Analysis of Alkaloids in Psilocybin Extract by HPLC-UV

	Peaks	t_R (min)
1.	N,N-desmethyl Psilocybin (Norbaeocystin)	1.671
2.	N-desmethyl Psilocybin (Baeocystin)	2.408
3.	Psilocybin	3.381
4.	4-hydroxy-N-Methyltryptamine (Norpsilocin)	3.937
5.	4-hydroxy-N,N-Dimethyltryptamine (Psilocin)	4.607

Column

Force Biphenyl (cat.# 962931E)

Dimensions:

100 mm x 3.0 mm ID

Particle Size:

3.0 µm

Pore Size:

100 Å

Guard Column:

Force Biphenyl EXP Guard Cartridge 5.0 mm, 3.0 mm ID (cat.# 962950253)

Temp.:

50 °C

Standard/Sample

Diluent:

Water

Inj. Vol.:

3 µL

Mobile Phase

Water, 10 mM ammonium formate, 0.1% formic acid

Methanol, 0.1% formic acid

Time (min)	Flow (mL/min)	%A	%B
0.00	0.8	95	5
4.00	0.8	65	35
4.01	0.8	5	95
5.00	0.8	5	95
5.01	1.0	95	5
7.50	1.0	95	5

Detector	UV/Vis @ 222 nm
Instrument	Agilent 1100
Sample Preparation	Extract was diluted 5x with HPLC grade water into a 2.0 mL amber short-cap vial (cat.# 21142) and capped with a 9 mm short cap (cat.#24497)
Notes	Guard cartridge was equipped with an UltraShield precolumn filter, 0.22 µm (cat.# 25809). System and column were passivated using LC Passivation Solution (cat.# 32475). The outlined method conditions were used to perform five injections of passivation solution (5 µL) on column, diverting to waste. Five warm-up injections were completed prior to analysis. Storage Conditions: Flush column using acetonitrile with 0.1% formic acid for 5 min.
Acknowledgement	We would like to thank 2 River Labs for their use and accommodations of lab space, instruments, and samples during collaboration.

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