexpress the NPY Y1 receptor. Therefore, the determined amount of BIBP 3226 was related to the levels of NPY Y1 receptor, demonstrating the method suitability to measure NPY Y1 receptor expression indirectly as an alternative to antibody-based fluorescence microscopy.

INSTRUMENT(S): Shimadzu instrument model

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture, Bone Marrow

SUBMITTER: Luisa Barreiros  

LAB HEAD: Luisa Barreiros

PROVIDER: PXD018157 | Pride | 2020-08-25

REPOSITORIES: Pride