The main aim of the study was the identification of major in vitro phase I metabolites of CUMYL‐PINACA, 5F–CUMYL‐PINACA, CUMYL‐4CN‐BINACA, 5F–CUMYL‐P7AICA, and CUMYL‐4CN‐B7AICA with a focus on the differentiation of the partly isomeric compounds. All parent compounds – especially the constitutional isomers 5F–CUMYL‐PINACA and 5F–CUMYL‐P7AICA as well as CUMYL‐4CN‐BINACA and CUMYL‐4CN‐B7AICA – could be clearly separated by retention time (Figures 2 and 3). However, due to extensive metabolism, the parent compounds of synthetic cannabinoids are usually not detectable in urine, which is the matrix of choice for screening purposes.6 Therefore, elucidation of the metabolism is essential in forensic toxicology. Interpretation of parent compound fragmentation built the prerequisite for further metabolite assignment. The main postulated fragmentation patterns of the parent compounds core structure are depicted in Figure 4. LC–ESI–HRMS fragmentation of CUMYL‐4CN‐BINACA was already discussed by Ozturk et al and explains fragments a, b, c, e, and f in Figure 4.12 Fragments e and h can be explained as products of the respective alpha cleavage. Fragment h can also be explained by a consecutive loss of carbon monoxide from fragment e. Loss of the side chain from fragment h leads then to fragment i. Within the group of the parent compounds, fragment d1 was only observed for 5F–CUMYL‐P7AICA and represents fragment c with further cleavage in the side chain. Fragments g result from fragment e with cleavage within the side chain. The fragment m/z 177.0457 of 5F–CUMYL‐PINACA – present in the parent compound, M9 and M10 – was not displayed in the figure. It might be the product of a fluorine migration at the side chain followed by an alkene elimination already discussed by Shevyrin et al. 18

Extracted ion chromatograms of (A) CUMYL‐PINACA, (B) 5F–CUMYL‐PINACA, and (C) CUMYL‐4CN‐BINACA and their metabolites in an incubation mixture of human liver microsomes with 10 μg/mL parent compound. Intensity of the parent compounds was scaled down to improve representation of less abundant metabolites [Colour figure can be viewed at wileyonlinelibrary.com

Extracted ion chromatograms of (D) 5F–CUMYL‐P7AICA and (E) CUMYL‐4CN‐B7AICA and their metabolites in an incubation mixture of human liver microsomes with 10 μg/mL parent compound. Intensity of the parent compounds was scaled down to improve representation of less abundant metabolites [Colour figure can be viewed at wileyonlinelibrary.com

3.1 Identification of metabolites

The identified in vitro phase I metabolites of CUMYL‐PINACA, 5F–CUMYL‐PINACA, CUMYL‐4CN‐BINACA, 5F–CUMYL‐P7AICA, and CUMYL‐4CN‐B7AICA are given in Table 1. The product ions were labeled using the letters a–i according to Figure 4. Identification of the metabolites was performed by comparing the mass fragments in the metabolite spectra with the corresponding fragments of the parent compounds. For instance, a metabolite with a mass increase of 16 u at the fragments a and b with unchanged m/z of the other fragments was interpreted as the hydroxylation product at the aryl moiety. An increase of 16 u at the fragments c and e with fragment f remaining unchanged was interpreted as hydroxylation at the tail structure.

