Studies on Fentanyl and Related Compounds IV. Chromatographic and Spectrometric Discrimination of Fentanyl and its Derivatives

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1 Studies on Fentanyl and Related Compounds V. Chromatographic and Spectrometric Discrimination of Fentanyl and its Derivatives Hikoto Ohta and Shinichi Suzuki* National Research nstitute of Police Science, 6-3-1, Kashiwanoha, Kashiwa-shi, Chiba , Japan Kunio Ogasawara Pharmaceutical nstitute, Tohoku University, Aobayama, Sendai-shi, Miyagi , Japan L Abstract Chromatographic and spectrometric discrimination of fentanyl and its 24 analogues are discussed. Twenty-three of 25 samples were discriminated from each other by gas chromatography (GC), and the combination of GC and mass spectrometry enabled discrimination of all 25 samples. Condensed-phase infrared spectrometry was also useful for the differentiation of structurally similar compounds, but vapor-phase infrared spectra were not useful for discrimination of fentanyls. ntroduction Drug abuse is a problem facing law enforcement agencies worldwide. n addition to the "classic" drugs (e.g., morphine, heroin, marijuana, and cocaine, etc.), totally synthesized drugs are sometimes encountered and seized at crime laboratories in Japan. These drugs are called "designer drugs". They have similar structures to original narcotics and stimulants and are also highly pharmacologically active. These drugs are presumed to be synthesized in well-equipped clandestine laboratories by highly trained chemists. These facts are clearly different from the classical simply modified drugs. For example, classically abused drugs, such as heroin and lysersic diethyl amide (LSD), are only esterifications of the original alkaloids. Making the problem even more serious, these new synthesized drugs are not currently covered by the drugcontrol laws. n Figure 1, fentanyl, meperidine, and amphetamine (above the line) are strictly controlled by the drug-control laws, but 9 Author t~l ~'. hcml corresp(mden( e Qu~uld be addre',sed their modified compounds, those below the line, were not restricted until quite recently9 Even now, p-fluorofentanyl is not controlled in Japan, but it has high analgesic activity compared with morphine or fentanyl itself. n this category, fentanyl analogues have high potency analgesic activities. Fentanyl itself is a useful and important analgesic used widely in surgical operations and is a controlled substance by law, but numerous reports have been published recently with modified fentanyl-related compounds abused as designer drugs (1-3). n this paper, 24 fentanyl-related compounds were synthesized, and chromatographic and spectroscopic discrimination of these compounds were examined. Materials and Methods Reagents Twenty-four fentanyl-related compounds were prepared by the original method of Van Bever et al. (4). Fentanyl citrate was obtained from Sankyo Co., Ltd. (Tokyo, Japan) and used as free base. The synthesized sample purities were confirmed by thin-layer chromatography (TLC) and direct-inlet electronimpact mass spectrometry (DEMS). Other chemicals used were analytical grade. The 24 synthesized compounds in this experiment and original fentanyl are summarized in Table. TC TLC was carried out on the glass precoated with silica gel 60F-254 (0.25-ram thick, Merck, Darmstadt, Germany), and four developing solvent systems and four detection methods were used. Developing solvents were methanol/28% aqueous ammonia (100:1.5, v/v), benzene/dioxiane/ethanol/28% 280 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

