生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 66-72.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0002
收稿日期:
2022-01-03
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
孙淑芳,女,硕士,研究方向:作物栽培与耕作;E-mail:基金资助:
SUN Shu-fang(), LUO Yong-li, LI Chun-hui, JIN Min(), XU Qian
Received:
2022-01-03
Published:
2022-10-26
Online:
2022-11-11
摘要:
建立一种利用UPLC-MS/MS对小麦茎秆木质素单体交联结构进行定性与定量分析的方法,为木质素的开发利用提供科学依据。采用ACQUITY UPLC®BEH C18(2.1 mm×100 mm,1.7 μm)色谱柱分离,以超纯水和乙腈为流动相进行梯度洗脱,选择MRM(多反应监测模式)进行检测。结果表明,9种木质素单体交联结构在相关线性范围内线性良好(R2=0.998 9),回收率为91.67%-100%,相对标准偏差(RSD)为0.24%-2.10%。将该方法应用于拔节后14 d小麦茎秆中9种木质素单体交联结构特性的检测,结果显示,样品中9种木质素单体交联结构的保留时间与标准品的保留时间相同,G(8-8)G是检出质量浓度最高的木质素单体交联结构,质量浓度范围为9.53-10.12 ng/g。该方法专一性强,灵敏度高,适用于对小麦茎秆9种木质素单体交联结构进行准确定性定量。
孙淑芳, 骆永丽, 李春辉, 金敏, 胥倩. UPLC-MS/MS测定小麦茎秆木质素单体交联结构的方法[J]. 生物技术通报, 2022, 38(10): 66-72.
SUN Shu-fang, LUO Yong-li, LI Chun-hui, JIN Min, XU Qian. Determination of Lignin Monomer Crosslinking Structures in Wheat Stems by UPLC-MS/MS[J]. Biotechnology Bulletin, 2022, 38(10): 66-72.
时间Time/min | 流速Flow/(mL·min-1) | 流动相A Mobile phase A/% | 流动相B Mobile phase B/% | 梯度变化Gradient |
---|---|---|---|---|
Initial | 0.4 | 95.0 | 5.0 | 线性Linear |
0.50 | 0.4 | 95.0 | 5.0 | 线性Linear |
3.00 | 0.4 | 75.0 | 25.0 | 线性Linear |
3.50 | 0.4 | 10.0 | 90.0 | 线性Linear |
4.00 | 0.4 | 10.0 | 90.0 | 线性Linear |
4.10 | 0.4 | 95.0 | 5.0 | 线性Linear |
6.00 | 0.4 | 95.0 | 5.0 | 线性Linear |
表1 液相方法表
Table 1 Liquid phase method
时间Time/min | 流速Flow/(mL·min-1) | 流动相A Mobile phase A/% | 流动相B Mobile phase B/% | 梯度变化Gradient |
---|---|---|---|---|
Initial | 0.4 | 95.0 | 5.0 | 线性Linear |
0.50 | 0.4 | 95.0 | 5.0 | 线性Linear |
3.00 | 0.4 | 75.0 | 25.0 | 线性Linear |
3.50 | 0.4 | 10.0 | 90.0 | 线性Linear |
4.00 | 0.4 | 10.0 | 90.0 | 线性Linear |
4.10 | 0.4 | 95.0 | 5.0 | 线性Linear |
6.00 | 0.4 | 95.0 | 5.0 | 线性Linear |
待测物 Analyte | ESI模式 ESI model | 保留时间 Retention time/min | 母离子 Parent ion/(m/z) | 子离子 Daughter ion/(m/z) | 锥孔电压 Cone/V | 碰撞能量 Collision/V |
---|---|---|---|---|---|---|
S(8-O-4)S(8-8)S | - | 4.08 | 643.00 | 225*/417.31 | 35 | 20/16 |
S(8-O-4)G(8-O-4)S | - | 4.00 | 631.00 | 209.17*/421.08 | 40 | 25/12 |
G(8-O-4)S(8-5)G | - | 4.04 | 583.00 | 195.02*/369.21 | 33 | 18/25 |
S(8-O-4)S(8-5)G | - | 4.04 | 613.00 | 225.15*/369.16 | 35 | 20/19 |
G(8-O-4)S(8-8)S | - | 4.08 | 613.52 | 195.12*/417.03 | 45 | 25/22 |
