Simultaneous determination nucleosides in marine organisms using ultrasound‐assisted extraction followed by hydrophilic interaction liquid chromatography–electrospray ionization time‐of‐flight mass spectrometry

A new method has been developed based on ultrasound‐assisted extraction (UAE) followed by hydrophilic interaction chromatography (HILIC) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF/MS) for the simultaneous determination of 16 nucleosides and nucleobases in medicinal extracts of various marine organisms. The separation was achieved on a Venusil HILIC column (250×4.6 mm id, 5 μm) and gradient elution using a solution of acetonitrile and buffer (0.20% formic acid and 20 mmol/L ammonium acetate) as the mobile phase. Identification of the 16 target nucleosides and nucleobases was based on the retention time, UV spectra, and mass measurements of the protonated molecules ([M+H]+) and main fragment ions (ESI‐TOF/MS). In addition, non‐target compounds of 2′‐deoxyinosine and four other amino acids were also tentatively identified by ESI‐TOF/MS. The 16 target compounds were quantified by HILIC‐ESI‐TOF/MS under optimized mass conditions. All calibration curves showed good linearity (r2>0.9951). The recoveries were 84.72–124.10%, and the limits of detection of the 16 target compounds were 0.6–130.0 ng/mL. The developed method was applied to quantify the target compounds in 15 batches of various marine organisms. The method has potential applicability for the identification and determination of highly polar and low‐concentration active compounds in marine organisms.