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1. 90 25 Ig 896 86 1800 1600 1400 1200 1000 800 600 Raman shift cm 1 Figure 3 1 Cocaine Hydrochloride reference spectra 54 3 2 KGHB in Alcohol 50 v v KGHB in ethanol was not detected using the TruScan device KGHB can be detected in ethanol using a DXR bench top Raman Spectrometer as a peak at 930cm is evident However this peak is more characteristic to GBL GBL and GHB are equilibrium when in a liquid Brewter e a considers peak 931cm to be GBL Brewster et al 2009 1KGHB in Ethanol 50 TruScan 12004 H HS fo H HE 1000 236 39 183 42 Raman intensity 1049 74 267 95 250 Ethanol Neat S 882 61 1095 82 Raman Intensity cps 051 41 Pi 39 o 8 a 97 m 76 E 1KGHB in ethanol 50 DXR a 8 881 67 1050 49 Raman Intensity cps 1093 75 s an 54 1276 39 50 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 S 80D 600 400 200 Raman shift cm 1 Figure 3 2 KGHB and Ethanol comparison 3 3 Mixture Analysis The TruScan device identified all three runs of the 0 25g powder composed 0 125g of cocaine and 0 125g of benzocaine as benzocaine This means it matched the mixture spectra to the benzocaine spectra contained in its library only It did not report a match to the cocaine spectra in its library Table 3 1 lists the peaks in the neat cocaine and benzocaine spectra and the peaks that occurr
2. Field Impairment Test g Grams GBL Gamma Butryolactone GC MS Gas Chromatography Mass Spectrometry GHB Gamma Hydroxybutyric acid IM Intramuscular IR Infrared IV Intravenous KGHB Potassium Gamma Hydroxybutyric acid LC MS MS Liquid Chromatography tandem mass spectrometry LOD Limit of detection LSD lysergic acid diethylamide MAO Monamine Oxidase MDE methylenedioxyethylamphetamine MDMA 3 4 methylenedioxymethamphetamine mg Milligrams mL Millilitre mW MilliWatts NA Noradrenalin NaGHB Sodium Gamma Hydroxybutyric acid ng Nanograms NMDA N methyl D aspartate OF Oral Fluid o t c over the counter ROSITA Roadside Testing Assessment RPM Revolutions per minute SCJS Scottish Crime and Justice Survey SERS Surfaced Enhanced Raman Spectroscopy SOP Standard Operating Procedure UK United Kingdom USA United States of America v v Volume Volume w v Weight Volume List of Figures Figure 1 1 Estimated number of drug users for each drug in 2009 Figure 1 2 Cocaine chemical structure Figure 1 3 Pathways of cocaine metabolism Figure 1 4 Mechanism of action for amphetamine Figure 1 5 Chemical structure of MDMA Figure 1 6 Chemical structure of ketamine Figure 1 7 Chemical structure of gamma hydroxybutric acid Figure 1 8 The three different types of Raman scattering Figure 1 9 KGHB in ethanol compared to neat ethanol spectr
3. 172891 EA ki Ji 1 o 8 S i sue uewey 1000 1800 2000 1200 1400 1600 2200 Raman shift cm 1 d ine an 2 compared with coca ine mix caffe ine benzoca ine 3 7 Cocai Figure DE 991 LELLE tzor Dt 206 D6 466 cv 919 82 669 ZE ELL C 818 ZO v98 GOLI VELEL ey LBGL EV MEN JE 696L OE pri 66 PLS 2 POS om pen o o FCL o 8 O peal a reest 0 Kaes m 3 o 2 o E o E N 2 5 Ej vy e9ez O S Aysuaqul ue uie 1000 4 50000 3 Caffeine Signature 66 456 c8 ecol 181201 SC LbvZL DEI E9 8cEl CD 6Svl 69 S681 890091 BB GG 66 8691 40000 30000 Aysuaqul ULE 30000 TBenzocaine POL ES IE DL cor 91 90 89719 Sc v9 96118 50 v98 EE LLLL viel OL 1821 LZLLEL pL BIEL Dt zbil 9 G GL 97091 Lv Zagat 10000 Aysuaqu uewea 3 1Cocaine HCI 98 968 ZL 0001 18 ECOL Ev 0OL 1000 ST9ILI LLEDZL 1200 Raman shift cm 1 caffe 47141 1400 86 sevi d Gi B6SL 1600 vc sut 2000 1800 2200 e o Asuaju uewey 3 compared with reference ine mix ine benzoca 3 8 Cocaine Figure spectra 63 The Truscan device identified all three runs of the mixture composing of 0 125g cocaine hydrochloride 0 125g benzocaine 0 125g
4. Smith and Dent 2005 One study was able to detect methamphetamine through plastic packaging such as polypropylene bags using FT Raman with little interference from the bag It was discovered the spectrometer could be used to distinguish between methamphetamine amphetamine sulphate and ephedrine It took around 1 minute for the compounds to be identified Methamphetamine dissolved to be concealed in water or sodium chloride could be detected down to 1 w w Tsuchihashi et al 1997 Dispersive Raman spectroscopy can be used to detect MDMA this has demonstrated good quality spectra and an acquisition of around 2 minutes using an 785 nm laser This can be used to distinguish between isomers and bulking agents so is useful in composition analysis Bell et a 2000 Raman spectroscopy has been utilised in the screening of large seizures of ecstasy tablets A study in 2000 demonstrated that Raman can be used to observe the inhomogeneity in tablets which had the same appearance and the same logo This means that testing just one area of the tablet will not give a complete picture of what the pill contains this much be considered when trying to obtain a full profile It was shown that 400 of the pills could be grouped using the excipients highlighting that a batch may looked the same but can be extremely variable Bell et al 2000 A larger study of pills was conducted in Northern Ireland This study tested 1500 pills from a variety of sources
5. 4000 2000 ER SERS MDMA in OF 12000 1675 89 71861 64 1667 02 1640 52 1628 23 8867 49 1487 89 1442 7651 62 1342 33 w a E z E 1507 74 10000 8000 200 24 1872 70 6000 Raman intensity 4000 2000 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Raman shift cm 1 Figure 3 21 Comparison of SERS spectra 2 75 Chapter 4 Discussion 4 1 Method Optimisation In order to obtain optimum results many variations of the method were attempted including different sizes of colloid 51 64 90 nm Different nose cones used and no nose cone attached Sampling from the supernatant instead of the pellet after centrifugation was also attempted For the Truscan run different methods attempted included drying the sample on a glass slide at room temperature pipetting the sample into a capillary tube and focussing the laser down the length of the capillary tube or focussing it through glass of the capillary tube The glass slide and capillary tube gave the same large glass hump with no peaks present in the spectra so were not suitable 4 2 Library Creating the library to gain reference spectra was a time consuming process It could take up to several hours to obtain a single reference spectra or signature as referred to in the TruScan s manual For use in the field the maximum amount possible of controlled substances would have to be added to the library As th
6. sample can be tiny particles or in bulk Smith and Dent 2005 A Raman spectrum can be obtained from a sample as small as less than 1um in diameter Jickells and Nergrusz 2008 Another advantage of Raman spectroscopy is that it is non destructive and samples inside glass containers can be analysed reducing contamination Smith and Dent 2005 As water gives a weak Raman signal analysis of solutions or moist items can be carried out West and Went 2010 Most Illicit drugs are strong Raman scatterers and give good spectra with good characteristic peaks Weyermann et al 2011 Portable Raman spectrometers offer in situ analysis which is ideal for investigating clandestine laboratories With Raman the analysts do not need to be in direct contact with any potentially dangerous substances it is a huge advantage if samples can be indentified without removing them from their packages Weyermann ef al 2011 1 5 3 Disadvantages of Raman Spectroscopy Fluorescence can be a problem when using Raman Fluorescence can mask some weak Raman bands West and Went 2010 The fluorescence can be caused by impurities present on the sample or the sample itself Anti Stokes scattering is preferred if this is the case as it avoids this interference Smith and Dent 2005 Another problem Raman faces is that the Raman effect is weak as only one in every 10 10 photons which scatter are Raman scattered so the instrument is required to be very sensitive an
7. London UK Clockwork Research LTD Transport Df 87 Jickells S Negrusz A 2008 Clarkes Analytical Forensic Toxicology London Pharmaceutical press Kalant H 2001 The pharmacology and toxicology of ecstasy MDMA and related drugs Canadian Medical Association Journal 165 7 917 928 Kang J Gu H Zhong L Hu Y amp Liu F 2011 The pH dependent Raman spectroscopic study of caffeine Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy 78 2 757 762 Karch S 2008 Drugs abuse handbook 2 edition CRC Press Kato K Hillsgrove M Weinhold L Gorelick D A Darwin W D amp Cone E J 1993 Cocaine and metabolite excretion in saliva under stimulated and nonstimulated conditions Journal of analytical toxicology 17 6 338 341 King L A 2009 Forensic chemistry of substance misuse a guide to drug control Royal Society of Chemistry Kintz P Goull J P Cirimele V amp Ludes B 2001 Window of Detection of y Hydroxybutyrate in Blood and Saliva Clinical chemistry 47 11 2033 2034 Klemenjak W Braun E Alvarez J Bernhoft M amp Fjerdingen L 2005 Final programme report IMMORTAL Li J Stokes S A amp Woeckener A 1998 A tale of novel intoxication a review of the effects of y hydroxybutyric acid with recommendations for management Annals of emergency medicine 31 6 729 736 MacLeod P Page L Kinver A llias
8. Rang and Dale 2003 24 Tolerance to amphetamine can develop with repeated doses as it is thought the stores of noradrenaline deplete Rang and Dale 2003 Amphetamine is also responsible for increase the levels of Dopamine and Serotonin in the synaptic cleft therefore heightening the effect of the post synaptic receptor in select areas in the brain which results in a temporary feel good effect Rang and Dale 2003 Amphetamine enters the presynaptic nerve terminal through the dopamine receptor the dopamine active transporter or DAT It then encourages dopamine to be released through DAT dopamine is usually released from synaptic vesicles and not through a receptor As with cocaine it is the high levels of dopamine which causes a behavioural effect Winger et al 2004 Amphetamine use can cause rhabdomyolysis pulmonary odema and acute myocardial infarction Rhabdomyolysis which is the breakdown of muscle fibres contents called myoglobin into the bloodstream this is toxic to the kidneys Karch 2008 1 4 2 1 Metabolism The half live of amphetamine is around 7 hours Around 30 of amphetamine is excreted from the body unchanged by the kidney however it does go through Phase and Phase II metabolism Winger et al 2004 Two enzyme systems are involved in Phase I these are cytochrome P450 and flavin monooxygenase In Phase Il the metabolites are conjugated and eliminated from the body Foye et al 2008 Methamphetamine is metabol
9. ST99LI LI eon LE G EL DC esr SL 866l EIER Cocaine HCI 44 iD o Aysuaqul uewey 400 600 1400 1200 1000 Raman shift cm 1 1600 1800 1 compared IX d paracetamol m ine an Caffe ine benzoca Cocaine 3 10 with reference spectra Figure 68 3 4 Oral fluid analysis 1 mL KGHB in 1 mL of oral fluid produced a KGHB spectra and the TruScan device reported a pass for GHB 10000 pr KGHB in 1ml OF Truscan 95001 9000 4 8500 4 8000 4 7500 4 700i 6500 1 sono 3 5500 Raman intensity 5000 4000 3500 3000 3 2500 4 1023 75 2000 3 15004 1000 4 soo 2200 2000 1800 1600 1400 1200 1000 800 600 400 Raman shift cm 1 Figure 3 11 The Truscan device showed peaks for 1 mL GHB in 1 mL of oral fluid 1059 92 931 38 877 28 1046 20 494 98 478 14 Z 107M83 49 1038 96 952 62 or 99 81 681 91 640 09 82225 1700 1600 1500 1400 1300 1200 1100 1000 900 soo 700 soo 500 Raman shift em 1 Figure 3 12 Pure KGHB Spectra taken from the authors previous study 69 Table 3 4 compares the significant peak numbers in the spiked oral fluid with the peak number present in neat KGHB The spiked oral fluid sample displays all of the same peaks as the neat oral fluid so it can be safely assumed the KHGB can be detected Table 3 4 Common Peaks Neat KGHB and KGHB in Oral Fluid 1 mL KGHB in 1 mL OF KGHB
10. Williams DA 2007 Foye s Principles of medicinal chemistry 6 edition Baltimore MD Lippincott Williams and Wilkins Gallardo E Barroso M amp Queiroz J A 2009 LC MS a powerful tool in workplace drug testing Drug testing and analysis 1 3 109 115 GHB Recipes www erowid org accessed 15 06 11 Gilhooly T C amp Daly A K 2002 CYP2D6 deficiency a factor in ecstasy related deaths British journal of clinical pharmacology 54 1 69 Hargreaves M D Page K Munshi T Tomsett R Lynch G Edwards H G M 2008 Analysis of seized drugs using portable Raman Spectroscopy in an airport environment a proof of principle study Journal of Raman Spectroscopy 39 7 873 880 Hoare 2009 Drug misuse declared Findings from 2008 2009 British Crime Survey England and Wales Home Office Statistical Bulletin London Home Office Holzgrabe U amp Malet Martino M 2011 Analytical challenges in drug counterfeiting and falsification The NMR approach Journal of pharmaceutical and biomedical analysis 55 4 679 687 Home Office http www homeoffice gov uk drugs drugs law Class a b c accessed 15 01 10 Hornfeldt C S Lothridge K amp Upshaw Downs J C 2002 Forensic science update gamma hydroxybutyrate GHB Forensic Science Communications 4 1 Jackson P amp Hilditch C 2010 A review of evidence related to drug driving in the UK a report submitted to the North review team
11. and oral fluid Therapeutic drug monitoring 26 2 200 205 Verstraete A G 2005 Oral fluid testing for driving under the influence of drugs history recent progress and remaining challenges Forensic Science International 150 2 143 150 Verstraete AG Raes E 2006 ROSITA 2 Project Final Report Belgium Ghent University Weiner A L Vieira L McKay Jr C A 8 Bayer M J 2000 Ketamine abusers presenting to the emergency department a case series The Journal of emergency medicine 18 4 447 451 West M J amp Went M J 2011 Detection of drugs of abuse by Raman spectroscopy Drug testing and analysis 3 9 532 538 Weyermann C Mimoune Y Anglada F Massonnet G Esseiva P amp Buzzini P 2011 Applications of a transportable Raman spectrometer for the in situ detection of controlled substances at border controls Forensic science international 209 1 21 28 Wills S 2005 Drugs of Abuse 2 edition London Pharmaceutical Press Winger G Woods JH Hofmann F 2004 A handbook on drug and alcohol abuse The Biomedical Aspects New York Oxford University Press 90 Appendix 1 GHB Recipes obtained from the internet How to make GHB You will need 1 135 grams 120 mL of gamma butyrolactone 2 63 grams of Sodium Hydroxide or 91 grams of Potassium Hydroxide 3 Papers to test pH 1 Place the content of the gamma butyrolactone bottle in a stainless steel or pyrex g
12. button to turn on the device Press login to start as directed on screen Select Jack_admin using the enter key Enter K as the password Select the appropriate attachment for the sample i e vial holder tablet holder or nose cone Sometimes no attachment is needed e g when sample is in thick 95 glass bottle Refer to Page 23 of Manual for Best Practise for making measurement Select Run from the main menu Select the Method most appropriate to the sample Enter Sample ID if required Select Go and wait for analysis Result will be a pass or fail If the result is a fail select Discover to see if any positive matches are found Click on any matches to view spectra and a library spectra comparison Transferring Run Spectra to Omnic on PC The CF card must be inserted into the card slot within the battery compartment at the bottom of the device Select Tools from the main menu Select Review Runs Select the Run from the list and select Export to Card from the pop up menu Once this is successful eject the card by pressing in the square button next to the card slot Insert the card into the USB card reader and insert into USB port on PC A folder will open on the PC when the card reader is connected select TruScan Runs Select the Run of interest The Runs do not have logical names other than the name of the person signed in They often have to be identified by time and date Right click the desired Run and s
