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.

中文翻译：

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技术说明：使用碳酸二甲酯萃取的气相色谱质谱法分析瘤胃液中的挥发性脂肪酸

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配备火焰离子化检测器 (FID) 的气相色谱仪 (GC) 来进行。水相样品存在很大问题，因为蒸汽体积大，可能导致峰形不佳和进样口污染，可能导致样品残留。使用水性样品的方法不太适合在质谱仪 (MS) 检测器系统中使用。该项目的目标是验证用于瘤胃 VFA 分析的碳酸二甲酯 (DMC) 提取工艺和 GCMS 方法。为进行提取，将 100 µL 样品、KHSO4 (500 g/L) 和 2-乙基丁酸（内标；8.5 mM）添加到微量离心管中（按顺序），然​​后添加 1 mL DMC。将混合物彻底涡旋并离心。移除有机层（顶部）并置于 GC 小瓶中。将 DMC 提取物注入 (0.5 µL) 到配备极性 DB-FFAP 色谱柱的 Agilent 5977B GCMS（8:1 分流进样）中。该柱在 105°C 下保持 5 分钟，以 10°C/分钟的速度升至 150°C，然后以 65°C/分钟的速度升至 240°C，并保持恒定 10 分钟。醋酸盐相对于内标的峰面积从大约 2 mM 到至少 130 mM 是线性的，并且包含其他 VFA 的瘤胃浓度预期值。在饲喂肥育日粮或放牧小麦牧场的阉牛的瘤胃液中，以三种浓度测试了加标瘤胃液中 VFA 的回收率。回收率不受动物饮食 (P > 0.10) 或醋酸盐、丙酸盐、异丁酸盐或丁酸盐的 VFA 掺入量 (P > 0.19) 的影响。VFA 加标量与动物饮食之间存在交互作用 (P = 0.021) 戊酸和异戊酸交互趋势 (P = 0.051)，因为 VFA 的回收率在中等加标量下较低在小麦牧场上的牛瘤胃液中。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%)。包括在每次运行结束时在 240°C 下保持 10 分钟可防止样品间的残留。这种方法似乎在 GCMS 系统中表现良好，并准确而精确地量化瘤胃液 VFA。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%)。包括在每次运行结束时在 240°C 下保持 10 分钟可防止样品间的残留。这种方法似乎在 GCMS 系统中表现良好，并准确而精确地量化瘤胃液 VFA。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%)。包括在每次运行结束时在 240°C 下保持 10 分钟可防止样品间的残留。这种方法似乎在 GCMS 系统中表现良好，并准确而精确地量化瘤胃液 VFA。