Table 1. Metabolites of CUMYL‐PINACA, 5F–CUMYL‐PINACA, CUMYL‐4CN‐BINACA, 5F–CUMYL‐P7AICA, and CUMYL‐4CN‐B7AICA including the underlying biotransformation, retention time, molecular formula, the accurate mass of the precursor ion [M + H] and the observed MS/MS product ions a–i Parent compound Metabolite # Biotransformation RT Formula M + H+ a b c d1 e f h Various CUMYL‐PINACA Parent compound 20.4 C22H27N3O 350.2227 91.0535 119.0850 232.1446 215.1223 145.0398 M5a‐b Dihydroxylation at pentyl moiety 12.3, 12.6 C22H27N3O3 382.2125 91.0540 119.0856 264.1350 175.0522 213.1184/ 229.1194/ 247.1385 145.0429 M6a‐d Hydroxylation and ketone formation 13.5, 13.95, 14.15, 14.8 C22H25N3O3 380.1969 91.0528 119.0842 262.1195 175.0479 245.0922 145.0394 M8 Hydroxylation at indazole moiety 18.6 C22H27N3O2 366.2176 91.0533 119.0850 231.1142 161.0342 M9a‐c Hydroxylation at pentyl moiety 14.9, 15.7, 16.5 C22H27N3O2 366.2176 91.0538 119.0851 248.1414/ 230.1525 (−H2O) 175.0524 231.1162/ 213.1073 (−H2O) 145.0397 M10a‐c Ketone formation 16.2, 17.1, 17.6 C22H25N3O2 364.2020 91.0538 119.0852 246.1235 229.1309 145.0395 M12 N‐desalkylation 13.3 C17H17N3O 280.1444 91.0542 119.0858 145.0438 M13a‐c N‐desalkylation and hydroxylation at indazole moiety 10.0, 11.0, 11.7 C17H17N3O2 296.1394 91.0534 119.0847 161.0343 5F–CUMYL‐PINACA Parent compound 19.0 C22H26FN3O 368.2133 91.0545 119.0868 250.1364 233.1159 145.0421 177.0457g5: 213.1078 M1 Dihydrodiol formation 12.6 C22H28FN3O3 402.2188 91.0546 119.0861 284.1441 267.1184 161.0352 (−H2O) g5: 247.1084 M2a‐d Dihydroxylation at aryl and pentyl moiety 12.0, 12.2, 12.4, 12.6 C22H26FN3O3 400.2031 107.0491 135.0802 266.1300 175.0506 249.1039/ 231.0928 145.0395 M3 Dihydroxylation at aryl moiety 14.6 C22H26FN3O3 400.2031 123.0439 151.0753 250.1345 233.1085 145.0396 g5: 213.1022 M4a‐b Dihydroxylation at pentyl and indazole moiety 13.7, 13.9 C22H26FN3O3 400.2031 91.0549 119.0861 282.1246 265.0989 161.0352 M7a‐c Hydroxylation at aryl moiety 12.5, 16.1, 16.3 C22H26FN3O2 384.2082 107.0494 135.0815 250.1352 233.1093 g5: 213.1027 M8a‐b Hydroxylation at indazole moiety 16.7, 17.5 C22H26FN3O2 384.2082 91.0539 119.0848 266.1298 249.1043 161.0345 g5: 229.0968 M9a‐c Hydroxylation at pentyl moiety 14.9, 15.1, 15.6 C22H26FN3O2 384.2082 91.0550 119.0856 266.1372 249.1109 145.0426 177.0461g5: 231.0959 M10 Ketone formation 16.8 C22H24FN3O2 382.1925 91.0532 119.0846 264.1163 247.0909 177,0479g5: 227.0816 M12 N‐desalkylation 13.3 C17H17N3O 280.1444 91.0557 119.0875 162.0687 145.0446 M13a‐c N‐desalkylation and hydroxylation at indazole moiety 10.7, 11.0, 11.7 C17H17N3O2 296.1394 91.0546 119.0860 161.0351 M20 Oxidative defluorination 15.0 C22H27N3O2 366.2176 91.0540 119.0850 248.1401 175.0504 231.1132/ 213.1027 145.0409 M21 Oxidative defluorination and carboxylation 14.7 C22H25N3O3 380.1969 91.0524 119.0865 262.1193/ 