LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies

Authors: O'NEILL, MAURA - Frederick National Laboratory For Cancer Research; CHAN, KING - Frederick National Laboratory For Cancer Research; JAYNES, JESSE - Tuskegee University; KNOTTS, ZACHARY - National Cancer Institute (NCI, NIH); XU, XIA - Frederick National Laboratory For Cancer Research; ABISOYE-OGUNNIYAN, ABISOLA - National Cancer Institute (NCI, NIH); GUERIN, THERESA - National Cancer Institute (NCI, NIH); SCHLOMER, JEROME - National Cancer Institute (NCI, NIH); LI, DANDAN - National Cancer Institute (NCI, NIH); Cary, Jeffrey; Rajasekaran, Kanniah - Rajah; YATES, CLAYTON - Tuskegee University; KOZLOV, SERGUEI - National Cancer Institute (NCI, NIH); ANDRESSON, THORKELL - Frederick National Laboratory For Cancer Research; RUDLOFF, UDO - National Cancer Institute (NCI, NIH)

Submitted to: Journal of Pharmaceutical and Biomedical Analysis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/1/2020
Publication Date: 1/2/2020
Citation: O'Neill, M.J., Chan, K., Jaynes, J.M., Knotts, Z., Xu, X., Abisoye-Ogunniyan, A., Guerin, T., Schlomer, J., Li, D., Cary, J.W., Rajasekaran, K., Yates, C., Kozlov, S., Andresson, T., Rudloff, U. 2020. LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies. Journal of Pharmaceutical and Biomedical Analysis. 181:113093. https://doi.org/10.1016/j.jpba.2020.113093.
DOI: https://doi.org/10.1016/j.jpba.2020.113093

Interpretive Summary: Naturally occurring and synthetic antimicrobial peptides are important sources for novel drugs and drug designs or used for plant protection against microbial diseases. These peptides are invariably subject to degradation in host cells. As such, quantitative assay for measurement of these highly charged peptides is extremely difficult. A novel method to assay for the defense peptides through the use of negatively-charged magnetic beads was developed to specifically capture and purify peptides in plasma or cytoplasm followed by successful quantification by liquid chromatography and mass-spectrometry (LC-MS/MS). Using this assay, three peptides were quantitatively measured in complex biological specimens successfully. This method should serve as a novel platform to conduct safety studies facilitating further development of this promising new class of antimicrobial agents.

Technical Abstract: Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206, reduces immune suppressive cues in solid tumors, and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid coated magnetic beads were used as an affinity capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1 x 100 mm, 3.5 µm XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring three different transition ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1 ng/mL to 1000 ng/mL in both mouse plasma and liver homogenate. The lower limited of detection for RP-182 was 1 ng in both matrixes with good intra- and inter-sample precision and accuracy in the plasma. Recovery ranged from 60% to 71% with minimum matrix affects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract from plasma and tissue.