RATIONALE Adenosine can induce various physiopathological effects and has been adopted as a drug to treat certain forms of supraventricular tachycardia. Adenosine is predominantly produced via fermentation that may generate many other bioactive compounds with similar structures at trace levels. Thus, it is necessary to identify these trace structurally related impurities. A new method using ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) is proposed to separate complex components at trace amounts; to obtain accurate mass measurements providing the elemental composition of unknown compounds; and MS/MS spectra for elucidation of structures. METHODS A UHPLC/QTOF-MS method was developed to separate and detect trace related impurities with structures similar to adenosine in formation samples. MS and MS/MS spectra of the impurities detected in real samples were acquired and used to propose their structures. Available reference standards were used to confirm the identification of some of the impurities detected. RESULTS Nine trace impurities of adenosine were separated and characterized. Of these nine, five were confirmed as 5'-adenylic acid, hypoxanthine, inosine, adenine, and 2'-deoxyadenosine by comparison with their available reference standards. The remaining four were proposed to be ADP-ribose, two S-epimers of 5'-deoxy-5'-methylsulfinyl adenosine, and 3'-α-glucosyl adenosine based on their MS and MS/MS spectra, and available literature. Additionally, the MS/MS fingerprints of the monosaccharide glycosyl groups were discovered and discussed. CONCLUSIONS A UHPLC/QTOF-MS method was established and used for the separation and characterization of nine trace related impurities of adenosine. Their structures were identified based on the MS and MS/MS spectra and retention times. In addition, the specific MS/MS fingerprints of the monosaccharide glycosyl moieties of the adenosine impurity analogs were summarized.

中文翻译：

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使用超高效液相色谱/电喷雾电离四极杆飞行时间串联质谱法鉴定腺苷产品中新的痕量相关杂质。

RATIONALE腺苷可诱导多种生理病理作用，已被用作治疗某些形式的室上性心动过速的药物。腺苷主要是通过发酵产生的，可以产生痕量水平上具有相似结构的许多其他生物活性化合物。因此，有必要鉴定这些痕量的结构相关杂质。提出了一种使用超高效液相色谱（UHPLC）结合四极杆飞行时间串联质谱（QTOF-MS / MS）的新方法来分离痕量复杂成分的方法。获得准确的质量测量值，以提供未知化合物的元素组成；和MS / MS光谱用于阐明结构。方法建立了UHPLC / QTOF-MS方法，以分离和检测痕量相关杂质，其结构与地层样品中的腺苷相似。采集了实际样品中检测到的杂质的MS和MS / MS光谱，并用于提出其结构。使用可用的参考标准来确认对检测到的某些杂质的鉴定。结果分离并表征了9种微量腺苷。在这九种中，与现有参考标准相比，五种被确认为5'-腺苷酸，次黄嘌呤，肌苷，腺嘌呤和2'-脱氧腺苷。根据其MS和MS / MS光谱，以及现有文献，其余4个提议为ADP-核糖，5'-脱氧-5'-甲基亚磺酰基腺苷的2个S-末端和3'-α-葡萄糖基腺苷。此外，发现并讨论了单糖糖基的MS / MS指纹图谱。结论建立了UHPLC / QTOF-MS方法，该方法用于分离和表征9种微量的腺苷相关杂质。根据MS和MS / MS光谱以及保留时间鉴定其结构。另外，总结了腺苷杂质类似物的单糖糖基部分的特异性MS / MS指纹。