Department of Animal and Food Sciences, Oklahoma State University, Stillwater, OK 74078, USA.
J Anim Sci. 2022 Aug 1;100(8). doi: 10.1093/jas/skac207.
Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ionization detector (FID). Aqueous samples are highly problematic because of the large vapor volume that can lead to poor peak shape and contamination of inlets, potentially causing sample carryover. Methods using aqueous samples are not well suited for use in a mass spectrometer (MS) detector system. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 µL of sample, KHSO4 (500 g/L), and 2-ethylbutyrate (internal standard; 8.5 mM) were added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture was thoroughly vortexed and centrifuged. The organic layer (top) was removed and placed in a GC vial. The DMC extract was injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105 °C for 5 min, increased at 10 °C/min to 150 °C, then 65 °C/min to 240 °C, and held constant for 10 min. The peak area of acetate relative to the internal standard is linear from approximately 2 mM to at least 130 mM and encompasses the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P > 0.10) or the amount of VFA spiked (P > 0.19) for acetate, propionate, isobutyrate, or butyrate. There was an interaction of amount of VFA spiked and the diet of the animal (P = 0.021) for valerate and a tendency for an interaction (P = 0.051) for isovalerate, due to the recovery of the VFA being lower in the medium spike amount in rumen fluid from cattle on wheat pasture. Overall, recovery was greatest for propionate (101.9 ± 1.67%) and lowest for valerate (95.7 ± 1.95%). Including the 10-min hold at 240 °C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantifies rumen fluid VFA.
瘤胃液挥发性脂肪酸（VFA）的分析通常通过将酸化的瘤胃液注入带有火焰电离检测器（FID）的气相色谱仪（GC）来进行。由于水蒸气的体积较大，水相样品会带来很多问题，可能导致峰形不佳和进样口污染，从而导致样品残留。使用水相样品的方法不适合用于质谱仪（MS）检测器系统。本项目的目的是验证一种用于瘤胃 VFA 分析的碳酸二甲酯（DMC）提取工艺和 GCMS 方法。为了进行提取，将 100 µL 样品、KHSO4（500 g/L）和 2-乙基丁酸（内标；8.5 mM）按顺序加入微量离心管中，然后加入 1 mL DMC。将混合物彻底涡旋并离心。去除有机层（顶部）并将其放入 GC 小瓶中。将 DMC 提取物（0.5 µL）注入配备极性 DB-FFAP 柱的安捷伦 5977B GCMS（8:1 分流注入）中。柱温保持在 105°C 5 分钟，以 10°C/min 升温至 150°C，然后以 65°C/min 升温至 240°C，保持 10 分钟不变。乙酸盐相对于内标物的峰面积与预期的瘤胃液浓度呈线性关系，范围约为 2 mM 至至少 130 mM，涵盖了其他 VFA 的预期值。在以精饲料或放牧小麦牧场饲养的牛的瘤胃液中，以三种浓度测试了从添加的瘤胃液中回收 VFA 的情况。回收率不受动物饮食（P > 0.10）或添加 VFA 量（P > 0.19）的影响，用于乙酸盐、丙酸盐、异丁酸和丁酸盐。由于在以小麦牧场饲养的牛的瘤胃液中，中等添加量的 VFA 回收率较低，因此存在添加的 VFA 量和动物饮食之间的相互作用（P = 0.021），以及异戊酸盐的相互作用趋势（P = 0.051）。总体而言，丙酸盐的回收率最高（101.9 ± 1.67%），戊酸盐的回收率最低（95.7 ± 1.95%）。在每次运行结束时，在 240°C 下保持 10 分钟可以防止样品之间的残留。该方法似乎在 GCMS 系统中表现良好，能够准确、精确地定量瘤胃液 VFA。
