3.6. UHPLC-MS/MS Method Development and Analysis

Chromatographic separation was performed on an Acquity UPLC instrument equipped with HSS C₁₈ column (2.1 × 100 mm, 1.8 µm particle size) attached to a VanGuard pre-column (2.1 × 5 mm) with similar packing material, all from Waters Corporation (Milford, MA, USA). Column temperature was maintained at 35 °C while HPLC autosampler compartment was at 10 °C. A binary gradient separation comprising of 0.1% formic acid in H₂O (Mobile phase A), and MeCN: H₂O (90:10, v/v—Mobile phase B), pumped at a flow rate of 0.6 mL min⁻¹ was employed. Analysis was carried out using a linear gradient: 0.0–1.10 min (90% A); 1.10–1.95 min (0–50% A); 1.95–2.45 min (50% A); 2.45–2.50 (50–90% A); and 2.50–3.50 (90% A) for column clean-up and equilibration. An injection volume of 5.0 µl was used for all the samples.

The Acquity UPLC instrument, was coupled to Micromass Quattro Premier Triple Quadrupole mass spectrometer, Waters Corporation (Milford, MA, USA) fitted with an electrospray ionisation probe. Conditions were optimised by teed infusion of a 500 ng mL⁻¹ standard solutions and mobile phase. NBPT and NBPTo were found to ionise in positive electrospray ionisation mode to form the pseudomolecular ion [M + H] ⁺. Following lower energy collision induced dissociation experiments, a satisfactory number of product ions were identified for NBPT and NBPTo (Table 3). The ion source parameters were as follows: capillary voltage 2.00 kV; cone voltage 20 V; inter channel delay 0.005 s; source temperature 120 °C; desolvation temperature 400 °C; cone gas flow 150 L h⁻¹; desolvation gas flow 1100 L h⁻¹. Instrument control and acquisition was performed with Waters MassLynx software, while data processing was done using the Waters TargetLynx software tool.

Mass Spectrometer settings for MRM of 5 mass pair in ESI+ mode.