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DATE DUE DATE DUE DATE DUE 11/00 c-lClRC/Datoouopes-p.“ ANALYSIS OF GAMMA-HYDROXYBUTYRATE (GHB) IN BEVERAGES By Scott James Tschaekofske A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Criminal Justice 2000 ABSTRACT ANALYSIS OF GAMMA-HYDROXYBUTYRATE (GI-1B) IN BEVERAGES By Scott James Tschaekofske The substance y-hydroxybutyrate (GHB) is an easily prepared, relatively new drug of abuse. It was not abused Si gnificantly until 1990 and did not become controlled federally until early 2000. The drug is abused both as a party drug and as a weapon of assault, often in “date rape” situations. In this Short history of abuse, the misuse of GHB has resulted in several deaths. The analysis of GHB is a significant challenge for the forensic chemist. In solution, GHB is in a delicate balance with its lactone, y-butyrolactone (GBL). GBL is not controlled in most states, nor is it controlled federally. Thus, the forensic chemist must identify GHB without disturbing this balance. The purpose of this research is to develop preliminary tests for GHB as well as methods of separation and identification that do not result in interconversion between GHB and GBL. The use of Silver nitrate to precipitate GHB from solution is the primary focus of the research. Specifically, a 5% Silver nitrate solution is added to GHB in a variety of beverages to form microscopic rectangular crystals. Silver nitrate is also added to GHB in various beverages to form an insoluble Ag-GHB salt. This salt is then removed from the beverage and identified instrumentally. The forensic chemist now has a variety of means to identify conclusively GHB in beverages. However, because the balance between GHB and GBL is affected by time in solution, the interpretation of this identification is not always straightforward. DEDICATION to Jennifer my loving wife, you are my everything. iii ' ACKNOWLEDGMENTS Throughout this project I have received assistance and direction in various forms from many individuals. A special thanks to Marv Szumlinski and the other members of the Northville drug identification unit. Marv welcomed me into the unit and presented me with the problem of GHB identification. Also, thanks to Dave Burke for having silver nitrate reagent at his bench. That bottle of reagent really made a difference in this project! I must also recognize my advisor, Jay Siege], who accepted me into his program and helped me fulfill my goal of becoming a forensic chemist. I also give thanks to the chemistry and criminal justice departments of Michigan State University for giving me financial support in the form of teaching and research assistantships. The assistantships made life much more bearable during my graduate work. And finally, thank you to my wonderful wife, Jennifer, who endured my frequent procrastination, who proofread many copies of this manuscript, and who traveled a thousand miles with me so that I could pursue this degree. iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES .......................................................................................................... viii INTRODUCTION ............................................................................................................... 1 Preparation of GHB ........................................................................................................ 2 Scientific and Legal History of GHB ............................................................................. 2 GHB AS a Street Drug .................................................................................................... 4 GHB As a Weapon of Assault ........................................................................................ 4 Interconversion Between GHB and GBL ....................................................................... 5 Analysis Problems For the Forensic Chemist ................................................................ 6 REVIEW OF LITERATURE .............................................................................................. 8 MATERIALS AND METHODS ...................................................................................... 11 Reagents ....................................................................................................................... l 1 Microscopes and Instrumentation ................................................................................ 11 Preparation of Na- and K-GHB .................................................................................... 12 Preparation of Ag-GI-IB ................................................................................................ 13 Evaporation of Aqueous Samples of GHB and GBL ................................................... 13 Derivatization with BSTFA .......................................................................................... 14 Derivatization of Standards .......................................................................................... 14 Extractions .................................................................................................................... 14 Testing of Dichloromethane Extraction Method .......................................................... 14 Silver Nitrate Microcrystalline Test ............................................................................. 15 Precipitation with Silver Nitrate for GC/MS Analysis ................................................. 16 Precipitation with Silver Nitrate for FI‘IR Analysis .................................................... 17 RESULTS AND DISCUSSION ....................................................................................... 19 Salt Forms of GHB ....................................................................................................... 19 Evaporation of Aqueous Samples of GHB and GBL ................................................... 21 Derivatization of Standards .......................................................................................... 