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Isoform‐specific quantification of endothelins in HUVEC culture supernatants by on‐line high‐performance liquid chromatography/electrospray mass spectrometry
Author(s) -
Carrascal Montserrat,
Schneider Klaus,
Peralta Carmen,
Escolar Gines,
Gelpi Emilio,
Abian Joaquin
Publication year - 2004
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.343
Subject(s) - chemistry , chromatography , detection limit , high performance liquid chromatography , mass spectrometry , endothelins , umbilical vein , quantitative analysis (chemistry) , electrospray , solid phase extraction , endothelin receptor , biochemistry , in vitro , receptor
A method for the quantitative analysis of endothelin peptides in human umbilical vein endothelial cell (HUVEC) culture supernatants is reported. The analysis is isoform‐specic and employs solid‐phase extraction and subsequent HPLC fractionation followed by HPLC‐ESIMS analysis. The peptide vasoactive‐intestinal‐contractor (VIC) was used as internal standard for the HPLC‐ESIMS analysis. Linearity of calibration curves was from 50 fmol to 25 pmol. The limit of detection of the HPLC‐ESIMS step using a buffer matrix was estimated at 50 fmol (S/N > 3). The overall limit of detection for supernatants of HUVEC was 500 fmol/mL. In HUVEC culture supernatants only ions of endothelin‐1 (ET1) were observed. Basal levels were determined to be 1.8 ± 0.3 pmol/mL. Quantitative results obtained for ET1 were in agreement with those obtained by using a standard addition method and by an ELISA method. Copyright © 2004 John Wiley & Sons, Ltd.