Analysis of Benzylpiperazine-Like Compounds Hiroyuki Inoue 1
鑑識科学，9(2)，165―184(2004) 165
―Technical Note―
Analysis of Benzylpiperazine-like Compounds
Hiroyuki Inoue1,YukoT.Iwata1, Tatsuyuki Kanamori1, Hajime Miyaguchi1, Kenji Tsujikawa1, Kenji Kuwayama1, Hiroe Tsutsumi2, Munehiro Katagi2, Hitoshi Tsuchihashi2 and Tohru Kishi1
National Research Institute of Police Science 631, Kashiwanoha, Kashiwa, Chiba 2770882, Japan1 Forensic Science Laboratory, Osaka Prefectural Police H. Q. 1318, Hommachi, Chuo-ku, Osaka, Osaka 5410053, Japan2
(Received 6 January 2004; accepted 6 March 2004)
1-Benzylpiperazine (BZP) and 1-(3-tri‰uoromethylphenyl)piperazine, newly controlled as narcotics in Japan on 2003, and their analogues were analyzed. The analytical data with color test, thin layer chromatography (TLC), infrared spectroscopy (IR), gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) are presented. The BZP-like compounds were less sensitive to Simon's reagent than amphetamine type stimulants on spot plates. Using on-site screening kit based on Simon's test (X-Checker), BZP indicated almost the same result as methamphetamine. For TLC, the solvent system, methanol －25 aqueous ammonia (100 : 1.5), was the best among the systems examined. Iodoplatinate reagent was the most sensitive one to detect BZP. The IR spectra showed su‹cient diŠerences to make identiˆcation. Trimethylsilylation was the most appropriate choice for the GC/MS analysis of BZP-like compounds in terms of the peak shapes, separation and stability (using a J&W DB-5MS column). In LC/MS analysis, the gradient elution (10 mM formic acid and acetonitrile) using a Waters Symmetry
Shield C18 column achieved discrimination of isomers except for 1-(2-‰uorophenyl) piperazine and 1-(4-‰uorophenyl)piperazine. The cone voltage of 30 V was recommended for the LC/MS screening. The information would be useful for identiˆcation of piperazines in conˆscated powders, liquids or tablets.
Key words: piperazine, TLC, IR, GC/MS, LC/MS
Introduction (2C-B), 4-methylthioamphetamine (4-MTA), 4- Recent information technology makes hydroxybutylic acid (GHB) and magic dramatic progress in popularization of the mushrooms (fungi containing psilocybin and/or Internet. Although the Internet is undoubtedly psilocin), and in recent years, these are our valuable source of knowledge, it also controlled under Narcotic and Psychotropic facilitates widespread of various kinds of drugs Drug Control Law in Japan. of abuse even in the young people. These drugs More recently, piperazine derivatives, 1- include 4-bromo-2,5-dimethoxyphenethylamine benzylpiperazine (BZP) and 1-(3-tri‰uoromethy- 166166 Hiroyuki Inoue et al lphenyl)piperazine (mTFMPP) (Fig. 1), have mTFMPP is a 5HT-releasing agent and binds to been found on the illicit market as a new group 5HT receptors in the brain10,11).Afatalcase of designer drugs14). They are often sold as associated with BZP and MDMA had been ``BZP'', ``A2'', ``legal E'' or ``legal X''. BZP reported in 200112). There are no recognized and mTFMPP, with their easy availability and therapeutic uses of these substances in Japan and their so-called legal status, are becoming drugs the United States. Four kinds of psychoactive of abuse worldwide. Both of piperazine piperazines, BZP, mTFMPP, 1-(4-methoxy- derivatives had been placed temporarily into phenyl)piperazine (pMeOPP), 1-(3-chloro- Schedule I of the Controlled Substances Act in phenyl)piperazine (mCPP), have been found in the United State on September 2002, in order to Japan and they are also sold as a mixed form avoid an imminent hazard to the public safety5). with BZP in some cases. The scheduling is still valid at the present A few study on identiˆcation of piperazines moment. BZP and mTFMPP are controlled in in tablets and powders as well as biological Japan after October 2003. matrices are reported1,1316),however,itshould BZP was ˆrst synthesized in 1944 as a be noted that there are several isomers in the potential anthelmintic agent. BZP also shows a piperazine derivatives which would have quite central serotoninomimetic action that involves 5- similar chromatographic and mass spectrometric
HT uptake-inhibition and 5-HT1 receptor properties to each other. In the present study, agonistic eŠects6).BZPactsasastimulant analytical data of BZP-like compounds similar to 3,4-methylenedioxymethamphetamine appeared on the illicit market and available (MDMA) or amphetamine, producing euphoria isomers in Japan using color tests, thin layer and inducing cardiovascular eŠects in humans, chromatography (TLC), infrared spectroscopy including increased heart rate and systolic blood (IR), gas chromatography/mass spectrometry pressure7,8). mTFMPP shows hallucinogenic (GC/MS) and liquid chromatography/mass eŠects similar to those produced by MDMA9). spectrometry (LC/MS) are presented.