21 Extractions .................................................................................................................... 24 Testing of Dichloromethane Extraction Method .......................................................... 24 Silver Nitrate Microcrystalline Test ............................................................................. 24 Precipitation with Silver Nitrate for GC/MS Analysis ................................................. 27 Precipitation with Silver Nitrate for FTIR Analysis .................................................... 28 CONCLUSIONS ............................................................................................................... 29 APPENDIX -- RECOMMENDED ANALYSIS OF GHZB SAMPLES ............................ 31 LIST OF REFERENCES .................................................................................................. 32 BIBLIOGRAPHY ............................................................................................................. 34 vi LIST OF TABLES Table l - GC/MS conditions ............................................................................................. 12 Table 2 - Samples used in silver nitrate microcrystalline test ........................................... 16 Table 3 - Solutions precipitated with silver nitrate and derivatized with BSTFA ............ 17 Table 4 - Solutions purified with silver nitrate precipitation/sodium chloride conversion ......................................................................................................... 18 Table 5 - Results from Silver nitrate microcrystalline test using Na-GI-IB standard ......... 26 vii LIST OF FIGURES Figure 1 - Lactonization of GHB / Hydrolysis of GBL ...................................................... 2 Figure 2 - Mass Spectrum obtained from a sample of GHB ................................................ 7 Figure 3 - Mass spectrum obtained from a sample of GBL ................................................ 7 Figure 4 - Derivatization of GHB with a silylating agent ................................................. 10 Figure 5 - Preparation of GHB salts from GBL ................................................................ 13 Figure 6 - Infrared spectrum of standard Na-GHB ........................................................... 19 Figure 7 - Infrared spectrum of prepared K-GHB ............................................................. 20 Figure 8 - Infrared spectrum of prepared Ag-GHB ........................................................... 20 Figure 9 - Mass spectrum of derivatized GI-IB .................................................................. 22 Figure 10 - Mass spectrum of derivatized AI-IB ................................................................ 22 Figure 11 - Mass spectrum of derivatized BI-IB ................................................................ 23 Figure 12 - Mass spectrum of derivatized 1,4-butanediol ................................................. 23 Figure 13 — Plate formation after addition of silver nitrate to 10 mg/mL Na-GHB in Pepsi solution (100x, polarized light) ....................................................... 26 viii INTRODUCTION y-Hydroxybutyrate, commonly referred to as GHB and more properly as 4- hydroxybutanoate salt, is an easily synthesized and relatively simple compound. Due to the overdoses associated with its ingestion and its use as a so-called "date rape drug", many state legislatures have banned the manufacture, distribution, or possession of GHB. In 1998, GHB became a Schedule I drug in Michigan. Recently, the Federal government amended the Controlled Substances Act to classify GI-IB as a Schedule I drug. Upon GHB becoming a controlled substance, forensic chemists have begun to look at methods of analysis for the drug. As GHB was scrutinized in more detail, problems with its analysis were uncovered. Much research has been performed recently regarding the analysis of GHB. Some of this research has led to methods that will be useful to forensic chemists, and some of the research is simply bad science. Due to the variability of many street samples and the chemical nature of GHB, samples must be purified or modified before most types of instrumental analyses can be performed. Because GHB is mixed in a variety of beverages, especially in “date rape” situations, this purification, and consequently the identification of GHB in the laboratory, is quite troublesome. The following research addresses the analysis problems concerning GHB. The focus of this work is to develop preliminary tests as well as methods of separation and identification for GHB that overcome the pitfalls of conventional techniques. Specifically, a microcrystalline test that permits preliminary identification of GHB in a variety of beverages is discussed. Established methods are investigated, and improvements to these methods are made. A separation scheme to remove GHB from beverages in a form pure enough to enable identification is developed. Lastly, a protocol for handling suspect GHB samples is detailed. These developments are significant advances in the analysis of GHB. Preliminary testing is more specific and in some cases more sensitive than previously developed methods. The separation scheme avoids converting GHB to a non—controlled substance, a serious weakness of some established procedures. These improvements give the forensic chemist a workable analysis scheme to identify GHB in a variety of beverages. Preparation of GHB GHB readily undergoes intramolecular esterification to form a five-membered ring (see Figure 1). This ester is a lactone known as y-butyrolactone or GBL. GBL is a solvent used in many applications such as paint removal and engine cleaning. Due to its widespread industrial and commercial use, GBL is not placed under the same restrictions as GHB in most states. The salt form of GHB can be prepared from the hydrolysis of the lactone under basic conditions. Thus, the preparation of GHB can be performed easily, using readily obtained chemicals. 