Q The project will be focused to scrutinise any observable changes in optical properties of alcoholic drinks Home, - Optical properties of different alcoholic beverages spiked with Gamma-hydroxybutyric acid INTRODUCTION Brief summary The project will be focused to scrutinise any observable changes in optical properties of alcoholic drinks mixed with a specific quantity of Gamma-hydroxybutyric acid. The Drug facilitated rape is a crime which is performed by spiking victim’s drink with any anaesthetic drug or substance. GBH is one the commonly used date rape drug due to characteristics such as colourless, odourless and tasteless. The subject of drug facilitated rape using GBH is very significant in forensics as it involves the use of illicit drug which can be measured only forensically by using analytical methods. The mainly analysed alcoholic beverages will be wines, beers and spirits as these are the most popular drinks among females in UK. The main optical feature analysed in this project will be absorbance by using Fluorescence spectrometer as detection instrument and High-Performance Liquid Chromatograph coupled with Mass Spectrometer (HPLC-MS) as a confirmatory analytical device. To quantify the amount of GHB added in each drink sample, a calibration curve will be plotted for each sample. All the absorbance values will be tabulated and calibration curves will be presented graphically. All data will be treated statistically by using arithmetic mean, standard deviation and % relative standard deviation. Research question Do the optical properties, such as absorbance, of alcoholic beverages alter if they are mixed by using GBH? Hypothesis Null Hypothesis (H_0): There is no observed change in optical properties of alcoholic beverages spiked with Gamma-hydroxybutyric acid (GHB). Alternative Hypothesis(H_1): There is an observable change in optical properties of different alcoholic drinks mixed with Gamma-hydroxybutyric acid (GHB). Aim of the Project The project will be oriented to achieve main 5 aims which are mentioned below: Firstly, the main aim of the project will be to examine the optical properties i.e. absorbance of alcoholic drinks spiked with Gamma-hydroxybutyric acid. Second aim will be to find the detectable amount of Gamma-hydroxybutyric in spiked drinks and to generate absorbance vs. concentration calibration curve. Another aim will be to compare the absorbance readings of each standard alcoholic drink sample with corresponding alcoholic sample spiked with GHB. Also, to evaluate the data and find that if any other components of the alcoholic drinks interfered with the fluorescence properties of GHB. Finally, the GHB added in alcoholic samples will be confirmed and quantified by using High Performance Liquid Chromatography coupled with Mass Spectrometer. Objectives: To achieve the aims of project following objectives will be followed: In order to examine absorbance properties of alcoholic drinks spiked with Gamma-hydroxybutyric acid, fluorescence spectrometer will be used to find the absorbance readings. By using different concentrations of GHB, least detectable amount of GHB in fluorescence spectrometer and HPLC-MS will be calculated. The absorbance readings and the concentration values of GHB added in alcoholic drinks will be used to construct the calibration curve for both fluorescence spectrometer and HPLC-MS. At least three absorbance readings will be taken for all samples to ensure the accuracy, precision and to get enough data which will be compared statistically. The absorbance readings will be compared by comparing the graphs of absorbance for each standard alcoholic beverage corresponding to spiked alcoholic drink with GHB. The samples will be applied to HPLC coupled with mass spectrometry to confirm the presence of the GHB in samples and to quantify GHB. For data evaluation, statistics used will be arithmetic mean, standard deviation and % relative standard deviation. BACKGROUND INFORMATION Drug facilitated rape is one of the majorproblematic subject area. In past 15-20 years, the benefaction of alcohols and drugs to sexual assaults has been increasing highly and it has gained a significant amount of attention from media, general public and professionals. According toButler Bernadette and Jan Welch’sDrug- facilitated sexual assault report 2009 (Canadian Medical Association Journal) provided significant data that about 20.9% of victims of rape cases encountered the drug facilitated rape criteria. Also, according to the Bureau of Justice Statistics (BJS) the