SCIJUS-0026; No of Pges Science nd Justice xxx (2011) xxx xxx Contents lists vilble t ScienceDirect Science nd Justice journl homepge: www.elsevier.com/locte/scijus Gs chromtogrphic method vlidtion for the nlysis of mjor components in illicit heroin seized in Mlysi Kr-Weng Chn,b,, Gun-Hut Tn, Richrd C.S. Wong Deprtment of Chemistry, University of Mly, 50603 Kul Lumpur, Mlysi b Deprtment of Chemistry Mlysi, Ministry of Science, Technology nd Innovtion (MOSTI), 46661, Petling Jy, Mlysi rticle info bstrct Article history: Received 7 December 20 Received in revised form 1 July 2011 Accepted 16 July 2011 Avilble online xxxx Keywords: GC Mjor components Heroin Profiling Method vlidtion Aprt from routine nlysis of totl morphine content required by the cril justice system, quntifiction of other mjor components in illicit heroin hs not been considered by the Mlysin enforcement lbortory. In order to quntify vrious other cutting gents in ddition to lkloids, gs chromtogrphic (GC) method ws developed to fcilitte simultneous quntifiction of eight trget nlytes commonly found in illicit heroin seized in Mlysi within 12 run time. The vlidtion results demonstrted high selectivity with the use of n HP Ultr 2 cpillry column. Different solvents were studied nd methnol:chloroform (1:9) proved best for smple dissolution. The method ws repetble nd reproducible. The study rnges covering 50 150% of the preferred concentrtions of the eight nlytes obtined r 2 N0.9997. Limits of detection up to 6 μg/ml were lso obtined nd the method chieved 99 2% recovery. The cpbility of the method in heroin profiling ws verified using smples from ten cse smples. 2011 Forensic Science Society. Published by Elsevier Irelnd Ltd. All rights reserved. 1. Introduction It is thought tht much of the heroin obtined in Mlysi is mnufctured outside the ntionl boundries of the country nd imported rther thn produced internlly. Trcing the distribution networks of the drug by the locl uthorities relies on lbortory nlysis. Recently, the profiling effort relting to street heroin in Mlysi hs been renewed to fcilitte smple-to-smple, cse-to-cse nd seizure-toseizure comprisons for intelligence purposes. There hve been mny studies on the instrumentl nlysis of illicit heroin for profiling purposes referred to in the literture [1 6]. Among ll of these, gs chromtogrphy coupled with flme ioniztion detector (GC FID) or mss spectrometer (GC MS) is widely employed. GC FID hs been shown to possess repetble nd reproducible impurity profiling results for sixteen peks in intr nd inter-lbortory nlyses in hrmoniztion study [7]. The FID is universl nd sensitive for most drug smples involving orgnic compounds, requiring smller smple mounts []. Zhng et l. [9] successfully clssified five hundred heroin smples seized in Chin into nine groups bsed on the heroin content nd other chrcteristic dulternts with the id of GC. A toxicologicl cse nlyzing textile smples bering sebceous excretion of opites ws lso successfully nlyzed with the id of GC MS [].High resolution within the chromtogrm is n ll-importnt criterion in drug profiling s resolved peks will enhnce the discrition of smples, Corresponding uthor t: Deprtment of Chemistry, University of Mly, 50603 Kul Lumpur, Mlysi. Tel.: +60 123627609; fx: +60 37951173. E-mil ddresses: chnkrweng@yhoo.com, chnkrweng@sisw.um.edu.my (K.