13. cocaine hydrochloride amphetamine sulphate ketamine hydrochloride methylenedioxyethylamphetamine hydrochloride MDE 3 4 methylenedioxymethamphetamine Hydrochloride MDMA and GBL benzocaine and caffeine also purchased from Sigma Aldrich were also used All the GHB used in this study was synthesised using an altered recipe obtained from the internet as the experiment aimed to mimic drinks spiked by GHB produced in a clandestine environment The recipe was obtained from www erowid org after typing GHB recipes into a Google search engine See Appendix 1 Potassium hydroxide was used for the synthesis The GHB was not bought commercially The GBL was obtained from Sigma Aldrich The colloid was obtained from Thermo Scientific All drugs used in this study were purchased made and stored under the strict home office conditions detailed in the previous chapter The university has a license from the home offence which states these conditions 2 2 Instrument The device used in this study was a Thermo Scientific TruScan Raman Spectrometer It operated using a 785nm laser with a maximum power output of 300mW It has a spectral range of 250 cm to 2875 cm Spectral data was transferred to a PC with OMNIC software using a data card 48 Figure 2 1 TruScan and DXR The purpose of this figure is to demonstrate the size difference between the two instruments 49 NOSE CONE Shield ALA H FRONT BACK Laser Aperture Las
14. g e 8 4000 2000 4 1KGHB in OF 60 10000 4 gt 5 z e 2 80004 5 a n z B8 i z e EJ E aul 3 K S E 8 E amp 6000 b 5 amp zm E z CH t ti 4000 CE E d 2000 4 1KGHB in OF 50 e 10000 S a 1 ci z S 2 s0004 5 1 E 6 6000 2 1 amp 4000 2000 4 1800 1500 1400 1300 1200 1100 1000 900 800 700 Raman shift cm 1 Figure 3 16 KGHB Limit of Detection 2 000 KGHB in OF 40 I o u ci 2 50004 s 5 I a S 4004 F 1 5 H x lane 3000 7 T KGHB in OF 30 6000 3 8 amp E E 3 5 5 50004 E t i 5 i 40004 i 1 x 3000 I KGHB in OF 25 6000 1 2 S 2 5 5000 1 5 5 S H 4000 1500 1400 1300 1200 1100 1000 900 800 700 Raman shift cm 1 Figure 3 17 KGHB Limit of Detection 3 The limit of detection of GHB in oral fluid is around 30 at the very lowest as the 931 and 803 cm peaks can still just be identified 72 3 6 SERS and Oral fluid analysis Spiking oral fluid with 0 5 mL of GHB and carrying out the SERS method produced characteristic peaks 1900 1SERS GHB in OF 1800 4 1700 4 8 8 421 68 1539 70 Raman intensity a 8 a S 5 1640 06 98 2000 1800 1600 1400 mn wu am om Raman shift cm 1 Figure 3 18 0 5 mL GHB in 0 5 mL oral fluid SERS method proved to be less ideal as although it provided several peaks for KGHB there is only one peak present which is common to K
15. ine an Caffe ine benzoca ine Coca 3 9 with reference spectra Figure pI S9L PG 106 16 882 v8 21 tL PGE ge See 09 car 8r bos 88 bes 96 GGG EL C86 08919 BE Ob9 og 089 Br ri 9186 c6 818 08 298 26 596 59 666 650201 E Lr ZGLL DEI 66 BEZ l A EL LIEU 26 6JEL 66 Z6EL 188vvl Lr E PI 8828 OZ POs 86 0591 e a 1000 4 1000 o 2000 Cocaine Benzocaine Caffeine Paracetamol Aysuaqul uewey pl esl EG II DL zur 91908 8919 sz ore 96218 wd w EE LLL PU ZIL OZ 1821 Le LIE Wes Ob pyL EIGISL 19 v09L 172891 Benzocaine Aysuaqul uewe 9c 891 S6 poz ENS BC 696 9c 16 le Spr Sr rer 66186 zeng S Wig EL 208 86 8c6 28 Z20L 18 L 0L d SC VCI LE GBZL E9BZEL 1809 Z0 65vl 89 aa 885591 668691 Caffeine Signature Alsuaqul uewey ZL est EI ble Or OEE PG Z6E er zip S Dp oop ve pos v9 G09 10 629 87259 ZB LL 81862 KR 11 696 62 LOL BE SOLL c6 89114 de LECI 299001 PERL ELGZEL BO ZIEL L 9bhL 65 GLGL 862951 6LEL9L Srel phen jAcetamino 50000 4 D ANSUSJU uewe 50000 62 PLL 80 PC S8 187 LYZE Sc D r 25 585 51919 68189 PP DEI 51982 19218 L6 698 98 968 220001 18 ECOI r 1401
16. it in the fridge the taste is better when it s cold 92 Appendix 2 TruScan Standard Operating procedures The following standard operating procedure for the TruScan Raman Spectrometer was devised in order to create the library Press button to turn on the device Press login to start as directed on screen Select Jack_admin using the enter key Enter K as the password Select the appropriate attachment for the sample i e vial holder tablet holder or nose cone Sometimes no attachment is needed e g when sample is in thick glass bottle Refer to Page 23 of Manual for Best Practise for making measurement Reference spectra are referred to as Methods on TruScan these have to be added to create the library 2 1 Creating a Method Select Tools from the main menu then click on Signature This opens a further menu select Acquire from this menu This can take some time It was found that the best and quickest way to obtain a Signature of a powder was to put a small amount of powder on a glass slide focus the laser on an even area on the powder with the nose cone attachment on then black out most of the surrounding light by resting black weighing boats against the plastic shield of the nose cone 93 a Test Sync Tools Ahura Scientific 06 Nov 2008 15 19 Figure 2 3 TruScan Main Menu Once the Signature run is finished collecting go to Tools in the main menu then Signature then Inactive click Activate a
17. they may also be missed by the survey Hoare 2009 The survey found that the most common drug used in 2009 was Cannabis with 7 9 of respondents admitting using the drug powder cocaine is the second most common 3 then ecstasy 1 8 amyl nitrate 1 4 and amphetamines 1 2 The Scottish Crime and Justice Survey SCJS and BCS have shown similarly figures in the prevalence of drugs The European Monitoring Centre for Drugs and Drug Addiction EMCDDA also report similar findings with Cannabis being 15 the most common drug followed by cocaine 3000000 2500000 2000000 1500000 1000000 500000 T 0 ii mm __ o 3 x gt e 5 Ap SU GU FES af uu am af ES ES o e gt 3 P oO ov ss ONEC s O Figure1 1 Estimated number of drug users for each drug in 2009 Hoare 2009 cocaine includes crack cocaine amphetamine includes methamphetamine hallucinogens include magic mushrooms and LSD and opiates include methadone and heroin According to the BCS cocaine use among young people aged 16 24 has seen a huge increase in use in one year it jumped from 5 1 of respondents admitting use to 29 The use of ketamine also increased Hoare 2009 1 2 2 Prevalence of drug use and driving The report Illicit Drugs and Driving produced by the Scottish Executive published in 2006 highlighted that 6 of the 17 39 year old drivers surveyed had drug driven It was found the most common drug used before driving under the influ
18. this increase is a reaction to the popularity of legal highs Mephedrone appeared on the legal high market in 2009 as a cheap substitute to cocaine as it was not cut with inert substances like benzocaine Mephedrone is still widely available despite being banned in April 2010 Police have also targeted the cutting agents market which could also have influenced the increased purity Daly 2010 19 HC dali Figure 1 2 Cocaine Chemical Structure Ravina et a 2006 Cocaine is classed as a stimulant Stimulants are used recreationally for their euphoric effects these effects create a distraction and this is not an idle state for a driver Acute effects can be feelings of elation powerful and superiority users can become agitated impatient and sometimes even violent An individual may take more risks whilst driving if under the influence of cocaine The most common way cocaine hydrochloride is administered is by nasal insufflation or snorting the user will often rub the remaining powder into their gums It is not very effective to smoke the hydrochloride form of cocaine as it has a high boiling point 197 C therefore the majority of the drug is wasted and a high amount would be required for effect which would be very expensive Crack cocaine is smoked as it has a much lower boiling 98 C It can be smoked in a pipe inhaled from heated foil or putting into a cigarette with tobacco Injecting cocaine is less common the hydrochlo
19. to others Nutt et a 2010 It was found that heroin crack cocaine and methamphetamine are the most harmful drugs to the user Alcohol heroin and crack cocaine were the most harmful drugs to others and alcohol followed by heroin and crack cocaine were the most harmful drugs overall Nutt et a 2010 The study calculated their findings correlations with the Misuse of Drugs Act 1971 as 0 04 and therefore no relation They concluded that current drug laws are not indicative of the level of harm a drug may cause Nutt et a 2010 1 4 Common Illicit Drugs 1 4 1 Cocaine Cocaine is extracted from the leaves of Erythroxylon coca and is produced either as a hydrochloride salt or a base crack 0 7 is the average concentration of cocaine produced per leaf Each coca shrub has a life expectancy of around 50 years and is harvested three or four times a year Karch 2006 The isolation and extraction process is fairly easy with no great technical knowledge or scientific equipment required The technique is mostly passed down through the generations Karch 2006 The purity of street cocaine is highly variable and can be as low as 1 Common cutting are sugars and other drugs such as amphetamine caffeine and codeine and procaine which has a similar anaesthetic property but is not a CNS stimulant However at the latter end of 2010 the Forensic Science Service noted that cocaine purity was increasing from around 17 to 26 on average It is thought that
20. 1 26 397 46 394 77 61 68 ZLC L 99 LBGL 16 OLEI PL LIEL 82 Orr 16 Z9PL cecus Ig P GL 6 CE91 052891 VOSLEL B Aysuajur ue ues 1000 Cocaine Benzocaine and Caffeine Mix 1 POL EG LIE 92099 SO 798 SELILI bre OL LBZL 69 G GL AER 172891 3 Cocaine HCI 30000 Benzocaine E Aysuaqul ue ues STSILI L EDGL LE G EL 8G 8Stl G 8651 S 2 a o sue uewe 66256 SL VL EL cO 8 8c 826 e 2201 iun SC wc LE SBZL EIBZEL LE OBEL 19 LOBI 8 1000 1200 Raman shift cm 1 Z0 Gr 69 G5GL 890091 1600 88 5591 668691 50000 Caffeine Signature ERRE Aysuaqul uewe th reference 1 compared wi ine mix caffe Ine benzocai Ine Cocai 6 igure 3 F spectra 62 060001 67 EZOL c9 LOL LEOLI 180821 DU D r pi pest 27091 67991 Cocaine Benzocaine and Caffeine Mix 2 1500 Aysuayu uewey 720001 18 ECOL Er 220L STILI LLEDZL LIEGE 86 SS S geslu ve ka Aysuaqul uewe Sv rar 66 9G SL pL lt E1 z08 EE Ze Zc0l 181201 SC We LE SBZL 9 8cEl 280981 9 LOBI Z0 G t 69 G66 830091 885591 66 8691 d 8 s 8 SOR sue uewey 30000 Benzocaine 20000 pi esl 96118 so a E LL PU ILL OL LBZI ZIEL bi B9EL Op zbil 9 SSL L9pO9L
21. 1 8 North Review The North review of the drink and drug driving law was published in 2010 It contains recommendations for the advancement of drug driving procedure The second recommendation is that the government should commission more research into the prevalence of drug driving Recommendation 11 states that type approval of an oral fluid device based on immunoassay or other test should be established quickly for use in police stations as a preliminary screen and used in accordance with the Road Traffic Act 1988 This would eliminate the need for a doctor or nurse to take the sample Further blood or urine confirmatory tests would be carried out after a positive result in oral fluid The review states that this process in police stations should be achieved within two years North 2010 Recommendation 17 then takes this further and states that the government should continue this technology and work on type approving a screening device to be used at the roadside A positive result at the roadside would result in the individual being arrested then they would be obliged to provide a blood or urine specimen for confirmatory testing The review considers that the device may only be suitable in a controlled setting such as the police station and the issue of environmental contamination must be addressed and overcome Other relevant recommendations from the North review include recommendation 13 14 and 15 Recommendation 14 considers what levels of
22. 18 BELEG 9G BEI 02870 LO vct 095621 66 EEL 8 6981 vG EBEL sda Ajsuaju ueue j 1200 1100 1000 900 800 700 Raman shift cm 1 1300 1400 1600 KGHB Liqui Figure A7 JE pr 1G 16h ve orl 200 67 LPE 18 182 66 EZE Be O6 E18 BE OLE OL pr 16966 56812 M 5 8 8 8 2888 SA RR Aysuajul ue uie 18000 8 Gre PETHE 1 Ee 98 LrOL 98 LIDL SC EDII P SELL EO 202 1200 Stach PU osi BZ DEL ZB EVEL r8 99EL S0 Dr GLOBE 1400 96 85vl D G tt EO ZOSL GE 8851 860191 1600 IG EES M M rereed eo 2 68 8 6 sa 5 SS R 16000 14000 12000 10000 4000 Raman shift cm 1 MDE Hydrochloride Solid Figure A8 100 59 E6L 66 257 U LE lt a LE Cre EL Lee 9801P oF Grr 6 pEb LC6LV OZ SES 92 909 92 EER Wu 69922 6C vI8 LC 888 16866 Dr Ezol Or 6601 Lv Eent VOZA OO SELI 99 86L1 Di BPEL v6 S0EL LVCEEL S6 0261 SG Z0bL D0 Err GO op 892051 802951 90 9551 ECH 68 EE9L 8 88 8 8 8 8 8 B 8 8 8 8 8 8 8 5 E 8 B 8 8 8 8 e P 8 Z s SS 9 8 B A RE S Aususju ueuies 200 1000 1200 1400 1600 Raman shift cm 1 MDMA Solid Figure A9 101
23. 40 1KGHB in ethanol 50 1454 54 Raman Intensity cps 8 1050 49 1093 75 3500 3000 2500 2000 1500 1000 500 Raman shift cm 1 Figure 1 9 KGHB in Ethanol compared to neat ethanol spectra Figure 1 10 demonstrates the limit of detection of KGHB in ethanol it shows that the 930cm 1 peak was present at 3 v v but could not be detected at a lower concentration 1KGHB in ethanol 10 801 Int 1051 53 1093 73 200 KGHB in ethanol 5 Int a s Pass des 54 135845094 150 KGHB 3 100 Int 79 61 1455 41 1276 08 1095 22 1053 09 e o a 200 1KGHB in ethanol 2 5 raj 1 m u a x E 100 A K 2 em RE i QD eo A N 1 200 KGHB in ethanol 1 A Ku 3 A D Bi og E eo 8 35 E 28 5 o 5 a A W 8 ji S 9 i E O 4 z Y T R i A 7 3500 3000 2500 2000 1500 1000 500 Raman shift cm 1 Figure 1 10 KGHB limit of detection in ethanol This study recognised that KGHB and GBL had very similar peaks Table 1 1 demonstrates this by showing a comparsion of the peaks both substances have in common 40 Table 1 1 KGHB and GBL peak comparison Peaks in common cm This led to the conclusion that KGHB is likely to be not fully unconverted from GBL unlike NaGHB which has been completely converted This is backed up by the presence of the 803cm peak as thi
24. 63 568 Day J S Edwards H G Dobrowski S A amp Voice A M 2004 The detection of drugs of abuse in fingerprints using Raman spectroscopy Il cyanoacrylate fumed fingerprints Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy 60 8 1725 1730 Diercks T Coles M amp Kessler H 2001 Applications of NMR in drug discovery Current opinion in chemical biology 5 3 285 291 Drummer OH 2001 The forensic pharmacology of drugs of abuse London Hodder Arnold Drummer O H 2006 Drug testing in oral fluid Clinical Biochemist Reviews 27 3 147 86 Dumont G J H Sweep F C G J Van der Steen R Hermsen R Donders A R T Touw D J amp Verkes R J 2009 Increased oxytocin concentrations and prosocial feelings in humans after ecstasy 3 4 methylenedioxymethamphetamine administration Social neuroscience 4 4 359 366 EMCDDA 2009 Annual report 2009 state of the drug problem in Europe Luxembourg Office for Official Publications of the European Communities Luxembourg EMCDDA 2009 Drugs in focus responding to drug driving in Europe Luxembourg Office for Official Publications of the European Communities Luxembourg Farquharson S Shende C Sengupta A Huang H Inscore F Rapid Detection and Identification of Overdose Drugs in Saliva by Surface Enhanced Raman Scattering Using Fused Gold Colloids 2011 Pharmaceutics 3 3 425 439 Foye WO Lemke TL
