Reverse‐Polynomial Dilution Calibration Methodology extends Lower Limit of Quantification and Reduces Error in Targeted Peptide Measurements in Blood Plasma. — ASN Events
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  3. Reverse‐Polynomial Dilution Calibration Methodology extends Lower Limit of Quantification and Reduces Error in Targeted Peptide Measurements in Blood Plasma.

Reverse‐Polynomial Dilution Calibration Methodology extends Lower Limit of Quantification and Reduces Error in Targeted Peptide Measurements in Blood Plasma. (#233)

Valerie C Wasinger 1 , Yunki Yau 2 , Xizi Duo 2 , Rupert W Leong 3
  1. School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia
  2. Mark Wainwright Analytical Centre, The University of NSW, Kensington, NSW, Australia
  3. Endoscopy, Concord Hospital, Concord, NSW, Australia

Matrix effect is the alteration of an analyte’s concentration‐signal response due to co‐existing ion components. It is one of the most common problems leading to inaccuracy and imprecision in quantitative Liquid Chromatography‐Tandem Mass Spectrometry (LC‐MS/MS). With Electrospray Ionization (ESI) as the ion source, matrix effects are believed to be a function of the relative concentrations, ionization efficiency, and solvation energies of the analytes within the ESI droplet. For biological matrices such as plasma (with ~107 unique proteins when unfractionated and non‐depleted), the interactions between droplet components is immensely complex and the subsequent effect on analyte signal response not well elucidated. This study comprised of three sequential quantitative analyses: We investigated whether there is a generalizable correlation between the range of unique ions in a sample matrix (complexity), the amount of matrix components (concentration), and matrix effect, by comparing an E.coli peptide‐digest matrix (an approximate 2600 protein proteome) with phospholipid depleted human blood plasma, and unfractionated, non‐depleted human plasma matrices (~107proteome) for six human plasma peptide Multiple Reaction Monitoring (MRM) assays. Our dataset demonstrated significant analyte‐specific interactions with matrix complexity and concentration properties resulting in significant ion suppression for all peptides (p<0.01), with 0>non‐uniform effects on the ion signals of the analytes and their stable‐isotope analogs. These matrix effects were then assessed for translation into error and precision affects in a low concentration (~0‐250ng/mL) range across no‐matrix, complex matrix (E.coli and phospholipid depleted plasma), and highly complex matrix (non‐depleted plasma), when a standard addition Stable Isotope Dilution (SID) calibration method was used. Back‐calculated error (%) and precision (CV%) across all matrices by SID were within <20%, however error in phospholipid depleted 20>20>and non‐depleted plasma matrices were significantly higher compared to no‐matrix and E.coli matrices (p=0.007). Finally a novel Reverse‐Polynomial Dilution (RPD) calibration method with and without phospholipid‐depletion was compared to SID for relative error and precision in a ~0‐250ng/mL range in plasma. RPD techniques extend the Lower Limit of Quantification and reduces error (p=0.005) in low‐concentration plasma peptide assays and is broadly applicable for verification phase Tier 2 multiplexed MRM assay development within the FDA‐National Cancer Institute (NCI) biomarker development pipeline.