Quantification of [1-(5-fluoropentyl)-1H-indol-3-yl](naphthalene-1-yl)methanone (AM-2201) and 13 metabolites in human and rat plasma by liquid chromatography-tandem mass spectrometry

Abstract

AM-2201 is a popular synthetic cannabinoid first synthesized in 2000. AM-2201 pharmacokinetic and pharmacodynamic data are scarce, requiring further investigation. We developed a sensitive method for quantifying AM-2201 and 13 metabolites in plasma to provide a tool to further metabolic, pharmacokinetic and pharmacodynamic studies. Analysis was performed by liquid chromatography-tandem mass spectrometry. Chromatographic separation was performed by gradient elution on a biphenyl column with 0.1% formic acid in water/0.1% formic acid in acetonitrile:methanol 50:50 (v/v) mobile phase. Sample preparation (75 μL) consisted of an enzymatic hydrolysis and a supported liquid extraction. The method was validated with human plasma with a 0.025 or 0.050 – 50 μg/L working range, and cross-validated for rat plasma. Analytical recovery was 88.8 – 110.1% of target concentration, and intra- (n = 30) and inter-day (n = 30) imprecision <11.9% coefficient of variation. Method recoveries and matrix effects ranged from 58.4 – 84.4% and −62.1 to −15.6%, respectively. AM-2201 and metabolites were stable (±20%) at room temperature for 24 h, at 4°C for 72 h, and after three freeze-thaw cycles, and for 72 h in the autosampler after extraction. The method was developed for pharmacodynamic and pharmacokinetic studies with controlled administration in rats but is applicable for pre-clinical and clinical research and forensic investigations. Rat plasma specimen analysis following subcutaneous AM-2201 administration demonstrated the suitability of the method. AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid concentrations were 4.8±1.0, 0.15±0.03, and 0.34±0.07 μg/L, respectively, 8h after AM-2201 administration at 0.3 mg/kg (n = 5).

1. Introduction

Synthetic cannabinoids are novel psychoactive substances eliciting subjective effects resembling Δ⁹-tetrahydrocannabinol (THC) [1]. These new compounds were first synthesized as research tools for investigating the endocannabinoid system, but now clandestine chemists synthesize these drugs for recreational purposes. Many countries have passed laws banning the sale, possession and use of these substances [2-5], spurring development of new analogs to circumvent legislation. In February 2015, 137 synthetic cannabinoids were monitored by the European Union Early Warning System [6]. Generally, there are no pharmacological or toxicological data available when these substances are first confiscated from the street drug market. Moreover, emerging synthetic cannabinoids are not detected by immunoassay, and new highly sensitive and specific methods are needed to quantify low concentrations of these compounds and their metabolites [7].

AM-2201 ([1-(5-fluoropentyl)-1H-indol-3-yl](naphthalene-1-yl)methanone) is a synthetic cannabinoid with an aminoalkylindole structure, first synthetized by Alexandros Makriyannis in 2000 (Fig. 1) [8]. AM-2201 is a full agonist at the cannabinoid CB₁ receptor, possessing a binding affinity 40 times greater than that for THC [8, 9]. Similarly, affinity at CB₂ receptors, responsible for cannabinoid peripheral effects, is 14 times that of THC’s [8]. In humans, reported blood and serum AM-2201 concentrations were < 0.1 to 12 μg/L [10-18]. Active smoked doses of AM-2201 in humans range from 250 μg to 3 mg [4] and induce cannabimimetic effects such as dry mouth, nausea, vomiting, drowsiness, confusion, convulsions, mydriasis, tachycardia, and psychotropic effects [10, 16, 18-20]. Psychiatric complications such as anxiety, elevated affect, and acute psychosis are expected at higher doses [13, 19]. AM-2201 abuse prevalence is difficult to estimate as few reports on synthetic cannabinoids intake are available. AM-2201 is a controlled substance in many European countries, in Japan, and in the United States [18]. From 2011 to 2013, AM-2201 was the most common finding among 862 positive synthetic cannabinoid cases in Norway [21]. However, the percentage of positive cases dropped after AM-2201 was scheduled, decreasing from 70% in February 2012 to 5% in January 2013. Similarly, AM-2201 was the predominant synthetic cannabinoid in United States, present from 2011 – 2012 [22].

Fig. 1.

Structures and m/z values of protonated molecules of AM-2201 and metabolites included in the method. The position of the hydroxyl group is specified in brackets. Cleavage sites for tandem mass spectrometry analysis are indicated by an arrow.

In 2012, in vitro experiments using metabolic enzymes, liver microsomes and urine samples identified seven major AM-2201 metabolites in humans [9, 23]. Cytochrome P450 (CYPs) CYP2C9 and CYP1A2 were determined as the main enzymes involved in AM-2201 metabolism, leading to oxidation and defluorination [9]. The major AM-2201 metabolites generated with human liver microsomes were AM-2201 N-(4-hydroxypentyl), JWH-018 N-(5-hydropentyl), JWH-018 N-pentanoic acid, and glucuronides [9]. Hutter et al. further confirmed these results with the identification of six major metabolites in human urine samples from authentic forensic cases: AM-2201 N-(4-hydroxypentyl), AM-2201 6′-hydroxyindole, JWH-018 N-(5-hydroxypentyl), JWH-018 N-pentanoic acid, JWH-073 N-(4-hydroxybutyl), and JWH-073 N-butanoic acid [11]. In the same study, human oral AM-2201 self-administration (0.07 mg/kg) led to maximum AM-2201 serum concentration of 0.56 μg/L, 1.5 h after intake. AM-2201 N-(4-hydroxypentyl), AM-2201 6′-hydroxyindole, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid were the only metabolites identified in serum, the last two reaching 0.73 and 0.42 μg/L, respectively [11]. Similarly, AM-2201 intraperitoneal administration in rats led to the formation of AM-2201 N-(4-hydroxypentyl), AM-2201 6′-hydroxyindole, JWH-018 N-(5-hydroxypentyl), JWH-018 N-pentanoic acid, and JWH-073 N-butanoic acid, detected in urine. However, AM-2201 6′-hydroxyindole and JWH-018 N-pentanoic acid were the main metabolites, while JWH-018 N-(5-hydroxypentyl) concentration was below the limit of quantification [24]. In a recent study, Banister et al. conducted the first AM-2201 pharmacodynamic study in rats [25]. The authors demonstrated a link between AM-2201 intraperitoneal injection and decreased body temperature, but the correlation between AM-2201 and metabolites’ blood concentrations and effects is yet to be determined. AM-2201 and metabolites in vivo kinetics are still unknown, with the exception of a single case of oral self-administration of AM-2201 [11]. Further research is needed, especially as AM-2201 N-(4-hydroxypentyl) and JWH-018 N-(5-hydroxypentyl) were shown to possess CB₁ affinity and full agonist activity [9, 26, 27].