25. 66 675 Road Traffic Act 1988 http www legislation gov uk ukpga 1988 52 Accessed 02 02 2011 S gm ller B Schwarze B Brehm G amp Schneider S 2001 Application of SERS spectroscopy to the identification of 3 4 methylenedioxy amphetamine in forensic samples utilizing matrix stabilized silver halides Analyst 126 11 2066 2071 Saunders N 1993 E for Ecstasy London Nicholas Saunders Smith E Dent G 2005 Modern Raman Spectroscopy A practical approach UK John Wily and Sons Smith F Siegel J 2010 Handbook of forensic drug analysis Elsevier Science and Technology Speedy T Baldwin D Jowett G Gallina M amp Jehanli A 2007 Development and validation of the Cozart DDS oral fluid collection device Forensic science international 170 2 117 120 Stafford P 1993 Psychedelics Encyclopaedia Ronin Publishing The Medicines Act 1968 http www legislation gov uk ukpga 1968 67 accessed 20 1 12 The Railways and Transport Safety Act 2003 http www legislation gov uk ukpga 2003 20 schedule 7 accessed 05 02 2011 TruScan Manual 2010 Ahura Scientific Tsuchihashi H Katagi M Nishikawa M Tatsuno M Nishioka H Nara A and Petty C 1997 Determination of methamphetamine and its related compounds using Fourier transform Raman spectroscopy Applied spectroscopy 51 12 1796 1799 89 Verstraete A G 2004 Detection times of drugs of abuse in blood urine
26. Acquisition time for the method used in this study was rapid at around 40 36 seconds By profiling the impurities it was possible to tell if batches of pills from different seizures were likely to have come from the same source Tablets with the same logos and appearance are assumed to be from the batch differed substantially in MDMA content and homogeneity This reiterates the dangers of users believing they are taking the same pill as they have previously tried this can lead to accidental overdose Bell et al 2003 As well as amphetamine type substances Raman spectroscopy has also demonstrated it can discriminate between crack cocaine and cocaine hydrochloride without issues arising from common adulterants such as lidocaine and benzocaine Carter et al 2000 A study also demonstrated that using thin layer chromatography TLC can separate out mixtures and spectra can be obtained from the TLC plate Angel et al 1999 Weyermann et alused a portable Raman spectrometer to investigate controlled substances in situ at border controls They found it to be a good screening device for powders and liquids due to it non destructive nature and its ability to penetrate containers It was noted that in order to acquire good spectra for illicit drugs dissolved in water or other liquids the concentration of the drug in most cases has to be very high therefore it is not sufficient for trace analysis Weyermann et al observed that focalisation was very im
27. C MS GHB can be detected to the nano gram level however an effective screening device with minimal sample preparation is desirable not just for biological samples but for bulk drug analysis and spiked alcohol analysis A screening device which could achieve this would save time and money 14 1 2 Prevalence 1 2 1 Prevalence of drug use The prevalence of drug use in the general population can only be estimated through the use of surveys this also applies to the prevalence of motorists driving under the influence of drugs The Scottish Crime and Justice Survey SCJS found that 8 4 of under 60 year olds have used cannabis in the year 2008 09 3 7 had taken cocaine 2 5 ecstasy and 1 4 amphetamine Wishart 2010 Since 2006 there appears to be a decrease in illicit drugs in Scotland the use of cocaine and the benzodiazepine temazepam however has remained unchanged Wishart 2010 A review by Jackson and Hilditch considers the British Crime Survey BCS as the most extensive drug use survey of England and Wales The survey s respondents lived in a household and were between the ages of 16 and 59 According to Hoare the survey is an underestimate as it is restricted to people who live in a household and does not include groups which have the potential to have high rates of drug abuse such as the homeless or prisoners Hoare also notes that opiate and cocaine addicts including crack cocaine addicts may lead such a chaotic lifestyle that
28. Development of a method for the detection of GHB and other drugs using a handheld Raman Spectroscopy Device Lauren O Connor A thesis submitted in partial fulfillment of the requirements of Edinburgh Napier University for the award of Master of Science by Research May 2014 Declaration It is hereby declared that this thesis and the research work upon which it is based were conducted by the author Lauren O Connor Lauren O Connor Acknowledgment would like to thank my supervisor Yvonne Cruickshank for all support and advice as well as Dr Kevin Smith for encouraging me to pursue a Masters by Research would also like to thank Raabell Shah for her help and the insightful discussions we have had which have been very valuable am also very grateful to Lora Stevenson for her assistance and Craig Dixon for his support help and understanding throughout The support of my parents and grandmother have made it possible for me to pursue a Masters by Research and am eternally grateful to them as well as my little sister Holly who was always available to encourage me make me laugh and help me with her brilliant word processing skills Abbreviations AEME Anhydroecgonine Methyl Ester BCS British Crime Survey CNS Central Nervous System DAT Dopamine transporter DFSA Drug facilitated sexual assault EMCDDA European Monitoring Centre for Drugs and Drug Addiction EME Ecgonine methyl ester FIT
29. Engine DecisionEngine is designed to eliminate false identification compare sample spectra to saved methods and if there is no significant Raman discrepancy then a PASS is reported when there is a discrepancy a FAIL is reported A p value of 0 05 or greater passes the method A p value of 0 001 and 0 05 are at moderate risk of passing as the sample has similar characteristics to the method p value is used to reject or confirm a hypothesis It is a measure of probability For clarity the TruScan names a reference spectrum a method This should not be confused which the experimental method 42 Method Measurement Si Determine if aspects of measurement statistically contradict Method PASS Suspect No Raman evidence to Raman evidence of believe otherwise discrepancy User level dependent gives results against stored Methods or optional Discovery Library Present solution Declare no resolution present similar library records Figure 1 11 TruScan PASS FAIL System TruScan user manual 2010 1 7 Oral fluid Oral fluid is defined as the fluid within the oral cavity this is made up mostly of saliva the secretions of the saliva glands but is also made up of small amounts of cellular debris blood food debris and gingival crevicular Oral fluid is primarily secreted by three glands called the parotid submaxillary and sublingual as well as other small glands Various factors affect the flow of saliva such as emotiona
30. GHB This is explained by comparing the blank dimple tray spectra to the SERS KGHB in oral fluid spectra Blank Dimple Tray Raman intensity iSERS GHB in OF 421 68 Raman intensity 266 45 253 25 1200 1100 1000 900 800 700 600 500 400 300 Raman shift cm 1 Figure 3 19 Blank dimple tray and SERS KGHB in OF comparison 73 Table 3 5 Common peaks to KGHB KGHB in oral fluid and SERS KGHB in oral fluid KGHB KGHBin OF SERS KGHB in OF 1039 80 1023 75 875 92 875 32 873 95 1296 94 1294 55 S 1239 73 z 641 98 E 421 68 266 45 403 18 S 253 25 74 Other drugs spiked in oral fluid did not produce any peaks when the SERS method was applied 1SERS GHB in OF 8 z amp 5 0 z s 2 RZ 5 E c LS Ed 8 amp 1004 2 28 NE z 83 H g 5 gt 3 RA I mm SERS Amphetamine Sulphate in OF P lx Bo S e Ee E 2 10007 8 35 3 a tej E E 5 smol GA S x ISERS Cocaine Hydrochloride in OF 8 5 B 150001 5 i 1 o N e E x 5 5 10000 Gos 3 amp 1 soon Ze A 15000 1 SERS Ketamine Hydrochloride in OF 1 8 z z 2 i 3 2 tomo g S 5 z 5001 SOG N 1400 1200 1000 800 600 400 200 Raman shift cm 1 Figure 3 20 Comparison of SERS spectra 1 SERS MDE in OF 12000 a o a E T867 90 1539 99 1341 98 10000 1421 48 8000 199 01 6000 Raman intensity
31. H of the oral fluid The oral fluid was disposed off and re collected if there were any pH changes 1 mLI of KGHB was used to spike 1 mL of oral fluid this was mixed for 10 seconds using a vortex mixer This was ran using the TruScan SOP and the vial attachment 52 2 mg of cocaine hydrochloride was used to spike 1mL of oral fluid this was mixed for 10 seconds using a vortex mixer This was analysed by following the TruScan SOP and using the vial attachment This was repeated for amphetamine sulphate and ketamine hydrochloride 2 8 SERS sample preparation and method A 0 1M sodium acetate buffer was prepared for the SERS method This was produced by dissolving 6 8g of sodium acetate in 400 mL of distilled water Hydrochloric acid was used to adjust the pH The volume was made up 500 mL using distilled water The final SERS sample preparation decided upon was to spike 0 5 mL of drug in 0 5 mL of oral fluid this was mixed with 1mL of buffer for 30 seconds 40 ul of this mixture was pipetted into an eppendorf tube 40 ul of 29 nm gold colloid was added and this was centrifuged for 10 minutes at 14000 RPM 40 ul of the pellet at the bottom of the eppendorf tube was pipette into a dimple tray and the pointed nose cone was attached to the Truscan device before analysis Many variations of this method were attempted including different sizes of colloid 51 64 90 nm Different nose cones used and no nose cone attached Sampling from the
32. Oral Fluid Neat 1408 23 1405 79 1294 55 1296 60 The Truscan device did not show any peaks for cocaine and amphetamine in oral fluid 11000 10500 H 10000 1 osno 4 am i 9500 4 s04 7500 H 7000 H 6500 1 tensity sono 1 E ml 56001 i 5 soi a 45004 4000 H 3600 H 3000 H 2500 4 2000 3 1600 4 1004 501 Figure 3 13 2 mg of Cocaine Hydrochloride in 1 mL oral fluid 70 I o 8 14000 4 E 1 a S 13000 12000 11000 10000 9000 8000 4 7000 Raman intensity 6000 5000 4000 1639 71 1374 32 1721 87 1872 10 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 Raman shift cm 1 Figure 3 14 2 mg amphetamine sulphate in 1 mL oral fluid No more than 2 mg of cocaine and amphetamine was added to oral fluid as high amounts would not give a realistic situation Metabolised drugs would not be as high a 2 mg in a user s oral fluid 3 5 Limit of Detection of KGHB in Oral Fluid The TruScan device reported a pass for GHB at 90 but failed at 80 and below 4000 11ml KGHB in 1ml OF Truscan z J a Z 3000 8 R 8 K a e 3 S E g B e 5 5 g 5 S E p we E aad ee S IKGHB in OF 90 z A 2 5 E S 5 x Raman intensity Raman shift cm 1 Figure 3 15 KGHB Limit of Detection 1 71 IKGHB in OF 70 10000 4 Z ewm o 2 1 s ki a D E 3 8 J E
33. a ine an 1 compared with cocai ine mix benzoca ine 3 3 Cocai reference spectra Figure icocaine benzocaine mixture 125mg 2 2000 4 1500 4 DI 9L 6c 98 120001 OL EZOL 896011 9LE LL 76 LOL W ZEL 66 89EL 020571 E G GL LLVOSL 257891 04 oi 8 8 8 Ajsuaqul uewey 504 4 Cocaine HCI 98 968 22 000 18 ECOI Ep OL 8C 9911 LOL LE ua 89 Es e Bia AED Aysuaqul uewey 25000 Benzecaine 105 20000 4 15000 3 vL 86l eS 21 arzor 31 905 89219 f GC 0v9 96718 SO v98 CELIH vi m nu ve i Le MEL bi 89 DP zbil iJ 1400 E9 G46 LVC89L 1000 800 600 400 200 Raman shift cm 1 1200 1600 1800 2000 somi m 8 Ii 10000 Meuse uewey ine d benzoca ine an ix 2 compared with coca ine m benzoca ine 3 4 Cocai reference spectra Figure 58 icocaine benzocaine mixture 125mg 3 Raman intensity ai Cocaine HCI g 5 E 2 5 5 174 29 25000 Benzocaine ine 1604 67 poem 75 20000 864 05 15000 4 Raman intensity 158 74 10000 1172 14 C 11138 2000 1800 en 1400 1200 1000 Raman shift em 1 Figure 3 5 Cocaine benzocaine mix 3 compared with cocaine and benzocaine reference spectra The Truscan device identified all three runs of the mixture compos
34. a Figure 1 10 KGHB limit of detection in ethanol Figure 1 11 TruScan PASS FAIL System Figure 2 1 TruScan and DXR Figure 2 2 TruScan portable Raman spectrometer Figure 2 3 TruScan focussed on white powder Figure 3 1 Cocaine hydrochloride reference spectra Figure 3 2 KGHB and ethanol comparison Figure 3 3 Cocaine benzocaine mix 1 compared with cocaine and benzocaine reference spectra Figure 3 4 Cocaine benzocaine mix 2 compared with cocaine and benzocaine reference spectra Figure 3 5 Cocaine benzocaine mix 3 compared with cocaine and benzocaine reference spectra Figure 3 6 Cocaine benzocaine caffeine mix 1 compared with reference spectra Figure 3 7 Cocaine benzocaine caffeine mix 2 compared with 16 20 23 24 26 28 29 33 39 40 45 49 50 52 54 55 58 58 59 62 reference spectra Figure 3 8 Cocaine benzocaine caffeine mix 3 compared with reference spectra Figure 3 9 Cocaine benzocaine caffeine and paracetamol mix 1 compared with reference spectra Figure 3 10 Cocaine benzocaine caffeine and paracetamol mix 2 compared with reference spectra Figure 3 11 Cocaine benzocaine caffeine and paracetamol mix 3 compared with reference spectra Figure 3 12 Pure KGHB spectra taken from previous study Figure 3 13 2 mg of Cocaine hydrochloride in 1 mL oral fluid Figure 3 14 2 mg amphetamine sulphate in 1 mL oral fluid Figure 3 15 KGHB limit of detection 1 Figure 3 16 KGHB limit of detection 2 Figur
35. above Standard Operating Procedure 0 125 g of paracetamol and 0 125 g caffeine were added to this mixture producing a white powder with the weight of 0 5 g with 25 w v of this being the illicit substance cocaine This is a crude representation of what a street sample could consist of Smith and Dodd reported that the mean purity of cocaine seized on the UK Street is 33 Smith and Dodd 2009 A method i e reference spectra for paracetamol and caffeine was saved on TruScan This 0 5 g mixture was analysed using the TruScan SOP Both 0 25 g and 0 5 g runs were carried out 4 times with the laser focussed on a different area of the powder each time 51 Figure 2 3 TruScan focussed on white powder This figure demonstrates how the powder was presented to the TruScan device It was put onto a glass slide with all the powder in one concentrated area of the slide and the nose cone was very close to the powder when a run was carried out 2 7 Oral Fluid Analysis The Oral fluid was collected from the donor by expectoration which means the oral fluid was spat out into a beaker The oral fluid was collected over a period two days and 10 mL was collected The oral fluid was stored in a sealed container in the fridge The author was the donor for ease and to be sure no drugs were present in the donors body The donor was not permitted to provide oral fluid within 10 minutes of eating or drinking Litmus strips were used to regularly check the p
