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Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined Using Liquid Chromatography#High Resolution Mass Spectrometry Bertram Yin Fong, Lin Ma, Geok Lin Khor, Yvonne van der Does, Angela Rowan, Paul McJarrow, and Alastair Kenneth H. MacGibbon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02200 • Publication Date (Web): 20 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016
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Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined Using Liquid
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Chromatography‒High Resolution Mass Spectrometry
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Bertram Y. Fong†, Lin Ma†, Geok Lin Khor#, Yvonne van der Does†, Angela Rowan†, Paul
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McJarrow†, and Alastair K. H. MacGibbon† *
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†
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North 4442, New Zealand
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#
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Bukit Jalil, 57000 Kuala Lumpur, Malaysia
Fonterra Research and Development Centre, Dairy Farm Road, Private Bag 11029, Palmerston
School of Health Sciences, International Medical University, No 126 Jln Jalil Perkasa 19,
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ABSTRACT
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Gangliosides (GA) are found in animal tissues and fluids, such as blood and milk. These
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sialo-glycosphingolipids have bioactivities in neural development, gastrointestinal tract, and the
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immune system. In this study, a HPLC‒MS method was validated to characterize and quantitate
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the GA in beef, chicken, pork, and fish species (turbot, snapper, king salmon, and island
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mackerel). For the first time, we report the concentration of GM3, the dominant GA in these
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foods, to range from 0.35 to 1.1 mg/100g and 0.70 to 5.86 mg/100g of meat and fish
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respectively. The minor GAs measured were GD3, GD1a, GD1b, and GT1b. Molecular species
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distribution revealed that the GA contained long to very long chain acyl fatty acids attached to
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the ceramide moiety. Fish GA contained only N-acetylneuraminic acid (NeuAc) sialic acid while
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beef, chicken, and pork contained GD1a/b species that incorporated both NeuAc and N-
31
glycolylneuraminic acid (NeuGc) and hydroxylated fatty acids
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KEYWORDS: Glycosphingolipids, fatty acids, sialic acid, mammalian tissue, multiple reaction
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monitoring
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___________________________________________________________________________
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INTRODUCTION
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Gangliosides (GAs) are a complex family of glycosphingolipids containing a variety of
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sialic-acid-containing oligosaccharide chains attached to a variety of ceramide structures. The
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common classification of GAs, and the nomenclature devised by Svennerholm,1 relies on the
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degree of sialylation and the glycosidic linkages of these oligosaccharide groups. However, GAs
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have extra complexity because of the variety of fatty acids (FAs) found in the ceramide, giving
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rise to numerous molecular species within each GA class (Figure 1). In addition, there are many
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types of sialic acid; however, only two types – N-acetylneuraminic acid (NeuAc) and N-
45
glycolylneuraminic acid (NeuGc) – are commonly incorporated into the oligosaccharide chain
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along with any modifications, such as O-acetylation. The type of GA, its sialic acid mix, and its
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FA molecular species profile vary with organism and tissue or fluid.
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GAs play important biological roles in neurological development, memory formation, and
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synaptic and transmembrane signal transduction, and are also implicated in regulating the
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immune system, supporting intestinal maturation, protecting against enteric pathogens, and
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promoting Bifidobacterium proliferation in the gut of infants.2-4 Different GAs are found in
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various biological tissues and fluids, and they are part of the milk fat globule membrane in
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mammalian milk.2, 5 There is growing evidence that dietary GAs, such as those from human
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breast milk, can have a variety of benefits, which has led to increased research in this area over
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the last few years.6-10 However, only very few studies have been carried out on the GA
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quantitation of other food sources such as meat and fish, despite the recognition that they are
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contributors of GAs and other sphingolipids to the human diet.11
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The majority of the GA work on animal tissues (including liver) was conducted using thin
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layer chromatography (TLC)-based methods.12-17 Although most of these studies were restricted
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to the characterization of total GAs, Nakamura et al.14 and Saito and Rosenberg16 did determine
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the concentration of a number of GA classes in hog and chicken muscle, respectively, using
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TLC-based methods.
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To date, only one study reports the GA content in several food products (milk, yogurt, beef,
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tuna, and egg) and this was used to estimate the GA intake for a small population group.18
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However, the total GA concentrations in the food products and the dietary intake were
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determined only as lipid-bound NeuAc (sialic acid) equivalents and NeuGc was not considered
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as a contributing sialic acid.
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The significant gaps in the quantitative data for the GAs in foods are probably due to the
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complex nature of these families of molecules. The traditional methods used to measure them
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were mainly TLC based, which is laborious. Also, the GA content was estimated using the
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indirect approach of measuring the lipid-bound sialic acid with no consideration of the variety of
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sialic acids, their responses to staining, sialic acid modification, or the difference in molecular
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weight because of the base and the FA length along with the presence of double bonds. With the
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advancement of high performance liquid chromatography–mass spectrometry (HPLC‒MS),
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there have been an increasing number of publications on the characterization and absolute
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quantitation of the GAs in biological fluids, such as blood and milk, using such techniques19-22
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and in biological samples23-25 but such HPLC‒MS-based methods have yet to be applied
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systematically to foods.
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In this study, we validated an HPLC‒high resolution MS-based method to characterize and
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quantitate the different GA classes in beef, chicken, pork, and fish. This method will provide a
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direct means of analyzing the GA concentration in foods, as opposed to measuring the GAs
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indirectly via their sialic acid concentration.
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MATERIALS AND METHODS Standards and Chemicals. GA standards [GM1a (bovine brain), GM2 (human brain), GM3
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(bovine buttermilk), GM4 (egg yolk), GD1a (bovine brain), GD1b (bovine brain), GD3 (bovine
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buttermilk), GT1b (bovine brain), and GQ1b (bovine brain)] were obtained from Matreya, LLC
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(Pleasant Gap, PA). All solvents were of liquid chromatography grade (Merck, Darmstadt,
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Germany), except for chloroform, which was analytical grade (ethanol stabilized).
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Samples. All food samples were purchased from a local supermarket in Palmerston North, New Zealand, except for the island mackerel, which was sourced from Malaysia. Lipid Extraction. All meat and fish samples (fillet including the skin) were cut into small
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pieces prior to freeze drying. A sample (20‒30 g) was weighed out and freeze dried. The freeze-
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dried sample was milled into a powder using a domestic coffee grinder. Approximately 0.1‒0.2
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g of freeze-dried powder was weighed into a Kimax tube (10 mL) together with water (0.4 mL).
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The sample was mixed and allowed to rehydrate for 10 min prior to lipid extraction.
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The lipids were extracted from the samples using a modified Svennerholm and Fredman26
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extraction protocol, as described previously.24 Briefly, a further volume of water (0.5 mL) was
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added to the homogenate, followed by methanol (2.7 mL) and chloroform (1.35 mL) at room
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temperature, and the sample was allowed to rock gently for 30 min on a rocker before
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centrifugation at 2,000 g for 20 min. The supernatant was transferred to a 15 mL Kimax tube
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and the pellet was re-extracted using water (0.5 mL) and chloroform:methanol (1:2, 2 mL). After
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mixing and centrifugation (30 min at 2,000 g), the supernatants were pooled and the pellet was
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discarded. Potassium chloride (0.01 M, 1.3 mL) was added to the pooled supernatant, vortex
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mixed, and centrifuged (2,000 g for 30 min). The upper phase was transferred to a 10 mL
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volumetric flask. Potassium chloride (0.01 M, 0.5 mL) was added to the remaining lower phase
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and any interfacial fluff (minimal) and was centrifuged (2,000 g, 20 min). The upper phase was
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pooled into the 10 mL volumetric flask without disturbing any interfacial fluff, and the flask was
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made up to the mark with methanol (50%). The upper phase was used for GA quantitation.
