A headspace-gas chromatograph (HS-GC) (external standard method) was established for the determination of the volatile fatty acid (VFA) contents in rumen fluid of ruminants. Under the optimized headspace sampling conditions (balance temperature at 85 ℃ and equilibrium time of 30 min), the method was verified and used to detect the VFA. The coefficient of determination of regression equation for each compound was not less than 0.999 7. The limit of detection (LOD) was 0.036-0.329 mmol/L. The recovery rates of three different concentrations of standards were 93.540%-108.130%. The detection results showed that the relative standard deviations (RSDs) of VFA in all samples were less than 2.3%, and the method was stable within three days. The developed method is simple, which has accurate quantity and good repeatability, and suitable for the quantitative analysis of VFA in rumen fluid of ruminants.
Junhong WANG,Ge GAO,Yuanyuan ZHANG,Ying SHAN,Weiren DONG. Determination of the volatile fatty acid contents in rumen fluid of ruminants by headspace-gas chromatograph method. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(5): 667-672.
Fig. 1 Relationship between balance temperature and response signal intensity
Fig. 2 Relationship between balance time and response signal strength intensity
挥发性脂肪酸
Volatile fatty acids
保留时间
Retention time/min
线性范围
Linear range/(mmol/L)
线性回归方程
Linear regression equation
R2
检出限
LOD/(mmol/L)
RSD/%
乙酸 Acetic acid
6.909
5.0～160.0
y=841.06x－1 091.112
0.999 8
0.329
2.917
丙酸 Propionic acid
8.182
0.5～16.0
y=2 860.22x－208.622
0.999 7
0.087
3.268
丁酸 Butyric acid
9.504
0.5～16.0
y=6 665.07x－490.210
0.999 7
0.036
3.464
Table 1Retention time, linear range, linear regression equation, and LOD of the volatile fatty acids
挥发性脂肪酸
Volatile fatty acids
样品浓度
Concentration of
sample/(mmol/L)
RSD/%
保留时间
Retention
time
峰面积
Peak area
乙酸 Acetic acid
43.503
0.647
1.609
丙酸 Propionic acid
13.821
0.589
3.045
丁酸 Butyric acid
12.847
0.542
3.240
Table 2Within-day precision
挥发性脂肪酸
Volatile fatty acids
1 d
2 d
3 d
样品浓度
Concentration of
sample/(mmol/L)
峰面积相对
标准偏差
RSD of peak
area/%
样品浓度
Concentration of
sample/(mmol/L)
峰面积相对
标准偏差
RSD of peak
area/%
样品浓度
Concentration of
sample/(mmol/L)
峰面积相对
标准偏差
RSD of peak
area/%
乙酸 Acetic acid
43.717
0.429
45.247
1.672
49.808
2.303
丙酸 Propionic acid
13.892
0.498
14.084
1.255
14.855
2.156
丁酸 Butyric acid
12.879
0.939
13.120
0.333
13.299
1.374
Table 3Between-day precision
挥发性脂肪酸
Volatile fatty acids
1 d
2 d
3 d
样品浓度
Concentration of
sample/(mmol/L)
峰面积相对
标准偏差
RSD of peak
area/%
样品浓度
Concentration of
sample/(mmol/L)
峰面积相对
标准偏差
RSD of peak
area/%
样品浓度
Concentration of sample/(mmol/L)
峰面积相对
标准偏差
RSD of peak
area/%
乙酸 Acetic acid
39.493
0.287
41.202
0.447
44.054
0.820
丙酸 Propionic acid
4.260
0.260
4.221
4.759
4.432
4.540
丁酸Butyric acid
3.897
1.772
4.136
3.314
4.072
8.318
Table 4Stability of the standards
挥发性
脂肪酸
Volatile
fatty acids
初始量
Initial
quantity/
(mmol/L)
加标量
Spike
quantity/
(mmol/L)
测定值
Measured
value/
(mmol/L)
回收率
Recovery
rate/%
乙酸
Acetic acid
24.817
19.853
43.497
94.090
24.817
51.651
108.130
29.780
55.855
104.220
丁酸
Butyric acid
4.321
3.456
7.789
100.350
4.321
8.363
93.540
5.185
9.306
96.140
Table 5Spike recovery rate of samples
Fig. 3 Chromatograms of the volatile fatty acids and other organic solvents1: Ethanol; 2: 1-propyl alcohol; 3: 1-butanol; 4: Acetic acid; 5: Propionic acid; 6: Butyric acid.
样品
Sample
乙酸 Acetic acid
丙酸 Propionic acid
丁酸 Butyric acid
浓度
Concentration/
(mmol/L)
RSD/%
浓度
Concentration/
(mmol/L)
RSD/%
浓度
Concentration/
(mmol/L)
RSD/%
奶牛瘤胃液 Rumen fluid of dairy cows
58.521
1.378
14.074
1.378
10.626
0.127
湖羊瘤胃液 Rumen fluid of Hu sheep
45.470
2.908
13.279
1.821
10.488
1.827
人工瘤胃发酵液
Artificial rumen fermentation fluid
34.099
3.333
9.994
1.038
8.768
2.298
Table 6Volatile fatty acid contents in three rumen fluids of ruminants
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