The pyridine nucleotides nicotineamide adenine dinucleotide (NAD) and nicotineamide adenine dinucleotide phosphate (NADP) are conserved coenzymes across all domains of life, and are involved in more than 200 different hydride transfer reactions supporting essential catabolic and anabolic functions. The intracellular levels of these metabolites, and the ratio of their oxidized to reduced forms regulate an extensive network of reactions ranging beyond metabolism. Hence, monitoring their intracellular levels provides information about, but not limited to, the metabolic state of a cell or tissue. Interconversion between oxidized and reduced forms, varying pH liability and varying intracellular concentrations of the different species leaves absolute quantification of the pyridine nucleotides analytically challenging. These polar metabolites are poorly retained on conventional reverseed-phase stationary phases without ion-pair reagents that contaminates the LC-system. Herein we demonstrate that zwitterionic HILIC-tandem mass spectroemtry can be applied to successfully resolve the pyridine nucleotides in biological extracts in a fast, robust and highly sensitive way. The presented method applies isotope dilution to compensate potential loss of these labile metabolites and is validated for low, medium and high biomass samples of two popular biological model systems; Escherichia coli and the human cell line JJN-3. High stability and rapid sample preparation without solvent removal allows for long sequence runs, making this method ideal for high-throughput analysis of biological extracts.

中文翻译：

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具有同位素稀释作用的两性离子HILIC串联质谱用于对生物提取物中的吡啶核苷酸进行快速，灵敏和可靠的定量。

吡啶核苷酸烟碱酰胺腺嘌呤二核苷酸（NAD）和烟碱酰胺腺嘌呤二核苷酸磷酸（NADP）是生命各个领域中保守的辅酶，参与200多种支持基本分解代谢和合成代谢功能的氢化物转移反应。这些代谢物的细胞内水平及其氧化形式与还原形式的比率调节了广泛的反应网络，其作用范围超出了代谢。因此，监测它们的细胞内水平可提供有关但不限于细胞或组织代谢状态的信息。氧化形式和还原形式之间的相互转化，不同物种的不同pH依赖性和不同细胞内浓度，使得对吡啶核苷酸的绝对定量分析具有挑战性。这些极性代谢物很难保留在常规的反相固定相上，而没有污染LC系统的离子对试剂。在本文中，我们证明两性离子HILIC串联质谱法可以快速，稳健和高度敏感的方式成功地解析生物提取物中的吡啶核苷酸。提出的方法应用同位素稀释来补偿这些不稳定代谢物的潜在损失，并已针对两种流行的生物模型系统的低，中和高生物量样品进行了验证。大肠杆菌和人细胞系JJN-3。高稳定性和快速样品制备而无需去除溶剂，可实现长序列运行，这使该方法成为生物提取物高通量分析的理想选择。