Development of a sensitive, accurate and robust liquid chromatography/mass spectrometric method for profiling of angiotensin peptides in plasma and its application for atherosclerotic mice

• Several LC/MS configurations were compared for determination of Ang peptides.
• Optimal LC/MS configuration was validated for analysis in mouse plasma.
• The method offered better sensitivity and analytical quality than any previous.
• The method was applied for angiotensin profiling in ApoE/LDLR (--/--) mice.
• Substantial changes in renin–angiotensin system in this mouse model were identified.

Quantification of angiotensin (Ang) peptides in biological matrices is a challenge due to their low picomolar (pM) concentration and poor analytical performance of current methods. This work aimed to select an optimal strategy for liquid chromatography/mass spectrometry (LC/MS) quantification of major angiotensins in plasma of wild type and atherosclerotic mice.Optimal LC/MS set-up for Ang quantification was chosen, based on analytical performance, from: nanoflow/orbitrap, nanoflow/triple quadrupole and preconcentration nanoflow/triple quadrupole. The best LC/MS configuration (preconcentration nanoflow/triple quadrupole) was validated and used for measurement of angiotensins (Ang I, II, III, IV and (1–7)) in plasma of 6-month-old atherosclerotic apolipoprotein E/LDL receptor double knock-outs (ApoE/LDLR (--/--)) and wild type C57BL/6J (WT) mice.The method established for Ang quantification was selective, accurate and highly sensitive with LLOQ of 5 pg mL−1. The peak area intra-day precisions for Ang II and Ang-(1–7) were in the range 3.0–5.1 and 3.5–5.8, respectively, with corresponding accuracy of 95.4–103.5% and 95.6–106.3%. Plasma angiotensin profile was substantially modified in ApoE/LDLR knock-out mice with increase in concentration of Ang II from 37.6 ± 21.3 pg mL−1 in WT to 200.2 ± 47.6 pg mL−1. Concentrations of Ang I, III and IV were also increased 3–10 fold in ApoE/LDLR (--/--) mice while that of Ang-(1–7) was unchanged.We conclude that the method developed could be effectively used for accurate, comprehensive profiling of angiotensin peptides in mouse plasma. We identified substantial changes in renin–angiotensin system in a genetic mouse model of atherosclerosis consistent with the overactivation of angiotensin converting enzyme (ACE) and the impairment of ACE2.