Fig. 1 Chemical structures of benzylpiperazine-like compounds used in this study. Analysis of Benzylpiperazine-like Compounds 167
Materials and Methods Pyridine (anhydrous) was obtained from Aldrich 1Chemicals Co. (Milwaukee, WI). HPLC-grade acetonitrile (1) BZP-like compounds was obtained from Wako Pure Chemical BZP and 1-(3-tri‰uoromethylphenyl) Industries. X-Checker(on-site screening kit for piperazine monohydrochloride (mTFMPP･HCl) MA, based on Simon's test) was obtained from were obtained from Lancaster Synthesis Taisho Rika (Saitama, Japan). Glass plates (20 (Lancashire, UK). BZP dihydrochloride was cm×20 cm) precoated to a depth of 0.25 mm prepared from BZP. mTFMPP, 1-(2-methoxy- with silica gel 60F254 was obtained from Merck phenyl)piperazine (oMeOPP), 1-(4-methoxy- (Darmstadt, Germany). A Milli-Q Simpli Lab- phenyl)piperazine dihydrochloride (pMeOPP･ UV system (Millipore, Billerica, MA) was used 2HCl), 1-(3-chlorophenyl)piperazine monohy- to prepare pure water throughout the study. All drochloride (mCPP･HCl), 1-(4-chlorophenyl) other chemicals used were analytical reagent piperazine monohydrochloride (pCPP･HCl), grade. 1-(2-‰uorophenyl)piperazine monohydrochlo- 2 Methods ride (oFPP･HCl) and 1-(4-‰uorophenyl) (1) Color tests piperazine dihydrochloride (pFPP･2HCl) were The samples (3 or 10 mg) were tested by obtained from Tokyo Kasei Kogyo (Tokyo, following reagents20).Blanktests(reagentsonly) Japan). 1-(2-Tri‰uoromethylphenyl)piperazine were also carried out. (oTFMPP) and 1-(4-tri‰uoromethylphenyl) (1)-1 Simon's reagent piperazine (pTFMPP) were from Fluorochem Solution A. 20 aqueous sodium (Derbyshire, UK). carbonate solution (2) Reference compounds Solution B. 50 ethanolic acetaldehyde d-Methamphetamine hydrochloride (MA･ solution HCl) was obtained from Dainippon Solution C. 1 aqueous sodium Pharmaceutical (Osaka, Japan). dl-MDMA nitroprusside solution hydrochloride (MDMA･HCl) was synthesized as a. Spots test previously reported17). 4-Bromo-2,5-dimethoxy- A sample was placed in a depression of spot phenethylamine hydrochloride (2C-B･HCl) was plates and mixed with 1 drop (20 mLapprox.)of synthesized as previously reported18). d- solutionA.OnedropofsolutionBandthatof Dimethylamphetamine hydrochloride (DMA･ solution C were added subsequently. HCl) was synthesized from l-norephedrine b. X-Checker hydrochloride (Wako Pure Chemical Industries, The kit is a capped plastic tube which Osaka, Japan) as previously reported19).Stock contains 2 sealed capillaries (enclosed Solution B solutions were prepared at concentrations of 1 andC)andasmallpieceofˆlterpapersoaked mg/mL in methanol. with Solution A. (3) Reagents Test was carried out according to an Sodium nitroprusside and hydrogen instruction manual attached to the kit21) (Fig. 2): hexachloroplatinate hexahydrate were obtained (1) 10 mg of sample is put into the plastic tube. from Kanto Chemical (Tokyo, Japan). p- (2) Bend the plastic tube to crack the sealed Dimethylaminobenzaldehyde (p-DMAB) was capillaries and the sample will be mixed with obtained from Kokusan Chemical (Tokyo, Solution B and C. The mixed solution will be Japan). Ferric chloride was obtained from soaked to the ˆlter paper. (3) If MA presents, the Nacalai Tesque (Kyoto, Japan). N,O- contents of the tube will turn blue in a few Bis(trimethylsilyl)tri‰uoroacetamide (BSTFA) seconds. was obtained from GL Sciences (Tokyo, Japan). (1)-2 Marquis reagent Trimethylchlorosilane (TMCS) was obtained The reagent was prepared by adding 1 drop from Pierce Chemical Co. (Rockford, IL). of formaldehyde to 1 mL of concentrated 168168 Hiroyuki Inoue et al