The aim of this study was to develop a sensitive method for quantifying AM-2201 and 13 potential metabolites in human and rat plasma. This highly sensitive method employing a small sample volume was developed for further pharmacodynamic/pharmacokinetic studies in rats. Only two methods for quantifying AM-2201 and metabolites in blood were previously published [11, 13]. But, neither method was fully validated for quantitative purposes and targeted fewer AM-2201 analytes than the 14 AM-2201 analytes in our current method. Patton et al. employed a small 100 μL blood volume that could be amenable for rat plasma specimens, however they did not include hydrolysis prior to analysis [13]. Hutter et al. included enzyme hydrolysis, however, a 500 μL blood volume was employed that is too large for supporting rat plasma pharmacokinetic studies [11]. Furthermore, validation parameters were not detailed in either report.

2. Materials and methods

2.1. Chemical and reagents

Working standards (AM-2201, AM-2201 N-(4-hydroxypentyl), AM-2201 6′- and 7′-hydroxyindole, JWH-018 N-(2-, 3-, 4-, and 5-hydroxypentyl), JWH-018 N-pentanoic acid, JWH-018 N-propanoic acid, JWH-073 N-(2-, 3-, and 4-hydroxybutyl), JWH-073 N-butanoic acid, JWH-018 N-(5-hydroxypentyl)-glucuronide, JWH-018 N-pentanoic acid-glucuronide, JWH-019 N-(6-hydroxyhexyl)-glucuronide, JWH-073 N-(4-hydroxybutyl)-glucuronide, and UR-144 N-(5-hydroxypentyl)-glucuronide)) and deuterated internal standards (IS) (AM-2201-d5, AM-2201 N-(4-hydroxypentyl)-d5, JWH-018 N-(5-hydroxypentyl)-d5, JWH-073 N-(4-hydroxybutyl)-d5, and JWH-073 N-butanoic acid-d5) were purchased from Cayman Chemical (Ann Arbor, MI, USA) and stored at – 20°C until use. AM-2201 for injection in rats was provided by the National Institute on Drug Abuse, Drug Supply Program (Rockville, MD, USA). LC-MS grade water, methanol, and formic acid (Optima^™ LC/MS), and hydrochloric acid (12.1 mol/L) and ammonium hydroxide (> 99.4%) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). LC-MS grade acetonitrile and HPLC grade tert-butyl methyl ether were acquired from Sigma-Aldrich^® (St. Louis, MT, USA). Distilled water was produced by an ELGA PURELAB^® Ultra Analytic purifier (Siemens Water Technologies, Lowell, MA, USA). BG-100 Red abalone enzyme solution from KURA Biotec (Puerto Varas, Chile) was diluted in distilled water to a concentration of 15,625 units/mL glucuronidase and 1,250 units/mL sulfatase. Ammonium acetate solution was prepared with ≥ 97% purity ammonium acetate salt (Sigma-Aldrich^®; St. Louis, MO, USA) dissolved in distilled water; pH was subsequently adjusted to 4.0 with glacial acetic acid (Fisher Scientific; Fair Lawn, NJ, USA).

2.2. Working solutions

Calibrator working solutions containing all 14 non-deuterated non-glucuronidated standards were prepared in methanol from 0.5 to 500 μg/L. Low, medium, and high quality control (QC) working solutions containing all 14 non-deuterated non-glucuronidated standards were prepared in methanol at 3, 20, and 400 μg/L for AM-2201 N-(4-hydroxypentyl), JWH-018 N-propanoic acid, and JWH-073 N-(2-hydroxybutyl) and 1.5, 20, and 400 μg/L for other analytes. IS solution with all five deuterated standards (Table 1) was prepared at 10 μg/L in methanol. A 20 mg/L in methanol glucuronide solution contained all five glucuronide standards. Solutions were stored in glass vials at – 20°C.

Table 1.

Mass spectrometry parameters for analytes and internal standards (IS). Glucuronide transitions were included during hydrolysis optimization only and scan speed was adjusted accordingly (between 15 and 20 scans per chromatographic peak).

Compound	IS	Q1 mass (m/z)	Q1 prerod bias (V)	Q3 masses (m/z)	Q3 prerod bias (V)	CE (V)	RT (min)
AM-2201	IS-1	360	−26	127 155	−23 −29	−47 −26	10.22
AM-2201 N-(4-hydroxypentyl)	IS-2	376	−26	127.10 155.10	−23 −29	−50 −24	8.63
AM-2201 6′-hydroxyindole	IS-4	376	−26	127 155	−23 −29	−50 −25	9.22
AM-2201 7′-hydroxyindole	IS-1	376	−26	127 155	−23 −29	−49 −28	10.47
JWH-018 N-(2-hydroxypentyl)	IS-5	358	−13	155 127	−29 −23	−25 −49	9.14
JWH-018 N-(3-hydroxypentyl)	IS-5	358	−25	155 127	−29 −23	−24 −48	8.91
JWH-018 N-(4-hydroxypentyl)	IS-5	358	−13	155 127	−29 −23	−22 −49	8.67
JWH-018 N-(5-hydroxypentyl)	IS-5	358	−13	155 127	−29 −23	−22 −45	8.76
JWH-018 N-pentanoic acid	IS-4	372	−27	155 127	−29 −23	−23 −48	8.73
JWH-018 N-propanoic acid	IS-4	344	−12	155 127	−29 −23	−25 −45	8.12
JWH-073 N-(2-hydroxybutyl)	IS-3	344	−12	155 127	−29 −23	−26 −44	8.67
JWH-073 N-(3-hydroxybutyl)	IS-3	344	−24	127 155	−23 −29	−45 −23	8.42
JWH-073 N-(4-hydroxybutyl)	IS-3	344	−12	127 155	−23 −29	−46 −22	8.35
JWH-073 N-butanoic acid	IS-4	358	−13	155 127	−29 −23	−24 −48	8.39
IS-1, AM-2201-d5		365	−13	127 155	−23 −29	−48 −26	10.20
IS-2, AM-2201 N-(4-hydroxypentyl)-d5		381	−14	127 155	−23 −29	−50 −25	8.60
IS-3, JWH-073 N-(4-hydroxybutyl)-d5		349	−25	155 127	−29 −23	−22 −43	8.32
IS-4, JWH-073 N-butanoic acid-d5		363	−13	155 127	−29 −23	−24 −48	8.36
IS-5, JWH-018 N-(5-hydroxypentyl)-d5		363	−13	155 127	−29 −23	−22 −50	8.73

CE, collision energy; RT, retention time;

^*, included during hydrolysis optimization only;

bold indicates quantification transitions.

