(W0930-06-34) Validation of an LCMS Hybrid Assay with EVOSEP Cleanup for the Quantitation of Islet Amyloid Polypeptide in Human Plasma

Purpose: Islet Amyloid polypeptide (IAPP) is a peptide hormone produced by the pancreas' beta cells and plays a role in regulating blood glucose. Research on IAPP and its role in diabetes is ongoing, and there is a need for a reliable method to accurately detect this hormone at clinically relevant levels and possibly the different forms of the peptide. We set out to validate a hybrid LC/MS assay for this biomarker that could be validated to an appropriate level to support clinical studies.

Methods: A 100 uL aliquot (calibrator or sample) is added to a previously prepared plate coated with anti-IAPP antibody. The anti-body is characterized for an LBA assay but was found to be non-specific but highly effective with a KD of at most 5.47E-12 M. The plate was incubated for 1.5 hours at ambient temperature to allow the capture. Once captured, the plate was washed 4x3 times with an automatic plate washer using a proprietary washing buffer, flipping the plate between each cycle. 100 uL of 100 mM acetic acid was then added to the plate, which was incubated for 15 minutes with stirring. The entire sample was then loaded on freshly prepared EvoTips 20 uL at a time. The Evotips were washed with 2x20 uL of 0.1% formic acid in water, preserved with the same, and injected using the EvoSep system on an EV1064 column with the 100 Sample Per Day High Organic Method. Data was collected using MRM-HR with the intact precursor and four B fragment ions. The +4-charge state (976.72 m/z) of the precursor provided a significant signal, and summing several +3-charge fragments B26 (921.782 m/z), B32 (1122.219 m/z), B25 (884.088 m/z), and B27 (959.479 m/z) provided quantification on the QTOF system. Importantly, this uses the intact IAPP peptide, and the richness of the QTOF data set should allow for the monitoring and identification of alternative forms of IAPP.

Results: By coupling the immune-precipitation cleanup with an Evosep One system, the detection limits are comparable with those of traditional immunocapture methodologies. We optimized the capture parameters, Evosep SPE, and Mass Spectrometer conditions separately and then combined them into a validated method. Quantitation was performed on a SCIEX 7600 Zeno TOF, where multiple transitions collected using MRM-HR, including the B26, B32, B25, and B27 ions, were summed. Calibrators were prepared in SigMatrix due to the presence of IAPP in human matrix. Endogenous quality control samples, in addition to SigMatrix quality control samples, were used to evaluate the accuracy and precision of the assay. The validation included three accuracy and precision runs, stability, and parallelism. We determined the optimal parameters of the 7600 ZenoTOF system for IAPP under a high organic micro-flow condition to maintain stable spray. The EvoSep tip loading and preparation were optimized away from the published recommended level to prevent over-pressure of the column while maintaining the signal of the analyte of interest. The use of individual trap columns (Evotips) eliminated carryover in the assay which is often present in traditional hybrid LC assays. Finally, we performed a rapid optimization of the LBA conditions, including selection of blocking buffer, aliquot volume, and release reagent.

Conclusion: A method of analysis for Islet Amyloid Polypeptide in human plasma was developed and validated to support regulated clinical studies. This method was validated following the principles outlined in the FDA M10 bioanalytical method validation guidance and from the AAPS “Biomarker Assay Validation by Mass Spectrometry” white paper. Included in the validation were experiments to measure accuracy and precision in an authentic and surrogate matrix, parallelism, and stability experiments.