Journal of Analytical Toxicology, Vol. 30, September 2006

Rapid Screeningfor and SimultaneousSemiquantitative Analysisof Thirty Abused in Human Samples Using Gas Chromatography- Mass Spectrometry Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021

Tomomi Ishida 1, Keiko Kudo 1, Hiromasa Inoue 1, Akiko Tsuji1, Takashi Kojima 2, and Noriaki Ikeda 1,* 1Department of Forensic Pathology and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan and 2Kanagawa Prefectural Institute of Public Health, Kanagawa 253-0087, Japan

Abstract[ signer drugs, , , and derivatives, are being widely abused among juveniles (1). Also, illicit use of opiates and the anesthetic (KET), In Japan, a wide variety of designer drugs became popular among which is known as or "Special is spreading (2,3). These juveniles because of their availability via the Internet and mobile "K" K', phones. Hence, it is necessary to develop simple and rapid designer drugs are sold as tablets or powder and are easily screening method for these drugs. We devised a rapid screening purchased via the Internet and mobile phones. method for and simultaneous semiquantitative analysis of 30 It is said that the classical , (AP) abused drugs, including , amphetamine-, and MA, mainly enhance -mediated neurotransmis- piperazine-, tryptamine-, and pbenethylamine-derived designer sion, leading to potent effects (4,5), whereas newer drugs and opiates in human urine. The urine sample was digested designer drugs such as amphetamine, piperazine, tryptamine, with urease, and the drugs were analyzed by gas chromatography- and phenethylamine derivatives mainly enhance - mass spectrometry in the scan mode after solid-phase extraction mediated neurotransmission, leading to psychedelic experi- with a FocusTM column and acetylation. The retention time ence and "feeling " (6-9). In recent years, a number of obtained with the use of a retention time locking technique and severe and even fatal intoxications attributable to these de- three qualifier ions were used to obtain positive results. As the signer drugs have been reported (10-18). Focus column requires only simple extraction steps and can retain various drugs of a wide range of polarity, screening of 30 abused Numerous methods for the determination of AP, MA, and drugs was feasible within 3 h. The calibration curves were linear amphetamine-derivatives using gas chromatography (GC), in the concentration range of 100-5000 ng/mL in most drugs with GC-mass spectrometry (MS), liquid chromatography (LC), correlation coefficients exceeding 0.99. The absolute recoveries and LC-MS have been reported and described in review articles for all drugs in urine samples were 6.9-125.4% at the (19,20). Several analytical methods for newer designer drugs concentration 1000 ng/mL. This method will be most useful such as piperazine (21-25), tryptamine (26,27), and phenethy- to confirm the presence of many abused drugs in urine in lamine (28,29) derivatives have been reported. However, no clinical and forensic cases. screening procedure for all drugs described is reported. De- signer drugs are often sold as a mixture of several drugs (2), and there are cases where results of immunoassay screening Introduction were negative but intoxication of these drugs is strongly sus- pected. In such cases, the analysis for a respective class of In Japan, (MA) has been the most ex- drugs is not easy. Therefore, it is necessary to develop a second tensively used illicit drug. In the latter half of the 1990s, the screening method that can detect a wide ranging class of drugs abuse of other drugs such as 3,4-methylenedioxymetham- simultaneously. Furthermore, it is more convenient if the phetamine (MDMA) and p-methoxyamphetamine (PMA) has method can roughly estimate the concentration of drugs at the also been on the rise. These drugs are generally called "club same time. drugs" or "designer drugs". More recently, varieties of de- We developed a rapid screening and semiquantitative method for 30 abused drugs (shown in Table I) in human

* Author to whom correspondence should be addressed. urine using solid-phase extraction (SPE) with a Focus column E-maih [email protected],ac,jp. followed by acetylation and GC-MS analysis.

468 Reproduction (photocopying)of editorialcontent of this journal is prohibitedwithout publisher'spermission. Journal of Analytical Toxicology, Vol. 30, September 2006

Experimental hydrochloride was purchased from Sankyo Co. (Tokyo,Japan). Codeine (COD) phosphate and dihydrocodeine (DCO) phos- Chemicals and reagents phate were purchased from Takeda Pharmaceutica] Co. (Osaka, MA hydrochloride was purchased from Dainippon Pharma- Japan). 1-(3-Trifluoromethyphenyl)piperazine (TFMPP) hy- ceutical Co. (Osaka, Japan). (PPA) hy- drochloride was purchased from Avocado Research Chemicals drochloride, 4-bromo-2,5-dimethoxy-[3-phenetylamine(-B) (Lancashire, U.K.). AP sulfate was a generous gift from De- hydrochloride, hydrochloride, and 5-methoxy-N,N- partment of Forensic Medicine, Fukuoka University School of dimethyltryptamine (5MeO-DMT) were purchased from Medicine. N-Methyl-l-(3,4-methylenedioxyphenyl)-2-bu- Sigma-Aldrich Co. (St. Louis, MO). Methylephedrine (ME), or tanamine (MBDB) was purchased from Cerilliant (Austin, TX). methyltryptamine (AMT), N- (BZP), and 1- Medazepam hydrochloride was provided by Shionogi & Co. (4-methoxyphenyl)piperazine (4MPP) were purchased from (Osaka, Japan). 1-(3-Chlorophenyl)piperazine (3CPP) hy- Aldrich Chemical Co. (Milwaukee,WI). 2,5-Dimethoxy-4-iodo- drochloride and KET hydrochloride were purchased from ~-phenethylamine hydrochloride (2C-I), 2,5-dimethoxy-4- Wako Pure Chemical Industries (Osaka, Japan).