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Solid Phase Extraction. Solid phase extraction (SPE) employed a C18 SPE cartridge
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(Strata, 500 mg/6 mL, 55 µm, 70Å, Phenomenex, Torrence, CA) and the SPE protocol as
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described by Sørensen.19 The SPE was conditioned with 3 mL of methanol followed 1 mL of
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60% methanol at a flowrate of 2-3 mL/min. The GA extract (10mL) was diluted with 1mL ACS Paragon Plus Environment
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disodium hydrogen phosphate buffer (30 mM, pH 9.2), followed 4mL of 60% methanol, prior to
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loading onto the SPE cartridge. The SPE cartridge was washed with 3 mL of 60% methanol and
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sucked dry under vacuum for 5 seconds. The GAs were eluted with 6 mL of methanol under
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vacuum. The eluent was dried down under a stream of nitrogen (30 °C) to near dryness, and
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was made up to a volume of 0.25 mL with 50% aqueous. methanol. The pre-concentrate extract
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was used for HPLC‒MS2 fragmentation and the characterization of minor level GAs (GD3,
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GD1a, GD1b, and GT1b).
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HPLC‒MS Quantitation of GAs. The HPLC separation of the GAs was conducted on an
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Agilent 1100 series HPLC system (Santa Clara, CA) equipped with a 150 mm x 2.1 mm i.d., 3
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µm, APS-2 Hypersil hydrophilic column ((Thermo Electron Corporation, Waltham, MA)
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coupled to a 10 mm x 2.1 mm i.d. APS-2 guard column. The GAs were separated using an
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acetonitrile:ammonium acetate buffer gradient as described previously.24
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The eluate from the HPLC system was introduced into an LTQ-Orbitrap mass spectrometer
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(Thermo Electron Corporation, Waltham, MA) using an electrospray ionization (ESI) probe
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inlet. Ions were generated and focused using an ESI voltage of ‒3750 V, a sheath gas flow of 30,
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an auxiliary gas flow of 10, and a capillary temperature of 300 °C. MS data acquisition was
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carried out with the mass spectrometer scanning in negative ion mode with a resolution of
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30,000 over a 700−1650 m/z range. The system was calibrated with GA standards obtained from
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Matreya, LLC (Pleasant Gap, PA). The HPLC run was diverted to waste for the first 9 min and
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after 18 min. The total run time was 25 min.
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Calibration Standard Preparation. The HPLC‒MS system was calibrated against
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commercial GA standards. GA stock standards [GM1a, GM2, GM3, GM4, GD1a, GD1b, GD3, GT1b,
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and GQ1b] were prepared at 1 mg/mL in methanol (50%), and serially diluted further in methanol
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(50%) to give a seven-point calibration curve ranging from 0.08 to 10 µg/mL.
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Identification of GAs Using Accurate Mass. A combination of GA calibration standard
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retention time and accurate mass was used to identify the GA molecular species in this study. ACS Paragon Plus Environment
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The resolving power of the LTQ-Orbitrap mass spectrometer was used to filter post analysis for
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known accurate masses of GA species present within each class of GA measured. Three factors
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were considered when compiling our extract mass lists for filtering post analysis.
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Sphinosine Group and Acyl FA Side Chains. The exact GA masses extracted were based on
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sphingosine d18:1 and dihydrosphingosine d18:0, the two most dominant,14 with acyl FA side
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chains C14:0 to C30:0, and their respective unsaturated and mono-hydroxy to tri-hydroxy FAs.
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Sialic Acid Moiety. GAs containing NeuAc and NeuGc sialic acids, and their respective O-
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acetylated moieties, were included in the mass list calculations.
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Charge State. The number of NeuAc/NeuGc residues present on a GA molecule determines
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the number of possible charge states that may be observed in the (electrospray) mass spectra of
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that GA. Where more than one charge state existed for a given class of GA, the charge state(s)
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observed in the greatest intensity were used for compiling our mass lists. The charge state(s)
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selected for each GA class were as follows: GM1a, [M–H]–; GM2, [M–H]–; GM3, [M–H]–; GM4,
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[M–H]–; GD1a, [M–2H]2–; GD1b, [M–2H]2–; GD3, [M–2H]2–; GT1b, [M–2H]2–, [M–3H]3–; GQ1b,
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[M–2H]2–, [M–3H]3–). GT1b and GQ1b displayed prominent species in their –2 and –3 charge
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states. Both charged states were included in the extract mass list.
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MS2 Fragmentation of GA Molecular Species. The identities of the major GA molecular
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species, particularly those that were observed in the samples but were not present in the
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calibration standards, were confirmed using HPLC–MS2 experiments. The HPLC–MS
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conditions were as described above, except that the LTQ-Orbitrap was set to acquire MS2 data in
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dependent data acquisition mode. The dependent data acquisition mode experiments were
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performed according to the following parameters: the full MS scan event 1 was carried out with
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a resolution of 30,000 in the Orbitrap followed by the MS2 scan event, which was carried out in
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the ion trap (LTQ). The dynamic exclusion time was set at 45 s for the most intense ions, with
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either collision-induced dissociation or pulsed Q collision-induced dissociation activation type
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parent masses were added to the mass list for MS2. The MS2 carbohydrate fragments were
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labelled using nomenclature as described by Domon and Costello27 and other standard
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carbohydrate nomenclatures.
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Accuracy and Repeatability. The accuracy of the method and matrix suppression effects
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were evaluated using spike recovery studies and standard addition, whereas the repeatability of
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the method was determined by repeated analysis of several samples in each batch of analyses
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carried out on different days over the course of this study, using the guidelines from Taylor.28
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The repeatability coefficient of variation (CV) was determined as the percentage of the standard
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deviation divided by the mean.
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In the standard addition protocol, the samples were spiked with increasing concentrations of a mixed GA standard solution (0.3, 0.6, and 1.2 µg/mL). Statistical Analysis. All statistical analysis was conducted using Minitab (Release 16.2.4,
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2013, Minitab Inc., State College, PA). Comparison between two groups of data was conducted
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using paired t-tests.
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RESULTS AND DISCUSSION Methodology. The majority of HPLC‒MS methods for GA quantitation are based on
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multiple reaction monitoring (MRM) MS techniques,7-9, 19, 21, 23 with scope limited to dairy, milk,
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and blood samples. Although the MRM mode of MS is specific and deemed to be more sensitive
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than accurate mass extract techniques, MRM methods require prior knowledge of the parent and
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fragment ions. Whereas MRM methods are easy to set up for known individual compounds,
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they are more complex for GAs, where multiple molecular species often exist for each GA class
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and these molecular profile differ between animal types and between tissues in the same animal.
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In this light the pre-investigatory work required to set up an MRM method makes this method
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less flexible and more time consuming. In contrast, an accurate mass extract technique using
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high resolution MS does not require prior knowledge of all the GA molecular species present
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before quantitation can proceed. Accurate mass extraction of parent ions is carried out post
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analysis, which also has the advantage of allowing the data to be re-mined at later stages as more
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information becomes available. Furthermore, data on the GA molecular species distribution is
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immediately available to the analyst.
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Typical HPLC‒MS extracted ion chromatograms, acquired in the mass range of m/z 700-
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1650, of the GA standards and the turbot fish sample are presented in Figure 2 and Figure 3,
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respectively. The GAs were separated on the amino-propyl column based on their sialic-acid-
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containing oligosaccharide moieties (or class), and were identified by their retention time,
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against calibration standards, and their accurate mass. In addition, MS2 fragmentation was used
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to confirm the identification of the GA molecular species that were present in the samples but
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not in the calibration standards. As there was no on-column separation between the GAs
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containing either an NeuAc or an NeuGc sialic acid moiety within each class of GA, NeuAc-
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and NeuGc-containing GA molecular species within each GA class were quantitated together.