2.3. Human plasma specimens

Blank human plasma was provided by the National Institutes of Health blood bank.

2.4. Rat plasma specimens

As proof of method, AM-2201 and metabolites were quantified in rat plasma samples after subcutaneous AM-2201 injection. Briefly, 14 male Sprague-Dawley rats were anesthetized with pentobarbital sodium (60 mg/kg intraperitoneal) and Silastic catheters (Dow Corning; Midland, MI, USA) were implanted into the jugular veins [28]. Rats received 0.1, 0.3, or 1.0 mg/kg subcutaneous AM-2201 in a volume of 1 mL/kg and 0.2 mL blood samples were withdrawn via the catheter at 8 time points, up to 24 h, following administration. Blood samples were collected into 1 mL tuberculin syringes, transferred to 1.5 mL heparinized (25 μL of 1000 iU/mL) plastic tubes on ice, and centrifuged. Plasma samples were decanted, 5 μL 250 mmol/L sodium metabisulfite added, and samples stored at – 80°C prior to analysis.

Blank rat plasma tested negative for all analytes. A pool of rat plasma collected up to 24 h after 1 mg/kg subcutaneous AM-2201 injection was prepared for hydrolysis optimization experiments.

Animal experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the National Institute on Drug Abuse Intramural Research Program Animal Care and Use Committee.

2.5. Sample preparation

Plasma samples (75 μL) were fortified with 7.5 μL standards (or methanol) and 7.5 μL IS solutions in polypropylene microcentrifuge tubes. After addition of 200 μL 400 mmol/L ammonium acetate solution, pH 4.0, and 16 μL β-glucuronidase solution (15,625 and 1,250 units/mL glucuronidase and sulfatase, respectively), tubes were capped, vortexed for 10 s, and incubated at 55°C for 1 h. A volume of 200 μL acetonitrile was added to the mixture and tubes were vortexed for 10 s and centrifuged at 4°C, 15,000 g for 5 min. An additional 500 μL ammonium acetate solution was added to samples and tubes were gently vortexed for 10 s, taking care not to solubilize the pellet, and centrifuged at 4°C, 15,000 g for 5 min. Supernatants were transferred into 1 mL ISOLUTE^® SLE+ cartridges from Biotage^® (Charlotte, NC, USA). Positive pressure was gently applied to the cartridges with fine pressure control (up to 25 mL/min through each cartridge), and cartridges allowed to equilibrate at ambient pressure for 5 min. Compounds were eluted with 2 × 3 mL tert-butyl methyl ether in conical glass tubes. Positive pressure was applied to complete elution. Eluates were evaporated to dryness under nitrogen at 40°C in a Cerex System 48 concentrator (SPEware Corporation; Baldwin Park, CA, USA). Samples were reconstituted with 150 μL mobile phase A:B 80:20 (v/v) and centrifuged at 4°C, 15,000 g for 5 min. Supernatants were transferred into autosampler vials containing glass inserts prior to injection onto the chromatographic system.

2.6. Hydrolysis optimization

2.6.1. Preliminary tests

Preliminary tests were performed to determine optimal hydrolysis of AM-2201 glucuronides in rat plasma. A single dose of 1.0 mg/kg AM-2201 was injected subcutaneously to five rats and plasma samples collected from 0.25 to 24 h after dosing. Samples were pooled and 75 μL were spiked with 7.5 μL methanol and 7.5 μL IS solution (10 μg/L) in duplicate and extracted following β-glucuronidase hydrolysis, and acid and base hydrolysis.

Following enzyme hydrolysis, samples were extracted as described in “Sample preparation”. For acid (or base) hydrolysis, 100 μL 12 mol/L HCl (or 100 μL 10 mol/L NaOH for samples undergoing base hydrolysis) were added to samples and tubes were capped, vortexed for 10 s, and incubated at 100°C for 45 min (base hydrolysis: 60°C for 20 min). Samples were neutralized with 100 μL 12 mol/L NaOH (or 100 μL 10 mol/L HCl for samples undergoing base hydrolysis). 200 μL acetonitrile were added, tubes vortexed for 10 s, and centrifuged at 4°C, 15,000 g for 5 min. Additional 500 μL 400 mmol/L ammonium acetate solution, pH 4.0, were added and tubes gently vortexed for 10 s, taking care not to solubilize the pellet. Samples were centrifuged at 4°C, 15,000 g for 5 min. SLE conditions were the same as described in “Sample preparation”.

2.6.2. Hydrolysis percentage

Optimal hydrolysis was assessed for five glucuronide analytes. Blank rat plasma (75 μL) was fortified with 7.5 μL methanol and 10 μL glucuronide solution (20 mg/L) in quintuplicate and extracted by three different methods: (1) samples were extracted as described in section 2.5; (2) samples were extracted as described in section 2.5 with distilled water instead of β-glucuronidase solution; (3) samples were extracted as described in section 2.5, with distilled water instead of β-glucuronidase solution, bypassing the incubation step. Residues were reconstituted with 1.5 mL instead of 150 μL at the end of the SLE to avoid saturation of the MS detector. Hydrolysis percentage at 55°C for 1 h, with and without β-glucuronidase, was calculated by comparing results from (1) and (3), and (2) and (3), respectively. For each glucuronide, % hydrolysis = (1 – MS peak area _hydrolyzed/ MS peak area _{not hydrolyzed}) × 100.

2.7. Instrumentation

LC-MS/MS analysis was performed on a Shimadzu LCMS-8050 mass spectrometer (triple quadrupole) equipped with an electrospray ionization source in positive ion mode (ESI+) and coupled with an LC-30AD HPLC system. Data were acquired with LabSolutions software version 5.72 (Shimadzu Corp; Columbia, MD, USA) and were processed with ASCENT software version 3.4 (Indigo BioAutomation; Indianapolis, IN, USA).

