Article pubs.acs.org/ac
Nano Liquid Chromatography Directly Coupled to Electron Ionization Mass Spectrometry for Free Fatty Acid Elucidation in Mussel Francesca Rigano,†,‡ Ambrogina Albergamo,† Danilo Sciarrone,† Marco Beccaria,‡ Giorgia Purcaro,‡ and Luigi Mondello*,†,‡ †
“Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali”, University of Messina, Polo Annunziata, viale Annunziata, 98168 Messina, Italy ‡ Chromaleont s.r.l., c/o, University of Messina, , Polo Annunziata, viale Annunziata, 98168 Messina, Italy S Supporting Information *
ABSTRACT: Recently the miniaturization of liquid chromatography (LC) systems and progresses in mass spectrometry instrumentation have enabled direct introduction of the effluent coming from a nanoLC column into the high-vacuum region of an electron ionization source. In the present research, a nanoLC system was directly coupled to an electron ionization mass spectrometer (EI-MS) without any interface or modification of the ion source. The advantage with respect to atmospheric pressure ionization techniques, normally coupled with LC, is major identification power because of a more extensive and reproducible fragmentation pattern, without any matrix effect or mobile-phase interference. In particular, a nanoLC/EI-MS method was developed for elucidation of the free fatty acid profile in mussel samples, avoiding a previous derivatization step, required when gas chromatographic analysis is involved. A total of 20 fatty acids were reliably identified through the comparison with commercial libraries. A quantitative determination was also carried out by using the response factors approach along with the internal standard method, allowing for quantification of 14 fatty acids. Among them, palmitic acid resulted the most abundant, followed by ω6 arachidonic acid. The quantitative data were compared with those obtained by a well-established technique, such as gas chromatography with flame ionization detection (GC-FID). Both nanoLC/ EI-MS and GC-FID methods were validated and similar results were obtained in terms of limit of detection and quantification, resulting in the picomole range, and sensitivity as well was not significantly different, as demonstrated by comparing the slope values of the calibration curves (p < 0.05, from a t-test).
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and among different instruments, and they can be heavily influenced by the matrix effect.5 For this reason, the aim to couple LC with EI-MS has never been completely successful. In fact, EI-MS can provide highly reliable, reproducible, and informative fragmentation patterns that can support fast and reliable characterization of unknown analytes. Earlier attempts were mainly based on splitting of the eluent flow to accomplish the coupling with EI-MS, but sensitivity was lost.2 In the 1980s, Browner and co-workers6,7 developed a particular interface called MAGIC-LC/MS (monodisperse aerosol generation interface for coupling LC with MS). The system was provided with a desolvation chamber and a multiple-stage momentum separator to introduce only dry particles into the ion source. These processes implied significant analyte losses that resulted in low sensitivity. Moreover, the desolvation process was often
ass spectrometry (MS) has been coupled with chromatography since the 1950s,1 and since then there has been an ever-increasing use of this powerful twodimensional combination. Due to its nature, the coupling with gas chromatography (GC) was more straightforward than with liquid chromatography (LC), because of low compatibility of the liquid phase with the vacuum region of an MS. Among the ion sources, electron ionization (EI) has been the most applied in GC/MS coupling. In LC/MS, although the first attempts at coupling were performed with an EI source,2 this was quickly replaced by atmospheric-pressure ionization (API) techniques and in particular electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI), in order to handle the high amount of liquid phase involved.3,4 However, API techniques generate poor mass fragmentation (often only the molecular weight information is noticeable) and a further fragmentation step is necessary for structure elucidation, such as the employment of tandem MS. Furthermore, the mass fragmentation patterns present low reproducibility over time © XXXX American Chemical Society