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The extracted ion chromatogram for each of the GA molecular species was generated post
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analysis using the accurate masses and was calibrated against NeuAc-containing calibration
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standards using peak areas as NeuGc-containing GA calibration standards were not available. As
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is common with other GA quantitative methods it was assumed that different acyl FA and sialic
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acid moieties within each GA class give the same response.23-25
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The ESI process during MS is prone to matrix effects, typically suppression of ionization.
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This was evaluated by comparing the standard addition results with the results obtained using
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the described external standard HPLC‒MS method. The standard addition results for GM3 were
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not significantly different from the external standard method results across the different sample
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matrices (beef, chicken, pork, and fish), (Table 1), giving us confidence that any major matrix
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effects, if present, were insignificant. The accuracy of the method was also evaluated using spike
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recovery experiments. GM3 was spiked with approximately twice the endogenous level, but
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GD3, GD1a, GD1b, and GT1b were spiked at approximately 0.1 mg/100g fresh product. The
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recoveries were 101‒104% for GM3, but were 90‒106% for GD3, GD1a, GD1b, and GT1b (Table
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2). No recovery was determined for GM2, which was detected only in island mackerel. These
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recoveries are consistent with those (96‒103%) that we reported previously for dairy products22
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using the same extraction protocol,26 giving us confidence that the protocol was robust.
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The repeatability of the method, determined as the CV, ranged from 2-10% for GM3.
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However, much higher CVs were measured for the minor GAs [GD3 (4‒20%), GD1a (5‒33%),
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and GT1b (33%)] because their concentrations were generally close to the LOQ (0.01 mg/100g
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fresh product equivalent) (Table 1).
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GA Molecular Species in Meat. The GA molecular species in the meats in our study were
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tentatively identified by d-value (the sum of the carbon atoms and unsaturated bonds attached to
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the sphingosine base and acyl FAs) (Figure 1), with d18:1 and d18:0 being assumed to be the
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major and minor sphingosine base FAs, respectively.29
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The GM3 and GD3 molecular species observed in beef, chicken, and pork samples (Figure 4)
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were also present in their respective bovine-milk-sourced calibration standards (Figure 2),
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although their relative distributions were different.
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The predominant GM3 molecular species observed in beef and pork was d36:1(m/z 1179),
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which could be tentatively identified as C18:1‒18:0, based on the d18:1 sphingosine being the
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major sphingoloid base that is commonly reported in mammalian muscle tissue.13,29, 30‒31 Based
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on the same d18:1 sphingosine, the minor GM3 molecular species were d38:1, d40:1, d41:1, and
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d42:1 and contained the very long chain acyl FAs C20:0, C22:0, C23:0, and C24:0, respectively.
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Pork and beef also contained small amounts of GM3 containing d18:0 sphingosine base, as
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observed by the presence of the d37:0 molecular species containing the odd chain C19:0 acyl FA
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(Figure 4). The HPLC‒MS GM3 molecular species distribution results for our pork sample are
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consistent with the TLC‒gas chromatography results reported by Namakura et al.14 in which the
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GM3 acyl FA composition contained predominantly C18:0 FA (76%), with minor levels of
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C20:0, C22:0, C23:0, and C24:0 FAs. The major sphingosine base was d18:1 (95‒97%),
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whereas the d18:0 sphingosine base was present in minor proportions (1‒3%). No FA
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composition data for beef muscle GAs are currently available in the literature for comparison
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with our observations.
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In chicken the GM3 dominant molecular species was d34:1 (m/z 1151), which could be
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tentatively identified as containing d18:1‒16:0 FAs. The minor chicken GM3 molecular species
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d36:1, d38:1, d40:1, d42:2, and d43:1 could be tentatively identified as containing the long to
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very long chain acyl FAs C18:0, C20:0, C22:0, C24:1, and C25:0, respectively, also on a d18:1
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sphingosine base. Similar to beef muscle, no GA molecular species data for chicken muscle
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could be located.
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All the GM3 molecular species that were identified across the three meat samples contained
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the NeuAc sialic acid moiety (Figure 5). However, using TLC methods, Nakamura et al.13,14
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reported the presence of NeuGc-containing GM3 in hog and bovine skeletal muscle at 4‒7.4%
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and 21% of the total GAs, respectively. We did not find any NeuGc-containing GM3 in our beef
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and pork samples. It is possible that either the NeuGc-containing GM3 molecular species were at
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concentrations below the limit of detection for our methodology or they were inherently absent
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from our samples because of the tissue origins. No NeuGc-containing GM3 (or GD3) was
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reported for chicken skeletal muscle by these researchers and, again, we did not detect them in
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our samples.
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For meat GD3 molecular species, although d34:1 (d18:1‒16:0, m/z 720 (doubly charged))
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was the most abundant molecular species, a number of other molecular species were also present
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in near similar abundance (Figure 4). Like the major GM3 molecular species, and GD3
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molecular species across the three meat samples were also identified from the MS2 data as
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containing NeuAc sialic acid moieties. Examples of the MS2 fragmentation of a GD3 molecular
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species containing an NeuAc sialic acid moiety are provided in Figure 5, showing their
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distinctive m/z 290 (NeuAc) and m/z 581(NeuAc‒NeuAc, for GD3) fragments.
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GD1a and GD1b were fully resolved using the HPLC separation conditions described (Figure
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2). Similar to the standards, the GD1a and GD1b mass spectra within each of the meat samples
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showed that they shared the same parent ion masses. For this reason, the pooled GD1a and GD1b
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mass spectrum for each of the meat samples is presented in Figures 4G‒4I, as GD1a/b. Accurate
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mass analysis of the GD1a/b molecular species in these samples indicated that they contained a
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combination of NeuAc and NeuGc sialic acid moieties (Figures 4G‒4I). Their presence was
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confirmed using MS2 experiments (Figure 6), in which GD1a shows the NeuAc (m/z 290) and/or
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NeuGc (m/z 306) fragment ions, and GD1b shows the characteristic NeuAc‒NeuAc (m/z 581) or
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NeuAc‒NeuGc (m/z 597) MS2 fragment ion. Together with other daughter fragment ions (Figure
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6), the accurate parent ion mass, and the retention time, tentative identification of the GD1
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species was made (Figure 4). This is an interesting observation, given that GD3 showed only
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NeuAc and GD3 is a precursor GA for GD1b biosynthesis. In addition to the different sialic acid
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moieties, we were also able to tentatively identify in the meat samples some of the GD1a/b
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containing hydroxylated FAs. These hydroxyl groups were tentatively identified to be located on
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the acyl FA chain. Based on their accurate mass, they were typically associated with very long
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chain acyl FAs (d40:0, d41:1, d42:0, d42:1, d42:2, d44:0) containing both NeuAc and NeuGc
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sialic acid moieties (Figures 4G‒4I). A GA with hydroxylated acyl FAs has not previously been
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reported in muscle tissues, but 2-hydroxylated acyl FAs are typically found in glucosyl-
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ceramides and lactosyl-ceramides in various mammalian tissues fluids and are typically
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associated with very long chain acyl FAs (C22:0, C23:0, and C24:0).12, 32-34 As lactosyl-
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ceramides are a precursor for GA biosynthesis, the presence of hydroxylated acyl FAs in GAs
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might be predicted. Skin tissues have also been reported to be rich in ceramides with ω-
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hydroxylated very long chain acyl FAs with up to 36 carbons that are generally unsaturated with
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one to two double bonds.35 Although we have tentatively identified the hydroxylated FAs to be
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associated with the acyl FAs, they could also be associated with the sphingoloid long chain base.