-W. Chn). imizing coincidentl mtches. After extrction, pproximtely seventy peks were detered with the method described by Neumnn et l. [11], utilizing minly FID. K et l. [12] lso illustrted tht sugrs including glucose, sucrose, lctose nd mnnitol which were not detected by other conventionl methods could be detected s their trimethylsilyl (TMS)-derivtives using GC with 15% Dexsil 300 column. Despite the dvncement in nlyticl techniques, profiling studies of illicit heroin smples seized in Mlysi hs not been previously reported. Insted of only quntifying the controlled drugs for prosecution purposes, co-quntifiction of other mjor components such s dulternts is lso importnt for intelligence purposes. For exmple, the use of unusul dulternts my suggest the emergence of new drug deler. The dtbses generted will lso provide vluble informtion for future smple trcking. The present study develops GC method tht fcilittes the quntifiction of up to eight mjor components present in heroin smples seized in Mlysi in 20. The method is economic nd rpid nd involves very little smple preprtion fcilitting high throughput. 2. Mterils nd method 2.1. Stndrds nd solvents Prcetmol nd dextromethorphn hydrobromide were supplied by Y.S.P. Industries (Mlysi). Acetylcodeine hydrochloride ws prepred by the Deprtment of Chemistry Mlysi (MOSTI). Codeine phosphte, morphine hydrochloride nd heroin hydrochloride were purchsed from Johnson Mtthey Mcfrln Smith. 6-monocetylmorphine (6-MAM) hydrochloride ws obtined from Lipomed nd 2,2,2 triphenyl 1355-0306/$ see front mtter 2011 Forensic Science Society. Published by Elsevier Irelnd Ltd. All rights reserved. doi:.16/j.scijus.2011.07.005 seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005
2 K.-W. Chn et l. / Science nd Justice xxx (2011) xxx xxx cetophenone (internl stndrd, ) from Aldrich Chemicl Compny. Cffeine ws purchsed from Merck. HPLC grde methnol nd nlyticl regent grde chloroform were obtined from Fisher Scientific. 2.2. Gs chromtogrphy flme ioniztion detector (GC FID) Quntittive nlysis of lkloids nd cutting gents ws performed on n Agilent N690 GC FID equipped with n utosmpler. Chromtogrphic seprtion ws chieved by employing two sets of GC prmeters s summrized in Tble 1. 2.3. Prtil method vlidtion An initil investigtion of the selectivity of nine compounds of interest (eight trget nlytes nd 2,2,2-triphenyl cetophenone s ) ws undertken using Option 1 nd 2 fter the conditions were optimized. Option 1 provided better pek resolution nd ws subsequently employed for method vlidtion. Chloroform (0%) nd methnol (0%) nd combintions of both in the rtio methnol: chloroform (1:9; 3:7; 5:5; 7:3; 9:1) were lso investigted using mixed stndrd of the nine compounds. The optimized solvent comprised methnol:chloroform (1:9) nd this ws used to prepre the eight nlytes of interest t their respective working concentrtions with the presence of 0.1 mg/ml. The mixed stndrd ws used to study within dy, between hour nd between dy precisions with ten injections of ech stndrd respectively. Linerity ws studied with initil concentrtions (prcetmol: ~0. mg/ml; cffeine: ~.0 mg/ml; dextromethorphn: ~0.3 mg/ml; codeine: ~0.2 mg/ml; morphine: ~0.2 mg/ml; cetylcodeine: ~0.1 mg/ml; 6-MAM: ~1.0 mg/ml; dimorphine (heroin): ~1.0 mg/ml) tht were diluted to obtin series of eight concentrtion levels with the held constnt t 0.1 mg/ml. The limit of detection of the instrument ws detered from the lowest detectble concentrtion prepred t 1 μg/ml, 5 μg/ml nd μg/ml, for ech component bsed on three times the signl-to-noise level. Three known levels (high, medium nd low) of the nlytes were spiked into the smple mtrix for recovery studies. Polypropylene centrifuge tubes nd volumetric flsks were investigted with three cse smples, ech weighing 50 mg, 60 mg, 70 mg nd 0 mg prepred with the presence of 0.1 mg/ml in solution. A preferred weight ws lso detered. Smple stbility Tble 1 GC prmeters for prtil method vlidtion. Condition Option 1 Option 2 Column J&W HP Ultr 2 (5% phenyl 95% methyl siloxne) J&W HP-5 (5% phenyl 95% methyl siloxne) Dimensions Length: 25 m Length: 30 m I.D.: 0.20 mm I.D.: 0.25 mm Film thickness: 0.33 μm Film thickness: 0.25 μm Crrier gs Helium Helium Pressure 239.1 kp 134.7 kp Totl flow 44.2 ml/ 3.7 ml/ Injection 1 μl 1μL volume Split rtio 40:1 40.5:1 Flow rte 1.0 ml/ 0.9 ml/ Injector temp. 290 C 20 C Temp. progrmg 240 C hold 1, rmp t 12 C/ to 270 C nd hold. 250 C hold 4, rmp t 7 C/ to 260 C nd hold 2, rmp t 6 C/ to 20 C. Detector temp. 290 C 20 C H 2 flow 30 ml/ 30 ml/ Air flow 300 ml/ 300 ml/ He mkeup flow 25 ml/ 25 ml/ Totl run time b12 b11 ws exed by pir of low nd high concentrtion smples monitored over month. Ten cse smples were nlyzed using the optimized method under different conditions (weight, dilution fctor nd difference in dily clibrtion) to evlute the method cpbility for profiling. 