ethylthio-[3-phenethylamine (2C-T-2) hydrochloride, Trifluoroacetic acid (TFA), urease from Jack Bean (activity, Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 2,5-dimethoxy-4-(n)-propylthio-~-phenethylamine (2C-T-7) 133 units/mg), and ethylacetate were purchased from Wako hydrochloride, 5-methoxy-cz-methyltryptamine (5MeO-AMT) Pure Chemical Industries (Osaka, Japan). Urease (200 rag) hydrochloride, and 5-methoxy-N,N- was dissolved in 10 mL of distilled water. Acetic anhydride (5MeO-DIPT) hydrochloride were synthesized by Chemical was purchased from Sigma-Aldrich Co. (silylation Soft R&D (Kyoto, Japan). 3,4-Methylenedioxyamphetamine grade) was purchased from Pierce (Milwaukee,WI). Focus was (MDA) hydrochloride, MDMA hydrochloride, dimethylam- purchased from Varian (Lake Forest, CA). The other chemicals phetamine (DMA) hydrochloride, PMA hydrochloride, p- were of analytical reagent grade. methoxymethamphetamine (PMMA) hydrochloride, 4MTA hydrochloride, and were synthesized in our laboratory Standard solutions using previouslypublished methods (29-31). (MOR) Most drugs (5 mg as free ) were dissolved in methanol, and the volume was adjusted to 5 mL to obtain a concentration of 1000 ng/tJL. This solution was further diluted in methanol Table I. Thirty Abused Drugs and Their Abbreviations to 100, 10, and 1 ng/IJL. PPA, EP, MOR, COD, and DCO were dissolved in 0.01M hydrochloric acid. ME was dissolved in Compound Abbreviation 0.01M hydrochloric acid containing 0.05% methanol.

Amphetamine AP Biological samples Methamphetamine MA DMA Urine samples were obtained from healthy Japanese volun- Phenylpropanolamine PPA teers and stored at -20~ until analysis. EP Methylephedrine ME Extraction and derivatization procedure 3,4-Methylenedioxyamphetamine MDA One milliliter of urine sample was mixed with 1 lJL IS solu- 3,4-Methylenedioxymethamphetamine MDMA tion (1 lJg medazepam) in a centrifuge tube (10 mE) and di- N-Methyl-l-(3,4-methylenedioxyphenyl)-2-butanamine MBDB gested with 200 units of urease at 37~ for 10 rain. One N-Benzylpiperazine BZP milliliter of 0.05M borate/0.1M potassium dihydrogen 1-(3-Trifluoromethylphenyl)piperazine TFMPP phosphate buffer (pH 9.0) was added, and the mixture was 1-(3-Chlorophenyl)piperazine 3CPP vortex mixed for 10 s and centrifuged at 850 xg for 5 min. The 1-(4-Methoxyphenyl)piperazine 4MPP supernatant was applied to Focus column conditioned se- 0~-Methyltryptamine AMT 5-Methoxy-~-methyltryptamine 5MeO-AMT quentially with 1 mL of methanol and I mL of distilled water. 5-Methoxy-N,N-dimethyltryptamine 5MeO-DMT The column was rinsed sequentially with 1 mL of distilled 5-Methoxy-N,N-diisopropyltryptamine 5MeO-DIPT water and 1 mL of 30% acetonitrile (ACN). The analytes were Psilocin eluted with 1 mL of ACN/distilled water/TFA (90:10:0.1, v/v). 4-Bromo-2,5-dimethoxy-~-phenethylamine 2C- Then the eluate was evaporated to dryness under a stream of 2,5-Dimethoxy-4-iodo-~-phenethylamine 2C-I . The residue was dissolved in 50 l~L of pyridine, and 2,5-Dimethoxy-4-ethylthio-~-phenethylamine 2C-T-2 50 lJL of acetic anhydride was added to the solution for acety- 2,5-Dimethoxy-4-(n)-propylthio-ig-phenethylamine 2C-T-7 lation. The mixture was kept at 60~ for 30 min, and then the p-Methoxyamphetamine PMA solvent was evaporated to dryness. The residue was dissolved in p-Methoxymetamphetamine PMMA 100 lJL of ethyl acetate, and a 2-1JL aliquot of the solution was 4-Methylthioamphetamine 4MTA injected into the GC-MS apparatus. Mescaline Morphine MOR Codeine COD GC-MS condition Dihydrocodeine DCO The apparatus used was an Agilent 6980 GC combined with Ketamine KET an Agilent 5973 MS. An HP-lms fused-silica capillary column (30 m x 0.25-mm i.d., 0.25-1Jm film thickness) coated with