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Sphingoloid bases with multiple hydroxylated groups have been reported but generally at minor
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levels.29 Future studies will examine the location of these hydroxylated FAs. In this study, these
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hydroxylated molecular species, and those containing either NeuAc or NeuGc, were quantitated
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together as either GD1a or GD1b.
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GA concentration in Beef, Chicken, and Pork. Table 1 summarizes the GA concentrations
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measured across the fish samples. GM3 was the major GA that was found in beef, chicken, and
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pork, making up 71‒96% of the total GAs in these samples, with the highest concentration (1.1
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mg/100g) in chicken thigh and the lowest concentration (0.35 mg/100g) in pork mince (Table 1).
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Data on meat GAs is sparse, with only two studies13, 14 reporting the GA composition in animal
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muscle. Of these two studies, one14 reported the GM3 concentration of pork (hog) at
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approximately 1 mg/100g (converted from 8.3 nmol/g using a GM3 molecular weight of 1180)
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using a TLC‒resorcinol‒HCl method. The other study, published by the same researchers,13
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reported GM3 as being the major GA in beef, chicken, and pork (hog), making up 71%, 52%,
309
and 34% of the total GA distribution. However, these results13, 14 were based on TLC
310
densitometry responses and were not corrected for GA mass. In this study, GM3 made up 96%
311
of the total GAs in the beef sample, 76‒79% of the total GAs in the two chicken samples and
312
71% of the total GAs in the pork sample.
313
GD3 is the dominant GA in bovine milk,22 but was measured at only a relatively low
314
proportion (4‒10%) of beef GAs and 4% of pork GAs, 0.02‒0.1 mg/100g and 0.02 mg/100g,
315
respectively (Table 1) in this study. Although absolute concentration data on beef GD3 was not
316
available from Nakamura’s TLC densitometry study,13 beef GD3 was estimated at 2% of the
317
total GAs. These researchers also estimated GD3 at 4% of the total GAs in pork, but they did not
318
find any GD3 in their second pork sample.14
319
Compared with pork and beef, chicken contained a significantly higher proportion of GD3, at
320
21% of the total GAs (0.2‒0.3 mg/100g) which is similar to the 16% reported by previously.13
321
However, the GA distribution in chicken changes over its developmental stages, with GD3 (50%
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14 322
of the total GAs) being the major GA in thigh and leg muscle during early development and
323
decreasing to ~ 15% (of the total GAs) beyond 90 days postnatal. Conversely, GM3 (60% of
324
total GAs) is dominant only after 20‒90 days postnatal.36
325
GD1a was typically at higher concentrations than GD1b in beef, chicken, and pork (Table 1).
326
In beef and chicken both GD1a/b were relatively minor GAs, being < 6% of the total GAs (up to
327
0.054 mg/100g) but in pork they were, a significantly higher proportion (16%, 0.08 mg/100g).
328
The GD1a concentration reported for hog muscle14 was 1.19 mg/100g (22% of the total GAs,
329
converted using a molecular weight of 1894), which was about 10 times higher than obtained in
330
this study. This discrepancy may be due to both analytical methodology and sample source.
331
Only relatively small proportions of GD1b (3% of the total GAs, 0.15 mg/100g) were reported by
332
these same researchers. Although the relative proportion of GD1b measured in this study was
333
nearly three times this, at 8% of the total GAs, the absolute concentration was much less, at 0.04
334
mg/100g (Table 1). No GD1a and GD1b concentration data are available in the current literature
335
for either beef or chicken, but the TLC densitometry response (uncorrected) has revealed GD1a
336
and GD1b together to be the second most abundant GA in beef, at 12% of the total GAs.13 No
337
GD1a or GD1b was detected in a chicken muscle sample by these researchers but they were
338
detected in a chicken thigh and leg muscle sample by Saito and Rosenburg36, at 15% of the total
339
GAs.
340 341
GA Molecular Species in Fish. The GM3 in fish was predominantly one molecular species, but
342
the exact molecular species differed across the four fish samples (Figures 3 and 7). The
343
dominant GM3 molecular species was d40:2 (tentatively identified as d18:1‒22:1) in turbot
344
(Colistium mudipinnis), d42:2 (tentatively identified as d18:1‒24:1) in snapper (Chrysophrys
345
auratus) and king salmon (Oncorhynchus tshawytscha), and d34:1 (d18:1‒16:0) in island
346
mackerel (Rastrelliger faughni). The island mackerel GM3 shorter FA chain is similar to that
347
observed for beef, chicken, and pork (Figure 4).
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Fish GD3 molecular species was observed to have an acyl FA composition that was
349
reflective of their GM3 counterpart, except for snapper, where d40:2 was the major GD3
350
molecular species, compared with d42:2 for GM3. In addition, turbot and snapper could contain
351
GD3 molecular species containing FAs with up to three unsaturated bonds [d43:4 (Figure 3),
352
d41:4 (Figure 7)].
353
Fish (except turbot) GD1a/b molecular species distributions were less reflective of GM3 and
354
GD3 (Figures 3 and 7). Furthermore, fish GD1a/b could also contain the molecular species d39:4
355
(d18:1‒21:3), d42:4 (d18:1‒24:3) and d43:4 (d18:1‒25:3) with highly unsaturated acyl FAs
356
(Figures 3 and 7).
357
In contrast to GD1a/b, the GT1b molecular species profiles (d-values) had an acyl FA
358
composition generally reflective of GM3 and GD3 in turbot (Figure 3) and salmon (Figure 7).
359
Snapper, on the other hand contained d-value molecular species (d47:2, d47:0, and d48:1)
360
(Figure 7) that were not observed with other GA classes. These molecular species (d47:2, d47:0,
361
and d48:1) could be tentatively identified as containing the very long chain acyl FAs d18:1‒
362
29:1, d18:1‒29:0 and d18:1‒30:0, respectively. As the fish samples were extracted with the skin
363
on, the presence of such very long chain acyl FAs cannot be ruled out given that FA chains up to
364
C30 have been reported in mammalian skin ceramides, 35 which is important for water proofing.
365
Unfortunately, in negative ion mode, it was difficult to generate secondary fragmentation of the
366
sphingosine base to determine the FA composition. GT1b was not detected in island mackerel.
367
GM2 was only detected in island mackerel, (Figure 7I), with an acyl FA composition that
368
was reflective of its GM3 and GD3 composition, and the d34:1 (d18:1‒16:0) molecular species
369
observed as the major molecular species. GM2 was not detected in any of the other fish or
370
animal muscle samples analysed in this study.
371
Unlike the beef, chicken, and pork samples, the fish GAs detected and measured in this
372
study contained only NeuAc sialic acid (Figure 3). This result is consistent with the total sialic
373
acid studies conducted by both Samraj et al.37 and Li and Fan,38 which showed that fish muscle,
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16 374
including salmon and mackerel, contained only NeuAc. However, both NeuAc and NeuGc have
375
been shown in fish liver15 so this could be tissue specific.
376 377
GA concentration in Fish. Table 1 summaries the GA concentration measured across the fish
378
samples. As with the animal samples, fish muscle contained predominantly GM3 (77‒97% of
379
total GAs,0.70‒5.86 mg/100g, Table 1), with the highest total GA concentrations measured in
380
island mackerel.
381
GD3 was present in the fish samples at low proportions of between 2 and 3% of the total
382
GAs (0.02‒0.23 mg/100g). Likewise, the total GD1a/b level in fish was also low (≤ 6% of total
383
GAs) except for turbot, at 14% of total GAs. Like the animal meat samples, GD1a was the major
384
GD1 present (Table 1). GT1b was detected only in small amounts (up to 3% of the total GAs) in
385
turbot, king salmon, and snapper with none in island mackerel. However, island mackerel was
386
the only fish sample analysed in this study that contained GM2, at 4% of the total GAs (0.24
387
mg/100g).
