Drug and Toxicology Laboratories in Forensic Science PDF

Forensic drug and toxicology laboratories also have defined analyte lists, SOPs, and instrumentation. Compound identifications are confirmed by the combination of tests and comparison to trustworthy reference standards. Because they are new on the illicit market, novel psychoactive substances are in...

Forensic drug and toxicology laboratories also have defined analyte lists, SOPs, and instrumentation. Compound identifications are confirmed by the combination of tests and comparison to trustworthy reference standards. Because they are new on the illicit market, novel psychoactive substances are initially uncharacterized. As information is obtained and reference standards become available, novel analytes are integrated into laboratory processes as known substances. Figure III.3 illustrates the migration process. Because chemical identification in forensic laboratories depends on reference standards, the typical target analyte approach is inadequate for uncharacterized substances. The novel drug must be isolated, purified, synthesized, and characterized by advanced MS, typically HRMS combined with NMR. Commercial and affordable reference standards are needed before laboratories can integrate the new compound into routine assays. Figure III.4 provides a framework for the next four chapters. At the center is the central nervous system (CNS), which is affected by drugs and abused substances. The six drug classes are groupings based on CNS effects; this chapter will address classification schemes. Structures shown in green represent traditional drugs, and those highlighted in yellow are examples of novel compounds in the same category. Analysis of traditional and novel seized drugs focuses on the first two chapters in the section. We examine drugs in the body in the third chapter, while the fourth delves into forensic toxicology. The compounds highlighted in orange in Figure III.4 are neurotransmitters (NT) involved in nerve transmission in the CNS. The gray boxes list corresponding receptor sites. We will discuss how abused drugs cause effects in the CNS and brain by mimicking or interfering with neurotransmitters. The blue boxes identify endogenous (native to the body) NTs that bind to receptors shown in the same category. Although overwhelming at first, the concepts and compounds should be comfortably familiar by the end of this section. We will take it a piece at a time. **Figure III.1** Most frequently identified drugs over the last 2 years for which complete data is available. This was taken from the National Forensic Laboratory Information System (NFLS, https://www.nflis.deadiversion.usdoj.gov/). This is a publicly available resource supported by the US Drug Enforcement Administration. NPSs are highlighted in yellow. (Adapted from U.S. Drug Enforcement Administration, Diversion Control Division. (2019). National Estimates for the Most Frequently Identified Drugs: 2019, Table 1. Springfield, VA: U.S. Drug Enforcement Administration.)Drugs and Poisons 213 **Figure III.2** Trends in overdoses in the last two decades. The citation of the source is included in the figure. **Figure III.3** Traditional and novel substances. Traditional drugs are well characterized while new substances are not. Overtime, novel drugs become integrated into standard analyses as data and reference standards become available.214 Forensic Chemistry **Figure III.4** The next four chapters will cover aspects shown in this figure. (Background image sourced and used with permission from Shutterstock.com.)DOI: 10.4324/9780429440915-9 215chapter 6 Overview of Drug Analysis CHAPTER OVERVIEW This chapter will explore the legislative and regulatory milestones that define and regulate abused substances pre-sented in the framework shown in Figure 6.1. The groupings shown are among many methods by which abused substances are classified. The substances are examples of traditional drugs in each category. In the next chapter, we will add examples of novel substances within these categories. Analytical methods will be discussed and described. Examples of online data sources and how they are exploited in forensic chemistry will be introduced but as with all things online, expect change and evolution. The goal is to supply the tools that will allow you to find sources as they develop and migrate. 6.1 CLASSIFICATION Drugs can be described and classified based on chemical characteristics. The presence or absence of ionizable centers (Chapter 3) allows the categorization of a molecule as acidic, basic, amphoteric, or neutral. Critical for analytical **Figure 6.1** Classes and examples of traditional abused drugs. The orange boxes supply a definition, and the gray boxes describe the effects of the drugs in the class on the central nervous system.216 Forensic Chemistry and toxicological work, these categories are not meaningful in the legal or regulatory context; forensic chemists understand many other descriptors. For example, the historical term **alkaloids** refer to substances extracted from seed plants and are natural products. Because these compounds are basic, they have an alkaline character. This group includes other plant-derived and related drugs such as **opiate alkaloids** (derived from the opium poppy) and **tropane alkaloids**, a group that includes cocaine. The term **opioid** is inclusive of opiates and synthetic compounds like fentanyl that cause the same physiological effects. Non-alkaloid basic compounds of forensic interest include the **phenethyl** (or **phenylethyl**) **amines** such as amphetamine, methamphetamine, and MDMA. Drugs can also be described as natural, semi-synthetic, or synthetic, although this differentiation is problematic in the age of advanced synthesis techniques. Natural drugs include morphine and cocaine which can be extracted directly from plant material. Heroin(diacetylmorphine) is semi-synthetic because it is produced by simple acetylation of morphine. Drugs like fentanyl and Valium® (a **benzodiazepine**) are synthetics. Some drugs are grouped based on how they are used and abused. Within a group, the chemical structures are typi-cally similar, as are the physiological effects. Not all are abused, and a substance can be a member of several catego-ries. Examples include: **Predator Drugs**: Also known as date-rape drugs and **drug-facilitated sexual assault** (DFSA) agents, these sub-stances are used to incapacitate a victim for sexual purposes. Current date-rape drugs, aside from alcohol, include ketamine, Rohypnol (flunitrazepam), and gamma-hydroxybutyrate (GHB). When the drug is mixed in a drink, the effects can range from disorientation to unconsciousness and short-term memory loss. Victims may awaken several hours after an assault with no memory of the event or the few hours leading up to it. Consequently, they may delay seeking treatment until the drug and metabolites are no longer detectable by toxicological assays. **Club Drugs**: These are drugs used at parties and clubs frequented by young people; many are also predator drugs. In addition to the compounds listed as predator drugs, Ecstasy (MDMA) is a club drug. Other hallucinogens, such as LSD and psilocin mushrooms, are sometimes included in this category, as are the stimulant-hallucinogens phencyclidine (PCP) and methamphetamine. **Human Performance Drugs**: Also referred to generically as **performance-enhancing substances** (PES), these drugs consist of substances that improve or impair one's performance, most notably anabolic steroids, and alco-hol. **Anabolic steroids** include dozens of drugs based on testosterone. These drugs are abused by athletes in attempt to increase their muscle mass and decrease the recovery time after strenuous training and competition. PESs are not just a concern in human athletes; for example, there are many toxicologists working in the horse racing industry. **Inhalants**: Unlike the other groups of drugs listed in this section, **inhalants** are ingested through breath to produce their desired effects. Examples are paint thinners, nitrous oxide (laughing gas), gasoline, cleaners, and nail polish. Any substance that has a volatile component can be used as an inhalant, and in general, these substances have depressant effects like those of alcohol. **Analgesics**: These are drugs that relieve pain. Among the common analgesics are aspirin, ibuprofen, naproxen sodium, and morphine. Aspirin and related drugs are nonsteroidal anti-inflammatory drugs (**NSAIDs**), which stop pain by reducing fever and inflammation. **Narcotics**: Narcotic drugs have analgesic effects and tend to depress the CNS and promote sleep. Opiate alkaloids (drugs derived from the opium plant) are the best-known narcotics. This group includes morphine, codeine, heroin, hydromorphone, oxycodone, and hydrocodone. Of all these ways to group and describe drugs, two schemes have the most utility in forensic drug analysis: classification by legal status and classification by physiological effect. Figure 6.1 shows groups by physiological effect, but these considerations alone do not dictate what substances are subject to legal and regulatory control. The drug classes we reference most often in these chapters are shown in Figures III.1 and 6.1, along with example structures. This chapter centers on traditional drugs (for lack of a better term). In some cases, these substances have existed in some cases for over a century. The next chapter addresses the novel synthetic substances. As we noted in the Overview for this section, traditional drugs still constitute most drug seizures and casework, so it is vital to be familiar with these groups and substances.Overview of Drug Analysis 217 Currently, the most frequently identified drugs include: methamphetamine, a synthetic stimulant marijuana (containing cannabinoids) cocaine, a semi-synthetic stimulant heroin, a semi-synthetic opiate fentanyl, a synthetic opioid, and a **diverted pharmaceutical** alprazolam (Xanax®), a synthetic sedative/hypnotic and diverted pharmaceutical. This last substance is classified as a benzodiazepine, a large group of synthetic drugs that includes diazepam (Valium®) and clonazepam. Forensic chemists must be fluent in this language and method of discussing and describing drugs. The next few sections provide the information and resources needed to hone your skills. 