LC optimization
SEPARATION
Chromatographic conditions were optimized to obtain a maximum intensity, and a high sensitivity with well-quantified chromatograms of each candidate analyte (AP1, AP3, AP4 and AP6). Addition of formic acid resulted in peak distortion and a decrease in negative signal intensity of the AP1 parent compound, as described by Xu et al.Finally, optimal chromatographic separation was achieved by using a mobile phase of Milli-Q water (A) and acetonitrile (B) following elution gradients: during 0–1.5 min maintained at 40%B, then increased to 100%B during 1.5–3.0 min, and maintained at 100%B for 3.0–4.5 min. The gradient was returned to 40%B at 4.6–6.0 min. Column temperature was maintained at 40 °C during the LC separation with a constant flow rate of 0.5 mL/min, and 10 μL of injection volume.