469 Journal of Analytical Toxicology, Vol. 30, September 2006

1

] 100 .s Z .~s DMA EP-2AC ~ "o~s < 91 15 148

, ,IL L .... ~...... ) ..... 9 i ..... I00 200 3O0 4OO I00 200 300 4OO m/z m/z 8

~ 118 AP-AC 8 ] ,,.C,s s

Ioo ~o ~ c~ o Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 < <

121

9 . .', ...... , 9 I00 200 3O0 40o .,[.,.,[ .I. 100 200 300 400 m/z m/z

9 [] [] O 8 o ME-AC < 771O5

h / / r L I >., ~l t ..... ! ...... 100 200 3~ 4o0 1(30 200 303 400 m/z m/z

,o []

~Hs ~ MA-AC TFMPP-AC i2~ ! :F3 < 27__Z

100 200 300 4o0 ...... k i I i I , ,[i.... , nffZ 1(~ 200 3o0 4O0 m/z

0 ,1 []

[] PPA-2AC 4MTA-AC

[ 107 134 z / 176 86

, bL , , , J...... u ) 100 200 3OO 4o0 100 200 3cO 40o affZ

" BZPAC C7 @ ., ' PAAc 146 O~

/ li 175 218 .LI. 1.1~ ~ ~.,[.~ .t.. ,..l ..... 1~ 200 300 400 100 200 300 4O0 rn/z m/z

Figure 1. El mass spectra and structures. The quantifier ions are boxed, and qualifier ions are underlined.

470 Journal of Analytical Toxicology, Vol. 30, September 2006

13 19 [] [] MDMA-AC ,j~ o?,3 2C-B-AC "o~ Ir

< e~ <

I00 200 300 400 . ,i, ~. ~ ,T .... u ,, I1 lO0 200 300 400 ndZ m/z 14

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I00 200 300 400 1~ 2~ 3~ 400 ndZ m&

15 21 162 []

5MeO-DMT ~'~s

13_5. 117 160 ~-~ .L, ,,,.!1!..,T.., ...... 100 200 300 400 I00 200 300 400 m/z ndZ

22 []

Mesealine-AC ~9 KET-AC 179 152 18i Is

125

...... 9 II ., I ~k ~1 .... IlL ,.tL 100 200 300 400 100 200 300 400 mlz m/z 17 [] 23 []

AMT-AC V, IC 0 3:10~3 2C-I-AC

216 349 103 I 275

., .J.,..~.,. , , .... j .I ...... , ...... , ..... 1oo 200 300 400 100 200 3(]0 400 m/z m/z 18 [] o ] ~ ...cHCH3~ "L'HCH3~ 5MeO-DIPT Psilocin-AC ~c ?~ w

Figure 1. (continued) El mass spectra and structures. The quantifier ions are boxed, and qualifier ions are underlined.

471 Journal of Analytical Toxicology, Vol. 30, September 2006

25 [] OCH3 oH:, 282 2C-T-2-AC T r~ ,o--( I 283 ocu3 .< COD-AC o I / 1298 ...... d ,], . [ 100 200 3OO 4OO I00 200 300 400 m/z m/z NCHI 26 [] [] 5MeO-AMT-AC 187 1~ ~ ~O~ =., Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 .< 246 ...., ,..i.~ .~..., ...... [ ...... , ...... , ...... ,T, ,,.. ~ 100 200 300 400 100 200 300 400 m/z m/z

27 [] IS [] 24_22[ ~.~ 2C-T-7-A~?,~ :HgHg;H~ o Medazepam(IS) I :I "0

297 <,.0

...... L. lI I H3C/ 100 200 3OO 4OO 100 200 300 400 m/z m/z 28 NCH3 []

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1 [ ...... L, ,~, L,L~.}L,,,~_,,J,L~L,~ IIL ~ .,w ,L. [ t00 200 300 400 m/z

Figure I. (continued) El mass spectra and structures. The quantifier ions are boxed, and qualifier ions are underlined.

100% dimethylpolysiloxanestationary phase was used. Splitless Screening procedure injection mode was selected with a valve off time of 2 min. The The presence of drugs was screened with two parameters. GC-MS conditions were as follows: the initial temperature One was the relative intensities of the two qualifier ions against 60~ was held for 2 min; the temperature was programmed to the quantifier ion, which were to be within + 20% of those ob- 300~ increased at a rate of 20~ this temperature being tained from the proper reference substance. The other was maintained for 5 min. Injection port and transfer line temper- the retention time of the specific drug, whose difference was atures were 250~ and 280~ respectively. The carrier gas within _+ 2.0% from mean value of five measurements under a was helium, and constant pressure mode was used. Retention different length of column. As the RTL method was used in our time was fixed using the retention time locking (RTL) tech- screening method, retention time, once fixed with the refer- nique: initially, the reference substance, medazepam (IS), was ence substance (medazepam in this study), was unchanged. measured with adequate pressure, then with pressures at -20%,-10%, +10%, and +20% from the initial pressure. The Preparation of calibration curves calibration curve was obtained by plotting these five-point Urine sample were prepared to contain (see Table I for ab- pressures versus respective retention times. We set the reten- breviations): AP, ]VIA, DMA, PPA, PMA, PMMA, MDA, MDMA, tion time of medazepam at 13.20 min, and the pressure ob- MBDB, 4MTA, TFMPP, 3CPP, 4MPP, 2C-B, 2C-I, 2C-T-2, 2C-T- tained from the calibration curve, 17.731 psi, was set as the 7, 5MeO-DIPT, MOR, COD, and DCO at concentrations of 50, RTL condition. The full-scan mode was used. One quantifier 100, 500, 1000, and 5000 ng/mL; ME, EP, BZP, mescaline,AMT, and two qualifier ions were selected for each drug. 5MeO-AMT,5MeO-DMT, and KET at 100, 500, 1000, and 5000