6.2 LEGISLATION AND REGULATION The term *drugs of abuse* applies to drugs and related compounds subject to regulations and laws because of their potential to be abused and cause harm to individuals and society. Abused drugs are usually addictive, causing physi-ological dependence, psychological dependence, or both. Addiction can drive behavior that is harmful to others, such as driving while intoxicated or committing crimes to fund addictive behaviors. Physical addiction is traceable to a biochemical or physiological change caused by repeated use of the substance. The potential for harm to self and harm to society drives legislative responses and regulations. The link between addiction and legislation traces back to the isolation of morphine from opium in the early 1800s \[1\]. The pain-relieving ability of the drug led to a fundamental change and advance in pain control. The accompanying dependence that often developed in users was first studied systematically in 1878 \[2\]. The report described the now familiar symptoms of user fixation on obtaining and using the drug, and withdrawal symptoms occurring if they stop. It was clear that addiction resulted in self-harm and could drive behaviors that would harm others. In subsequent years, addiction to other natural and semi-synthetic substances such as cocaine and heroin, in addition to opium and morphine, drove passage of the Harrison Anti-Narcotics Act of 1914, the first in a long history of laws related to drug control. A summary of relevant federal drug legislation in the forensic context is summarized in Table 6.1. Laws and regulations in other countries follow similar patterns. In the United Kingdom, there are two overarch-ing laws related to abused drugs. The Misuse of Drugs Act was passed in 1971 which established the legislative framework. It also instituted categories (called Classes) to group drugs based on how they are used medically and in research. For example, Class A substances include cocaine and heroin, Class B includes amphetamine, and Class C includes steroids and benzodiazepine sedatives like Valium®. The Psychoactive Substances Act of 2016 addressed novel substances and will be discussed in the next chapter. A good source of information across countries and regions is the United Nations Office on Drugs and Crime (UNODC). The UNODC Early Warning Advisory on New Psychoactive Substances (www.unodc.org/LSS/Home/ NPS), which we will discuss and use in the next chapter, has a link called Legal Resources that provides an alphabeti-cal list of drug laws by country. The CSA of 1970 and subsequent legislation that amended it are the framework for drug control legislation in the United States. The Drug Enforcement Administration (DEA), under the Department of Justice (DOJ) is tasked with implementing it. The text of the Act and how DEA implements it can be found on the DEA website (https://www.dea. gov, last accessed January 2022) and associated publications available there.218 Forensic Chemistry Scheduling a substance refers to placing it on one of five lists or Schedules (Table 6.2) based on considerations including: Risk to the public Utility in medical treatment Abuse potential Current scientific knowledge of the substance Potential use as a precursor to a controlled substance The DEA is tasked with making Scheduling recommendations, also available on the website. The DEA does not decide whether drugs are made available by prescription -- this is the responsibility of the Food and Drug Administration (FDA). The CSA has been amended several times since 1970. The federal **Anti-Drug Abuse Act** of 1986 expanded the list to include **designer drugs**, synthetically produced analogs of controlled substances. At the time, the list of such drugs was relatively small and included substances such as PCP (phencyclidine) and methamphetamine. The act also defined penalties for possession. The term designer drugs is still seen in some contexts relating to NPSs. **Table 6.1** Drug control legislation and in the United States Year Legislation Key requirements Enforcement agency 1914 Harrison Anit-Narcotics Act Registration requirements for importers, distributors, and manufacturers Prescription controls Treasury Dept 1937 Marijuana Tax Act Taxation; de facto ban Federal Bureau of Narcotics (FBN), Treasury Department 1970Controlled Substances Act (CSA) Scheduling to dictate penalties and access; comprehensive legislative approach Drug Enforcement Administration (DEA), Department of Justice 1984Comprehensive Crime Control ActCSA amended to allow temporary scheduling 1986Federal Controlled Substances Analogue Enforcement Act (CSAEA) CSA amended to address analogs Established sentencing for possession 1986 Anti-Drug Abuse Act Further expansions 1988 Chemical Diversion and Trafficking Act (part of Anti-Drug Abuse Act of 1988) CDTA Limit access to precursors and chemicals used in illicit synthesis 1993Domestic Chemical Diversion Control ActClarified listing for ephedrine products Established registration system related to listed chemicals 1996Domestic Comprehensive Methamphetamine Control Act Establish regulations related to methamphetamine and amphetamine precursors 2000 Methamphetamine Anti-Proliferation Act (MAPA) Enhanced penalties for manufacturing and trafficking 2003 Illicit Drug Anti-Proliferation Act Targeted phenethylamine synthetics (MDMA, etc.) 2005Combat Methamphetamine Epidemic Act (CMEA) Limit access to precursors (PPA, ephedrine, pseudoephedrine) 2012 Synthetic Drug Abuse Prevention Act (SDAPA) Extended time on temporary scheduling Placed 26 NPS on Schedule I *Sources:* 1. Brown, K.E. \"Stranger Than Fiction: Modern Designer Drugs and the Federal Controlled Substances Analogue Act.\" Arizona State Law Journal 47, no. 2 (2015). 2. Sacco, L.N. \"Drug Enforcement in the United States: History, Policy, and Trends.\" Washington, DC: Congressional Research Service, 2014. 3. USDOJ. \"Drugs of Abuse: A DEA Resource Guide 2016.\" edited by Drug Enforcement Administration Department of Justice. Washington, DC, 2016.Overview of Drug Analysis 219 The CSA has also been amended to combat illicit synthesis of drugs by including many of the key precursor chemi-cals needed for making methamphetamine and other clandestinely produced drugs. Rather than list all precursors as controlled substances, the **Chemical Diversion and Trafficking Act (CDTA)** was passed in 1988 and amended in 1993. This legislation created two lists of regulated chemicals controlled to deter diversions of these compounds for clandestine synthesis. The lists are also available on the DEA website. Notable among the chemicals on List I are "ephedrine, its salts, optical isomers, and salts of optical isomers," as well as "phenylpropanolamine, its salts, opti-cal isomers, and salts of optical isomers." All these precursor substances are used in the synthesis of amphetamine and methamphetamine. Note the inclusion of "all salts and optical isomers," typical wording in the regulations. Compounds such as iodine, sulfuric acid, and diethyl ether are on List II. These are necessary ingredients for the synthesis of many clandestine drugs but have legitimate uses. Section 6.4.3 delves into clandestine synthesis. The federal **Methamphetamine Anti-Proliferation Act** (MAPA) of 2000 and the Combat Methamphetamine Epidemic Act (2005) addressed methamphetamine precursors. In 2003, the Illicit Drug Anti-Proliferation Act tar-geted compounds related to methamphetamine such as MDMA ("Ecstasy," "Molly," 3,4-methylenedioxymetham-phetamine). In particular, the act addressed places where manufacturing and use occur (i.e., parties). The wave of NPSs that have appeared in the last 15 years illustrated shortcomings in the CSA and Scheduling regula-tions. These compounds' structures are purposely altered to evade existing laws, while providing users with the same or similar effects as the drugs they mimic such as morphine, fentanyl, or THC. Many are created by simple chemical changes to molecular skeletons such as adding a halogen or lengthening an alkyl chain. Despite what might seem like a minor structural change, the mechanism of action and toxicity are unpredictable. Toxicologists may first find them because of an emergency room visit or death, and there is inevitably a lag between detecting the presence of a new drug and definitive characterization of the structure. The road from NPSs to integration into routine testing in forensic labs (Figure III.3) can take years. To combat this issue, the 2012 Synthetic Drug Abuse Prevention Act (SDAPA) was passed. This act added several NPSs (cannabinoids and cathinones) to the CSA. Also, temporary scheduling was made more flexible to address what the legislation referred to as "imminent harm." The bill included a pharmacological definition of a **cannabimimetic agent** as "any substance that is a cannabinoid receptor type 1 (CB1 receptor) agonist as demonstrated by binding studies and functional assays within any of the following structural classes." A list of known novel cannabinoids fol-lowed. Regulation and control of novel substances are migrating from structural to pharmacological definitions; this is why forensic chemists need basic knowledge of action mechanisms. The wording highlights a topic we will encounter again regarding how to define compounds and structures. When the legislation referred to the CB1 receptor (Figure III.4) and compounds as agonists (ones that bind to that receptor), this is a **pharmacological definition**. Any compound that binds with this receptor and falls into one of the categories listed is a cannabimimetic agent. This language provides flexibility that the original CSA lacked in that compounds had to be specifically listed. As a side note, the use of the term "cannabimimetic agent" has been superseded by "cannabinoid." **Table 6.2** Drug schedules in the United States Schedule Accepted medical use Controls on prescriptions Required security Potential for abuse Addiction potential Example **I** None Research only Vault or safe Highest Severe Heroin **II** Some accepted uses with restrictions Written prescription with no refills Cocaine Fentanyl **III** Accepted uses Written or oral (phone in), limits on refills and time Secured area Moderate to low Ketamine **IV** Accepted uses Written or oral (phone in), limits on refills and time Secured area Ambien **V** Accepted uses Over the counter, written, or oral (phone in), limits on refills and time Secured area Low Codeine preparations220 Forensic Chemistry **EXHIBIT 6.1 WASTED EVIDENCE** Wastewater epidemiology was used during the height of the Covid-19 pandemic to check the wastewater from college dormitories and other sources for the virus. Monitoring wastewater can also shed light on illicit drug use and clandestine laboratories. The resulting data provides forensic intelligence used for investigative purposes rather than legal proceedings. Recent analysis of wastewater across Europe (using LC-MSn) obtained data on drugs and metabolites in several categories. The report highlighted regional differences as well as interesting temporal trends. For example, the amphetamine concentrations were higher during the weekends compared to weekdays, while methamphetamine levels were more consistent across the week. Also noted was the development of analytical methods capable of distinguishing enantiomers, and ingested vs. dumped drugs. Another study, published in 2019, focused on wastewater treatment plants in Sydney, Australia, using a similar analytical scheme. The two figures here summarize the findings of this study. The authors noted that wastewater analysis could be combined with other investigative tools better to understand the clandestine drug market and consumption patterns. **Exhibit 6.1 Figure 1.