Gefitinib (Iressa) dissolved in methanol was spiked into human plasma to create a calibration curve from 0.75–1000 ng/mL. Protein precipitation was performed on 100 µL of each sample in triplicate using 1:3 methanol. 250 µL of the supernatant was transferred to a LCMS vial and 10 µL of the sample was injected onto LC-MS/MS system. The LC-MS/MS methodology used for this separation is the same as described by Tanna et al.2 Briefly, separation was performed on an HSS T3 Column using a linear gradient employing water and ACN containing 0.1% formic acid as mobile phases A and B (5–75% B, 2.5 mins). For Gefitinib, MRM transition 448.22 → 128.03 was monitored for quantification. This same sample set was injected on both the ACQUITY Premier with MaxPeak HPS Column as well as conventional UPLC with conventional column hardware.
The LLOQ achieved on both systems was 0.75 ng/mL and the calibration curve was linear from 0.75–1000 ng/mL on both systems. However, as shown in Figure 1, the ACQUITY Premier System and MaxPeak HPS Column showed a superior peak shape and higher area counts for gefitinib at the LLOQ level. On the conventional system and column, although the LLOQ peak at 0.75 ng/mL is quantifiable there is slight tailing observed which could cause higher variability in integration resulting in higher %CV. This phenomenon is illustrated in Figure 2 which compares the %CV at the lower concentration levels on the novel system and column combination vs the conventional system and column. For the two lowest points on the calibration curve, the %CV observed for on the ACQUITY Premier System and MaxPeak HPS Column is <5%. The %CV for the LLOQ (0.75 ng/mL) and 1 ng/mL levels on the standard system/column combination is 17.5% and 11.5% respectively. This improved performance is most significant at the lower concentrations. Since there are fewer analyte molecules present at the low concentration levels, any loss of these molecules on the analytical system due to metal interactions is exacerbated, resulting in higher variability in results at the lowest concentrations. The ACQUITY Premier System/Column minimizes these unwanted interactions, thereby resulting in more consistent area counts and peak shape, even at the lowest concentration levels, thereby reducing the variability in measurement as demonstrated by the lower %CV observed in Figure 2. Although the %CV values observed on the conventional system/column are acceptable based on the industry accepted bioanalytical method validation guidelines, the higher variability can be a cause of concern, especially when dealing with late stage clinical samples where the matrix variation is higher than safety assessment FTIH studies.
