Highly sensitive and quantitative analysis of polyeptides using a new gradient system based on an abrupt change in adsorption of polypeptide to the reversed‐phase column packing

Abstract

During a study of 100 microL aliquots of urocortin containing various acetonitrile contents, we hypothesized that a change in the acetonitrile content in the solution across a specific content of acetonitrile (critical threshold) causes an abrupt change in adsorption capacity to the column packing. Circular dichroism measurements suggest that the conformational change induced by acetonitrile in the solution causes the abrupt change in adsorption capacity, and this solvent-induced conformational change is reversible across the critical threshold. This hypothesis can apply to various polypeptides with molecular weights range from 1007 to 6789 and to other organic solvents. A new gradient system utilizing the instant recovery of the adsorption capacity across the critical threshold was designed, and applied to the analysis of a 100 microL aliquot of various polypeptide solutions. The results suggest that use of a solution containing organic solvents more than the critical threshold allows successful dilution of polypeptides up to picomolar concentration range without any loss due to its adsorption during handling procedures and loading onto the LC system, and that a new gradient system enables quantitative analysis of polypeptides at picomolar concentrations in such solutions.

Transition of average drug‐to‐antibody ratio of trastuzumab deruxtecan in systemic circulation in monkeys using a hybrid affinity capture liquid chromatography‐tandem mass spectrometry

“…The 100‐fold diluted plasma samples were digested by 8 μg/mL trypsin for 5 h at 37°C. The HC68 peptide fragment in the digested samples was detected and quantified by peptide adsorption‐controlled (PAC)‐LC/MS (Goda & Sudo, 2007, 2008), consisting of Shimadzu Nexera MP (Shimadzu, Kyoto, Japan) and a QTRAP 6500 mass spectrometer (AB Sciex LCC, Framingham, MA, USA). The analytical column was Inertsustain AQ‐C18 (100 × 4.6 mm i.d., 5 μm; GL Sciences, Tokyo, Japan) and the temperature of the column oven was 60°C.…”

Superiority of a new gradient system utilizing a critical threshold for the adsorption capacity of polypeptides to column packing when compared with a standard gradient system for the simultaneous and quantitative analysis of polypeptides

“…In our previous study (Goda and Sudo, 2007), we investigated the effects of the amount of acetonitrile in solutions containing urocortin (molecular weight 4696) on its adsorption to various materials using 100 μL of urocortin solution with varying acetonitrile content and a standard gradient system. Our initial results indicated that the peak area of urocortin retained on the column was dramatically changed by the acetonitrile content, despite loading the same concentration of urocortin onto the LC system.…”