An eco-friendly and rapid HPLC method for simultaneous determination of adenosine and ergothioneine in ten kinds of Cordyceps spp. samples with blue applicability grade index assessment

This study developed a rapid and eco-friendly HPLC method for simultaneous determination of adenosine and ergothioneine in Cordyceps spp. samples. The extracted protocol involved dispersing samples in diatomaceous earth followed by vortex extraction with 80% methanol. Chromatographic separation was achieved within 5 minutes using a Shim-pack Velox HILIC column with a gradient mobile phase of 0.1% formic acid aqueous solution and acetonitrile solution containing 0.1% formic acid and detection at 260 nm. Both analytes demonstrated excellent linearity ( r > 0.9999) with average recoveries of 102.82% (adenosine) and 100.10% (ergothioneine), accompanied by RSD s below 2%. The contents of adenosine and ergothioneine in 46 batches of Cordyceps spp. samples were 0.000~2.700 mg/g dry weight (DW) and 0.000~0.703 mg/g DW, respectively. The Ophiocordyceps sinensis and its related species were successfully classified by hierarchical cluster analysis. This study determined the content of ergothioneine in Cordyceps spp. samples for the first time, and identifies ergothioneine as a critical marker for differentiating authenticated Ophiocordyceps sinensis from its adulterants. Results of BAGI assessment (score=87.5) confirmed the superior practicality and environmental friendliness of the developed method. This rapid, accurate, and reliable method provided a scientific foundation for enhancing quality evaluation technology of Cordyceps spp. species samples. • Rapid, eco-friendly HPLC method for adenosine and ergothioneine. • Ophiocordyceps sinensis has the highest ergothioneine (>0.20 mg/g DW). • Ergothioneine can distinguish Ophiocordyceps sinensis from commercial adulterants.