G(8-O-4)G(8-5)G | - | 4.01 | 553.00 | 194.88*/339.00 | 50 | 23/19 |
S(8-O-4)S | - | 4.01 | 435.00 | 198.08*/225.00 | 44 | 24/18 |
G(8-8)G | - | 4.03 | 357.00 | 357.00 | 35 | 25/23 |
G(8-5)H | - | 4.05 | 327.00 | 309.00*/190.73 | 40 | 26/24 |
表2 各木质素单体交联结构的质谱参数表
Table 2 Mass spectrum parameters of crosslinking structures of lignin monomers
待测物 Analyte | ESI模式 ESI model | 保留时间 Retention time/min | 母离子 Parent ion/(m/z) | 子离子 Daughter ion/(m/z) | 锥孔电压 Cone/V | 碰撞能量 Collision/V |
---|---|---|---|---|---|---|
S(8-O-4)S(8-8)S | - | 4.08 | 643.00 | 225*/417.31 | 35 | 20/16 |
S(8-O-4)G(8-O-4)S | - | 4.00 | 631.00 | 209.17*/421.08 | 40 | 25/12 |
G(8-O-4)S(8-5)G | - | 4.04 | 583.00 | 195.02*/369.21 | 33 | 18/25 |
S(8-O-4)S(8-5)G | - | 4.04 | 613.00 | 225.15*/369.16 | 35 | 20/19 |
G(8-O-4)S(8-8)S | - | 4.08 | 613.52 | 195.12*/417.03 | 45 | 25/22 |
G(8-O-4)G(8-5)G | - | 4.01 | 553.00 | 194.88*/339.00 | 50 | 23/19 |
S(8-O-4)S | - | 4.01 | 435.00 | 198.08*/225.00 | 44 | 24/18 |
G(8-8)G | - | 4.03 | 357.00 | 357.00 | 35 | 25/23 |
G(8-5)H | - | 4.05 | 327.00 | 309.00*/190.73 | 40 | 26/24 |
待测物Analyte | 含量Content/(ng·g-1) |
---|---|
S(8-O-4)S(8-8)S | 0.03 |
S(8-O-4)G(8-O-4)S | 0.04 |
G(8-O-4)S(8-5)G | 0.55 |
S(8-O-4)S(8-5)G | 0.05 |
G(8-O-4)S(8-8)S | 0.29 |
G(8-O-4)G(8-5)G | 0.03 |
S(8-O-4)G | 0.05 |
G(8-8)G | 9.79 |
G(8-5)H | 0.15 |
表3 拔节后14 d小麦茎秆中9种木质素单体交联结构的含量
Table 3 Contents of crosslinking structures of 9 lignin monomers in wheat stems at 14 d after jointing
待测物Analyte | 含量Content/(ng·g-1) |
---|---|
S(8-O-4)S(8-8)S | 0.03 |
S(8-O-4)G(8-O-4)S | 0.04 |
G(8-O-4)S(8-5)G | 0.55 |
S(8-O-4)S(8-5)G | 0.05 |
G(8-O-4)S(8-8)S | 0.29 |
G(8-O-4)G(8-5)G | 0.03 |
S(8-O-4)G | 0.05 |
G(8-8)G | 9.79 |
G(8-5)H | 0.15 |
待测物 Analyte | 含量 Content/(ng·g-1) | 相对标准偏差 Relative standard deviation/% | ||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 平均值Average | ||
S(8-O-4)S(8-8)S | 0.02 | 0.03 | 0.04 | 0.03 | 0.03 | 0.03 | 0.03 | 2.10 |
S(8-O-4)G(8-O-4)S | 0.04 | 0.04 | 0.03 | 0.04 | 0.05 | 0.05 | 0.04 | 1.80 |
G(8-O-4)S(8-5)G | 0.50 | 0.52 | 0.56 | 0.58 | 0.57 | 0.54 | 0.55 | 0.57 |
S(8-O-4)S(8-5)G | 0.05 | 0.04 | 0.06 | 0.05 | 0.05 | 0.04 | 0.05 | 1.55 |
G(8-O-4)S(8-8)S | 0.27 | 0.32 | 0.26 | 0.28 | 0.32 | 0.29 | 0.29 | 0.87 |
G(8-O-4)G(8-5)G | 0.04 | 0.02 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 2.10 |
S(8-O-4)G | 0.04 | 0.05 | 0.06 | 0.05 | 0.05 | 0.07 | 0.05 | 1.93 |
