Margret Thorsteinsdottir (Presenter)
University of Iceland
Bio: Associate professor of Pharmaceutical Analytical Chemistry at the Faculty of Pharmaceutical Sciences, University of Iceland and the R&D director of ArcticMass a spin-off company from a Bioanalytical Laboratory at deCode Genetics. I built up and directed the laboratory for the past 10 years, supporting the entire compound development process from drug discovery through post-marketing trials. My current research interest includes development of analytical methods for quantification of clinical biomarkers utilizing liquid chromatography coupled to mass spectrometry (LC-MS/MS). I have extensive experience in the field of mass spectrometry, especially in development of assays for metabolite profiling and quantification of biomarkers in different cell lines and other biofluids utilizing chemometric approaches. I have been using design of experiments for many years and was co-chairman of the Scandinavian Symposium on Chemometrics (SSC) and a member of the scientific committee for SSC. I was chairmen of the board of the Icelandic Chemical Society and I am a scientific advisor in the Rare Kidney Stone Consortium (RKSC), responsible for implementation of UPLC-MS/MS assays for routine diagnostics. I am a principal investigator of a Marie Curie program, BluePharmTrain and I have supervised several Ph.D. students as well as M.Sc. students.
Authorship: Margret Thorsteinsdottir (1,2)
(1) University of Iceland, Reykjavik, Iceland, (2) ArcticMass, Reykjavik, Iceland
| Short Abstract Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique for quantification of biomarkers in various biological matrices for support of clinical diagnosis and therapeutic drug monitoring. Method optimization involves many experimental factors which need to be simultaneously studied to obtain maximum sensitivity and selectivity at minimum retention time. This paper will illustrate that using design of experiments (DoE) for optimization of LC-MS/MS methods is much more efficient with only fraction of experiments that would been required by changing one-factor-at-time (OFAT) approach. Examples will be given to illustrate how DoE works for optimization of LC-MS/MS clinical diagnostic assays. |
Long Abstract
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is becoming a routine technique in clinical laboratories for support of clinical diagnosis and therapeutic drug monitoring. The LC-MS/MS has the capacity to analyze several analytes in the same assay with ultimate selectivity and sensitivity needed for quantification of biomarkers in biological matrices. The method development consists of several integrated steps from sample preparation, chromatographic separation, mass spectrometry detection as well as data analysis. The LC-MS/MS is a two stage process, liquid introduction and analytes ionization. The goal is to transfer the analytes from condensed phase to gas phase and maintain conditions that are compatible for both the LC and the MS. These processes involve many experimental factors which need to be simultaneously optimized to obtain maximum selectivity and sensitivity at minimum retention time. Optimization of experimental conditions for LC-MS/MS methods is usually performed by changing one-factor-at-time (OFAT) approach. However this procedure can be very ineffective and possible interactions between experimental factors studied are not taken into account. A much more effective strategy for optimization of quantitative LC-MS/MS clinical diagnostic methods is to implement a chemometric based technique such as design of experiments (DoE). DoE is a systematic approach where experiments are performed according to predefined plan in as few experiments as possible and modelling is by empirical functions and graphical visualization. This paper illustrates the benefits of utilizing DoE for method development and optimization of quantitative LC-MS/MS methods. Examples will be presented from selected assays utilizing this chemometric strategy for optimization of LC-MS/MS methods for evaluation of biomarkers for gene-to-clinic drug discovery, clinical diagnostic and therapeutic drug monitoring.
A chemometric approach was implemented for optimization of simultaneous quantification of biomarkers in various biological matrices with high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) coupled to an electrospray tandem mass spectrometer (MS/MS). The chemometric software MODDE 11 (MKS Data Analytical Solutions) was used to set up the design of experiments. Experimental screening of significant variables was performed by fractional factorial design to reveal the most influential experimental factors. When multi-levels qualitative factors were included in the screening experiments D-optimal design was applied. Optimization of significant factors were studied via central composite design and related to sensitivity, resolution and retention time utilizing partial least square (PLS)-regression.
DoE was utilized for optimization of a quantification method of leukotriene B4 (LTB4) in plasma with HPLC-MS/MS and UPLC-MS/MS. Historically, various immunoassay methods have been used for the analysis of LTB4, however these methods are susceptible to interference and are not able to resolve LTB4 from its isomers. A specific high through-put LC-MS/MS method for simultaneous quantification of LTB4 and its isomers in plasma was successfully developed and was used for support of a clinical development process. The results showed that the biomarker concentration decreased significantly with the dose of the clinical candidate.
D-optimal design was used in another study to develop a sensitive, specific and reliable UPLC-MS/MS assay for simultaneous quantification of urinary 2,8-dihydroxyadenine (DHA) and adenine. This assay has been implemented for clinical diagnosis and therapeutic drug monitoring of patients with the inborn error of purine metabolism, adenine phosphoribosyltransferase (APRT) deficiency.
In another study, optimization of HPLC-MS/MS and UPLC-MS/MS methods for simultaneous quantification of cortisol, cortisone and glycyrrhetinic acid in plasma was performed by utilizing DoE. Results showed that many interaction factors were significant so these variables could not be independently controlled to obtain optimal conditions. Baseline separation was achieved between the biomarkers and the method was implemented for analyses of human plasma samples from individuals with and without liquorices consumption. The assay was used for support of clinical diagnosis of liquorice induced hypertension and evaluation of 11 β-hydroxysteroid dehydrogenase type 2 (11βHSD2) enzyme activity in patients.
These selected studies show that design of experiments (DoE) can be used to ensure that selected experiments contain maximum information and optimization of these LC-MS/MS clinical assays is achieved efficiently with only fraction of the experiments that would be required by changing one-factor-at-time (OFAT) experiments.
References & Acknowledgements:
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