472 Journal of Analytical Toxicology, Vol. 30, September 2006 ng/mL; or psilocin at 3000, 5000, 7000, and 10000 ng/mL, buffer solution resulted in the highest average recovery for each containing 1000 ng IS. These samples were extracted in most of the drugs. Thus, we selected pH 9.0 buffer solution for the same manner as described previously.The calibration curve effectivelyanalyzing most of the basic drugs. However, extrac- was obtained by plotting the peak-area ratio of the specific tion efficienciesof EP, ME, PPA, MOR, psilocin, and KET tended drug to IS versus the amount of that drug. to be lower probably because of the hydroxyl group in EP, ME, PPA, and MOR, and the lower PKa value of KET (PKa 6.46). In Estimation of recovery, precision, and retention time these drugs, a higher recovery was obtained with use of buffer Recovery of each drug was measured by comparing the peak solution of lower pH (pH 7.5 or pH 8.0). area of acetylated standard (1000 ng each except for psilocin, 5000 ng was used for this drug) with that of acetylated ex- Selection of SPE column tracts from urine spiked with 1000 ng/mL drug (5000 ng/mL SPE has become an effective approach to isolate and con- for psilocin). Quality control samples were prepared at con- centrate analytes in biological samples. It is said that SPE has centration of 1000 ng/mL, except for psilocin (5000 ng/mL), a number of advantages over liquid-liquid extraction, such as and analyzed as described previously. Within-day precisions cleaner extracts, no emulsion formation, large selection of sol- [as relative standard deviation, RSD (%)] were calculated based vents available for use, reduction of sample preparation time, Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 on the prepared calibration curves. and easier automation of preparation (34-36). It is also im- The retention times of 30 drugs were measured with five dif- portant that SPE can require a smaller sample volume be- ferent lengths of column. Mean value, standard deviation (SD), cause a sample is sparse in forensic cases. and RSD (%) were calculated for each compound. We, therefore, extracted 30 drugs using several types of SPE columns. Silica-based mixed-mode (non-polar C8 and a strong cation exchanger) sorbent and polymer-based column (poly- Results and Discussion merized hydrophilic and hydrophobic monomer) were exam- ined. Obtaining clean extracts and high recoveries was Extraction procedure Urine was selected as matrix for this screening procedure be- Table II. Peak No., Retention Time (RT), and Relative cause urine was easily collected in both clinical and forensic Response Acetylated Drugs cases and contained high concentrations of drugs. In some urine samples, a large amount of urea interfered with analysis PeakNo. Drug RT (min) RelativeResponse of drugs. Shinka et al. (33) successfully removed urea by urease for the analysis of 4-hydroxybutyric acid (GHB), and when we 1 DMA 7.46 72(100) 91(7.1) 115(1,3) applied their method we obtained a sharp and symmetrical 2 AP-AC 8.88 86(100) 118(79) 177(2.7) peak of each drug. As the pH of urine after urease treatment 3 ME-AC 8.93 72(100) 77(2.3) 105(1.4) varied significantly because of different amounts of 4 MA-AC 9.47 58(100) 100(71) 191(1) produced from different urine samples, a pH adjustment step 5 PPA-2AC 10.38 86(100) 107(17) 134(13) was required with buffer solution. Different buffer solution 6 PMA-AC 10.46 148(100) 121(31) 86(14) with pH 7.5, 8.0, 9.0, 10.0, and 11.0 were examined, and pH 9.0 7 EP-2AC 10.79 58(100) 100(86) 117(4.3) 8 PMMA-AC 10.97 58(100) 148(72) 100(39) 9 MDA-AC 11.06 162(100) 135(24) 77(13) 10 TFMPP-AC 11.51 200(100) 188(39) 132(33) 3,0 x 10 6 z~ A 14 19 11 4MTA-AC 11.54 164(100) 137(18) 86(18) 2.5 x 10 6 12 BZP-AC 11.54 91(100) 146(49) 132(22) 2.0 X 10 6 ~ 4 It 13 MDMA-AC 11.57 58(100) 162(76) 100(32) ,6 21 2~ ~ 1.5 x 10 6 14 MBDB-AC 11.91 72(100) 176(56) 114(30) 1.0 x 106 15 17. Ig 2Z '?'3 15 5MeO-DMT 12.04 58(100) 218(11) 160(5,8) 5.0xlO s 16 Mescaline-AC 12.16 194(100) 179(46) 253(18)