2.4. Dt nlysis All dt were nlyzed using Microsoft Excel. Multivrite nlysis ws performed using Minitb 15. 3. Results nd discussion 3.1. Choice of trget compounds The eight mjor components were chosen minly becuse they were often detectble in the smples seized in Mlysi. As the smples were highly diluted (cut), ppverine nd noscpine were normlly bsent nd if present, were treted s or rther thn mjor components. The choice of the ws bsed on previous work [13]. 3.2. Choice of column nd selectivity A slightly polr sttionry phse is widely ccepted for universl use nd in drug nlysis, the use of 5% polr column is routine nd therefore it ws chosen in this study. An initil investigtion performed on two GC cpillry columns using mixture of eight trget compounds nd n (ech t bout 0.1 mg/ml) demonstrted different resolutions. The components were first seprted using the prmeters in Option 1 nd HP-5 column. All peks were eluted quickly, however seprtion between cetylcodein nd 6-monocetylmorphine ws poor with tiling observed in the prcetmol pek (Fig. 1). Under the conditions in Option 2 the chromtogrm in Fig. 1b illustrtes tht ll the compounds were well resolved but pek tiling is still evident in the prcetmol pek. Further optimiztion of the split rtio, flow rte nd temperture did not resolve this problem. This suggested smple overloding nd thicker film ws required in the column. Chnging the column to n HP Ultr 2 with Option 1 resulted in good selectivity nd pek resolution without significnt pek tiling even though higher rmping rte ws used nd this is illustrted in Fig. 1c. In ddition, the retention time for the first three compounds ws longer, thus promoting better seprtion. A slightly higher injector temperture (290 C) ws lso used s the thicker film within the column ws ble to retin lrger mount of prcetmol nd dextromethorphn, improving the sensitivity. Notwithstnding this, 3-monocetylmorphine co-eluted with 6-monocetylmorphine, nd the quntittive results re used to represent both components. For the quntittive detertion of mjor components in illicit heroin, Option 1 with the HP Ultr 2 column ws selected nd ws used for the subsequent vlidtion. 3.3. Solvent studies Methnol nd chloroform re two commonly used solvents in drug nlysis. The combintion of these two solvents provides n idel medium for both polr nd non-polr compounds present in the smple mtrix to be extrcted. Hence, both of these solvents were considered. With 0% chloroform s the solvent, dissolution of the slts of codeine, morphine nd 6-monocetylmorphine ws often problem nd t higher concentrtions of drug, colloidl solution or suspensions were observed. To compre methnol:chloroform solvent mixtures ech component ws prepred t pproximtely 0.1 mg/ml resulting in cler solution for ech solvent type. Thus, series of seven solvents contining equl compositions of the nine trget compounds ech in single mixture were nlyzed using seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005
