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Online HPLC-ESI-HRMS Method for the Analysis and Comparison of Different Dissolved Organic Matter Samples Claudia Patriarca, Jonas Bergquist, Per J. R. Sjöberg, Lars Tranvik, and Jeffrey Alistair Hawkes Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04508 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017
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Online HPLC-ESI-HRMS Method for the Analysis
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and Comparison of Different Dissolved Organic
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Matter Samples
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Claudia Patriarcaa,*, Jonas Bergquista, Per J. R. Sjöberga, Lars Tranvikb, Jeffrey A. Hawkesa
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a
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Sweden
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b
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ABSTRACT: Natural dissolved organic matter (DOM) is an ultra-complex mixture that is
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essential to global carbon cycling, but is poorly understood because of its complexity. The
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most powerful tool for the DOM characterization is high-resolution mass spectrometry
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(HRMS) generally combined to direct infusion (DI) as sample introduction. Liquid
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chromatography (LC) represents a compelling alternative to DI; however state-of-the-art
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techniques involve only offline LC-HRMS approaches, which have important logistical
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drawbacks that make DOM analysis more challenging. This study introduces a new method
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based on online coupling of liquid chromatography to high resolution mass spectrometry, able
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to overcome the disadvantages of usual approaches. It is characterized by high reproducibility
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(%Bray-Curtis dissimilarity among replicates ≈ 2.5%), it reduces transient complexity and
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contaminant interferences, thus increasing the signal to noise ratio (S/N), leading to the
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identification of an overall larger number of formulas in the mixture. Moreover, the
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application of an in silico fractionation prior to the statistical analysis allows an easy, flexible,
Department of Chemistry – BMC, Analytical Chemistry, Uppsala University, Uppsala,
Department of Ecology and Genetics, Limnology, Uppsala University, Uppsala, Sweden
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fast and detailed comparison of DOM samples from a variety of sources with a single
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chromatographic run.
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INTRODUCTION
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Dissolved organic matter (DOM) is one of the most heterogeneous mixtures in the
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environment1. Despite its crucial role in the global carbon cycle, it is still poorly characterized
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due to its extreme complexity2–6.
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Advances in mass spectrometry (MS) technology have allowed resolution of thousands of
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individual formulas from DOM6,7. High field strength (>7T) Fourier transform ion cyclotron
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resonance (FTICR-) MS associated with direct infusion (DI) sample introduction is the most
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popular technique for DOM analysis1,3,8–16, and Orbitrap technology is beginning to be
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utilized for the same purpose17–19. The homogeneity of a natural DOM sample in direct
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infusion experiments leads to a ionization suppression of many compounds at the same time,
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and the absence of separation means that compounds having the same chemical formula but
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different chemical structure are overlaid in the mass spectrum, therefore making it impossible
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to discriminate which compound is responsible for the signal4,6.
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Chromatography and other separation techniques facilitate the fractionation of compounds,
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improving our ability to examine the nature of DOM5,8,9,20,21. Unfortunately, most separation
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methods applied to DOM analysis are time consuming, as they involve fractionation and
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collection steps with further sample treatment prior to mass spectrometric analysis. Generally,
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researchers have aimed to isolate individual compounds from the complex natural mixture,
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making it necessary to apply multiple separations on the material20,22. Most online, MS
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coupled chromatographic studies of DOM to date have used low resolution mass spectrometry
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without the ability to separate the numerous extant isobaric molecular masses at each nominal
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mass. Moreover, few studies to date have considered using a broad chromatographic
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separation to simply add a polarity dimension to the data before using the usual statistical
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techniques for interpreting the data. This study describes the first online high resolution
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method that we are aware of, and is an easy, fast, reproducible and flexible method that can be
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adopted for deeper investigation of aquatic DOM by in silico fractionation of the data before
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interpretation by multivariate statistics and data visualization. We applied the method to the
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study of two terrestrial and one marine sample to demonstrate the new insights and
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capabilities that it provides.
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MATERIALS AND METHODS
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Chemicals and samples
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All reagents were of analytical or high-performance liquid chromatographic grade. Three
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isomers with molecular formula C16H18O10 and deprotonated mass 369.08272, were selected
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as model compounds. In this study, three samples from different natural sources were
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considered. The first was a marine sample from the North Pacific Ocean, the second was
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taken from a brown water lake in Sweden, and the last sample was from the reference material
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Suwannee River Fulvic Acid (SRFA). Marine and lake samples were solid phase extracted
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with PPL cartridges as described elsewhere23, while the reference material (SRFA) powder
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was simply diluted before the analysis. More information about chemicals, samples location
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and preparation are described in details in the Supporting Information.
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Sample analysis
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The chromatographic system was an Agilent 1100 system, equipped with a binary pump and a
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100x2 mL well-plate autosampler. The chromatographic separation was conducted using an
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Agilent PLRP-S poly(styrene/divinylbenzene) column (1.0x150 mm, 3 µm bead size, 100Å
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pore size) fitted with a pre-column filter (0.5 µm, Supelco Column Saver).
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The mass spectrometer was a LTQ-Velos-Pro Orbitrap MS (Thermo Scientific, Germany)
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equipped with an electrospray ionization source (ESI) that was operated in negative mode.
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In order to test the analytical reproducibility, each sample was injected three times and a
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blank, consisting of initial mobile phase, was injected between samples to monitor and
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minimise carry-over (Figure S1). 20 µL sample was injected and a three step gradient (Table
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S1) consisting of A) 18 MΏ deionized water + 0.1% formic acid and B) acetonitrile were
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used. The initial flow rate was set to 100 µL min-1 to minimize the processing time. After 2.1
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minutes the flow rate was decreased to 50 µL min-1 until the end of the run. Directly after the
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injection, the acetonitrile percentage was increased from 5 to 20% over 2 min and maintained
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constant for 10 minutes followed by an increase to 40% at 13 min and held isocratic until 22
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min. In the last step, to achieve the complete elution of the most strongly retained material,
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the acetonitrile percentage was increased up to 90% and kept steady for 10 min. Lastly, the
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initial mobile phase composition was restored and maintained for 10 minutes until the next
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injection. The abovementioned gradient was delayed by 6 min (200 µL) due to the system
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dead volume. Sample breakthrough and retention was tested at different loading volumes
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using the model compounds.
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The mass analyzer was externally calibrated for mass accuracy on the day of analysis using
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the manufacturer’s guidelines and negative calibration solution. The ESI source and the
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instrument ion optics settings were optimized as follows: ESI spray voltage: -3.0 kV; sheath
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gas flow rate: 28; capillary temperature: 275 ºC; S-lens RF level: 68.7%. The ion optics was
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tuned to maximize the signal of the base peak in SRFA. 4 ACS Paragon Plus Environment
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The automatic gain control setting was used to trap 1 x 106 ions in the Orbitrap, with a
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maximum accumulation time of 50 msec. Spectra were recorded between 150-1000 m/z at
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resolution setting 100000 (m/∆m50%) and each spectrum was internally calibrated in lock mass
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mode using a three expected compounds (369.0827, 269.0667 and 425.1089m/z), providing
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suitable accuracy and precision (