0 O HO H+ ‘ O ‘ OH + H20 OH Figure 1. Lactonization of GHB / Hydrolysis of GBL. Scientific and Legal History of GHB GHB is a central nervous system depressant. Research has Shown that small quantities of GHB are present in the mammalian brain and other tissues. It has been identified as a metabolite of the neurotransmitter 4-aminobutanoic acid. However, the mechanism by which GI-IB works in the body is not fully understood. In the 1950’s, GHB was developed and used to induce sleep as an adjunct to anesthesia. Its use was discontinued because of side-effects and its lack of analgesic properties. More recently, it has been studied in the treatment of narcolepsy and opiate and ethanol addictions. The abuse of GHB was first reported in the summer of 1990, approximately six months after the Food and Drug Administration (FDA) banned the sale of l-tryptophan, a popular sleep aid. Initially, GHB was abused by body builders. It was marketed in health food stores as a Sleep aid and a “steroid alternative” because a study showed that GHB increased the release of growth hormone.1 Until 1990, GHB was classified as an “unapproved food additive” by the FDA. However, the FDA changed this status to “investigational new drug” and banned its sale after a rash of abuse and overdose reports. By the end of 1999, more than 20 states had passed legislation to control GHB. The majority of these states placed the drug in Schedule I. In February 2000, Congress followed the lead of the state governments and updated the Controlled Substances Act to place GHB under the restrictions of a Schedule I drug. At the same time, the Federal government classified GBL as a List I chemical to increase regulation of its manufacture and distribution. However, most states, even those with laws controlling GHB, have not scheduled GBL. Wisconsin, a notable exception, has recently amended its legislation to place the lactone under the same control as GHB. GHB As a Street Drug GHB has now become a drug of abuse by teenagers and young adults, especially at “rave” parties. On the Internet, kits are sold which contain the lactone and a base, along with instructions for producing GHB. The kit is sold under the guise of being a chemistry experiment that demonstrates an exothermic reaction. GI-IB is normally taken orally in a dosage one-half to three teaspoons (approximately 1.5 to 9 g) of the salt dissolved in water or another beverage. Thus, it most often arrives at the laboratory for analysis in some type of solution, although the salt is sometimes confiscated. On the street it is known as Somatomax PM, Grievous Bodily Harm (GBH), great hormones at bedtime, easy lay, liquid X, liquid ecstasy, salty water, and water. It is sometimes sold as ecstasy (3,4-methylenedioxymethamphetamine or MDMA) and is claimed to have the effect of alcohol without the hangover. The effects of GHB use vary widely from individual to individual and according to dose and use of other drugs. Extreme drowsiness, irritation, confusion, nausea, reduced blood pressure, and decreased respiration are some of the more common adverse effects of GHB abuse. As the popularity of GHB has risen, the nationwide number of reported emergency room visits related to GHB has increased from 54in 1994 to 764 in 1997.2 Recently, five teenagers from Michigan were hospitalized after consuming GHB at a party.3 GHB use with alcohol and other depressants is quite dangerous because of the synergistic effect. This type of abuse has resulted in several deaths. GHB As a Weapon of Assault Another form of abuse of GHB has been as a "date rape drug". Since GHB can cause deep, unarousable sleep as well as amnesia in high doses, it can be a dangerous weapon of assault. AS with other "date rape drugs", GHB is typically mixed in the victim’s drink at a bar or party to render the person physically and mentally incapacitated. Several instances of this type of assault have been reported, including one in Michigan that resulted in the death of the victim.4 Interconversion Between GHQnd GBL GBL readily hydrolyzes to GHB in basic conditions. Hydrolysis of the lactone can also be acid-catalyzed, even though the lactone is the favored form under acidic conditions. At an initial hydrochloric acid concentration of 0.01M and an initial lactone concentration of 0.1M, researchers reported the equilibrium constant for the hydrolysis reaction to be 0.347.5 Until recently, little information was known about this equilibrium at more neutral conditions. A study by the FDA showed a 1% conversion to GHB by a GBL in de- ionized water solution after one day. This conversion was approximately 3% after Sixteen days, and it was 33% after six months.6 Conversion of GBL to GHB was found to be much faster in acidic and basic solutions than in pure water. This FDA study indicated that pH and the amount of time since the drug solution was prepared are important considerations. For example, an individual adds pure GBL to a beverage. The person then uses the spiked beverage to incapacitate a victim for the purpose of committing rape. Whether or not the forensic chemist detects GHB in the sample depends on the pH of the sample and how much time has passed since the GBL was added to the beverage. If the individual added GBL to pure water, no GHB would be found if the sample was analyzed immediately. However, if the sample remained in a police or crime laboratory property room for six months before being analyzed, the identification result would be much different. Analysis Problems for the Forensic Chemist Analyses of GHB samples are complicated by the few functionalities present for manipulation, the facile interconversion of GHB and GBL, and the variability of sample matrices. Because GBL is not controlled in most states, chemists must ensure that they do not convert the lactone into the hydroxyacid. Since GBL appreciably hydrolyzes under acidic and basic conditions, an acidic/basic extraction cannot be used to purify a sample. This severely limits the separation scheme. Gas chromatography coupled with mass spectrometry (GC/MS) is often the method of choice in drug identification laboratories. However, there is a problem with GHB analyses through this route as well. The mass spectrum obtained from