Simultaneous and Rapid Determination of Four Pyridine Nucleotide Coenzymes in Cells by Ultra Performance Liquid Chromatography-Mass Spectrometry

doi:10.19894/j.issn.1000-0518.210033

Chinese Journal of Applied Chemistry ›› 2022, Vol. 39 ›› Issue (02): 332-339.DOI: 10.19894/j.issn.1000-0518.210033

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Simultaneous and Rapid Determination of Four Pyridine Nucleotide Coenzymes in Cells by Ultra Performance Liquid Chromatography-Mass Spectrometry

Ying-Yi HUO¹^,², Maripat XAMXIDIN², Min WU¹^,²()

^1.National Demonstration Center for Experimental Biology Education (Zhejiang University), Hangzhou 310058, China
^2.College of Life Sciences, Zhejiang University, Hangzhou 310058, China

Received:2021-01-20 Accepted:2021-06-07 Published:2022-02-10 Online:2022-02-09
Contact: Min WU
Supported by:
Basic Work Project of the Ministry of Science and Technology(2017FY100300);Key Construction Experimental Teaching Demonstration Center of Zhejiang Province During the 13th Five Year Plan

Abstract

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

CLC Number:

O657.6

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.

Figures/Tables 9

References 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

Simultaneous and Rapid Determination of Four Pyridine Nucleotide Coenzymes in Cells by Ultra Performance Liquid Chromatography-Mass Spectrometry

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待测物 Analyte	HeLa细胞内浓度 Concentration in HeLa（fmol／cell）	大肠杆菌细胞内浓度 Concentrationin E.coli（fmole／μg wet mass）
待测物 Analyte	HeLa细胞内浓度 Concentration in HeLa（fmol／cell）	不加葡萄糖 Without glucose	添加葡萄糖 With glucose
NADP⁺	0.15±0.01	126.20±4.19	92.72±9.30
NADPH	0.06±0.01	41.28±10.31	6.56±0.58
NAD⁺	5.80±0.55	1053.47±112.94	2747.51±37.19
NADH	0.92±0.07	14.79±4.25	10.37±3.70

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