0 17 AMT-AC 12.58 130(100) 157(82) 216(11) 8 9 10 I1 12 13 14 15 18 Psilocin-AC 12.67 58(100) 146(7.1) 246(0.4) Time (rain) 19 2C-B-AC 12.75 242(100) 229(26) 148(24) 3.5 x 10 6 20 3CPP-AC 12.76 166(100) 238(43) 138(21) 3.0 x 10 6 21 4MPP-AC 12.81 234(100) 162(96) 135(26) 2.5 x 10 6 22 KET-AC 12.87 216(100) 208(86) 125(15) ~ 2.0 x 10 6 IS Medazepam 13.21 ~ 1.Sx10 6 23 2C-I-AC 13.25 290(100) 349(18) 275(12) 1.0xl0 6 24 5MeO-DIPT 13.34 114(100) 160(7) 274(0.4) 5.0x10 s 25 2C-T-2-AC 13.34 224(100) 211(33) 283(28) 0 8 9 10 II 12 13 14 26 5MeO-AMT-AC 13.57 160(100) 187(77) 246(16) Time (rain) 27 2C-T-7-AC 13.69 238(100) 225(29) 297(27) Figure 2. Total ion chromatogram of derivatized extracts from urine 28 DCO-AC 14.34 343(100) 284(31) 300(31) spiked with 5000 ng/mL each of 30 drugs and IS (A) and from blank urine 29 COD-AC 14.48 341(100) 282(64) 229(31) (B). 30 MOR-2AC 15.05 327(100) 369(75) 268(56)

473 Journal of Analytical Toxicology, Vol. 30, September 2006 unsatisfactory; thus, we tried to a use Focus column, which was by the solvent containing TFA and recoveries were the best. recently developed and had multifunctional polymer as sor- As there was no difference in recovery between a mixed sol- bent. Colorless extracts were obtained by simple extraction vent of methanol/ACN and ACN only, so ACN was used as or- steps, and the recovery was satisfactory in most drugs. Various ganic solvent. drugs of a wide range of polarity (from polar to non-polar drug) were retained by polar-enhanced sorbent with a hy- Derivatization procedure drogen-bond donor and acceptor site, dipole-dipole, and hy- Screening methods with several derivatizations, heptafluo- drophobic interactions. robutylation (HFB) (37,38), tert-butyldimethylsilylation (TBDMS), trimethylsilylation (TMS) (39), and 2,2,2- Focus column protocols trichloroethylcarbamation (3Clec) (40), have been reported. We The extraction protocol by the Focus column was a modifi- thought that drugs having a higher molecular weight such as cation of that described in the manual provided by Varian opiates would have too a large molecular weight after HFB or (http://www.varianinc.com). Several concentrations of ACN 3Clec derivatization in GC-MS analysis, although they are were examined as rinse solvent, and 30% ACN led to the sensitive and suitable for analysis of amphetamine, piperazine, Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 highest recoveries. The eluting solvent recommended was a and phenethylamine derivatives. There is also a possibility that mixture of methano], ACN, distilled water, and acid. According derivatives are incompletely formed because of steric hin- to the manual, distilled water, ACN, and methanol disrupts drance between bulky trimethylsilyl, heptafluorobutyl, and the hydrogen bond acceptor, dipole-dipole,and hydrophobic in- tert-butyldimethylsilyl groups and drugs. teraction, respectively. Adding acid, bonding between the hy- TMS and acetylation were examined for the derivatization of drogen donor and analytes having hydrogen acceptor is 30 drugs. Comparing with acetylated extracts, TMS-deriva- dissociated. The effects of acid were investigated using TFA, tized extracts gave interfering peaks probably because of bio- formic acid, and acetic acid. Clean extracts were obtained only logical matricies on the chromatogram, which tended to give

Table III. Column Length and Retention Time

Retention Time (min) by Column Length Peak No. Analyte 30.0 m 29.5 m 29.0 m 28.5 m 28.0 m Mean (min) SD RSO (%)

