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Automated Parallel Derivatization Strategy with Broad Metabolite Coverage Coupled to SWATH/MS Data Acquisition for Qualitative and Quantitative Analysis
Poster Title: Automated Parallel Derivatization Strategy with Broad Metabolite Coverage Coupled to SWATH/MS Data Acquisition for Qualitative and Quantitative Analysis
Submitted on 29 Jun 2018
Author(s): David Ruskic, Maria Fernanda Cifuentes Girard; Renzo Picenoni, Guenter Boehm, Gérard Hopfgartner
Affiliations: University of Geneva Department of Analytical and Inorganic Chemistry Life Sciences Mass Spectrometry Geneva Switzerland; CTC Analytics AG Zwingen Switzerland
This poster was presented at ASMS 2018
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Poster Information
Abstract: Introduction
Diversity in metabolite polarity and electrospray ionization efficiency challenge the development of a single method. Chemical derivatization can significantly improve chromatographic retention time and MS response. We propose a workflow including an automated fast parallel derivatization of amines, phenols, aldehydes, alcohols and ketones followed by QUAL/QUANT SWATH/MS analysis for broad metabolite coverage.

Dansyl-chloride (DanCl), dansyl-hydrazine (Dan-N2H3) and their 13C label analogs were used as derivatization agents. Samples (49 analyte mix and urine) were derivatized with 12C reagents while standards were derivatized with 13C labeled regents using PAL RTC autosampler (CTC Analytics). The two fractions were mixed and injected onto a column-swtiching LC system. MS acquisition was performed on a TTOF6600 (Sciex) using SWATH acquisition.

Polar metabolites are converted to more hydrophobic products (DanCl and Dan-N2H3 derivatives), enabling them to be separated on reverse phase liquid chromatography. The presence of a basic p-amino group on both reagents increase the electrospray response factor by a factor of 10 to 200. Primary alcohols, phenols, primary and secondary amines are derivatized with DanCl and Dan-N2H3 transforms ketones and aldehydes to hydrazones. The automated parallel derivatization enables reproducible derivatizations in a single workflow including light labeling of sample and heavy 13C labeling of metabolite mix in 15 minutes during the LC analysis of the previous sample. Collision induced dissociation generates fragments specific for the analyte, and for the light and heavy derivatized analytes and for tags (XIC m/z=171 and m/z=173). LC-SWATH/MS which collects all precursors and all fragments allowed the screening and the relative and absolute quantification (n=45) of metabolites in urine.

Labelled reagents enable to generate adequate standards for amines, phenols, aldehydes, alcohols and ketones. Automation was found to be key for reproduciblility and is performed on-line prior LC analysis. In a batch, sample preparation and sample analysis are overlaid resulting in a significant gain of time. Dansyl-chloride and dansyl-hydrazine were found to be ideal for light/heavy labeling, improved LC retention, improved MS response and MS/MS tag
Summary: Parallel derivatization with Dansyl-Cl and Dansyl-N2H3 covers broad range of polar metabolites
Improved retention on RP LC by ~10 min for a total run time of 25 minutes
Enhanced signal to noise (S/N) by a factor of 10 to 200
Automated derivatization with on-line C18 SPE based fractionation improves reaction kinetics control for quantitative analysis
QUAL/QUANT SWATH/MS data analysis using (13C) labelled derivatization agents
References: D. Siegel, A.C. Meinema, H. Permentier, G. Hopfgartner, R. Bischoff, Anal Chem, 86 (2014) 5089-5100
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