Track: Bioanalytics - Biomolecular - Bioanalytical Innovations and Applications
Category: Late Breaking Poster Abstract
Evaluation of MRM Based Peptide Quantification, Using a Novel Triple Quadrupole Instrument with Optimized Ion Source E-Lens Technology and D Jet Ion Guide
Purpose: Quantification of peptide/protein therapeutics in biological matrix, play a critical role in the drug discovery and development process. LC-MSMS has been routinely adopted for quantification of this molecular class in the bioanalytical Laboratories, serving as an orthogonal technology to the traditional ligand binding assays (LBAs). The ability to quantify low concentration analytes, in line with current LBAs, as well as limited sample amount has continued to drive the need for sensitivity improvements for LC-MS assays. In this work, a novel triple quadrupole mass spectrometer is employed for quantitation of signature peptides derived from a digested monoclonal antibody or ADC. This new triple quadrupole system has optimized desolvation and ion transmission within the ion source, based on an E-Lens Technology. Further improvement is derived, based on a new D Jet Ion Guide within the entrance region of the system. The combination of these new elements provides a notable signal to noise improvement, enhanced CVs as well as improved LDR on the low end of range. Methods: Two types of samples, synthetic peptides spiked in processed biological matrices, and trastuzumab emtansine spiked in rat plasma, are used for system evaluation. In the first sample set, various peptide standards, including signature peptides from a monoclonal antibody CDR region, synthetic peptide biomarkers and heavy isotopically labeled synthetic peptides, were spiked into the different biological matrices and diluted to cover concentrations over 4 orders of magnitude. In the second sample set, trastuzumab emtansine was spiked into rat plasma with concentrations covering wide range. The plasma samples were processed through immuocapture using streptavidin beads with immobilized anti-human IgG antibody, followed by protease digestion using trypsin-lysine-C. All prepared samples were analyzed both on a standard triple quadrupole/linear ion trap instrument as well as on the modified system. Both regular flow and micro flow HPLC systems were used for analyte separation using chromatographic gradients of water and acetonitrile containing 0.1% formic acid. The MRM transitions for each peptide were optimized to achieve the best S/N after extraction from biological matrix. Samples were analyzed in triplicate on both MS systems. Results: Hardware modifications made to the triple quadrupole system provided enhanced ionization and facilitated a significantly larger number of target precursor ions to transmit into the instrument; whereby the modified triple quadrupole system showed significant improvement in sensitivity when quantifying peptides in biological matrices. In general, a 2 to 4-fold LOQ improvement was observed when using this novel modified MS system compared to existing platforms. The peptides were quantitated over a wide linear dynamic range covering over 4 orders of magnitude with precision and accuracy that meets current guidance for regulated bioanalytical assays. Conclusion: Hardware modifications to a triple quadrupole system have demonstrated ~3X better sensitivity and quantitative performance for peptide quantification vs. a SCIEX 6500+ system.