36. aliva after 20 minutes Kintz et al 2001 This means that in theory if the saliva sample was taken quickly after administration i e 20 minutes or under it is possible for the Raman method detailed in this study to detect it However this depends on variables such as the dose of GHB administered The dose required would almost certainly sedate the individual This is hugely limited by the short time frame 80 4 7 SERS and oral fluid analysis The TruScan device has clearly picked up peaks from the dimple tray therefore the laser has by passed the solution that was intended to be analysed This is a potential issue with the TruScan device it is very difficult to understand exactly what the laser is focussing on This is could also explain why containing samples in capillary tubes would not work as it was very difficult to focus the laser on the desired area As the DXR Raman has a microscope where the laser is focussed is very apparent and the user has more control over the precise area of the sample All other drugs did not give any peaks at all A likely explanation to the failure of SERS is that the method detailed in this study failed to combine the drug molecules with the gold colloid molecules to achieve an enhanced effect It is likely that the colloid and equipment used were not the most suitable 4 8 Further Research This study has shown that the TruScan device does not have any real potential in the detection of illicit d
37. as been counted in table 4 2 this peak is common to both cocaine and benzocaine Every run detailed in the table also peaked at the 1000cm region This is a characteristic cocaine peak however it is not exclusive to cocaine as it also occurs in amphetamine and other drugs therefore its presence alone is not enough to conclude a cocaine result Other characteristic peaks must also be present 78 Table 4 3 Number of peaks common to benzocaine reference spectra Number of Run Mixture 2 Composition Cocaine and p Rus benzocaine o c 14 11 11 o es 3 o Cocaine TE O benzocaine 9 15 13 15 x and caffeine o Cocaine 5 O benzocaine 3 13 13 13 3 caffeine and o paracetamol lt 5 The above table also included the 817cm peak that is common to cocaine and benzocaine Peaks in the region of 1604 1281 864 cm are the most intense peaks on the mixture spectras These peaks are common to benzocaine The TruScan software obviously considers these peaks important in identification The intensities of these peaks are weighted more than the occurrence of common peaks as the tables show i e 16 common cocaine peaks to 13 benzocaine peaks The careful wording of raw material in the manual makes perfect sense as the device is not capable of identifying simple mixtures 79 4 5 Oral fluid analysis KGHB was the only drug to be detected at a high concentration in oral flu
38. ates the opportunity to adulterate the sample 1 7 3 Disadvantages of oral fluid Stimulating the production of oral fluid by chewing gum or other agents will alters the pH and therefore the concentration of the drug Stimulating oral fluid has been shown to reduce the concentration of the drug from two to four fold for methamphetamine and five fold for cocaine Hillsgrove et al 1993 Some drugs such as MDMA reduce the secretion of oral fluid This can mean that collecting just 1mL of oral fluid can take up to several minutes Having a dry mouth due to improper hydration or the anxiety of the test can also hinder the process of drug testing in this way The ROSITA project also noted that sometimes oral fluid was too viscous and therefore could not be used with some devices Sometimes in this case a different sample such as blood may have to be taken Drummer 2006 An article published in 2011 details a study carried out by Real Time Analyzers Inc an American company that make design and market Raman Spectrometers establishing a method of detecting drugs in saliva for the use of identifying offending overdose drugs in a hospital setting They successfully employed SERS to identify numerous drugs in saliva at the ng mL concentration within 10 minutes A Solid Phase Extraction capillary was combined with SERS 45 active capillary connected to a syringe driven sample system A portable Raman Spectrometer was used Farquharson et al 2011
39. caffeine and 0 125g paracetamol as benzocaine This means it matched the mixture spectra to the benzocaine spectra contained in its library only It did not report a match to the cocaine caffeine or paracetamol spectra in its library Table 3 3 lists the peaks in the neat cocaine benzocaine caffeine and paracetamol spectra and the peaks that occurred in each run of the mixture The lists allow the peaks to be compared easily and common peaks to cocaine and benzocaine can be identified quickly 64 Table 3 3 Cocaine benzocaine Caffeine Paracetamol mix run results Cocaine Benzocaine Caffeine Paracetamol Run 1 Run 2 Run 3 1716 24 1715 89 1715 90 1698 99 1682 47 1682 31 1682 83 1682 30 1655 88 1649 83 1648 15 1650 98 1652 60 2 2 1613 19 8 S 1604 67 1600 68 1603 93 1604 20 1601 72 1575 63 gt 1574 89 1575 03 1575 33 1515 59 151642 1458 58 1459 02 1455 75 1454 78 1447 40 gt 1446 91 1448 87 1368 74 1360 37 1372 08 1368 82 1371 40 1328 63 1325 13 1327 40 1326 75 1311 21 1312 46 1311 73 1281 76 1285 37 1278 34 1281 37 1281 84 1279 12 1241 25 1237 37 1236 77 1238 99 1236 75 65 1203 71 1205 98 1202 86 1166 25 E 1168 92 1077 43 1071 81 1070 62 1077 17 1000 72 1001 36 999 65 1000 87 869 57 z z E 858 73 E 817 96 817 61 818 08 818 52 819 74 798 18 796 70 798 16 74175 74248 s z 711 82 709 61
40. cation of the illicit substances Day et al 2003 In the cyanoacrylate fumed fingerprints the illicit substances were detected successfully as under normal sampling conditions the sample was photo bleached in order to reduce fluorescence interfering bands were present in the spectra due to the polymer however they did not prevent identification of drugs of abuse Day et al 2003 Hargreaves et al demonstrated that Raman Spectroscopy was able to identify a number of suspect powders in their containers were identified as illicit drugs in situ in an airport environment This study was carried out using two different Raman Spectrometers They concluded good quality spectra could be obtained 35 from Custom and Excise samples in 30 seconds and under providing there was not a highly fluorescent cutting agent Hargreaves et al 2008 Raman spectrometers involved in the detection of drugs are often Fourier transform FT and dispersive FT spectrometers using a near intra red NIR laser and is often coupled with a microscope for trace samples An advantage of FT Raman is that it uses a 1064 nm laser which is less prone to fluorescence as most compound do not have excited states low enough in energy to fluoresce with this laser This does mean scattering is weaker however Weaker scattering results in longer detection time as the acquisition time is longer Dispersive can use a UV visible or NIR laser and a charge coupled detector ccd detector
41. d z H v pe GH NI A d a RZE OOCH L n H T gt RZ AE 1 NL OH _oco jonine methy ester EME r da Py o Et A pa CH CH N COOCH C00 H r Sr f F7 A h FEN RA INN v N OGO Nd Anhydroecgonine methyl ester AEME Cocaethviene CA 77 22 Figure 1 3 Pathways of Cocaine metabolism Drummer 2001 1 4 2 Amphetamines Amphetamines are illegally synthesised usually by a process known as the Leuckart reaction This involves the condensation of phenyl 2 propanone with formamide then a hydrolysis of N formylamphetamine and finally purified by steam distillation The Leuckart reaction does not produce any hazardous chemicals and produces a good yield which may explain its popularity Jickells and Negrusz 2008 Street amphetamines are bought in small wraps and have the appearance of an off white powder they are cut with adulterants such as caffeine to provide a stimulant effect and mask the low level of drug Sugars are another common diluent Methamphetamine is more popular in the USA and Japan than the UK and is usually sold as methamphetamine hydrochloride Jickells and Negrusz 2008 If amphetamine is injected smoked or vaporised the effects can be quick as a few seconds giving a rush of euphoria Snorting does not give the same heighten effect of euphoria and takes minutes for the effect to take hold it may take up to 20 minutes for any effects to kic
42. d highly optimized Smith and Dent 2005 34 1 5 4 Surface Enhanced Raman Spectroscopy Surfaced Enhanced Raman Spectroscopy is technique which enhances Raman Scattering Molecules are absorbed on rough metal surfaces to create this enhancement The technique can be used to detect a single molecule as it the enhancement factor can be as much as 10 to 10 Blackie et al 2009 As SERS is sensitive to the metallic surface employed the shape and size of the nanoparticles used strongly affects the enhancement The ideal size of metal nanoparticles or colloid used varies depending on what molecules are targeted This means trial and error may have to be adopted in a SERS experiment The enhancement effects exact mechanism is still debated in literature Arocha 2006 1 5 5 Previous Research There is a great deal of past research on the subject of detecting illicit drugs using Raman spectroscopy Controlled substances are usually strong Raman scatterers and therefore produce characteristic peaks and informative spectra Weyermann et al 2011 In 2003 Day et al carried out a study investigating the detection of drugs in latent and cyanoacrylate fumed fingerprints they concluded Raman spectroscopy was successful in detecting drugs of abuse in sweat rich latent fingerprints using photo bleaching to reduce fluorescence background Sebum rich latent fingerprints presented some interfering bands however these bands did not interfere in the identifi
43. dence relating to drug facilitated sexual assaults GHB is heavily associated with drink spiking as it is difficult to detect and its sedative effects are exacerbated by alcohol making it an ideal date rape drug A study demonstrated that GHB and its precursor GBL can be identified in alcoholic drinks in a number of containers such as glass and plastic using Raman spectroscopy GBL is as important to detect as GHB as when in solution the two inter convert The study was able to detect the drugs lower than the common dose Brewster ef a 2009 However this study was limited as it did not include drink mixers such as soft drinks Surface enhanced Raman spectroscopy SERS has also demonstrated its use in the detection of drugs Studies have shown it can be an effective way to identify amphetamine powder and ecstasy pills S gm ller et al 2001 The active ingredient of the drug was extracted using cyclohexane this extraction worked well as components such as the colouring in the tablets were not present in the cyclohexane phase This means there was less interference or fluorescence from excipients SERS has also been utilised successfully for the detection of 2 5 dimethoxy 4 bromoamphetamine DOB which is potent at low doses The use of a silver colloid enables DOB to be detected in a tablet down to a concentration of 15 ug However if this technique cannot to used if MDMA is also in the pill as MDMA is present at a much higher concentration and t
44. der to determine information about the chemical bonds of the substance in question Jickells and Nergrusz 2008 1 5 1 Basic theory of Raman Spectroscopy The light emitted from the laser impinges on the molecule and causes the electron cloud around the nucleus to distort and create a brief state known as the virtual state This state can quickly reverse back and is not stable Excitation happens in the visible or near infrared range Smith and Dent 2005 The photons emitted from the molecule may scatter at the same frequency and wavelength as the laser frequency and wavelength This is what happens with the majority of photons Jickells and Nergrusz 2008 This is called elastic or Rayleigh scattering No analytical information is obtained from Rayleigh scattering Bell 2006 Not all of the photons will scatter like this however few scatter with a frequency which differs from the laser When the molecule relaxes back from the virtual state by emitting a photon and it relaxes back to a higher vibrational energy this is inelastic scattering The 32 emitted photon has a longer wavelength than the incident light so therefore it has a lower frequency This is Stokes Raman scattering Jickells and Nergrusz 2008 If the molecule relaxes back to a lower vibrational state emitting a photon which has a greater energy therefore a higher frequency than the incident light this is referred to as anti Stokes Raman scattering Jickells and Ner
45. drugs and their active metabolites should be considered impairing and these prescribed levels should be included in the legislation Recommendation 14 states that if the driver if found to have drugs in their body above the prescribed limit as they had taken a drug in accordance with medical advice a stationary defence should be available Recommendation 15 states that if no scientific census on the impairment level can be reached a policy of zero tolerance should be introduced North 2010 46 1 9 Aim The aim of this study is investigate the use of Raman Spectroscopy as a screening method for drugs of abuse Raman Spectroscopy was chosen as in published literature it is praised for its quickness and its minimal sample preparation i e No extraction step required It must be established if Raman can qualitatively detect drugs of abuse in mixtures and in oral fluid for individual drug testing which could be applied to suspected drug drivers The drugs used in this study were chosen for their stimulant effects which are likely to cause risky behaviour such as driving under the influence GHB was chosen as it is a notoriously difficult drug to detect in the body and may be administered to for DFSA Previous work by the author can also offer a brief comparison between portable and bench top Raman instruments 47 Chapter 2 Materials and Method 2 1 Materials The drugs used in this study were purchased from Sigma Aldrich The drugs used were
46. e 3 17 KGHB limit of detection 3 Figure 3 18 0 5 mL GHB in 0 5 mL oral fluid Figure 3 19 Blank dimple tray and SERS KGHB in OF comparison Figure 3 20 Comparison of SERS spectra 1 Figure 3 21 Comparison of SERS spectra 2 65 65 68 68 69 69 70 71 71 72 72 73 73 75 75 List of Tables Table 1 1 KGHB and GBL peak comparison Table 3 1 Cocaine and benzocaine mix run results Table 3 2 Cocaine benzocaine caffeine mix run results Table 3 3 Cocaine benzocaine caffeine paracetamol mix run results Table 3 4 Common peaks neat KGHB and KGHB in oral fluid Table 3 5 Common peaks to KGHB KGHB in oral fluid and SERS KGHB in oral fluid Table 4 1 Handheld and bench top Raman comparison Table 4 2 Number of peaks common to cocaine reference spectra Table 4 3 Number of peaks common to benzocaine reference spectra 41 56 60 65 70 74 77 78 79 Abstract The aim of this study was to investigate the potential of a portable Raman spectroscopy device TruScan for the screening of illicit drugs The study aimed to establish if the device could be used as a presumptive test on the spot in order to determine if a bulk sample alcohol or oral fluid has an illicit substance in it Should this device be successful in its detection and be easy to use police would be able to utilise it in situations such as clandestine laboratories and on suspicion of drug driving A review of the North report demonstrated a great need for
