4.5.2. LC/MRM-MS

All samples were analyzed in duplicate with an HPLC-MS system consisting of an ExionLC™ (UHPLC system (ThermoFisher Scientific, Waltham, MA, USA) coupled online to a SCIEX QTRAP 6500+ triple quadrupole mass spectrometer (SCIEX, Toronto, ON, Canada). LC-MS parameters, such as the LC gradient and the MRM parameters (Q1 and MRM scans) were adapted and optimized based on previous studies [53].

The sample volume loaded was 10 μL per injection. HPLC separation was carried out using an InfinityLab Poroshell 120 HILIC-Z (HILIC, 2.1 mm × 150 mm, 1.9 µm) (Agilent, Santa Clara, CA, USA) with gradient elution. Mobile phase A was 10% of 200 mM ammonium formate (pH = 3) formic acid in water; mobile phase B was 10% of 200 mM ammonium formate (pH = 3) in acetonitrile. LC separations were performed at a flow of 0.6 mL/min using a 10 min gradient from 100 to 60% of mobile phase B. Mass-spectrometric measurements were carried out using the multiple reaction monitoring (MRM) acquisition method. The electrospray ionization (ESI) source settings were as follows: ion spray voltage 4000 V, temperature 450 °C, ion source gas flow 40 L/min.

Two BLM forms, A2 and B2, were detected and analyzed in MRM experiments with the corresponding transition list (Q1/Q3) masses: BLM-A2 (m/z 708/551 and 708/399) and BLM-B2 (m/z 713/624 and 713/598) at the retention time (RT) of 3.5 min. Optimization of collisional energy (CE) was performed for each transition with 10 V step from 10 to 70 V. The optimal values were 50 and 60 V for BLM-A2 (m/z 708/551 and 708/399) and 50 V for both transitions for BLM-B2 (m/z 713/624 and 713/598).

The most intensive signal which was used for plasma sample analysis was observed for BLM-A2 with transition (Q1/Q3)—m/z 708/399 and was further used for plasma sample analysis.