Isoform-specific quantification of endothelins in HUVEC culture supernatants by on-line high-performance liquid chromatography/electrospray mass spectrometry |
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Authors: | Carrascal Montserrat Schneider Klaus Peralta Carmen Escolar Gines Gelpi Emilio Abian Joaquin |
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Institution: | Structural and Biological Mass Spectrometry Unit, Department of Medical Bioanalysis, IIBB-CSIC, IDIBAPS, Rossello 161, 08036-Barcelona, Spain. |
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Abstract: | A method for the quantitative analysis of endothelin peptides in human umbilical vein endothelial cell (HUVEC) culture supernatants is reported. The analysis is isoform-specific 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. |
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Keywords: | endothelin peptides human umbilical vein epithelial cell HPLC‐ESIMS selected ion monitoring |
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