1 DMA 7.471 7.412 7.563 7.366 7.493 7.461 0.076 1.015 2 AP-AC 8.858 8.86 8.901 8.873 8.896 8.878 0.020 0.225 3 ME-AC 8.919 8.923 8.954 8.916 8.941 8.931 0.016 0.182 4 MA-AC 9.458 9.456 9.484 9.473 9.485 9.471 0.014 0.146 5 PPA-2AC 10,359 10.359 10.402 10,372 10,394 10,377 0,020 0.192 6 PMA-AC 10,451 10,445 10,486 10,459 10,48 10,464 0,018 0,172 7 EP-2AC 10,772 10,771 10,793 10,793 10,8 10,786 0,013 0,124 8 PMMA-AC 10.974 10.972 10.997 10.99 11.001 10,987 0,013 0,120 9 MDA-AC 11,043 11,036 11.082 11,053 11,076 11.058 0,020 0.183 10 TFMPP-AC 11.499 11,495 11.533 11.51 11,526 11.513 0.017 0.144 11 4MTA-AC 11.522 11,515 11,561 11.528 11,55 11,535 0,019 0.169 12 BZP-AC 11.527 11,526 11,559 11.54 11,554 11,541 0,015 0.131 13 MDMA-AC 11.556 11.553 11,587 11.567 11,582 11,569 0,015 0.131 14 MBDB-AC 11.891 11.903 11,917 11,906 11,917 11,907 0.011 0,091 15 5MeO-DMT 11,983 12,12 12.113 11,964 11.995 12.035 0.075 0,625 16 Mescaline-AC 12,151 12,141 12,183 12,159 12.182 12.163 0.019 0.154 17 AMT-AC 12,575 12,551 12,609 12,568 12.602 12.581 0.024 0,192 18 Psi[ocin-AC 12,639 12.63 12.79 12.62 12.658 12.667 0.070 0.552 19 2C-B-AC I2.743 12.731 12,77I 12.749 12.769 12.753 0.017 0.135 20 3CPP-AC t2.747 12.735 12.77 12,755 12.769 12.755 0.015 0,117 21 4MPP-AC 12,796 12.787 12.818 12,805 12.821 12.805 0.014 0,112 22 KET-AC 12.858 12.855 12.886 12.874 12.889 12.872 0.016 0.121 23 2C-I-AC t3,238 13.227 13.259 13,247 13.26 13.246 0.014 0.106 24 5MeO-DIPT 13,297 13.343 13.409 13.31 13.324 13.337 0.044 0.329 25 2C-T-2-AC 13.328 13.317 13.352 13,337 13.351 13.337 0.015 0.113 26 5MeO-AMT-AC 13,619 13.549 13.549 13.576 13.575 13.574 0,029 0.211 27 2C-T-7-AC 13.638 13.678 13.707 13~697 13.709 13.686 0,029 0.215 28 DCO-AC 14,321 14.323 14.36 14.34 14.345 14.338 0.016 0.113 29 COD-AC 14.463 14.459 14.493 14.48 14.485 14,476 0,015 0.100 30 MOR-2AC 15.032 15.032 15,07 15.051 15.054 15,048 0.016 0.107 IS Medazepam 13.196 13.193 13.218 13.213 13,219 13,208 0,012 0.094