47. e results of this study show once cutting agents and adulterants are added it is not always likely that the TruScan software will report the result as the controlled substance present in the sample For this reason a library of common cutting agents and adulterants should be included also further adding to the time consuming process The controlled drug may not be reported by the software but the cutting agent result gives a clue it is likely an illicit drug is there This however requires inspection of the spectra on Omnic software on a PC and this cannot be performed on the TruScan device The TruScan manual claims the installed software DecisonEngine on the device takes environmental factors such as light into consideration when a spectra is being obtained However covering the sample up whilst the laser is on reduces the time taken This study used items as black weighing boats to 76 cover up the sample in order to speed up the process This applied to obtaining a signature used to create the reference spectra as well as performing a simple run This also applied to the vial attachment although it appears enclosed and not subject to external light interference covering it up reduced the time considerably 4 3 KGHB in alcohol 50 v v KGHB in ethanol is not detectable using the TruScan device The DXR Raman spectrometer shows a characteristic 930 37cm peak denoting the presence of KGHB There is no way of distinguishing the TruScan Sp
48. ectra from the neat ethanol spectra This raises questions about TruScans sensitivity Its failure to identify a substance defining peak causing a false negative is concerning as the DXR easily picks up the peak Table 4 1 Handheld and Bench top Raman comparison Specification Comparison Specification TruScan Raman Spectrum Range 50 and 3300 cm 250cm to 2875cm Laser Excitation Wavelength 780 nm Laser output Maximum 14mW Maximum 300mW The high laser output seems excessive at 300mW On a number of occasions the powder samples were burned black holes were present on the sample where the laser was focussed and there was a burning smell No way of adjusting the laser power was identified from reading the manual as well as investigating the menus present on the device 77 4 4 Mixture analysis Three runs were carried out on each mixture each on a different location of the powder as the cocaine content will vary between areas as crudely cutting the substance in this way would not to produce a uniform mixture This is evident in table 4 2 Table 4 2 Number of peaks common to cocaine reference spectra Number of Run Mixture 2 Composition Cocaine and o z e c benzocaine o 3 5 4 8 z Cocaine o h Ke benzocaine D i 6 16 5 x and caffeine o Cocaine o O benzocaine 3 16 6 16 3 caffeine and 5 paracetamol gt Every run featured a peak at 817cm which h
49. ed in each run of the mixture The lists allow the peaks to be compared easily and common peaks to cocaine and benzocaine can be identified quickly 55 Table 3 1 Cocaine and benzocaine mix run results Cocaine Benzocaine Run 1 Run 2 Run 3 1716 24 1713 79 1716 77 1682 47 1682 46 1683 52 1682 34 1604 67 1605 45 1604 77 1604 66 1575 63 1575 46 1574 47 1458 58 3 1433 31 1447 40 1446 65 1368 74 1367 65 1367 96 1311 21 1311 19 1312 41 1310 55 1281 76 1281 98 1281 94 1280 41 1275 31 1203 71 1172 14 1171 57 1173 16 1171 95 1111 33 1110 50 1077 43 1075 75 1023 81 1023 76 1024 33 1000 72 999 17 1000 71 1001 71 896 86 869 57 864 05 863 13 863 29 863 64 817 96 817 61 817 12 820 27 818 50 oo o o o o o o o oo 57 EL 698 OS OLbL LG bill 86 LBZL sr HEN 59 LSEL S9 opp 9v 5 GL Sv 5091 9v z891 G EL U DU 1821 cocaine benzocaine mixture 125mg 1 1000 4 500 4 Aysuaqul ue uie 2 0 Cocaine HCI 25 585 51 919 68189 19218 1G 698 zi 0001 18 ECOL EF 2201 56991 L LUED LE G ZL ec pr ENSCH en H H e o e D o 2 Asusjul uewesy Benzocaine 89 G GL 29 p091 20000 10000 89219 Sc Droa 9618 S0 v98 Minis Peli s 021821 Le LIEL PL B9EL or ima uy e Aysuaqul ue uie 1600 1400 1200 1000 800 600 Raman shift cm 1 1800 2000 ine d benzoc
50. elect openOmnicFiles from the pop up menu The Spectra will appear in the Omnic program window and can be saved 96 Appendix 3 Reference Spectra All spectra in this appendix are of the neat compound and have been acquired by the TruScan device Acetaminophen 214 63 24000 4 22000 412 19 20000 392 54 153 12 18000 858 73 16000 4 1256 53 330 40 14000 4 1325 13 Raman intensity 1237 37 798 18 652 48 504 94 sA 12000 4 10000 4 1278 34 834 99 1105 38 1017 29 Raman shift cm 1 Figure A1 Acetaminophen paracetamol Solid 1d Amphetamine Sulphate Sig 1003 05 8 8 8 976 81 Raman intensity 837 35 B 8 1607 39 1210 16 1181 20 1031 57 827 50 462 47 1101 60 500 01 436 42 1336 95 602 31 E 02 2000 1800 1600 1400 1200 1000 800 600 Raman shift cm 1 Figure A2 Amphetamine Solid In literature characteristic amphetamine peaks are 1030 cm 1003 cm and 970 cm Weyermann et al 2011 97 36000 Benzocaine 34000 6 36 32000 30000 i 28000 26000 1 24000 22000 J 20000 4 1604 67 18000 4 Raman intensity 16000 4 864 05 14000 1682 47 1281 70 12000 4 10000 4 e000 soon 4000 2000 0 i 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 Raman shif
51. ence was cannabis Ecstasy was the second most common drug followed by cocaine and then amphetamines Myant et al 2006 1 3 Legislation 1 3 1 Misuse of Drugs Act 1971 The main piece of legislation which addresses illicit drugs in the UK is the Misuse of Drugs Act 1971 The Act was designed to prevent the use of drugs which are capable of having harmful effects sufficient to constitute a social problem North 2010 16 Drugs are divided into three categories Class A B or C Class A drugs are considered to be the most dangerous and carry the highest penalties In the UK it is an offence to e Possess a controlled drug e Possess a controlled drug with the intent to supply e Offer to supply a controlled drug e Produce manufacture or cultivate a controlled drug e Import or export controlled drugs e Allow premises to be used for use supply or production of controlled drugs Class A drugs include heroin cocaine crack cocaine and ecstasy Possession of a Class A drug can result in a prison sentence of up to seven years and an unlimited fine Life imprisonment and an unlimited fine is the maximum penalty for the supply of Class A drugs Misuse of Drugs Act 1971 Class B drugs include amphetamine and cannabis If a Class B drug is prepared for injection it becomes a Class A drug this is common with amphetamine Possession of a Class B drug can lead to a prison sentence of up to five years and an unlimited fine Supply of a C
52. er ON Indicator Lights Bar Code Reader Display Arrow key Login Key Escape key Wake Sleep Key Enter Key Bar Code Key KEYPAD Serial Number Battery Compartment Door Figure 2 2 TruScan Portable Raman Spectrometer TruScan user manual 2010 2 3 Creating a Library In order to use the TruScan Raman Spectrometer as an identification tool a library of materials was created This was achieved by following the devised standard operating procedure see appendix 2 2 4 Production of liquid GHB 120 mL of GBL was added to a Pyrex glass container with 91 grams of Potassium hydroxide 250 mL of warm distilled water was then added and the container was covered for the reaction to take place The solution was slowly heated for an hour on a hot plate taking care not to overheat or burn the solution The solution was topped up with water to 1000 mL and 50 75 mL of vinegar was added until the pH was below 7 5 50 2 5 KGHB in alcohol To compare the TruScan device to the DXR Raman microscope used in a previous study 0 5 mL of KGHB was added into a vial containing 1 mL of ethanol this was mixed for 30 seconds using a vortex mixer then analysed using the vial attachment on the TruScan 2 6 Mixture Analysis A 0 25 g white powder was made by mixing 0 125 g cocaine and 0 125 g benzocaine together A Method for both cocaine and benzocaine was saved on the TruScan A Run was carried out on this mixture sample using the
53. es a very high dose and sample to be taken 20 minutes after administration There is a danger of missing concentrations of KGHB in the range of 4 300 mg L KGHB in alcohol would go undetected with TruScan The device is unable to report a positive for the illegal substance cocaine in a white powder mixture Analysis of spectra is required this cannot be done by police and the spectra have to be transferred to a PC 83 with Omnic software as the TruScan screen is unable to show detailed spectra and peak numbers 84 Chapter 6 References Advisory Council on Misuse of Drugs http drugs homeoffice gov uk drugs laws acmd accessed 17 01 10 Angel S M Carter J C Stratis D N Marquardt B J amp Brewer W E 1999 Some new uses for filtered fiber optic Raman probes in situ drug identification and in situ and remote Raman imaging Journal of Raman spectroscopy 30 9 795 805 Anyu C Lin H Jinghua L ZiJian C Yi J Dian Q amp Hong W 2009 January Detecting Narcotic Usage Using Surface Enhanced Raman Spectroscopy on Saliva Samples In World Congress on Medical Physics and Biomedical Engineering September 7 12 2009 Munich Germany pp 71 74 Springer Berlin Heidelberg Aroca R 2006 Surface enhanced vibrational spectroscopy England John Wiley amp Sons BBC News Italian River full of cocaine http news bbc co uk 1 hi world europe 4746787 stm accessed 25 01 2011 Bell S E B
54. found to be carrying nearly 4kg of benzoylecgonine daily This is the equivalent of 40 000 doses daily in the 21 region which puts the street value of cocaine consumed in the area at around 84 million per year Italian river full of cocaine 2005 Other cocaine metabolites are produced such as ecgonine methyl ester EME ecgonine and nor cocaine The enzyme butyrlcholinesterase in the blood converts cocaine to ecgonine methyl ester and benzoylecgonine these are relatively inactive metabolites Winger et al 2004 Norcocaine is further metabolised to N Hydroxynorcocaine and this may be responsible for cocaine s toxicity of the liver Drummer 2001 Anhydroecgonine methyl ester AEME is only produced when cocaine is smoked as it is not produced from metabolism but from pyrolysis This makes it a helpful and unique marker See Fig 1 3 Another marker is cocaethylene which is only formed when the user is consuming both cocaine and alcohol Greater intoxication has been reported by users when cocaine is combined with alcohol This is thought to be due to cocaethylene binding to dopamine receptors as well as the cocaine molecule therefore dopamine reuptake is further blocked leading to a greater build up of dopamine heightening the euphoric effects Wills 2005 CH CH i N N X A nnr COOH B EL j Re SE BE J ka E OH d c TCO G e ma Ecgonine Benzoyl ecg BZE n air A e d D A NI
55. grusz 2008 It is typically the Stokes region of the spectrum that is used as they are more intense Stokes scattering occur from the higher energy which is less frequent in molecules The spectrum is usually shown as Raman intensity versus Raman shift Raman Intensity is the amount of photon per second and Raman Shift is the shift in frequency of the emitted photon Jickells and Nergrusz 2008 Virtual state 1 vibrational excited state Ground State Figure 1 8 The three different types of scattering Adapted from Bell 2006 There is no different in frequency in Rayleigh scattering A molecule which is already in an excited state is further excited and relaxes back to the lower frequency in Anti stokes scattering this emits a photon of a higher energy In Stokes scattering the molecule relaxes back to a higher frequency emitting a photon of a lower frequency 33 1 5 2 Advantages of Raman Spectroscopy Raman Spectroscopy has many favourable properties as a technique it is easy to carry out and has a large range of uses with no or very little sample preparation required other techniques such as GC MS require the drug to be extracted from the sample matrix in order to be identified Jickells and Nergrusz 2008 A spectrum can be obtained within seconds making it an extremely quick technique Smith and Dent 2005 Analysis can be done on practically any type of sample organic or inorganic liquid solid or vapour The
56. he enhancement will have effected both compounds The MDMA signal will drown out the weak DOB signal Bell et al 2007 Another study showed that comparing the SERS spectra of drug heroin methamphetamine and methadone users with non users a characteristic peak at 1030 cm 1 was present in the 38 users but not in the non users therefore users could be swiftly identified non invasively Anyu et al 2009 1 5 6 Previous research by the Author In a previous study carried out by the author KGHB that is GHB manufactured in a clandestine laboratory using potassium hydroxide as a starting material was detected in ethanol and some alcoholic drinks to a concentration as low as 3 v v using a bench top DXR Raman Spectrometer NaGHB manufactured using sodium hydroxide as a starting material was not detected in alcohol using a DXR Raman Spectrometer GBL GHB precursor in alcohol was detected down to a concentration as low as 0 25 v v This study did not include mixers in any of the alcoholic drinks tested which is a great limitation most people do not drink spirits straight and the addition of mixers increases the volume of the drink therefore diluting the drug further Figure 1 9 shows a neat ethanol spectrum compared to an ethanol spiked with KGHB spectrum The 930cm 1 peak was what determined the presence of KGHB in ethanol 180 Ethanol 5 Mins 160 1404 1204 cu o D 5 S E i 5 Raman Intensity cps 1453 97 1
57. id equivalent of 300 mg L 4 mg L is the optimum detection limit for GHB in oral fluid 1 mL GHB in 1 mL oral fluid give clearly defined peaks which are common to pure KGHB As noted in a previous study the 803 cm peak is present in the pure KGHB spectra as well as in KGHB in oral fluid spectra This peak is likely to corresponding to a ring structure As GHB does not have a ring within its structure it is likely to be unconverted GBL or GHB and GBL in equilibrium GBL is considered a stronger Raman scatterer than GHB This possible explains why a spectra containing characteristic peaks have not be obtained Adding a liquid to a liquid also may be a factor All other drugs used in this study were powder dissolved in oral fluid 4 6 Limit of detection in oral fluid KGHB can be detected down to 3096 v v in oral fluid This is still a high concentration as it corresponds to a limit of around 300 mg L in saliva Oral fluid tests need to be sensitive enough to detect the presence of a drug down to the ng level It is established in the literature that detection limit cut off level of GHB in oral fluid is around 4 mg L Verstraete 2004 this is something to aim for in screening devices however this is well below the 300 mg L detection limit established in this country A study carried out by Kintz et a found that if an individual was administered 60 mg kg of GHB on an empty stomach it would be expected that around 257 mg L GHB would be present in s