K.-W. Chn et l. / Science nd Justice xxx (2011) xxx xxx 3 50 45 40 35 30 25 20 1.311 PC 1.2 2.243 DM 3.313 4.637 AC 4.979 5.42 5.590 6.592 7.529 15 0 2 4 6 50 b 1.470 45 40 DM 35 3.614 30 25 20 PC 2.009 2.357 5.534 6.016 AC 6.777 6.923.33 9.536 15 0 2 4 6 50 c 1.16 45 DM 40 PC 4.2 35 30 25 20 2.065 2.629 6.033 6.554 AC 7.292 7.464 9.001.479 15 0 2 4 6 Fig. 1. Chromtogrphic selectivity shown by () HP-5 using prmeters in Option 1, (b) HP-5 in Option 2 nd (c) HP Ultr 2 in Option 1. Seprtion is improved between PC nd in (c). All components were t ~0.1 mg/ml. PC=prcetmol; =cffeine; DM=dextromethorphn; =codeine; =morphine; AC=cetylcodeine; =6-monocetylmorphine, =heroin; =internl stndrd (2,2,2-triphenyl cetophenone). Option 1. Two seprte sets of nlyses with 6 replicte injections of ech mixture were performed on different dys. Tble 2 summrizes the reltive stndrd devitions (%RSD) of ll the compounds expressed s pek re rtios (reltive to ) obtined from the two sets of nlyses. All combintions demonstrted good performnce (RSDb2%) prt from 0% chloroform in which inconsistent redings for morphine were significnt presumbly due to its reltively high polrity [14] nd very low solubility of the compound in the solvent. Since the presence of methnol cn cuse trnscetyltion of prcetmol nd morphine s well s fcilitting the decomposition of dimorphine [1,15], it ws decided to use methnol:chloroform (1:9) s the solvent for this study. Some use of methnol ws required to fcilitte the dissolution of more polr compounds. 3.4. Repetbility nd reproducibility A mixed stndrd contining eight nlytes t working concentrtions (rnging from 0.05 to.00 mg/ml depending on the nlytes) nd n (0.1 mg/ml) were used to evlute within dy, between hour nd between dy precisions using chromtogrphic conditions in Option 1. The mixed stndrd ws injected ten times consecutively nd the pek res of ll the nlytes nd the showed excellent repetbility seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005
4 K.-W. Chn et l. / Science nd Justice xxx (2011) xxx xxx Tble 2 %RSD of re rtios (pek reltive to ) for eight trget compounds ech t bout 0.1 mg/ml in different solvent combintions (n=6). PC DM AC Men b Precision M (0%) 0.37 0.50 0.79 0.41 0.90 0.34 0.52 0.20 0.51 0.23 ^(0.23) (0.29) (0.30) (0.11) (0.59) (0.13) (0.27) (0.16) (0.26) (0.15) M:C (9:1) 0.1 0.33 0.1 0.16 0.41 0.07 0.29 0.14 0.22 0.11 (0.24) (0.22) (0.29) (0.27) (1.37) (0.2) (0.42) (0.26) (0.42) (0.39) M:C (7:3) 0.13 0.21 0.21 0.17 0.49 0.14 0.14 0.17 0.21 0.12 (0.27) (0.21) (0.13) (0.22) (0.73) (0.1) (0.33) (0.22) (0.29) (0.19) M:C (5:5) 0.23 0.16 0.12 0.23 1.7 0.16 0.49 0.2 0.44 0.59 (0.25) (0.31) (0.16) (0.16) (0.46) (0.14) (0.22) (0.0) (0.22) (0.12) M:C (3:7) 0.20 0.35 0.17 0.12 0.70 0. 0.23 0.17 0.26 0.19 (0.0) (0.27) (0.11) (0.06) (0.56) (0.9) (0.15) (0.11) (0.2) (0.30) M:C (1:9) 0.42 0.37 0.22 0.20 0.72 0.15 0.26 0.96 0.41 0.29 (0.41) (0.36) (0.19) (0.21) (0.4) (0.37) (0.21) (0.26) (0.31) (0.11) C (0%) 1.00 0.23 0.23 0.20.36 0.15 0.49 0.16 1.35 2.5 (0.42) (0.62) (0.17) (0.12) (6.31) (0.36) (0.2) (0.15) (1.05) (2.13) M=methnol; C=chloroform. Men of ech row. b Stndrd devition of ech row. Series 1. ^ Series 2. Tble 3 %RSD of pek res nd re rtios (pek reltive to ) for mixed stndrd injected within 2 h. PC DM AC Pek re 9.75.09 9.63 9.1.62.41 9.23 9.65 9.1 0.51 0.72 0.1 0.90 2.90 1.00 1.29 0.70 (RSDb1.5%). A single mixed stndrd ws repetitively injected over 2 hour period nd the %RSD of the pek res nd pek re rtios (reltive to ) re summrized in Tble 3. Lrge devitions in pek re cn be ttributed to solvent evportion, cusing n increse in pek res over time. These errors were corrected when ll the trgeted nlytes were normlized to the (RSDb3%). This ws n importnt fctor in order to ssure system stbility for high throughput nlysis when the smples re prepred nd left unttended overnight on GC smple try. Between dy reproducibility is good mens of evluting system stbility nd other uncontrolled vribles over time. The system ws evluted using the mixed stndrd on ten different dys, nd ll mjor pek re rtios (reltive to ) showed RSDb5% except for morphine with RSDb9% most likely due to its reltively low solubility in the presence of chloroform. For smple-to-smple comprison, ll mjor compounds were sufficiently reproducible nd stble. However, morphine should be nlyzed seprtely with more suitble solvent if more rigorous nlysis is required. 