Determination and Confirmation of Psychedelic and Psychoactive Compounds in Botanical Materials by UPLC-ESI-MS/MS: Single-Laboratory Validation

Abstract

Background

A multi-analyte method was developed for determination and confirmation of 26 natural and synthetic psychedelic and psychoactive compounds from botanical sources using a simple extraction procedure and UPLC-ESI-MS/MS analysis.

Objective

The objective of this work was to develop and validate the method for analysis of psychedelic and psychoactive compounds in botanicals (fungi, cactus, leaf), extract (brewed tea), and synthetic and simulated finished product (using maltodextrin).

Methods

The method uses an acidified methanol solution for extraction and diphenhydramine as an internal standard. Extracts are subjected to UPLC with tandem mass spectrometry using a phenyl-hexyl column and acidified mobile phase with acetonitrile gradient. Validation studies followed US Pharmacopeia <1225> and Official Methods of Analysis^SM Appendix K guidelines to assess specificity, accuracy (recovery), repeatability and intermediate precision, limits of detection and quantification, calibration curve linearity, system suitability, and robustness.

Results

Average recovery of spiked replicates in matrix and surrogate matrixes varied between 88 and 124% for analytes in fungi, cactus, and leaf matrixes; 81–106% in brewed tea matrix; and 81–174% across all 26 analytes in maltodextrin. Norbaeocystin and harmaline were the only analytes outside the acceptance criterion (79–126%) in maltodextrin. The 90% upper confidence limit on repeatability was ≤33% for all analytes and matrixes except for norbaeocystin. Repeatability precision for analysis of matrixes with native analytes met the acceptance criterion of ≤33% in all cases and the 90% upper confidence limit on intermediate precision was ≤35% in all cases. LOQ values fell below the lowest calibration standard (0.016 µg/mL) for all 26 analytes.

Conclusion

The method met the predetermined acceptance criteria for recovery and precision in nearly all cases. Calibration curve linearity and system suitability measures were established.

Highlights

A method for 26 psychedelic/psychoactive compounds was validated for analysis of botanical (fungi, cactus, leaf), extract (brewed tea), and synthetic and simulated finished product (using maltodextrin) using native and surrogate botanicals.

Psychedelic is derived from the two root Greek words: psyche and delos. Psyche refers to the mind, soul, or intelligence. Delos means to manifest or make clear. Thus, psychedelics are “mind-manifesting” or “making clear the mind’s content.” Psychedelics are a class of psychoactive substances that produce changes in perception (hallucinogens), mood, and cognitive processes (1). Certain botanicals and fungi naturally contain psychedelic compounds that have different pharmacological activities and health benefits. Other plants contain psychoactive substances that affect how the brain works and cause changes in mood, awareness, thoughts, feelings, or behaviors but do not produce hallucinations. All psychedelics are psychoactive, but not all psychoactive compounds are psychedelic.

Botanicals and botanical products containing psychoactive compounds have different health benefits and pharmacological activities. The objective of this study is to develop and validate an analytical method for psychedelic and psychoactive compounds derived from plants and fungi. Traditionally, analytical methods for psychedelic compounds focused on clinical and forensic analyses of urine, blood, and hair (2–5) and, more recently, wastewater (6, 7), due to the illegal use and abuse of these natural and synthetic drugs. However, recent publications (8, 9), which were released after the completion of this method development and validation, have also described the direct analysis of these compounds in plant material. This further illustrates the growing need for robust analytical methods tailored to botanical matrixes.

Psychedelic drug treatments are showing promising benefits in clinical research, especially for mental health conditions that are resistant to traditional therapies. The recognition of therapeutic uses of psychedelics in the treatment of various addictions and mental disorders has led to the emergence of psychedelic-assisted therapy (10, 11), which is now legal and regulated in the state of Oregon (12). Other states have or are working to decriminalize possession of small quantities of these drugs (13). Ayahuasca, ibogaine, and psilocybin show promise for treatment of addiction and substance use disorders. Psilocybin has potential for the treatment of resistant depression and MDMA-assisted therapy for reducing symptoms of post-traumatic stress disorder (PTSD) and trauma recovery. In 2023, the US FDA released a draft guidance on clinical trials with psychedelic drugs (14).

While current clinical methods for detection of drugs and their metabolites are useful for monitoring clinical drug trials, a validated multitarget method for characterization of botanical sources, extracts, and finished products containing psychedelic compounds is needed for determining accurate dosages in controlled clinical trials or psychedelic-assisted therapy. Dosage control is critical for efficacy and patient safety concerns because psychedelics often have potent effects at very low (µg to mg) doses. Incorrect dosing of psychedelics, whether too high or too low, can lead to a range of negative effects, both physiological and psychological. A validated method for determination and confirmation of 26 psychedelic/psychoactive compounds by UPLC-ESI-MS/MS in botanicals and botanical products is described. We report here the validation of the method for use with botanical sources, extracts, and simulated finished products. Maltodextrin was chosen to simulate a finished product because it is a commonly used stabilizer, carrier, and bulking agent in dietary supplements. The psychedelic compounds and botanical sources for these analytes are listed in Table 1. The validation followed the guidelines outlined in USP <1225> (15) and AOAC INTERNATIONAL Official Methods of Analysis^SM Compendium Appendix K (16).

Table 1.

Psychedelic or psychoactive compounds, their sources, MS/MS conditions, and m/z transitions

