Are Psilocin and Psilocybin Tripping up your LC Method Development?

"Magic mushrooms" is the street name for mushrooms that contain hallucinogenic indole alkaloids, PC (Psilocin) and PB (Psilocybin) (Fig. 1). Psilocybin structurally resembles the neurotransmitter serotonin. Once ingested, psilocybin is rapidly metabolized by intestinal alkaline phosphates and nonspecific esterases to psilocin, which is responsible for psychoactive effects.

Fig. 1: Chemical structures of (a) psilocin and (b) psilocybin

Currently, psilocybin and psilocin are defined as Schedule 1 illicit drugs under the United States Controlled Substances Act. However, in June 2019 and January 2020, the possession of psychotropic mushrooms has been decriminalized in Oregon, Denver, Colorado, Washington, D.C and the cities of Oakland and Santa Cruz, California. This is because psilocybin has been repurposed and investigated for the treatment of cluster headache, obsessive compulsive disorder, anxiety, depression, and alcohol abuse. While the selling and purchase of “magic mushrooms” remains illegal, we can see this as a roadmap to eventual legalization, similar to that of cannabis in the United States.

Due to the increased interest in these compounds, robust and sensitive analytical methods are needed to determine potency. However, both psilocin and psilocybin are highly polar in nature and require derivatization in order to be retained on a reverse phase LC column. Additionally, psilocybin is dephosphorylated into psilocin due to collision induced dissociation in the mass spectrometer; proving that the baseline separation is important for accurately identifying these compounds.

Here, we present a method that can separate both psilocin and psilocybin, without derivatization, in 3 minutes with Raptor HILIC-Si column (Table 1 & 2 and Fig. 2).

Fig 2: (2a) TIC Separation of Psilocin and Psilocybin in 10mM Ammonium Formate in 90% Acetonitrile: 10% Water (v/v) sample diluent at 100ng/mL, (2b) XIC of Psilocin demonstrating the low intense daughter ion and (2c) XIC of Psilocybin for both the daughter ions.

Instrument Conditions:

Analysis of psilocin and psilocybin was performed on a Shimadzu Prominence HPLC equipped with a SCIEX API 4000 MS/MS. Instrument conditions can be found in Table 1 and analyte transitions are provided in Table 2.

Table 1:

Column:	Raptor HILIC-Si 50x2.1mm, 2.7µm
Flow Rate:	0.5 mL/min
Column Temp:	40 °C
Mobile Phase A:	10mM Ammonium Formate in Water
Mobile Phase B:	10mM Ammonium Formate in 90% Acetonitrile: 10% Water (v/v)
Sample Diluent:	10mM Ammonium Formate in 90% Acetonitrile: 10% Water (v/v)
Gradient:	Time	%B
	0.01	100
	0.2	100
	1.70	5
	1.71	100
	3.00	STOP
Injection Volume:	2 µL
Detector:	MS/MS, Positive ESI

Table 2: MRM transitions and Retention times for Psilocin and Psilocybin by LC-MS/MS.

No.	Analyte	Retention time (Minutes)	Q1	Q3
1	Psilocin.1	1.12	205.1	160.1
2	Psilocin.2	1.12	205.1	115.0
3	Psilociybn.1	1.73	285.1	240.0
4	Psilociybn.2	1.73	285.1	205.2

Can we discuss about sample diluent mismatch????

It is important to note that a sample diluent mismatch can often cause poor chromatography and affect the separation especially under HILIC conditions. A typical extraction of psilocin and psilocybin from magic mushrooms uses methanolic extraction, where the mushrooms are dried, ground and extracted sing 100% methanol and analyzed to determine the levels of the hallucinogens, Here, we have 2 choices: 1) We can extract the mushrooms using methanol, dry under nitrogen, and reconstitute using our mobile phase B to match our starting gradient conditions (Figure 2, Table 1) or 2)_We can use direct injection of the methanol extract (Figure 3, Table 3).

In the second step of this work, we analyzed psilocin and psilocybin in a methanol standard to confirm that these two alkaloids can be detected and separated without the need for drying and reconstitution of the sample when extracted with methanol.

As shown in Figure 3, both psilocin and psilocybin demonstrated good separation and identical retention times in methanol, compared to Fig. 2, with the standard prepared using our B mobile phase B (10Mm Ammonium Formate in 90% ACN).

Fig 3: (3a) TIC Separation of Psilocin and Psilocybin in 100% Methanol sample diluent at 100ng/mL, (3b) XIC of Psilocin demonstrating the low intense daughter ion and (3c) XIC of Psilocybin for both the daughter ions.

Instrument Conditions:

Table 3:

Column:	Raptor HILIC-Si 50x2.1mm, 2.7µm
Flow Rate:	0.5 mL/min
Column Temp:	40 °C
Mobile Phase A:	10mM Ammonium Formate in Water
Mobile Phase B:	10mM Ammonium Formate in 90% Acetonitrile: 10% Water (v/v)
Sample Diluent:	10mM Ammonium Formate in 100% Methanol
Gradient:	Time	%B
	0.01	100
	0.2	100
	1.70	5
	1.71	100
	3.00	STOP
Injection Volume:	2 µL
Detector:	MS/MS, Positive ESI

Table 4: MRM transitions and Retention times for Psilocin and Psilocybin by LC-MS/MS.

No.	Analyte	Retention time (Minutes)	Q1	Q3
1	Psilocin.1	1.13	205.1	160.1
2	Psilocin.2	1.13	205.1	115.0
3	Psilociybn.1	1.75	285.1	240.0
4	Psilociybn.2	1.75	285.1	205.2

To summarize, here are some highlights of the method described here:

• This technique can separate both psilocin and psilocybin under 3 minutes without the need for derivatization.
• Suitable for samples extracted using methanol, without the need for dilution or drying and reconstitution.
• Simple Mobile phases.

With the ever-changing laws, I hope this blog is helpful for labs looking to analyze potency of psilocin and psilocybin containing mushrooms to be used for therapeutic purposes.