2 aqueous ammonia (50:40:5:5, v/v/v/v), chloroform/acetone (2:1, v/v), and chloroform/benzene/methanol (10:2:1, v/v/v). Detection methods used were platinum chloride/potassium iodide reagent, Dragendorff reagent, 1% iodine/methanol solution, and ultraviolet (254 nm) absorption. Gas chromatography (GC) A Shimadzu GC-17A GC equipped with a flame-ionization detector was used. The capillary column was nonpolar, Hi-Cap CBP-1 (50 m x 0.2-ram i.d., 0.25-pro film thickness, Shimadzu Co., Ltd., Kyoto, Japan). Oven temperature was programmed at ~ (10~ and injection temperature and detector temperature were 280~ and 300~ respectively. The carrier gas was nitrogen, and linear velocity was 22.5 mm/s. The split ratio was 80:1. The injection amount of each sample was 0.5 pg. Direct inlet mass spectrometry (MS) Direct inlet electron impact MS was conducted on the Shirnadzu QP-1000 (Shimadzu Co., Ltd., Kyoto, Japan), with direct inlet sample introduction techniques. onization voltage and ionization current were 70eV and ll0]~a, respectively. on Fentanyl X~ O'~-CH2CH 3 ~H2CH2-N~'N, 3-Methylfentanyl CH3~--_~ 9 CH3 d-methylfentanyl O'~-CH2CH 3 CO2CH2CH3 H3C-N~,~ Meperidine OCOCH2CH 3 ~H2CH~N~ / pcoch2ch3 H3C-N,~ Amphetamine [~CH2CHCH3 CT~ CH2CHCH3 ~H 2 Methylenedioxyamphetamine (MDA) ~ CH2CHCH3 NHCH 3 Methylenedioxymethamphetamine (MDMA) p-fluorofentanyl '~F Figure 1. Controlled drugs and their uncontrolled (in Japan) derivatives. Table. Synthesized Fentanyl and its 25 derivatives. R3 ~'~X No R1 R2 R3 X No R1 R2 R3 X 1 ~-CH2CH 2- -~13 H H acethylfenumyi 14.H3CO ~-~2CH(CH 3 ) -CH2~ 3 H H 2 ~-CH2CH 2- -CH2CH 3 H H femany[ 15 ~-CH2CH(CH 3 ) -CH 3 H H 2- -CH2CH2CH 3 H H 3) -CH2CH 3 H H 2- -CH(CH3) 2 H H 3) -CH2CH2CH 3 H a 2- -CH 3 H F CH 3 ) -CH(CH 3 )2 H H 2 - -CH2CH 3 H F p-fluorofeut4m:y[ 19 CH 3 - -CH 3 H H 7 ~-CH2CH 2- -CH2CH2CH 3 H F 20 CH 3- -CH2CH 3 H H 8 ~-CH2CH 2- -CHtCH3) 2 H F 21 CH 3- -CH2CH2CH 3 H H 9 ~-CH2CH 2- -CH2CH 3 H CL p-chlorofentmzyl 22 ~)-CH 2- -CH 3 H H 2- -C'2CH2CH 3 H CL 2- -CH2CH 3 H H 11 ~-CH2CH 2- -~(~"13) 2 H CL 24 ~-CH 2- -~2~2~13 H H ~z ~>-c, zc, z- -c,~3, % ~toly~=.=.~ z5 ~-c, zc,2- -c,z% -%, 13 ~.-CH2CH 2- -CH2CH 3 H OCH 3 ~-mm~.ceth~m~ ~-m~fmsnyl N-m~yff~tanyi N-m~,~m,~ 3-~mh,~mu~ 281