Plant source	Analyte	Parent mass	Daughter massa	Dwell	Cone voltage (V)	Collision energy
Psilocybe cubensis mushroom	Psilocin	205.2	160.1	Auto	20	15
		205.2	58.1	Auto	20	18
	Psilocybin	285.33	58.19	Auto	25	20
		285.33	205.37	Auto	25	20
	Norpsilocin	191.2	114.86	Auto	35	30
		191.2	159.67	Auto	35	20
	Norbaeocystin	257.2	239.93	Auto	20	20
		257.2	114.82	Auto	20	30
	Baeocystin	271.2	191.05	Auto	25	20
		271.2	239.78	Auto	25	15
Inocybe aeruginascens and Pholiotina cyanopus mushrooms	Aeruginascin	300.3	115.91	Auto	25	40
		300.3	142.88	Auto	25	30
San Pedro cactus (Trichocereus pachanoi)	Mescaline	212.2	195.2	Auto	35	10
		212.2	180.2	Auto	35	20
Apocynaceae family (Tabernanthe iboga, Voacanga africana, and Tabernaemontana undulata)	Ibogaine	311.34	174.2	Auto	20	30
		311.34	44.04	Auto	20	40
Papaver somniferum (opium poppy)	Codeine	301.29	58.06	Auto	25	30
		301.29	200.23	Auto	25	30
Erythroxylum coca and Erythroxylum novogranatense	Cocaine	304.26	82.09	Auto	25	30
		304.26	182.16	Auto	25	20
Synthetic	d-Methamphetamine	150.36	119.16	Auto	25	10
		150.36	91	Auto	25	20
	MDMA	194.23	163.17	Auto	25	10
		194.23	105.16	Auto	25	20
	Ketamine	238.14	125.06	Auto	25	30
		238.14	179.18	Auto	25	20
	LSD	324.3	223.2	Auto	25	24
		324.3	208.2	Auto	25	31
Ayahuasca (Psychotria virdis & Banisteriopsis caapi)	DMT	189.2	58.4	Auto	10	14
		189.2	144.1	Auto	10	17
	Harmine	213.1	198.1	Auto	10	22
		213.1	170.1	Auto	10	29
	Harmaline	215.1	200.1	Auto	10	23
		215.1	172.1	Auto	10	30
	Harmol	199.1	103.2	Auto	10	37
		199.1	131.1	Auto	10	31
	Harmalol	201.1	160.1	Auto	10	16
		201.1	185.1	Auto	10	32
	Tetrahydroharmine	217.1	188.1	Auto	10	14
		217.1	200.1	Auto	10	12
	5-MeO-DMT	219.2	58.4	Auto	10	14
		219.2	174.1	Auto	10	16
	DMT-N-Oxide	205.2	144.1	Auto	10	15
		205.2	115.25	Auto	10	40
	N-Methyltryptamine	175.2	144.1	Auto	10	12
		175.2	117.2	Auto	10	29
	2-MTHBC	187.2	144.1	Auto	10	12
		187.2	44.4	Auto	10	37
Some mushrooms & psychoactive toads	5-OH-DMT	205.2	58.4	Auto	10	14
		205.2	160.1	Auto	10	16
Various	Tryptamine	161.18	144.09	Auto	10	10
		161.18	115.03	Auto	10	30
Internal standard	Diphenhydramine	256.3	167.14	Auto	25	20
		256.3	152.17	Auto	25	40

^aThe top ion mass is the Quantitative and the bottom ion mass is the Confirmatory.

Experimental

Standards and Reagents

Powdered reference standards of 1-methyl-7-hydroxy-β-carboline (harmol), tetrahydroharmine (THH), 5-methoxydimethyltryptamine (5-MeO-DMT), N, N-dimethoxytryptamine-N-oxide (DMT-N-Oxide), N-methyltryptamine (NMT, as HCl salt), 2-Methyl-1,2,3,4-tetrahydro-ß-carboline (2-MTHBC), and 5-hydroxydimethyltryptamine (5-OH-DMT, bufotenine as HCl salt) were purchased from Toronto Research Chemicals (North York, ON, Canada), and standards of 1-methyl-7-methoxy-3,4-dihydro-β-carboline (harmaline), 1-methyl-4,9-dihydro-3H-β-carbolin-7-ol (harmalol, as HCl salt) were purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth Germany). Powdered standard of tryptamine was purchased from Sigma-Aldrich (St. Louis, MO), cocaine hydrochloride from US Pharmacopeia (Rockville, MD), N, N-dimethyltryptamine (DMT), 5-(aminomethyl)-3-isoxazolol (muscimol), 4-hydroxy-N-methyltryptamine 4-phosphate (baeocystin), 4-hydroxytryptamine 4-phosphate (norbaeocystin), 4-hydroxy-N-methyltryptamine (norpsilocin), N, N, N-trimethyl-4-phosphoryloxytryptamine (aeruginascin), and ibogaine from Cayman Chemical (Ann Arbor, MI), 1-methyl-7-methoxy-β-carboline (harmine) from Chromadex (Longmont, CO), and diphenhydramine hydrochloride salt (Internal Standard, IS) from Spectrum Chemical (New Brunswick, NJ). Powder reference standards were accompanied by a Certificate of Analysis (CoA), and weights were adjusted to account for hydration or hydrochloride salts. Reference solution standards (1.0 mg/mL in methanol or acetonitrile) of psilocin, psilocybin, mescaline, d-methamphetamine, ketamine, lysergic acid diethylamide, and codeine were purchased from Cerilliant (Round Rock, TX), and 3,4-methylenedioxymethamphetamine (MDMA) from Cayman Chemical. All solution standards were accompanied by a Certificate of Analysis. Certified deuterated reference standards including psilocin-d₁₀ in acetonitrile, psilocybin-d₄ in 1:1 acetonitrile: water, mescaline-d₃ in methanol, cocaine-d₃ in acetonitrile, N, N-dimethyltryptamine-d₄ (DMT-d4) in methanol, and harmine-d₃ in methanol were purchased from Cerilliant (Round Rock, TX) as 100 µg/mL solutions with Certificate of Analyses.

Reference standard solutions were prepared by dissolving powdered standards in diluent (70% methanol with 0.5 M acetic acid and 5 µg/mL IS) to a concentration of 100 µg/mL. Mixed standard solutions were prepared by combining standard solutions in diluent to appropriate concentrations. Calibration curves consisted of mixed standards in diluent with each compound at 0.016, 0.166, 0.333, 0.500, 0.666, 0.833, and 1.00 µg/mL, referred to as level 1 to level 7, respectively. Matrix effects were evaluated using deuterated standards and matrix-matched calibration curves prepared using the primary psychedelic compounds for comparison to the solvent-based calibration curves described above.

Ammonium formate (AmF), acetonitrile (ACN), acetic acid (AcA), formic acid (FA), and methanol (MeOH) were MS grade and sourced from major laboratory suppliers. Water was ultrapure.

