Analysis of toxins of magic mushrooms Psilocybe species

For analysis of hallucinogenic toxins, such as psilocybin and psilocin, GC, GC/MS, LC and LC/MS are being used. The authentic standards of psilocybin and psilocin are not commercially available in Japan; the solution vials of psilocin can be imported after an appropriate procedure from Sigma, USA.

i. HPLC

For HPLC, a spectrophotometric detector or an electrochemical detector (ECD)c can be used. If LC/MS or LC/MS/MS is available, analysis with much higher sensitivity and reliability can be realized. Here, an HPLC method with a relatively cheap and highly sensitive ECD detector is described [5].

Column: Inertsil ODS-3 (150 x 4 mm i.d., particle size 5 |im, GL Sciences); mobile phase: pH 3.8 buffer solution (300 mL of 0.1 M citric acid solution + 160 mL of 0.1 M sodium di-hydrogenphosphate solution)/ethanol (9:1); its flow rate: 1.0 mL/min; detector: ECD (+1.0 V).

After ingestion of psilocybin, it is easily metabolized into psilocin in human bodies. In a recent report [6], psilocin is said to exist in the glucuronide-conjugated form in human samples; they have insisted that enzymatic hydrolysis with glucuronidase is required before analysis. Psilocybin is dephosphorylated into psilocin in an injection chamber of GC at high temperature; TMS derivatization is required for GC or GC/MS analysis. The readers can refer to the reference [6] on the details of the method.

Scan range: m/z 50-550; retention index: 2,099; psilocin-di-TMS: m/z 290, 291 and 348.

iii. Extraction from a mushroom [5]

i. A 300-mg aliquot of a mushroom is mixed with 30 mL methanol and homogenized.

ii. After shaking for 24 h, the homogenate is passed through a paper filter.

iii. The clear solution is evaporated to dryness under a stream of nitrogen; the residue is dissolved in 3.0 mL methanol and a 10-|L aliquot of it is injected into HPLC.

iv. Extraction from a dried mushroom [7]

i. A 100-mg aliquot of a dried mushroom is mixed with 9 mL methanol and extracted by sonication for 120 min.

ii. The volume of the mixture is adjusted to 10 mL and centrifuged at 1,000 g for 15 min. An aliquot of the supernatant solution is injected into HPLC.

v. Extraction from cerebrospinal fluid (CSF) [5]

i. A 5-mL volume of CSF is mixed with 0.35 mL of 70 % perchloric acid solution, and centri-fuged at 1,000 g for 30 min.

ii. After decanting the supernatant solution, its pH is adjusted to 12 by adding 45 % KOH solution with cooling, followed by centrifugation at 1,000 g for 5 min.

iii. The supernatant solution is mixed with 1 g NaCl, extracted with 6 mL dichloromethane by shaking for 15 min and centrifuged. The organic phase is transferred to another tube, and 6 mL dichloromethane is again added to the aqueous phase; the same extraction procedure is conducted. The resulting organic phases are combined.

iv. The combined extract is dehydrated with anhydrous Na2SO4 and centrifuged at 1,000 g for 5 min.

v. The organic extract is evaporated to dryness under a stream of nitrogen, and the residue is dissolved in 200 p,L methanol. An aliquot of the solution is injected into HPLC.

vi. Extraction from blood or urine [7]

i. A 1-mL volume of blood or urine is mixed with 10 p,L of ^-glucuronidase (E. coli origin, Sigma) and incubated at 45 °C in a water bath with shaking for 1 h.

ii. The mixture is diluted with 5 mL of 0.1 M potassium phosphate-NaOH buffer solution (pH 8) and poured into a Bond Elut Certify LRC 300 mg column (Varian, Harbor City, CA, USA). The column had been activated by passing 2 mL methanol and 2 mL of 0.1 M potassium phosphate-NaOH buffer solution (pH 8) in advance.

iii. The above sample solution is poured into the column at a flow rate of 1-2 mL/min. Thereafter, nitrogen gas is passed through the column to dry it.

iv. The column is washed with 2 mL water, 2 mL of 0.2 M acetic acid-sodium acetate buffer solution (pH 4) and 2 mL of 30 % methanol aqueous solution.

v. After passing nitrogen gas through the column to dry it up, 2 mL of methanol/concen-trated ammonia solution (98:2) and 1 mL of the same solution are passed for elution of the target compound.

vi. After both solutions are combined, they are evaporated to dryness under a stream of nitrogen with warming at 40 °C.

vii. The residue is mixed with 50 p,L of N-methyl-N-trimethyl- silyltrifluoroacetamide (MSTFA), capped airtightly and heated at 80 °C for 15 min.

viii. After cooling to room temperature, an aliquot of the solution is injected into GC/MS.

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