HPLC-MS Urinalysis: Synthetic Cannabinoid 5F-AKB-48 Metabolites

Synthetic cannabinoids are illicit designer drugs whose use may result in serious health symptoms such as tachycardia, nausea and vomiting; overdose may result in overt and even fatal toxicity. One emerging designer cannabinoid, N-(1-Adamantyl)-1-(5-ﬂuoropentyl)-1H-indazole-3-carboxamide (also known as 5F-AKB-48, or APINACA), has been metabolically characterized in cryopreserved human hepatocytes; however, until now, it has not been profiled in authentic urine samples derived from patients actually experiencing symptoms of intoxication. In this paper, Holm et al. (2014) report on the complete metabolic profile of 5F-AKB-48 via high-performance liquid chromatography (HPLC) and mass spectrometry (MS), revealing potential biomarkers for drug intake with real-world applications for clinical and forensic scientists.¹

To accomplish this, the team performed in vivo analysis of liquid–liquid extracted urine samples and compared the results with in vitro analysis of human liver microsomal (HLM) incubations. They performed HPLC on both sample types, using Dionex UltiMate 3000 UHPLC instrumentation coupled with a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). For chromatographic separation, they relied upon a 2.6-μm Phenyl-Hexyl 100 Å column (50 mm x 2.1 mm, 0.3 mL/min flow rate). For detection, they conducted full scans (m/z 200 to 800; 70,000 resolution) with subsequent MS/MS scans (17,500 resolution) of the eight most intense ions. The team used TraceFinder software (revision 3.1, Thermo Scientific) for MS instrument control and Xcalibur software (Thermo Scientific) for data processing.

Using the authentic urine sample, Holm et al. determined that parent compound 5F-AKB-48 possesses a protonated molecular weight of 384.2446 and a retention time of 5.53 minutes. The limit of detection was 0.5 ng/mL, with a 5-μL injection volume. They detected no actual parent compound in the urine sample, indicating that the synthetic cannabinoid completely metabolizes. The team identified sixteen 5F-AKB-48 metabolites (see below table). The major metabolites underwent monohydroxylation (M12), dihydroxylation (M4, M10), and trihydroxylation (M2) of the adamantyl group, often with simultaneous hydroxylation of the N-fluoropentylindazole moiety. Other metabolic activities included oxidative defluorination with mono- or dihydroxylation of the adamantyl ring (M3, M5, M6, M7, M8, M14, M15). By comparison, the HLM incubations produced mostly mono-, di- and trihydroxylation of the adamantyl ring without clinically significant oxidative defluorination.

The researchers posit that this information, coupled with their proposed metabolic pathway, may assist clinicians in identifying screening targets for urinalysis. Given the complete metabolization of synthetic cannabinoids (as confirmed in this study), the current compilation of metabolite-specific information for 5F-AKB-48 represents a key step in advancing clinical and forensic screening for this designer drug.

Metabolite	Activity	Product Ions (m/z)	Retention Time (min)
M1	N-dealkylation, monohydroxylation	133, 151	0.95
M2	trihydroxylation	131, 149, 167, 249, 267	1
M3	defluorination, dihydroxylation, carboxylation	131, 149, 167	1.09
M4	dihydroxylation	133, 151, 251	1.44
M5	defluorination, monohydroxylation, carboxylation	133, 151	1.56
M6	defluorination, dihydroxylation	133, 151	1.56
M7	defluorination, monohydroxylation, ketone formation	133, 151	1.73
M8	defluorination, monohydroxylation, carboxylation	133, 151	1.81
M9	N-dealkylation, monohydroxylation	135	1.91
M10	dihydroxylation	131, 149, 167, 233, 251	2.22
M11	N-dealkylation, monohydroxylation	135	2.23
M12	monohydroxylation	133, 151	2.99
M13	monohydroxylation	135	3.90
M14	defluorination, monohydroxylation, ketone formation	135	3.95
M15	defluorination, monohydroxylation	135	3.97
M16	monohydroxylation	135	4.96

Reference

1. Holm, N.B., et al. (2014, May) “Metabolites of 5F-AKB-48, a synthetic cannabinoid receptor agonist, identiﬁed in human urine and liver microsomal preparations using liquid chromatography high-resolution mass spectrometry,” Drug Testing and Analysis, doi: 10.1002/dta.1663 [e-pub ahead of print].

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.