Vanguard 2.1 5 mm

AMP was assayed with a previously validated method [23 (link)] using an ACQUITY UPLC H-Class System (Waters; Milfors, MA, USA) with ACQUITY PDA detector (Waters; Milfors, MA, USA). The column used was ACQUITY^® BEH C18 column (2.1 × 50 mm, 1.7 μm, Waters) at a temperature of 30 °C with a VanGuard 2.1 × 5 mm (Waters; Milfors, MA, USA) pre-column. The separation was achieved using a gradient elution composed of ammonium formate 20 mM pH = 6.5 (mobile phase A) and methanol/acetonitrile (75:25) (mobile phase B) with the gradient applied at a constant flow of 0.35 mL/min (Table 1). The detection wavelength was 225 nm. Data were collected and processed with Empower 3 Software (2002–2019 Waters; Milfors, MA, USA) Application Manager.
At each study time, the first 1 mL of the AMP solution from EIPs was discarded, and another 2 mL was collected on ice for analytical determination. Collected samples were accurately diluted 1:100 with cold LC-MS-grade water. Injection volume was set to 1 μL and injections were performed in triplicate.

Only PQ and its metabolites, CPQ and PQCG, were detectable in plasma samples collected here. All compounds were analysed using the liquid chromatography–mass spectrometry (LC–MS) method of Avula et al. [14 (link)], modified to employ an ACQUITY UHPLC™, BEH Shield RP18 column (100 mm × 2.1 mm I.D., 1.7 mm) equipped with an LC-18 guard column (Vanguard 2.1 × 5 mm, Waters Corp, Milford, MA, USA). PQ, CPQ and PQCG were separated and eluted within 10-min retention time. The mobile phase, run at a flow rate of 0.25 ml/min, consisted of 0.05% formic acid in water (A) water and 0.05% formic acid in acetonitrile (B) and was applied in a linear gradient elution. The proportion of solvent A decreased from 90 to 63% during the first 5 min, then from 63 to 37% during minutes 5–8, then from 37 to 0% during minutes 8–10. A 3-min wash with 100% B and a 3.5 min equilibration period of 90% A followed each run. Samples were injected at 10 μL volume. The limits of quantification in plasma were 5 ng/mL for PQ and PQCG, and 1 ng/mL for CPQ. Quality control samples at 20 ng/mL were run at beginning and end of the batch analyses in duplicate. Intra-assay variability was less than 10%.