Received: January 25, 2016 Accepted: March 3, 2016
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DOI: 10.1021/acs.analchem.6b00328 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
puriss (glacial), and n-hexane (GC grade) were all purchased from Sigma−Aldrich. Butylated hydroxytoluene (BHT, FG grade), BSTFA [N,O-bis(trimethylsilyl) trifluoroacetamide] + 1% TMCS (trimethylchlorosilane) kit, and pyridine anhydrous (purity grade 99,8%) were purchased from Sigma−Aldrich. A standard mixture stock solution containing myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), stearic acid (C18:0), palmitoleic acid (C16:1ω7), oleic acid (C18:1ω9), linoleic acid (C18:2ω6), α-linolenic acid (C18:3ω3), and arachidonic acid (C20:4ω6) (Sigma−Aldrich) was prepared with a concentration of 1000 mg/L for each component in water/2-propanol (1:2 v/v). A working solution at 100 mg/L was used for method optimization. Lauric acid (C12:0) was used as the internal standard (IS) for quantitative purposes. A 500 mg/6 mL Bond Elut NH2 solid-phase extraction (SPE) cartridge was purchased from Agilent Technologies (Santa Clara, CA). Sample and Sample Preparation. Adult specimens of Mytilus galloprovincialis were obtained from an aquaculture farm in the lake of Faro (Messina, Italy) and transported alive to the laboratory. Ten organisms were randomly selected, and total lipids were extracted from their tissues previously dissected from the shells. The method of Bligh and Dyer,28 recommended by the technical guidelines of the Organization forEconomic Cooperation and Development (OECD) for complete extraction of total lipids from marine species,29 was used. Briefly, mussel tissues were pooled, homogenized, and weighed (∼10 g). The sample was extracted with 30 mL of a chloroform/methanol mixture (1:2 v/v), containing 50 μg/mL of antioxidant agent BHT. Chloroform (10 mL) and distilled water (18 mL) were added, and the mixture was rehomogenized for 1 min. After centrifugation at 3000 rpm for 15 min, the lower chloroform phase was removed carefully and transferred to a flask. The polar phase was extracted again with 20 mL of 10% (v/v) methanol in chloroform. Then the two extraction phases were pooled together and evaporated at 30 °C by use of a rotary evaporator (Hei-VAP Precision, Heidolph). To the dried lipid extracts was added a known amount of IS (50 μg/mg), and these were reconstituted in methanol/chloroform/hexane (2:1:1 v/v/v) prior to isolation and purification of the FFA fraction by SPE.30 The SPE cartridge was preconditioned with n-hexane, and then about 70 mg of lipid extract was loaded. Neutral lipids (triglycerides, diglycerides, monoglycerides, cholesterol, and cholesterol esters) were first eluted with 4 mL of chloroform/2-propanol (2:1 v/v); the following fraction, containing FFAs, was collected with 8 mL of diethyl ether/ acetic acid (98:2 v/v), while phospholipids were retained in the cartridge. FFAs were dried under a stream of cold nitrogen and reconstituted in acetonitrile/2-propanol (1:1 v/v) to be analyzed by nanoLC/EI-MS and GC-FID. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures were in accordance with guidelines for the protection of animal welfare, in compliance with the Italian National Bioethics Committee (INBC) (European Community Council Directive of November 24, 1986-86/609/EEC). NanoLC/EI-MS Instrumentation and Software. The instrument consisted of a nano prominence HPLC (Shimadzu, Kyoto, Japan) system coupled to a GCMS-QP2010nc Ultra system (Shimadzu). The nanoLC configuration was equipped with two LC-20AD nano pumps, a CBM-20A controller, a
incomplete, especially when a high concentration of water was used in the mobile phase, thus causing contamination of the ion source and further sensitivity reduction. Miniaturization of LC systems, which has been a continuously increasing trend over recent decades,8 has made LC/EI-MS coupling more feasible. In the 1990s, Cappiello et al.9,10 proposed a miniaturized version of the particle beam interface previously presented by Browner and co-workers6,7 by introducing a capillary-scale particle beam interface, which handled a flow rate of 1 μL/min. However, sensitivity was still low and controversial linearity results were obtained. Further reduction of the LC flow rate to the nanoliters per minute range ( 0.05 by t-test), with overall variability