3.5. Linerity Linerity defines the working rnge within which the nlyte should fll. In illicit heroin, ech nlyte hs its self-defined rnge tht vries from those of other nlytes present in the sme smple. Using mixed stndrd confers n dvntge of mimicking the smple mtrix. In this study, series of eight levels of concentrtion covering 50 150% of the working rnge were prepred from single mixed stndrd with the held constnt t 0.1 mg/ml. Six repetitive injections were mde for ech concentrtion. Eight individul clibrtion curves were constructed from the series nd their results re shown in Tble 4 nd Fig. 2. As indicted by the linerity index, the liner rnge of interest ws sufficient for routine quntittive detertion. In line with the work done by Anstos et l. [3], the negtive y-intercept vlues my be the suppression effect rising from the mixture. This effect however did not ffect the nlyticl results. The limit of detection (LOD) of the GC ws detered using mixture of stndrds ech prepred t 1 μg/ml, 5 μg/ml nd μg/ml ( constnt t 0.1 mg/ml). The lowest detected concentrtion ws used to detere the LOD bsed on pek height reltive to three times the signl-to-noise rtio. The instrument ws found to be sufficiently sensitive to detect nlyte mounts between 1 nd 6 μg/ml. In cses where the concentrtions of the mjor components re too close to the LODs, the weight of the smple ws incresed. 3.6. Accurcy mesured by recovery For evidentil purposes, the bsolute mount of drug present in the smple is of mjor concern. Recovery studies re necessry to evlute the level of ccurcy tht the method cn offer. A known mount of ech trget nlyte ws spiked into smple mtrix to yield high spiked smple from which two dilutions were prepred to obtin medium nd low spiked smples nd the recovery test ws Tble 4 Linerity of the study rnge nd LOD of the instrument. Concentrtion rnge covered (mg/ml) Linerity function Correltion coefficient, r 2 RSD for re rtio (%) b Linerity index (%) LOD (mg/ml) Prcetmol 0.04 0.0 y =3.44x 0.026 0.999 0.17 0.69 0.9 1.05 0.0021 Cffeine 0.50.0 y =2.11x 0.121 0.999 0.05 0.45 0.9 1.05 0.0006 Dextromethorphn 0.02 0.30 y =5.30x 0.012 0.9997 0. 0.55 0.9 1.05 0.0004 Codeine 0.01 0.20 y =4.27x 0.005 0.999 0.13 0.54 0.99 1.02 0.0005 Morphine 0.01 0.20 y =3.36x 0.0 0.9997 0.65 1.91 0.9 1.03 0.0061 Acetylcodeine 0.05 0. y =4.37x 0.022 0.999 0. 0.56 0.99 1.03 0.000 6-monocetylmorphine 0.05 1.00 y =3.95x 0.032 0.9997 0.24 0.74 0.9 1.03 0.0019 Heroin 0.05 1.00 y =3.73x 0.016 0.9999 0.0 0.55 0.99 1.02 0.0013 The rnge of RSD vlues indictes the precisions of the six injections ech t the eight concentrtion levels. Clcultion is bsed on pek reltive to. b Percent difference between the known stndrd concentrtion nd clculted concentrtion from the experimentl dt using the eqution obtined from the lest squres fit. All lkloids show very good fits of b±5% linerity indices. seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005
K.-W. Chn et l. / Science nd Justice xxx (2011) xxx xxx 5 3.00000 2.50000 y = 3.44x - 0.026 R 2 = 0.999 25.00000 2 b y = 2.11x - 0.121 R 2 = 0.999 2.00000 1.50000 15.00000 1 5.00000 0.0000 0.00 0.2000 0.3000 0.4000 0.5000 0.6000 0.7000 0.000 0.9000 0.0000 2.0000 4.0000 6.0000.0000.0000 12.0000 1.0000 1.60000 c y = 5.30x - 0.012 R 2 = 0.9997 0.90000 d y = 4.27x - 0.005 R 2 = 0.999 1.40000 0.0000 1.20000 0.0000 0.70000 0.60000 0.40000 0.60000 0.30000 0.40000 0.20000 0.20000 0.000 0.0000 0.0500 0.00 0.1500 0.2000 0.2500 0.3000 0.3500 0.0000 0.0500 0.00 0.1500 0.2000 0.2500 0.90000 0.0000 e y = 3.36x - 0.0 R 2 = 0.9997 4.50000 4.00000 f y = 4.37x - 0.022 R 2 = 0.999 0.70000 3.50000 0.60000 3.00000 0.40000 2.50000 2.00000 0.30000 1.50000 0.20000 0.000 0.0000 0.0500 0.00 0.1500 0.2000 0.2500 0.0000 0.2000 0.4000 0.6000 0.000 1.0000 5.00000 4.50000 g y = 3.95x - 0.032 R 2 = 0.9997 4.50000 4.00000 h y = 3.73x - 0.016 R 2 = 0.9999 4.00000 3.50000 3.50000 3.00000 3.00000 2.50000 2.00000 2.50000 2.00000 1.50000 1.50000 0.0000 0.2000 0.4000 0.6000 0.000 1.0000 1.2000 0.0000 0.2000 0.4000 0.6000 0.000 1.0000 1.2000 Fig. 2. Linerity curves for () prcetmol, (b) cffeine, (c) dextromethorphn (d) codeine, (e) morphine, (f) cetylcodeine, (g) 6-monocetylmorphine nd (h) heroin. performed t high, medium nd low concentrtion levels for ech compound. The men recoveries for the eight trget nlytes re displyed in Tble 5. All nlytes show men recovery between 99 nd 2% using Option 1. 