Aripovsky A V, Kolesnik P O, Kulagina T P, Titov V N
State Scientific Center of Applied Microbiology and Biotechnology, Obolensk town, Moscow region, Russian Federation, 142279.
Uzhgorod National University, Uzhgorod, Transcarpathia, Ukraine, 88000.
Klin Lab Diagn. 2018;63(3):141-147. doi: 10.18821/0869-2084-2018-63-3-141-147.
Different methods of sample preparation and derivatization were compared from the point of view of product yield, speed and convenience of the technique used. Fatty acid determination in absolutely dry objects (biochemical preparations, food protein isolates, lyophilized microbial biomass) may be performed easily with the use of Folch method provided that 4-component system "chloroform/ methanol/water/acetic acid" was employed. Nevertheless, we ould not find any real advantages of classical Folch or Hara-Radin extraction method variants when compared to simple non-extraction technique (which consists in direct trans-esterification of dried biomaterial due to sequential sample treating with sodium methoxide and boron trifluoride methanolic solutions). The letter method, being completely universal, provides considerable increase of fatty esters yield, sample preparation is noticeably simplified and accelerated (becoming much more economical). It's "dry blood spot" variant (using cellulose or, preferably, fluoroplast filter paper disks) seems to be extremely convenient for laboratory routine analysis of liquid biological samples, allowing to exclude not only their liquid-liquid extraction but also the stage of vacuum drying. Unlike the methods of Folch and Hara-Radin, the non-extraction method does not necessarily require the homogenization of the biological material, that is, it's grinding to fragments of micron size. Direct derivatization method provides noticeably better parameters of fatty acids yield even for relatively large particles - 0.2-1.0 mm - of the test material (in comparison with those parameters observed upon extraction of micron size homogenizates by the Folch method in its most advanced modifications).
从产物产率、所使用技术的速度和便利性的角度,对不同的样品制备和衍生化方法进行了比较。对于绝对干燥的物体(生化制剂、食品蛋白分离物、冻干的微生物生物质)中的脂肪酸测定,只要采用“氯仿/甲醇/水/乙酸”四组分体系,使用Folch方法就可以轻松完成。然而,与简单的非萃取技术(该技术通过用甲醇钠和三氟化硼甲醇溶液依次处理样品,对干燥的生物材料进行直接酯交换)相比,我们没有发现经典的Folch或Hara-Radin萃取方法变体有任何实际优势。后一种方法完全通用,能显著提高脂肪酸酯的产率,样品制备明显简化且加速(变得更经济)。其“干血斑”变体(使用纤维素或,优选氟塑料滤纸片)对于液体生物样品的实验室常规分析似乎极其方便,不仅可以排除液-液萃取,还可以省去真空干燥步骤。与Folch和Hara-Radin方法不同,非萃取方法不一定需要生物材料的匀浆,即磨碎至微米大小的碎片。即使对于测试材料中相对较大的颗粒 - 0.2 - 1.0毫米 - 直接衍生化方法也能提供明显更好的脂肪酸产率参数(与在最先进的改进方法中用Folch方法萃取微米大小的匀浆时观察到的参数相比)。