Validation Materials

Native botanical matrixes with naturally occurring analytes including Psilocybe cubensis mushrooms, San Pedro cactus (Trichocereus pachanoi), coca leaves (Erythroxylum coca), and ayahuasca decoction (tea) were analyzed with and without spiking. Other matrixes included maltodextrin (Sigma-Aldrich, St. Louis, MO) as a representative stabilizer, carrier, and bulking agent commonly used for dietary supplements; reishi mushrooms (Ganoderma lingzhi), prickly pear cactus cladode (Opuntia ficus-indica), sage leaf (Salvia officinalis), and brewed green tea (Camellia sinensis) as surrogate matrixes for spiking. The surrogate matrixes were selected to represent botanical material closely related to those that naturally contain the psychedelic or psychoactive analytes of interest. Because the surrogate materials do not contain the target analytes, they provide a clean background for spiking experiments enabling assessment of method recovery, accuracy, and precision. The native and surrogate matrixes were sourced from the botanical reference material in the Alkemist Labs Herbarium collection. Maltodextrin was spiked with all analytes. Each surrogate matrix was spiked only with those analytes expected to occur in the similar native matrix. Spiking was performed at 16.6 and 83.3 mg/kg per analyte. To each 100 mg test portion in a 15 mL polypropylene tube, 166 or 833 µL of spike stock solution (10 µg/mL each analyte in 70% MeOH with 0.5M AcA and 5 µg/mL IS) was added, extracted, and analyzed according to the method.

Sample Preparation

Botanical samples were mixed well, or ground if needed in an IKA Tube Mill (Hampton, NH) disposable grinding chamber. Test portions (∼100 mg) were accurately weighed and transferred to 15 mL polypropylene conical tubes. Ten (10.0) mL diluent was added, and tubes were vortex-mixed for 30 seconds, and then sonicated in an ultrasonic bath (VWR Symphony, Radnor, PA) at 35 kHz for 30 min at room temperature (20–30°C). Resulting extracts were centrifuged for 5 min at 3200 × g, and then a portion of the supernatant liquid was filtered through a 0.2 µm syringe filter into a clean 15 mL tube. If needed, the filtrate was diluted with diluent to achieve analyte concentrations within the standard curve.

Analysis

Analyses were performed using an Acquity H-Class UPLC system with Xevo TQ-S Micro MS/MS detector equipped with an electrospray ionization source (Waters Corporation, Milford, MA) and fitted with an XSelect CSH Phenyl-Hexyl column (2.5 µm, 2.1 × 50 mm, Waters) with matching guard column (XSelect CSH Phenyl-Hexyl XP VanGuard Cartridge, 2.5 µm, 2.1 × 5 mm). A mobile phase gradient using 5 mM AmF in 0.1% FA (Mobile phase A) and 0.1% FA in ACN (Mobile phase B) consisted of 0–1 min 0% mobile phase B, 1–5 min 0–25% mobile phase B, 5–8 min 25–95% mobile phase B, 8–9 min 95% mobile phase B, 9–9.1 min 95–0% mobile phase B, 9.1–15 min 0% mobile phase B. LC conditions included column temperature, 50°C; autosampler temperature, 10°C; flowrate 0.4 mL/min; run time 15 min; and injection volume, 0.1 µL. MS conditions and corresponding m/z transitions are listed in Table 1. Compound optimization was performed through acquisition of mass fragmentation data to support selection of appropriate multiple reaction monitoring (MRM) transitions for each compound as shown in Figures 1 and 2.

Figure 1.

Representative chromatograms for psilocin, psilocybin, norpsilocin norbaeocystin, baeocystin, aeruginacin, DMT, harmine, harmaline, harmol, harmalol, and THH.

Figure 2.

Representative chromatograms for 5-MeO-DMT, DMT-NO, N-methyltraptamine, 5-OH-DMT, mescaline, ibogaine, methamphetamine, MDMA, ketamine, LSD, cocaine, and codiene.

The injection sequence consisted of 1 MRM window placement solution; 1 diluent; 1 level 1 standard; 1 level 2 standard; 1 level 3 standard; 6 level 4 standards; 1 level 5 standard; 1 level 6 standard; 1 level 7 standard; up to 10 unknown extracts; 1 check standard (level 4 standard); up to 10 unknown extracts; 1 check standard (level 4 standard). The sequence continued with one check standard after a maximum of every 10 unknown extract injections. The MRM window placement solution is typically the level 4 standard solution and is used to determine the retention times of each analyte for that analytical sequence. The check standard is the level 4 standard solution.

The peak areas of each analyte and IS in each standard, check standard, and unknown test portion extract were measured. A calibration curve was constructed from plotting the analyte: internal standard peak area ratio versus concentration of the calibration standards and performing weighted (1/x) least squares linear regression. Analyte concentrations for unknown test portions were determined using Equation 1 and the regression equation from the calibration curve.

$$\mathit{Amount}\mathrm{ }\left(\mathrm{\%}\right)=\frac{\left[\frac{PA\mathrm{ }\mathit{Analyte}}{PA\mathrm{ }IS}\mathrm{ }x\mathrm{ }IS\mathrm{ }\mathit{conc}\mathrm{ }\left(\frac{\mathrm{\mu }g}{mL}\right)\right]-\mathit{Intercept}}{\mathit{Slope}\mathrm{ }x\mathrm{ }\mathit{Sample}\mathrm{ }\mathit{Concentration}\mathrm{ }\left(\frac{\mathrm{\mu }g}{mL}\right)}\times 100\mathrm{\%}\mathrm{ }$$ (1)

If the analyte response was above the highest calibration standard, the extract was diluted with diluent and retested along with calibration standards and check standards.

Confirmation of analytes was achieved by comparing the ion ratio (ratio of determinative to confirmatory ion transitions) in the unknown test portions to the mean ion ratio of the calibration standards from the same analytical batch. To ensure reliable identification and quantitation, the ion ratio of an unknown test portion must be ±30% of the mean ion ratio of the calibration standards to confirm a given analyte. This criterion was applied to the chromatographic data obtained for four example botanical samples illustrated in Figures 3–5.

Figure 3.

Example chromatograms for Psilocybe cubensis mushroom.

Figure 4.

Example chromatograms for ayahuasca decoction (tea).

Figure 5.

Example chromatograms for San Pedro cactus (Trichocereus pachanoi) and coca leaf (Erythroxylum coca).

