Determination of Psilocybin and Psilocin in Hallucinogenic Mushrooms by HPLC with Diode Array ...

112 Pages·2008·0.86 MB·English

by  David Kieber

Determination of Psilocybin and Psilocin in Hallucinogenic Mushrooms
by HPLC with Diode Array and MS Detection

by

Raymond C. Van Orden

A thesis
submitted in partial fulfillment
of the requirements for the
Master of Science Degree

State University of New York
College of Environmental Science and Forestry
Syracuse, New York

April, 2008

Approved:
Faculty of Chemistry

______________________________ ______________________________
David J. Kieber, Major Professor Patrick Lawler, Chair, Examining
Committee

______________________________ ______________________________
Arthur J. Stipanovic, Faculty Chair Dudley J. Raynal, Dean, Instruction
and Graduate Studies
1456711

1456711
2008
ACKNOWLEDGMENTS

I thank my advisor, Dr. David J. Kieber, who gave me much appreciated
guidance and knowledge during the research part of this thesis. I especially want to
thank him for his patience, guidance, and understanding during the writing of this
study. Without his help and review, this project never would have come to fruition.
I thank my steering committee, Dr. Greg Boyer and Dr. David L. Johnson for
their guidance and timely review of this project and discussions regarding this thesis.
I especially want to thank my laboratory director from the Onondaga County Center
for Forensic Sciences, Dr. Kathleen Corrado, who allowed me the resources and time
to accomplish this project. I also wish to thank Samuel VanDee and Jennifer Wilson,
my colleagues at the Center for forensic Sciences, for their hard work and time that
allowed me to pursue this project. I also want to thank my interns, Mark Jordan,
Jamie Tull, Joe Gagnon, Jeremy Kerling, Ryan Peck, and Nicole Canfield, for their
hard work and dedication of running the numerous samples when I was busy doing
lab assignments. Finally, I wish to thank Mike Satchwell and Jennifer Liberatore for
their help in the enzymatic testing of samples in the Biochemistry lab.
Special recognition goes to my wife, Joyce, who played taxi for my two kids
while I was busy writing this project and who was instrumental in pushing me to a
successful completion. And finally, my two kids, Kaitlyn and Bryan, for
understanding the importance of this project to Dad, and allowing me to occasionally
miss some functions.

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TABLE OF CONTENTS

Page

Acknowledgments…………………………………………….……………… i

Table of Contents…………………………………………………………….. ii

List of Figures………………………………………………..………………. iii

List of Tables…………………………………………………………………. vi

Glossary of Terms………………………………………………………….… vii

Abstract……………………………………………………………….………. ix

Chapter 1. Introduction…… ……………………………………… ………… 1

Chapter 2. Background……… …………………………………… ………… 4

Chapter 3. Optimization of Chromatographic Separation of Psilocin and
Psilocybin.………………………………………………………... 14

Chapter 4. Extraction and Identification of Psilocin and Psilocybin..………... 53

Chapter 5. Thesis Summary…….…… ……………………………………….. 92

Chapter 6. References………………………………………………………..... 95

Vita………………………………………………………………………….…. 101

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List of Figures

Page

Figure 3.1 Chemical structure of indole alkaloids………………………… 17

Figure 3.2 Effect of percent organic phase in the mobile phase on the capacity
factor of psilocybin, bufotenine, psilocin, and tryptamine…...…. 26

Figure 3.3 Average capacity factor for psilocybin, bufotenine, psilocin,
and tryptamine plotted for each buffer type used in the mobile
phase………………………………………………………….…. 30

Figure 3.4 Average number of theoretical plates calculated for psilocybin,
bufotenine, psilocin, and tryptamine plotted for each buffer
type used in the mobile phase…………………………………… 31

Figure 3.5 Chromatograms of psilocybin, bufotenine, psilocin, and
tryptamine employing TCA and acetate as the buffer in the mobile
phase………………………………………...………………..…. 34

Figure 3.6 Asymmetry factor calculated for each compound in all buffers... 35

Figure 3.7 Effect of buffer pH on the capacity factor for psilocybin,
bufotenine, psilocin, and tryptamine in 50 mM citrate buffer...… 37

Figure 3.8 Effect of citrate buffer concentration (at pH 2.5) on the capacity
factor for psilocybin, bufotenine, psilocin, and tryptamine …….. 38

Figure 3.9 Ultraviolet spectrum of psilocybin standard and the unknown peak as
they eluted from the RP-HPLC…………………………………. 40

Figure 3.10 Total ion chromatogram and extracted ion chromatogram of
mushroom extract……………………………………………….. 41

Figure 3.11 Mass spectrum of peak (7.0 min) obtained from Figure 3.9A…... 42

Figure 3.12 Mass spectra of peaks (6.93 and 7.09 min) obtained from
Figure 3.9B…..………………………………………………….. 44