2.7.1. LC conditions

Separation was performed on a Raptor^™ LC biphenyl column (core-shell technology; length: 100 mm, internal diameter: 2.1 mm, particle size: 2.7 μm) combined with a 10 × 2.1 mm guard column of identical phase from Restek^® (Bellefonte, PA, USA). The run time was 18 min with a gradient mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in methanol:acetonitrile 50:50 (v/v) (B) at a flow rate of 0.7 mL/min. Initial conditions were 20% B, held for 2 min, increased to 81.4% B within 9 min, increased to 100% B within 0.2 min, held for 4.3 min, returned to 20% B within 0.1 min, and then held for 2.4 min. LC flow was directed to waste the first 7.6 min (7.1 min when glucuronides were included, during hydrolysis optimization) and after 11 min. The column was extensively washed by increasing flow to 1.2 mL/min between 11.2 and 15.5 min. Autosampler and column oven temperatures were 4°C and 40°C, respectively. The injection volume was 20 μL (5 μL when glucuronides were included).

2.7.2. Low-energy collision induced dissociation-tandem mass spectrometry and multiple reactions monitoring (MRM) scans

The mass spectrometer operated in scheduled multiple reaction monitoring (MRM) mode with two transition ions for each analyte (scan time: retention time ±0.2 min). Cleavage sites are depicted in Figure 1. Scan speed was optimized to produce 15 to 20 scans per chromatographic peak. MS parameter settings were optimized by injecting neat standards individually in A:B (50:50, v/v) and ramping Q1 and Q3 pre-rod bias voltage and collision energy (Table 1). Glucuronide transitions were included during hydrolysis optimization only and scan speed was adjusted accordingly. Both quadrupoles were set to unit resolution. ESI parameters were optimized in the chromatographic conditions of the analysis: nebulizing gas flow rate = 2 L/min, heating gas flow rate = 10 L/min, drying gas flow rate = 6 L/min, interface temperature = 350°C, DL temperature = 200°C, heat block temperature = 400°C. Nebulizing gas was nitrogen produced by a Genius 1051 nitrogen generator from Peak Scientific^® (Billerica, MA, USA). Collision gas was Ultra-High Purity argon (99.999%).

2.8. Method validation

The method was validated for linearity, limit of detection and quantification, analytical recovery, and imprecision, interferences, recovery and matrix effect, carryover, dilution integrity, and stability in human plasma according to the Scientific Working Group for Forensic Toxicology (SWGTOX) forensic toxicology recommendations [29]).

2.8.1. Linearity

Dynamic range with the most appropriate calibration points, calibration model, and weighting for each analyte were determined with preliminary experiments. Calibration curves were calculated by linear least squares regression with six to seven calibrators, defining the lower (LLOQ) and the upper (ULOQ) limits of quantification. Linearity was assessed with six calibration curves across six different days. Calibrators were required to quantify within ±15% of target concentration (±20% for LLOQ) and precursor/product ion ratios were required to be within ±20% of the average calibrator ion ratio.

2.8.2. Sensitivity

Limit of detection (LOD) was determined with three different sources of plasma in duplicate over three different days (n = 18). LOD criteria were fulfilled if a peak eluted within ±0.1 min of average calibrator retention time with signal:noise ratio ≥3:1 and a quantifier:qualifier ratio within ±20% of mean calibrator ratio.

2.8.3. Interferences

Matrix interferences were evaluated in quintuplicate across five different days. Samples were fortified with 15 μL methanol instead of the standards and the internal standards (blank samples, n = 5). Interferences were considered negligible if no chromatographic peak was detected at the retention time of the analytes, if a peak was detected but presented a signal to noise ratio ≤3:1, or if the quantifier/qualifier ratio was outside ±20% of mean calibrator ratio.

Interferences from the internal standards solutions were evaluated in quintuplicate across five different days. Samples were spiked with methanol instead of the analytes (negative samples, n = 5). Interferences were considered negligible if the samples quantified below the LOD.

Interferences from standards solutions were evaluated by analyzing samples fortified with one analyte at a time at the highest calibrator concentration (50 μg/L). Interferences were considered negligible if samples quantified below the LOD for analytes not included in the fortified samples.

2.8.4. Analytical recovery (bias) and imprecision

Intra- and inter-day analytical recovery and imprecision were determined at 3, 20, and 400 μg/L for AM-2201 N-(4-hydroxypentyl), JWH-018 N-propanoic acid, and JWH-073 N-(2-hydroxybutyl) and 1.5, 20, and 400 μg/L for the other analytes. Analytical recovery and imprecision were evaluated with quadruplicate on five different days at each concentration (n = 30, at low, medium, and high QC concentrations). Each QC was required to quantify within ±20% of target and fulfil LOD criteria. Imprecision was expressed as a coefficient of variation (CV) of calculated concentrations. Intra- and inter-day differences were tested via one-way analysis of variance.

2.8.5. Extraction efficiency and matrix effect

Extraction recovery and matrix effect were evaluated at low and high QCs with ten plasma sources (n = 10). Three sets of samples were prepared: A) samples fortified and extracted as described in “sample preparation”; B) samples fortified with analytes and internal standards after SLE; C) neat standards fortified into 6 mL elution solvent; all sets were completely evaporated under nitrogen and reconstituted in 150 μL mobile phase A:B 80:20, v/v. Recovery was calculated by dividing the chromatographic peak area of set A by the chromatographic peak area of set B. Matrix effect was calculated by dividing the chromatographic peak area of set B by the chromatographic peak area of set C.

2.8.6. Carryover

Carryover was evaluated in triplicate by injecting a high calibrator followed by a negative sample. Carryover was considered negligible if negative samples quantified below the LOD.

2.8.7. Dilution integrity

Dilution integrity was demonstrated at a high QC concentration in triplicate. Samples were spiked at 400 μg/L, diluted tenfold in blank plasma and extracted as described in “Sample preparation”. QCs were required to quantify within ±20% of target (40 μg/L).

2.8.8. Stability

Analyte stability was determined at room temperature for 24 h, at 4°C for 72 h, after three freeze and thaw cycles, and after 72 h on the 4°C autosampler. Internal standards were added immediately before extraction. Experiments were prepared in triplicate, or quadruplicate for stability on the autosampler, at a low and high QC concentration.