244.1085 227.0821 (−H2O) 145.0413 217.1013 M22a‐b Oxidative defluorination and carboxylation and hydroxylation at aryl moiety 12.0, 12.2 C22H25N3O4 396.1918 107.0479 135.0816 262.1180/ 244.1076 217.0966 M24a‐b Oxidative defluorination and hydroxylation at indazole moiety 12.7, 13.7 C22H27N3O3 382.2125 91.0541 119.0852 264.1338 191.0445 247.1075 161.0350 g5: 229.0969 M25a‐b Oxidative defluorination and hydroxylation at pentyl moiety 12.0, 12.2? C22H27N3O3 382.2125 91.0528 119.0842 264.1355 175.0505 247.1101 145.0391 g5: 229.0973 CUMYl‐4CN‐BINACA Parent compound 16.9 C22H24N4O 361.2023 91.0553 119.0870 243.1269 226.1004 145.0413 Amide hydrolysis 13.0 C13H13N3O2 227.0817 145.0389 M1 Dihydrodiol formation 10.7 C22H26N4O3 395.208 91.0524 119.0849 227.1298 260.1016/ 242.0911 161.0324 (−H2O) M2 Dihydroxylation at aryl and pentyl moiety 11.1 C22H24N4O3 107.0485 135.0797 242.0918/ 226.0974 145.0391 M3a‐b Dihydroxylation at aryl moiety 12.5, 14.1 C22H24N4O3 393.1921 123.0419 151.0741 243.1233 226.0977 145.0374 M7a‐c Hydroxylation at aryl moiety 10.3, 13.9, 14.1 C22H24N4O2 377.1972 107.0469 135.0798 243.1219 226.0983 145.0390 M8a‐b Hydroxylation at indazole moiety 14.6, 15.5 C22H24N4O2 377.1972 91.0534 119.0843 242.0924 226.1045 161.0358 M9 Hydroxylation at pentyl moiety 15.2 C22H24N4O2 377.1972 91.0537 119.0851 259.1215 242.0937 145.0401 g4: 215.0811 M11 Loss of nitrogen and carboxylation 14.8 C22H25N3O3 380.1969 91.0535 119.0850 262.1186/ 244.1100 175.0497 245.0920/ 227.0828 145.0390 217.0977 M12 N‐desalkylation 13.3 C17H17N3O 280.1444 91.0524 119.0837 162.0680 145.0434 M13a‐c N‐desalkylation and hydroxylation at indazole moiety 10.7, 11.0, 11.6 C17H17N3O2 296.0530 91.0530 119.0846 161.0339 M14 Nitrile hydrolysis 14.0 C21H25N3O2 352.2020 91.0541 119.0858 234.1257 175.0505 217.1019/ 199.0856 145.0431 M15a‐c Nitrile hydrolysis and hydroxylation at aryl moiety 11.1, 11.3, 11.4 C21H25N3O3 368.1969 107.0586 135.0803 217.0978 145.0394 M16 Nitrile hydrolysis and hydroxylation at indazole moiety 12.8 C21H25N3O3 368.1969 91.0531 119.0840 233.0920 161.0330 M17 Nitrile hydrolysis and further oxidation to carboxylic acid 14.0 C21H23N3O3 366.1812 91.0535 119.0853 248.1015/ 230.0908 231.0773/ 213.0644 145.0393 203.0803 M18 Nitrile hydrolysis and further oxidation to carboxylic acid and hydroxylation at aryl moiety 11.2 C21H23N3O4 382.1761 107.0482 135.0796 230.0918 (−H2O) 231.0760/ 213.0651 145.0388 5F–CUMYL‐P7AICA Parent compound 15.9 C22H26FN3O 368.2133 91.0551 119.0816 250.1525 174.0750 233.1241 145.0455 207.1417 d5: 230.1286i5: 187.1228131.0609 M12 N‐desalkylation 10.1 C17H17N3O 280.1444 91.0542 119.0845 162.0374 145.0401 M20 Oxidative defluorination 12.1 C22H27N3O2 366.2176 91.0540 119.0864 248.1556 174.0729 231.1256 145.0439 205.1447 i5: 187.1222131.0603 M21 Oxidative defluorination and carboxylation 12.1 