UPLC-MS/MS测定小麦茎秆木质素单体交联结构的方法

doi:10.13560/j.cnki.biotech.bull.1985.2022-0002

摘要/Abstract

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。

关键词: UPLC-MS/MS, 小麦, 茎秆, 木质素单体交联结构, 回收率

Abstract:

A method for the qualitative and quantitative determination of lignin monomer crosslinking structures in wheat stems by UPLC-MS/MS was established，providing a scientific basis for the development and utilization of lignin. The chromatographic separation was carried out on an ACQUITY UPLC^® BEH C18（2.1 mm×100 mm，1.7 μm），ultrapure water and acetonitrile were used as the mobile phase to conduct gradient elution，and MRM（multiple reaction monitoring）were employed for detection. The results showed that the crosslinking structures of 9 lignin monomers had good linearity in the linear correlation range（R²=0.998 9），the recovery was 91.67%-100%，and the relative standard deviations was 0.24%-2.10%. The method was applied to detect the crosslinking structure characteristics of 9 lignin monomers in wheat stems 14 d after jointing. The results revealed that the retention times of the crosslinking structures of 9 lignin monomers in the sample were the same as that of the standard one. G（8-8）G was the crosslinking structure of lignin monomers with the highest mass concentration，and the mass concentration range was 9.53-10.12 ng/g. In conclusion，the method is of strong specificity and high sensitivity. It is suitable for accurate qualitative and quantitative analysis of the crosslinking structures of nine lignin monomers in the wheat stems.

Key words: UPLC-MS/MS, wheat, stem, crosslinking structure of lignin monomer, recovery

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.

图/表 7

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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

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

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