MSACL 2016 EU Abstract

Throughput and Sample Preparation Improvements for Endocrine Research by Microflow LC-MS/MS

Daniel Blake (Presenter)
SCIEX

Bio: Dan Blake is currently the Technical Applications Manager for Clinical Research and Diagnostics, EMEA at ABSCIEX, with responsibilities for application portfolio development and management within EMEA and wider afield. Dan holds a BSc in Biochemistry from the University of Lancaster, UK and a MSc in Clinical Biochemistry from the University of Manchester, UK. Dan has been with ABSCIEX, in clinical and application development focussed roles for 14 years. Prior to roles at ABSCIEX, Dan held positions of increasing responsibility at AstraZeneca UK and Syngenta UK.

Authorship: Daniel Blake (1)
(1) SCIEX, Warrington, UK

Short Abstract

Simple and rapid analysis of endocrine samples is necessary in a clinical research laboratory. Additionally, as financial and environmental concerns become more prevalent, heavy use of solvents for high flow LC-MS/MS methods, particularly those involving online extraction, can be an issue. Microflow LC offers a solution to these concerns, whilst greatly enhancing on column sensitivity and chromatography performance, reducing sample consumption and improving system robustness and uptime. As the sample injection volume is minimized, robustness and reproducibility of the entire method is dramatically increased as matrix effects are significantly reduced.

Long Abstract

Introduction

Simple and rapid analysis of endocrine samples is necessary in a clinical research laboratory. Additionally, as financial and environmental concerns become more prevalent, heavy use of solvents for high flow LC-MS/MS methods, particularly those involving online extraction, can be an issue. Microflow LC offers a solution to these concerns, whilst greatly enhancing on column sensitivity and chromatography performance, reducing sample consumption and improving system robustness and uptime. As the sample injection volume is minimized, robustness and reproducibility of the entire method is dramatically increased as matrix effects are significantly reduced.

Methods

The sample preparation consists of a simple pre-treatment step using a zinc sulfate precipitation reagent, followed by centrifugation and direct injection of very low volume of supernatant. HPLC methodology consists of a water/methanol/formic acid phase with a very short gradient on a 0.5mm ID Microflow LC column, using the SCIEX M3 MicroLC System. Mass Spectrometry analysis was carried out using the SCIEX Triple Quad™ 5500 LC/MS/MS System, running MRM, giving quantitative results for the compounds in question.

Results

Using this method it was possible to analyse the steroids Testosterone, Androsteinedione and 17-Hydroxycortisol and their corresponding labeled internal standards, in a single injection with an injection - injection time of 4 minutes. Sensitivity for all compounds has been shown to be <0.03nMol/L in serum.

Robustness has been demonstrated on commercially available calibrators and controls and shows accuracies within 10% of nominal at the LOQ and %CV of +/- 10%, also at the LOQ. Linearity (r2) has been shown for all compounds to be > 0.99

Conclusions

A fast, robust, and reliable method for the determination of steroid concentrations in serum using Microflow chromatography is proposed. High performance, rapid and robust Microflow LC with high sensitivity detection using a SCIEX QTRAP® 5500 LC/MS/MS system using Multiple Reaction Monitoring (MRM). The method has been demonstrated to show a significant reduction in consumable cost due to the direct injection process and the significantly reduced solvent use due to the low flow rates characteristic of Microflow LC. Performance data has shown accuracy and precision and long term reproducibility of the proposed method.


References & Acknowledgements:


Financial Disclosure

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