most occurring rape cases are the drug facilitated rapes. DRUG FACILITATED SEXUAL ASSAULT (DFSA): Drug facilitated sexual assault is a sexual assault or rape which is performed on a person who is incapable to consent to any sexual activity or is incapacitated due to being under the effect of any drug, substance or alcohol. The drug facilitated rape has given different names such as drug and alcohol facilitated rape, incapacitated rape, date rape and drug rape. The term ‘Date rape’ means to commit the crime of rape or sexual assault while on a date. The drug facilitated rape consists of all types of non-consensual penetrative sexual activity.The substance or drug used for date rape are normally anaesthetics and are also known as date rape drugs. A date rape drug is intentionally added by the defender in victim’s drink andthat drink is consumed byvictim without any knowledge of it. The victim mostly passes out or is awake but unable to control his or her behaviour under the influence of the given drug.The phenomenon of mixing or placing drugs into drinks to change someone’s behaviour with mainly an intention to perform a crime is called drink spiking. Date rape, as clear from its name, mainly starts from a bar, restaurant or pub and the perpetrator gives the drug deliberately to the victim by spiking their drink, mainly victims are females but however some cases of drug facilitated sexual assault have been reported in males as well. The drugs mainly used for date rape purposes are colourless and odourless, so it is very hard for the victim to detect whether their drink is spiked or not. These drugs mainly emit their effects after 15 to 30 minutes. The time taken by spiked drinks to act also depend upon the amount of the drug added to the drink and the amount of drink consumed. The age of the consumer, body weight and size also influences the response given out by date rape drugs. The victim may not realise that his or her drink was spiked until they lose consciousness. The reasons behind this problem can be because of great advancement in drugs, lack of awareness among people, socialisation and miscommunication but there is no direct or main cause of it.This crime is punishable under common law of England. There is no definite sentence for this crime as factors such type of assault, circumstances affect the type and duration of sentence. The drug facilitated rape holds a great importance in forensics as this crime is based upon the use of drugs and the evidence such as leftover spiked drink consumed by the victim are tested forensically only. As the date rape drugs are mainly not visible in drinks and odourless as well, a number of chemical analytical techniques are used in this subject to analyse and quantify the amount of the drug present in a sample submitted to a forensic laboratory. LITERATURE REVIEW: Over the last ten years, the numbers of reports of drug facilitated rapes have been increasing all over the world and mainly in Australia, the United States of America and the United Kingdom according to Drug Facilitated Sexual Assault (DFSA) report-2007. The British crime Survey 2001 showed that about 43% victims of sexual assault were either unable to consent to sexual activity or unconscious due to the influence of drugs or alcohol. The data collected from the Crime Survey for England and Wales in 2009-10, 2010-11 and 2011-12 showed that, on average, 2.5% of women and 0.4% of men had been victims of drug facilitated sexual assault in last 12 months. In these reports, it was also observed that the main victims of drug facilitated sexual assaults are women. Young females between the age group of sixteen and twenty-four are at the high risk for being raped (Feminist Majority Foundation, 2005).This age group people are the most targeted, as the young people are easy victims for perpetrators. The drug facilitated rape is performed by spiking victim’s drink using anaesthetic or central nervous system depressing drug. For analysing this problem, it is vital to study the drinking patterns in women. According to Office for National Statistics [ONS] 2013, the type of alcohols consumed by females In this project, I will be analysing wine, spirits and beer as these alcoholic drinks are most popular among women. However, these drinks are generally consumed as mixture but all the samples tested will be in pure or neat form. DATE RAPE DRUGS: According to ACMD’s Drug facilitated sexual assault (DFSA) Report 2007, drugs mainly used for date rape are divided into two groups- central