2.3. LC/MS Measurement of Angiotensin Peptides

Metabolites of ANG I were analyzed by liquid chromatography-mass spectrometry (LC/MS) method, developed in our previous studies [10, 11], with analytical conditions optimized to current type of samples. Separation of peptides was performed on a reversed-phase HPLC system, equipped with a quaternary high pressure pump L-7000 (Merck, Germany), using a Purospher STAR RP C18e column (125 mm × 2 mm ID, 5 μm particle size) with an appropriate guard C18 column (4 mm × 4 mm ID, 5 μm particle size). Samples were injected onto chromatographic column in a volume of 50 μL. The optimized mobile phase solvents were 5% acetonitrile in a buffer of 4 mM ammonium formate with 4 mM of formic acid (phase A) and 90% acetonitrile in the same buffer (phase B). Angiotensin peptides were separated at a flow rate of 0.25 mL/min with a linear gradient of B in A.

Mass spectrometric detection was performed using an LCQ ion-trap mass spectrometer (Finnigan, San Jose, USA) with an electrospray (ESI) source. All experiments were carried out in the positive ion mode (ion spray voltage 5 kV; capillary voltage 46 V; capillary temperature 200°C; nitrogen flow rate 65 psi) and selective ion monitoring (SIM) mode; m/z values of monitored ions were the following: 450 for ANG (1–7) (MW = 899,02), 466 for ANG III (MW = 931,11), 524 for ANG II (MW = 1046,19), 592 for ANG (1–9) (MW = 1183,34), 649 for ANG I (1296,51), 665 for ANG (1–5) (664,76), and 775 for ANG IV (774,92). Acquired data were analyzed by Xcalibur Software v. 1.2.

Samples for calibration curves of each examined peptide (mixture of standards) were analyzed in the same mode as above. Concentrations of ANG I metabolites were calculated using the standard calibration curves. They were constructed by linear regression analysis with plotting of peak area versus angiotensin concentration. Calibration curves were prepared for each examined peptide at a concentration range of 20 pM–100 nM.