Carvalho Ana P, Malcata F Xavier
Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal.
J Agric Food Chem. 2005 Jun 29;53(13):5049-59. doi: 10.1021/jf048788i.
Assays for fatty acid composition in biological materials are commonly carried out by gas chromatography, after conversion of the lipid material into the corresponding methyl esters (FAME) via suitable derivatization reactions. Quantitative derivatization depends on the type of catalyst and processing conditions employed, as well as the solubility of said sample in the reaction medium. Most literature pertinent to derivatization has focused on differential comparison between alternative methods; although useful to find out the best method for a particular sample, additional studies on factors that may affect each step of FAME preparation are urged. In this work, the influence of various parameters in each step of derivatization reactions was studied, using both cod liver oil and microalgal biomass as model systems. The accuracies of said methodologies were tested via comparison with the AOCS standard method, whereas their reproducibility was assessed by analysis of variance of (replicated) data. Alkaline catalysts generated lower levels of long-chain unsaturated FAME than acidic ones. Among these, acetyl chloride and BF(3) were statistically equivalent to each other. The standard method, which involves alkaline treatment of samples before acidic methylation with BF(3), provided equivalent results when compared with acidic methylation with BF(3) alone. Polarity of the reaction medium was found to be of the utmost importance in the process: intermediate values of polarity [e.g., obtained by a 1:1 (v/v) mixture of methanol with diethyl ether or toluene] provided amounts of extracted polyunsaturated fatty acids statistically higher than those obtained via the standard method.
生物材料中脂肪酸组成的测定通常通过气相色谱法进行，即先通过适当的衍生化反应将脂质材料转化为相应的甲酯（FAME）。定量衍生化取决于所使用的催化剂类型和处理条件，以及所述样品在反应介质中的溶解度。大多数与衍生化相关的文献都集中在替代方法之间的差异比较上；尽管有助于找出针对特定样品的最佳方法，但仍迫切需要对可能影响FAME制备各步骤的因素进行更多研究。在这项工作中，以鱼肝油和微藻生物质为模型系统，研究了衍生化反应各步骤中各种参数的影响。通过与AOCS标准方法比较来测试所述方法的准确性，而通过（重复）数据分析的方差来评估其重现性。碱性催化剂产生的长链不饱和FAME水平低于酸性催化剂。其中，乙酰氯和BF₃在统计学上彼此等效。与单独使用BF₃进行酸性甲基化相比，在酸性甲基化之前对样品进行碱性处理的标准方法提供了等效的结果。发现反应介质的极性在该过程中至关重要：中等极性值[例如，通过甲醇与乙醚或甲苯的1:1（v/v）混合物获得]提供的提取多不饱和脂肪酸量在统计学上高于通过标准方法获得的量。