3.7. Dissolution vessels, smple weight nd smple stbility Dissolution vessels nd smple weight ply n importnt role in ccurte quntifiction work. Using one-point clibrtions, the suitbility seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005
6 K.-W. Chn et l. / Science nd Justice xxx (2011) xxx xxx Tble 5 % Recovery for eight trget compounds. Low Medium High Men SD Prcetmol 99.90 99.45 0.74 0.03 0.66 Cffeine 1.2 9.66 9.30 99.59 1.94 Dextromethorphn 99.60 99.6 99.76 99.6 0.0 Codeine 9.30 99.01 99.77 99.03 0.73 Morphine 99.74 1.70 1.94 1.13 1.21 Acetylcodeine 0.37 99.25 1.34 0.32 1.04 6-Monocetylmorphine 9.79 0.66 1.2 0.42 1.53 Heroin 0.34 1.37 1.53 1.0 0.65 Tble 6 Totl % nlyte content±stndrd devition for three smples prepred in PTs nd VFs. Smple A Smple B Smple C Weight (mg) PT VF PT VF PT VF 50.4 90.0 3.6 6.1 92.5 94.2 60 9.3 9.3 5.5 7.5 92.2 94.9 70.6.4 6.5 9.2 91.7 94.0 0.6 7.5 1.9 5.2 92.5 93.6 Men.7. 4.4 7.0 92.2 94.2 Stndrd devition 0.4 1.1 2.1 1.7 0.4 0.5 of polypropylene centrifuge tubes (PT) nd volumetric flsks (VF) for smple preprtion ws exed using three rndomly chosen cse smples ( A C ). Ech pir of 15 ml PT nd ml VF contined specific weight: 50 mg, 60 mg, 70 mg nd 0 mg of the smples with four pirs prepred for ech of the cse smples. All weighed substnces were dissolved in ml of methnol:chloroform (1:9) with the presence of the. Ech liquot ws injected six times. Ech component is expressed s percentge nlyte. At ech weight level prepred in PT nd VF, the totl percentge nlyte representing the sum of the eight components in the vessel ws clculted (Tble 6). The smll stndrd devition in ech cse revels tht the weight used did not hve much effect. However, the PTs show lower totl percentge nlyte content compred to the corresponding VFs (especilly in smples B nd C ), suggesting loss of nlytes when using the plstic vessels. In ddition, rtifcts were observed in the smples extrcted in the PTs nd these which becme significnt when the liquots were nlyzed on dy 4 fter cold storge. These rtifcts hd greter impct on the elution times of prcetmol nd dextromethorphn. In contrst, the chromtogrms of the smples prepred in the VFs re illustrted in Fig. 3b nd d show reltively constnt bselines. From the six nlyses t ech concentrtion, the smple weight rnge of 50 mg 0 mg illustrted n RSDb3%. Lower weights however my run risk of hving undetectble nd unquntifible nlytes (such s codeine nd morphine). Therefore, it ws decided to 1 16 14 AC 12 0 2 4 6 1 b 16 14 AC 12 0 2 4 6 1 c 16 14 AC 12 0 2 4 6 1 d 16 14 AC 12 0 2 4 6 Fig. 3. Chromtogrms of smple prepred in PTs nd VFs. () nd (b): smples in PT nd VF respectively nlyzed on the dy of smple preprtion. (c) nd (d): smples in PT nd VF respectively nlyzed on dy 4. Artifcts re significnt in () nd (c). seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005
K.-W. Chn et l. / Science nd Justice xxx (2011) xxx xxx 7 Tble 7 Cumultive %RSD of re rtios (pek reltive to ) of the eight mjor components. 