474 Journal of AnalyticalToxicology, Vol. 30, September2006 only one large peak on the mass spectra and were easily de- After analyses of numerous samples in routine screening, the composed. Also, two peaks being attributed to free drug and leading edge of the column becomes tainted, which causes derivative drug appeared on the chromatogram in a many peak broadening and decreased sensitivity. In such cases, cut- drugs. Therefore, we considered that acethylation was appli- ting the leading edge of column is often conducted as mainte- cable to various drugs with wide molecular weight. DMA, nance. Therefore, retention times obtained in the past 5MeO-DMT, 5MeO-DIPT,and IS were not acetylated. measurement often change. The RTL technique is reportedly to overcome this problem and give consistent retention times GC-MS analysis with good precision (41). Table III shows values of retention The election impact (EI) mass spectra of 30 drugs and IS is times, standard deviation, and %RSD for 30 drugs with RTL shown in Figure 1. Figure 2 shows total ion chromatogram of technique after every cutting of the column by 0.5 meter. Re- derivatized extracts from urine spiked with 5000 ng/mL of tention times of 30 drugs were almost constant, and the RSD each of the 30 drugs and IS (Figure 2A) and from blank urine (%) was below 1.0% in all drugs except DMA, which was (Figure 2B). Table II shows retention time, quantifier and two 1.015%. Therefore, consistent retention times for each drug qualifier ions, and relative response of 30 drugs. Sharp peaks was obtained. Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 attributed to each drug were obtained. There were some peak Table IV shows calibration ranges, correlation coefficients, overlappings: peaks 10, 11, and 12, attributed to TFMPP-AC, precision (RSD%) and recoveries (%) of all drugs. The cali- 4MTA-AC, and BZP-AC, and peaks 19 and 20, attributed to 2C- bration curves were liner in the concentration range from B-AC and 3CPP-AC, had nearly same retention times (11.51, 50-5000 ng/mL for DMA, AP, MA, PPA, PMA, PMMA, MDA, 11.54, and 11.54 min and 12.75 and 12.76 rain, respectively). MDMA, MBDB, 4MTA, 2C-B, 3CPP, 4MPP, 2C-I, 5MeO-DIPT, These drugs were easily differentiated by selecting specific ions 2C-T-2, 2C-T-7, MOR, COD, and DCO; 100-5000 ng/mL for for each drug. It is another advantage that GC-MS analysis EP, BZP, AMT, 5MeO-DMT, mescaline, 5MeO-AMT and KET; using full scan mode makes possible spectral identification of and 3000-10,000 ng/mL for psilocin in urine with correlation drugs not included in this study. coefficients exceeded 0.99 in almost all drugs (over 0.98 for 4MTA, MDMA,and 5MeO-AMT).Within-day precision (RSD%) of this method was 2.43-15.76%. The calculated recoveries Table IV. Calibration Range, Correlation Coefficient (R2), were above 70% for 18 drugs (DMA, AP, MA, PMA, PMMA, Extraction Recovery, and Precision (RSD%) MDA, TFMPP, 4MTA, BZP, MDMA, MBDB, 3CPP, 4MPP, 2C-I, Peak Calibration Recovery Precision 5MeO-DIPT, 2C-T, 2C-T-7, and COD), from 40% to 70% for six No. Analyte Range(ng/mL) R2 (%) (RSD%) drugs (ME, PPA, 5MeO-DMT, 2C-B, and DCO), and below 40% for six drugs (EP, mescaline, psilocin, KET, 5MeO-AMT, and 1 DMA 50-5000 0.9983 103.6 5.60 MOR). The calculated mean recovery for 30 drugs was 69.31%. 2 AP-AC 50-5000 0.9973 125.4 2.61 Some fatal and nonfatal poisoning cases by controlled drugs 3 ME-AC 100-5000 0,9967 44.8 2.43 and designer drugs have been reported (10-18). Although the 4 MA-AC 50-5000 0,9904 112.5 7.16 drug concentration of the urine sample was rarely determined 5 PPA-AC 50-5000 0,9971 44.8 6.16 in these reports, we assume that this screening method would 6 PMA-AC 50-5000 0,9987 73.6 7.57 7 EP-2AC 100-5000 0.9979 24.0 10.50 be applicable for confirmation of intake of 30 drugs in emer- 8 PMMA-AC 50-5000 0,9906 85.0 8.28 gency and forensic toxicology. This method required less than 9 MDA-AC 50-5000 0.9988 76.6 4.42 3 h from sample preparation to instrumentalanalysis. 10 TFMPP-AC 50-5000 0.9982 75.6 6.83 Thus, this screening method is useful in terms of 1. rapid 11 4MTA-AC 50-5000 0.9845 99.8 6.21 confirmation and semiquantitative analysis; 2. simple prepa- 12 BZP-AC 100-5000 0.9929 91.9 6.13 ration procedure using a Focus column for a broad range of 13 MDMA-AC 50-5000 0.9881 79.3 11.12 drug; and 3. reliable identification using mass spectra and the 14 MBDB-AC 50-5000 0,992 84.4 7.43 RTL technique. 15 5MeO-DMT 100-5000 0.9953 64.5 10.70 16 Mescaline-AC 100-5000 0,9998 38.6 7.31 17 AMT-AC 100-5000 0,9993 48.8 4,58 18 Psilocin-AC 3000-10000 0,9949 33.5 9.07 Conclusions 19 2C-B-AC 50-5000 0,9975 68.0 5,61 20 3CPP-AC 50-5000 0.9966 91.0 6.42 We developed a rapid method for screening and simulta- 21 4MPP-AC 50-5000 0.9982 99,8 5.70 neous semiquantitative analysis of 30 abused drugs in human 22 KET-AC 100-5000 0,992 6,9 15.76 urine using GC-MS. This may be the first screening method for 23 2C-I-AC 50-5000 0.9995 70.4 5.52 30 drugs, which can be used in clinical and forensic toxico- 24 5MeO-DIPT 50-5000 0,9917 81.9 12.03 logical cases. 25 2C-T-2-AC 50-5000 0.9995 74.7 6.18 26 5MeO-AMT-AC 100-5000 0.9847 31.9 9.34 27 2C-T-7-AC 50-5000 0.9996 71.9 6.18 28 DCO-AC 50-5000 0,9992 67.6 7.01 Acknowledgments 29 COD-AC 50-5000 0.99 74.8 15.49 30 MOR-2AC 50-5000 0.9987 33.8 12.81 This work was supported by a Research Foundation for Safe Society and Grant-in-Aid for Scientific Research (No.

475 Journal of Analytical Toxicology, Vol. 30, September2006

16390194) from the Ministry of Education, Sciences, Sports 17. R. Meatherall and P. Sharma. Foxy, a designer tryptamine hallu- and Culture. We thank Dr. R. Hanajiri of National Institute of ciongen. J. Anal. Toxicol. 27:313-317 (2003). 18. H. Long, L.S. Nelson, and R.S. Hoffman. AIpha-methyltryptamine Health Sciences, Dr. O. Shirota of the Faculty of Pharmaceu- revisited via easy internet access. Vet. Hum. Toxicol. 45:149 tical Sciences at Kagawa Campus Tokushima Bunri University, (2003). and Dr. K. Hara of the Department of Forensic Medicine, 19. M.R. Moeller, S. Steinmeyer, and T. Kraemer. Determination of Fukuoka University School of Medicine for their helpful drugs of abuse in blood. J. Chrornatogr. B 713:91-109 (1998). comments as well as M. Ohara (Fukuoka, Japan) for language 20. T. Kraemer and H.H. Maurer. Determination of amphetamine, methamphetamine and amphetamine-derived designer drugs or assistance. medicaments in blood and urine. J. Chromatogr. B 713:163-187 (1998). 21. D.D. Boer, I.J. Bosman, E. Hidv~gi, C. Manzoni, A.A. BenkO, L.J.A.L.D. Reys, and R.A.A. Maes. Piperazine-like compounds: a References new group of designer drugs-of-abuse on the European market. Forensic Sci. Int. 121: 47-56 (2001). 1. M. Katagi, H. Tsutsumi, A. Miki, K. Nakajima, and H. Tsuchihashi. 22. R.F. Staack, G. Fritschi, and H.H. Maurer. Studies on the

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477 Journal of Analytical Toxicology, Vol. 31, January/February 2007

I l e, at.m I

There was a problem with the chemical structures in Figure 1 of"Rapid Screening for and Simultaneous Semiquantitative Analysis of Thirty Abused Drugs in Human Urine Samples Using Gas Chromatography-Mass Spectrometry" by T. Ishida, K. Kudo, H. Inoue, A. Tsuji, T. Kojima, and N. Ikeda [J. Anal. Toxicol. 30(7): 468-477 (2006)].

