Simultaneous analysis of angiotensin peptides by LC-MS and LC-MS/MS: metabolism by bovine adrenal endothelial cells

Abstract

Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed to simultaneously determine the concentrations of angiotensin (Ang) II, Ang 1-7, Ang III, and Ang IV in biological samples. The samples were extracted with C18 solid-phase extraction cartridges and separated by a reverse-phase C18 column using acetonitrile in water with 0.1% formic acid as a mobile phase. Ang peptides were ionized by electrospray and detected by triple quadrupole MS in the positive ion mode. (M+3H)(3+) and (M+2H)(2+) ions were chosen as the detected ions in the single ion recording (SIR) mode for LC-MS. The limits of detection (signal/noise [S/N]=3) using SIR are 1 pg for Ang IV and 5 pg for Ang 1-7, Ang III, and Ang II. Multiple reaction monitoring (MRM) mode was used for LC-MS/MS. The limits of detection (S/N =3) using MRM are 20 pg for Ang IV and 25 pg for Ang 1-7, Ang III, and Ang II. These methods were applied to analyze Ang peptides in bovine adrenal microvascular endothelial cells. The results show that Ang II is metabolized by endothelial cells to Ang 1-7, Ang III, and Ang IV, with Ang 1-7 being the major metabolite.

Figure 1

The mass spectra of Ang 1-7, Ang III, Ang II and Ang IV under low cone voltages (10–15 V) by LC-MS.

Figure 2

LC-MS and LC-MS/MS chromatograms of extracted angiotensin peptides using the SPE protocol, SIR mode for LC-MS (m/z 300.6 for Ang 1-7, m/z 311.8 for Ang III, m/z 349.6 for Ang II, m/z 388.8 for Ang IV and m/z 391.8 for internal standard), and MRM mode for LC-MS/MS (m/z 300.6→109.6 for Ang 1-7, m/z 311.3→228.4 for Ang III, m/z 349.6→255.2 for Ang II, m/z 388.8→263.4 for Ang IV and m/z 391.8→269.2 for internal standard).

Figure 3

Calibration curves of Ang 1-7, Ang III, Ang II and Ang IV by (A) LC-MS and (B) LC-MS/MS.

Figure 4

The loss of angiotensin peptides with storage as stock solution water:acetonitrile (84:16) with 0.1% formic acid.

Figure 5

LC-MS and LC-MS/MS chromatograms of Ang 1-7, II, III, IV and internal standard in supernatant samples from bovine adrenal microvascular endothelial cells incubated for 30 min with 10⁻⁷M Ang II.

Figure 6

Concentrations of Ang 1-7, Ang II, Ang III and Ang IV in bovine adrenal microvascular endothelial cells incubated with Ang II (10⁻⁷M) for 0, 5, 15, 30, 60 min. Values= mean±S.E. (n=3).