a GHB sample analyzed using GC/MS is indistinguishable from a spectrum collected from a sample of lactone (see Figures 2 and 3). Because the high temperature of the injection port converts GHB to GBL, this method cannot be used to distinguish the two substances without the use of some type of protecting group. Laboratories also must distinguish GHB from the related compounds, 0t- and B- hydroxybutanoate salts (AHB and BHB, respectively). Further, because the uncontrolled substances GBL and 1,4-butanediol are converted to GHB in the body and thus give the same type of response, laboratories will begin to see these substances more frequently as abusers manipulate the laws. Methods for identifying these substances have not been researched by most drug laboratories. Abundance 35000‘ 300004 1 zsoooi 200003 150001 i 100004 J 39 32 if. 56 53. l A 86 Y—Yfi'IVVTY 37 1.1., .11. l l 425 30 35 40 r,..v.,...., 45 50 55 "IVVV 60 ' l 65 VT‘Y .I....,... 70 75 Figure 2. Mass spectrum obtained from a sample of GHB. Abundance 120000{ 110000§ 100000? 90000; 00000; 70000§ 60000? sooooi 40000§ 30000§ 200003 rooooé 3 O 39 37 311 l ‘ 43 L. TV] 5 s? i l 6 57 86 87 an—> ..,... 25 I V'YUT‘TYI'I 30 35 40 45 U V I I I 50 U U I 'l 55 fiT‘TY r 60 Y‘TTTY'IVU'II'V' 75 65 70 VIYYYVIUIYY 80 85 Figure 3. Mass spectrum obtained from a sample of GBL. V—rr‘l Y W 'eb" 85 90 REVIEW OF LITERATURE Development of methods for preliminary testing of GHB has been largely unsuccessful. It has been reported that no reaction is obtained using the test reagents Marquis, Scott, Ehrlich’s, Liebermann’s, or 5% silver nitrate.7 Crystals of the sodium salt do not fluoresce under short or long wave ultraviolet (UV) light, and aqueous solutions of GHB do not absorb in the UV region.7 The salt form of GHB as well as aqueous solutions of it do react with Jones reagent (chromium(VI) in sulfuric acid), a test for primary and secondary alcohols.8 A positive reaction is noted as a color change from orange to green. Screening can also be done using iron(III) chloride.9 Formation of a brown precipitate indicates a positive response. Thin layer chromatography (TLC) is a possible preliminary test for GHB. Visualization of GHB on TLC plates using iodine fuming or spraying with Fast Blue BB has been successful.lo More recently, color tests utilizing 1% cobalt nitrate to indicate the presence of GHB and cobalt thiocyanate to indicate the presence of GBL have been described.9 While there is now a battery of spot tests for GHB, most are lessened in value due to the false positives the tests give with substances such as sugars, ethanol, or alkaline solutions. An acidic extraction procedure has now been described that utilizes the cobalt thiocyanate color test to eliminate the possibility of the sample containing GBL.9 After obtaining a positive color test for GHB and a negative test for GBL, the sample is acidified and converted to the lactone. The GHB is later precipitated to separate it from sample impurities. A microcrystalline technique for GHB has been detailed recently. 11 The authors used a reagent consisting of 1% copper (II) nitrate and 1% silver nitrate to form large rectangular crystals when viewed under appropriate magnification. The detection limit for the technique was given as 2 mg/mL. Thus far, most research into the separation and identification of GHB has been dependent on the instrumentation available at the laboratory where the study was conducted. The Drug Enforcement Agency (DEA) has published work involving analysis of a liquid sample containing GHB using Fourier transform infrared spectrophotometry (FT IR) and Fourier transform nuclear magnetic resonance spectrometry (FI‘NMR).12 The sample used in the study was evidently pure as the only sample preparation performed was evaporation of the solvent to obtain the GHB salt. The FDA also has analyzed GHB samples using FI‘IR. In one study, addition and subtraction of spectra was used to identify GHB and GBL in various sample matrices.13 The FDA has published a study involving the separation of GHB and GBL along with the identification of the two substances using high performance liquid chromatography (HPLC)/thermospray mass spectrometry.14 These techniques have been successful, but there are still problems. Purification of the sample is necessary in many cases before FTIR analysis can be performed. Further, many laboratories do not have the instrumentation available to perform the more exotic techniques discussed above. The remaining work has focused heavily on derivatization of GHB for analysis using GC/MS.7’10"5 In all instances, the sample was extracted, the extract was evaporated to dryness, and the residue was derivatized using bis-(trimethylsilyl) trifluoroacetamide (BSTFA, see Figure 4). O O BSTFA HOM > ”MM OH OTMS TMS: trimethylsilyl Figure 4. Derivatization of GHB with a silylating agent. The crucial element in this method is not the derivatization; it is the extraction. Two papers from the Illinois State Police have described using dichloromethane to extract GHB for derivatizationm’lS Because GHB in salt form is insoluble in dichloromethane, the use of dichloromethane as an extraction solvent is counterintuitive. Evaporation techniques have been successful in separating GI-IB from beverages, except with more acidic drinks such as orange juice.16 The GHB solution was evaporated to dryness and extracted with methanol. The methanol was evaporated and acetone was added. A stream of air was blown gently across the sample on a watchglass. Upon evaporation, an outer ring of relatively pure GHB formed on the watchglass. The purified sample was then analyzed using FI‘IR. However, the concentration of GHB must be high (tested samples were lOOmg/mL or greater) to produce sample pure enough for instrumental analysis. 