Sample Extraction. Samples were extracted following the Garret Lab acid hydrolyzed fatty acid extraction procedure (Ulmer, et al., 2015). Briefly, approximately 10 mg of each sample was weighed out into clean glass tubes. 100 µl acetonitrile with 100 mg/L BHT was added to each sample followed by 50 µL of 37% (v/v) hydrochloric acid. Samples were mixed on a vortex mixer and sonicated for 5 min. Samples were heated at 80-90°C for 2 h in a heating block. After allowing samples to cool down to room temperature, 200 µl hexane was added, vortexed, and incubated at 4° C for 10 min. Sample were centrifuged at 3260 x g for 5 min at room temperature. 150 µl of the hexane layer was transferred into a clean tube for drying under nitrogen. Samples were reconstituted with 50 µl of 80:20 acetonitrile: 5 mm ammonium acetate (v:v) and transferred into clean LC vials. 1µl of fatty acid internal standard mixture was spiked into each sample and mixed well.

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LC-MS Analysis. Total fatty acid profiling was performed on a Thermo Q-Exactive Orbitrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on a Waters Acquity UPLC HSS T3 150 x 2.1 mm, 1.8 µm column with mobile phase A as 1 mm ammonium acetate and mobile phase B as acetonitrile with 0.1% acetic acid. The flow rate was 500 µL/min with a column temperature of 30° C. Samples were injected at 4 µL.

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Results

Our laboratory received one excel file for the targeted analysis that contained quantitative & qualitative data. Fatty acid peak identification was done by SECIM personnel using the Garret Metabolite Standard library. Quantitative data included the peak area, the percentage of fatty acid (FA) peak area, moles present, and the percentage of moles.

In the targeted analysis, all fatty acid peaks present in the pig and ding samples were identified. Saturated fatty acid peaks matched for fatty acids C7-C9, C11-C18, and C22. Caproic acid (C6), found naturally in animal fats, was not present in the ding sample, and capric acid (C10), found in the milk of various mammals, was absent in both samples.

Of specific interest was the relative proportion of palmitic acid (C16) to stearic acid (C18) in the two samples (Table 1). According to Evershed, et al., (1997, 2002), the relative proportion of C16 is higher than C18 in mongastric animals, i.e., pigs, a finding that led to the identification of porcine fats in dripping dishes. In addition, the unsaturated fatty acid (see below), oleic or octadecenoic acid (C18.1), in its single isomer ω-9 configuration, is consistent with an origin from monogastric animals (Evershed, et al., 1997).

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Table 1. Amount* and percent of palmitic and stearic acid in the pig and ding sherd samples.

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Matched unsaturated fatty acids in the cooked pig bone and ding samples came from the omega 3, 5, 7, 9, and 11 groups.

• ω-3: α-Linolenic (18.3), Steariodonic (18.4), Eicosapentanoic (20.5), Docosa-pentanoic (22.5), Docosahexanoic (22.6)

• ω-6: Linolenic (18.2), γ-Linolenic (18.3), Arachidonic (20.4), Docosapentanoic (22.5), Adrenic (22.4)

• ω-7: Palmitoleic (16.1)

• ω-9: Oleic (18.1), Gondoic (20.1), Eicosatrienoic (20.3), Erucic (22.1), Nervonic (24.1)

• ω-11: Gadoleic (20.1)

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