G(8-8)G | 9.70 | 9.53 | 10.12 | 9.60 | 9.77 | 10.00 | 9.79 | 0.24 |
G(8-5)H | 0.14 | 0.13 | 0.18 | 0.17 | 0.14 | 0.16 | 0.15 | 1.28 |
表4 拔节后14 d小麦茎秆中9种木质素单体交联结构含量的精度分析
Table 4 Accuracy analysis of crosslinking structure contents of 9 lignin monomers in wheat stems at 14 d after jointing
待测物 Analyte | 含量 Content/(ng·g-1) | 相对标准偏差 Relative standard deviation/% | ||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 平均值Average | ||
S(8-O-4)S(8-8)S | 0.02 | 0.03 | 0.04 | 0.03 | 0.03 | 0.03 | 0.03 | 2.10 |
S(8-O-4)G(8-O-4)S | 0.04 | 0.04 | 0.03 | 0.04 | 0.05 | 0.05 | 0.04 | 1.80 |
G(8-O-4)S(8-5)G | 0.50 | 0.52 | 0.56 | 0.58 | 0.57 | 0.54 | 0.55 | 0.57 |
S(8-O-4)S(8-5)G | 0.05 | 0.04 | 0.06 | 0.05 | 0.05 | 0.04 | 0.05 | 1.55 |
G(8-O-4)S(8-8)S | 0.27 | 0.32 | 0.26 | 0.28 | 0.32 | 0.29 | 0.29 | 0.87 |
G(8-O-4)G(8-5)G | 0.04 | 0.02 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 2.10 |
S(8-O-4)G | 0.04 | 0.05 | 0.06 | 0.05 | 0.05 | 0.07 | 0.05 | 1.93 |
G(8-8)G | 9.70 | 9.53 | 10.12 | 9.60 | 9.77 | 10.00 | 9.79 | 0.24 |
G(8-5)H | 0.14 | 0.13 | 0.18 | 0.17 | 0.14 | 0.16 | 0.15 | 1.28 |
待测物Analyte | 样品中含量Found/(ng·g-1) | 加标量Added/(ng·g-1) | 测定量Measureed/(ng·g-1) | 回收率Recovery/% |
---|---|---|---|---|
S(8-O-4)S(8-8)S | 0.08 | 0.07 | 0.15 | 100.00 |
S(8-O-4)G(8-O-4)S | 0.10 | 0.09 | 0.19 | 100.00 |
G(8-O-4)S(8-5)G | 0.73 | 0.80 | 1.47 | 92.50 |
S(8-O-4)S(8-5)G | 0.12 | 0.12 | 0.23 | 91.67 |
G(8-O-4)S(8-8)S | 0.38 | 0.40 | 0.77 | 97.50 |
G(8-O-4)G(8-5)G | 0.04 | 0.04 | 0.08 | 100.00 |
S(8-O-4)G | 0.11 | 0.10 | 0.20 | 90.00 |
G(8-8)G | 11.44 | 11.00 | 21.75 | 93.73 |
G(8-5)H | 0.27 | 0.30 | 0.55 | 93.33 |
表5 不同加标水平的木质素单体交联结构回收率
Table 5 Recovery of crosslinking structure of lignin monomer at different spiked levels
待测物Analyte | 样品中含量Found/(ng·g-1) | 加标量Added/(ng·g-1) | 测定量Measureed/(ng·g-1) | 回收率Recovery/% |
---|---|---|---|---|
S(8-O-4)S(8-8)S | 0.08 | 0.07 | 0.15 | 100.00 |
S(8-O-4)G(8-O-4)S | 0.10 | 0.09 | 0.19 | 100.00 |
G(8-O-4)S(8-5)G | 0.73 | 0.80 | 1.47 | 92.50 |
S(8-O-4)S(8-5)G | 0.12 | 0.12 | 0.23 | 91.67 |
G(8-O-4)S(8-8)S | 0.38 | 0.40 | 0.77 | 97.50 |
G(8-O-4)G(8-5)G | 0.04 | 0.04 | 0.08 | 100.00 |
S(8-O-4)G | 0.11 | 0.10 | 0.20 | 90.00 |
G(8-8)G | 11.44 | 11.00 | 21.75 | 93.73 |
G(8-5)H | 0.27 | 0.30 | 0.55 | 93.33 |
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