** Influx of substances into three wastewater treatment plants in Sydney, Australia. Benzoylecgonine is a metabolite of cocaine; EDDP is a novel psychoactive substance we will discuss in the next chapter. (Reproduced with permission from open access Taylor and Francis publication Bannwarth, A., et al., The use of wastewater analysis in forensic intelligence: Drug consumption comparison between Sydney and different European cities, *Forensic Sciences Research* 4 (2) (2019) 141--151. DOI: 10.1080/20961790.2018.1500082.)Overview of Drug Analysis 221 6.3 DATA SOURCES There are many resources available for obtaining data on seized drugs. For years, the primary resources were large desk reference books such as the Physician's Desk Reference (PDR), Clarke' Handbook of Drugs and Poisons, and the Merck Manual. Some of these reference titles are available electronically, while others are discontinued. In the last decade, the availability of free resources on the internet has been a welcome added source that expands and improves every year. This chapter explores methods (and shortcuts) to find physicochemical properties and seized drug analysis data. These resources evolve and migrate, so it is essential to develop searching and evaluation skills. Critical appraisal of the quality and reliability of data sources is as important as finding them in the first place and citing them. You will discover that data will not always agree exactly; you may find pKa values from two slightly different sources (8.6 vs. 8.7 for example). How do you know which is right? Look for source notations such as the refer-ence to peer-reviewed articles or a notation such as EST, which would mean that the value is estimated (EST) based on simulation. This may be all that is available, but consensus across sources is an indicator of reliability. It is good practice to cite your sources in calculations. Table 6.3 lists frequently used resources for data relevant to forensic chemistry. Types of data that analysts use often include structure, molecular weight, exact mass, acid-base character and pKa, logP, and solubility. Codes related to structure and classification are also useful, as are chromatographic and spectral data. The best place to start is usually PubChem, as it is a central repository that links to many other resources. Sources: 1. Perspectives on Drugs: Wastewater analysis and drugs: a European multi-city study. 2019, www.emcdda. europa.eu/topics/pods, last accessed March 2021. 2. Bannwarth, A., et al., The Use of Wastewater Analysis in Forensic Intelligence: Drug Consumption Comparison between Sydney and Different European Cities, Forensic Sciences Research 4 (2) (2019) 141--151. DOI: 10.1080/20961790.2018.1500082. **Exhibit 6.1 Figure 2.** Patterns of influx over time at WWTP-1. (Reproduced with permission from open access Taylor and Francis publication Bannwarth, A., et al., The use of wastewater analysis in forensic intelligence: Drug consump-tion comparison between Sydney and different European cities, *Forensic Sciences Research* 4 (2) (2019) 141--151. DOI: 10.1080/20961790.2018.1500082.)222 Forensic Chemistry To illustrate how easily data can be obtained, we start with a simple example and use resources from Table 6.3. Assume that a seizure arrives at the lab with 5 oval capsules, half turquoise blue, half white. The encapsulated material is opaque and is labeled as "MUTUAL 866." This is called the **imprint**. With materials that appear to be commercial pharmaceutical preparations, the first step is to seek information regarding the identification of the formulation. We can use the PDR phone app (mobilePDR) and enter information about the color, shape, and imprint. A search yields Temazepam, 7.5 mg capsule. This type of information, physical descriptors of the items, is referred to as **pharmaceutical identifiers**. They include color, shape, size, and imprint. Thus, at this point, we have a tentative identification and a hypothesis to confirm or refute; many counterfeit pharmaceuticals look similar to legitimate commercial products. Next, you might search CHEMID Plus to learn more about temazepam (Figure 6.2). The structure and formula weight are provided along with several information categories, starting with classifications. Notice that this sub-stance is listed on Schedule IV of the CSA. The remaining data obtained at CHEMID is shown in Figure 6.3. The top **Table 6.3** Resources for chemical and biochemical data Resource URL\* Data available Notes PubChem pubchem.ncbi. nlm.nih.gov/ Links to all kinds of chemical, biochemical, toxicological, and analytical data The single best curated resource for chemical and pharmacological information currently available ChemID Plus chem.nlm.nih. gov/chemidplus/ Links to structure, toxicology, and physicochemical descriptors Good source for 3D structures NIST Webbook https://webbook. nist.gov/ chemistry/ Chemical data and descriptors including spectral data Source for mass spectra of a range of compounds SWGDRUG monographs https://www. swgdrug.org/ monographs.htm Detailed information about drugs of abuse Excellent source of spectral and analytical data and novel substances ENFSI https://enfsi.eu/ See Documents link Many publications related to seized drugs; extensive cross-listing with SWGDRUG UNODC https://www. unodc.org/LSS/ Home/NPS Focus on novel drugs and global perspective The links to other portals have good general information Drugbank go.drugbank.com/ Extensive links to pharmacological information Accessible through Pubchem EMCDDA www.emcdda. europa.eu Drug abuse trends, information, and reports from the European Union Great source for information on trends and current topics Physician's / Prescribers Desk Reference (PDR) https://www.pdr. net/ Mobile app is useful for finding pharmaceutical identifiers. Need to set up account (free). The Physician's Desk Reference was retired in 2017; this is the successor. The website has some information, but the mobile app is the best source for identifiers National Forensic Laboratory Information System https://www.nflis. deadiversion. usdoj.gov/ Data collected from forensic laboratories in the US Current trend data and reports; includes toxicology labs and Coroner/Medical Examiners \**:* as of March 2021.Overview of Drug Analysis 223 frame shows links to resources, with the ones of most interest highlighted. The PubMed links take you to compila-tions of published papers, including open access sources. Toxicological information and references are found in the second frame in Figure 6.2. Finally, the lowest frames provide data on physical properties. The notation "EXP" refers to experimentally derived data. **Figure 6.2** (a) Two pills received as evidence and submitted to the PDR app for tentative identification. (b) Results of a ChemID search for the active ingredient temazepam.224 Forensic Chemistry **Figure 6.3** Remaining results from the ChemID search.Overview of Drug Analysis 225 Next, you could search PubChem for additional information. This provides structures, including 3D versions. As seen in Figure 6.4, a list at the right provides links to many data sources, including available spectra. The pharmacol-ogy and biochemistry data are useful in toxicology and for understanding mechanisms of action. For this compound, available data includes retention indices, EI-MS (from the NIST Webbook), other MS spectra from other databases, MS/MS information, HRMS, and FTIR and Raman spectra. One of the advantages of PubChem is that many of the resources include dates entered and updated. The best way to learn how to use these resources is to try your own search and see where it leads. As was noted above, data resides in more than one place and, while not necessarily identical, should be consistent. For example, more than one study may have been published on pKa, LogP, or solubility; variation is expected. Use this type of data for designing assays or evaluating physicochemical or toxicological characteristics. However, you would not, for example, use a mass spectrum downloaded from one of these web references as the sole method of confirming the identification of temaze-pam. Instead, you would obtain a reference standard and proceed as per laboratory standard practice. You need to be resourceful in finding data and information, while understanding the proper role of such data in an analytical scheme. Finally, as an example of how this retrieved data could be used, suppose you were going to design a simple extraction targeting temazepam so that you could confirm or refute the hypothesis that the pills seized as evidence are what the pharmaceutical data suggests. As discussed in Chapter 3, taking advantage of acid-base character is one way to approach the problem. Is temazepam acidic, basic, or neutral? The structure (Figure 6.2) shows three potential ion-ization sites -- two nitrogen atoms on the rings and one hydroxyl group. The ChemID data (Figures 6.2 and 6.3) does not list a pKa but PubChem lists predicted values for the strongest acidic site as 10.68 and strongest basic site as −1.4. Both values were calculated rather than determined experimentally. How should this be interpreted? The stronger the acid, the larger the Ka, and the more negative the pKa. Here, the strongest acidic pKa is \>10 (Drugbank), which suggests that the acid site (--OH) is not easily ionized. Conversely, the strongest basic site has an estimated pKa of −1.5 which suggests that the basic site defines whatever ionization occurs. Confirmation of this deduction can be found in the typical salt form of the drug. The drug exists as a sulfate or hydrochloride salt, confirming its basic character. A drugs' salt form provides a handy shortcut for determining acid-base character. If no salt forms are listed, and no pKa values are found across references, this suggests a neutral drug. At this point, you would have enough information to develop an extraction for GC-MS analysis to confirm or refute your hypothesis that the capsule contains temazepam. There will be reliable library spectra for this compound, and **Figure 6.4** PubChem search results for temazepam. Links to information are shown in the highlighted box including proper citation and download boxes. 226 Forensic Chemistry the available reference standard for comparison since this is traditional drug. A small amount of powder from a capsule could be placed in water adjusted to a basic pH to drive the drug to the neutral B form and extracted with a solvent like hexane. A simple solvent **dilute-and-shoot** (with filtering or centrifuging) is also practical employing methanol. The drug is soluble in water (\~164 mg/L as per Figure 6.3), so methanol would be appropriate in sufficient amounts. A limited number of solvents are commonly used in forensic settings, often dictated by instrument compat-ibility. Methanol, ethanol, acetonitrile, hexane, or ethyl acetate are typical. For mass spectral and chromatographic data, the NIST Webbook is an excellent place to start. The MS data for EI mass spectra are also in the commercial libraries, but this format is useful on occasion. Chromatography data is sup-plied but may be dated. The Scientific Working Group for Seized Drug Analysis (SWGDRUG) hosts a collection of "Drug Monographs" with mass spectra and other relevant data. Finally, an excellent and often unappreciated data source is technical or application notes at vendor websites. 