2.8.9. Human-rat plasma cross-validation

Due to limited access to blank rat plasma, the method was validated in human plasma. Human-rat plasma cross-validation was performed to ensure applicability to rat plasma. Validation parameters included linearity, matrix interferences, analytical recovery and imprecision, extraction recovery, and matrix effect.

Interferences, analytical recovery, imprecision, extraction recovery, and matrix effect were evaluated in rat plasma using a calibration curve prepared with blank human plasma. Interferences from matrix components were evaluated by extracting a blank sample as described in 2.8.3. Analytical recovery and imprecision experiments were prepared in quadruplicate at a low, medium, and high QC concentration in human (n = 4, at low, medium, and high QC concentrations) and rat plasma (n = 4, at low, medium, and high QC concentrations). Recovery and matrix effect experiments were prepared in quadruplicate at a low and high QC concentration (n = 4, at low and high QC concentrations). Experiments were the same as described in 2.8.5. QCs from the analytical recovery and imprecision evaluations and from set A) of recovery evaluation were required to quantify within ±20% of target and present a quantifier/qualifier ratio within ±20% of mean calibrator ratio.

3. Results

3.1. Hydrolysis optimization

No significant differences in metabolite plasma concentrations were observed between nonhydrolyzed and hydrolyzed rat samples, regardless of enzymatic, acid, or base hydrolysis.

Percentages of hydrolysis under the extraction conditions (enzymatic hydrolysis with 250 and 20 units glucuronidase and sulfatase, respectively, for 1 h, at 55°C) were 97.3, 96.6, 98.1, 97.7, and 97.7% for JWH-018 N-(5-hydroxypentyl)-glucuronide, JWH-018 N-pentanoic acid-glucuronide, JWH-073 N-(4-hydroxybutyl)-glucuronide, JWH-019 N-(6-hydroxyhexyl)-glucuronide, and UR-144 N-(5-hydroxypentyl)-glucuronide, respectively.

3.2. Validation parameters

Chromatograms of a negative human plasma sample and a human plasma sample fortified at the LLOQ are shown in Figure 2. The chromatogram of an authentic rat plasma sample, 8 h following AM-2201 subcutaneous injection, is shown in Figure 3.

Fig. 2.

Extracted Ion Chromatograms (XIC) obtained from AM-2201 (A1), AM-2201 N-(4-hydroxypentyl) (A2), AM-2201 6′-hydroxyindole (A3), AM-2201 7′-hydroxyindole (A4), JWH-018 N-(2-hydroxypentyl) (A5), JWH-018 N-(3-hydroxypentyl) (A6), JWH-018 N-(4-hydroxypentyl) (A7), JWH-018 N-(5-hydroxypentyl) (A8), JWH-018 N-pentanoic acid (A9), JWH-018 N-propanoic acid (A10), JWH-073 N-(2-hydroxybutyl) (A11), JWH-073 N-(3-hydroxybutyl) (A12), JWH-073 N-(4-hydroxybutyl) (A13) and JWH-073 N-butanoic acid (A14) quantification transitions in blank human plasma and in blank human plasma fortified with all analytes at the lower limit of quantification (B1 to B14). Plain black line indicates raw data signal; dashed horizontal line indicates signal baseline; blue area indicates the actual peak integration; arrows indicate expected retention times; CPS, count per second. ¹transition 360 – 127, ²376 – 127, ³358 – 155, ⁴372 – 155, ⁵344 – 155, ⁶344 – 127

Fig. 3.

Extracted Ion Chromatogram (XIC) obtained from an authentic rat plasma specimen. Blood was collected 8 h after 0.3 mg/kg AM-2201 subcutaneous injection. AM-2201 (quantification transition A1 and qualification transition A2), JWH-018 N-(5-hydroxypentyl) (B1 and B2), and JWH-018 N-pentanoic acid (C1 and C2) concentrations were 6.5, 0.2, and 0.4 μg/L, respectively. Plain black line indicates raw data signal; dashed horizontal line indicates signal baseline; blue area indicates the actual peak integration; CPS, count per second.

3.2.1. Linearity and sensitivity

Calibration ranges were from 0.1 to 50 μg/L for AM-2201 N-(4-hydroxypentyl), JWH-018 N-propanoic acid, and JWH-073 N-(2-hydroxybutyl) (n = 6, 6 calibrators) and from 0.05 to 50 μg/L for the other analytes (n = 6, 7 calibrators). All regression curves were linear with 1/x weighting. Calibrators quantified within ±15% of target. LODs ranged from 0.025 to 0.1 μg/L (n = 18) (Table 2).

Table 2.

Validation parameters for AM-2201 and metabolites: limit of detection (LOD), working range, accuracy and imprecision. Low, medium, and high quality control working solutions contained all 14 standards at 3, 20 and 400 μg/L for AM-2201 N-(4-hydroxypentyl), JWH-018 N-propanoic acid, and JWH-073 N-(2-hydroxybutyl) and 1.5, 20 and 400 μg/L for other analytes.