10 MATERIALS AND METHODS Because bases other than sodium hydroxide can be used to convert GBL to GHB, both the sodium and potassium salts of GHB were produced for further studies. Initially, various dry and liquid/liquid extractions were attempted to purify samples of GHB in beverages. The use of Silver nitrate to precipitate GHB from solution as part of a microcrystalline test and also as a separation step before instrumental analysis was the primary focus of the research. Reagents Sodium salt standards of AHB, BHB, and GHB were purchased from Sigma Chemical Co. Sodium, potassium, and silver salts of GHB were prepared as described below. GBL and 1,4-butanediol were obtained from Aldrich Chemical Co. The purity of all standards was confirmed using FTIR. The derivatizing agent, BSTFA, was obtained from Sigma Chemical Co. A variety of beverages typical of what might be encountered in a date rape Situation were purchased for use in testing. These included several soft drinks, juices, wine, beer, and hard liquor. The specific beverages are listed in Table 2. Microscopes and Instrumentation An American Optics Microstar polarized light microscope was used for all microcrystalline examinations. An American Optical Corporation Spencer comparison microscope equipped with a Sony CCD-Iris/RGB color video camera, a Sony CMA-D2 camera adaptor, a Sony Trinitron monitor, and a Sony UP-1200A color video printer was used to capture and print photomicrographs. ll The GC/MS instrument system consisted of a Hewlett-Packard GCD1800B GC/MS system. The conditions used are Shown in Table 1. FT IR analyses were performed using a Nicolet Protégé 460 FT IR spectrophotometer equipped with a MCT detector. For each sample analyzed, 100 background and 100 sample scans were collected at a resolution of 4 cm”. Table l. GC/MS conditions. Carrier Gas helium Flow Rate 1.0 mUminute Injection Volume 2 ”L (split), l ”L (splitless) Injection Port Temperature 250°C Initial Oven Temperature, Time 60°C, 0.4 minutes Ramp Rate 25°C/minute Final Temperature, Time 250°C, 2.0 minutes Detector Temperature 280°C Solvent Delay 2.1 minutes Scan Range 45 - 450 m/z (30 — 200 m/z for GHB/GBL) Preparation of Na- and K-GHB In an Erlenmeyer flask 30 g sodium hydroxide were added to 300 mL methanol. While stirring, 100 mL GBL were added over a two hour time period. After complete addition of the GBL, the cloudy solution was stirred for one additional hour. Because the solution was still highly alkaline, it was refluxed for one hour in a round-bottomed flask fitted with a condenser to increase the yield of GHB. After this, the nearly clear solution was poured into a large beaker. This was placed in the hood to promote solvent evaporation. The white solid was removed using vacuum filtration. The solid was dried in an oven at 60°C. The product was analyzed using FT IR. This was repeated by adding 30 mL GBL to 20 g potassium hydroxide in 100 mL methanol. The dry solid obtained was also analyzed using FTIR. 12 O o CH3OH 0H + NaOH ———> O'Na” (KOH) (K+) Figure 5. Preparation of GHB salts from GBL. Preparation of Ag—GHB A solution of GHB was prepared by dissolving 2.0 g of the sodium salt in 10 mL de- ionized water. A silver nitrate solution consisting of 3.0 g solid in 10 mL de-ionized water was added to the GHB solution, resulting in the formation of a white solid. The mixture was cooled in an ice-water bath. The solid was removed by vacuum filtration. The white solid was placed in an Erlenmeyer flask with a minimal amount of de-ionized water. The mixture was heated in a hot-water bath until the solid was fully dissolved. The solution was cooled at room temperature, and the recrystallized product was removed by vacuum filtration. The dry solid was analyzed using FT IR. Evaporation of Aqueous Samples of GHB and GBL A solution of 0.50 g Na—GHB standard, 2 mL GBL, and 10 mL de-ionized water were placed on a watchglass. The solution was evaporated on a hot-water bath. After drying the solid fully in an oven at 60°C, it was analyzed using FTIR. A solution of 2 mL GBL and 10 mL de-ionized water were placed on a watchglass. The solution was evaporated on a hot-water bath. No solid remained for FTIR analysis. 13 Derivatization with BSTFA To each sample in screw-top and snap-top tubes was added 100 ML BSTFA. The contents were mixed. The tubes were sealed and heated in an oven for 30 minutes at 60°C. After this time, approximately 200 p.L acetonitrile was added to each tube. Derivatization of Standards Standards of AHB, BHB, GHB, GBL, and 1,4-butanediol were derivatized with BSTFA. Prepared samples of Na-, K-, and Ag-GHB were also derivatized. All derivatized samples were analyzed using GC/MS. Extractions A 100 mg/mL Na-GHB standard in Pepsi solution was heated on a steam bath to remove all water. Methanol was added to the residue. The mixture was stirred and the methanol was decanted. The solvent was evaporated, leaving a sticky residue. FI‘IR analysis of the final residue was not attempted. This was repeated with isopropanol and n-butanol. Again, FTIR analysis was not attempted on the final residue. Ethyl acetate was also used as an extraction solvent. Extraction of a dried GI-IB in Pepsi solution as described above was attempted. A liquid/liquid extraction of a non- dried GHB in Pepsi solution was performed. A 100 mg/mL Na-GHB standard in Pepsi solution was extracted with two portions of ethyl acetate. The ethyl acetate was evaporated, leaving a sticky residue. No FTIR analysis was performed. Testing of Dichloromethane Extraction Method A 5 mL solution of 10 mg/mL N a-GHB standard in de-ionized water was placed in a centrifuge tube with 5 mL dichloromethane. The tube was capped and rotated for 15 14 minutes. After centrifuging the contents, the dichloromethane layer was transferred to a clean tube with a pipet. Following evaporation of the dichloromethane, the sample was derivatized with BSTFA and analyzed using GC/MS. A small amount of dichloromethane was saturated with a Na-GHB standard. This solution was analyzed using GC/MS. Silver N Etc Microcrystalline Test Samples were prepared using the Na-GHB standard, except where noted otherwise. One drop of the test solution (see Table 2) and one drop of a 5% silver nitrate solution were placed on a microscope slide. The slide was examined under polarized light at lOOX. After 5 minutes and 30 minutes, the slide again was examined. Plate formation was noted as "none", "small", or "large". Distortion of plates was also noted. Testing with 1% and 10% silver nitrate solutions was tried on some samples in an attempt to increase sensitivity. Blank solutions also were tested for every beverage. 