6.4 DRUGS AS PHYSICAL EVIDENCE **6.4.1 Five P's** The analysis of materials suspected to be or to contain controlled substances is the largest portion of the workload in most forensic laboratories. These examinations are called **seized drug analysis** or **solid dose analysis**. When sus-pected controlled substances are submitted as physical evidence (**exhibits**), the forensic chemist must identify and, in some cases, quantify the controlled substances present. The most common forms of drug evidence can be summa-rized as the "five P's": powders, plant matter, pills, precursors, and paraphernalia. Powders include colored powders from crystalline white to resinous brown, and many, such as heroin and cocaine, are derived directly or indirectly from plants. Some submitted solids are oily and odiferous, and some are best described as "goo." Hashish, for exam- ple, a concentrated form of marijuana, lies between plant and powder. Typical plant matter exhibits are marijuana, mushrooms, and cactus buttons. As biological evidence, plant matter is stored to prevent rotting and degradation before analysis; failure to do so can generate the goo. Pills, such as prescription medications or clandestinely synthesized tablets, are common forms of physical evidence. In cases where the evidence looks like commercial (over the counter (OTC) or prescription) drugs, tentative iden-tifications are made visually using pharmaceutical identifiers as discussed above. In other cases, the pills may have different markings, such as crosses or other imprints. Amphetamines and methamphetamine are often sold in pill form, although the pills are typically cruder than those produced commercially. **Precursors** are compounds or materials used in the clandestine synthesis of drugs such as methamphetamine. Some precursors are controlled and listed on Schedules, while others are not. For example, illicit methamphet-amine was once predominantly made from phenylacetone (phenyl-2-propanone, or P2P), now listed on Schedule II. Methamphetamine can be made from pseudoephedrine, an ingredient in over-the-counter cold and allergy rem-edies. Given that access to these materials is now limited, clandestine chemists turn to **distant precursors** to synthe-size the necessary immediate precursors. **Immediate precursors** require one or two simple steps to convert to the controlled substance; distant precursors require added steps. We explore this topic in detail in Section 6.4.3. Lysergic acid and lysergic amide, precursors of LSD, are listed on Schedule III. Other precursors are not necessarily controlled but are identified as part of investigations of clandestine synthesis. Drug **paraphernalia** is the implements and equipment used in the preparation and ingestion of drugs. Typical items include syringes (a biohazard to the analyst) and cookers used to prepare heroin and other drugs; pipes and bongs (water-filled vessels used in smoking marijuana); and razor blades, mirrors, and straws, used for snorting cocaine. Such items are both a sampling, and an analytical challenge since only traces of material may remain. If needed, the items are rinsed with a solvent to extract the residues. Although effective, this destroys the evidence. If it is unavoid-able, analysts must adhere to any laboratory or legal requirements regarding preserving extracts. **EXAMPLE PROBLEM 6.1** Three tablets are received at a laboratory. The pharmaceutical identifiers indicate that they contain naproxen. You must develop a simple extraction to confirm or refute this tentative identification. Describe your approach and the online resources used.Overview of Drug Analysis 227 *Answer:* The best place to start is PubChem. If you enter the search term naproxen, notice that the drug and the com-mon formulation of naproxen sodium appear (top frame of the figure). Automatically you know the drug is acidic because of the salt form, as discussed in Chapter 3. Take any free information you can get. Sodium salts are soluble, which is not the issue here; you need intrinsic solubility, so select naproxen as the compound. Notice there are several options; read these to ensure that the top match is the best one, as it is here, shown in the second frame of the figure. From there, navigate to *Chemical and Physical Properties* and find solubility. Above the menu bar, you see a citation and a download option. You should cite this page if you used data obtained here; when in doubt, select the APA style. Of the many solubility listings (Figure 2), the one linked to the HSDB is most useful. Naproxen is "freely soluble" in alcohol. Accordingly, you can crush the tablet, transfer a few milligrams to a small test tube, add methanol, mix thoroughly, filter or centrifuge, and draw off a sample for GC-MS injection.228 Forensic Chemistry **6.4.2 Adulterants, Cutting Agents, and Impurities** Illicit drug samples, particularly small seizures and individual dose units, contain substances other than the drug of interest. Several terms describe other materials present including cutting agents, excipients, diluents, and contami-nants. Here, the terms are defined consistent with the literature \[3, 4\] although in practice you may see them used interchangeably. **Diluents** (**thinners** or **cutting agents**) are not drugs and have no pharmacological properties. They are usually inexpensive, easily obtained, and added purposefully. Baking soda and sugars fall into this category. Cutting agents are added to drugs for many reasons, including adding bulk, stretching the supply, and enhancing the effect when ingested. **Adulterants** are pharmacologically active and typically cause effects similar to the drug. Caffeine is added to cocaine because both are stimulants. Similarly, cocaine is a topical anesthetic, so it should be no surprise that cocaine is often cut with local anesthetics such as procaine, lidocaine, or tetracaine. Injected drugs like heroin may contain one of the local anesthetics. Samples often contain more than one cutting agent or adulterant. A recent study \[5\] evaluated over 500 seized drug exhibits from two states (Kentucky and Vermont) and reported on the drug and adulterants found. The results illustrated how drug trends vary among states and over time. Of the sam-ples analyzed, 36.6% had 1--4 cutting agents. Samples from Kentucky were dominated by cocaine and methamphet-amine with levamisole (16.5%) the most frequently identified cutting agent. Diphenhydramine, caffeine, quinine/ quinidine, and acetaminophen followed in order. From Vermont, opioids were the most frequently detected with caffeine (48.8%) as the most common diluent, followed by quinine/quinidine, procaine, and lidocaine phenacetin. Many other diluents were identified, and the authors emphasized their potential toxicity. Rather than purposeful addition, impurities (contaminants) arise from the processing and production of the drug. Plant-derived drugs such as cocaine and heroin undergo several processing steps and are susceptible to the intro-duction of impurities. For example, the detection of lead in heroin and methamphetamine samples is probably attributable to lead vessels and containers used in processing \[3\]. Solvent residues and impurities from reagents also contribute to contaminants; the more steps and ingredients used, the greater the potential for contamination. Diluents of cocaine and heroin often include sugars (sucrose and glucose as examples), starch (such as corn starch), and baking soda/baking powder \[3,4,6\]. Just as there are trends in drug abuse patterns, diluents and cutting agents change over time. In the 1980s, cocaine was typically cut with lidocaine and sugars. Now the list includes caffeine, levamisole, phenacetin, diltiazem, hydroxyzine, and procaine. Levamisole is an interesting addition to this list; it Overview of Drug Analysis 229 treats parasitic worm infections. Removed from the US market by the year 2000, it is still used in veterinary medi-cine. This seems an odd choice for dilution of cocaine, but there is some evidence indicating it enhances the effect of cocaine \[3,7\]. Levamisole and its isomer dexamisole are now common cutting agents \[8\]. Knowledge of cutting agents and impurities can supply **investigative information** useful in profiling (discussed in the next section) and understanding toxicity. Investigative information has no bearing on the legal aspects of the case. Still, it can help investigators identify drugs from the same shipment or the synthetic route, for example. Additionally, many cutting agents are toxic \[5\] alone or in combination with the drug in the sample. For example, a heroin sample cut with fentanyl will have significantly different physiological effects when ingested compared to heroin cut with lidocaine. Analytically, it is essential to know if adulterants will interfere with sample preparation, analysis, or identification. Figure 6.5 illustrates how investigative information regarding a sample was collected using two different analytical methods (GC-MS and LC-QTOF). In the LC-QTOF data (bottom frame), coelution and thus potential interfer-ences with heroin appear at 0.56 and 0.71 minutes. Given that these interferents are chemically related to heroin, spectral interferences such as shared precursor ions are possible and would have to be addressed in method valida-tion (Chapter 2). The same observations hold for the GC-MS data in the top frame. Ingestion of the mixed sample containing several pharmacologically active opioids such as fentanyl and heroin could lead to an overdose death. In summary, cutting agents, diluents, and contaminants can impact forensic analysis. They must be considered in the design and execution of any seized drug assay even though the presence of non-controlled substances has no legal implications. In forensic toxicology, the diluents can impact judgments regarding toxic effects \[9\], medical treatment, degree of impairment, and cause of death determinations. **6.4.3 Clandestine Synthesis** Methamphetamine and amphetamine are two of the easiest drugs to synthesize. This section explores meth-amphetamine as an example of clandestinely synthesized drugs and how the methods used to make a drug can provide useful chemical and investigative information. As laws are changed to control access to precur-sors, methods of synthesis evolve. New precursors may be identified, or more distant precursors synthesized, or entirely new approaches may be developed. The continuous cat-and-mouse struggle applies to