Protein kinase inhibitor analysis with the core–shell separation technique

Abstract

Here, we describe the application of the core-shell separation technology in the field of protein kinase inhibitor analysis with the HPLC-diode-array detector (DAD) technique. A Kinetex 2.6 μm C8 100 Å, 150 × 4.6 mm column maintained at 30 ± 1 °C was used for the separation, and further, a connection of two core-shell HPLC columns with a total column dimension of 250 × 4.6 mm was also applied. The analytes were eluted with a mobile phase consisting of 0.05 M H(3)PO(4)/KH(2)PO(4) (pH = 2.3)-acetonitrile (7:3, v/v), with a flow rate of 0.7 mL/min. A liquid-liquid extraction with 1-chlorobutane was used for the sample preparation. The validation of the canertinib analytical method resulted in recoveries in the range of 74-79 % and in relative standard deviation and accuracy lower than 15 %, both for between- and within-batch calculations. A very good linearity in the validated range (5-10,000 ng/mL) and a limit of quantification (LOQ) of 5 ng/mL were achieved. Peak width, height and peak area of six protein kinase inhibitors analysed with a core-shell column and with a conventional fully porous particle column were compared. The results showed that, for most inhibitors analysed with the core-shell analytical column, the peaks were about two to three times narrower and two to three times higher, with areas that remained almost unchanged. The presented study revealed that the application of this separation technology is a very cost-effective way to bring the LOQ of the protein kinase inhibitor analysis with an HPLC closer to the possibilities of the LC-MS/MS technique.