388 389
Molecular Species Diversity Across GA Classes. The acyl FA composition observed for the
390
GM3 molecular species for all the samples analyzed was not retained across the other GA classes
391
for their respective samples, although less so for fish. This observation was unexpected, given
392
that GM3 is the precursor for the biosynthesis of other GAs (Supporting Information Figure 2),
393
and there was an expectation that the acyl FA composition would be retained in subsequent GA
394
classes. This suggests that the glycosyl-transferase enzyme involved in each of the biosynthesis
395
pathways may be selective based on the acyl FA chain, hence resulting in a different molecular
396
species profile for each GA class. Although there is limited understanding of the reasons behind
397
the great diversity in the GA molecular species, recent research has shown that ceramides (GA
398
precursors) with specific acyl FA chain lengths have different biological functions.39 The
399
heterogeneous nature of the GAs across different animal species and the tissue-specific
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17 400
expression of GAs make quantitation of the GAs across different samples complex. However,
401
the HPLC‒high resolution MS technique based on accurate mass extract post analysis gives this
402
method great flexibility, such that prior knowledge of the different GA molecular species is not
403
required and allowing for future reanalysis of the data.
404
Although we have characterized and measured GM3, GD3, GD1a, GD1b, GT1b, and GM2 in
405
the samples, and tentatively assigned them with an acyl FA composition, based on a d18:1
406
sphingosine base, the presence of other GAs, such as O-acetylated GAs, in these samples cannot
407
be ruled out. However, they are likely to be present in low concentrations.
408 409
In conclusion, using this method, for the first time, we were able not only to profile and
410
characterize the different GA molecular species present in the beef, chicken, pork, and fish
411
samples but also to quantitate them. Given that these GAs play an important role in various
412
biological processes including brain development, it is important to have a good understanding
413
of not only the concentration but also the type of GA class and their molecular species that are
414
found in the foods consumed. This methodology will enable future studies on dietary GA intake
415
to be conducted for toddlers and young children. This is the first step in understanding what the
416
recommended daily intakes of such complex lipids should be for supporting proper growth and
417
cognitive development. On the other end of the age spectrum, GAs may also be vital for the
418
elderly population, as cognitive decline is a common occurrence. Understanding dietary GA
419
intake in this population group may also provide some insights into their cognitive degeneration.
420
In both population groups, the development of functional foods containing enriched sources of
421
complex lipids requires data studies that are similar to this study.
422
423
ABBREVIATIONS USED
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18
Cer, ceramide; CV, coefficient of variation; FA, fatty acid; GA, ganglioside; Gal, galactose;
424 425
GalNAc, N-acetylgalactosamine, Glc, glucose; LOQ, limit of quantitation; MRM, multiple
426
reaction monitoring; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid.
427
Gangliosides (where M, D, T and Q refer to 1 to 4 NeuAc attached)
428
GM4
NeuAcα2,3Gal-Cer
429
GM3
NeuAcα2,3Galß1,4Glc-Cer
430
GM2
GalNAcß1,4(NeuAcα2,3)Galß1,4Glc-Cer
431
GM1
Galß1,3GalNAcß1,4(NeuAcα2,3)Galß1,4Glc-Cer
432
GD3
NeuAcα2,8NeuAcα2,3Galß1,4Glc-Cer
433
GD2
GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-Cer
434
GD1a
NeuAcα2,3Galß1,3GalNAcß1,4(NeuAcα2,3)Galß1,4Glc-Cer
435
GD1b
Galß1,3GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-Cer
436
GT1b
NeuAcα2,3Galß1,3GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-Cer
437
GQ1b
NeuAcα2,8NeuAcα2,3Galß1,3GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-
438
Cer
439 440
AUTHOR INFORMATION *Corresponding author (Tel: +64 6 3504649; Fax: +64 6 356 1476; E-mail:
441
[email protected]),
442 443 444
ACKNOWLEDGMENTS This work was supported by the New Zealand Primary Growth Partnership post-farm
445
gate dairy programme, funded by Fonterra Co-operative Group and the New Zealand Ministry
446
for Primary Industries. The authors would like to thank Maree Luckman for statistical analysis
447
and guidance, and Claire Woodhall for editing the manuscript. The cooperation of Snigdha
448
Misra, Sangeetha Shyam and Megan Chong for the procurement and shipment of the samples
449
from Malaysia is much appreciated.
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19 451 452
SUPPORTING INFORMATION DESCRIPTION Table S1. GA accurate masses used for post analysis to generate extracted ion
453
chromatograms. Figure S1. Schematic of the biosynthetic pathway of GAs. This material is
454
available free of charge via the internet at http://pubs.acs.org.
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20 455
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derived glycan promotes inflammation and cancer progression. Pro Natl Acad Sci USA 2015,
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HPLC-FLD. Open Journal of Preventive Medicine 2014, 4, 57-63.
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560
561
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FIGURE CAPTIONS
563
Figure 1. The structure of GM3 d40:2 consisting of a ceramide, with a d18:1 sphingosine base
564
and a C22:1 fatty acid, and sugar groups; glucose, galatose and sialic acid.
565
Figure 2. Extracted HPLC‒MS traces of GA calibration standards and their respective
566
molecular species profiles in negative ion mode. Molecular species are identified using their d-
567
values. The ganglioside structures are represented by symbols as follows, ceramide (Cer),
568
glucose (●), galactose (○), GalNAc (□) and NeuAc (
569
Figure 3. Extracted HPLC‒MS traces in negative ion mode of GAs (A) GM3, (B) GD3, (C)
570
GD1a/b , and (D) GT1b in turbot fish, followed by their respective mass spectra (E-H), and the
571
MS2 fragmentation spectra of the d40:2 major molecular species (I-L). Fragment ions are
572
annotated using carbohydrate fragment ion nomenclature devised by Domon and Costello.27
573
Where possible, the abbreviated sugar residue description is also provided. The NeuAc fragment
574
(B1α, B1α”) from multiple regions of GD1a/b or GT1b is labelled as B1.
).
575 576
Figure 4. Mass spectra in negative ion mode of GA [(A-C) GM3, (D-F) GD3, and (G-I) GD1]
577
molecular species profiles for (A,D,G) beef, (B,E,H) chicken, and (C,F,I) pork. The molecular
578
species were tentatively identified using their d-values (the sum of carbon atoms and the number
579
of double bonds attached to the dihydroxy sphingosine base and their fatty acid moieties). All
580
GD1a/b species were tentatively identified as containing NeuAc sialic acid except those denoted
581
by an asterisk, which also contained NeuGc sialic acid. Some molecular species were tentatively
582
identified as containing extra hydroxyl (‒OH) groups.
583
Figure 5. Examples of MS2 fragmentation (in negative ion mode) of some key GM3 and GD3
584
molecular species found in beef, chicken, and pork from Figure 1. Only identified fragments
585
have been labelled.
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26
Figure 6. MS2 fragmentations (in negative ion mode) of some key GD1a/b molecular species found in beef, chicken, and pork, showing the presence of fragments containing NeuAc and NeuGc sialic acid moieties. 586
Figure 7. Mass spectra (in negative ion mode) of (A-C) GM3, (D-F) GD3, (G-I) GD1a/b, and
587
(J,K) GT1b molecular species found in (A,D,G,J) snapper, (B,E,H,K) king salmon, and (C,F,I,L)
588
island mackerel. (L) GM2, but not GT1b, was detected in island mackerel.