reagents. b. DragendorŠ reagent Preparation of the reagent is as described in the section ``13 DragendorŠ reagent''. c. Simon's reagent The reagent was prepared by mixing equal volumes of the Solution A and the Solution C described in the section ``11 Simon's reagent''. The plates were exposed to acetaldehyde gas Fig. 2 Schematics of how to use X-Checker. after spraying the reagent. d. Iodoplatinate reagent Solution A: aqueous 10 hydrogen sulfuric acid. A sample was placed in a hexachloroplatinate hexahydrate solution depression of a spot plate, and 3 drops of the Solution B: aqueous 4 potassium iodide reagent were added. solution. (1)-3 DragendorŠ reagent The Solution A, B and water were mixed in One gram of bismuth subnitrate was the ratio of 1 : 25 : 24 by volume. dissolved in a small amount of concentrated e. 1 Iodine-methanol solution hydrochloric acid; 25 aqueous ammonia was f. p-DMAB solution added until the precipitation (Bi(OH)3 )ˆnished; 0.125 g of p-DMAB was dissolved in 100 the supernatant was removed and the residue mL of 65 sulfuric acid and 0.1 mL of 5 waswashedwithwater;theresiduewasdissolved ferric chloride was added to this solution. in 1 mL of concentrated hydrochloric acid; 3 g (3) Infrared (IR) absorption spectroscopy of potassium iodide dissolved in 1 mL of water IR absorption spectroscopy was performed wasadded;48mLof70 aqueous acetic acid with a JIR-WINSPEC50 infrared spectropho- was added. tometer (JEOL, Tokyo, Japan) utilizing the A sample was placed in a depression of a standard KBr method except for the liquid spot plate and added 3 drops of the reagent. samples (BZP, mTFMPP, oTFMPP and (2) Thin layer chromatography (TLC) oMeOPP)whichwerepreparedasthinˆlms The tested compounds were dissolved with between two NaCl disks. methanol at a concentration of 0.01, 0.1, 1 and (4) Gas chromatography/mass spectrometry 10 mg/mL as standard solutions for TLC. (GC/MS) Five diŠerent solvent systems were (4)-1 Extraction examined for the development in TLC: Solvent BZP-like compounds obtained as A, 2-butanone dimethylformamide 25 hydrochloride salts were converted to their free aqueous ammonia (13 : 0.9 : 0.1, v/v/v); Solvent bases. One mg of each salt dissolved in 1 mL of B, 2-propanol 25 aqueous ammonia (95 : 5, distilled water was basiˆed to pH 12 with 1 M v/v); Solvent C, acetone toluene 25 sodium hydroxide solution, and then extracted aqueous ammonia (20 : 10 : 1, v/v/v); Solvent with 3 mL of diethylether. The organic layer was D, methanol 25 aqueous ammonia (100 : transferred into other glass tubes and evaporated 1.5, v/v); Solvent E, 1-butanol acetic acid to dryness under a stream of nitrogen. The water (2 : 1 : 1, v/v/v). After the developing residues were dissolved in 1 mL of methanol and solvents were dried, the spots on chromatograms stored at －20°C. In the following study, these were detected as follows. solutions were used as 1 mg/mL stock solution. a. UV light (4)-2 Derivatizations for GC/MS analysis All the plates were observed under UV light Derivatization was performed for free bases (254 nm) and sprayed with the following of BZP-like compounds. Analysis of Benzylpiperazine-like Compounds 169
Acetylation: acetylation was performed by quadrupole mass spectrometer (Waters) was adding 80 mL of acetic anhydride and 20 mLof used for LC/MS measurements. The column pyridine to 5 mgofeachstandard.Themixture used for the separation was a Symmetry Shield was heated at 45°C for 30 min. Excess reagents C18 (Waters) 3.5 mm, 15 cm×2.1 mm i.d. A 5-mL were evaporated under the nitrogen stream and injection was made via the built-in autosampler. the residue was redissolved in 100 mLofethyl Mobile phase for gradient separations was acetate containing 50 mg/mL n-eicosane. prepared by low-pressure mixing of 10 mM Tri‰uoroacetylation: tri‰uoroacetylation formic acid (A) and acetonitrile (B) scheduled as was performed using the method reported by de follows: 02min,5 B; 23min,510 B; 35 Boer et al.1) with a minor modiˆcation. Fifty min, 10 B; 510 min, 1050 B; 1015 min, microliters of tri‰uoroacetic anhydride and 50 50 B; 1515.5 min, 505 B; 15.545 min mL of ethyl acetate were added to each standard (equilibration step), 5 B. The ‰ow rate was set (5 mg), and the mixture was heated at 55°Cfor20 at 0.2 mL/min and chromatography was carried min. After evaporation of excess reagents, the out at 40°C. residue was redissolved in 100 mL of ethyl acetate Electrospray ionization was carried out in containing 50 mg/mL n-eicosane. the positive mode using a Z-spray module Trimethylsilylation: each standard (5 mg) attached to the mass spectrometer. Seven was derivatized in the mixture of 50 mLof acquisitions with various cone voltages (from 10 BSTFA containing 1 TMCS (99 : 1, v/v) and V to 70 V, 10 V intervals; scan time 0.2 s) were 50 mLofacetonitrileat70°Cfor30min. performedineachseparationofthestandard (4)-3 GC/MS analysis solutions although the cone voltage and the scan GC/MSanalysiswasperformedwitha time were set to 30 V and 1 s respectively when GCMSQP5050A (Shimadzu, Kyoto, Japan) measuring the standard mixture. Other equipped with a DB5MS capillary column (30 conditions were as follows: capillary voltage, 3 m length, 0.25 mm i.d. and 0.25 mmˆlm kV; source temperature, 100°C; desolvation thickness). The temperature of the injector and temperature, 300°C; cone gas (N2 ) ‰ow, 50 L/h; the interface was set at 280°Cand300°C, desolvation gas (N2 ) ‰ow, 350 L/h; scan range, respectively. The oven temperature was held at m/z 70300. 90°C for 1 min, then raised to 300°Cat15°C/min and held for 5 min. Helium was used as the Results and Discussion carrier gas (head pressure 72.3 kPa, total ‰ow 50 1 Color tests ml/min). The mass spectrometer was operated Reagents commonly used in Japan for under the electron ionization (EI) mode at an identiˆcation of MA were applied to the color ionization energy of 70 eV and under the tests. Besides BZP-like compounds, phenethyla- chemical ionization (CI) mode with a reagent gas minessuchasMA,MDMA,2C-BandDMA of isobutane. One microliter of the samples was were also tested as reference compounds. injected in the splitless mode. (1)-1 Simon's reagent (Fig. 3(a)(c) and Table (5) Liquid chromatography/mass spectrometry 1) (LC/MS) A blue color indicates the present of Each standard solution of the analytes (1 secondary aliphatic amine in most cases by mg/mL in 5 methanol/water) and a standard Simon's reagent. The indicated color of the mixture solution containing all of the analytes (1 BZP-like compounds was changed from blue to mg/mL each in 5 methanol/water) were yellowish blue gradually. Simon's reagent was prepared from the methanolic stock solutions less sensitive to BZP-like compounds than described previously. reference drugs such as MA or MDMA. A 2690 liquid chromatograph (Waters, Comparing mTFMPP with mTFMPP･HCl, the Milford, MA) connected with a ZQ single free base form was less sensitive than the 170170 Hiroyuki Inoue et al
Fig. 3 (a) Photograph of BZP-like compounds and reference compounds (3 mg) tested with Simon's reagent. Numbers: 1, BZP; 2, mTFMPP; 3, mTFMPP･HCl; 4, oTFMPP; 5, pTFMPP; 6, oMeOPP; 7, pMeOPP･ 2HCl;8,mCPP･HCl; 9, pCPP･HCl; 10, oFPP･HCl; 11, pFPP･2HCl; 12, MA･HCl; 13, MDMA･HCl; 14, 2C-B･HCl; 15, DMA･HCl. (b) Photograph of BZP-like compounds (10 mg) tested with Simon's reagent. Numbers are as described in (a). (c) Photograph of BZP-like compounds (10 mg) tested with X-Checker. Numbers are as described in (a). (d) Photograph of BZP-like compounds and reference compounds (3 mg) tested with Marquis reagent. Num- bers are as described in Fig. 3(a). (e) Photograph of BZP-like compounds and reference compounds (10 mg) tested with Marquis reagent. Num- bers are as described in Fig. 3(a).
hydrochloride form. One of the reasons of less reagent. sensitivity could be that the free bases were not We also examined BZP and mTFMPP, and completely dissolved in or mixed with the the isomers of mTFMPP, with X-Checker,on- Analysis of Benzylpiperazine-like Compounds 171
Table 1 Indications of Simon's reagent.