3 Journal of Analytical foxicologv, Vol. 23, July/August 1999 Table. Rf Values of Fentanyl and ts Analogues in Several Developing Systems Rf Values Compound Solvent system t no.* Remarks l acetylfentanyl fentanyl p-fluorofentanyl p-chlorofentanyl p-tolylfentanyl cr ~-methylfentanyl , , N-methylfentanyl B N-benzylfentanyl } }-methylfentanyl source temperature was 250~ and direct inlet temperature was programmed from 100 to 250~ (20~ nfrared spectrometry Condensed-phase infrared spectra were measured by KBr sample pellet preparation. Nicolet 750 infrared spectrometer (Nicolet Co., Ltd., Madison, W) with triglycerin sulfide (TGS) as a detector was used for the measurement of spectra of monomethylated fentanyl analogues, such as n-propylfentanyl (compound 3), i-propylfentanyl (compound 4), p-tolylfentanyl (compound 12), cr (compound 16), and 3- methyl-fentanyl (compound 25). Results TtC The Rf values of 25 fentanyl and its analogues are shown in Table. Their Rf values were very similar except for the substituents of piperidine 1 position of fentanyl to methyl group and benzyl group. n this examination, the chloroform/benzene/methanol (10:2:1, v/v/v) solvent mixture was the most suitable for discrimination of these compounds. As a detecting reagent, Dragendorff reagent was specific and had a high sensitivity, an approximately 3-1Jg spot was enough to detect the compounds. Three other reagents also showed positive results, but it was difficult to specify the analogues by only TLC, and further instrumental analyses were required. *CompoLmd number<~ { orrespond to Talfle. +Sokeni,,~qelm"r as tl>llm~,:, methanol/28't, aqueous ammonia (lo0:l.3, ~, ~. ; 2 bt'rllt'r]u/lic~',<ian~>/uthanol J-~ ". a(tff'{)tls,/rlmoigi<t[~():4():~:~, V/\/\!v); ~, (hl(illil'liri/ <1( ('hlrlt' fj:lr ~.i\ i; arll] 4 ( hlirlilljrrl/~)t'll/l'llt'in}l'lh</rlol (10:2:1,,,/,,/,, i. r i GC GC had high discriminating power for these analogues. Typical gas chromatograms of the mixture of 0.5 pg of each analogue are shown in Figure 2, and retention indices of these compounds are shown in Table. Optimal separation conditions using a non- ~ o polar long capillary column were as previously ~1[ ~/~ ~" stated. n a previous report, an abused fentanyl anar ~ logue called bogus "China White" was first conol ~..,- sidered 3-methylfentanyl, which was a fentanyl it ~ " derivative with one methyl group introduced in the 3 position of the piperidine ring of fentanyl. t was only presumption from the analytical data of infrared spectroscopy (R), MS, and nuclear magnetic resonance spectrometry (NMR) (5). Two monomethylated fentanyl analogues were synthesized, detailed mass spectra were compared, and its structure was confirmed to _ k.,.. / be (x-methylfentanyl (6). Furthermore, the ~ ~ 1] 1' most widely abused fentanyl analogues were Retention time (rain) monomethylated fentanyls, so five monomethylated fentanyls were synthesized and Figure 2. Gas chromatogram of fentanyl and its derivatives. See compound numbers in Table for their retention times and retention indices peak identification, were compared by GC (7). n this report, 25 fentanyl analogues were 282

4 Table. Retention ndices of Fentanyl and ts Analogues Compound no.* Remarks Retention ndices 19 N-methylfentanyl N-benzylfentanyl * t acethylfentanyl p-fluorofentanyl fentanyl c-methylacetylfentanyl methylfentanyl r p-tolylfentanyl p-chlorofentanyl t t *Compound numbers correspond to Table. +Not separated. synthesized and discrimination of these compounds by GC was examined. Of these compounds, only compounds 24 and 8 and 13 and 10 were not able to be separated; all other compounds were separated by a single injection. N-Methylfentanyls (compounds 19, 20, 21) showed very short retention times. Other fentanyl analogues showed similar retention times, and their retention times were distributed between 12.5 and 17.0 min. Direct-inlet MS n the direct-inlet electron-impact mass spectra, molecular ion (M peaks were not generally observed except for N-methylated and N-benzylated analogues, and the highest mass ion peaks were M-91 (elimination of tropilium ion) as base ion peaks (cleavage a). Other diagnostic ions originated from the 2-3 and 1-6 bond cleavages of the piperidine ring and the elimination of the propionyl group (cleavage c in Figure 3), for example, m/z 146, which corresponds to fentanyl and (~-methylfentanyl, and m/z 160, which corresponds to 3-methylfentanyl. These results are summarized in Table V. The comparison of cleavage of five monomethylated fentanyl analogues and their discrimination by mass spectra are shown in Figure 3. R spectroscopy R spectra of the fentanyl and its five monomethylated analogues are shown in Figure 4. They were quite similar except regions of and cm -1, but their discrimination was possible by the comparison of these regions in the R spectra. C"CH, CH 3 N.1 b" 'c 2 : Fentanyl,ll 16 :o-methyl 46c a : tolyl 3:n-Pr 160 c # 2O3! a 11 25: 3-methyl \ 160! i,216 4:i-Pr ill., il,jl., O m/z m/z Figure 3. Mass spectra of fentanyl and its 1-methylated fentanyl analogues and comparison of the fragmentation of the compounds.! i

