The US Food and Drug Administration (FDA) recommends in their Metabolites in Safety Testing (MIST) guidelines that the safety of drug metabolites should be evaluated if they are present in human plasma at concentrations greater than 10% of the parent drug (or API) at steady state. In addition, the need to rapidly evaluate multiple, diverse drug candidates and metabolites in a discovery or preclinical setting requires methodologies that are generic, fast and robust. This application note highlights the utility of Ostro 96-well plates and XBridge BEH eXtended Performance (XP) 2.5 µm columns for accurate metabolite ratio determination. Ten pairs of drugs and metabolites were accurately analysed within a 2.5 min analysis time with almost no phospholipid build up and column back pressures of less than 3500 psi, allowing the flexibility to use XP columns on any HPLC, UHPLC or UPLC system.
Instrumentation & Consumables
Sample Preparation: Ostro 96-well plates
Part Number: 1186005518
Instrument: ACQUITY UPLC
Column: XBridge BEH C18 XP Column, 130 Å, 2.5 µm, 2.1 × 50 mm
Part Number: 186006029
Instrument: XEVO TQ-S
Ion Source: ESI+
Results & Discussion
Figure 1 shows a chromatogram of 10 pairs of drugs and metabolites that were analysed in under 1.5 min. Peak widths were under 2.5 s and the maximum column back pressure was 3500 psi. Analysis of phospholipid (PL) accumulation revealed that greater than 99% of phospholipids were eliminated compared to protein precipitation (PPT) prepared samples and PL build-up on the column was prevented.
Figure 1: 10 pairs of drugs and metabolites separated on an Xbridge BEH C18 XP column.
Figure 2 shows that calculated response ratios for metabolites spiked at 10% of parent drug concentration versus those at 100% of parent drug concentrations were within 2–3% of the theoretical ratios expected.
Figure 2: Measured response ratios for metabolites spiked at 10% versus 100% of parent drug concentrations. The red line indicates the theoretical response.
The generic nature of Ostro sample preparation plates makes them ideal for use in a discovery or preclinical setting where one must analyse a variety of compounds. The removal of phospholipids prevents PL accumulation on the analytical column and minimizes the risk of matrix effects due to PLs. Expected responses from analytes spiked at different concentrations are very close to theoretical targets, allowing for accurate quantification. Finally, XP columns provide excellent performance and peak capacity at back pressures that enable their use on any LC system, at analysis speeds conducive to high throughput production.
For the full application, go to www.waters.com/720004453en
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