Compound	LOD (μg/L) (n = 18)	Linear range (μg/L)	Intra-day accuracy (%) ± CV (%)			Inter-day accuracy (%) ± CV (%)
			Low (n = 30)	Mid (n = 30)	High (n = 30)	Low (n = 30)	Mid (n = 30)	High (n = 30)
AM-2201	0.025	0.05 – 50	103.3 ± 3.8	98.5 ± 1.8	98.7 ± 1.8	104.3 ± 5.1	100.5 ± 2.1	99.5 ± 2.5
AM-2201 N-(4-hydroxypentyl)	0.050	0.10 – 50	107.9 ± 5.7	110.1 ± 2.6	102.5 ± 3.8	109.6 ± 4.5	108.8 ± 3.5	103.8 ± 3.9
AM-2201 6′-hydroxyindole	0.025	0.05 – 50	91.8 ± 4.6	104.5 ± 6.2	106.6 ± 2.4	100.7 ± 10.0	107.2 ± 5.6	106.8 ± 8.0
AM-2201 7′-hydroxyindole	0.050	0.05 – 50	102.7 ± 1.2	107.5 ± 7.0	99.0 ± 3.0	100.8 ± 8.9	103.8 ± 9.5	100.0 ± 11.9
JWH-018 N-(2-hydroxypentyl)	0.025	0.05 – 50	100.5 ± 10.1	106.8 ± 10.1	103.2 ± 4.8	101.7 ± 10.2	104.4 ± 7.0	105.2 ± 7.0
JWH-018 N-(3-hydroxypentyl)	0.050	0.05 – 50	104.8 ± 1.6	105.6 ± 3.5	105.7 ± 5.8	103.9 ± 6.2	104.3 ± 3.6	105.8 ± 6.0
JWH-018 N-(4-hydroxypentyl)	0.025	0.05 – 50	97.2 ± 2.3	102.3 ± 1.3	98.1 ± 3.7	100.6 ± 4.4	100.5 ± 3.2	97.5 ± 3.9
JWH-018 N-(5-hydroxypentyl)	0.050	0.05 – 50	97.0 ± 1.6	105.2 ± 5.5	102.6 ± 3.8	101.4 ± 5.1	99.7 ± 4.5	101.6 ± 5.1
JWH-018 N-pentanoic acid	0.050	0.05 – 50	104.8 ± 5.5	101.3 ± 0.9	102.1 ± 3.8	102.9 ± 7.1	103.7 ± 3.2	104.7 ± 5.2
JWH-018 N-propanoic acid	0.100	0.10 – 50	105.5 ± 5.2	102.1 ± 1.5	103.0 ± 2.6	103.2 ± 6.1	101.5 ± 3.8	104.5 ± 4.7
JWH-073 N-(2-hydroxybutyl)	0.050	0.10 – 50	104.8 ± 5.5	101.3 ± 0.9	102.1 ± 3.8	107.7 ± 6.4	109.4 ± 5.4	103.4 ± 7.0
JWH-073 N-(3-hydroxybutyl)	0.050	0.05 – 50	91.7 ± 6.3	100.9 ± 6.0	99.9 ± 2.4	97.5 ± 8.3	104.1 ± 3.9	102.6 ± 4.7
JWH-073 N-(4-hydroxybutyl)	0.050	0.05 – 50	88.8 ± 4.2	99.1 ± 4.3	99.4 ± 0.3	92.3 ± 9.6	99.5 ± 5.5	99.0 ± 2.1
JWH-073 N-butanoic acid	0.050	0.05 – 50	93.7 ± 4.8	102.1 ± 2.8	103.0 ± 3.8	101.2 ± 6.4	104.2 ± 2.7	102.6 ± 3.2

CV, coefficient of variation.

3.2.2. Interferences

No interferences were detected, either from matrix components, standards solutions, or internal standards solutions. All low QCs quantified within ±20% of target concentration when potential interferences were added.

3.2.3. Bias and imprecision

Inter- and intra-day bias ranged from 88.8 – 110.1% and 92.3 – 109.4%, respectively. Inter- and intra-day imprecision ranged from 0.3 – 10.7% and 2.1 – 11.9%, respectively (%CV) (Table 2).

3.2.4. Extraction efficiency and matrix effect

Extraction recoveries ranged from 58.4 – 84.4% and matrix effects ranged from −62.1 to −15.6% (n = 10) (Table 3). Analytes from set A quantified within ±20% of target in ten plasma specimens from different sources.

Table 3.

Validation parameters for AM-2201 and metabolites: method recovery, and matrix effect. Low and high quality control working solutions contained all 14 standards at 3 (low) and 400 (high) μg/L for AM-2201 N-(4-hydroxypentyl), JWH-018 N-propanoic acid, and JWH-073 N-(2-hydroxybutyl) and 1.5 and 400 μg/L for other analytes.

Compound	Recovery (%)		Matrix effect (post peak areas CV) (%)
	Low (n = 10)	High (n = 10)	Low (n = 10)	High (n = 10)
AM-2201	65.7	61.3	−57.6 (64.0)	−47.7 (65.0)
AM-2201 N-(4-hydroxypentyl)	75.7	84.4	−37.3 (55.7)	−35.8 (52.7)
AM-2201 6′-hydroxyindole	67.7	70.4	−46.0 (61.0)	−42.1 (59.2)
AM-2201 7′-hydroxyindole	65.2	58.4	−62.1 (85.2)	−55.5 (83.8)
JWH-018 N-(2-hydroxypentyl)	69.2	71.1	−44.2 (62.8)	−38.4 (60.4)
JWH-018 N-(3-hydroxypentyl)	71.4	76.7	−45.2 (59.3)	−40.9 (55.9)
JWH-018 N-(4-hydroxypentyl)	75.8	81.5	−35.3 (48.7)	−21.5 (47.0)
JWH-018 N-(5-hydroxypentyl)	74.0	79.4	−33.3 (54.1)	−23.8 (53.5)
JWH-018 N-pentanoic acid	73.3	79.0	−22.9 (47.1)	−16.4 (45.6)
JWH-018 N-propanoic acid	73.4	78.5	−32.5 (34.5)	−30.4 (34.3)
JWH-073 N-(2-hydroxybutyl)	69.0	80.5	−37.6 (52.9)	−32.1 (49.1)
JWH-073 N-(3-hydroxybutyl)	78.5	81.4	−46.7 (49.1)	−38.7 (46.5)
JWH-073 N-(4-hydroxybutyl)	77.5	84.4	−32.5 (44.9)	−28.6 (41.9)
JWH-073 N-butanoic acid	74.4	78.6	−26.6 (37.9)	−15.6 (34.7)

3.2.5. Carryover

AM-2201, AM-2201 6′-hydroxyindole, and AM-2201 7′-hydroxyindole carryover was detected after the injection of samples spiked at 50 μg/L. The negative samples quantified below the LLOQ for the three compounds. There was no carryover for the other analytes (n = 3).

3.2.6. Dilution integrity

High QC accuracies were within 13.2% of diluted target concentration after 1:10 dilution (n = 3) (Table 4).

Table 4.

Validation parameters for AM-2201 and metabolites: dilution integrity and stability. Low and high quality control working solutions contained all 14 standards at 3 (low) and 400 (high) μg/L for AM-2201 N-(4-hydroxypentyl), JWH-018 N-propanoic acid, and JWH-073 N-(2-hydroxybutyl) and 1.5 and 400 μg/L for the other analytes.

