Identification of new trace related impurities in adenosine products using ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

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'-alpha-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.