Nondestructive quantitative determination of docosahexaenoic acid and n−3 fatty acids in fish oils by high‐resolution 1H nuclear magnetic resonance spectroscopy

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Abstract
By using a 500 MHz proton nuclear magnetic resonance (1H NMR) spectrometer we have developed a quantitative method for determining the contents of docosahexaenoic acid (DHA) in fish oils (mg/g), the molar proportions (mol%) of DHA to all other fatty acids composing the fish oils, and the molar proportions of total n‐3 fatty acids to all other non‐n‐3 fatty acids in the fish oils. After examining the suitability of ethylene glycol dimethyl ether (EGDM), methanol, and 1,4‐dioxane as internal standards, experimental conditions were optimized by mainly using EGDM as an internal standard. By setting the pulse repetition time at 30 s, five times longer than the longest T1 of the 1H NMR signals of fish oils, good reproductibility of data and analytical times less than 10 min were achieved. The use of the internal standard also allowed us to quantify DHA on a weight basis (mg/g). Verification of the method was carried out in an interlaboratory study between Japan and Norway on bonito, tuna, and salmon oils. The relative errors in the 1H NMR data between Japan and Norway were 0.57–5.29% for quantification of DHA, 0.7–2.09% for the molar proportion of DHA, and 0.1–1.41% for the molar proportion of total n‐3 fatty acids. Good agreement was observed between the 1H NMR data and those obtained by gas chromatography (GC). The sample preparation before 1H NMR measurements required only two steps: sample weighing and preparation of an internal standard solution. Based on the high reproducibility, simplicity of the procedure, and clarity of principle, the proposed 1H NMR method was judged to be a promising alternative to the GC method in quantification of DHA and n‐3 fatty acids in fish oils.