nervous system depressants and central nervous system stimulants. The central nervous system depressants drugs are used to change victim’s behaviour, unconsciousness and to cause memory loss. The mainly used depressants drugs are alcohol, gamma-hydroxy butyric acid (GHB), benzodiazepines (mainly diazepam and rohypnol)and ketamine. However, the central system stimulants are mainly used to enhance sexual behaviour of the victim and to cause loss of inhibitions to enhance risky sexual behaviour such as cocaine, methylamphetamine and MDMA(ecstasy). Methylamphetamine is used rarely in England as compared to USA according to Advisory Council on the misuse of drugs(ACMD) Drug Facilitated Sexual Assault report (2007). My focus will be analyzing the detection and optical properties of Gamma-hydroxybutyric acid. GAMMA-HYDROXYBUTYRIC ACID (GHB): GHB also known as 4-Hydroxybutanoic acid, is a fatty acid linked to gamma-aminobutyric acid (GABA)which is a natural existing neurotransmitter and it delivers effects similar to a mixture of alcohol and ecstasy such as drowsiness, loss of control, lowered anxiety and increased sensuality (Galloway et al.,2000).GHB is a psychotropic drug and used as a narcotic as it introduces unconsciousness without causing any crucial heart or respirational problem and it can result in short-term memory loss. GHB drug is a central nervous system depressant and the working mechanism of this drug is different from all alcohols and benzodiazepines [Advisory Council on the misuse of drugs(ACMD) UK Drug Facilitated Sexual Assault report (2007)].GHB has no colour and odour to it and gives nearly no taste or very slightly salty taste. This drug is also known with other names such as gamma-OH and gamma hydroxybutyrate. GHB was first produced and utilized for medicinal purposes in 1960.Initially it was used as an anaesthetic and for treating alcohol problems and cataplexy in medical field. But due to popularity of GBH as a drug of abuse and its weak analgesic properties the clinical use of it as an anaesthetic has been reduced. Also, in two scientific researches where GHB was used for treating alcoholism,it was discovered that 10 to 15% patents became dependant on GHB (Addolorato et al., 1996; Gallimberti et al., 2000).This drug is now widely used as a recreational drug and date rape drug (McCusker, Rachel R., etal.,1999).GBH is rapidly absorbed with peak plasma concentration and starts emitting its effect after 30 to 45 minutes. It is metabolised in about 30 minutes and cannot be detected in urine after 12 hours. [Advisory Council on the misuse of drugs UK (ACMD) Drug Facilitated Sexual Assault report (2007)]. Gamma-hydroxy butyrate (GHB) is classified as Class C drugs under the Misuse of Drugs Act 1971 in UK and it was brought under this act in 2003. Gamma butyrolactone(GBL) and 1,4-BD (1,4-butanediol) chemicals are transformed into GHB as they enter inside human body, these drugs were started to be used for GHB purposes. But in 2009 GBL and 1.4-BD were also classified as Class C drugs under the Misuse of Drugs Act. The amount of GHB used for spiking is very important as this drug has a steep dose-response curve which means even a slight increase in dose can results in toxicity. According to BBC report 2003, GHB is available in UK in the form of liquid in a 30-ml plastic bottle which costs about £15. Because GBH is a drug of abuse, it is found in the form of oily liquid and powder but the purity of the drug is hard to predict. A teaspoon or a capful amount i.e. approximately 5ml of GBH is normally used for spiking purposes but as it is an illicit drug, it is hard to predict the safe dosage and is very easy to overdose. As GBH is a drug of abuse, it needs to be handled with care. According to safety sheet(SIGMA), this drug has no flammability and no reactivity. All the safety measures will be adopted during project according to the MSDS - Lab safety sheet (The Sigma-Aldrich library of regulatory and safety data) PAST REPORTS RELATED TO THE SUBJECT: Various previous reports have been done on the topic of drug facilitated rape, date rape drugs analyzation and identification etc. Latest reports in this field are such as Drug detection straw (2009), Drug detection device (2011), Drug detection in nail polish (2014), System and method for detection of a contaminated beverage (2014) and Methods and kits for detection of drugs (2016). These reports mainly focus on the detection of drugs to check if the drink is spiked or not. But to detect, quantify and confirm the presence