Spot test X-Checker Compounds 3mgsample 10mgsample 10 mg sample (1) BZP ＋ [Z (2) mTFMPP －±＋partially (3) mTFMPP･HCl － Z ＋ partially (4) oTFMPP －＋＋partially (5) pTFMPP －±± (6) oMeOPP ＋ Z NT (7) pMeOPP･2HCl ＋＋NT →yellowish (8) mCPP･HCl ± Z NT →yellowish (9) pCPP･HCl ±＋NT →yellowish (10) oFPP･HCl ＋ Z NT →yellowish (11) pFPP･2HCl Z ＋ NT →yellowish (12) MA･HCl [ NT [ (13) MDMA･HCl [ NT NT (14) 2C-B･HCl － NT NT (15) DMA･HCl － NT NT ＋, Z and [, positive (blue color). The number of ``＋'' relatively indicates the extent of coloration; ±, purple (slightly changed blue); －,negative;→, change with time; NT, not tested. site screening kit sold in Japan. BZP showed ThereagentwouldbeeŠectivetodistinguishthe almostthesameresultwithMA･HCl (Fig 3(c)). BZP-like compounds from ATS. The result indicates that BZP could be (1)-3 DragendorŠ reagent (Table 3) misidentiˆed as MA on site and investigators An orange, red-orange, or brown-orange have to beware of misidentiˆcation. precipitate suggests the presence of an alkaloidal (1)-2 Marquis reagent (Fig. 3(d), (e) and Table base. In many cases, tertiary amines show 2) strongly positive results but the BZP-like Various colors are given by Marquis compounds were not as sensitive as reagent with a large number of compounds such dimethylamphetamine. as heterocyclic compounds which consist of S, O For on-site drug screening, Simon's and or N in element, or other compounds which Marquis reagents are commonly carried out by consistofC,HandN.DiŠerentcolorsare investigators involved in drug enforcement in obtained with compounds of various types using Japan. Using both reagents at the same time sulfuric acid alone22). Thus three drops of wouldbeeŠectivetodistinguishtheBZPfrom sulfuric acid were also applied directly to the ATS. sample as reference reaction. 2TLC The BZP-like compounds showed negative Table 4 shows the Rf values of the tested results or faint color. The reactions were almost compounds in ˆve developing solvents. Solvent thesameasthatofsulfuricacidexceptfor B and E developed slowly as compared with the pTFMPPandoMeOPP.AsshowninFig.3(d), other solvents. The times required to travel 10 amphetamine type stimulants (ATS) show cm were one hour for solvent B and two hours positive results strongly with Marquis reagent. for solvent E. The Rf values of all spots were 172 Hiroyuki Inoue et al
Table 2 Indications of Marquis reagent.
Marquis Conc. H SO Compounds 2 4 3mgsample 10mgsample 10 mg sample (1) BZP precipitate, white precipitate, white precipitate, white →brownish green →brownish green →dark green (with fume) (with fume) (with fume) (2) mTFMPP precipitate, white precipitate, white precipitate, white →pale brown →pale brown (with fume) (3) mTFMPP・HCl －a) －a) － (4) oTFMPP precipitate, white precipitate, white precipitate, white →pale brown →pale brown (5) pTFMPP pale brownish red pale brownish red － solution (gradually) solution (gradually) (6) oMeOPP pale pink solution pale pink solution － (gradually) (gradually) (7) pMeOPP・2HCl －a) －a) －a) (8) mCPP・HCl －a) －a) －a) (9) pCPP・HCl －a) －a) －a) (10) oFPP・HCl －a) －a) －a) (11) pFPP・2HCl －a) －a) －a) (12) MA・HCl brown NT NT (13) MDMA・HCl black NT NT (14) 2C-B・HCl green NT NT (15) DMA・HCl brown NT NT a), dissolved with ˆzz; －, negative; →, change with time; NT, not tested.
Table 3 Indications of DragendorŠ reagent. Compounds 3 mg sample 10 mg sample (1) BZP ±＋ (2) mTFMPP ±＋ (3) mTFMPP・HCl ±± (4) oTFMPP ＋ ＋ precipitate, precipitate, vermilion→partially metallic vermilion→partially metallic (5) pTFMPP ±＋ (6) oMeOPP ＋＋ (7) pMeOPP・2HCl ＋ Z (8) mCPP・HCl ＋ Z (9) pCPP・HCl ＋ Z (10) oFPP・HCl ＋＋ (11) pFPP・2HCl ＋＋ (12) MA・HCl ± NT (13) MDMA・HCl ± NT (14) 2C-B・HCl ＋ NT precipitate, vermilion (15) DMA・HCl Z NT ＋ and Z, positive (red precipitate). The number of ``＋'' indicates the relative extent of precipitation; ±, slightly precipitated; →, change with time; NT, not tested. Analysis of Benzylpiperazine-like Compounds 173
Table 4 Rf values of the tested compounds in the Table 5 Detection limits (mg) of the tested com- ˆve solvent systems. pounds by various detection reagents. Compounds A B C D E Compounds a b c d e f BZP 0.03 0.15 0.13 0.25 0.66 BZP 5 0.5 3 0.05 0.05 ― mTFMPP 0.11 0.37 0.36 0.38 0.78 mTFMPP0.510521― oTFMPP 0.11 0.41 0.36 0.33 0.80 oTFMPP11051 0.5― pTFMPP 0.11 0.37 0.36 0.33 0.77 pTFMPP0.510511― oMeOPP 0.05 0.26 0.18 0.28 0.74 oMeOPP 0.5 5 2 10 0.2 ― pMeOPP 0.05 0.25 0.20 0.28 0.72 pMeOPP 0.5 5 3 10 0.05 ― mCPP 0.11 0.38 0.37 0.32 0.77 mCPP0.53520.5― pCPP 0.07 0.30 0.30 0.27 0.77 pCPP 0.3 3 5 0.5 0.5 ― oFPP 0.12 0.40 0.36 0.28 0.74 oFPP 0.5 5 5 10 0.5 ― pFPP 0.07 0.30 0.25 0.24 0.74 pFPP155100.5― MA 0.09 0.37 0.32 0.21 0.76 MA 10 2 0.1 10 0.1 ― DMA 0.25 0.42 0.51 0.28 0.70 DMA 10 0.5 ― 10.1― MDMA 0.09 0.36 0.32 0.21 0.74 MDMA 5 2 0.1 10 0.1 ― Solvent system: A, 2-butanonedimethylformam- ―, not detected ide25 aqueous ammonia (130.90.1);B,2- Detection reagents: a, UV light; b, DragendorŠ propanol25 aqueous ammonia (955);C,ace- reagent; c, Simon's reagent; d, iodoplatinate rea- tonetoluene25 aqueous ammonia (2010 gent;e,1 iodine-methanol solution; f, p-DMAB 1); D, methanol25 aqueous ammonia (100 solution. 1.5); E, 1-butanolacetic acidwater (211).