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27 Table 1. GA Concentration in Measured Various Animal Meat and Fish Samples. The GA Results Determined Using Standard Addition Are Also Provided for Beef, Chicken, Pork, and Turbot Fish. sample
n
GM3 mg/100g fresh product (CV,%) 0.35 ± 0.02a (6) 0.34 ± 0.01a
GD3 mg/100g fresh product (CV,%) 0.02 ± 0.002 (10)
GD1a mg/100g fresh product (CV,%) 0.08 ± 0.01 (13)
GD1b mg/100g fresh product (CV,%) 0.04 ± 0.00
GT1b mg/100g fresh product (CV,%) ND
total GAs mg/100g fresh product (CV,%) 0.49
0.02 ± 0.003 (15)
< LOQb
< LOQb
ND
0.48
0.05 ± 0.01 (20) < LOQb
< LOQb
ND
0.95
< LOQb
ND
0.95
0.01 ± 0.00
ND
1.44
0.04 ± 0.00
0.03 ± 0.01 (33)
0.94
pork mince (10% fat)
external standard
6
(Sus scrofa domesticus)
standard addition
2
beef mince (10% fat)
external standard
6
(Bos taurus)
standard addition
4
0.46 ± 0.03a (6) 0.41 ± 0.04a
4
0.80 ± 0.05 (6)
0.1 ± 0.01 (10) 0.20 ± 0.02 (10)
beef blade steak (Bos taurus) chicken breast
external standard
6
(Gallus gallus domesticus)
standard addition
2
0.75 ± 0.04a (5) 0.79 ± 0.02a
4
1.10 ± 0.10 (9)
0.3 ± 0.02 (7)
0.72 ± 0.02a (3) 0.80 ± 0.03a
0.02 ± 0.004 (20)
chicken thigh (with skin) (Gallus gallus domesticus) turbot fish
external standard
6
(Colistium nudipinnis)
standard addition
4
0.03 ± 0.01 (33) 0.13 ± 0.02 (15)
c
island mackerel 2 5.86 ± 0.09 (2) 0.23 ± 0.01 (4) 0.38 ± 0.02 (5) 0.01 ± 0.002 ND (Rastrelliger faughni) (20) king salmon 2 1.09 ± 0.10 (9) 0.03 ± 0.002 < LOQb < LOQb < LOQb (Oncorhynchus tshawytscha) (7) snapper 2 0.70 ± 0.07 0.02 ± 0.003 0.01 ± 0.003 0.03 ± 0.003 < LOQb (Chrysophrys auratus) (10) (15) (30) (10) a Superscript letters denote that paired t-test results between the external standard and standard addition were not significantly different (p > 0.05). b
c
GA levels above the limit of detection (0.004 mg/100g fresh product) but below the limit of quantitation (LOQ).
GM2 was measured at 0.24 ± 0.04 mg/100g.
ND: not detected.
ACS Paragon Plus Environment
6.48 1.12 0.76
Journal of Agricultural and Food Chemistry
Page 28 of 37
28
Table 2. Recovery (%) of GA after Spiking into the Sample. sample pork mince (10% fat) beef prime mince (10% fat) chicken breast turbot fish (Colistium nudipinnis)
n 3 4 3 4
GM3 103 ± 3 101 ± 3 104 ± 9 102 ± 6
GD3 99 ± 8 94 ± 10 93 ± 3 101 ± 4
GD1a 103 ± 4 100 ± 4 101 ± 6 106 ± 3
GD1b 92 ± 4 91 ± 4 92 ± 1 100 ± 5
ACS Paragon Plus Environment
GT1b 94 ± 4 93 ± 6 90 ± 6 99 ± 2
Page 29 of 37
Journal of Agricultural and Food Chemistry
29
Ceramide
Glucose
Figure 1
ACS Paragon Plus Environment
Galactose
Sialic acid
5e+3
0 1e+5
GM4 0
5 10 3e+4
4e+4
2e+4
0 8e+5
GD1a 6e+4
4e+5
2e+5
0 8e+5 GD3 0 5e+4
6e+5 4e+4
4e+5 3e+4
2e+5 2e+4
0 6e+5 GT 1b
0 4e+5
3e+5 GQ1b
2e+5 8e+3
1e+5 4e+3
0 15
Time (min) 20 25 890
1e+4
4e+5
2e+5 2e+4
8e+3
0
790 800
2e+4
1e+4
1040
900
2e+4 720 740
1040 1060
810
1150
0
GD1b 1080
4e+4
2e+4
910 920
760
1080
0 820 830
m/z
Figure 2
ACS Paragon Plus Environment
1249.8120 (d41:1)
1550
1200
1120
930
1e+4
1200
1277.8440 (d43:1)
1400 1263.8270 (d42:1)
1410.8419 (d38:1)
1500
1222.58621 (d38:1) 2[M-2H]
1e+5 1221.7822 (d39:1)
1572.8929 (d38:1)
1544.8620 (d36:1)
2e+3
1235.7970 (d40:1)
5000
775.9484 (d42:2) 776.9564 (d42:1) 783.9677 (d43:1)
1e+4
931.4898 (d38:1)
2e+5
1208.5711 (d36:1) 2[M-2H]
6e+5
4e+3
1117.7704 (d43:0)
15000
769.9472 (d41:1)
3e+5
1209.7799 (d38:0)
1350
1115.7523 (d43:1)
8e+3
1382.8103 (d36:1)
2e+4
762.9399 (d40:1)
8e+4 2e+4 1179.7336 (d36:1)
4e+4
1207.7659 (d38:1)
6e+4
1077.0374 (d38:1)
GM3
1089.7437 (d41:0)
1450
1103.7576 (d42:0)
0 5e+5
917.4746 (d36:1)
4e+5 2e+4
749.9340 (d38:0) 755.9326 (d39:1)
2e+4
748.9259 (d38:1)
GM2
1063.0223 (d36:1)
8e+4
814.7186 (d38:1) 3[M-3H]
0 1e+5
1151.7030 (d34:1)
10000
734.9095 (d36:1)
GM 1
720.8947 (d34:1)
6e+4
Intensity
20000
805.3758 (d36:1) 3[M-3H]
Intensity
Journal of Agricultural and Food Chemistry Page 30 of 37
30
25000
0 1600
2e+4 0 1450 1500
1250 1300
1160 1200
940
1225
950
0 780 800
1100 1120
1250
3e+3
5
2e+3
10
C 5e+4
GD1a
GD1b
0
15 Time (min) 20
20 25
D GT1b
1e+3
25
4e+3 4e+4
3e+4
2e+4
1e+4
860
6e+3
4e+3
2e+3
1020 880
0 1040 1060
6e+4
G
900
1e+4
8e+3
H
750
920
760 770
940 960
0 0
1080 1100
780
980
1120
790
1140
800
0