2.63 Week Smple PC DM AC 0 (n=3) Low 1.74 0.44 0.11 0.16 0.40 0.15 0.12 0.12 High 0.32 0.27 0.47 0.43 0.33 0.53 0.45 0.65 1 (n=6) Low 1.4 1.4 1.02 1.04 3.2 0.96 1.3 0.73 High 1.22 0.45 0.66 0.79 1.44 0.76 1.24 0.67 2 (n=9) Low 6.05 1.41 1.2 1.66 2.6 1.32 2.52 1.40 High 4.99 1.03 1.61 2.16 3.12 1.7 2.56 1.96 3 (n=12) Low 13.17 3.24 3.53 7.07 17.60 4.36 7.44 3.49 High 12.02 3.43 3.71 5.49.5 4.30 5.91 3.71 4 (n=15) Low 14.42 3.5 3.9 6.75 15.65 4.36 7.45 3.63 High 13.2 3.92 4.17 5.79.65 4.56 6.37 4.16 use higher weight (N70 mg) s strting weight. The weight should be incresed or decresed for components tht re too low or too high in their concentrtions. Smple stbility ws further exed by nlyzing pir of low nd high content smples. A smple contining reltively high nlyte contents ws diluted to yield low content smple. Both were nlyzed (three injections ech) on the first dy nd every seventh dy for month. Tble 7 shows the cumultive %RSD of the pek re rtios of the eight mjor components. Discrepncies in the pek re rtios becme greter s the nlysis time incresed. Both the smples performed very well within the first week (RSDb4%). The performnce strted to drop with subsequent weeks. Prcetmol nd morphine were more ffected s the smples ged suggesting tht smples should be nlyzed within week. 3.. Anlysis of cse smples A vlid method should be robust nd cpble of withstnding environmentl chnges nd other uncontrolled fctors. To ssess the method cpbility, ten smples were prepred ccording to Tble. The smple ws weighed in volumetric flsk nd ml of methnol: chloroform (1:9) contining 0.1 mg/ml ws dded. Anlysis ws performed using Option 1. For smple-to-smple comprison t the distribution level, the GC redings were trnsformed to percentge nlyte (clled vrible-i) nd ech of the eight compounds ws stndrdized (vrible-i divided by stndrd devition-i) prior to hierrchicl cluster nlysis (HCA) using single linkge method with Eucliden distnce mesure (Fig. 4). All ten clusters were obtined t similrity level of 92.59 illustrting tht the method ws cpble of providing reproducible results irrespective of the inter-dy vritions. Using rtios of the lkloid compounds imizes the dulternt effects nd experimentl errors [1,9]. To ssess smple origin, the GC redings in mg/ml were normlized to chieve five rtios bsed on AC/, AC/, AC/(+), /(++) nd AC/(+ + ). These quotients were tenttively chosen insted of normliztion the pek res reltive to the sum of the pek res becuse they re ble to compenste the decomposition effects [1]. These multivrite dt in correltion mtrix were nlyzed using principl component nlysis (PCA) nd the clusters re shown in Tble Description of cse smples considered for nlysis. Smple ID Description 1 2 5 smples (40 mg, 50 mg, 60 mg, 70 mg nd 0 mg) prepred nd nlyzed on dy 1, 1 3 4 smples (50 mg, 60 mg, 70 mg, nd 0 mg) prepred nd nlyzed on dy 2. 1, 4 1 smple (0 mg) prepred nd nlyzed on dy 3 1 diluted smple (from dy 3) prepred nd nlyzed on dy 4 Anlyses were performed s such due to the limited vilbility of the mount of the smple. Similrity 52.42 76.21 92.59 0.00 Fig. 5. The smples within ech individul cluster nlyzed under different conditions show insignificnt differences in the nlyticl results except for smple 1 presumbly becuse of the bsence of heroin in smple 1. Smples 3 nd 4 prepred nd nlyzed t different concentrtions with different clibrtion curves illustrted extremely close greements in their respective clusters. The GC system proved robust nd very insensitive to environmentl chnges. Slight seprtions between the units within the individul clusters re theoreticlly due to rndom errors nd in generl this method ws ble to cluster ll the smples into their respective groups correctly. 4. Conclusion 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 4 4 Smples 5 5 6 6 7 7 3 3 3 3 9 9 Fig. 4. Hierrchicl cluster nlysis of ten smples nlyzed under different conditions. % nlytes were used to evlute the cpbility of the method to cluster similr smples t distribution level. Quntifiction of the mjor components in heroin is fesible with GC FID using n HP Ultr 2 column nd the chromtogrphic conditions specified in option 1. The method ws prtilly vlidted nd found to be simple, ccurte nd precise for mjor component nlysis of illicit heroin smples. To enhnce the relibility of the results, it is proposed tht in the illicit smple weight is in excess of 