of DragendorŠ reagent was lower than that of lower in solvent A, B, C and D than in solvent E. iodoplatinate reagent. The compounds tested All of the compounds tested were not separated were not detected by p-DMAB solution, which is suitably in any developing solvents. In sensitive to compounds with indole25).Simon's particular, it was di‹cult to separate isomers of reagent is reported to be speciˆc for secondary BZP-like compounds. Considering the amines like MA26) and MDMA27).Allofthe separation of spots and the rapidity of tested compounds except DMA are secondary development, solvent D was the best among ˆve amines, but the sensitivity was not as high as solvents. those of MA and MDMA. In terms of speciˆcity Table 5 shows the detection limits by and sensitivity, iodoplatinate reagent was most various detection reagents. The colors of spots eŠective to detect BZP. had few diŠerences among the compounds. They 3 IR absorption spectroscopy were typical colors which were usually indicated IR spectra of the free bases (BZP, by the respective reagents, i.e., orange, dark mTFMPP, oTFMPP, pTFMPP and oMeOPP) blue, purple and brown by DragendorŠ reagent, and of the hydrochlorides (BZP ･ 2HCl, Simon's reagent, iodoplatinate reagent and mTFMPP･HCl, pMeOPP・2HCl, mCPP･HCl, iodine-methanol solution. Therefore, it was pCPP･HCl, oFPP･HCl and pFPP･2HCl) are impossible to identify compounds with the color. shown in Fig. 4. Three isomers of tri‰uoro- Iodine-methanol solution was the most sensitive methylphenylpiperazine (mTFMPP, oTFMPP reagent to detect all spots on chromatograms, and pTFMPP) are clearly distinct from one but not speciˆc. Iodoplatinate, which is sensitive another. The notable diŠerences between these to tertiary amines23), was the most sensitive three spectra are in the aromatic CH out of reagent to detect BZP, but less to the other BZP- plane deformation region (6001000 cm－1). like compounds. DragendorŠ reagent is also 4GC/MS sensitive to tertiary amines24). But the sensitivity Fig. 5 shows total ion chromatogram (TIC) 174174 Hiroyuki Inoue et al
Fig. 4 IR spectra of BZP-like compounds. Analysis of Benzylpiperazine-like Compounds 175175 and mass chromatograms of the molecular ions Most of the derivatized BZP-like of non-derivatized BZP-like compounds under compounds were relatively stable. Any the EI mode, and their retention indices were derivatives except for N-acetyl-pMeOPP kept summarized in Table 6. Except for mCPP, three the peak area ratio (PAR) to the internal kinds of the psychoactive piperazines (BZP, standard (for acetylation and tri‰uoro- mTFMPP and oMeOPP) were separated from acetylation) or peak area (for trimethylsilylation) their isomers and BZP. The EI mass spectra are more than 80 at 4 hr after derivatization in illustrated in Fig. 6. The fragmentation patterns comparison with the initial injection. The PAR of the compounds were previously proposed by of N-acetyl-pMeOPP decreased to 55 of the de Boer et al.1). The spectra showed weak initial injection. molecular ions and relatively simple spectra. TheEImassspectraofderivatizedBZP-like Chromatograms of derivatized BZP-like compounds are shown in Fig. 1012. compounds are shown in Fig. 79, and these Fragmentation patterns of acetylated and retention indices are summarized in Table 6. tri‰uoroacetylated compounds were previously Any derivatizations used in this study improved proposed by de Boer et al.1). We proposed that peak shapes and separation of CPP isomers. of trimethylsilylated compounds, as shown in However, with the exception of Fig. 13. Trimethylsilylated BZP-like com- trimethylsilylation, other two derivatizations pounds, except for N-trimethylsilyl-BZP, impaired separation between BZP and showed common fragmentation patterns. The EI mTFMPP, whose non-derivatized forms were mass spectra of all derivatized compounds separated from each other. potently showed the molecular ions. In particular, in trimethylsilylated derivatives except for N-trimethylsilyl-BZP, the molecular ions were the base peak in the mass spectra. However, there was no improvement in terms of distinction of the isomers. It was considered that trimethylsilylation was the most appropriate choice for the analysis of BZP-like compounds in terms of the peak shapes, separation and