Compound	Dilution integrity 1/10 (% target)	24 h, room temperature (% target)		72 h, 4° C (% target)		3 freeze & thaw cycles (% target)		72 h after extraction, autosampler (% target)
	High (n = 3)	Low (n = 3)	High (n = 3)	Low (n = 3)	High (n = 3)	Low (n = 3)	High (n = 3)	Low (n = 4)	High (n = 4)
AM-2201	89.3	102.9	99.5	107.3	98.9	112.9	99.8	102.5	100.0
AM-2201 N-(4-hydroxypentyl)	98.1	105.3	101.8	109.8	102.6	108.3	102.5	113.8	104.3
AM-2201 6′-hydroxyindole	93..2	102.2	99.8	104.7	106.4	106.7	105.3	93.2	98.3
AM-2201 7′-hydroxyindole	96.7	106.9	103.5	107.8	96.0	103.6	99.9	114.0	116.5
JWH-018 N-(2-hydroxypentyl)	94.3	101.8	101.0	105.6	107.0	107.1	99.2	110.0	107.5
JWH-018 N-(3-hydroxypentyl)	90.7	103.6	105.3	95.3	104.6	106.9	100.3	105.7	107.1
JWH-018 N-(4-hydroxypentyl)	89.7	97.6	95.8	97.3	97.9	99.3	96.6	108.7	101.9
JWH-018 N-(5-hydroxypentyl)	89.4	98.7	97.5	99.6	99.6	101.1	100.6	107.5	102.0
JWH-018 N-pentanoic acid	92.3	97.6	101.7	91.8	100.2	109.3	101.2	101.7	107.0
JWH-018 N-propanoic acid	86.8	102.2	110.0	110.1	109.1	112.3	109.1	111.6	106.3
JWH-073 N-(2-hydroxybutyl)	98.1	111.9	104.2	110.7	103.5	107.6	97.4	115.1	107.4
JWH-073 N-(3-hydroxybutyl)	97.2	89.3	100.6	100.9	103.7	97.6	104.9	92.2	98.3
JWH-073 N-(4-hydroxybutyl)	91.3	91.8	96.7	94.2	98.9	87.3	96.3	89.2	96.6
JWH-073 N-butanoic acid	91.5	98.0	102.4	91.3	101.8	102.7	101.9	105.5	107.6

3.2.7. Stability

Analytes were stable for 24 h at room temperature in plasma with 89.3 – 111.9% analytical recovery (n = 6), for 72 h at 4°C in plasma with 91.3 – 110.7% analytical recovery (n = 6), after three freeze and thaw cycles in plasma with 87.3 – 112.9% analytical recovery (n = 6), and after 72 h following extraction in the autosampler at 4°C with 89.2 – 116.5% analytical recovery (n = 8).

3.2.8. Human-rat plasma cross-validation

No interferences were detected from matrix components in rat plasma. Extraction recoveries and matrix effects in rat plasma (n = 8) are reported in Table 3. Analytes from set A quantified within ±20% of target. Accuracies were 93.1 – 103.0% and imprecision was 2.0 – 9.6% (%CV) (n = 12).

3.2.9. Proof of method

As proof of method, a single dose of 0.3 mg/kg AM-2201 was injected subcutaneously to 5 rats and AM-2201 and metabolites were quantified in plasma 8 h after dosing. AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid were detected at 4.8±1.0, 0.15±0.03, and 0.34±0.07 μg/L, respectively. A chromatogram from one authentic specimen is shown in Figure 3.

4. Discussion

Several methods for quantifying AM-2201 and metabolites in urine have been previously published [11, 24, 30-35]. Until now, only two methods are available for AM-2201 and metabolites’ quantification in blood [11, 13]. Patton et al. quantified AM-2201 and three metabolites and identified another one in 100 μL postmortem blood. Concentrations of AM-2201 and an oxidated metabolite were 12 and 2.5 μg/L, respectively [13]. The same year, Hutter et al. developed an LC-MS/MS method for quantifying AM-2201 and 15 potential metabolites in human urine and serum with 500 μL sample volume [11]. LODs for AM-2201 and metabolites were 1 and 50 ng/L, respectively. These two studies were primarily designed for identifying AM-2201 metabolites with preliminary experiments quantifying AM-2201 and metabolites; a complete method validation was not presented. We present a fully validated method quantifying AM-2201 and 13 metabolites in 75 μL plasma. We attempted to include all commercially available AM2201 metabolites; AM-2201 2′-hydroxyindole and AM-2201 5′-hydroxyindole were eliminated from the method, as they didn’t fulfill sensitivity and analytical recovery requirements. Neither of these analytes were identified as major metabolites during in vitro experiments [9, 23], preclinical studies [24, 36], or in authentic human samples [9, 11, 20]. The method was sensitive, with LLOQs from 50 – 100 ng/L, for in vivo quantification in human and rat specimens (0.05 – 12 μg/L in blood) [10-18]. This is currently the most sensitive method for the quantification of AM-2201 metabolites in biological specimens, considering the low volume sample (75 μL plasma) and LODs (0.025 – 0.100 μg/L).

4.1. LC-MS analysis

Chromatographic resolution of positional isomers was critical, as MS fragments and physical and chemical properties were similar. A biphenyl column was suitable for AM-2201 and metabolites with a naphthoylindole core, due to the ability to separate polar aromatic compounds based on π-π interactions. A Kinetex^® biphenyl column from Phenomenex (length: 100 mm, internal diameter: 2.1 mm, particle size: 1.7 μm) was evaluated but peak shapes were not Gaussian. A C₁₈ column also was tested, as it was employed in a previous study [9] and referenced in an application note from Phenomenex [37], but separation of AM-2201 hydroxyindole and JWH-018 N-hydroxy metabolites could not be achieved with multiple mobile phases, including those containing acetonitrile, methanol, and isopropanol. Higher proportions of methanol in mobile phase B diluted with acetonitrile improved AM-2201 5′- and 6′-hydroxyindole and JWH-018 N-(4- and 5-hydroxypentyl) separation but reduced JWH-018 N-(3- and 5-hydroxypentyl) and JWH-073 N-(3- and 4-hydroxybutyl) separation. JWH-073 N-hydroxybutyl and AM-2201 5′- and 6′-hydroxyindole metabolites separation was optimal with 100% methanol mobile phase B, while JWH-018 N-hydroxypentyl metabolites separation was optimal with 60% methanol. A similar phenomenon was observed with the column oven temperature; a high temperature favored separation of JWH-018 N-(2- and 3-hydroxypentyl) and JWH-073 N-(4- and 5-hydroxybutyl) but decreased separation of JWH-018 N-(3- and 4-hydroxypentyl) metabolites. A compromise was found with a methanol:acetonitrile 50:50 (v/v) mobile phase B and a 40°C column oven temperature.

