Alkaloid content was determined by liquid
chromatography with mass spectrometric detection
using electrospray ionization. (Table 1 contains a
detailed description of the instrumentation and
parameters.) Stock solutions of tryptamine
hydrochloride (99%; Aldrich Chemical, Milwaukee,
WI), serotonin (96.7%; Sigma), N,N-dimethyltryptamine
(Alltech), 5-methoxy-dimethyltryptamine
(Sigma), and bufotenine (Radian Analytical Products,
Austin, TX) were prepared individually. Bufotenine
and DMT were dissolved in methanol; tryptamine in
ethanol; and 5-HT and 5-MeODMT in water acidified
to a pH of 3.2 in acetic acid with a few drops of
methanol to assist dilution. A five-point curve (R2 ≥
0.998) of mixed standards was freshly prepared in
ethanol from the individual stocks each day; each
contained 20 μg mL-1 of caffeine to match the sample
extracts.
Qualitatively, the positive confirmation of a
compound’s identity requires comparison of the
retention time and the pattern of fragmentation ions of
the sample and the standard. These must all agree
within some limit of acceptability, generally, 0.2-0.3
min for the retention time and 5-10% for the ion
proportions. Due to the similarity in structures among
this particular group of compounds, close retention
times and the sharing of fragmentation ions were not
uncommon. There was also the reasonable concern
that one of the many previously unrecognized
analogues could appear in the extracts. To ensure
identification of any target compound believed to be
present, that sample extract was spiked to a level of 20
μg mL-1 with a standard of the suspect analyte and reanalyzed.
The unspiked and spiked spectra were then
compared against each other, as well as against the
corresponding standard. While identification depended
on multiple ions, mass spectra of the standards and
samples were also collected in selected-ion mode
(single-ion monitoring) to maximize sensitivity for
quantification. The [M + H]+ ions at m/z 161 for
tryptamine, the [M + H] + ions at m/z 177 for 5-HT,
the [M + H] + ions at m/z 205 for bufotenine, the [M +
H] + ions at m/z 189 for DMT, and [M + H] + ions at
m/z 219 for 5-MeODMT were selected for
quantification.
Table 1. Description of instrumentation and
parameters.
HPLC System
Instrumentation: Hewlett-Packard HP1100 system
with autosampler, degasser, binary
pump modules, and variable
wavelength UV detector (Hewlett-
Packard Company, Wilmington, DE)
Column: Adsorbosil C8 5 μm (4.6 mm ID,
150 mm length; Serial #97032088;
Alltech)
Mobile Phase: Solvent A: 20 mM ammonium
acetate in water
Solvent B: methanol
Gradient: 13% Solvent B for 2 min;
13% increased to 100% Solvent B
over 14 min; hold 8 min; 100%
decreased to 13% Solvent B over 2
min; equilibrate 5 min
Column Temp: 25 °C
Flow Rate: 1 mL/min
Injection Vol.: 50 μL
UV Detection: 280 nm
Mass Spectrometer
Instrumentation: Finnigan LCQ Ion Trap Mass
Spectrometer (Finnigan MAT, San
Jose, CA)
Range: 100-500 m/z full scan mode
Source: Electrospray Ionization
Spray Volt: 4.20 kV
Capillary Volt: 20 V
Capillary Temp: 220 °C
