超高效液相色谱-质谱联用法同时快速测定细胞中的4种吡啶核苷酸辅酶

doi:10.19894/j.issn.1000-0518.210033

摘要/Abstract

摘要：

建立了超高效液相色谱-质谱联用法同时测定细胞内4种吡啶核苷酸辅酶（烟酰胺腺嘌呤二核苷酸（NAD⁺）和烟酰胺腺嘌呤二核苷酸磷酸（NADP⁺）以及它们的还原态（NADH和NADPH））的方法。样品经甲醇低温快速提取后，在新型混合模式Atlantis PREMIER BEH C₁₈AX色谱柱上分离，10 mmol／L甲酸铵-乙腈作为流动相在8 min内完成梯度洗脱分离，采用质谱多反应检测（MRM）模式，负离子［M－H］^－扫描检测。对于4种吡啶核苷酸辅酶，本方法均在较宽浓度范围内呈良好线性关系，检出限和定量限分别在0.03～0.30 pmol和0.06～1.20 pmol范围内；检测准确度在92.7%～107.6%之间，相对标准偏差在1.98%～7.57%之间。该方法操作简便、分析快速、准确度高、灵敏度好，适用于人和微生物等多种细胞样品中吡啶核苷酸辅酶的快速定量测定，为细胞生理代谢研究提供可靠的技术支持。

关键词: 超高效液相色谱, 质谱, 吡啶核苷酸辅酶, NADPH, NADP⁺, NADH, NAD⁺, 细胞

Abstract:

An ultra-performance liquid chromatography-mass spectrometry method (UPLC-MS) was developed for the simultaneous determination of four pyridine nucleotide coenzymes (NADPH, NADP⁺, NADH and NAD⁺) in cells. The analytes were rapidly extracted with methanol at low temperature, separated by Atlantis PREMIER BEH C₁₈AX column with a gradient elution with 10 mmol/L ammonium formate and acetonitrile, and detected in negative ion [M-H] ^-scanning under the multiple reaction monitoring (MRM) mode. For the four pyridine nucleotide coenzymes, the method shows good linear relationships in wide ranges of concentrations, the limits of detection and quantification are in the range of 0.03~0.3 pmol and 0.06~1.2 pmol, respectively, and the detection accuracy is 92.71%~107.65% with the relative standard deviation of 1.98%~7.57%. The method is simple, rapid, accurate and sensitive. It is suitable for the rapid quantitative determination of pyridine nucleotide coenzymes in both human and microbial cells, and provides reliable technical support for the research of cell physiological metabolism.

Key words: Ultra performance liquid chromatography, Mass spectrometry, Pyridine nucleotide coenzyme, NADPH, NADP⁺, NADH, NAD⁺, Cellular

中图分类号:

O657.6

霍颖异, 谢木西丁·买热帕提, 吴敏. 超高效液相色谱-质谱联用法同时快速测定细胞中的4种吡啶核苷酸辅酶[J]. 应用化学, 2022, 39(02): 332-339.

Ying-Yi HUO, Maripat XAMXIDIN, Min WU. Simultaneous and Rapid Determination of Four Pyridine Nucleotide Coenzymes in Cells by Ultra Performance Liquid Chromatography-Mass Spectrometry[J]. Chinese Journal of Applied Chemistry, 2022, 39(02): 332-339.

图/表 9

参考文献 16

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待测物	母离子	子离子	解簇电压	入口电压	碰撞能量	碰撞出口电压
Analyte	Precursor ions （m／z）	Daughter ions （m／z）	Declustering potential／V	Entrance potential／V	Collisionenergy／eV	Cell exit potential／V
NADP⁺	742.4	620.3^＃	－60	－10	－22	－10
?	742.4	79.0	－60	－10	－110	－10
NADPH	744.4	79.0^＃	－60	－10	－110	－8
?	744.4	159.0	－60	－10	－72	－16
NAD⁺	662.3	540.2^＃	－55	－10	－20	－10
?	662.3	79.0	－55	－10	－100	－10
NADH	664.3	79.0^＃	－100	－10	－45	－10
?	664.3	408.2	－60	－10	042	－10

待测物	母离子	子离子	解簇电压	入口电压	碰撞能量	碰撞出口电压
Analyte	Precursor ions （m／z）	Daughter ions （m／z）	Declustering potential／V	Entrance potential／V	Collisionenergy／eV	Cell exit potential／V
NADP⁺	742.4	620.3^＃	－60	－10	－22	－10
?	742.4	79.0	－60	－10	－110	－10
NADPH	744.4	79.0^＃	－60	－10	－110	－8
?	744.4	159.0	－60	－10	－72	－16
NAD⁺	662.3	540.2^＃	－55	－10	－20	－10
?	662.3	79.0	－55	－10	－100	－10
NADH	664.3	79.0^＃	－100	－10	－45	－10
?	664.3	408.2	－60	－10	042	－10

待测物	线性范围	回归方程	相关系数	检出限	定量限
Analyte	Linear range／pmol	Regression equation	Correlation coefficient（r）	LOD／pmol	LOQ／pmol
NADP⁺	0.03～500	Y＝5870X　+4769	0.995 2	0.03	0.12
NADPH	0.15～2500	Y＝1656X+3188	0.995 8	0.30	1.20
NAD⁺	0.03～250	Y＝8205X+11994	0.996 4	0.03	0.06
NADH	0.03～500	Y＝5010X +3817	0.993 0	0.03	0.12

待测物	线性范围	回归方程	相关系数	检出限	定量限
Analyte	Linear range／pmol	Regression equation	Correlation coefficient（r）	LOD／pmol	LOQ／pmol
NADP⁺	0.03～500	Y＝5870X　+4769	0.995 2	0.03	0.12
NADPH	0.15～2500	Y＝1656X+3188	0.995 8	0.30	1.20
NAD⁺	0.03～250	Y＝8205X+11994	0.996 4	0.03	0.06
NADH	0.03～500	Y＝5010X +3817	0.993 0	0.03	0.12

待测物 Analyte	保留时间 Retention time		准确度 Accuracy
待测物 Analyte	平均 Mean／min	相对标准偏差 RSD／%	平均 Mean／%	相对标准偏差 RSD／%
NADP⁺	1.478	0.45	92.71	4.32
NADPH	2.198	0.41	104.50	1.98
NAD⁺	2.984	0.22	107.65	7.57
NADH	3.167	0.31	104.00	4.56