C22H25N3O3 380.1969 91.0534 119.0873 262.1355/ 244.1075 145.0423 A26 Oxidative defluorination and methylation 14.9 C23H29N3O2 380.2333 91.0533 119.0837 262.1561 174.0659 245.1296 145.0393 219.1497 i5: 187.1230i1: 131.0607 M10 Ketone formation 13.9 C22H24FN3O2 382.1925 91.0535 119.0834 264.1146 247.0876 221.1080 i1: 131.0605 M23a‐b Oxidative defluorination and hydroxylation at aryl moiety 9.5, 10.0 C22H27N3O3 382.2125 107.0478 135.0803 248.1405 174.0658 231.1128 145.0413 205.1332 M7a‐b Hydroxylation at aryl moiety 13.0, 13.2 C22H26FN3O2 384.2082 107.0495 135.0809 250.1360 174.0664 233.1085 M9a‐b Hydroxylation at pentyl moiety 12.2, 12.7 C22H26FN3O2 384.2082 91.0532 119.0863 266.1469 174.0733 248.1183/ 233.1078 145.0431 205.1125 i1: 131.0607 M2a‐d Dihydroxylation at aryl and pentyl moiety 9.4, 9.6, 10.0, 10.2 C22H26FN3O3 400.2031 107.0493 135.0804 248.1196/ 266.1302 174.0663 145.0398 M3 Dihydroxylation at aryl moiety 11.6 C22H26FN3O3 400.2031 123.0446 151.0764 250.1392 174.0665 233.1087 145.0401 207.1296 M13 N‐desalkylation and hydroxylation 7.5 91.0539 119.0950 178.0611 145.0397/ 161.0574 CUMYL‐4CN‐B7AICA Parent compound 13.8 C22H24N4O 361.2023 91.0550 119.0821 243.1277 226.0993 145.0406 200.1219 M3a‐b Dihydroxylation at aryl moiety 9.6, 11.4 C22H24N4O3 393.1921 123.0425 151.0750 243.1250 226.0979 145.0392 200.1187 M7a‐c Hydroxylation at aryl moiety 7.7, 10.9, 11.1 C22H24N4O2 377.1972 107.0514 135.0844 243.1332 226.1050 145.0414 200.1244 M8 Hydroxylation at azaindole moiety 12.0 C22H24N4O3 377.1972 91.0446 119.0794 259.1372 242.1107 161.0332 216.1232 M9 Hydroxylation at pentyl moiety 11.7, 12.4 C22H24N4O2 377.1972 91.0535 119.0845 259.1196 242.1005 145.0415 M11 Loss of nitrogen and carboxylation 12.0 C22H25N3O3 380.196 91.0531 119.0843 262.1183/ 244.1076 145.0387 201.1015/ 219.1121 i4: 173.1065 M12 N‐desalkylation 10.1 C17H17N3O 280.1444 91.0550 119.0842 162.0703 145.0421 M13 N‐desalkylation and hydroxylation 7.4 C17H17N3O2 296.1394 91.0534 119.0843 178.0651/ 161.0560 145.0390 M14 Nitrile hydrolysis 11.2 C21H25N3O2 352.2020 91.0556 119.0872 234.1398 145.0418 191.1244 i4: 173.1101 M15a‐b Nitrile hydrolysis and hydroxylation at aryl moiety 8.6, 8.8 C21H25N3O3 368.1969 107.0411 135.0767 234.1393 145.0380 191.1251 i4: 173.1094 M17 Nitrile hydrolysis and further oxidation to carboxylic acid 11.3 C21H23N3O3 366.1812 91.0555 119.0871 248.1235/ 230.0914 213.0647 (−H2O) 145.0422 205.0959 g3: 187.0854 M18a‐b Nitrile hydrolysis and further oxidation to carboxylic acid and hydroxylation at aryl moiety 7.8, 8.7 C21H23N3O4 382.1761 107.0484 135.0795 248.1029/ 230.0913 213.0653 (−H2O) 145.0390 205.0960 g3: 187.0859 M19 Nitrile hydrolysis and further oxidation to carboxylic acid and hydroxylation at pentyl moiety 9.7 C21H23N3O4 382.1761 91.0530 119.0858 264.0972 247.0695 145.0389 221.0910 g3: 203.0797/ 185.0696