NPSs as well. Methamphetamine is the only drug for which we cover synthetic methods in detail. First, methamphetamine remains one of the most common forms of physical evidence in seized drugs (Figures III.1 and III.2). Secondly, the methods are well characterized and are straightforward. This is not always the case, particularly with NPSs. Methamphetamine synthesis serves as an instructive model of clandestine methods, their evolution, and how they can provide useful chemical and investigative information. For more information, there are several excellent reviews and articles available \[10--13\]. Methamphetamine syntheses utilize reductions or **reductive amination** (the addition of an amine group) to a phenethylamine skeleton \[14\]. Chemical methods for synthesizing other related phenethylamines are analogous. Given that dozens of methods are used in clandestine laboratories, a detailed discussion is beyond the scope of the text. Instead, this section highlights current methods while providing the basis for further study. Methamphetamine has two immediate precursors: phenyl-2-propanone (also called P2P, phenyl acetone, and benzyl methyl ketone) and ephedrine/ pseudoephedrine (which are all isomers related through stereochemis-try). Figure 6.6 illustrates the generic chemical conversions for each precursor that lead to methamphetamine. Starting with P2P, the clandestine chemist must reduce the carbonyl group and add a methyl and amine group. The term reductive describes the process. The route from ephedrine or pseudoephedrine is simpler, requiring reduction only. Until the 1990s, domestic clandestine methamphetamine laboratories relied on P2P, a common and versatile sol-vent with many legitimate uses. P2P and related compounds also have potent smells that can betray the location of a clandestine facility. The DEA added P2P to Schedule II of the controlled substances list as part of the Chemical Diversion and Trafficking Act (CDTA) of 1988. Review Section 6.2 for more information. In 2006, the Combat Methamphetamine Epidemic Act (CMEA) was signed into law in the United States. The Act limited access to prod-ucts containing ephedrine and pseudoephedrine.230 Forensic Chemistry **RAPID REVIEW 6.1 STEREOCHEMISTRY** The figure below provides a summary of terms related to isomers and stereochemistry. Of most interest in clan-destine synthesis are stereoisomers. The naming of enantiomers is based on the Cahn Ingold Prelog system. The diagram shows how this is done. The atom at the center is the chiral center. The numbers refer to the priority of each attached atom, highest atomic number first. Arrange the lowest priority atom pointing away from you as viewed in three dimensions, with the highest priority atom pointed upward. The direction of rotation starting at 1 and moving through 2 and 3 dictates the R (clockwise or right) or S (counterclockwise or left) designation.Overview of Drug Analysis 231 **Figure 6.5** Two chromatograms from GC-MS (top) and LC-QTOF (bottom) obtained from the same sample. (Reprinted from Fiorentin, T. R., A. J. Krotulski, D. M. Martin, et al., Detection of cutting agents in drug-positive seized exhibits within the United States. *Journal of Forensic Sciences* 64 (3) (copyright 2019): 888--96, with permission from Wiley.)232 Forensic Chemistry Figure 6.7 illustrates common methods of clandestine synthesis. We focus on two methods that start with ephedrine/ pseudoephedrine as precursors. The **Nagai**/**red cook** method reduces the -OH group to hydrogen via an alkyl halide and is fast with high yields \[15--17\]. One variant involves reflux while another is "cold" or mildly heated \[13,16,17\]. Hydriodic acid is used in conjunction with red phosphorus obtained from matches or road flares. The cold cook method differs in that the reactants are not heated as aggressively (or not at all), and the process uses HI generated from iodine (I2), water, and red phosphorus. The second common synthetic route is called the **Birch** method, Birch reaction, or Birch reduction \[18\]. The reac-tion is not a traditional Birch reduction described in organic textbooks and references in which a benzene ring is reduced, resulting in two double bonds rather than three. Here, "Birch" may have arisen from the reactants used in the procedure. The descriptor "Birch reduction" is still seen, but "Birch method" or "Birch reaction" is preferred. The Birch method is also called the "Nazi" method, although there are conflicting reports regarding this name's origin. Thus, this synthetic method's naming could not get much more confusing, even if the chemistry is clear (or, rather, blue...). Figure 6.8 shows the procedure and an image showing the deep blue color that forms during the reaction. The solution turns a grayish color as the reaction proceeds. The methamphetamine generated is converted to its salt by bubbling HCl(g) through the solvent. The Birch method's advantages are the ability to boil off excess ammonia and the simple decomposition of residual lithium metal by water. The disadvantage is the need for anhydrous ammo-nia, which can be hazardous, explosive, and corrosive. One-pot methods \[11,13\] circumvent this problem by generat-ing ammonium in-situ through the reaction of sodium hydroxide and ammonium nitrate \[11\]. Stereochemistry (Rapid Review 6.1) plays a role in synthesis and product determination. Because metham-phetamine has a chiral carbon at the ß position relative to the benzene ring, d- and l-isomers exist. Figure 6.9 shows the two optical isomers of methamphetamine (top frame) and the variants of the precursors. The d-form **Figure 6.6** Outline of two synthetic routes to methamphetamine. Overview of Drug Analysis 233 of methamphetamine is the more active, so clandestine syntheses favor d-methamphetamine (also referred to as (+) methamphetamine, the S enantiomer. The synthesis route dictates the stereochemistry of the products. The Nagi method produces only d-methamphetamine \[13\]. The Birch and iodide methods yield (+) methamphetamine if the starting material is either (−) ephedrine or (+) pseudoephedrine. Specialized chiral chromatographic col-umns can separate enantiomers. **Figure 6.7** Details and names of common clandestine syntheses of methamphetamine. (Reprinted with permission from Stojanovska, N., S. L. Fu, M. Tahtouh, T. Kelly, A. Beavis, K. P. Kirkbride, A review of impurity profiling and synthetic route of manu-facture of methylamphetamine, 3,4-methylenedioxymethylamphetamine, amphetamine, dimethylamphetamine and p-methoxyam-phetamine, *Forensic Science International* 224 (1--3) (2013) 8--26. Copyright Elsevier.) **Figure 6.8** Details of the Birch/Nazi/Nagi method. The solution at right shows the deep royal blue color.234 Forensic Chemistry The plethora of synthetic methods and conditions used to create methamphetamine implies that different cook methods generate detectable differences in the final product. In turn, this information can be used by law enforcement as investigative information. Stereochemistry is one example; contaminants and by-products are another. Collectively, the analysis of drugs to obtain information regarding origin, history, and synthesis is called **profiling**. **6.4.4 Profiling** Profiling a drug sample involves analyzing the sample's composition beyond identifying and quantitating the con-trolled substance(s) present. Profiling data is used to categorize drug samples into similar groups to provide inves-tigative information, such as origin, processing, and history. This type of analysis of evidence is a source of forensic intelligence \[19--22\]. Additional goals of profiling can include: Identification of diluents, adulterants, and impurities Elucidation of the extraction and preparation method Elucidation of the synthetic pathway Identification of the drug's geographic origin for plant-derived exhibits Figure 6.10 shows how extraneous compounds are incorporated into a sample at every step. In the case of a plant- based drug (starting at the bottom left of the figure) such as cocaine or heroin, the first step is solvent extraction. Extractions inevitably capture dozens of components in addition to the substance of interest. **Figure 6.9** The stereoisomers of methamphetamine and ephedrine/pseudoephedrine precursors.Overview of Drug Analysis 235 Ingredients and reagents are of variable purity. The synthesis generates by-products that are often characteristic of that synthetic pathway; residual solvents and unreacted components will also be present. In some cases, chiral products or by- products are generated. Every processing step, be it a chemical reaction, extraction, crystallization, or purification, contributes to the sample through sources including by-products, materials leached from containers or glassware, and impurities in reagents such as acids or solvents. Over time, storage and environmental factors such as heat or cold can generate by- products or induce degradation, adding to sample complexity. All these contributions are in addition to diluents, adulter-ants, and cutting agents discussed in the last section. Thus, even carefully produced and processed illicit drug samples carry evidence of the production methods and history. While many of the added compounds are present in trace and ultra-trace concentrations, current analytical methods can detect an impressive number and variety of substances present in a sample. Adulterants and diluents are part of profiling. Figures 6.11 and 6.12 show data from a multiyear study of adulterants and diluents in hundreds of heroin and cocaine samples \[19\]. Almost all the heroin samples were cut with a mixture of acetaminophen (paracetamol) and caffeine, while cocaine samples showed more variety. Over the years studied, the purity of heroin samples was in the range of \~30%--45%, while cocaine was \~10%--20%. One interpretation of these results is that the cocaine distribution system is more diverse than that of heroin. Such findings demonstrate how investigative information is provided by chemical analysis beyond the detection of illegal or controlled substances. Cocaine and heroin are derived from plant matter. The first processing step is solvent extraction of the harvested materials. Residual solvents can become trapped (occluded) in the final powder product and are detectable using headspace or related methods \[23--29\]. An example of an analysis of cocaine samples is shown in Figure 6.13. In this study, samples from three countries in South America were characterized by HS-GC-MS \[25\]. Once extracted, the base form was analyzed (left side). Next, the base was converted to the hydrochloride salt and to purified basic form (crack cocaine) shown at right. Note how everyday products, such as gasoline and kerosene, become extraction solvents. Compound numbers 6, 9, 14, 23, and 42 are internal standards added for quantitation. The other volatiles identified were acetone (number 7), ethyl ether (12), benzene (33), n-propyl acetate (38), and toluene (43). Occluded