5 Table V. Diagnostic ons of Fentanyl Analogues in Electron mpact Mass Spectrometry Diagnostic ions Compound M* Cleavage a Cleavage c no.* Remarks M*-CTH7 1 acethylfentanyl fentany{ p-fluorofentanyl p-chlorofentanyl p-tolylfentanyl r r N-methylfentanyl N-benzylfentanyl methylfentanyl *Compound numbers correspond to Table. * not observed. Conclusions Fentanyl itself is an important analgesic medicine used in surgical operations, but its derivatives are synthesized in clandestine laboratories and abused illegally. These compounds are called "designer drugs" and have not been controlled by drug control laws. n this report, 25 fentanyl derivatives were synthesized and analyzed by, TLC, GC, GC-MS, and condensedphase Fourier-transform infrared spectroscopy. Detailed examination of fentanyls by NMR has been previously reported (8). n this paper, more conventional methods generally used in forensic science laboratories were applied to the discrimination of these compounds. When the drug bogus "China White" was synthesized and abused, the compound was considered to be 3-methylfentanyl (5). However, monomethylated fentanyl derivatives were synthesized and their mass spectra were compared carefully with each other. By this comparison, its structure was finally confirmed to be c~-methylfentanyl (6). These compounds were distinguished from each other not only by their mass spectra but also by their retention indices (7). n the future, if the fentanyl analogues that have high euphoric activity are abused, the compounds could be identified quickly by these data. These kinds of drugs have to be synthesized before their abuse, and forensic science laboratories have to prepare for the identification of designer drugs, including fentanyl and other kinds of analgesic medicine or stimulants, such as meperidine or amphetamines. Furthermore, newly discovered abused drugs must be controlled quickly by changing drug-control laws. Fentanyl ~ t/~,! p-tolyl J { c~-methyl n-propy~ ~'~ 3-methyl ~ ~' J-propy~,,~ 32~0 28'00 le~ 12b0 8bo Figure 4, nfrared spectra of fentanyl and its 1-methylated fentanyl analogues. ~]~00 ~ 1(500 ~X) ~0 cm

6 References 1. R.M. Baum. New variety of street drugs poses growing problem. Chem. Eng News Sep. 9:7-16 (1985). 2. S. Suzuki and T. noue. New abused drug "China White". Forensic Toxicol. News 5:5-12 (1987). 3. J.A. Heagy, dentification of 4-propionoxy-4-pheny-Nmethylpiperidine. Microgram 15: (1982). 4. W.F.M. Van Bever, C.J.E. Niemegeers, and RA.J. Janssen. Synthetic analgesics. Synthesis and pharmacology of diastereoisomers of N- [3-methyl-1 -(2-phenylethyl)-4-piperidyl]-N-phenylpropanamide and N-[3-methyl-1-(1-methyl-2-phenylethyl)-4-piperidyl]-Nphenylpropanarnide. J. Med. Chem. 17: (1974). 5. S. Stinson. Structure of bogus "China White" solved. Chem. Eng. News 19:71-72 (1981 ). 6. S. Suzuki, T. lnoue, and C. Kashima. Studies on 1-(2-phenethyl)- 4-(N-propionyl-anilino)piperidine (Fentanyl) and related compounds.. Spectrometric and chromatographic analyses of 3-methylfentanyl and o~-methylfentanyl. Chem. Pharm. Bull. 34: (1986). 7. S. Suzuki. Studies on fentanyl related compounds:. Spectrometric discrimination of five mono methylated fentanyl isomers by gas chromatography/fourier transform-infrared spectrometry. Forensic Sci. nt. 43:15-19 (1989). 8. D. Cooper, M. Jacob, and A. Allen. dentification of fentanyl derivatives. J. Forensic 5ci. 31 : (1986). Manuscript received August 3, 1998; revision received October 5,

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