Determination of psilocin in magic mushrooms and rat plasma by liquid chromatography with fluorimetry and electrospray ionization mass spectrometry

Abstract

The sensitive determination of psilocin was carried out by reversed-phase liquid chromatography (HPLC) coupled with fluorimetry (FL) and electrospray ionization mass spectrometry (ESI–MS). Psilocin and bufotenine, used as an internal standard (IS), were labeled with excess amounts of 4-( N, N-dimethylaminosulfonyl)-7-(2-chloroformylpyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-Pro-COCl) at 60 °C for 10 min in the presence of pyridine as the scavenger of HCl produced in the solution. The resulting derivatives were separated by a Mightysil RP-18 GP column (150 mm × 4.6 mm, i.d., 3 μm) with an acidic mobile-phase containing 0.1% trifluoroacetic acid (TFA) and detected at 560 nm (excitation at 440 nm). Under the conditions for derivatization, separation and detection, a good linearity of the calibration curve of psilocin was observed by the HPLC–FL method. On the other hand, the derivative, separated by a Mightysil RP-18 GP (100 mm × 2.0 mm, i.d., 3 μm) using 50 mM AcONH 4–CH 3CN (73:27), was also determined by HPLC–ESI–MS. The mass spectrometer was operated in the selected-ion monitoring (SIM) mode for the protonated-molecular ion [M + H] + ( m/ z = 527). The calibration curve in the SIM mode was also linear in the range of 0.16–4.08 ng psilocin, similar to the HPLC–FL method. The coefficient of variation (CV) was 5.15% (0.16 ng injection, n = 6). The quantitation limit was 0.64 ng/mg dried mushroom. The amounts of psilocin in six magic mushrooms using HPLC–MS were lower than 12.67 ng/mg samples. The developed method was also successfully applied to the determination of psilocin in rat plasma after a single i.p. administration of psilocybin. The proposed method provides good precision and trace detection of psilocin in actual samples, suggesting that these analytical techniques are usable for the determination of psilocin in various specimens.