solvents may also arise from contaminants of other reagents or solvents used in the entire process from extraction to final crystallization of the salt form \[26\]. The relative amounts and concentrations are thus important considerations in analyzing results. Profiling of samples' elemental constituents is possible using ICP-MS \[30,31\], but this is less com-mon in seized drug analysis. **Figure 6.10** How and where drug samples become contaminated or mixed with other substances. Profiling can exploit these materials.236 Forensic Chemistry As we noted in Section 6.4.3, profiling is an invaluable tool for elucidating synthetic pathways of drugs ranging from methamphetamine \[15,17,18,32--39\] and amphetamine to NPSs. The previous section demonstrated the variety of methods used for synthesis, each of which leaves evidence in the final product. Chiral products also reflect the differ-ent synthetic methods \[12,34,36,40\]. Isotope ratio mass spectrometry (IRMS, Chapter 4) has also been used in profiling \[41--47\]. Once again, methamphet-amine provides an excellent example to illustrate the value of the technique. A 2018 report discussed the application **Figure 6.11** Compounds detected in heroin and cocaine. (Reproduced with permission from Broseus, J., S. Baechler, N. Gentile, and P. Esseiva. Chemical profiling: A tool to decipher the structure and organisation of illicit drug markets an 8-year study in western Switzerland. *Forensic Science International* 266 (2016)18--28. Copyright Elsevier.) **Figure 6.12** Circle graph showing the preponderance of cutting agents in heroin and cocaine. (Reproduced with permission from Broseus, J., S. Baechler, N. Gentile, and P. Esseiva. Chemical profiling: A tool to decipher the structure and organisation of illicit drug markets an 8-year study in Western Switzerland. *Forensic Science International* 266 (2016) 18--28. Copyright Elsevier.)Overview of Drug Analysis 237 of IRMS to nearly 1,000 case samples to learn the likely synthetic method used \[44\], the same goal as in the previ-ous example. The difference here is that the study considered methamphetamine syntheses that do not start with an immediate precursor but instead start with the synthesis of ephedrine that is then converted to methamphetamine. As illustrated in Figure 6.14, ephedrine can be extracted from a plant (*ephedra*, a grass), made via fermentation of sugar with benzaldehyde (semi-synthetic), or synthesized from chemical precursors \[42,44\]. There is a method shown in Figure 6.14 that we did not discuss in Section 6.4.3. This approach (lower left) starts with benzyl cyanide and the intermediate **APAAN** (α-phenylacetoacetonitrile). It is another method that is becoming more common as controls on precursors force changes in clandestine syntheses. The authors analyzed plants from two locations and synthetic and semi-synthetic ephedrine and were able to dis-tinguish the two regional samples as well as natural ephedrine from synthetic ephedrine. They noted that in natural ephedrine, the nitrogen source is ammonia or nitrate found in the soil, and soil nitrogen composition varies by loca-tion. Methylamine is the nitrogen source for synthesized ephedrine. Figure 6.15 shows the results of IRMS analysis of 871 methamphetamine samples synthesized using the Emde method (Figures 6.7 and 6.14), which utilizes H2(g) and a catalyst to cause the reduction. The green triangles rep-resent samples based on natural ephedrine extracted from grass, and the black squares represent samples from synthetic ephedrine. The samples represented by the red circles could not be assigned to either synthetic route. Thus, using two different methods, we have seen how profiling can provide invaluable investigative information, but that the process for obtaining it requires many samples and significant effort. Profiling is an addition to routine case analysis, described next. **Figure 6.13** Chromatograms obtained using headspace GC-MS. The goal of the study was to characterize residual solvents as a means of profiling. (Reproduced with permission from Colley, V. L., J. F. Casale, Differentiation of South American crack and domestic (us) crack cocaine via headspace-gas chromatography/mass spectrometry, *Drug Testing and Analysis* 7 (3) (2015) 241--246. Copyright Wiley.)238 Forensic Chemistry **Figure 6.14** Top frame (a) Three methods to make ephedrine. Lower frame (b) Common methamphetamine syntheses. (Reproduced with permission from Liu, C. M., P. P. Liu, W. Jia, Y. F. Fan, Carbon and nitrogen stable isotope analyses of ephedra plant and ephedrine samples and their application for methamphetamine profiling, *Journal of Forensic Sciences* 63 (4) (2018) 1053--1058. Copyright Wiley.) **EXHIBIT 6.2 WASTED EVIDENCE II** Drugs and related compounds, including metabolites, precursors, and by-products in wastewater, are more than intelligence sources. In one case in the Netherlands, an influx of drug waste disabled a small wastewater treatment plant. These plants remove solids and oxygenate wastewater before discharge but cannot cope with large influxes of organic chemicals or other pollutants. Initial treatment exploits bacterial degradation, and when this process is disrupted, plants are effectively disabled. In 2016, a small treatment facility saw ammonia levels spike along with a drop in pH of the incoming water to \~2. Investigators attempted to find the source by backtracking and testing pH but were unable to locate the source. The conditions killed bacteria and required inoculation with fresh bacteria before operations could resume. Samples collected at the time indicated that direct disposal of amphetamine to the sewer system was the culprit. A second incident Overview of Drug Analysis 239 6.5 OVERVIEW OF CHEMICAL ANALYSIS OF ILLICIT DRUGS The flow of chemical analysis for seized drugs typically starts with a screening assay or presumptive test followed by instrumental analysis for confirmation of identification based on comparison to trusted reference standards (Figure 16.16). The laboratory process is called the **analytical scheme**. Schemes in this context include screening or other preliminary tests followed by more selective analysis that leads to identification. The tests used in schemes can vary, but the goals of qualitative identification (and occasionally quantitation) are the same. We discussed sample prep-aration and separations in Chapter 3 and instrumentation in Chapters 4 and 5, so we will not delve into detail here. **Figure 6.15** Plot of nitrogen and carbon values for 871 methamphetamine samples. See text for details. (Reproduced with permis-sion from Liu, C. M., P. P. Liu, W. Jia, Y. F. Fan, Carbon and nitrogen stable isotope analyses of ephedra plant and ephedrine samples and their application for methamphetamine profiling, *Journal of Forensic Sciences* 63 (4) (2018) 1053--1058. Copyright Wiley.) **Figure 6.16** Overview of analytical schemes for seized drug analysis. The analysis progresses from less specific to confirmation of identification using mass spectrometry. occurred in 2017, and this time, law enforcement found a clandestine amphetamine laboratory with direct sewer access. Numerous waste containers, including one holding more than 10,000 L were found at the site. Source: Emke, E., et al., Wastewater-Based Epidemiology Generated Forensic Information: Amphetamine Synthesis Waste and Its Impact on a Small Sewage Treatment Plant, Forensic Science International 286 (2018) E1--E7. DOI: 10.1016/j.forsciint.2018.03.019.240 Forensic Chemistry **EXHIBIT 6.3 BACKGROUND LEVELS OF DRUGS IN FORENSIC LABORATORIES** We discussed the use of blanks in Chapter 2 as a method of ensuring that solvents, equipment, and the instrument do not contain any target compounds or other interfering substances. Where might these types of contaminants arise? Improper cleaning of equipment or contamination of reagents, solvents, and standards is another possibility. The laboratory environment can also be a source of contaminants. In the past, when procedures and instruments were not as advanced, trace residuals from past cases might not have been at detectable levels, but that is no longer the case. A study published in 2019 demonstrated that residuals from past cases are often detectable and highlight the need for vigilant cleaning and other practices to prevent contamination from impacting case results. In the study, the authors collected samples from 20 forensic laboratories. They used dry wiping and sampled numerous surfaces, including balances, keyboards, phones, laboratory benches, and microscopes. The wipes were analyzed using an LC-MS/MS method targeting 18 drugs. **Exhibit 6.3. Figure 1.** The percentage of samples containing the labeled drugs. The size of the colored circles corresponds to amount detected as shown in the scale at right. (Reprinted with permission Sisco, E. and M. Najarro, A multi-laboratory investigation of drug background levels, *Forensic Chemistry* 16 (2019). DOI: 10.1016/j.forc.2019.100184. Copyright Elsevier.) **Exhibit 6.3. Figure 2.** Amount of six of the target drugs recovered. (Reprinted with permission from Sisco, E. and M. Najarro, A multi-laboratory investigation of drug background levels, *Forensic Chemistry* 16 (2019). DOI: 10.1016/j.forc.2019.100184. Copyright Elsevier.)Overview of Drug Analysis 241 In seized drug analysis, the most common screening test involves using reagents that cause a color change when added to a sample containing the drug or similar compounds (a **color test**). These tests are not specific and cannot be used for identification for reasons that will become clear (if not already) as we discuss the chemistry behind them. In the field, color tests can be used by law enforcement officers to provide sufficient information to suspect that an illicit drug is present. The results may help narrow down what that substance may be or what class of drug it may belong to, but that is the extent of the information such screening tests provide. Because of their importance in many schemes, we will discuss color tests in more detail in Section 6.5.2. Tests become increasingly selective, sensitive, and specific from left to right across the analytical scheme (see Chapter 2 to review the meaning of these figures of merit). The final instrumental technique is usually GC-MS coupled with refer-ence spectra and trusted reference standards. This instrument provides two pieces of information regarding the substance detected -- a chromatographic retention time and an EI mass spectrum. All results must be **internally consistent** for definitive identification to be assigned. This means that all results in the analytical scheme must be consistent. If there is a discrepancy (for example, if a color test result is not consistent with GC-MS results), additional testing or analysis is needed. For example, suppose a sample contains a mixture of drugs, causing a false negative color test for one of the drugs. GC-MS analysis shows this drug to be present, creating the inconsistency of results. Additional testing is needed to address this inconsistency since the substance missed by the color test has not met the whole scheme's requirements. The complexity of biological samples such as blood necessitates more involved sample preparation and extractions in forensic toxicology. However, both disciplines rely on hyphenated chromatography-mass spectrometry instrumental analysis for compound identification. GC-MS is most frequently used in seized drugs, while in toxicology, LC-MS and LC-MSn methods predominate. Current practice relies on reference spectra and the availability of reference compounds for definitive identification. With novel compounds, this may not be possible given the lack of library spectra and reference materials. In some cases, a compound is tentatively identified for intelligence and investigative purposes, but the designation carries no legal weight. **6.5.1 What Is Definitive Identification?