The amended figure is reproduced here and continues on the next two pages.

The Journal regrets the error. Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021

m [] m

! Medazcpam(IS)165. 1 <

.. . -.. ,: ,,= .:< ~-~ : .1~ ~ ,L...t . . . I00 200 3OO 400 4

f-OH3 /CH3 [] 100 MA-AC DMA CH3 O < 91 115 148 100 200 300 4O0 100 200 30O 400 ~z m/z

./.H o ~ PPA-2AC H3C,'~O i 118 AP-AC CH3 O

< < o 177 e . ,LL , , , 1,,...... J , , 100 200 300 400 100 200 3OO 4OO m/Z m/:

3 0 [] o o~ Nyo.,/H ME-AC N~ CH~ 121 [ PMA-AC207

.7.2 10S 9~,( "~ ...... 1(13 200 300 400 I00 200 300 4O0 m/z ~z

Figure 1. El mass spectra and structures. The quantifier ions are boxed, and qualifier ions are underlined.

66 Journal of Analytical Toxicology,Vol. 31, January/February2007

13 o [] Z DMA'AC /OH= H

I EP-2AC i <

I00 200 ~z ndz 8 14 148 PMMA-AC ~/-~ NCOlll [] M2~DB-AC C ,yo.,

OHiO ~.,,~'~ CH~ O Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 <

.-, .... ~ , ,[.. , .t ...... , ..... 3oo 4oo 2111 300 400 m/z 15 ] MoAAc C 'yH oH OHm,.. ~ ~ ~N.~ o ~.1~ o~ o w 131 <

10o 100 200 300 400 m/z m/z

,o [] 18 N ~H OH~ Mescaline-AC ~ OHiO~~.~ y OF~ OH=O" T 179 01%0 232 < [ IL L ..... It[ J l ~t 3O0 4o0 100 200 300 400 mlz 1, [] 17 []

u /H t ....H 4MTA-AC 1S7 AMT-AC C yT .< .~l,.86 J,!~L~ [ I 2? 1 I0O 200 3OO 400 I00 200 3Q0 400 m/Z mlz

18 12 [] BZP-AC ~I~AN/'-.~ j [] .,o-~ io., i ,< Psilocin-AC ~ N\OH~ J60 4oo L . ,. :, , .. ~ 1. I . . . , .... [ ...... IQO 200 3(]0 IGO 200 300 400 ,w'z ndz

Figure I. (continued)El mass spectra and structures.The quantifier ions are boxed,and qualifier ions are underlined.

67 ournal of Analytical Toxicology, Vol. 31, January/February 2007

,~ [] C~HI

) 2C-B-AC ~N?CH= o 2C-T-2-AC OH3CHzS" T == OOH= < 14g Or" ~H~ / 283

...... ,.1 ~ .L ~ L I00 200 3(]0 400 ?l ~ lO0 200 3~0 m/Z ,n/Z =e [] /H 8 3CPP-AC Ol 5MeO-AMT-AC ':"--~ N': OH~ 0 23S i H Downloaded from https://academic.oup.com/jat/article/30/7/468/711520 by guest on 01 October 2021 .J'~l 195 l 246 .... L~ L tO0 200 300 400 tO0 200 300 400 ~Z

21 ~ []

4MPP-AC CH,O~~/N.._.~O Ol"l,~HtCH~ OOH= i / / ~ ]91 i ..,, ,,..~J.i. ... !.., ...... J_L. . [ lOO 200 300 400 I O0 2OO 30O 40O .dZ m/:

22 28 NCH= [] 20S 'a KET-AC 'i0 o 152 t v'>~ ..j HaG N\ ] cH~o~0 "-~oCH3 284 /300 <

J. !., .,,, L: ~ :..L~..,~...~..L ~.~ .,~..= ,l .. L...... 100 200 300 400 1oo 200 30o 4oo m/Z m/z

23 OCHi 29 NCHa N CHa o 2C-I-AC 1 "or ] OCH3 c.~ / \o / ~O--~o < < ~49 COD-AC t\229 |29g .~ ...... L .[= [ 100 200 300 400 m/Z 100 2OO 30O 4OO m/Z NCHa ] ICH(CH~)= [] o 5MeO-DIPT CHIO~ N~'CH(CH3~I CH ) MOR-2AC 215 268/'1 \

100 200 300 400 m/z IOO 200 TJ30o 4oo m/z Figure 1. (continued) El mass spectra and structures. The quantifier ions are boxed, and qualifier ions are underlined.

68