高效液相色谱法测定猪油甘油三酯中的脂肪酸位置分布

Analysis of Positional Distribution of Fatty Acids in Triacylgly-cerols from Lard by High Performance Liquid Chromatography

建立了一种采用高效液相色谱法(HPLC)分析猪油甘油三酯中的脂肪酸组成及其位置分布的方法。利用sn 1,3位专一性脂肪酶对甘油三酯sn 1,3位上的脂肪酸进行水解,形成sn 2位甘油单酯和游离脂肪酸;之后,通过甘油三酯中脂肪酸总含量和sn 1,3位上脂肪酸含量之间的差值计算出sn 2位上的脂肪酸含量。利用 2 溴苯乙酮仅同游离脂肪酸作用的特点,将脂肪酸酯化为苯乙酰甲酯,然后进行HPLC分析。分析所用色谱柱为ZORBAXSBC18柱,以十七酸作为内标,甲醇 乙腈 水为流动相,采用梯度洗脱 (梯度洗脱程序为甲醇 乙腈 水由 80∶10∶10(体积比,下同)在 35min内线性变化到 86∶10∶4,然后在 5min内恢复到起始比例,流动相流速为 1mL/min),通过测定苯乙酰甲酯在 254nm处的吸光度值来测定脂肪酸含量。结果表明,猪油甘油三酯中的脂肪酸主要是棕榈酸和油酸(分别占总量的 26 61%和 43 18% ),其中油酸主要分布于sn 1,3位上,而棕榈酸分布于sn 2位上。这些测定结果与传统气相色谱法的测定结果相吻合。该方法简单可行,省去了传统测定中费时的薄层色谱分离步骤,可成为一种有效的实验室分析方法。

A high performance liquid chromatographic (HPLC) method for the determination of fatty acid positional distribution on triacylglycerols from lard has been established. The analysis was carried out with an sn-1, 3 specific lipase to deacylate the fatty acid residues esterified at sn-1 and sn-3 positions of triacylglycerols, forming sn-2 monoglycerides and free fatty acids. After lipase action, it was possible to determine the fatty acid esterified at the sn-2 position by the substraction of the results of the (sn-1,3) analysis from an overall composition analysis based on complete saponification of the original sample. The free fatty acid mixtures were converted to phenacyl esters via 2-bromoacetophenone and analyzed on an Agilent 1100 series high performance liquid chromatograph, equipped with a ZORBAX SB C18 column (4.6 mm i.d.×250 mm, 5 μm) and a UV detector. Elution was performed at a flow rate of 1 (mL/min), with a gradient of methanol-acetonitrile-water starting at 80∶10∶10 (v/v), increasing linearly to 86∶10∶4 (v/v) in 35 min, then returning to the initial conditions in 5 min. Elution of phenacyl esters was monitored by absorbance at 254 nm. Pentadecanoic acid was used to be an internal standard. The results showed that palmitic and oleic acids were the major components in lard, (accounting) for 26.61% and 43.18% respectively. Among these, palmitic acid mainly locates at sn-2 position, while oleic acid at sn-1,3 positions. These results were consistent with those obtained via gas chromatographic method. The new method is simpler and easier to use as it eliminates time-consuming thinner-layer chromatography used in standard regioselective analysis. It is possible to be an effective laboratory analytical method.