Rapid quantification of Psilocybin with reversed-phase HPLC and single-wavelength detection

26 October 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


The alkaloid psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) and the neurologically active psilocin (4-hydroxy-N,N-dimethyltryptamine) are the foremost compounds of pharmaceutical interest in Psilocybe mushrooms. As these compounds are infrequently analyzed in analytical labs, validated methods for rapid purity analysis are lacking. Newfound therapeutic use has invigorated academic and commercial interests in the molecules and new methods of production and available products are expanding. As a result, high-throughput methods of analysis for psilocybin must be improved to promptly determine chemical differences between mushroom genera or other sources of psilocybin and psilocin, as well as refined product purity. To address this, we developed an inexpensive HPLC technique for the efficient quantification of psilocybin and psilocin by using readily available equipment and dilute reagents. Aqueous ammonium formate (0.143 mM) was found to be preferable over techniques with much higher buffer concentrations or stronger acids for controlling psilocybin Zwitterion resolution. The chromatographic run time satisfied high-throughput analytical requirements with an efficient total runtime under 2 minutes. A standard octadecyl silica (C18) column provided excellent resolution between psilocybin and psilocin signals. The quality of the method was validated using certified analytical reference standards and was found to be accurate (3.5% bias, Psilocybin), reliable (0.32% RSD), and efficient (Psilocybin k’ = 1.78).


Method development
High performance liquid chromatography (HPLC)
High-throughput experimentation (HTE)


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