Pinho O, Ferreira I M P L V O, Ferreira M A
CEQUP/Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, Portugal.
Anal Chem. 2002 Oct 15;74(20):5199-204. doi: 10.1021/ac020296m.
This work describes a method for quantification of the major free fatty acids of ewe cheese that contribute to its distinct and strongly marked flavor. A headspace SPME method in combination with GC/MS was used for the extraction, identification, and quantification of butanoic, hexanoic, octanoic and decanoic acids in ewe cheeses. The method used for sample preparation was simple. A fiber coated with 85-microm polyacrylate film was chosen to extract the free fatty acids. To perform a reliable quantification, several factors were taken into consideration for reliable quantification, namely, (i) the influence of addition of water, of an electrolyte or of a hygroscopic salt, on the release of free fatty acids from the matrix; (ii) the linear relationship between the amount of analyte adsorbed by the SPME polymer film and the initial concentration of the analyte in the cheese sample; and (iii) the competition for adsorption by fiber. Water removal with sodium sulfate promoted a more efficient extraction of volatile free fatty acids; biases due to competition or linear range excesses were controlled by choosing the appropriate amount of sample for each ewe cheese. The method of standard additions was used with success for the quantification of free fatty acids. Calibration curves that were constructed for the major short-chain free fatty acids (butanoic, hexanoic, octanoic, and decanoic acids) spiked into cheese followed linear relationships with highly significant (p < 0.001) correlation coefficients (r > 0.999). Coefficients of variation of <7.9% indicated that the technique was reproducible. A marked increase in concentration of short-chain free fatty acids was observed during cheese ripening, ranging from 0.35 to 9.33 mg/100 g for butanoic acid, 0.363 to 4.34 mg/100 g for hexanoic acid, 0.343 to 2.0 mg/100 g for octanoic acid, and 1.291 to 3.85 mg/100 g for decanoic acid. The limits of quantification were registered at levels of parts per million. The absolute quantification of butanoic acid was also carried out by using isotope dilution assays (IDA). The levels of acid obtained with this method were similar to those obtained by the standard additions method.
本研究描述了一种定量分析母羊奶酪中主要游离脂肪酸的方法，这些游离脂肪酸赋予了母羊奶酪独特且浓郁的风味。采用顶空固相微萃取（SPME）结合气相色谱/质谱联用（GC/MS）的方法，对母羊奶酪中的丁酸、己酸、辛酸和癸酸进行提取、鉴定和定量分析。所采用的样品制备方法简单。选用涂覆有85微米聚丙烯酸酯薄膜的纤维来萃取游离脂肪酸。为了进行可靠的定量分析，考虑了几个因素以确保可靠的定量，即：（i）添加水、电解质或吸湿盐对游离脂肪酸从基质中释放的影响；（ii）SPME聚合物膜吸附的分析物量与奶酪样品中分析物初始浓度之间的线性关系；以及（iii）纤维吸附的竞争情况。用硫酸钠去除水分可促进挥发性游离脂肪酸的更有效萃取；通过为每种母羊奶酪选择合适的样品量，可控制竞争或线性范围过量导致的偏差。标准加入法成功用于游离脂肪酸的定量分析。针对添加到奶酪中的主要短链游离脂肪酸（丁酸、己酸、辛酸和癸酸）构建的校准曲线呈线性关系，相关系数高度显著（p < 0.001）（r > 0.999）。变异系数<7.9%表明该技术具有可重复性。在奶酪成熟过程中，观察到短链游离脂肪酸浓度显著增加，丁酸浓度范围为0.35至9.33毫克/100克，己酸浓度范围为0.363至4.34毫克/100克，辛酸浓度范围为0.343至2.0毫克/100克，癸酸浓度范围为1.291至3.85毫克/100克。定量限为百万分之一水平。还通过同位素稀释分析法（IDA）对丁酸进行了绝对定量分析。用该方法获得的酸水平与标准加入法获得的结果相似。
