Evaluation of analytical performance and reliability of direct nanoLC-nanoESI-high resolution mass spectrometry for profiling the (xeno)metabolome Andrew J. Chetwynd, † Arthur David, † Elizabeth M. Hill and Alaa Abdul-Sada* Mass spectrometry (MS) profiling techniques are used for analysing metabolites and xenobiotics in biofluids; however, detec- tion of low abundance compounds using conventional MS techniques is poor. To counter this, nanoflow ultra-high-pressure liquid chromatography-nanoelectrospray ionization-time-of-flight MS (nUHPLC-nESI-TOFMS), which has been used primarily for proteomics, offers an innovative prospect for profiling small molecules. Compared to conventional UHPLC-ESI-TOFMS, nUHPLC-nESI-TOFMS enhanced detection limits of a variety of (xeno)metabolites by between 2 and 2000-fold. In addition, this study demonstrates for the first time excellent repeatability and reproducibility for analysis of urine and plasma samples using nUHPLC-nESI-TOFMS, supporting implementation of this platform as a novel approach for high-throughput (xeno)metabolo- mics. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: nanoflow; nanoESI; metabolomics; mass spectrometry; plasma; urine Introduction Metabolomic analyses consist of profiling of the many endoge- nous metabolites present in biological matrices (i.e. the metabo- lome), in order for example, to discover potential biomarkers of disease or toxicant exposure. [1] In addition, xenobiotics and their metabolic by-products can also be analysed in the same sam- ples, and these compounds are referred to as the xenometa- bolome. [2–4] Analysis of the (xeno)metabolome may increase scientific understanding of the impact of toxicants and lifestyle factors on human and wildlife health. [5,6] Ultra-high performance liquid chromatography-electrospray ionisation-time-of-flight mass spectrometry (UHPLC-ESI-TOFMS) enables the detection of polar to apolar (xeno)metabolites at high mass resolution and has been extensively used for profiling of small organic molecules in sample extracts. [7,8] However, using this technique, the detection of very low abundance metabolites such as signalling compounds or chemical contaminants is limited as they can suffer from poor ionization efficiency or ion suppression from co-eluting metabo- lites. [7] As a result, more efficient separation and ionization tech- niques are required to undertake profiling of trace level metabolites in sample extracts. In recent years, new technological advances have paved the way for nano scale chemical analysis in the form of nanoflow UHPLC-nanoESI-MS (nUHPLC-nESI-MS). [9–13] The key advance- ment of this technique is the improved sensitivity as a result of using lower flow rates and nanospray emitters. [9] Nanospray emitters utilize small internal diameters, thus producing very fine spray droplets which are 100–1000 times smaller than those formed using traditional ESI emitters. Consequently, the small droplet size benefits from a greater charge to volume ratio, increased surface area and reduced specific heat capacity. As such, fewer desolvation cycles are required before charged ions are released into the mass analyser, resulting in increased ioniza- tion efficiency. [9,12] To date, nUHPLC-nESI-MS techniques have been used primarily for proteomic analyses [14–16] and have been for the most part overlooked for metabolomics applications. [17] The increased ionization efficiency of nanoscale systems offers significant improvements for small molecule analysis and is now starting to be implemented for some non-targeted applications. How- ever, many of the nanoflow systems described use traditional LC setups and split the flow prior to entry to the MS. Compared with split flow, direct injection into nanoflow LC could enhance sensitivity by reducing sample loss and should result in more reproducible nanoflow rates into the source. [18] Therefore, the use of direct nanoflow coupled with nESI-MS could be advanta- geous to improve the detection of low levels of many biologically important signalling compounds during metabolite profiling of extracts of tissue or biofluids. These compounds include, for instance, estrogenic steroids which are poorly ionized by ESI, or prostanoids present at pg/ml concentrations in blood plasma, or xenobiotics which can often accumulate as low concentrations * Correspondence to: Alaa Abdul-Sada, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK. E-mail: a.abdul-sada@sussex.ac.uk † The first two authors contributed equally. School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK J. Mass Spectrom. 2014, 49, 1063–1069 Copyright © 2014 John Wiley & Sons, Ltd. Application note Received: 17 April 2014 Revised: 17 June 2014 Accepted: 29 June 2014 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3426 1063