** An interesting and pertinent question in any chemical analysis, especially in forensic chemistry, is what constitutes a *definitive identification* of a substance. How can you, as a forensic chemist, for example, be confident that your analysis has identified heroin as the controlled substance in a sample? How many tests are needed in the analytical scheme? Can you be sure of the identification? How sure? In Chapter 2, we discussed the concept of uncertainty in quantita-tive analysis; now, the forensic and analytical chemistry community's attention is turning to qualitative analysis and certainty. In most seized drug cases, the laboratory is tasked with providing a binary result -- presence or absence of a controlled or regulated substance and its identification. Forensic analysis is not exhaustive, nor does it have to be (pro-filing excluded). The laboratory does not identify every component in the sample, just controlled or regulated ones. The universe of interest to forensic laboratories is a constricted space, defined by an extensive but fundamentally lim-ited list of compounds. Analytical schemes work within the boundaries of that space. The forensic questions are: *are there controlled substances present? Which ones*? If needed, quantitative analysis follows identification, not precedes it. Chapter 2, Section 2.2 (Figures of Merit and Method Validation) discussed how the LOD is established for analytical methods. We can rephrase the presence/absence questions in these terms: *is/are controlled substances present above the analytical scheme's detection limit(s)*? Any jurisdictional or legal requirements would be considered at this stage, More than 80% of the samples contained cocaine or heroin, and over half contained methamphetamine. Look at Figure III.1, and this mix makes sense. The second figure breaks down the amounts of drugs found. Frame A (left) shows the amounts recovered from the wipes in ng per cm2 of the surface wiped. At the high end, over 180 ng was detected (\>0.18 mg). Frame B (right) shows amounts of three novel synthetic opiates, all of which we will discuss in the next chapter. These substances can present a health hazard due to their toxicity as well as a potential contamination source. The authors made several recommendations, such as using disposable bench coverings and weighing drugs in secondary containers. Source: Sisco, E. and M. Najarro, A Multi-Laboratory Investigation of Drug Background Levels, Forensic Chemistry 16 (2019). DOI: 10.1016/j.forc.2019.100184.242 Forensic Chemistry but that would be a separate question. Thus, the laboratory will determine presence or absence of a controlled sub-stance or substances at a threshold determined from the LOD and report the results accordingly. This still leaves the question of how many individual tests are needed to support the results. This question of what constitutes definitive identification is not unique to forensic chemistry and the topic is of current interest and debate. SWGDRUG and ENFSI publish recommendations to the community and are now joined in US by NIST OSAC com-mittees working in seized drugs, toxicology, and other forensic disciplines. SWGDRUG documents are available to the public at the website (SWGDRUG.org), and many of these are joint efforts with ENFSI. The 2019 (Version 8.0) of the *Recommendations* document includes a discussion of analytical schemes in the context of seized drugs and identification of compounds. The document provides guidance on combing methods to reach a "scientifically sup-ported conclusion." The wording in this and other documents is critical and challenging to craft. Terms like *definitive identification*, *scientifically supported*, and *to the exclusion of all others* are continually debated in the forensic com-munity. There is no right answer, just competing thoughts, opinions, and ideas that must be studied and discussed until consensus is reached. In drug analysis, schemes are designed as laid out in Figure 6.16 and combine tests from least to most specific. To guide laboratories, SWGDRUG and ENFSI have adopted a category system organized in tabular form. Category C methods are the least selective and specific, while Category A are the most. Category B techniques are in the middle. Color tests fall into Category C, GC, and LC into Category B (retention time data such as from LC-UV), and MS, IR, and NMR in Category A. The document recommends how to combine tests from these categories to support results. For example, a Category A method such as IR must be combined with at least one additional test to support the results. Hyphenated methods such as GC-MS can be considered as both Category A and Category B (retention time plus mass spectrometry). The most recent SWGDRUG recommendations add to this approach by discussing specific molecular characteristics targeted in a given technique \[48\]. For example, gas chromatography is based on intramolecular forces dictating interactions between the stationary phase and compound, while mass spectrometry provides structural information through fragmentation patterns. These are molecular characteristics independent of each other to the extent pos-sible within a given molecule. FTIR and Raman probe vibrational modes of bonds, which is structural information obtained without fragmentation. 1H-NMR reveals structural information through proton interactions. On the other extreme, color tests provide general information regarding the classification of substances. The analytical scheme is designed to combine tests in such a way as to provide and support identification by probing different molecular properties. GC-MS accomplishes this with a single instrument. Thus, a color test combined with GC-MS data would approach identification from three directions -- specific structural (MS), intramolecular forces and interactions (GC), and class information (color test). This combination is commonly used in seized drugs analytical schemes. In summary, as was the case with method validation and estimation of uncertainty, there are many viable analytical schemes to characterize seized drugs. There is no one right way. The criteria used to evaluate a given scheme comes down to the same criteria we discussed in relation to method validation, sampling plans, and uncertainty estima-tions. The scheme must be reasonable, defensible, and fit for purpose. Reliability and utility are foundational. **6.5.2 Chemistry of Color Tests** Color tests are a mainstay of drug analysis and, as such, warrant a detailed discussion. A color change is the outward evidence of a chemical reaction, just as the evolution of gas indicates chemical decomposition. Appearance or change of color points to an alteration in chemical bonding that accompanies a reaction. Some surprisingly complex reac-tion chemistry causes this change. Many of these tests date back more than a century. Several reports published in the 1970s and 1980s \[49--52\] provided a foundation for proposing and assessing reaction mechanisms. An article by Philp and Fu published in 2018 \[53\] provides a concise and comprehensive review of common color test reagents and proposed mechanisms of color change. We use this to illustrate the mechanisms of some of these tests as categorized in Figure 6.17. The basics of spectroscopy, described in Chapter 5, apply here. Color is perceived when our eyes detect light in the visible region (VIS, 400--700 nm). If you hold a test tube of water with red food coloring up to the light, you perceive the color as red; this is an absorptive interaction. The white light that illuminates the liquid is changed when the dye molecules absorb photons in the blue region while allowing photons in the red region to pass through. This process is an example of a subtractive interaction based on absorption. Analogously, red ink dried on paper appears red because Overview of Drug Analysis 243 the blue portions are absorbed, and red light reflects back to the eye. The absorption process occurs because the energy of a photon matches that of an energy gap between orbitals. Absorption promotes the electron, and thus, that photon is prevented from reaching your eyes. In the context of presumptive color tests, these energy gaps arise from two sources -- differences in energies between molecular orbitals and from differences in atomic orbitals, specifically d-orbitals of transition metals. For a color change to occur, either molecular orbitals are altered by a chemical reac-tion or d-orbitals are altered by complexation. This alteration facilitates absorption in the visible range manifested as color formation or color change. **Figure 6.17** Grouping of common color tests by pH range and color formed. (Reproduced with permission from Philp, M., S. L. Fu, A review of chemical "Spot" tests: A presumptive illicit drug identification technique, *Drug Testing and Analysis* 10(1) (2018) 95--108. Copyright Wiley.) **RAPID REVIEW 6.2 MOLECULAR ORBITALS** Molecular orbitals form by combining the atomic orbitals of atoms in the bond. The \* indicates non-bonding (anti-bonding) molecular orbitals; the others are bonding orbitals. They are filled analogously to atomic orbitals. Here the 2s and 2p overlap is shown. The 1s combination forms the same pattern as the 2s. Energy increases upward in the levels.244 Forensic Chemistry Within organic molecules, visible light can excite two kinds of molecular orbital transitions -- n → π\* and π → π; thus, the molecule must have π electrons to interact with visible light. Reactions with reagents fall into two categories -- those that result in polymerization and those that react with specific functional groups on the drug molecule. The reactions alter the molecular orbitals in such a way as to produce or change the perceived color, typically through increased conjugation (alternating double bonds). An example is shown in Figure 6.18. In this study \[54\], the authors evaluated the absorption and relaxation characteristics of carotenoids, including β-carotene, which imparts the familiar orange color to carrots. On