15 Table 2. Samples used in silver nitrate microcrystalline test. Beverage/Solvent Chemical Added (Concentration mg/mL) de-ionized water I standard Na-GHB (100, 50, 10, 5, 2, 1, 0.1) -- fresh, 1 day old, 1 week old prepared Na-GHB (10, 5) prepared K-GHB (10) standard Na-AHB (10) standard Na-BHB (10) standard GBL (1000, 200, 20) standard 1,4—butanediol (20) tap water Standard Na-GHB (10, 1) 95% ethanol standard Na-GHB (10, 5, 1) I standard Na-GI-IB (100, 50, 10, l) -- fresh, 1 day old, 1 week old Pepsi I prepared Na-GHB (100, 50, 10) I Standard GBL (1000, 200, 20) -- fresh, 1 day old, 1 week old Diet Pepsi (caffeine free) standard Na-GI-IB (10) Coca-Cola standard Na-GHB (50, 10, 1) Cherry Coke standard Na-GHB (10, 5, 1) Dr. Pepper standard Na—GHB (10, 1) Mt. Dew standard Na-GHB (50, 10) Country Time lemonade I standard Na-GHB (100, 50, 10) I standard GBL (1000, 200, 20) Mei jer cranberry apple juice cocktail standard Na-GI-IB (50, 10) Kroger pasteurized orange juice standard Na-GI-IB (100, 50, 10) Mogen David concord wine standard Na-GHB (100, 50, 10) Molson beer standard Na-GHB (100, 50, 10) E & J brandy standard Na-GHB (10, 1) Seagram’s gin standard Na-GHB (10, 1) Jack Daniel’s whiskey standard Na-GHB (10, 1) Precipitation with Silver Nitrate for GC/MS Analysis For each test solution prepared using Na-GHB or GBL standards (see Table 3), 1 mL was placed in a micro-centrifuge tube. To this was added 0.1 g silver nitrate. The contents were mixed, chilled in an ice-water bath, and spun down. The liquid was decanted. A methanol wash was performed on each sample. After the wash was 16 decanted, the samples were dried in an oven at 60°C. The samples were derivatized as described previously and analyzed using GC/MS. Blank solutions also were tested for every beverage. Table 3. Solutions precipitated with silver nitrate and derivatized with BSTFA. Beverage/Solvent Chemical Added (Concentration Jig/mL) de-ionized water - GBL(105) - GHB(1000)andGBL(105) Pepsi GI-IB (1000, 100, 10, l) Diet Pepsi (caffeine free) GI-IB (1000, 100, 10, 1) I GHB (1000, 100) Mogen David concord wine I GBL (105) - GHB (1000) and GBL (105) Molson beer GHB (1000, 100) E & J brandy GHB (1000, 100) Seagram’s gin GHB (1000, 100) Jack Daniel’s whiskey GHB (1000, 100) Precipitation with Silver Nitrate for FTIR Analysis A 10 mL solution of 10 mg/mL Na-GHB standard in Pepsi was prepared. After the addition of 1.0 g silver nitrate, the sample was poured into two centrifuge tubes. The contents were spun down, and the liquid was decanted. A wash of 5 mL methanol was added to each tube. After the methanol wash was removed, a 5 mL acetone wash was performed. To each sample was added 4 mL de-ionized water and 0.1 g sodium chloride. The contents were mixed, spun down, and decanted onto a watchglass. The sample was dried over a hot-water bath. The solid was dried fully in an oven at 60°C and was analyzed by FTIR. This was repeated with a 10 mL solution of 10 mg/mL Na-GI-IB standard in Diet Pepsi (caffeine free). 17 A measured amount of Na—GI-IB or GBL standard was added to 10 mL of the test beverage/solvent (see Table 4). The sample was precipitated by the addition of 1.0 g silver nitrate. The cloudy mixture was stirred and cooled in an ice-water bath. The solid was removed by vacuum filtration and rinsed with a small amount of de-ionized water. The solid was dried in an oven at 60°C. The dry solid was placed in an Erlenmeyer flask with 2 mL de-ionized water. The contents were heated to approximately 70°C. To the hot mixture was added 0.15 g sodium chloride. The mixture was vacuum filtered. The filtrate was transferred to a watchglass and evaporated on a hot-water bath. The solid was dried fully in an oven at 60°C and was analyzed by FTIR. Table 4. Solutions purified with silver nitrate precipitation/sodium chloride conversion. Beverage/Solvent Chemical Added (Concentration mg/my de-ionized water I GHB (25) I GBL (100) I GHB (25) and GBL (100) Pepsi I GHB (50, 25, 10) I GBL (100) Diet Pepsi (caffeine free) GHB (50,25, 10) Mogen David concord wine I GHB (25) I GBL (100) Molson beer GHB (25) E & J brandy GHB (25) Seagram’s gin GHB (25) Jack Daniel’s whiskey GHB (25) 0.1 M HCl GHB (25) 18 RESULTS AND DISCUSSION S; Forma of GHB The spectra of standard Na—GHB and prepared K-GHB and Ag-GHB are shown in Figures 6, 7, and 8. While the spectra for K-GHB and Ag—GHB are highly similar, there are minor spectral differences between these two salts and Na—GHB. 0.9 i 0.8 J 0.7 i 0.6 ‘ eoueqrosqv 0.5 * 0.4 ‘ 0.3 ‘ 0.2 ‘ 0.1 ‘ 0.0 . 4000 3500 3000 2500 2000 1500 1000 500 Waventmbers (om-1) Figure 6. Infrared spectrum of standard Na-GHB. l9 aoueqrosqv 1.8 “l 1.6 " 1.2 " l 1.0 ‘l l 0.8 ‘ 0.4 1’ 0.2 ' 0.0 L _—'7 3500 3000 2500 2000 Wavenurnbers (cm-1) 1560 Figure 7. Infrared spectrum of prepared K-GHB. aoueqrosqv 1.5 1: 1.4 1 1.31E 1.2 1’ 1.1 E} 1.0 1 0.9 1: 0.8 1 0.7 1 0.6 1 0.5 1 0.4 1 0.3 1 0.2 1 0.1 1 0.0 j 3000 f 2500 2000 Wavenumbers (cm-1) 1500 1000 Figure 8. Infrared Spectrum of prepared Ag-GHB. 20 Eyapfioration of Aqueous Samples of GHB and GBL Upon evaporation over a hot-water bath of a solution of GHB and GBL in de-ionized water, a white solid remained. This was identified as GHB using FT IR. Further, recovery was nearly quantitative. Similar evaporation of a solution containing only GBL in de-ionized water resulted in no detectable solid. Thus, GHB mixed with GBL, de- ionized water, or a combination of the two may be removed by simple evaporation of the solvent. Derivatization of Standafi The mass spectra obtained from the derivatization of AHB, BHB, GHB, and 1,4- butanediol standards are in Figures 9, 10, 11, and 12. Due to the facile loss of a methyl radical from trimethylsilyl ethers, a molecular ion is not present in the spectra for any of these compounds. Derivatization of a GBL standard yielded mutiple peaks in the chromatogram, one of which was identified as the GHB derivative. This may have come from trace amounts of GHB in the GBL standard. Due to the intensity of the GI-IB derivative peak in the chromatogram, it is more likely that the derivatizing agent, BSTFA, reacted with the lactone to form the GHB derivative. 