Figure 3 presents chromatograms for Psilocybe cubensis mushroom, showing five identified tryptamine compounds: psilocin, psilocybin, norpsilocin, baeocystin, and norbaeocystin. Figure 4 displays the chromatographic profile of an ayahuasca decoction (tea), highlighting the presence of DMT from Psychotria viridis and five β-carboline alkaloids from Banisteriopsis caapi. Figure 5 includes chromatographic examples of mescaline from San Pedro cactus (Trichocereus pachanoi) and cocaine from coca leaf (Erythroxylum coca). In each case, the ion ratios of the detected compounds were within the acceptable range, confirming their identity and supporting the robustness of the analytical method.

System Suitability

System suitability measures included bounds on the recovery (90–110%) and precision (≤5% RSD_r) of the standard response and precision (≤2% RSD_r) of the retention times of the analytes for the consecutive level 4 standard injections. These are determined from the initial six replicate injections of the level 4 standard in the analytical sequence. In addition, the correlation coefficient (r²) for the calibration curve must be ≥0.990. Finally, the analytes from unknown test portions must meet the confirmatory criteria.

Statistical Analysis

Data were analyzed for accuracy (recovery), precision (repeatability and intermediate standard deviations), specificity, limit of detection (LOD), and limit of quantitation (LOQ). Confidence intervals (95% and 90%) were determined on the recovery and relative standard deviation estimates. Acceptance criteria were 79–126% recovery and ≤33% RSD_r (15). To be acceptable, the 90% confidence intervals on recovery and precision must be contained entirely within the acceptance criteria.

Results and Discussion

Specificity

The specificity of the method was evaluated by analysis of diluent and non-spiked surrogate matrixes according to the method. Diluent, maltodextrin, green tea, prickly pear cactus, and sage leaf demonstrated no detectable response for any of the 26 analytes. Reishi mushroom (Ganoderma lingzhi) produced one peak in the tryptamine MRM window with an ion ratio of the quantitative transition to the confirmatory transition within ±30% of the standards in that batch. Therefore, tryptamine was confirmed to be present in the matrix and no interfering peaks were observed. Confirmatory ion ratios for all analytes were verified and met the ±30% criteria.

Calibration and System Suitability

Calibration curves for 26 target compounds and the internal standard were analyzed by weighted (1/x) linear regression and system suitability parameters. The regression equations from the calibration curves are used to determine analyte concentrations for unknown test portions using Equation 1. Results for linear regression coefficient of determination, residuals, system suitability recovery, concentration RSD_r, and retention time RSD_r are shown in Table 2. The correlation coefficients ranged from 0.9931 to 0.9999, meeting the acceptance criterion of ≥0.990. The size of the residuals at each concentration was determined and ranged from –18.3% to +20.0%, meeting the criterion of residuals ±20%. The demonstration of random residuals above and below zero in Table 2 indicate the weighted linear regression is an appropriate model for the data. Weighted linear regression was used to avoid potential heteroscedasticity due to the large calibration curve concentration range between 0.016 and 1.00 µg/mL. System suitability parameters were met as demonstrated by standard response RSD_r ≤5%, retention time RSD_r ≤2%, and mean recovery of the Level 4 standard of 90–110%.

Table 2.

Calibration curve evaluation for linearity and system suitability

Analyte	R2	Residuals							Level 4 Standard
		L1	L2	L3	L4	L5	L6	L7	Mean recovery	Concentration RSDr	Retention time RSDr
Psilocin	0.9995	5.3	−3.9	−4.1	−0.4	3	−0.3	0.4	96	3.0	0.04
Psilocybin	0.9976	−12.8	7.9	10	0.2	0.5	−5.4	−0.4	93	5.8	0.1
Norpsilocin	0.9993	3.2	−5	−2	2.8	3.2	−0.3	−1.8	92	5.4	0.000
Norbaeocystin	0.9931	ND	−8.7	10.6	−1.8	4.3	−2	−2.4	91	9.6	0.2
Baeocystin	0.9957	−11.6	9.4	4.1	−3.2	9.7	−5.1	−3.4	99	2.9	0.000
Aeruginacin	0.9990	−1.3	1.1	4.4	−6.2	1.8	−0.2	0.4	97	6.1	0.2
Mescaline	0.9994	7.8	−6.1	−4	−1.6	2.1	1.2	0.6	100	1.6	0.2
Ibogaine	0.9983	−11.2	5.6	9.2	1.2	0.5	−2.8	−2.4	102	1.6	0.0
Codeine	0.9994	3.2	−6.3	1.3	1.5	2.6	−2.3	0	104	5.4	0.2
Cocaine	0.9984	−11.8	7.1	8.9	0.4	−0.3	−2.3	−2.1	103	5.4	0.04
d-Methamphetamine	0.9987	−10.5	6.5	6	1.2	2.3	−2.8	−2.7	93	3.0	0.1
MDMA	0.9995	−2.6	0.2	0.9	2.8	2.3	−2.5	−1.1	96	5.8	0.000
Ketamine	0.9996	3.9	−1.8	−3.3	−0.5	1.3	−1.5	2	92	5.4	0.1
LSD	0.9998	0.5	−1.7	1.6	0.8	−1.3	−0.9	0.9	106	9.6	0.03
DMT	0.9998	−1.3	−0.6	1.5	1.6	0.7	−1.4	−0.5	101	0.9	0.1
Harmine	0.9951	20	−13.1	−16.2	2.9	0.9	1.7	3.8	87	5.4	0.04
Harmaline	0.9969	16.2	−15.4	−18.3	0.5	−2	4.8	5.4	85	7.7	0.04
Harmol	0.9997	4.1	−4	−2.1	−0.2	2.1	0.4	−0.3	99	1.2	0.1
Harmalol	0.9991	6.4	−6	−3.9	−0.3	4.3	0.3	−0.7	97	2.2	0.1
THH	0.9979	10.7	−4.9	−11.7	0.6	2.4	1.6	1.3	95	7.3	0.1
5-MeO-DMT	0.9999	0.6	−0.8	0	−0.3	1.4	−0.9	0.1	99	0.9	0.1
5-OH-DMT	0.9997	0.3	0.8	0.2	−2.6	1.2	−1.4	1.4	101	1.2	0.2
DMT-N-Oxide	0.9995	3.8	−3.5	−3.3	0.7	3.5	−0.5	−0.6	96	1.6	0.1
N-Methyltryptamine	0.9992	−3.7	1.7	−0.3	2.9	3.7	−1.3	−2.9	98	1.8	0.1
Tryptamine	0.9989	−4.4	3	−1.6	3.9	4.1	−2.1	−2.8	102	2.9	0.000
2-MTHBC	0.9993	0.8	−1.8	−2.2	1.8	4.1	−0.6	−2.1	99	1.3	0.1

The matrix effects from other substances in the samples, which can potentially interfere with the measurement by enhancing or suppressing the signal, are minimized through dilution. Given the large dilution factor used during sample preparation and the sensitivity of the method, any residual matrix effects were expected to be negligible. Evaluation of the extent of matrix effects were assessed using deuterated standard spike recoveries in Table 3, matrix-matched calibration curve slope comparison in Table 4, and analyte quantitation comparison between solvent-based versus matrix-matched calibrations curves in Table 5.

