Direct Analysis - No Sample Preparation - of Bioavailable Cortisol in Human Plasma by Weak Affinity Chromatography (WAC; Affinity LC/MS) Sten Ohlson (Presenter) Nanyang Technological University Presenter Bio: Prof. Sten Ohlson is a biochemist graduated with a doctor degree from Lund University, Sweden in 1980. After academic studies, Sten Ohlson joined the biotechnology- and life science industry in Sweden (Gambro, Perstorp) and USA (HyClone Labs) where he spent thirteen years as manager and director of research. In 1993, he returned to Academia where he was a founding associate professor of the Biomedical Chemistry Centre of the University of Kalmar in Sweden. In 1999, Sten Ohlson was appointed a full professor in Applied Biochemistry and head of the biotechnology division at the University of Kalmar and later in 2010 he was a professor and head of the biotechnology group of Linnaeus University of Sweden. In 2013 he became a visiting professor followed by a full professorship in Applied Biochemistry at the School of Biological Sciences (SBS) at Nanyang Technological University (NTU), Singapore. He is recognized for his pioneering contributions in cell culture engineering (whole-cell catalysts and novel cell culture growth media), in biosensor techniques for studies on transient biological interactions and most importantly the introduction of novel techniques in affinity chromatography ((High Performance Liquid Affinity Chromatography (HPLAC) and Weak Affinity Chromatography (WAC)). Of special importance has been the application of WAC for drug discovery and clinical diagnostics. Further, he has introduced the concepts of transient drugs and continuous biosensors based on dynamic biological interactions. In 2017, he was the recipient of the ISMR Affinity Award for outstanding contributions to the field of affinity technology. Sten Ohlson is also a bioentrepreneur and he has founded four life-science companies in Sweden (Perstorp Biolytica, ProLiff, KalBiotech and Transientic Interactions).

Authors: Sten Ohlson
Nanyang Technological University, Singapore 637551

Pre-analytical treatment of blood plasma is a time consuming and often rate limiting step in the workflow of LC/MS analysis. We present in this study a new approach for quantitative LC/MS based clinical analysis based on Weak Affinity Chromatography (WAC) making sample preparation obsolete.

Introduction

In this presentation, I will introduce Weak Affinity Chromatography (WAC) as a tool for clinical analysis where direct and fully integrated analysis of blood plasma can be performed on a HPLC-MS/MS platform without the need for sample preparation. By exploiting the weak- or transient affinities between the analytes of interest and the immobilized target, simple isocratic chromatography conditions can be applied. Albumin is a major protein in plasma that naturally binds a variety of small hydrophobic compounds, including steroid hormones. Albumin has a natural on-off dynamic binding affinity for these compounds in the affinity range from low mM to µM. We have taken advantage of this fact and used a highly concentrated immobilized albumin support directly integrated with a HPLC-MS/MS platform for straight analysis of biological samples. We have applied this approach for the direct analysis of the steroid hormone cortisol (hydrocortisone) from clinical plasma samples representing various pathological conditions and covering a wide range of cortisol concentrations.

Methods

A WAC unit with saturating, immobilized albumin as a prototypic weak binder was used in combination with an ion-funnel MS/MS detector to perform zonal affinity chromatography of cortisol directly from a plasma sample, followed by quantitative multiple reaction monitoring (MRM).

Results

The WAC procedure allowed us to determine the amount of bioavailable cortisol in a clinical plasma sample which should be of significant therapeutic interest. This WAC-MS approach showed an excellent correlation (R2 = 0.86 (P < 0.0001 (highly significant); n = 60) with a state-of-the-art, clinical competitive immunoassay procedure for plasma cortisol analysis.

Conclusions & Discussion

By integration of WAC into the LC/MS workflow, it was possible to both accelerate and improve assay performance by eliminating the sample extraction step. Preliminary data with other steroid hormones indicate that WAC-MS can be applied to various biomolecules using a plasma transport protein such as albumin.

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1. Ohlson S, Kaur J, Raida M, Niss U, Bengala T, Drum CL, Boehm B and Torres AR (2017) Direct analysis – no sample preparation – of bioavailable cortisol in human plasma by weak affinity chromatography (WAC). Journal of Chromatography B 1061-1062:438-444. doi: 10.1016/j.jchromb.2017.07.035