58. ied The MS detector can specifically scan for pre selected masses which are characteristic for the substance in question this is called selected ion monitoring SIM or it can scan in full scan mode which gathers all the ions in the mass range given Both LC MS and GC MS require the drug to be extracted from the matrix which is time consuming and adds to the expense of the analysis Some drugs may need to be derivatised to be made more suitable for GC MS analysis This requires a specific chemical to be added to the extracted sample under heated conditions this also adds time and money onto the analysis The gold standard in drug detection in biological matrices was considered to be GC MS Jickells and Nergrusz 2008 however LC MS has become increasingly favourable in recent years due to its ability to detect drugs in biological matrices at low concentrations A study by Gallardo et a discusses the how LC MS can be very advantageous for work place drug testing where hair oral fluid or sweat could be used LC MS has the sensitivity to detect the low concentrations of drugs present in this matrices Another advantage of LC MS is there is no need to derivatize samples which reduces sample preparation time However LC MS can be susceptible to matrix effects and this can vary between specimens this is a factor which must be considered during the validation of a method as this can affect the accuracy of the quantitation Gallardo et al 2009 Using G
59. increasing muscle and improving physical performance Illicit GHB is usually sold in small containers such as eye dropper bottles or vitamin bottles Mixing the colourless liquid with another drink often water or orange juice is a typical ingestion method GHB is sold in a variety of different concentrations and its chemical composition varies highly in its illicit form this makes it dangerous and difficult to dose correctly GHB and GBL could be purchased with ease on the internet before classification Some suppliers provided a pipette for accurate dosing for recreational use GBL is sold as a chemical cleaner marketed as a 99 99 solvent cleaner which cleans car wheels removes graffiti and glue The Misuse of drugs act 1971 was amended in 2009 to include GBL as a dangerous drug The Misuse of Drugs Act 1971 Amendment Order 2009 Since the amendment to the legislation in December 2009 some websites selling GBL under the pretence that they are selling a car cleaning product such as www alloycleaner com ceased selling the product in compliance with the law It is still possible to buy GBL from many websites as well as 1 4 Butanediol which is another GHB precursor that can also be converted into the drug When GHB is unavailable GBL or 1 4 Butanediol is sometimes consumed recreationally as the body will convert these substances to GHB so the same effect is achieved GBL is a chemical which comes with the warning Not for human consum
60. ing of 0 125g cocaine hydrochloride 0 125g benzocaine and 0 125g caffeine as benzocaine This means it matched the mixture spectra to the benzocaine spectra contained in its library only It did not report a match to the cocaine or caffeine spectra in its library Table 3 2 lists the peaks in the neat cocaine benzocaine and caffeine spectra and the peaks that occurred in each run of the mixture The lists allow the peaks to be compared easily and common peaks to cocaine and benzocaine can be identified quickly 59 Table 3 2 Cocaine benzocaine Caffeine mix run results Cocaine Benzocaine Caffeine Run 1 Run 2 Run 3 1716 24 1715 01 1715 66 1715 02 1698 99 1682 47 1682 50 1682 33 1682 24 1655 88 1604 67 1604 08 1602 77 1604 78 1598 75 1575 63 1574 81 1574 74 1574 99 S 1555 69 1458 58 1459 02 1462 91 1460 10 S 1447 40 1440 78 1447 30 z 1407 67 p 2 1368 74 1360 37 z 1370 88 1369 36 z 1328 63 z 1311 21 E 1310 91 1310 71 1311 17 1281 76 1285 37 1281 66 1280 81 1281 43 z 1241 25 s 5 2 1275 31 s z s z S 1203 71 s z 5 z 1172 14 1170 64 1170 77 1171 71 1111 33 1109 11 1112 09 60 1077 43 1071 81 1077 62 1000 72 1001 57 1000 90 1002 12 893 64 896 01 863 45 863 00 864 02 741 75 742 28 736 98 681 89 683 33 614 97 616 86 616 42 557 99 557 57 558 06 557 90 490 95 S 487 52 445 41 445 28 450 77 39
61. ique allows the structure to be determined non destructively which is huge advantage over mass spectroscopy however a larger amount specimen is required Isotopes of atoms can be studied using NMR spectroscopy NMR technology is invaluable in pharmaceutical quality control and assurance for structure identification and to check for impurities from formation processes or degradation Diercks et al 2001 A paper from 2011 discussed how NMR is an effective tool in the discovery of counterfeit drugs NMR technology was able to distinguish between genuine and counterfeit sildenifil In this case the packets and tablets look identical looking the Pfizer pharmaceutical logo but the tablet composition was different In a similar case a Chinese natural sexual enhancement product which claimed to be completely natural with no sildenifil was found to contain sildenifil through NMR technology Holzgrabe and Malet Martino 2011 Drugs in biological matrices are typically analysed using Gas Chromatography Mass Spectroscopy GC MS or Liquid Chromatography Mass Spectroscopy LC MS These instruments allow the compound to be separated 13 out by chromatography using gas or liquid as a carrier through a column The time it takes for a substance to elute from the column is the retention time and this gives an identifying feature to the substance The mass spectrometry MS part fragments the effluent ions in a reproducible pattern allowing the drug to be identif
62. ised to amphetamine which is then metabolised to benzoic acid Winger et al 2004 The metabolism of amphetamine is dependent on urine pH if the pH is unregulated around 15 of the drug is excreted in urine unchanged In acidic urine the drug is trapped as it is a basic compound its ability to be reabsorbed into the blood is reduced and clearance is more rapid than usual Alkaline urine therefore delays clearance from the body Users often take a substance that alkalises urine such as sodium bicarbonate to extend the effects of the drug The cytochrome P450 enzyme CYP2D6 is involved in the metabolism Wills 2005 25 1 4 3 MDMA 3 4 methylenedioxymethamphetamine MDMA was first synthesised by a German company known as Merck in 1913 Users may not always receive MDMA when they buy ecstasy it may be another psychedelic amphetamine such as MDEA or MDA These similar chemicals are reported to give very similar stimulant effects but it s MDMA which is associated with the feeling of empathy and warmth Repeated use of one of these chemicals results in the user becoming tolerant to their effects However there does not appear to be any cross tolerance between the chemicals therefore if a user is tolerant to MDMA they will still get a high if they take MDEA Saunders 1993 B u NN l MDMA Figure 1 5 Chemical Structure of MDMA Farquharson et al 2011 Ecstasy is almost always taken orally injecting the drug has been reported but is
63. k in if it is taken orally Wills 2005 Common effects users experience includes alertness self confidence very talkative impulsive and increased stamina The psychoactive effects of amphetamine usually last for around 4 hours the effects of methamphetamine can last for around 12 hours if snorted or taken orally Wills 2005 Smoking the crystals of methamphetamine is the purest form of the drug The same crystals can be reheated several times and will still produce the same high due to their high melting point Wills 2005 A brownish yellow powder with a waxy appearance is more common form of methamphetamine This appearance is caused by impurities Wills 2005 23 Amphetamines main mechanism of action is to stimulate the release of certain neurotransmitters heightening their natural effect See Fig 1 4 Amphetamines act as indirect sympathomimetics Amphetamine enters the nerve by the noradrenaline NA transporter and then into the synaptic vesicles by the vesicular monoamine transporter in exchange for Noradrenaline which gathers in the cytosol Monoamine oxidase MAO degrades some of the NA within the cell and some is released from the cell via the NA transporter in exchange for amphetamine The realised NA acts on the postsynaptic receptors the action of realised Na is enhanced as amphetamine also reduces NA reuptake Rang and Dale 2000 Amphetamine Amphetamine Figure 1 4 Mechanism of action for Amphetamine
64. l state hunger and drug use Saliva flow ranges from 0 to 6 43 mL per minute Oral fluid can be tested to detect recent drug use This is very relevant for testing motorists as only recent drug use is of interest Oral fluid will not replace the need for urine drug testing where in cases a more historic view is required or hair drug testing in cases where a long term picture of drug use is required Specimens collected by expectoration and by placing absorbants in the oral cavity are defined as oral fluid specimens Jickells and Nergrusz 2008 1 7 1 ROSITA The first Roadside Testing Assessment ROSITA discovered that oral fluid was the most promising alternative specimen compared to sweat and urine for a roadside drug screen Urine testing gave satisfactory results as overall accuracy was over 95 and sensitivity and specificity was over 90 compared with a reference method but no device scored highly for all drug categories Urine testing would require facilities such as a sanitary van to be able to take the sample at the roadside Most countries who took part in ROSITA preferred Oral fluid testing with only One country favouring urine and one country favouring sweat Verstraete and Raes 2006 ROSITA 2 concentrated on oral fluid only It states for a test to be fit for use it must be 95 accurate and over 90 sensitive and specific All 9 devices tested in ROSITA 2 fell short of this and a very high number of failures were reported wi
65. lass B drug can result in a prison sentence of up to 14 years and an unlimited fine Drugs controlled under Class C include Benzodiazepines Ketamine and GHB Possession of a Class C drug can result in the maximum of two years imprisonment and an unlimited fine Supply of a Class C drug can result in a maximum of 14 years imprisonment and unlimited fine The Misuse of Drugs Act 1971 Any company who needs exemption from these laws such has a university carrying out drug research can apply for a Home Office domestic licence This study was conducted under this licence Home Office 2012 17 1 3 2 Misuse of Drug Regulations 2001 The Misuse of Drug Regulations 2001 divides illicit drugs into five schedules which reflect their medicinal use and their potential for misuse Schedule 1 includes the drugs cannabis ecstasy LSD and raw opium Schedule 1 drugs are not authorised for any medicinal use and should only be possessed supplied and administered under a Home Office licence Schedule 1 drugs have a high potential for abuse Schedule 2 drugs have a medicinal use but a possession is only legal with a prescription Strict storage requirements and recordkeeping must be employed with these drugs in circumstances such as a hospital setting Schedule 2 drugs include morphine cocaine amphetamines and dihydrocodeine The potential for abuse is still high Schedule 3 drugs are not required to be kept under strict storage and a register is n
66. lass saucepan Do not use aluminum cookware to make GHB 2 Place the content of the NaOH or KOH bottle in the same saucepan 3 Put SLOWLY around a half cup of warm distilled water in it Put a cover fast the reaction may be immediate on it but not tight 4 Wait a little it will start reacting on itself If it doesn t after 2 3 minutes heat it a LITTLE once it reacts remove it from the stove 4 5 If there is some NaOH not dissolved stir it up till it is 5 This step is optional some like it like that and others prefer to heat the solution a little After it s finished Start heating it slowly You will see it starting boiling Don t overheat It can burn Do it for one hour Don t forget to add water if you make it boil for a long time 5 5 Between step 4 and 6 you might see a white compound on the side of the saucepan it doesn t happen everytime Don t throw it away it s GHB When you will add water it will dissolve 6 When you are finished put it in a measuring cup Pyrex and fill it with water when I m in a hurry to taste it use ice to 1000 mL a little more than 4 cups That way you ll have around 1 grams per teaspoon 91 7 Measure the PH If it s higher than 7 5 add vinegar to lower it to below 7 5 It can take 50 75 mL of vinegar 9 To store it use a mason glass jar with a plastic cover draw the poison logo on it very important you don t want a kid to take a full glass of GHB place
67. mes and it is thought that this may mean they are at greater risk of an ecstasy related death should they take the drug Gilhooly and Daly 2001 The half live of MDMA is around 8 hours so elimination from the blood is relatively slow as it takes 5 half lives for a drug to be 95 elimination from the body therefore it takes around 40 hours for the drug to be eliminated Users report some effects the day after which may be due to active metabolites such as MDA Kalant 2001 1 4 4 Ketamine Ketamine is structurally related to phencyclidine and gives the same anaesthetic and analgesic effects without causing cardiac or respiratory depression Ketamine is used in human and veterinary medicine when sold illicitly it has usually come from a diverted legitimate supply or theft of a legitimate supply Commercial ketamine is a racemic mixture composed of both R and S isomers S ketamine has four times the affinity for the NDMA receptor than R ketamine It also binds to the opioid receptors Mu and Kappa Weiner et al 2000 Ketamine is N Methyl D aspartate NMDA receptor antagonist The NMDA receptor allows the transfer of electrical signals from the brain and the spinal cord Glutamate and Glycine are the substrates required by the the receptor to open the channel to allow transfer Ketamine is a non competitive antagonist which binds to allosteric sites blocking the ion channel 27 Cl NH Hc 0 Figure 1 6 Chemical Structu
68. mphelamines ass 23 14 2 1 MetaboliSM T t 25 Eqs oe CAFE RE E O E 26 LES Melab len an 27 144 Ketam a a ie a e aE 27 1 4 4 1 Metabolism ee 28 1 4 5 Gamma Hydroxybutyric Acid 29 1 5 Raman Spectroscopy EE 32 1 5 1 Basic theory of Raman Spectroscopy sse 32 1 5 2 Advantages of Raman Gpechoscop eene 34 1 5 3 Disadvantages of Raman Spectroscopy see 34 1 3 5 Previous Research anne 35 1 5 6 Previous research by the Author 39 1 6 Ta a BA KGOUNA ett rH 41 1 6 1 How TruScan workS ri e AS EA AEEA AEAEE 42 1 7 el DEE 43 Prope 44 1 7 2 Advantages Of oral fluid WE 44 1 7 3 Disadvantages of oral fluid EE 45 nn 46 DO PT Gozo ae Aig OG ie PURI NETS 47 Chapter 2 Materials and Method nana annnnnanna 48 2 1 Mailera EE 48 2 2 Instrument use keine 48 2 3 Greating a BIDEA EE 50 2 4 Production of liquid GHB ER Ro b WE 50 25 En Ee EE 51 2 7 Oral Fluid Analys s aussi ea 52 2 8 SERS sample preparation and method AA 53 Chapter E 54 El Le a EBIA E 54 32 SC og Ee 4 6 EE 55 3 3 MIX U E ANA SIS eege eegene 55 34 Ora GIO Analysis meet 69 3 5 Limit of Detection of KGHB in Oral Fluid 71 3 6 SERS and Oral fluid analysis route essc ttt cep ep he AA ape i PEERS 73 ET DISCUSSION EE 76 AZ B ori n ET EUN 76 2 3 I Cable in A SON OL aote cce ete bud attese eA ae andthe haben ayes 77 4 4 Mixture analysis tse ee Te sort t