Table 3.

Deuterated standard in matrix spike recovery

Analyte	16.6 mg/mL spike		83.3 mg/mL spike
	Recovery, %	RSDr, %	Recovery, %	RSD, %
Psilocin-d10	88	4	84	4
Psilocybin-d4	101	2	101	5
Mescaline-d3	89	4	92	2
Cocaine-d3	124	7	104	5
DMT-d4	85	14	103	15
Harmine-d3	103	9	100	3

Table 4.

Calibration curve slope comparison

Analyte	Matrix	Slope	% Difference
Psilocin	Solvent-based	0.0811	0.9
	Psilocybe mushroom	0.0803
Psilocybin	Solvent-based	0.0320	3.7
	Psilocybe mushroom	0.0308
Mescaline	Solvent-based	0.1520	8.9
	San Pedro stem	0.1656
Cocaine	Solvent-based	0.8757	2.9
	Coca leaf	0.9012
DMT	Solvent-based	0.3014	0.3
	Ayahuasca tea	0.3023
Harmine	Solvent-based	0.3785	8.5
	Ayahuasca tea	0.3464
5-OH-DMT	Solvent-based	0.4447	4.0
	Reishi mushroom	0.4268
Codeine	Solvent-based	0.0075	2.5
	Salvia leaf	0.0077

Table 5.

Analyte quantitation curve comparison

Analyte	Solvent-based curve	Method of standard addition	% Difference
	Amount, mg/kg
Psilocin	837	913	9.2
Psilocybin	103	103	0.1
Mescaline	2104	2058	2.2
Cocaine	6033	5986	0.8
DMT	2851	2606	8.6
Harmine	7655	7712	0.7

Deuterated standards were spiked into corresponding matched matrixes, Psilocybe cubensis mushrooms, San Pedro cactus, coca leaves, and ayahuasca decoction (tea) at 0.166 and 0.833 µg/mL concentrations. Five replicates at each spike concentration level were analyzed for the six deuterated compounds. Table 3 shows the mean recovery and RSD_r using the solvent-based calibration curve. The percent recovery ranges from 84 to 124% and the RSD_r from 2 to 15%, which are within the acceptance criteria.

Matrix-matched calibration curves were developed for psilocin and psilocybin in psilocybe mushroom, mescaline in San Pedro cactus, cocaine in coca leaf, DMT and harmine in ayahuasca decoction (tea), and 5-OH-DMT in reishi mushroom. The slopes of the seven-point calibration curves in solvent-based and matrix-matched were compared in Table 4. The percentage difference between the solvent-based curves and matrix-matched curves range between 0.3 and 8.9%.

By applying the method of standard addition to the matrix-matched calibration curves, the native analyte concentrations were determined. Analyte quantitation comparison between the use of solvent-based versus matrix-matched calibration curves is shown in Table 5. The percent difference between the solvent-based and matrix-matched quantities varied between 0 and 9%.

Due to the demonstrated minimal matrix effect, the use of a single solvent-based, mixed multi-analyte calibration curve presents several advantages for the simultaneous quantification of analytes from diverse botanical matrixes. This approach enables the concurrent determination of multiple analytes across different botanical samples, significantly reducing the number of individual calibration curves required. As a result, it enhances laboratory efficiency, increases sample throughput, and lowers overall analytical costs. Furthermore, applying a unified calibration model across varied matrixes ensures consistent quantification criteria, thereby improving method robustness, reproducibility, and reliability.

Accuracy (Recovery) and Repeatability Precision

The accuracy of an analytical procedure is the closeness of test results obtained by that procedure to the true value and should be established across the method range. Repeatability precision is a measure of the random error from a set of replicate test portions analyzed in one batch by one analyst. Accuracy and repeatability were determined using appropriate surrogate matrixes and maltodextrin (simulated finished product) spiked at 16.6 and 83.3 mg/kg. Five replicate test portions at each concentration were extracted and analyzed. Table 6 shows the results of the appropriate analyte/matrix combinations. Acceptance criteria were defined for spike recovery as 79–126%, with repeatability (RSD_r) ≤33%, and 90% confidence intervals encompassing the full range of recovery and precision data (17). The 90% confidence intervals for recovery met the criterion for most analytes and matrixes. The exceptions included norbaeocystin in maltodextrin, norbaeocystin at 16.6 mg/kg in reishi mushrooms, and harmaline in maltodextrin spiked at 16.6 mg/kg. The upper 90% confidence limit on repeatability was ≤33% for all analytes and matrixes except for norbaeocystin at both spike levels in maltodextrin and at the low spike level in reishi mushrooms.

Table 6.

