Comparison of tandem quadrupole and high resolution MS for quantification of Trastuzumab in plasma using surrogate peptide and intact approaches, including measurement of detection limits and linear dynamic range.
For the surrogate peptide approach Trastuzumab was spiked into rat plasma along with an infliximab internal standard and immuno-purified using Protein A plate. Samples were then prepared for LC-MS analysis using commercially available digest and SPE kits following supplied protocols. Resulting signature peptides were analyzed using tandem quadrupole (Xevo TQ-XS) and high resolution (Xevo G2-XS QTof) MS. For quantification at the intact level, Trastuzumab was spiked into mouse plasma and purified using magnetic bead based anti-human FC immuno-purification. The prepared sample was analyzed using the Vion QTof platform.
Using the surrogate peptide approach the LLOQ ranged from 10-25 ng/mL using tandem quadrupole MS and 25-50 ng/mL for high resolution MS. The linear dynamic range was >4 orders of magnitude for both platforms. Using the intact approach, the LLOQ was 3 ng/mL in BSA solution with a linear dynamic range of >2.4 orders of magnitude. In mouse plasma a LLOQ of 250 ng/mL and a linear dynamic range of >2 orders of magnitude was achieved.
Sensitive detection of mAb concentration in plasma was achieved using both the surrogate peptide and intact approach. The importance of sample preparation for achieving high sensitivity, linearity and accuracy is highlighted. While high resolution mass spectrometry offer excellent sensitivity and flexibility to study large molecules at both the peptide and intact level, tandem quadrupole MS remains the most sensitive platform for surrogate peptide based quantification. Recent progress in intact level analysis suggests that the sensitivity gap between surrogate peptide and intact level quantification could be closed further with improved sample preparation and MS technologies.