AOACSPIFANMethods-2017Awards

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212 G olay & M oulin : J ournal of AOAC I nternational V ol . 99, N o . 1, 2016

( 3 ) Capillary column .—Cyanopropyl-polysiloxane phase (or equivalent polarity) capillary columns with 100 m length × 0.25 mm id, 0.2 μm film thickness. Note : Traces of oxygen and humidity will damage the polar phase of the column. When pure gas is not available, use a gas purifying filter device.

omega-6, and omega-9] in milk products, infant formula, and adult/pediatric nutritional formula containing milk fat and/or vegetable oils, supplemented or not supplemented with oils rich in long-chain PUFAs. The determination is performed by direct transesterification of food samples without prior fat extraction and, consequently, it is applicable to liquid samples or reconstituted powder. Products containing <1.5% fat can be analyzed after preliminary fat extraction using a suitable fat-extraction reference method (i.e., ISO/IDF-AOAC). In the case of products supplemented or enriched with PUFAs having fish-oil or algae origins, the extraction solvents must be evaporated at a maximum of 40°C. Addition of the internal standard solution to the sample, preparation of FAMEs by direct transesterification with methanolic sodium methoxide for liquid samples and fat extracted from food; dissolution (i.e., reconstitution) in water for powder sample and direct transesterification with methanolic sodium methoxide. Separation of FAMEs using capillary GLC. Identification of peak by comparison with the retention time of pure standards and quantification as fatty acids by reference to an internal standard (C11:0 FAME) and instrument response factors. Verification of the transesterification performance using a second internal standard [C13:0 triacylglycerols (TAG)]. Common laboratory equipment and, in particular, the following: (a)  Analytical balance .—Capable of weighing to the nearest 1 mg, with a readability of 0.1 mg. (b)  One-mark volumetric flasks .—50, 100, 250, 300, and 500 mL. (c)  One-mark volumetric pipets .—2, 5, 10, 25, and 50 mL; class AS (ISO). (d)  Two-mark pipet, volumetric.— 2 and 5 mL; class AS (ISO). (e)  Micropipet .—200 μL. (f)  Dispensers .—2, 5, and 10 mL. (g)  Test tube .—26 mm (diameter) × 100 mm (length), fitted with PTFE-lined screw cap. (h)  Test tube mixer .—Vortex Genie Scientific Industries, Inc., Bohemia, NY, or equivalent. (i)  Laboratory centrifuge .—Equipped with adapters for test tubes with external diameter of 26 mm. (j)  Gas–liquid chromatograph .—Equipped with flame ionization detector and capillary split-injection system or on-column. Autosampler and integration system preferably computerized. Note : Use of the cleanest possible glassware and caps is required to avoid impurities in the FAME chromatogram. ( 1 ) Carrier gas.— Hydrogen or helium. Purity ≥99.9997%. Note : The use of hydrogen or helium affects principally the chromatography duration but does not have any significant impact on the chromatographic resolution. ( 2 ) Other gases .—Free from organic impurities (C n H m <1 ppm), nitrogen and hydrogen, purity at least ≥99.995%, and compressed pure air. B. Principle C. Apparatus and Materials

D. Chemicals and Reagents

Use only reagents of recognized analytical grade, unless otherwise specified. (a)  Water .—HPLC grade or equivalent quality. (b)  Sodium methoxide solution (CH 3 ONa) .—Dissolved in methanol 30% (w/v; ca 5.4 M). (c)  Transesterification solution .—Sodium methoxide solution 5% in methanol. Into a 300 mL volumetric flask, pipet 50 mL sodium methoxide solution, D ( b ), and complete gently with 250 mL methanol using a magnetic stirrer. Remove the magnetic stirrer, then cool to room temperature, and dilute to the mark with methanol. Stored in the dark at 4°C, this solution is stable for 1 week. Allow the solution to come to room temperature before use. Perform the transesterification reaction at ambient temperature (20–25°C). (d)  Disodium hydrogen citrate sesquihydrate [HOC(COOH) (CH 2 COONa) 2 ·1.5H 2 O]. (e)  Sodium chloride (NaCl). —Puriss. (f)  Neutralization solution .—Disodium hydrogen citrate sesquihydrate 10%, sodium chloride 15% in water. Weigh 50.0 g disodium hydrogen citrate sesquihydrate, D ( d ), and 75.0 g sodium chloride, D ( e ), in a 500 mL volumetric flask, C ( b ). Dissolve in 450 mL water using a magnetic stirrer. Remove the magnetic stirrer, and dilute to volume with water. Stored in the dark at 4°C, this solution is stable for 1 month. Salt crystals may appear in the solution during storage but disappear after shaking. Allow the solution to come to room temperature before use. (g)  tert-Butyl methyl ether (MTBE) . (h)  Methyl undecanoate (C11:0 FAME) .—Purity ≥99% mass fraction. (i)  Tritridecanoin (C13:0 TAG) .—Purity ≥99% mass fraction. (j)  C11:0 FAME/C13:0 TAG standard solution.— Into a 250 mL volumetric flask, weigh to the nearest 0.1 mg about 500 mg tritridecanoin, D ( i ), and 500 mg methyl undecanoate, D ( h ). Dissolve and dilute to the mark with MTBE. Stored in the dark at 4°C, this solution is stable for 1 wk. Allow the solution to come to room temperature before use. (k)  Octadecenoic acid methyl ester .— Cis/trans isomer mixture of C18:1 with trans -4 to trans -16 (all isomers) and principal cis isomers. Concentration 2.5 mg/mL in methylene chloride. Note : This standard is commercially available from the Supelco brand of Sigma-Aldrich St. Louis, MO (Cat. No. 40495-U). (l)  Linoleic acid methyl ester .— Cis/trans isomer mixture of C18:2 with trans -9, trans -12-octadecadienoic acid (50%); cis -9, trans -12-octadecadienoic acid (20%); trans -9, cis -12- octadecadienoic acid (20%); and cis -9, cis -12-octadecadienoic acid (10%). Concentration 10 mg/mL in methylene chloride. Note : This standard is commercially available from the Supelco brand of Sigma-Aldrich (Cat. No. 47791).