Accuracy and repeatability precision in spiked bulking agent and spiked surrogate plant materials (results are based on analysis of five replicate test portions at each concentration)

Matrix	Analyte	Spiked concentration, mg/kg	Mean recovery, %	Confidence intervals on recovery		RSD, %	Confidence intervals on RSDr
				95%	90%		95%	90%
Mushroom analytes
Maltodextrin	Psilocin	16.6	95	92, 98	93, 97	2.4	1, 7	2, 6
		83.3	91	88, 95	88, 94	3.2	2, 9	2, 8
	Psilocybin	16.6	97	86, 108	89, 106	9.3	6, 29	6, 23
		83.3	90	87, 94	88, 93	3.2	2, 9	2, 8
	Norpsilocin	16.6	99	94, 103	95, 102	3.9	2, 11	3, 9
		83.3	99	96, 102	97, 102	2.7	2, 8	2, 6
	Norbaeocystin	16.6	174	99, 249	117, 232	35.0	23, 156	25, 115
		83.3	136	96, 176	105, 167	24.0	17, 96	18, 75
	Baeocystin	16.6	112	93, 130	97, 126	13.3	8, 41	9, 33
		83.3	100	93, 108	95, 106	5.6	3, 17	4, 14
	Aeruginacin	16.6	98	84, 113	87, 109	11.9	7, 33	7, 27
		83.3	98	92, 103	93, 102	4.5	3, 13	3, 11
Reishi mushrooms (Ganoderma lingzhi)	Psilocin	16.6	91	86, 95	87, 94	3.7	2, 11	2, 9
		83.3	91	88, 94	88, 93	2.7	2, 8	2, 6
	Psilocybin	16.6	93	91, 94	91, 94	1.6	1, 5	1, 4
		83.3	95	94, 97	94, 97	1.4	1, 4	1, 3
	Norpsilocin	16.6	96	94, 97	94, 97	20.0	1, 4	1, 4
		83.3	91	88, 93	89, 93	8.0	1, 6	1, 5
	Norbaeocystin	16.6	92	70, 115	75, 109	1.5	12, 63	13, 51
		83.3	88	79, 96	81, 94	2.2	4, 21	5, 17
	Baeocystin	16.6	98	91, 105	93, 104	5.8	4, 17	4, 14
		83.3	98	92, 103	94, 102	4.6	3, 13	3, 11
	Aeruginacin	16.6	98	95, 100	96, 100	2.2	1, 6	1, 5
		83.3	97	92, 102	93, 101	4.2	3, 12	3, 10
Cactus Analytes
Maltodextrin	Mescaline	16.6	108	104, 111	105, 110	2.5	2, 7	2, 6
		83.3	101	98, 104	98, 103	1.5	1, 4	2, 6
	Ibogaine	16.6	103	101, 104	101, 104	1.2	1, 3	1, 3
		83.3	100	97, 104	98, 103	0.7	0.4, 2	2, 7
Prickly pear cactus	Mescaline	16.6	99	95, 103	96, 102	3.2	2, 9	2, 8
		83.3	96	94, 97	94, 97	1.5	1, 4	1, 4
	Ibogaine	16.6	99	103, 107	103, 107	3.2	1, 5	1, 4
		83.3	101	98, 103	99, 103	2.0	1, 6	1, 5
Leaf Analytes
Maltodextrin	Codeine	16.6	106	100, 112	101, 111	5	3, 14	3, 11
		83.3	102	96, 108	98, 106	4	2, 12	3, 10
	Cocaine	16.6	105	103, 106	104, 106	1	1, 3	1, 2
		83.3	101	97, 105	98, 104	1	1, 3	2, 7
Salvia officinalis leaf	Codeine	16.6	104	94, 115	96, 112	8	5, 23	5, 19
		83.3	100	97, 104	98, 103	3	2, 8	2, 7
	Cocaine	16.6	106	104, 108	104, 107	2	1, 5	1, 4
		83.3	101	100, 102	100, 101	1	0, 2	0, 1
Synthetic Analytes
Maltodextrin	d-Methamphetamine	16.6	92	89, 95	90, 94	2.3	1, 6	1, 5
		83.3	83	83, 84	83, 84	0.7	0, 2	0, 2
	MDMA	16.6	96	92, 99	93, 98	2.9	2, 8	2, 7
		83.3	86	85, 88	85, 87	1.2	1, 3	1, 3
	Ketamine	16.6	96	95, 97	95, 97	1.1	1, 3	1, 3
		83.3	89	88, 90	89, 90	0.9	1, 3	1, 2
	LSD	16.6	108	105, 111	106, 110	2.0	1, 6	1, 5
		83.3	102	95, 108	97, 107	5.1	3, 15	3, 12
Ayahuasca Analytes
Maltodextrin	DMT	16.6	96	93, 99	94, 98	2	1, 7	2, 5
		83.3	104	100, 108	101, 107	3	2, 8	2, 7
	Harmine	16.6	84	80, 89	81, 88	4	2, 12	3, 10
		83.3	88	86, 89	86, 89	2	1, 5	1, 4
	Harmaline	16.6	81	75, 87	76, 85	6	3, 16	4, 14
		83.3	86	83, 90	84, 89	3	2, 9	2, 7
	Harmol	16.6	94	91, 98	92, 97	3	2, 8	2, 6
		83.3	103	100, 105	101, 104	2	1, 5	1, 4
	Harmalol	16.6	87	84, 91	84, 90	3	2, 10	2, 8
		83.3	97	96, 97	96, 97	0	0.3, 1	0, 1
	THH	16.6	90	84, 96	85, 95	6	3, 17	4, 14
		83.3	96	90, 101	92, 100	4	3, 13	3, 10
	5-MeO-DMT	16.6	94	91, 98	92, 97	3	2, 8	2, 7
		83.3	103	100, 106	101, 105	2	1, 7	1, 5
	5-OH-DMT	16.6	94	90, 99	91, 98	4	2, 12	3, 10
		83.3	104	100, 107	101, 106	3	2, 8	2, 7
	DMT-N-Oxide	16.6	93	92, 94	93, 94	1	1, 3	1, 2
		83.3	98	96, 99	97, 99	1	1, 3	1, 3
	N-Methyltryptamine	16.6	93	92, 94	92, 94	1	1, 2	1, 2
		83.3	98	97, 100	97, 100	1	1, 3	1, 3
	Tryptamine	16.6	106	98, 113	100, 111	6	3, 17	4, 14
		83.3	99	96, 102	97, 101	2	1, 7	2, 6
	2-MTHBC	16.6	94	93, 95	93, 95	1	1, 3	1, 2
		83.3	99	97, 101	98, 100	1	1, 4	1, 3
Brewed green tea	DMT	16.6	102	100, 103	100, 103	1	1, 4	1, 3
		83.3	105	102, 108	102, 107	3	1, 7	2, 6
	Harmine	16.6	85	82, 88	83, 88	3	2, 9	2, 7
		83.3	87	83, 91	84, 90	3	2, 10	2, 8
	Harmaline	16.6	82	78, 87	79, 86	5	3, 13	3, 11
		83.3	87	83, 91	84, 90	3	2, 11	1, 5
	Harmol	16.6	100	98, 103	98, 102	2	1, 6	1, 5
		83.3	104	101, 106	102, 106	2	1, 6	1, 5
	Harmalol	16.6	96	93, 99	93, 98	3	2, 8	2, 6
		83.3	100	96, 103	97, 102	3	2, 8	2, 6
	THH	16.6	94	86, 102	88, 100	7	4, 19	4, 16
		83.3	100	96, 103	97, 103	3	2, 9	2, 7
	5-MeO-DMT	16.6	100	99, 102	99, 102	2	1, 5	1, 4
		83.3	105	101, 108	102, 107	3	2, 8	2, 6
	5-OH-DMT	16.6	103	101, 105	101, 105	2	1, 5	1, 4
		83.3	105	103, 107	103, 107	2	1, 5	1, 4
	DMT-N-Oxide	16.6	98	95, 100	96, 100	2	1, 6	1, 5
		83.3	98	97, 100	97, 100	1	1, 4	1, 3
	N-Methyltryptamine	16.6	97	96, 99	97, 98	1	1, 3	1, 2
		83.3	99	97, 102	97, 101	2	1, 6	1, 5
	Tryptamine	16.6	106	103, 109	104, 108	2	1, 6	1, 5
		83.3	101	99, 103	99, 103	2	1, 5	1, 4
	2-MTHBC	16.6	98	97, 100	97, 100	1	1, 4	1, 3
		83.3	100	98, 102	99, 102	2	1, 4	1, 4