of these drugs reports such as Analysis of gamma-hydroxybutyrate (GHB) in urine by gas chromatography-mass spectrometry (Rachel R., et al. 1999), Clinical features and management of gamma-hydroxybutyrate (GHB) withdrawal (Michael, et al.2004) and Analysis of biofluids for gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) by headspace GC-FID and GC-MS (Marc A., et al. 2000) are remarkable reports in forensic field. As this project will be focusing at looking optical properties of GHB in different alcoholic drinks, the main optical property analysed will be absorbance. It will also be analysed does the absorbance of different concentration GBH analytes follow Beer lambert law or not. ABSORBANCE: Absorbance is the measurement of the ability of a sample or substance to absorb light of a specific wavelength. According to Gold, V., et al. 1997, the absorbance is equal to the logarithm of the ratio of incident to the transmittance radiant power through a sample. absorbance is dimensionless and has no units as it the ratio of two similar quantities. Absorbance can be expresses mathematically as, A= L?og ?_10(I_i/I_t) Here, I_i is the radiant flux incident and I_tis the radiant flux transmitted. BEER LAMBERT LAW: The Beer-Lambert lawis the linear relationship between the absorbance and concentration of absorbing analytes. According to Calloway, et al. 1997, the beer-lambert law can be expressed as: A= ?bc [here, ? is molar absorptivity, b is the path length and c is molar concentration] This law is applied to study the absorbance pattern in a sample with different concentration of analytes. METHODOLOGY In order to carry the project, the samples analysed will be: 1. Wine - red wine, rosé and white wine 2. Spirits- vodka, whisky and dark rum 3. Beer- stout, larger and ale Water will be used as blank and at least three readings will be taken for each sample in order to maintain accuracy. DETECTION USING FLUORESCENCE SPECTROMETER: To study the absorbance of all alcoholic samples mixed with GHB, Fluorescence spectrometer will be used. The fluorescence spectrometer analyses a sample and gives out fluorescence intensity or absorbance reading. Before analysing any sample, the apparatus will be calibrated by using water. Initially, different amount of GHB will be added into the samples to construct a calibration curve. By constructing calibration curve, the lowest and the highest detectable quantity of GHB will be calculated as well.After this each sample will be analysed without any GHB and then three readings of each sample will be taken by adding specific amount of GHB. All samples will be analysed to obtain absorbance difference in samples with GHB and without GHB. This method will be used as detection method. HPLC-MS will be used as a confirmatory test. However other methods such as Gas chromatography coupled with mass spectrometer can also be used for confirmation purposes. But this instrument can be problematic as GHB gets converted into GBL due to high temperature inside the apparatus. Also, the detection method involves acidification steps coupled with extraction. Such acidification can result in any GHB present conversion to GBL which is problematic as detection is not very conclusive (McCusker et al. 1999). CONFIRMATION BY USING HPLC-MS: The samples analysed by using detection method of Fluorescence spectrometer will be finally analysed by using HPLC coupled with mass spectrometer. In this method, the samples will be analysed to quantify the amount of GHB present in them by plotting calibration curve of absorbance against concentration. This method is conclusive and more accurate as the mass spectrometer detects compounds in a sample by measuring molecular weight and no two different compounds share same molecular weight. DATA INTERPRETATION The results of Fluorescence Spectrometer and HPLC-MS will be presented as following: The absorbance value for each sample will be tabulated corresponding to the concentration of GHB in each sample. Calibration curves for both florescence spectrometer and HPLC-MS will be constructed by plotting absorbance values against concentration values of GHB. From calibration graph, lowest quantifiable GHB concentrations and saturation points will be determined. Three readings will be taken for each sample and data for each sample will be treated statistically by applying arithmetic mean, standard deviation and % relative standard deviation. The variance in absorbance readings for each sample with GHB and without it will be compared by using tables.