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Page

Figure 3.13 Optimized gradient elution chromatograms for psilocybin,
bufotenine, psilocin, and tryptamine in 50 mM TFA and
TCA – methanol mobile phases…………..………………….. 45

Figure 3.14 First order kinetic plot of psilocin in air-saturated water and
methanol, and deaerated water……………………………….. 50

Figure 4.1 Optimized gradient elution chromatograms for psilocybin,
bufotenine, psilocin, and tryptamine in 15 mM ammonium
formate – methanol mobile phase…………………………….. 67

Figure 4.2 Mass spectra of psilocybin, bufotenine, psilocin, and
Tryptamine generated from Figure 4.1 with the fragmentor
voltage set at 130 V……………..……………………………. 68

Figure 4.3 Mass spectra of psilocybin, bufotenine, psilocin, and
tryptamine generated from Figure 4.1 with fragmentor voltage
set at 250 V…………………………………………………... 70

Figure 4.4 Optimized total ion chromatogram of mushroom material
sequentially extracted in methanol followed by 50:50 (v/v)
methanol and 50 mM citrate buffer………….……………….. 73

Figure 4.5 Mass spectra generated from mushroom material of
psilocybin and psilocin from Figure 4.4 with fragmentor
voltages set at 130 and 250 V.……………………………….. 74

Figure 4.6 Mass of psilocybin and psilocin extracted from 500 mg
ground mushroom using different percentages of 50 mM,
pH 2.5 citrate buffer………………………………………….. 78

Figure 4.7 Percent psilocybin and psilocin extracted from 300 mg
ground mushroom as a function of time with different
extraction solutions…………..………………………………. 80

Figure 4.8 Percent psilocybin and psilocin extracted in 2 h with
different volumes of methanol from 500 mg ground
mushroom using different extraction techniques………….…. 81

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Page
Figure 4.9 Mass and percentage of psilocybin and psilocin extracted from
different amounts of ground mushroom sequentially
extracted in methanol followed by 50:50 (v/v) methanol
and 50 mM citrate buffer……….…………………….………. 84

Figure 4.10 Box and Whisker plot showing the average percentage of
psilocybin and psilocin extracted in separate forensic samples
analyzed over several years using three different extraction
methods……………………………………………………….. 88

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List of Tables
Page

Table 3.1 Separation factor (a ) for psilocin, psilocybin, bufotenine
and tryptamine in different mobile phase buffers…………….. 28

Table 3.2 Mass spectral abundances of psilocybin, psilocin, bufotenine
and tryptamine in the different mobile phase buffers………… 47

Table 3.3 Regression parameters (slope, r2) and half-life of psilocin
determined from first order kinetic plots of psilocin in water and
methanol…………………………………………..………….. 51

Table 4.1 Peak area generated for the protonated molecular ion
(M + H) of psilocybin with different mass spectrometer
settings……………………………………………….……….. 66

Table 4.2 Average percent psilocybin and psilocin extracted with
25 mL of different organic solvents for 2 h by rotary
inversion……………………………………………….……… 76

Table 4.3 Phosphatase activity and specific activity in different mushroom
extracts..…………………………………………………….… 85

Table 4.4 Weight percent (w/w %) of psilocybin and psilocin in mushroom
samples……………………………………………………….. 89

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Glossary of Terms

Symbol Unit
Length of perpendicular line drawn from center vertical line of the
a min
peak to right side of peak at 10% of the peak height
ACN Acetonitrile
API-ES Atmospheric pressure ionization-electrospray
a Separation factor
amu Atomic mass unit
Length (min) of perpendicular line drawn from center
b min
vertical line of the peak to left side of peak at 10% of the peak height
CID Collision-induced-dissociation
DL Detection Limit
GC-MS Gas chromatography-mass spectrometry
IS Internal Standard
k Capacity factor
LC/MS Liquid Chromatography Mass Spectrometry
M + H Molecular ion plus hydrogen
M + Na Molecular ion plus sodium
MS Mass Spectrometer
m/z Mass to charge ratio
MeOH Methanol
N Theoretical plates
m mole
P Phosphatase activity
ase min-1
pNPP p-nitrophenate
RP-HPLC Reverse Phase High Performance Liquid Chromatography
Reverse Phase High Performance Liquid Chromatography Mass
RP-HPLC-MS
Spectrometry
Reverse Phase High Performance Liquid Chromatography Mass
RP-HPLC-MS2
Spectrometry2
TCA Trichloroacetic acid
SID Source-induced dissociation
TFA Trifluoroacetic acid
vii
t Retention time min
r
t Void volume min
v
UV Ultraviolet
V Total assay volume mL
f
v/v Volume per volume ratio
w Width of peak at half height min
0.5

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