"Testing Trippy Samples"? Check this Psilocin and Psilocybin analysis in Urine by LC-MS/MS

The active ingredients in "magic mushrooms", psilocin (PC) and psilocybin (PB), are responsible for its psychoactive effects when ingested. These compounds are getting a lot of attention in laboratories these days, as several counties have decriminalized micro-dosing of magic mushroom extracts due to their potential therapeutic properties for treating conditions such as cluster headaches, anxiety, and depression. In a previous blog, I had discussed an approach to test for the potency of these highly polar, difficult-to-retain compounds in mushroom extracts.

As magic mushroom derived products are being commercialized, there is an emerging need to also test for the presence of these active ingredients from a clinical perspective. PB and PC are polar in nature and can be difficult to retain on a typical reverse phase LC column. Additionally, psilocybin is dephosphorylated into psilocin due to in-source fragmentation, thus requiring baseline separation for accurate identification of these compounds. Here, we present two analyses for the separation of psilocybin and psilocin in urine using a simple solvent protein precipitation: 1. A simple way to add these compounds to the Restek “Big Pain” method, with 231 drugs of abuse and 2. A standalone method to analyze just psilocin and psilocybin in urine.

The First Step is Simple Sample Preparation

 A 500 ng/mL standard mix of psilocin and psilocybin was prepared in pooled urine to get a representative clinical matrix. A 50 µL aliquot was taken from the standard and mixed with 10 µL of internal standard (psilocin-D10, 20 µg/mL) and 100 µL of methanol. The mixture was vortexed at 3000 rpm for 10 seconds and centrifuged at 4300 rpm for 10 minutes at 10 °C.  After centrifugation, I added a dilution step to eliminate the all-too-common problem of solvent mismatch.  Because these compounds elute quickly, sample diluent mismatch can cause loss in resolution and poor peak shapes.  So, I diluted the extract 20x with mobile phase A (Water, 0.1% formic acid + 2 mM ammonium formate).  Thus, 100 µL of the supernatant was diluted with 900 µL (20-fold dilution) of water containing 0.1% formic acid and 2 mM ammonium formate (mobile phase A).  It is important to note, the addition of ammonium formate to the sample diluent improved sensitivity when compared to 0.1% formic acid alone.

Option 1 – Quickly add these compounds to your Pain Panel

For labs that have adopted our single method for drugs of abuse and pain management analytes, this sample preparation and analyte set can be directly applied to your method.  The sample, post extraction (sample prep described above), was run using the exact conditions of our previously published “Big Pain” panel method that accommodates the analysis of 231 analytes [1].  As demonstrated in Fig. 1, both the analytes are well separated, with retention factor >5, and selectivity of the Raptor Biphenyl column allowed these new compounds to be added to an existing method, providing labs with a seamless way to improve efficiency and productivity. The analysis of psilocin and psilocybin was performed on a Shimadzu Prominence HPLC equipped with a SCIEX API 4000 MS/MS. Instrument conditions and analyte transitions are provided below (Tables 1 & 2).

Fig 1: Separation of Psilocin and Psilocybin using the Restek “Big Pain” method at 500 ng/mL in pooled human urine.

Table 1: Instrument Conditions

Analytical Column:	Raptor Biphenyl 100x2.1mm, 2.7µm (PN: 9309A12)
Guard Column	Raptor Biphenyl EXP guard column cartridge 5.0, 2.1mm, 2.7µm (PN: 9309A0252)
Flow Rate:	0.6 mL/min
Column Temp:	30 °C
Mobile Phase A:	Water, 0.1% formic acid + 2 mM ammonium formate
Mobile Phase B:	Methanol, 0.1% formic acid + 2 mM ammonium formate
Sample Diluent:	Water, 0.1% formic acid + 2 mM ammonium formate
Gradient:	Time	%B
	0.01	5
	9.00	100
	10.00	100
	10.01	5
	12.00	STOP
Injection Volume:	5 µL
Ion Source	Electrospray, Positive ESI
Detector:	MS/MS

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

No.	Analyte	Retention time (Minutes)	Precursor Ion. Q1	Quantitation Ion. Q3	Qualifier Ion. Q3
1	Psilocybin	2.09	285.1	205.1	240.0
2	Psilocin-D10	2.74	215.1	66.1
3	Psilocin.1	2.78	205.1	160.1	115.0

Option 2 – A standalone method for the analysis of psilocybin and psilocin in urine

For labs, interested in developing a fast method for PC and PB, I also developed a 2-minute assay with a total cycle time of 3.5 minutes.  Remember, we need baseline separation of these analytes due to in-source fragmentation. A Raptor Biphenyl column was chosen to achieve a selective separation of these polar analytes. I chose a 50mm column length, a 2.1mm inner diameter and a 2.7µm particle size. An advantage of reducing the column length, is that the number of theoretical plates in the column will be reduced, thereby increasing throughput of the method. Optimizing these dimensions increases the throughput of the method, while maintain analyte resolution and keeping the system backpressure within the limits of HPLC, making this a fast, robust method that can fit any instrumentation. Therefore, a 50x 2.1mm, 2.7µm, Raptor Biphenyl column was selected for the separation of psilocin and psilocybin only in urine under 2 minutes (Figure 2, Tables 3 &4).

If you are interested in scaling down your current LC methods to a smaller column format, to reduce run time, improve sample throughput, save solvent costs and improve overall efficiency of your assays, look into our Pro EZLC method translator.

Fig 2: Separation of Psilocin and Psilocybin at 500ng/mL in pooled human urine by LC-MS/MS.

Table 3: Instrument Conditions

Analytical Column:	Raptor Biphenyl 50 x 2.1mm, 2.7µm (PN: 9309A52)
Guard Column	Raptor Biphenyl EXP guard column cartridge 5.0 x 2.1mm, 2.7µm (PN: 9309A0252)
Flow Rate:	0.5 mL/min
Column Temp:	35 °C
Mobile Phase A:	Water, 0.1% formic acid + 2 mM ammonium formate
Mobile Phase B:	Methanol, 0.1% formic acid + 2 mM ammonium formate
Sample Diluent:	Water, 0.1% formic acid + 2 mM ammonium formate
Gradient:	Time	%B
	0.01	5
	0.20	5
	2.50	95
	2.51	5
	3.50	STOP
Injection Volume:	5 µL
Ion Source	Electrospray, Positive ESI
Detector:	MS/MS

Table 4: MRM transitions and Retention times for Psilocin and Psilocybin in an independent method by LC-MS/MS

No.	Analyte	Retention time (Minutes)	Precursor Ion. Q1	Quantitation Ion. Q3	Qualifier Ion. Q3
1	Psilocybin	1.36	285.1	205.1	240.0
2	Psilocin-D10	1.58	215.1	66.1
3	Psilocin	1.60	205.1	160.1	115.0

I hope you find this blog helpful in navigating the ever-changing world of Drug Monitoring & Toxicology.  As psilocybin and psilocin are en route to decriminalization or legalization, you can prepare to analyse these compounds with the approach that works best for your laboratory.

References:

S. Lupo, “The Big Pain”: Development of Pain-Free Methods for Analyzing 231 Multiclass Drugs and Metabolites by LC-MS/MS, Restek Corporation