70 mg to ensure tht the nlytes re detectble nd quntifible. Volumetric flsks should lso be used to imize rtifct formtion. All prepred smples should be nlyzed within week. Sttisticl nlysis of the results indicted good cpbility of the method to link smples together correctly. The proposed method ws lso ble to detect ppverine, noscpine nd chloroquine. As our smples did not contin the former two lkloids Second Component 2 1 0-1 -2-2 -1 0 1 First Component 2 3 Cse 1 2 3 4 5 6 7 9 Fig. 5. Principl component nlysis of ten smples nlyzed under different conditions. Five rtios of normlized lkloids were used to evlute the cpbility of the method to cluster similr smples t mnufcturing level. Smples 3 nd 4 respectively show overlpped symbols, indicting extremely close greement between the smples in their clusters. seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005
K.-W. Chn et l. / Science nd Justice xxx (2011) xxx xxx which elute fter 12, the nlysis time ws thus shortened to bout 12(option1).Chloroquineisoftenddedsndulterntinthe locl smples in Mlysi, however due to difficulties in obtining stndrd this compound ws excluded for this study. Future studies will include these compounds. Acknowledgements The uthors would like to thnk the University of Mly for the PPP grnt (PS360 20A), nd fcilities supplied by the Deprtment of Chemistry Mlysi (MOSTI) nd stndrds of prcetmol nd dextromethorphn provided by Y.S.P. Industries (M) Sdn. Bhd. to crry out this project. References [1] A. Zelkowics, A. Mgor, M.D. Rvreby, R. Levy, Anlysis of simulted heroin distribution chin by HPLC, J. Forensic Sci. 50 (4) (2005) 49 52. [2] H. Huizer, Anlyticl studies on illicit heroin: II. Comprison of smples, J. Forensic Sci. 2 (1) (193) 40 4. [3] N. Anstos, S.W. Lewis, N.W. Brnett, J.R. Person, K.P. Kirkbride, The rpid nlysis of heroin drug seizures using micellr electrokinetic chromtogrphy with short-end injection, J. Forensic Sci. 50 (1) (2005) 37 42. [4] N. Anstos, N.W. Brnett, S.W. Lewis, J.R. Person, K.P. Kirkbride, Rpid detertion of crbohydrtes in heroin drug seizures using cpillry electrophoresis with short-end injection, J. Forensic Sci. 50 (5) (2005) 39 43. [5] M. Rvreby, Quntittive detertion of cocine nd heroin by Fourier Trnsform Infrred Spectrophotometry, J. Forensic Sci. 32 (1) (197) 20 37. [6] M. Chirotti, N. Fucci, C. Furnri, Comprtive nlysis of illicit heroin smples, Forensic Sci. Int. 50 (1991) 47 55. [7] L. Strömberg, L. Lundberg, H. Neumnn, B. Bobon, H. Huizer, N.W. vn der Stelt, Heroin impurity profiling: hrmoniztion study for retrospective comprisons, Forensic Sci. Int. 114 (2000) 67. [] V. Dufey, L. Dujourdy, F. Bescier, H. Chudron, A quick nd utomted method for profiling heroin smples for tcticl intelligence purposes, Forensic Sci. Int. 16 (2007) 117. [9] D. Zhng, X. Shi, Z. Yun, H. Ju, Component nlysis of illicit heroin smples with GC/MS nd its ppliction in source identifiction, J. Forensic Sci. 49 (1) (2004) 1 6. [] A. Trcqui, P. Kintz, B. Ludes, C. Jmey, P. Mngin, The detection of opite drugs in nontrditionl specimens (clothing): report of ten cses, J. Forensic Sci. 40 (2) (1995) 263 265. [11] H. Neumnn, M. Gloger, Profiling of illicit heroin smples by high-resolution cpillry gs chromtogrphy for forensic ppliction, Chromtogrphi 16 (192) 261 264. [12] E. K, K. Bent, Impurities, dulternts nd diluents of illicit heroin in Denmrk (Jutlnd nd Funen), Forensic Sci. Int. 31 (196) 195 2. [13] Austrlin Government Ntionl Mesurement Institute, Chemicl proficiency testing, proficiency study AQA 07 01, nlysis of heroin, NMI report No: 333, My 2007. [14] F. Medin III, The identifiction of heroin nd three structurlly relted isoheroins, J Forensic Sci. 34 (3) (199) 565 57. [15] H. Huizer, A.J. Poortmn, Some spects of the gs chromtogrphic (GC) nlysis of heroin, United Ntions (Februry 199) 1 16. seized in Mlysi, Sci. Justice (2011), doi:.16/j.scijus.2011.07.005