768.9333 (d41:2 770.9308 (d41:0) 775.9406 (d42:2) 780.9328 (d43:4) 784.9454 (d43:0)
Intensity 250
200
K
150
100 50
1000 180
160
140
L
120
100 80
60
40
20
200
200
m/z
0
Figure 3
ACS Paragon Plus Environment
400
0 400
500
600
600
800
1000 m/z
800
1000
1500
1304.1771 (0, 2 X3 )
1233.5164 (Y1 )
942.35693 (Y2 )
780.5163 (Y1 )
762.0204 [M-2H]2-
618.5025 (Y0 )
-
581.1588 [(NeuAc-NeuAc)-H20-H]
290.0130 [NeuAc-H20-H]- (B1)
0 1000
1200
1589.9156 [M-NeuAc-H]-
740 1000
1307.5948 [M-2NeuAc-H]-
730 2000
1126.7465 (Gal-GalNAc-GalNeuAc-NeuAc-H)
720
J
944.4088 [M-2H]2-
710
4000
1248.7855 (d41:2) 1251.7803 (d40:0) 1261.8003 (d42:2) 1271.7846 (d43:3) 1279.8106 (d43:0)
-
762.6729 (Y1-H20) 780.5163 (Y1 )
1000
1400
1215.3685 [M-H20-H]1233.5936 [M-H]-
1189.4852 (1,3 X1)
1012.0654 (0, 2X1)
942.3974 (Y2)
618.5025 (Y0 )
290.0130 [NeuAc-H20-H] (B1)
800
1890.3517 [M-NeuAc-H]-
6e+3 0 600
1598.5676 [M-2NeuAc-H]-
8e+3 700
3000
400
833.8568 (Glc-Gal-NeuAc-NeuAc)
0
200
1378.9830
-
1e+4
0
618.5025 (Y0 )
2e+4
1300
581.1588 [(NeuAc-NeuAc)-H20-H]
4e+4
500
1307.9830 [M-3NeuAc-H]- (Y3a')
15 25
F 1250
1000
944.5958 [M-NeuAc-2H]2-
-
GD3
I
581.1648 [(NeuAc-NeuAc)-H20-H]
3e+4
1200
290.0330 [NeuAc-H20-H]- (B1)
1e+3 1150 1500
511.3790 (NeuAc-GalNac)
10 20
1100
951.4966 (41:2) 958.5049 (d42:2) 963.4968 (d43:4)
0
2000
364.2633
2e+3 15
E 1219.7582 (d39:2) 1233.7710(d40:2)
4e+4
290.0530 [NeuAc-H20-H]- (B1)
5 5e+4
1205.7417(d38:2)
0
754.9180 (d39:2) 761.9270 (d40:2)
5e+4
1104.0524 (d42:2)
10 25
747.9118 (d38:2)
2e+3 20
1097.0457 (d41:2)
5 15
1077.0298 (d38:1) 1084.0399 (d39:1) 1090.0372 (d40:2)
10
903.4514 (d34:1)
5e+4
917.4668 (d36:1) 922.4596 (d37:3) 931.4813 (d38:1) 938.4886 (d39:1) 944.4906 (d40:2)
1e+4 1123.6632 (d32:1)
1e+5
1063.0145 (d36:1) 11068.0058(d37:3)
2e+4
1048.9982 (d34:1)
B 2e+5
720.8876 (d34:1)
3e+4
706.8729 (d32:1)
GM3
713.3432(d33:1)
5 2e+5
875.4206(d30:1) 882.4287 (d31:1) 889.4364 (d32:1)
0 Intensity
A
1034.9802 (d32:1)
Intensity
Page 31 of 37 Journal of Agricultural and Food Chemistry
31
1200
1200 1400
1600
2000
Intensity 2e+3
880
G
2e+3
1e+3
3e+3
5e+2
900 920 940 m/z 960 980
0 1000 6e+3
4e+3
2e+3
880 900
8e+3
H
920 940 m/z 960
Figure 4.
ACS Paragon Plus Environment
2e+3
F
0 780
0 0
980 1000
1e+3
800 700
4e+3
3e+3
I
2e+3
1e+3
880
5e+2
720
900 920
2e+3
740
940 m/z 960
1200
760
776.9555 (d42:1)
775.9405 (d42:2)
1150 762.9329 (d40:1)
748.9177 (d34:1)
1100
734.9020 (d36:1)
720.8864(d34:1)
1300
984.5042 (d42:0)* (+2 OH- )
Intensity
1179.7236 (d 36:1)
1151.6928 (d 34:1)
Intensity
1277.7953 (d 43:1)
1261.8014 (d 42:2)
1235.7865 (d 40:1)
1207.7548 (d 38:1)
1263.8134 (d42:1)
1235.7855 (d40.1)
1207.7549 (d38:1)
1195.7212 (d37:0)
1179.7228 (d36:1)
1151.6939 (d34:1)
2e+3
975.5103(d43:0)*
760
783.9372 (d 43:1)
755.9413 (d 42:2)
762.9344 (d 40:1)
8e+3 4e+3
966.5125 (d42:2)*
740
1250 6e+3
954.5034 (d40)* (+ OH-) 958.5150 (d42:2)
720
748.9189 (d 38:1)
0
C
939.4900 (d38:1)* 945.5055 (d40:1)
700
734.9027 (d 36:1)
1e+4
8e+3
925.4748 (d35:2)* 931.4899 (d38:1)
800 2e+4
720.8879 (d 34:1)
Intensity
1263.8160 (d42:1)
1249.8003 (d41:1)
1235.8015 (d40:1)
1221.7835 (d39:1)
1207.7552 (d38:1)
1195.7177 (d37:0)
1165.7112 (d35:1) 1179.7244 (d36:1)
1e+5
903.4505 (d34:1) 911.4588 (d35:0) 917.4762 (d36:1)
790
E 1200
Intensity
Intensity 3e+4
1150
996.5271 (d 44:2)* (+2 OH-) 998.4935 (d 44:0) (+3 OH-)
780
1100
984.4960 (d 42:0)* (+2 OH-)
770
1300
968.5096 (d 41:1)* (+ OH-) 970.4793 (d 40:0)* (+2 OH-)
760
2e+4
958.5052 (d 42:2)
0 4e+4
954.4853 (d 40:0), (+ OH-)
750 1250
6e+4
940.4696 (d 38:0)*
740
8e+4
931.4882 (d 38:1)
730 776.9478 (d42:1)
0
B
917.4733 (d 36:1)
720
1e+5
903.9516 (d 34:1)
710
Intensity
2e+3
-
4e+3 1200
984.4961 (d42:0)* (+2 OH )
6e+3
762.9334 (d40:1)
1150
-
2e+4
962.4841 (d40:0)* (+ OH )
4e+4
1151.6950 (d34:1)
6e+4
-
1100
734.9020 (d36:1)
8e+4
954.4867(d40:0) (+ OH )
D 720.8874 (d34:1)
A
925.4639 (d36:1)*
700
903.4523(d34:1)
intensity 1e+5
911.4483(d35:0) 917.4673 (d36:1)
Intensity
Journal of Agricultural and Food Chemistry Page 32 of 37
32
1e+4
0 1250 1300
0 780
980
800
1000
Intensity 80
60
40
20
200 400 600 800
0 1000 1200 400
200
1200
2 D. MS of GM3 m/z 1249.7 (d41:1) from beef 140 200 400 600 800 1000 2 F. MS of GD3 m/z 762.8 (d40:1) from beef
120
100
80
60
40
20
200 400
m/z
Figure 5.