21 Abundance 147 25000001 1 20000001 15000001 1 1 10000001 . 73 117 1 5000001 233 204 rss 131 L 1111 A a. A V I V717 T Y—T 10 133 59 88 13 I 0 ‘1 v ‘vlllv‘rlrli 1‘; r v llr 111 It!" f 7 r r v ' I J r I I I. .. ., . r' .. nflz-> 40 60 80 100 120 140 160 180 200 220 Figure 9. Mass spectrum of derivatized GHB. Abundance 13 I 2500001 1 147 2000001 73 1500001 1 1000001 500001 66 190 205 233 81 ‘7 .11.“ 1. 9.5.1.9331? i 11311 161 175 r L. 219 L 0 71111 ‘ri‘nv—rwlrrrr r11fTvvrrlrrVYI rrrlvrrrlvvrrz—zgr 1 1 r an"> 40 60 80 100 120 140 160 180 200 220 Figure 10. Mass spectrum of derivatized AHB. 22 Abundance 1 1 47 1 1 2500004 . 2000001 150000 1 73 117 1000001 191 500001 88 130 233 7 59 101 '1. l 204 L 4 [Ill 11 11 1 i 1 1 — —. p—g— -1 LALI .1. 1 JLL. o vvrv'rrvr vvrrlrrrr YIIT Yr—fr rift rIvr Irrrlrrvr 1 l nflzn> 4D 60 80 100 120 140 160 180 200 220 Figure 11. Mass spectrum of derivatized BHB. Abundance 1 147 4 1 30000009 1 1 25000001 1 1 20000004 1 1 116 15000001 ‘ 73 10000003 4 4 5000001 101 1 133 177 219 1 .1 l 1.44 .41. o 11-“.llil 1,1111 I 1817. 1'1 [1 I'l 163 11I 1 AA A FIFTY VVVVVVVVVVVV T IIIIIIIIII V I YYYYYYYYYYYYYYYYYYYY V'YY V an~> 40 50 60 70 80 90 100 110 120 130 1i0 150 160 170 180 190 200 210 2201' Figure 12. Mass spectrum of derivatized 1,4-butanediol. 23 Extractions None of the extractions attempted with solutions of GHB were successful. Dry extractions with methanol, isopropanol, and n-butanol performed on evaporated samples of GHB in Pepsi yielded brown sticky residues that were unsuitable for FT IR analysis. A dry extraction with ethyl acetate dried with sodium sulfate on a similar sample resulted in no residue of any kind. Ethyl acetate that was not dried with sodium sulfate did extract some brown residue similar to the alcohols due to water being in the extraction solvent. A liquid/liquid extraction with ethyl acetate on a non-dried sample of GHB in Pepsi also resulted in a brown sticky residue. This was again due to water in the ethyl acetate. The extractions using ethyl acetate also did not produce samples suitable for FTIR analysis. Testing of DichloromethJale Extraction Method A method for extracting GHB from beverages and urine using dichloromethane has been publishedm‘ls The purpose of this portion of the study was to verify this method. No GHB derivative was detected using GC/MS analysis on an extracted and derivatized sample of 10 mg/mL N a-GHB standard in de-ionized water. Further, no GBL was found upon GC/MS analysis of a sample of dichloromethane saturated with Na-GHB standard. This was not surprising as Na-GHB is insoluble in dichloromethane. After these failed attempts, the author of this extraction method was contacted. She stated that the laboratory does not use this technique because "it no longer works."17 Silver Nitrate Microcrystalline Test The addition of silver nitrate to samples containing GHB resulted in the formation of microscopic plates as shown in Figure 13. The detection limits for GHB in a variety of 24 beverages for this microcrystalline test are shown in Table 5. Solutions made with prepared forms of GHB gave the same response as those made with a Na-GHB standard. Solutions prepared with Na-GHB and K-GHB also gave the same response. Further, no difference was found for GHB solutions that were tested when they were fresh, one day old, and one week old. Also, the test was equally responsive when the solvent used was tap water versus de-ionized water. Some solutions were determined to have much poorer detection limits than others. All of these samples precipitated substantially with the addition of silver nitrate, and it is believed that it was the heavy formation of other insoluble silver salts that prevented good plate formation. The detection limits for the solutions that precipitated heavily such as orange juice and beer were not improved by the use of 1% or 10% silver nitrate reagent. Solutions containing a large amount of ethanol tended to evaporate before plates developed fully in size. To give these samples adequate time for plate development, the technique of adding "nose grease" (wiping a portion of a microscope slide across one’s nose)18 was sometimes used to maintain a compact drop and slow solvent evaporation. While many of the blank solutions formed a precipitate with the addition of silver nitrate, no crystal formation was detected in any of these samples. Further, no GBL samples at any concentration yielded crystals. Likewise, solutions of 1,4-butanediol, AHB, and BHB did not result in crystal formation. 25 Figure 13. Plate formation after addition of Silver nitrate 10 mg/mL Na-GHB in Pepsi solution (100x, polarized light). free Coca—Cola Coke Dr Mt. Dew Time lemonade cocktail David wine Molson beer E & J Jack 26 Precipitation with Silver Nitrate for GC/MS Analysis The GHB derivative was detected in all solutions containing GHB with a concentration of 1mg/mL or higher. Splitless injections were tried for samples below this concentration. All samples tested in this manner including the beverage blanks contained a peak in the chromatogram with the same retention time as the GHB derivative. Although the corresponding mass spectrum had peaks associated with column bleed as well as unexplained peaks, the spectrum was very similar to the GHB derivative. Analysis of acetonitrile blanks between runs did not show the presence of any GHB derivative. However, when a blank consisting of BSTFA was analyzed, the quasi- spectrum of the GHB derivative was obtained. This was again tried with a freshly opened ampoule of BSTFA with the same results. It is believed that the instrument was contaminated with GHB, and that this GHB was derivatized by the injected BSTFA and eventually detected. This contamination was only detectable with splitless injection. All samples containing GHB that were run with split injection showed no sign of the GHB derivative below the lmg/mL level. The mixed GHB and GBL samples that were analyzed followed the 1mg/mL detection limit. The GBL did not hinder the analysis. Analysis of the solutions containing only GBL did not show presence of the GHB derivative because the GBL was not carried over in the precipitation step. Due to time contstraints, some of the soft drinks and the juices used in the microcrystalline portion of the study were not tested with this method. It is expected that GHB mixed in the beverages not tested would follow the same concentration limits as shown here because the precipitation of GHB is the key step in the procedure. 