Norbaeocystin and harmaline exhibited inherently low MS signal intensity in the calibration curves, which, combined with the matrix effects, low spike concentration, possible spiking errors, and spike recovery efficiency contributed to the observed deviations from the acceptance criteria. The use of stable isotope-labeled internal standards for these analytes is recommended to compensate for matrix and variability during sample preparation and analysis.

For the psychedelic mushroom-related analytes, the mean recovery ranged from 90 to 174% in maltodextrin and from 88 to 98% in reishi mushroom matrix. Repeatability ranged from 2.4 to 35% RSD_r in maltodextrin and from 1.4 to 20% in reishi mushroom matrix. The 90% confidence intervals for recovery and repeatability for all analytes met the acceptance criteria except for norbaeocystin in maltodextrin at both spike levels. The results were much better in the mushroom matrix, with only the low level 90% confidence intervals of recovery and repeatability exceeding the acceptability bounds. Additional work is needed to identify the reason for these aberrant results for norbaeocystin, especially in maltodextrin.

For mescaline and ibogaine, recovery was slightly higher in maltodextrin than prickly pear cactus, but all 90% confidence intervals were within the acceptance criterion. Repeatability precision point estimates were ≤3.2% in all cases. The leaf-based analytes all met the acceptance criteria for recovery and repeatability precision.

The synthetic analytes spiked into maltodextrin demonstrated acceptable recovery, ranging from 83 to 108%, and repeatability was also acceptable, ranging from 0.7 to 5.1%. Finally, 12 ayahuasca-related analytes were spiked into maltodextrin and brewed green tea. Recoveries from maltodextrin ranged between 81 and 106% and those from tea ranged between 82 and 106%. Repeatability was also very similar between maltodextrin (0.5–6% RSD) and green tea (1–7%). The 90% confidence intervals on recovery and repeatability all met the acceptance criteria except harmaline at the low level, which slightly exceeded the low end of the acceptable recovery range.

Intermediate Precision

Matrixes containing native analytes were analyzed on two days by different analysts on the same instrument. Each analyst, on different days, prepared calibration curves from the same standard stock solutions and analyzed five replicate test portions of each matrix. Table 7 presents the repeatability and intermediate precision results. Repeatability precision (RSD_r) was similar between the two analysts and was ≤11.2%. In most cases, intermediate precision (RSD_i) was higher than RSD_r, as expected, and all estimates were ≤20.4%. While there is no acceptance criterion for intermediate precision, many of the 90% CIs on RSD_i meet the RSD_r criterion, and all meet the RSD_R criterion of ≤55%, so are deemed acceptable.

Table 7.

Intermediate precision in matrixes with native analyte concentrations

Analyte	Analyst 1, Day 1, N = 5		Analyst 2, Day 2, N = 5		Grand mean, g/kg		RSDi, %	90% Confidence intervals on RSDi
	Mean, g/kg	RSDr, %	Mean, g/kg	RSDr, %
Mushroom (Psilocybe cubensis)
Psilocin	3.061	1.2	3.807	2.9	3.434		11.7	9, 19
Psilocybin	10.510	3.0	7.121	1.5	8.815		20.4	15, 35
Norpsilocin	0.041	4.4	0.061	1.9	0.051		20.1	15, 35
Norbaeocystin	0.058	5.2	0.046	9.2	0.052		14.5	11, 24
Baeocystin	0.251	6.4	0.173	5.0	0.212		20.2	15, 34
Aeruginascin	0.034	9.2	0.029	11.2	0.031		12.7	9, 21
San Pedro cactus (Trichocereus pachanoi)
Mescaline	3.047	4.0	4.084	2.6	3.565		16	12, 26
Ayahuasca tea
DMT	2.435	2.8	2.309	4.8	2.400		5	3, 8
Harmine	1.642	3.2	2.123	4.9	1.882		14	10, 23
Harmaline	0.089	6.1	0.117	6.5	0.103		15	11, 26
Harmol	0.058	2.8	0.062	2.8	0.060		5	3, 7
Harmalol	0.004	0.0	0.004	0.0	0.004		4	3, 7
THH	0.761	6.3	0.971	3.0	0.866		14	10, 23
5-MeO-DMT	<LOD	<LOD	<LOD	<LOD	<LOD		<LOD	N/A
5-OH-DMT	0.001	0.0	0.001	5.8	0.001		20	15, 35
DMT-N-Oxide	<LOD	<LOD	<LOD	<LOD	<LOD		<LOD	N/A
NMT	0.072	2.1	0.071	2.9	0.072		2	2, 4
Tryptamine	<LOD	<LOD	<LOD	<LOD	<LOD		<LOD	N/A
2-MTHBC	0.001	4.1	0.001	4.0	0.001		4	3, 6

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ were determined in solution from six replicate injections of decreasing concentrations of each analyte. LOD was estimated as the lowest concentration tested with a signal-to-noise ratio of ≥3. LOQ was estimated as the lowest concentration tested with RSD_r ≤20%. Table 8 presents the LOD and LOQ estimates for each analyte. LOQ concentrations are all below the lowest calibration standard, L1 (1.6 mg/kg).

