Metabolite analysis and nucleoside determination using reproducible UHPLC-Q-ToF-IMS in Ophiocordyceps sinensis

Abstract

A fast, reliable and reproducible UHPLC-IMS method was developed and validated for simultaneous determination of eight nucleosides (adenine, adenosine, cordycepin, guanosine, inosine, thymidine, thymine and uracil) in natural Ophiocordyceps sinensis. Present study was conducted to determine the marker nucleoside contents in low quantities using the developed method. Methanol and water in combination were used as extraction solvents due to hydrophilic nature of nucleosides. These nucleosides showed good linearity (r2>0.99), LOD (0.28–0.97ng/mL), LOQ (0.79–3.20 ng/mL), recoveries (88.3 and 103.2%), reproducibility, stability and intra- and inter-day precision (<2.87%). Method was validated using three samples of natural Ophiocordyceps collected from different geographical region. PCA analysis depicted that adenine, adenosine, guanine, guanosine, uracil and uridine were major components contributing to total variance. Highest nucleosides content (5124 µg/g) was detected in STD-1. The presence of cordycepin, an important marker compound was identified for first time using ion mobility mass spectrometry (IMMS) technique. Metabolomics approach resulted 18, 12 and 9 metabolites in three samples (STD-1, STD-II and STD-III, respectively) using METLIN database which opened the new avenues to identify and explore more novel biomarkers in Ophiocordyceps. We anticipate that these methods may be applied to verify/certification of Ophiocordyceps containing marker nucleosides for their dietary intake.