The Ostro 96-well plate was used to remove residual PLs prior to LC-MS/MS analysis. Utilizing the generic, simple protocol provided by the manufacturer (Figure 2), a group of analogous compounds were extracted. The resulting analysis demonstrated an average recovery of 86% for the group of structural analogs in plasma (Figure 4), which is equal to or better than traditional PPT. To assess PL removal, eight individual PLs were summed. Results showed that Ostro plates remove >99% of the 8 PLs relative to traditional PPT (Figure 5). In addition, the MRM transition, 184>184, was monitored to visually demonstrate the significant decrease in residual PLs using Ostro plates compared to traditional PPT (Figure 6). One of the primary reasons to eliminate PLs is to improve method robustness. Overnight runs of both Ostro plates and traditional PPT samples were carried out using the generic gradient and PLs were monitored continuously. Figure 7 shows the LC-MS trace for a representative PL at the beginning and end of the runs. When Ostro plates are used, the PL levels are negligible and no build-up occurs. When PPT is used, a significant amount of PLs are present and accumulate throughout the run. The result of this undesirable build-up is a continuous downward trend in area counts throughout the duration of the run (Figure 8). This in turn results in high signal variability relative to samples prepared with Ostro plates, 33% using PPT vs. 9% for Ostro samples. In addition, area counts decrease by 57% from the first injection to the final injection when PPT is used. In discovery bioanalysis, high throughput is of utmost importance. If one tries to increase throughput by shortening gradient time, the impact of residual PLs is further magnified. To demonstrate the negative effect PLs have on analytical throughput, the gradient time was decreased by half. Flow rate was increased and organic content was ramped from 50 to 98% in 0.5 minutes. 200 Ostro samples and 200 PPT samples were injected using the shorter gradient. The MRM transition 184>184 was monitored to reveal overall PL build-up and elution in the shortened gradient window (Figure 9). Using the 2 minute gradient, PLs elute within 0.2 minutes of the end of the gradient. Under the truncated gradient conditions PLs continue to elute for more than 1 minute after the end of the gradient and well into the reequilibration phase and beginning of the next injection. These resultant chromatograms demonstrate the inability to shorten gradient time with PPT due to PLs which continue to elute significantly after the gradient ends at 0.5 minutes. Overall, the Ostro plate allows for increased method robustness and reduced variability. Additionally both improved instrument uptime and the ability to significantly shorten run times are realized through the elimination of PLs, all of which are highly desirable in a discovery setting. Calibration curves from 1-500 ng/mL for each of the 26 structural analogs had a resulting average r2 value of 0.925, sufficient for discovery screens.