ACS Paragon Plus Environment 600 800
m/z 1000
-
600
1235.7495 [M-NeuAc-H] (Y3)
800
-
1600 -
1179.7722 [M-NeuAc-H] (Y3)
888.4603 [M-2NeuAc-H] (Y2)
-
735.0123 [M-2H]
2-
1800
944.5053 [M-2NeuAc-H] (Y2)
1000
2-
1200
-
-
-
1151.7078 [M-NeuAc-H] (Y3)
-
2-
860.5734 [M-2NeuAc-H] (Y2)
721.0252 [M-2H]
581.0749 [2NeuAc-H]
536.7307 (Y0)
20
762.8301 [M-2H]
1400 581.1108 [2NeuAc-H]
1200
290.1490 [NeuAc-H]
40
732.3531
0 60
-
0 80
290.1490 [NeuAc-H]
Intensity
100
-
-
1179.7789 [M-H]
120
-
1000 Intensity
-
140
581.1428 [2NeuAc-H] 620.3694 (Y0)
800 1151.4955 [M-H]
888.6679 [M-NeuAc-H] ( Y2)
726.8030 [M-Hex-NeuAc-H] (Y1)
564.6516 [M-2Hex-NeuAc-H] (Y0)
336.5716
-
2 A. MS of GM3 m/z 1179.8 (d36:1) from pork
290.1257 [NeuAc-H]
600
-
400 -
200 400 600 800 1000 2 C. MS of GM3 m/z 1151.5 (d34:1) from chicken
Intensity
200
-
200 860.4360 [M-NeuAc-H] ( Y2)
1200
1249.6851 [M-H]
400
-
600
958.6159 [M-NeuAc-H] ( Y2)
800
698.3844 [M-Hex-NeuAc-H] (Y1)
10
536.4629 [M-2hex-NeuAc-H] (Y0)
20
408.1899
30
290.0682 [NeuAc-H] (B1)
40
778.7771 [M-Hex-NeuAc-H20-H] (Y1)
100 -
1000
290.0282 [NeuAc-H] (B1)
50
634.6345 [M-2Hex-NeuAc-H] (Y0)
0
-
Intensity 60
290.1127 [NeuAc-H] (B1)
Intensity
Page 33 of 37 Journal of Agricultural and Food Chemistry
33
2 B. MS of GD3 m/z 721.0 (d34:1) from pork
0 200 400 600 800 1000 2 D. MS of GD3 m/z 735.0 (d36:1) from chicken 1200
0 1200
1200
1400
0 1400
200
150
100
50
0 200 400
250
600
0 800 1000 1200 1400 1600 m/z 50
30
20
10
0 200 400 600
40
800 1000
747.4907
1200
1200 1400
Intensity
1336.5126 (Y2α')
970.3803 (Y2β - B1α')
0
1600
40
20
1400
D. MS2 for GD1b m/z 925.55 (d36:1) from beef d 7
0
0 200
200
6
5
4
3
2
1 400
m/z
Figure 6.
ACS Paragon Plus Environment
400
600
600
800
800
m/z
1677.6537 [M-NeuAc-2H]
1662.9182 [M-NeuGc-H]
1371.1302 [M-(NeuAc+NeuGc)-H]
0
2984.8369 [M-2H] 1122.4702
655.1985 (NeuAc-Gal-GalNAc) 725.2111753.0759 831.4360
306.0586 (NeuGc)
290.0526 (NeuAc)
80
-
1000
2 E. MS for GD1a m/z 984.5 (d42:0) beef
e
1678.8748 [M-NeuAc-H]
800
60
2 F. MSf for GD1b m/z 984.5 (d42:0) beef 902.4890 951.7296 2984.4490 [M-2H] 1030.8717
600
896.4900 940.4160 [M-2H-H20]2958.5100 [M-2H]2-
100
615.7296 (NeuAc+NeuGc+H20)
400
597.2180 (NeuAc+NeuGc)
200
563.4926 (B1α' - H 0) 2 580.9960 (B1α')
C. MS2 for GD1b m/z 958.5 (d42:2) from pork
c
Intensity
0
1398.9877 (Y3β − NeuAc)
1200
365.2612 (Gal-GalNAc)
290.1149 (NeuAc)
10
1542.5532 (Y2α'' -H20) 1561.6600 (Y2α'')
1253.3721 (Y2β ) Intensity
1181.9064
20
1253.8434 (Y2α)
B. MS2 for GD1a m/z 998.5 (d47:5) from Chicken b 30
925.3836 [M-2H]2-
1000
40
705.4937
800
50
597.0471 (B1α')
0
306.1629 (B1α'')
600
Intensity
400 936.4963 [M-2H-H20]2954.4929 [M-2H]2-
25
1706.9444 [M-NeuAc-H]-
200 709.0954 726.0886 (Y1-2H) 729.2063 (Y1) 790.3869
A. MS2 for GD1a m/z 954.5 (d40:0) from chicken
a
1483.8480 1544.9579 (Y3β )
5
1050.8145 (Y2β − NeuAC)
10
888.3132 (Y1) 954.3987 976.8137 998.6851 [M-2H]2-
15
290.2999 (NeuAc)
20
589.5417
Intensity 30
290.1656 (NeuAc)
300
187.1945
Intensity
Journal of Agricultural and Food Chemistry Page 34 of 37
34
1000 1200 1400 1600
1000 1200 1400 1600 1800 2000
Intensity 5e+2
1020
3e+2
2e+2
0
1040 1060 1080 940
1100
m/z 960
6e+2
J
4e+2
3e+2
1120 980
2e+2
1e+2
0 1140 1160 1180 5e+3
1000
1020
1e+4
8e+3
6e+3
720
4e+3
H
3e+3
920 940
1040
740
2e+3
1060
760
960
2e+3
K
1e+3
1080
780
980 1000
5e+2
0 1100 1120 1140
Intensity
4e+3
0
6e+3
3e+3
F
2e+3
800 700
1e+4
1020 840
2e+3
1e+3
8e+2
4e+2
m/z 1300
Figure 7.
ACS Paragon Plus Environment 860
734.9089 (d36:1)
1e+3
720
880
1350
4e+3
2e+3
740
I
9e+3
6e+3
2e+3 900 920
L
1400 m/z
940
775.9476 (d42:2)
1200
769.8797(d41:1)
1150
761.9319(d40:2) 762.9381 (d40:1)
739.9001(d37:3)
1e+4 720.8928 (d34:1)
1100
943.4044 (d39:4)
E 1300
916.4761 (d35:3)
700
1250
Intensity
1261.8010 (d42:2)
Intensity
1271.7871 (d43:4) 1279.8114 (d43:0)
760
960
1450
1261.8115 (d42:2)
1249.8129 (d41:1)
1233.7808 (d40:2) 1235.7963 (d40:1)
1189.7185 (d37:3)
1179.7334 (d36:1)
1151.7025 (d34:1)
1123.6700 (d32:1)
2e+3
1482.9002 (d43:0)
G 800
1200 4e+3
1464.8904 (d42:2) 1466.9030 (d42:1)
4e+2 780
0
6e+3
1436.8579 (d40:2) 1438.8738 (d40:1)
6e+2 5e+3
C
1392.7975 (d37:3)
1e+3
0 920 760
1e+4
8e+3
1354.7798 (d34:1)
1e+2
900 740
2e+4
1326.7529 (d32:1)
0 1150
Intensity
720 1e+4 1104.5553 (d42:2)
6e+3
B
Intensity
1e+3 1100
979.5418(d45:2)
2e+3 1300
958.5046 (d42:2)
3e+3 1250
Intensity
0
963.4976 (d43:4)
4e+3 2e+4
1104.5553 (d42:2)
880 1200 1261.8067 (d42:2)
A
Intensity
700 1233.7747 (d40:2)
1e+4
Intensity
1150
790.9540 (d44:1)
1100
1141.5778 (d47:0) 1147.0700 (d48:1)
5e+2
D 761.9477d40:2)
2e+3 1151.7000 (d34:1)
4e+3
766.9401 (d41:4)
Intensity 6e+3
958.5095 (d42:2) 963.5026 (d43:4)
5e+3
956.4958 (d42:4)
Intensity 8e+3
1139.0653 (d47:2)
Intensity
Page 35 of 37 Journal of Agricultural and Food Chemistry
35
1e+4
0 1250 1300
0
2e+4
780 800
3e+3
0 980 1000
0 1500
Journal of Agricultural and Food Chemistry
Page 36 of 37
36
For Table of Contents Only
ACS Paragon Plus Environment
Page 37 of 37
Journal of Agricultural and Food Chemistry
80x45mm (298 x 298 DPI)
ACS Paragon Plus Environment