Table 6 Retention indices of non-derivatized and derivatized BZP-like compounds. derivatization Compounds non Ac TFA TMS BZP 1514 1979 1728 1686 mTFMPP 1540 1972 1724 1700 oTFMPP 1428 1847 1597 1583 pTFMPP 1605 2043 1791 1768 oMeOPP 1653 2102 1837 1813 pMeOPP 1782 2239 1958 1953 mCPP 1771 2234 1954 1942 Fig. 5 TIC and mass chromatograms of non-deriva- pCPP 1778 2222 1967 1951 tized BZP-like compounds in the EI mode. A, oFPP 1489 1928 1665 1659 BZP; B, mTFMPP; C, oTFMPP; D, pTFMPP; pFPP 1534 1979 1723 1706 E, oMeOPP; F, pMeOPP; G, mCPP; H, pCPP; non, non-derivatized; Ac, N-acetylation; TFA, N- I,oFPP;J,pFPP. tri‰uoroacetylation; TMS, N-trimethylsilylation. 176176 Hiroyuki Inoue et al
Fig. 6 EI mass spectra of non-derivatized BZP-like compounds. Analysis of Benzylpiperazine-like Compounds 177177
Fig. 7 TIC and mass chromatograms of acetylated Fig. 8 TIC and mass chromatograms of tri‰uoroa- (Ac-) BZP-like compounds in the EI mode; A, cetylated (TFA-) BZP-like compounds in the EI N-Ac-BZP; B, N-Ac-mTFMPP; C, N-Ac- mode; A, N-TFA-BZP; B, N-TFA-mTFMPP; oTFMPP; D, N-Ac-pTFMPP; E, N-Ac- C, N-TFA-oTFMPP; D, N-TFA-pTFMPP; E, oMeOPP; F, N-Ac-pMeOPP; G, N-Ac-mCPP; N-TFA-oMeOPP; F, N-TFA-pMeOPP; G, N- H, N-Ac-pCPP; I, N-Ac-oFPP; J, N-Ac-pFPP. TFA-mCPP; H, N-TFA-pCPP; I, N-TFA- oFPP; J, N-TFA-pFPP. stability. Protonated molecules ([M＋H]＋ )inCI protonated or deprotonated from the molecules. mass spectra of BZP-like compounds were Meanwhile, increase in cone voltage (also called summarized in Table 7. In addition to proton- skimmer voltage for other spectrometer) ated molecules, trimethylsilylated compounds facilitates fragmentation of the ions. This showed the other peaks that corresponded to the phenomenon, called in-source collision-induced ions at m/z of [M＋H－72]＋. dissociation, is helpful to identify compounds 5LC/MS because a fragment pattern re‰ects on the Chromatographic conditions of BZP-like molecular structure. compounds were developed (Fig. 14). The C18 LC/MS measurements were performed bonded phase of the Symmetry Shield column under the multiple settings of cone voltage to embedded polar group practically suppressed examine the relationship between cone voltage peak tailing under the acidic condition, although and mass spectrum of the target analytes. Cone the combination of neutral mobile phase and voltage of 30 V or less commonly gave the conventional C18 bonded phase columns gave spectra for all the analytes that correspond signiˆcant peak tailing (data not shown). The almost exclusively to protonated molecules (Fig. gradient elution achieved discrimination of 15, left). With respect to the protonated isomers except for oFPP and pFPP. molecules, cone voltage of 40 V was the best to Electrospray ionization generally produces gain the largest peak for most of the analytes, molecular-related ions that are usually but some fragment ions were also emerged (data 178178 Hiroyuki Inoue et al
the detail mechanism can not be provided by this study.
Conclusion The analytical data of BZP-like compounds with color tests, TLC, IR spectroscopy, GC/MS and LC/MS were presented. The information would be useful for identiˆcation of piperazines in conˆscated powders, liquids or tablets.