27 Precipitation with Silver Nitrate for FT IR Analysis The precipitation of GHB as a silver salt followed by conversion to Na-GHB using sodium chloride was an adequate method to purify GHB in all the beverages tested at a concentration of 25 mg/mL or higher except for the wine sample. It is believed that components in the wine also precipitated out of solution and contaminated the final product. The mixed GHB and GBL solutions also resulted in purification and identification of GI-IB. The GBL did not hinder the analysis as it was lost in the filtrate following the precipitation. Inadequate residue was obtained from the solutions containing only GBL so that further analysis was not possible. The 10 mg/mL GHB in Pepsi sample that was centrifuged rather than filtered also was successfully purified and identified. It is believed that less sample was lost by avoiding filtration. However, this method was unsuccessful in purifying the sample consisting of GHB in Diet Pepsi, most likely because the beverage contained more organic salts or other components that precipitated out of solution than the Pepsi sample. Again, due to time constraints, some of the soft drinks and the juices used in the microcrystalline portion of the study were not tested with this method. It is expected that purification of GHB mixed in the beverages not tested would not be as successful as most of those discussed above because like the wine, these beverages produced heavy precipitate with the addition of silver nitrate. This precipitate would likely remain as contamination throughout the remainder of the procedure. 28 CONCLUSIONS The analysis of GHB is greatly complicated due to the facile interconversion of GBL and GHB. The methods outlined here provide forensic chemists with sound techniques to identify GHB to the exclusion of all other substances including GBL. The interpretation of the results obtained by following these methods, however, is sometimes less definite. Was GHB originally placed in the beverage or was it GBL only? Did conversion of GBL to GHB take place while the suspect had possession of the beverage or did it take place solely in a police property room? These are questions that are difficult to answer conclusively. Better data, such as that presented in the study by the ‘IFDA6, is needed for a clearer picture of interconversion between GBL and GHB. A second note of caution involves research involving GHB by some forensic laboratories. The dichloromethane extraction procedure10 investigated in this work was presented at a Midwestern Association of Forensic Scientists meeting. The authors of the microcrystalline test previously discussed stated, “An earlier experiment had shown no crystal growth with a reagent of silver nitrate alone.” 11 This study was published in the Journal of Forensic Sciences. Incorrect work is sometimes presented and published. Forensic chemists must question and test new methods before they are used in actual casework. The caseload of the forensic chemist in most laboratories is such that efficiency in screening samples and carrying out analyses is crucial. And with the increased media attention regarding GHB, more suspect GHB samples are being submitted to laboratories. This research and the work of others has provided the forensic chemist with various 29 means to thoroughly and efficiently analyze suspect GI-IB samples. A step-by-step protocol for such analyses is detailed in the appendix. While much research has been performed recently regarding the analysis of GHB, there are questions remaining to be answered, and, as the saying goes, there is always room for improvement. In regard to the methods detailed here, one area requiring improvement is the loss of Ag-GHB following precipitation. A technique that would make Ag-GHB less soluble in aqueous solution would greatly increase the sensitivity of the method. A second adjustment that has the potential to improve detection limits is evaporation of the beverage before analysis. This may result in the recovery of more Ag- GHB. In the case of GC/MS analysis, the use of other derivatizing agents may be beneficial, especially since it is unclear whether BSTFA derivatizes not just GHB but GBL as well. 30 APPENDIX -- RECOMIVIENDED ANALYSIS OF GHB SAMPLES Screening of Suspected GHB Samples 1) solids and liquids (except residues) —9 place portion of sample in dilute acid and extract with toluene, chloroform, or another appropriate solvent, run on MS and look for GBL/GHB 2) solids and liquids —> use silver nitrate microcrystalline test to screen for GHB and cobalt thiocyanate color test to screen for GBL Separation/Identification (After Positive Screening) Residues —> derivatize directly with BSTFA, run on MS Solids —9 direct [R if possible; otherwise place portion in small amount of de- ionized water, precipitate with silver nitrate, filter, convert with sodium chloride, and analyze with IR; if insufficient solid is recovered, precipitate a second portion with silver nitrate, derivatize with BSTFA, and run on MS Pure aqueous—> evaporate if appearance of only water, then handle as solid Other liquids —> precipitate with silver nitrate, filter, convert with sodium chloride, and analyze with IR; if insufficient solid is recovered, precipitate a second portion with silver nitrate, derivatize with BSTFA, and run on MS 31 REFERENCES lTakahara, J ., Yunoki, S., Yakushiji, W., Yamauchi, J ., Yunane, Y., & Ofuji, T. 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