69. orno ease tpi rere obese deme 78 45 Oral lid analysis ero ke 80 4 6 Limit of detection in oral fluid a ette te tette ttt ehe 80 4 7 SERS and oral fluid analysis EE 81 4 8 Further Research a ak eer EE dte ete ue lavet pai t feli eeu 81 Chapter 5 GoOneluslOfk auc tii eq ee 83 Chapter 6 Beferencs coser A WIO reae pide EES 85 Appendix 1 GHB Recipes obtained from the internet A 91 Appendix 2 TruScan Standard Operating procedures 93 Appendix 3 Reference Spectra essent nnn 97 12 Chapter 1 Introduction 1 1 The identification of drugs Many different scientific techniques have been applied to the identification of drugs from simple colour change reactions to the use of sophisticated analytical instruments Identification techniques can be presumptive or confirmatory and can quantify the concentration or be qualitative An example of a simple spot test is the Marquis reagent a mixture of formaldehyde and concentrated sulphuric acid which results in a colour change indicative of a drug when it comes into contact with a substance This is a presumptive test which is not very specific different drugs may produce similar colour changes Jickells and Nergrusz 2008 Nuclear magnetic resonance spectroscopy NMR can be used for bulk drug analysis NMR is a technique which is based on the physical phenomenon where a magnetic field causes a nuclei to absorb and re emit electromagnetic radiation This techn
70. ot required Schedule 3 drugs include barbiturates and temazepam Schedule 4 is divided into two parts part 1 includes most of the benzodiazepines Possession is legal under prescription but supply is illegal Part 2 includes anabolic steroids which can be possessed for medicinal purposes without a prescription however supply to others is illegal Schedule 5 includes over the counter drugs such as cough medicines and mild painkillers The risk of misuse is reduced with these drugs as any controlled substance used in their preparation is at a low level Misuse of Drugs Regulations 2001 1 3 3 The Medicines Act 1968 The Medicines Act 1968 controls the distribution of medicines Medicines can be prescription only and therefore supplied by a pharmacist only when in receipt of a doctor s prescription Pharmacy medicines can be bought from a pharmacist without a prescription and general sales medicines can be bought from a variety of shops with no need for prescription or pharmacist These are called over the counter medicines o t c The Medicines Act 1968 18 1 3 4 Drug Harm and the law In 2010 a study carried out by Nutt et a was published on drug harm in the UK This study was carried out by an interactive workshop attended by the members of the Independent Scientific Committee The participants were asked to score 20 drugs on 16 harm related criteria Nine criteria related to how the drug affects the user and seven on the harm it causes
71. ov A 2010 Scottish Crime and Justice Survey 2008 9 Drug Use Scottish Government Social Research McBride R 2010 Thermo Fishers 145M buyout of Ahura Scientific good news for Arch Castile and other venture backers www xconomy com Accessed 17 11 11 Misuse of Drugs Act 1971 Amendment Order 2009 http www opsi gov uk si si2009 draft ukdsi 9780111486610 en 1 accessed 26 01 2011 Misuse of Drugs Regulations 2001 http www legislation gov uk uksi 2001 3998 introduction made accessed 3 10 11 Myant K Hope S Mcintosh J O Brien T McKeganey N Stradling S 2006 Illicit drugs and driving Scottish Executive Social Research Nicholson K L amp Balster R L 2001 GHB a new and novel drug of abuse Drug and alcohol dependence 63 1 1 22 88 North P 2010 Report of the Review of drink and drug driving law Great Britain Department for transport Nutt D J King L A 8 Phillips L D 2010 Drug harms in the UK a multicriteria decision analysis The Lancet 376 9752 1558 1565 O Connor L 2010 The detection of GHB and GBL in alcohol using Raman Spectroscopy Edinburgh Napier University Unpublished Rang H P Dale M M Ritter J M amp Moore P K 2003 Pharmacology New York Churchill Livingstone Ravna A W Sylte l Kristiansen K 8 Dahl S G 2006 Putative drug binding conformations of monoamine transporters Bioorganic amp medicinal chemistry 14 3 6
72. portant in obtaining good quality spectra this study used a probe and it was found that the focalisation varied from drug to drug therefore a standard distance from the container was not adopted The probe was pressed against the container then moved gradually away from it until optimal distance was found Weyermann et al 2011 Burnett et al conducted a study into the concealment of cocaine in bottles of Rum for smuggling purposes It was noted that confiscated samples typical contain between 50 80 cocaine w v This study used a portable 785nm Raman as well as a 1064nm bench top instrument The glass of the bottles had two chances to react with the radiation as the beam must travel through the glass to the sample then back through again to the detector doubling the opportunity to affect the spectra The colour of the glass also affects the spectra green glass will fluoresce at 785nm 37 The study showed that colourless and brown glass give good results down to a concentration of 6 w v Plastic bottles had the same result Green glass produced a spectra which completely masked the cocaine peaks on the 785nm instrument however the FT Raman gave clear cocaine peaks down to 6 w v The study concluded that cocaine in concentrations of 8 w v and above in rum can be identified and therefore concentrations of 50 80 should present no problem Burnett et a 2011 The detection of drugs in drinks is also very useful in the prevention or evi
73. ption Once GBL is purchased illicit GHB can be synthesised in a clandestine laboratory using Potassium or Sodium Hydroxide Recipes to make GHB can be found on the internet be doing a simple Google search GHB is a central nervous system CNS depressant in high doses its actions are similar to the hypnotics Benzodiazepines In combination with alcohol another CNS depressant the sedative effect is exacerbated GHB also causes relaxation of the voluntary muscles Nicholson and Balster 2001 30 GHB is rapidly absorbed from the gastrointestinal tract and the intoxicating effects can be observed in around 5 to 15 minutes after a dose Drummer 2001 Peak plasma concentrations are reached around 30 45 minutes after administration Drummer 2001 The half life of a dose of GHB is around 27minutes Li et al 1998 One half life is the amount of time it takes for the drug to degrade by a half Five half lives equal 97 elimination of the drug from the body Jickells and Nergrusz 2008 This means that it is theoretically possible for all trace of the drug to have left the body 2 hours 15 minutes after administration GHB is oxidised by hepatic enzymes to succinic semialdehyde and then succinic acid this is able to enter the Krebs cycle and therefore the ultimate end product of metabolism is carbon dioxide and water Elimination occurs mainly through the lungs in the form of carbon dioxide however it is a urine or blood sample which is
74. rare Ecstasy is supplied in tablet form which are often embossed with logos Wills 2005 MDMA increases the secretion and reuptake of the serotonin dopamine and nor adrenaline in the brain causing feelings of euphoria and increased energy MDMA can cause the users judgement to be impaired which results in dangerous behaviour Dehydration hypertension and hyperthermia are short term health risks associated with using MDMA in the long term permanent disruption of serotonin in the CNS can lead to depression Jickells and Negrusz 2008 A placebo controlled study using human volunteers showed that MDMA indirectly stimulates the hormone Oxytocin through agonising the 5HT A receptor Oxytocin is the hormone naturally produced after hugging and childbirth it facilitates bonding and trust This is the reason users feel connected and warmth towards others Dumont et a 2009 26 1 4 3 1 Metabolism MDMA is absorbed from the gut and reaches peak plasma concentration around 2 hours after oral administration It is mainly metabolised by the liver mainly using the enzyme CYP2D6 Several other enzymes are involved in the metabolism however these appear to be saturated at fairly low concentrations the higher the dose results in the higher affinity becoming saturated This means that the risk of toxicity increases greatly as the dose is increased just slightly Kalant 2001 Around 5 10 of the Caucasian population are deficient in the CYP2D6 enzy
75. re of Ketamine Stafford 1992 Ketamine is a short acting drug and the hallucinatory experience sought after will only last around 2 hours when ingested and around one hour if snorted or injected The bioavailability of IV IM nasal and oral administration are 90 90 50 and 20 respectively Stafford 1992 At low doses the user may feel dissociative effects such as outer body experiences at higher doses 60 125 mg IM 100 250 mg insufflated users can experience vivid hallucinations memory loss and mimic the symptoms of schizophrenia These effects are known as the K Hole Curran and Monaghan 2001 It has been suggested that there are six broad categories of experiences caused by ketamine abuse based on reports from users This are 1 Dissociation such as out of body or near death experiences 2 Entry into information networks 3 The ability to enter alternative realities 4 The ability to communicate with aliens 5 Enhancement of sexual experiences 6 Creative and personal problem solving skills increased Stafford 1992 1 4 4 1 Metabolism Ketamine is mostly metabolised through N demethylation to norketamine which is an active metabolite CYP2B6 in the liver is major route of clearance although other routes are involved 9096 of the dose is cleared from urine within 5 days Methadone and diclofenac inhibit the conversation of Ketamine to norketamine 28 as they are CYP2B6 and CYP2C9 substrates 90 of Ketamine is excre
76. ride form is generally used as it is more soluble than crack Wills 2005 Cocaine is not commonly taken orally as the onset of effects is slow and produces a low blood concentration opposed to other routes of administration this may be due to first pass metabolism to ecgonine methyl ester EME Drummer 2001 Smoking and injecting produces similar effects the onset time is quick but peaks within minutes Oral and insufflation take longer to peak oral may take up to an hour and insufflation 15 30 minutes Wills 2005 By inhibiting sodium influx into the cells cocaine acts as a local aesthetic this is likely responsible for the vasodilatory action seen with cocaine use Cocaethylene is formed when cocaine and alcohol are consumed at the same time it is estimated that around half of users consume both together Cocaethylene is a more potent sodium 20 channel blocker than cocaine and the effects of this may cause sudden death Karch 2008 The reuptake of dopamine noradrenaline and serotonin is inhibited by cocaine at the nerve synapses thereby prolonging their effects The behavioural effects of cocaine are more associated with the accumulation of dopamine then the other two neurotransmitters Cocaine blocks the dopamine transporter DAT this is located along the presynaptic nerve terminal walls When the DAT is blocked the rapid termination of the effects of dopamine are prevented and the release of dopamine is increased in the synaptic
77. rugs and therefore no further work using this device would be recommended Raman Spectroscopy could be a potential drug detection device as it has many favourable properties Further research on other Raman devices may be of value Other devices may have the sensitivity required and prove promising as a first screening technique before destructive testing is carried out A method of detecting drugs of abuse in saliva using SERS has already been discussed in a published article however the article was vague and difficult to understand how the method could be reproduced However Real Time Analyzers Raman manufacturers who published the article claim to have a successful technique which could be promising If this research could be replicated independently and successfully this would be a great step forward 81 The SERS area of this research could be replicated using commercially available SERS kits which are available from companies such as Thermo Scientific These kits would possibly be more reactive with the drug molecules and produce indentifying Raman peaks This could be further improved upon by using a different Raman Spectrometer which allows the operator to focus the laser beam precisely 82 Chapter 5 Conclusion The study concluded that TruScan is able to identify a pure drug i e not a powder containing a percentage of an illegal drug when reference spectra have been saved in the TruScan library Creating a library is ver
78. s z 652 48 z m 66 616 46 616 80 616 53 557 62 555 96 557 34 490 95 s s z i 466 46 468 66 E 402 10 317 53 315 16 317 33 317 84 318 07 2 22495 E z 153 12 15128 67 86 LSL 80 vL 62 607 Dee DE 126 98 LOOL OE STOL SE P OL 08 60LL B EELL SCH I 86 SOZI LL 96CV JE 1821 nord CH 896L SL SGL SG opt KI 68 F GL 6 E09L i GOSLEH 5 E ZS 3 E 5 Z z S s 8 2 3 g s 2 z E 8 S R 1000 D Aysuaqul uewe 1000 05 SEI LOL LE G EL 86 Or ER SLB6SL POSLI AA Cocaine HCI 2 Asuaju uewey estes lcor 91906 89219 Sc Kei 96218 50 Pop EE HL eni OZ 1821 IZ MEN be 8961 t App EIGISL 19 P0391 iv Mies Benzocaine 40000 7 D Aysuaqul uewey 97891 56 rze DUGLE 80 696 Ergi Lr spr SP Per 66155 A SL Ga S IP SEO E ke ZB zzo IB LOL SC MCI LE GBZL EIBZEL LE 0961 rege 89 onak 895591 668691 j 60000 Caffeine Signature 2 8 8 8 S R Aysuayul uewe BL 862 L era 11 696 6c 2101 BE SOLL c6 83117 LE LECI E9 9921 PE BL EL GZEL BO ZIEL L opp 6SSISL 862951 6VELSL 8 691 wm LAcetaminophen 20000 Aysuaqul uewey 1400 1200 1000 Raman shift cm 1 1600 1800 1 compared IX d paracetamol m
79. s and give the sample an appropriate name Connect the TruScan device to the PC using the CF Ethernet adapter and cable The adapter fits in to the card slot in the battery compartment at the bottom of the device The cable fits into this and a USB port on the PC Once the device is connected Open the Web Admin Utility which is labelled New Internet Shortcut on the PC desktop Log in to this program the same way as logging into TruScan device see above Select Method Management from the list then select Add New Method Enter a name of the Method into the Method name field and click Enabled in Status field Highlight the corresponding Signature in the Unattached Signatures window and click the arrow between the two windows to attach Signature to the Method 94 Click Save Changes It is now possible to run a sample against this Method e Method Editor Windows Internet Explorer E t3 oo v le http ts1403 methodedit asp vie Favorites Method Editor TruScan Administration Serial TS1403 On Device Administrator Home Method Management Edit Method Method name Menthol 10000106 Barcode Field 10000106 SamplelD Prefix 10000106 lot Info Files none Manage Status enabled e Signatures in Method Unattached Signatures Menthol U1869 Save changes Figure 2 4 Method Management on PC 2 2 Running a Sample Press