For metabolites with very low abundance, IDA acquisition did not always lead to generation of MS/MS spectra. For these metabolites SWATH acquisition was more successful. However, considering the risk of gaining mixed mass spectra due to a Q1 window width of 30 u using SWATH, IDA acquisition was used for metabolite identification as first choice.

In the negative controls, no metabolites were detected except for M20 (oxidative defluorination) and A26 (oxidative defluorination and methylation) of 5F–CUMYL‐P7AICA. M20 and A26 could have been formed during the storage of 5F–CUMYL‐P7AICA in methanol. As a methylated metabolite was not detected in any other cumyl‐derivative assay, formation should be mainly artefactual and rather not occur in vivo. Nevertheless, a formation of M20 in vivo is probable, as oxidative defluorination was previously observed for many other fluorinated synthetic cannabinoids.6

The highest number of metabolites was detected in the assays containing a substrate concentration of 10 μg/mL (27–29 μM). Major metabolites of CUMYL‐PINACA were M5a‐b (dihydroxylation at the pentyl moiety), followed by M6b‐c (further oxidation of one of the hydroxy groups of M5a‐b to a ketone), M10a (further oxidation of the hydroxyl group of M9a to the corresponding ketone) and M9a (hydroxylation at pentyl moiety). For 5F–CUMYL‐PINACA major metabolites were M9a‐b (hydroxylation at pentyl moiety) and M21 (defluorination and carboxylation), followed by M10 (oxidation of the hydroxyl group of M9 to the corresponding ketone). Major metabolites of CUMYL‐4CN‐BINACA were M17 (nitrile hydrolysis and carboxylation), M14 (nitrile hydrolysis), M11 (carboxylation at position 5 of the pentyl moiety) and M7c (hydroxylation at the aryl moiety) (Figure 2). Ozturk et al also identified M17, M14, and hydroxylation at the aryl moiety as major metabolites in their HLM experiments; however, M11 was neither identified in vitro nor in vivo.12 For 5F–CUMYL‐P7AICA, major metabolites were M3 (dihydroxylation at aryl moiety) and M21 (defluorination and carboxylation), followed by M7a and M9b (hydroxylation at aryl and pentyl moiety, respectively). For CUMYL‐4CN‐B7AICA, the most abundant metabolites were M17 (nitrile hydrolysis and carboxylation) followed by M7b (hydroxylation at aryl moiety), M3a (dihydroxylation at aryl moiety) and M14 (nitrile hydrolysis) (Figure 3).

Some of the metabolic transformations lead to structurally identical metabolites, which should consequently not be used as marker for the intake of a particular parent compound. Among the most abundant metabolites, oxidative defluorination to carboxylic acid of 5F–CUMYL‐PINACA (M21) and loss of nitrogen with carboxylation of CUMYL‐4CN‐BINACA (M11) lead to the same metabolite; as well as M6d of CUMYL‐PINACA, which was only a minor metabolite. Among the minor metabolites, N‐desalkylation led to identical metabolites for CUMYL‐PINACA, 5F–CUMYL‐PINACA and CUMYL‐4CN‐BINACA (RT 13.3 min) as well as for 5F–CUMYL‐P7AICA and CUMYL‐4CN‐B7AICA (RT 10.1 min).

Next to structurally identical metabolites, several structurally similar metabolites with identical [M + H] were detected. Oxidative defluorination of 5F–CUMYL‐PINACA and 5F–CUMYL‐P7AICA lead to constitutional isomers with similar mass spectra (M20), and should therefore rather be differentiated based on their retention time and mass spectra and not based on their mass spectra alone. This is also important for M7a‐c and M9a‐c (hydroxylation at aryl or pentyl moiety), dihydroxylation (M3), oxidative defluorination and carboxylation (M21) and several minor metabolites (M2 and M10). CUMYL‐4CN‐BINACA and CUMYL‐4CN‐B7AICA also showed metabolites with identical [M + H], for example the metabolites formed by nitrile hydrolysis (M14 and M17), the hydroxylated (M7, M8, M9) and the dihydroxylated metabolites (M3).

Besides constitutional isomers, isobaric metabolites with similar mass spectra were observed for 5F–CUMYL‐PINACA and 5F–CUMYL‐P7AICA, for example. M10 (ketone formation) and M25 (oxidative defluorination and hydroxylation at pentyl moiety) lead to metabolites with m/z 382.1925 and 382.2125, respectively. Identification of these metabolites was only possible using high resolution MS, as also the product ion masses only differed by 0.02 u.