Table 8.

LOD and LOQ of analytes in solution

Analyte	LOD conc., mg/kg	Average signal to noise	LOQ conc., mg/kg	RSDr, %b	Analytical range, mg/kga
Psilocin	0.05	19	0.4	7	0.4–100
Psilocybin	0.8	13	0.8	10	0.8–100
Norpsilocin	0.2	5	1.6	ND	1.6–100
Norbaeocystin	1.6	23	1.6	ND	1.6–100
Baeocystin	1.6	1511	1.6	ND	1.6–100
Aeruginascin	1.6	8	1.6	ND	1.6–100
Mescaline	0.2	4	1.6	ND	1.6–100
Ibogaine	0.1	18	0.2	4	0.2–100
Codeine	1.6	15	1.6	ND	1.6–100
Cocaine	0.05	55	0.1	4	0.1–100
d-Methamphetamine	0.05	31	0.05	9	0.05–100
MDMA	0.05	26	0.05	7	0.05–100
Ketamine	0.05	29	0.1	6	0.1–100
LSD	0.05	1323	0.05	12	0.05–100
DMT	0.05	27	0.2	2	0.2–100
Harmine	0.05	958	0.05	14	0.5–100
Harmaline	0.1	9	0.8	6	0.8–100
Harmol	0.1	387	0.1	10	0.1–100
Harmalol	0.1	10	0.8	13	0.8–100
THH	0.1	33	0.2	16	0.2–100
5-MeO-DMT	0.05	41	0.8	2	0.8–100
5-OH-DMT	0.05	3968	0.2	8	0.2–100
DMT-N-Oxide	0.05	104	0.1	4	0.1–100
N-Methyltryptamine	0.1	2031	0.1	5	0.1–100
Tryptamine	0.1	14	0.8	7	0.8–100
2-MTHBC	0.05	33	0.05	13	0.05–100

^aNote upper range can be extended with sample dilution.

^bND = Not determined.

Robustness

Method robustness was evaluated by altering two method conditions: (1) removal of the guard column, and (2) removal of AmF from mobile phase A. Only these two parameters were changed to evaluate robustness. Each parameter was altered individually, and the effect was evaluated by comparison of the concentration precision from the system suitability series of six injections of the level 4 mixed standard solution. Table 9 presents the results of the study. Removal of the guard column had very little effect on the system suitability precision, while removing AmF from mobile phase A caused many peaks to shift outside the MRM window and, therefore, not be detected. The pH of the mobile phase affects the retention time of analytes on the column due to zwitterionic, weak acid, or weak basic properties of the compounds, thereby affecting the interaction of the compounds with the solid phase of the phenyl-hexyl column.

Table 9.

Robustness study results

Analyte or IS	Concentration RSDr of system suitability injections
	Method as written	LC without guard column	Mobile phase A without AmF
Diphenhydramine (IS)	0.03	0.07	Not Detected
Psilocin	0.02	0.03	0.04
Psilocybin	0.03	0.04	0.02
Norpsilocin	0.02	0.04	Not Detected
Norbaeocystin	0.15	0.13	0.13
Baeocystin	0.08	0.05	0.04
Aeruginascin	0.06	0.05	0.09
Mescaline	0.01	0.02	Not Detected
Ibogaine	0.02	0.02	Not Detected
Codeine	0.05	0.02	Not Detected
Cocaine	0.03	0.04	Not Detected
d-Methamphetamine	0.03	0.04	Not Detected
MDMA	0.02	0.03	Not Detected
Ketamine	0.02	0.02	Not Detected
LSD	0.02	0.01	Not Detected
DMT	0.01	0.05	Not Detected
Harmine	0.06	0.08	Not Detected
Harmaline	0.08	0.09	Not Detected
Harmol	0.01	0.03	Not Detected
Harmalol	0.02	0.02	Not Detected
THH	0.05	0.08	Not Detected
5-MeO-DMT	0.02	0.05	Not Detected
5-OH-DMT	0.02	0.04	Not Detected
DMT-N-Oxide	0.02	0.02	Not Detected
N-Methyltryptamine	0.02	0.02	Not Detected
Tryptamine	0.03	0.04	Not Detected
2-MTHBC	0.02	0.02	Not Detected

Conclusions

Given the promising therapeutic potential of psychedelic drug treatments in clinical research for mental health conditions, there is a critical need for a validated, multitarget analytical method to characterize botanical sources, extracts, and finished products containing psychedelic compounds. Accurate dosage determination is essential to ensure consistency in controlled clinical trials, support psychedelic-assisted therapy, and safeguard patient safety. To address this need, this study was to develop and validate an analytical method for the quantification of psychedelic and psychoactive compounds derived from plants and fungi. An UPLC-ESI-MS/MS method was developed and validated in accordance with USP <1225> (15) and AOAC OMA Appendix K (16) guidelines for the simultaneous determination of 26 psychedelic compounds across a range of botanical matrixes, including mushrooms, cactus, and leaves, as well as processed forms such as brewed tea and finished products formulated with maltodextrin as a bulking agent. The method was designed to meet predefined acceptance criteria using simple and objective design rules (17) and was successfully validated, demonstrating its suitability for accurate and reliable analysis across diverse botanical matrixes.

Credit Author Statement

Torey French (Data curation [Equal], Formal analysis [Equal], Methodology [Equal], Validation [Lead], Writing—review & editing [Equal]), Anthony Fontana (Conceptualization [Equal], Formal analysis [Equal], Methodology [Equal], Project administration [Lead], Validation [Equal], Writing—review & editing [Equal]), Robert A. LaBudde (Formal analysis [Equal]), Sharon L. Brunelle (Writing—original draft [Lead], Writing—review & editing [Equal]), and Sidney Sudberg (Conceptualization [Lead], Formal analysis [Equal], Writing—review & editing [Equal])

Conflict of Interest

A.J.F., T.F., and S.S. work for Alkemist Labs and declare no conflict of interest. S.B. is an independent consultant and received payment from Alkemist Labs.