Despite LC condition optimization, JWH-018 N-(4- and 5-hydroxypentyl) and JWH-073 N-(3- and 4-hydroxybutyl) co-eluted approximately 10% in the highest calibrator. This co-elution potentially affected analytical recovery and precision. To overcome this issue, data were processed with ASCENT software, which employs exponentially modified Gaussian model and is able to predict the shape of two co-eluting chromatographic peaks (Fig. 2) [38]. Validation experiments demonstrated that the presence or absence of JWH-018 N-(4-hydroxypentyl) in the sample did not interfere with JWH-018 N-(5-hydroxypentyl) quantification and vice versa (QC at 50 μg/L within ±20% of target even though both compounds were present in calibrators). The same assessment was made for JWH-073 N-(3- and 4-hydroxybutyl), confirming the ability of the software to accurately predict peak shape.

Trace AM-2201 and AM-2201 6′- and 7′-hydroxyindole carryover was observed after injection of the highest calibrator concentration with carryover in the ensuing negative sample equivalent to the LLOQ. Column rinse time at 100% B was extended; however, no significant carryover reduction was observed and only a between-injections rinse of the outside and the inside of the needle, the injection port, and the sample loop with acetonitrile:isopropanol:water 45:45:10 (v/v/v) decreased carryover to an acceptable limit (signal < 50% of LLOQ after injection of the highest calibrator concentration).

AM-2201 2′-hydroxyindole was initially included in the method but presented a different fragmentation pattern with low intensity ions m/z 127 and 155 compared to ions m/z 252 and 270 produced by N-dealkylation and loss of one or two H₂O groups. It was hypothesized that the hydroxyl group forms a tautomerism equilibrium with the ketone function that stabilizes the ketone. Consequently, AM-2201 2′-hydroxyindole signal intensity was low and led to a lack of sensitivity and the metabolite was removed from the method.

4.2. Sample preparation

Several buffers at different pH (pH = 3, 4, and 5) and organic phases were evaluated for optimizing SLE recoveries. tert-Butyl methyl ether achieved similar recoveries to ethyl acetate. Acetonitrile was added to samples before SLE (20%, v/v) to increase recovery of AM-2201 and AM-2201 6′- and 7′-hydroxyindole, the three most nonpolar analytes. AM-2201, AM-2201 N-(4-hydroxypentyl), JWH-018 N-(5-hydroxypentyl), JWH-073 N-(4-hydroxybutyl), and JWH-073 N-butanoic acid had matching deuterated IS. JWH-018 N-(5-hydroxypentyl)-d5, JWH-073 N-(4-hydroxybutyl)-d5, and JWH-073 N-butanoic acid-d5 were employed as internal standards for JWH-018 N-hydroxypentyl, JWH-073 N-hydroxybutyl, and JWH-018 N-carboxylic acid metabolites respectively, as they share physicochemical characteristics. JWH-018 pentanoic-d5 IS was initially chosen for acidic compounds but it interfered with the AM-2201 N-(4-hydroxypentyl) signal, as the two compounds partially co-eluted and the quadrupoles were set to unit resolution (JWH-018 pentanoic-d5 and AM-2201 N-(4-hydroxypentyl) masses are 376 and 375, respectively). JWH-018 5′- and 6′-hydroxyindole-d9 IS were tested for AM-2201 hydroxyindole metabolites quantification, but AM-2201-d5 appeared more suitable for AM-2201 7′-hydroxyindole, as they eluted nearer, and JWH-073 N-butanoic acid appeared more suitable for AM-2201 6′-hydroxyindole. QCs for all target analytes quantified within ±20% of target when using plasma samples of different origins, indicating internal standards compensated effectively to achieve reliable analyte quantification. AM-2201 5′-hydroxyindole, which was initially included in the method (retention time: 9.07 min), was excluded as matrix effect was not compensated by IS and several QCs quantified −54.1 – 57.6% target when fortified into different plasma lots.

In vitro studies and authentic urine specimen analysis had demonstrated the formation of AM-2201 glucuronidated metabolites [9, 23]. However, preliminary hydrolysis experiments never achieved ≥9% increase in peak areas of non-glucuronidated metabolites in plasma samples from rats that received an AM-2201 subcutaneous injection. Our experiments with fortified glucuronide standards demonstrated >96.6% hydrolysis efficiencies suggesting efficient hydrolysis should have occurred. Thus, the most likely reason for insignificant peak area changes after hydrolysis is the absence of or low concentration of glucuronidated AM-2201 and metabolites in rat plasma. This may be due to a rapid elimination of glucuronides in urine or the use of a pooled positive sample. Glucuronidation may occur at a specific time following administration, and pooling the samples may have diluted metabolites. As glucuronidation reactions were expected [9, 23] and in order to increase the sensitivity of the method, it was validated with an enzymatic hydrolysis step.

4.3. Proof of concept

The method was human-rat plasma cross-validated to ensure its applicability to rat plasma. Consequently, the method can be applied to human and rat plasma for further pre-clinical, clinical, or forensic investigations. As a proof of concept, AM-2201 and metabolites were quantified in rat plasma after AM-2201 subcutaneous injection. Major metabolites JWH-018 N-(5-hydroxypentyl) and JWH-018 N-pentanoic acid were detected, as expected in light of previous studies [9, 11, 20, 23, 24]. AM-2201 6′-hydroxyindole was not identified, although it had been detected as a major metabolite in rat urine [24]. AM-2201 6′-hydroxyindole may be rapidly excreted into urine after formation, therefore, we did not detect it in our samples despite our highly sensitive method (AM-2201 6′-hydroxyindole LLOQ = 0.05 μg/L).

5. Conclusion

A fully validated method for quantifying AM-2201 and 13 metabolites in human and rat plasma is presented. The chromatographic separation of isobaric compounds and the high required sensitivity, in regards to previously reported blood concentrations, were the most important challenges encountered during the method development. This is currently the most sensitive method reported in the literature, with a low volume sample, for quantifying AM-2201 metabolites in blood. It can be applied to both human and rat species for further pre-clinical, clinical, or forensic investigations. Analysis of AM-2201 dosed rat plasma specimens demonstrated the suitability of the method in authentic cases.

Highlights.

• Little pharmacokinetic or pharmacodynamics data are available for AM-2201

• We developed a method for quantifying AM-2201 and 13 metabolites in plasma

• The most sensitive method for quantifying AM-2201 metabolites in biological samples

• Rat plasma specimens following sc AM-2201 injection were analyzed