Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid c
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RESEARCH PAPER
Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometry Luisa Barreiros 1,2 & Eduarda M. P. Silva 1 & Inês S. Alencastre 3,4 & Meriem Lamghari 3,4,5 & Marcela A. Segundo 1 Received: 11 June 2020 / Accepted: 16 July 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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. 5-Carbamimidamido-2-(2,2-diphenylacetamido)-N-[(4hydroxyphenyl)methyl]pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized application in bone regeneration studies, requiring quantification at picogram levels. Hence, BIBP 3226 determination is proposed here by a validated HPLC-MS/MS method, based on a reversed-phase Kinetex® core-shell C8 column (2.6 μm, 150 × 2.1 mm) at 30 °C, elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25 mL min−1, 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 determined in cell extracts and supernatants obtained from internalization assays. Using similar exposure conditions, the amount of BIBP 3226 found in breast cancer cells (MCF7) was 72 to 657 times higher than that found in bone marrow cells (Wt C57BL/6 mice), providing an indirect indicator of NPY Y1 receptor expression. Keywords Neuropeptide Y . Y1 receptor antagonist . BIBP 3226 . Mass spectrometry . Receptor expression
Introduction Published in the topical collection featuring Female Role Models in Analytical Chemistry. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02825-z) contains supplementary material, which is available to authorized users. * Marcela A. Segundo [email protected] 1
LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
2
Escola Superior de Saúde, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida 400, 4200-072 Porto, Portugal
3
i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 280, 4200-135 Porto, Portugal
4
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 280, 4200-135 Porto, Portugal
5
ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
Neuropeptide Y (NPY) is a 36-amino acid peptide that was first isolated from the porcine brain [1] and it belongs to a family of peptides
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