References 1) deBoer,D.,Bosman,I.J.,Hidvegi,E., Manzoni, C., Benko, A. A., dos Reys, L. J. and Maes, R. A.: Piperazine-like com- pounds: a new group of designer drugs-of- abuse on the European market. Forensic Sci. Int., 121,4756 (2001). 2) Unusual Piperazine Mixture in Alliance, Ohio. Microgram Bull., 35, 190 (2002). 3) Piperazine mixture tablets in Chicago, Fig. 9 TIC and mass chromatograms of trimethyl- Illinois. Microgram Bull., 36,67 (2003). silylated (TMS-) BZP-like compounds; A, N- 4) Piperazine mixture tablets in the Balearic TMS-BZP; B, N-TMS-mTFMPP; C, N-TMS- Islands (Spain). Microgram Bull., 36,710 oTFMPP; D, N-TMS-pTFMPP; E, N-TMS- oMeOPP; F, N-TMS-pMeOPP; G, N-TMS- (2003). mCPP; H, N-TMS-pCPP; I, N-TMS-oFPP; J, 5) Recent Notiˆcations Concerning BZP, N-TMS-pFPP. TFMPP, 2C-T-7, and GBL. Microgram Bull., 35,177182 (2002). 6) Tekes, K., Tothfalusi, L., Malomvolgyi, not shown). Thus we recommend the cone B., Herman, F. and Magyar, K.: Studies on voltage of 30 V for the LC/MS screening in the biochemical mode of action of EGYT- order to simplify the chromatograms and the 475, a new antidepressant. Pol. J. mass spectra. Pharmacol. Pharm., 39,203211 (1987). Mass spectra of the analytes at 60 V are 7) Bye, C., Munro-Faure, A. D., Peck, A. W. shown in Fig. 15 (right). Fragmentation was and Young, P. A.: A comparison of the observed at the cone voltage of 50 V or more. eŠects of 1-benzylpiperazine and The signals that correspond to the ions at m/z of dexamphetamine on human performance [M＋H－43]＋ (e.g. m/z 188 in the mTFMPP tests. Eur. J. Clin. Pharmacol., 6, 163169 spectrum) were commonly observed except for (1973). BZP and oTFMPP. This may involve the 8) Campbell, H., Cline, W., Evans, M., elimination of ethyleneimine (C2H5N) from the Lloyd, J. and Peck, A. W.: Comparison of piperazine moiety. The ion at m/z 91 in a BZP theeŠectsofdexamphetamineand1- spectrum seems benzyl cation. The fragment benzylpiperazine in former addicts. Eur. J. pattern of oTFMPP was considerably diŠerent Clin. Pharmacol., 6,170176 (1973). from those of the isomers. The elimination of 9) BZP and TFMPP: Chemicals used to ‰uoride together with an adjacent proton at the mimic MDMA's eŠects. Microgram Bull., imine nitrogen would provide the fragment ion 35,123126 (2002). at m/z 211 in the oTFMPP spectrum, although 10) Pettibone, D. J. and Williams, M.: Analysis of Benzylpiperazine-like Compounds 179179
Fig. 10 EI mass spectra of acetylated BZP-like compounds. 180180 Hiroyuki Inoue et al
Fig. 11 EI mass spectra of tri‰uoroacetylated BZP-like compounds. Analysis of Benzylpiperazine-like Compounds 181181
Fig. 12 EI mass spectra of trimethylsilylated BZP-like compounds. 182182 Hiroyuki Inoue et al
Fig. 13 Proposed structures of characteristic ions of trimethylsilylated BZP-like compounds; N-
TMS-BZP (A) and other 9 analytes (B). R1,R2
and R3 depend on the compounds.
Table 7 Protonated molecules in CI mass spectra of Fig. 14 Mass chromatograms of the standard mix- non-derivatized and derivatized BZP-like com- ture solution containing 1 mg/mL of each ana- pounds. lyte. The cone voltage was set to 30 V. derivatization Compounds non Ac TFA TMS receptor involvement in the stimulus BZP 177 219 273 249(177) mTFMPP 231 273 327 303(231) properties of 1-(m-tri‰uoromethylphenyl) oTFMPP 231 273 327 303(231) piperazine (TFMPP). J. Pharmacol. Exp. pTFMPP 231 273 327 303(231) Ther., 237,369377 (1986). oMeOPP 193 235 289 265(193) 12) Balmelli, C., Kupferschmidt, H., Rentsch, pMeOPP 193 235 289 265(193) K. and Schneemann, M.: Fatal brain edema mCPP 197 239 293 269(197) after ingestion of ecstasy and pCPP 197 239 293 269(197) benzylpiperazine. Dtsch. Med. Wochenschr., oFPP 181 223 277 253(181) 126, 809811 (2001). pFPP 181 223 277 253(181) 13) Staack, R. F., Fritschi, G. and Maurer, H. non, non-derivatized; Ac, N-acetylation; TFA, N- H.: Studies on the metabolism and tri‰uoroacetylation; TMS, N-trimethylsilylation. toxicological detection of the new designer The numbers in parentheses show fragment ions. drug N-benzylpiperazine in urine using gas chromatography-mass spectrometry. J. Chromatogr. B, 773,3546 (2002). Serotonin-releasing eŠects of substituted 14) Staack, R. F., Fritschi, G. and Maurer, H. piperazines in vitro. Biochem. Pharmacol., H.: New designer drug 1-(3-tri‰uoro- 33, 15311535 (1984). methylphenyl) piperazine (TFMPP): gas 11) Cunningham, K. A. and Appel, J. B.: chromatography/mass spectrometry and Possible 5-hydroxytryptamine1 (5-HT1) liquid chromatography/mass spectrometry Analysis of Benzylpiperazine-like Compounds 183183
Fig. 15 ESI mass spectra of each analyte with the cone voltage of 30 V (left) and 60 V (right).
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