80. s is probably a ring structure GHB does not have a ring in it structure but GBL does 1 6 TruScan Background This study used a portable Raman Spectrometer called TruScan This device was designed by a company called Ahura Scientific which Thermo Fisher Scientific bought over in 2010 McBride 2010 www xconomy com The user manual produced by Ahura Scientific claims the instrument is light and portable as the unit weighs less than 4lbs rugged and can be used in the field sampling is easy as non contact analysis is possible which improves safety and reduces contamination of the sample method development is fast and the device can easily be used by non technical staff TruScan User Manual 2010 Ahura Scientific and now Thermo Fisher Scientific claim that with TruScan and TruScan products No matter how complex your raw material identification challenge we put the solution in the palm of your hand This sounds promising but as illegal drugs are a mixture of a variety of substances adulterants and 41 cutting agents the device would need to be able to identify the illicit drug in these mixtures if it was to be used in drug identification based on its PASS FAIL system which allows non technical users to use the instrument 1 6 1 How TruScan works TruScan shines a 785nm laser on the sample to receive a Raman measurement The software which analyses the spectra and gives the PASS FAIL result is a patent pending package called Decision
81. supernatant instead of the pellet after centrifugation was also attempted For the Truscan run different methods attempted included drying the sample on a glass slide at room temperature pipetting the sample into a capillary tube and focussing the laser down the length of the capillary tube or focussing it through glass of the capillary tube The glass slide and capillary tube gave the same large glass hump with no peaks present in the spectra so were not suitable 53 Chapter 3 Results Raman Spectroscopy causes vibrations which produce spectra highlighting the covalent bonds and the structural carbon elements of the molecule This can be used to give a chemical fingerprint of the molecule Comparing a spectrum to reference spectra is how a substance is identified The numbers assigned to each peak assists in the identification 3 1 Creating a Library The library of chosen drugs was successfully created this was confirmed by comparing all spectra to spectra in the literature Weyermann et al attributed peaks at 1003cm and 1022 cm to cocaine which corresponds to the double peak in the spectrum in figure 3 1 The reference spectra s or Methods were all saved successfully See appendix 2 for reference spectra for all drugs used in this study including details of the corresponding peaks in mentioned in the literature 869 57 Raman intensity 1716 24 1598 75 1000 72 817 61 616 15 1203 71 1023 81 730 49 585 52
82. t cm 1 Figure A3 Benzocaine Solid 85000 4 18 80000 6 87 75000 70000 N 65000 soon i 55000 50000 45000 Raman intensity 557 99 40000 35000 1328 63 30000 25000 484 45 1698 99 1285 37 20000 1071 81 369 28 15000 10000 5000 4 0 mr 2800 2500 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 Raman shift cm 1 Figure A4 Caffeine Solid In literature characteristic caffeine peaks are 1327 cm and 555 cm Kang et al 2011 98 i 869 57 a RI fej o 5 817 61 a i Raman intensity 1800 1600 1400 1200 1000 800 600 Raman shift cm 1 Figure A5 Cocaine Hydrochloride Solid In literature characteristic cocaine peaks are 1022 cm 1003 cm and 869 cm Weyermann et al 2011 Ketamine Hydrochloride Sig 90000 85000 80000 75000 204 33 70000 4 458 64 85000 152 84 60000 55000 1044 90 326 66 172 52 50000 441 91 45000 Raman intensity 40000 4 656 43 35000 4 1157 24 1090 43 1022 98 526 45 414 65 358 65 290 48 a A m 5 e E 5 30000 4 25000 2 20000 4 389 08 2 1450 16 15000 3 1722 91 10000 4 5000 4 1600 1400 1200 1000 800 600 400 200 Raman shift cm 1 Figure A6 Ketamine Hydrochloride Solid 99 821
83. ted in the urine Curran and Monaghan 2001 1 4 5 Gamma Hydroxybutyric Acid Gamma Hydroxybutyric acid or GHB C4HaO3 is a short chain carboxylic acid which occurs naturally in the mammalian body See Figure1 7 It is soluble in water and is formed from gamma butryolactone GBL It is also known as liquid ecstasy GHB has no medicinal use except when found in Xyrem which is a treatment for narcolepsy Doses of 6 to 9 g are recommended nightly for the treatment of narcolepsy This dose is not taken all at once however Winger et al 2004 The lack of hangover effects is a property which makes GHB an attractive recreational drug GHB is the precursor to the gamma aminobutyric acid GABA neurotransmitter which is an inhibitory neurotransmitter which promotes sleep and relaxation Bennet and Steiner 2009 O PER a HO m CH CH CH C cn HO O Figure 1 7 The chemical structure of gamma hydroxybutyric acid Brewster et al 2008 GHB can cause mild euphoria increased sensuality lower inhibitions cause memory loss and lack of consciousness Bennet and Steiner 2009 These effects are dose dependent A low dose which is defined as between 0 5 and 1 gram gives these desirable effects and has led to the drug being taken recreationally Higher doses between 2 5 and 4 grams give the sedative effect desired for its use in DFSA Brewster et a 2008 29 GHB was initially sold in health food stores for the purpose of burning fat
84. terminal The mesolimbic dopamine system area responsible for memory and emotion is the part of the brain cocaine effects in particular This is what causes the feeling of euphoria Wills 2005 Winger et al 2004 When cocaine is administered locally the conduction of the axon potential is prevented as sodium channels on the neuron membrane are blocked by the drug The vasoconstriction effect is due to the increasing levels of norepinephrine which acts on the alpha adrenergic receptors on the blood vessels Winger et a 2004 Thickening of the heart muscle or myocardial hypertrophy is associated with stimulant drug use Coronary artery reserves decline as ventricular mass increases leading to impaired myocardial contractility this means the heart is under strain and this may also be responsible for sudden cocaine related death Karch 2008 The left ventricles are larger in laboratory rats and rabbits that are treated with cocaine and the hearts of cocaine users are heavier than those of controls by around 10 Cocaine use can also cause coronary atherosclerosis and thrombosis Karch 2008 1 4 1 1 Metabolism The methyl ester of cocaine is hydrolysed chemically and by enzymes to produce the primary enzyme detected in blood and urine after use benzoylecgonine In 2005 the River Po in Italy was tested for benzolylecgonine in order to determine if any had polluted the river by getting into the sewage system via users urine The river has
85. th some devices This may have due to the viscosity of the saliva tested or the malfunctioning of the device Verstraete and Raes 2006 1 7 2 Advantages of oral fluid A huge advantage of oral fluid drug testing is that it is quick and non invasive Drummer 2006 Only small samples are required and can be analysed by LC MS MS this adds the high sensitivity and specificity of a mass spectrometer to the liquid chromatograph This technique is a confirmatory technique but can also be used as a preliminary test for drugs Drummer 2006 This includes kits that can be used for onsite drug testing Drug testing at the side of the road allows police offers to confidently arrest those driving under the influence and 44 reduces expensive laboratory analysis This also eliminates whose who are not under the influence with minimal inconvenience Verstraete 2005 Another advantage of oral fluid is there is a relationship between oral fluid concentration and blood plasma concentration This can be calculated using the pH of the oral fluid and blood the pKa of the drug and the protein binding of drug The equilibrium favours blood for the acidic drugs and favours oral fluid of basic drugs Drummer 2001 Theoretical ranges for some drugs have been calculated For example the saliva plasma for cocaine varies from 2 73 0 44 as saliva pH varies from 5 0 7 8 Jickells and Nergrusz 2008 Oral fluid is collected under direct supervision so this elimin
86. the latter It must be emphasised that this study only attempted to consider the device as a presumptive screening device and does not intrude on the confirmatory drug testing domain which includes instruments such as GC MS The devices ability to detect KGHB in alcohol was investigated by spiking ethanol then analysing This offered a brief comparison of the TruScan and DXR bench top Raman instrument Bulk samples of mixed powder were produced to replicate street drugs in order to establish the devices ability to detect drugs in a mixture Blank oral fluid was spiked in order to establish the devices ability to detect drugs in saliva SERS analysis was also attempted on spiked oral fluid samples It was found that the TruScan device was not effective in detecting drugs in alcohol or mixtures but it was able to detect KGHB in oral fluid down to a 30 v v concentration A successful SERS method was not established for the oral fluid analysis Contents Chapter 1 Introduction E 13 1 1 The identification of drugs une EEG GREGA ai 13 12 1 Eege e RI 15 1 2 2 Prevalence of drug use and driving 16 1 3 LOGS uai D e 16 1 3 1 Misuse of Drugs Act 1971 m 16 1 3 2 Misuse of Drug Regulations 2001 aa GE E HERE QE UC AE bU VEU RS 18 1 3 3 The Medicines TIME 18 1 3 4 Drug Harm and KT 19 1 4 Common Illicit Drugs ua ee 19 14 1 ea O ENE A O RR DANA 19 1 4 1 1 Metabo 21 1 4 2 A
87. ules Journal of the American Chemical Society 131 40 14466 14472 Brewster V Edwards H G Hargreaves M D amp Munshi T 2009 Identification of the date rape drug GHB and its precursor GBL by Raman spectroscopy Drug testing and analysis 1 1 25 31 Burnett A D Edwards H G M Hargreaves M D Munshi T amp Page K 2011 A forensic case study the detection of contraband drugs in carrier solutions by Raman spectroscopy Drug testing and analysis 3 9 539 543 Carter J C Brewer W E amp Angel S M 2000 Raman Spectroscopy for the in Situ Identification of Cocaine and Selected Adulterants Applied Spectroscopy 54 12 1876 1881 Ciolino L A Mesmer M Z Satzger R D Machal A C McCauley H A amp Mohrhaus A S 2001 The chemical interconversion of GHB and GBL forensic issues and implications Journal of forensic sciences 46 6 1315 1323 Curran H V amp Monaghan L 2001 In and out of the K hole a comparison of the acute and residual effects of ketamine in frequent and infrequent ketamine users Addiction 96 5 749 760 Daly M 2010 Drug gangs boost cocaine purity to compete with internet highs Drugscope 25 155 156 Day J S Edwards H G Dobrowski S A amp Voice A M 2004 The detection of drugs of abuse in fingerprints using Raman spectroscopy latent fingerprints Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy 60 3 5
88. urns D T Dennis A C amp Speers J S 2000 Rapid analysis of ecstasy and related phenethylamines in seized tablets by Raman spectroscopy Analyst 125 3 541 544 Bell S E Burns D T Dennis A C Matchett L J amp Speers J S 2000 Composition profiling of seized ecstasy tablets by Raman spectroscopy Analyst 125 10 1811 1815 Bell S E J Beattie J R McGarvey J J Peters K L Sirimunthu N M S Speers S J 2004 Development of sampling methods for Raman analysis of solid dosage forms of therapeutic and illicit drugs Journal of Raman Spectroscopy 35 409 417 Bell S E J Beattie J R McGarvey J J Peters K L Sirimuthu N M S and Speers S J 2004 Development of sampling methods for Raman analysis of solid dosage forms of therapeutic and illicit drugs Journal of Raman Spectroscopy 35 409 417 Bell S 2006 Forensic Chemistry New Jersey Pearson Prentice Hall Bell S E Fido L A Sirimuthu N Speers S J Peters K L amp Cosbey S H 2007 Screening Tablets for DOB Using Surface Enhanced Raman Spectroscopy Journal of forensic sciences 52 5 1063 1067 85 Bennett M J amp Steiner R R 2009 Detection of Gamma Hydroxybutyric Acid in Various Drink Matrices via AccuTOF DART Journal of forensic sciences 54 2 370 375 Blackie E J Ru E C L amp Etchegoin P G 2009 Single molecule surface enhanced Raman spectroscopy of nonresonant molec
89. used to detect the presence of GHB Li et a 1998 1 5 of GHB is excreted unchanged in the urine If the urine is particularly acidic less will be excreted Hornfeldt et a 2002 Endogenous GHB which is GHB which is naturally present in the body is found in the serum of the human body unbound to protein The basal ganglia is where the greatest concentration of endogenous GHB is found in the human body Binding sites are found in several parts of the brain including the cortex midbrain and the hippocampus Areas such the cerebellum and medulla do not contain binding sites Li et a 1998 31 1 5 Raman Spectroscopy Raman Spectroscopy is a type of vibrational spectroscopy used to determine information on chemical structure of a substance it is used as an identification tool and can be used to determine quantitatively or semi quantitatively the amount of a particular substance in a sample Smith and Dent 2005 Raman Spectroscopy is complimentary to IR spectroscopy in the sense that Raman highlights the covalent bonds and structural carbon elements and IR highlights the functional groups West and Went 2010 A Raman spectrum is obtained by focussing a laser of monochromatic light on the area of the sample that is being analysed Monochromatic light refers to light of a single wavelength Bell 2006 Unlike Infrared and ultraviolet spectroscopy which uses absorbed and transmitted radiation Raman Spectroscopy uses scattered light in or
90. y time consuming KGHB which is easily indentified using the 930cm peak in an ethanol spectra on the DXR cannot be identified on the TruScan The TruScan is therefore less sensitive and less favourable than other Raman spectrometers KGHB was the only drug tested that was identified in oral fluid using the TruScan However the concentration was high around 75 times higher than an optimum cut off level for a screening device i e 300 mg L compared to 4 mg L Applying this to a real life situation the oral fluid would have to be tested around 20 minutes after a high dose of KGHB had been administrated GHB is quickly eliminated from the body and the TruScan is not sensitive enough to detect GHB in oral fluid down to the ng level With regard to mixture analysis it appears TruScan will report the substance in a mixture with the most intense peaks as a positive Peak intensity is favoured over occurrences of common peaks The SERS method employed in this study only gave peaks for KGHB It did not give peaks for any of the other drugs tested The peak numbers were not peak numbers usually associated with GHB however The exact same method was carried out for all drugs The police would not be able to use the TruScan device for drug screening or roadside testing of oral fluid for several reasons The device is not able to identify even high concentrations of drugs in oral fluid with the exception of KGHB even this is limited as it requir

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