the left are the structures evaluated and a count of the conjugated C=C bonds and C=O bonds. The top two substances with similar conjugation also have similar perceived colors (shown in the spectra) and similar absorption patterns. The addition of two conjugated C=O bonds results in a shift in absorbance to the right (less energetic, redder) and a change in the perceived colors to a blue/green hue. The **Marquis** reagent (formaldehyde in sulfuric acid) is the most versatile and widely used color test in drug anal-ysis. Colors result from increasing conjugation through polymerization and carbocation formation \[49--51,53,55\]. Figure 6.19 shows examples of proposed reaction mechanisms for methamphetamine, amphetamine, and morphine. One important but often underappreciated factor in color testing is contact time, which is critical with the Marquis reaction. Sulfuric acid discolors within a few minutes in a spot plate, even with no other substances present. The best practice with color tests requires interpretation within a short time from the reagent addition to the sample. The **Duquenois--Levine** (D--L) reagent for cannabinoids in marijuana also falls into this category of reagents. Figure 6.20 shows the reaction, colors, and structures. The test differs from the Marquis test in that as a final step, the colored compound (the Duquenois chromophore) is trapped in a chloroform layer to provide further confidence of the color change observed. This is Levine modification. This test is also one of the few for which the chromophore structure is confirmed with HRMS \[56,57\]. The first step in the test is to extract the sample (plant, paste, etc.) into a solvent such as petroleum ether. The solvent evaporates quickly, leaving a residue to which the Duquenois reagent is added (Figure 6.20, upper left). The solution is acidified with hydrochloric acid and mixed. As the acid is added, the bluish-purple color develops. The example in the figure shows a test with THC only and not plant extract, so the colors are not always as crisp as shown here. The final step is the addition of the water-insoluble chloroform, which becomes the bottom layer. This step aids in the interpretation of colors and removing some of the backgrounds from plant extracts. The mass spectrum was obtained using a DART-TOF instrument (Chapter 4). The peak at *m/z* 315.2336 is unreacted THC, but unreacted vanillin (part of the reagent) is also present. The study's authors used preparative TLC to isolate the chromophore from residuals for MS analysis. **Figure 6.18** VIS spectra of selected carotenes. (Reproduced with permission from Takaya, T., M. Anan, and K. Iwata, Vibrational relaxation dynamics of B-carotene and its derivatives with substituents on terminal rings in electronically excited states as studied by femtosecond time-resolved stimulated Raman spectroscopy in the near-IR region. *Physical Chemistry Chemical Physics* 20 (5) (2018) 3320--3326. Copyright American Chemical Society.)Overview of Drug Analysis 245 **Figure 6.19** Proposed reaction mechanisms for the Marquis reagent and morphine (right), methamphetamine (lower left), and amphetamine (upper left). Notice the difference between methamphetamine (orange) and amphetamine (brownish orange). If left too long, the orange methamphetamine color can darken and complicate interpretation. **Figure 6.20** The Duquenois test for THC. The structure of the chromophore has been confirmed with HRMS as shown. (The spectrum was reproduced with permission from Jacobs, A. D., R. R. Steiner, Detection of the Duquenois-Levine chromophore in a marijuana sample, *Forensic Science International* 239 (2014) 1--5. Copyright Elsevier.)246 Forensic Chemistry Aside from reactions that form highly conjugated compounds, the other color-producing mechanism seen in forensic chemistry regents is based on transition metals and d-orbital splitting (Figure 6.21). **Coordination complexes** arise from a Lewis acid-base interaction between the d orbitals of a metal cation such as cobalt and an atom with unshared electrons (the ligand). Ligands include water, a halogen, or, in the case of drugs, basic nitrogen found in alkaloids amines. **Ligand field theory** (LFT) explains d-orbital alterations, color, and magne-tism of transition-metal complexes. Simplified, as a ligand approaches and forms an association with a metal ion, the two different electron density environments of the d orbitals are observed (Figure 6.21). The approach along an axis is impeded by two of the five d orbitals, with the electron density repelling the approaching ligand's electrons. The other three d orbitals have no electron density along the axis resulting in weaker repulsion. Because the three orbitals are symmetric, their energy is degenerate and lower than that of the other two. Put another way, an unshared pair of electrons on oxygen that resides in the metal's dx2-y2 orbital are repelled significantly more from metal-ion electrons than would the pair of electrons on oxygen residing in the dx-z orbital. The new gap facilitates d-electron transitions in the visible range. The degree of d-orbital splitting depends on the ligand's relative strength, described as the **spectrochemical series**. This abbreviated series is CO \> CN− \> NO2− \> NH3 \> H2O \> OH− \> Cl−. Of these, NH3, H2O, and Cl− are of the most interest in forensic chemistry and presumptive color testing. In this abbreviated series, CO is the strongest ligand and creates the largest gap, while chloride is the weakest and produces the smallest gap. This concept is illustrated in Figure 6.22. Any charge on the metal ion also affects splitting. All else being equal, an octahedral ammonia complex has a larger d orbital gap than a water complex of the same structure around the same central atom. A larger gap means that light of higher energy (bluer) is absorbed, leading to a reddish appearance. Of the transition metals, cobalt, as part of two common reagents (cobalt thiocyanate and Dilli-Koppanyi) is the most versatile in forensic testing. Cobalt has the electron structure 3d74s2, whereas the cation, depending on oxida-tion state, has a 3d7(2+) or 3d6(3+) structure. In an aqueous solution, the cobalt ion appears light pink due to the water complex. The same is true of copper, which is light blue in solution owing to a water complex. In both cases, the water ligands are arranged in an octahedral pattern around the central cation. Any observed color change is a result of a change in the d orbital splitting pattern. As charged and stable entities, complex ions can act as cations or anions and form coordination compounds or tightly associated ion pairs. These ion pairs may be solids, or they may form stable neutral species extractable into an organic solvent such as chloroform. The cobalt thiocyanate test for cocaine is illustrated in Figure 6.23. As an alkaloid and a tertiary amine, cocaine is basic, with a pKa of 8.6. In acidic aqueous solutions, cocaine exists in the protonated BH+ form. The BH forms the ion pair solid according to the reaction: ( ) ( ) ( ) + \_\_ \_\_ \_ \_\_ \_\_ + − 2NHR Co SCN NHR Co SCN 3 2 aq,pink 32 4s,blue (6.1) **Figure 6.21** Empty d-orbitals are degenerate (same energy) but split upon the approach of a ligand. The size of the gap depends on the ligand.Overview of Drug Analysis 247 **Figure 6.22** Example cobalt complexes with ligands of different strength. Water causes greater splitting and thus must absorb higher energy (bluer) photons for the transition. Chloride is a weaker ligand creating a smaller gap that requires a less energetic photon (redder) for the transition. **Figure 6.23** The cobalt thiocyanate test with cocaine. The colors are shown in the spot plate image at the bottom. Notice that the blue substance produced is a solid.248 Forensic Chemistry where the product is a neutral and extractable compound. The pH is critical since the BH+ forms the solid. The col-ored solid results from an ion-pair compound formed from the cationic cocaine and the anionic cobalt complex. In one modification of the test (**Scott test**), the blue solid is extracted into a chloroform layer as further evidence of a tightly associated ion pair. We saw the same thing in the Duquenois--Levine test, where the chromophore is extracted into chloroform. 6.6 CURRENT ISSUES: MARIJUANA Seized drug analysis is straightforward, and for most forensic laboratories, routine assays are qualitative. The pro-gression from screening tests (color tests) through instrumental analysis, primarily GC-MS, works well for most submitted cases. Weighing samples is integrated into the process where applicable (powders or solids) because the amount of a controlled substance is critical information that applies to the charges and, if convicted, the penalties assessed. The need for quantitative analysis varies based on the jurisdiction. One area of rapid change in seized drug testing is marijuana analysis. We noted in the section overview how mari-juana laws are changing as societal norms evolve. Still, marijuana represents a significant number of cases submitted for seized drug analysis. Marijuana is all parts of the plant *Cannabis sativa*, excluding the stalk and sterilized seeds. **Hashish** and hash oil are derivative products of the marijuana plant. **Hashish** is the resinous material derived from the flowering tops, and the oil is a potent solvent-extracted variant. The marijuana plant is also known as hemp, which is an agricultural crop cultivated for its fibers. The active ingredients of marijuana and its derivatives are cannabinoids, summarized in Table 6.4. Marijuana contains many more cannabinoids; these are the ones most referenced in forensic work. Two naming conven-tions exist, but the dibenzofuran method is more common in the forensic context and is used throughout this text. Cannabinoids are oily and insoluble in water but soluble in solvents such as chloroform and petroleum ether. They are unusual among plant-derived controlled substances in that none contain nitrogen; thus, none are alkaloids. However, marijuana plant and extracts contain a variety of alkaloid bases, as is typical of any plant extract. The analytical scheme for marijuana began with an examination of the plant's morphology. A microscopic exami-nation is also required, as the leaves have characteristic (but not definitive) features. The most important of these features are **bear claws** or **cystolithic hairs** found on the lower leaf surface. The presumptive test is the Duquenois-- Levine test described above. In some laboratories, the analysis stops with the D--L test, but many add TLC with stan-dards, using the dye Fast Blue B or Fast Blue BB as a developer. Fast Blue B gives the constituents distinctive colors: THC turns red, CBN purple, and CBD orange. An example of a TLC analysis of marijuana appears in Chapter 3 (Figure 3.24). Marijuana samples are weighed, which can be more complicated than it sounds. Pills and powders are usually dry, and weighing requires transferring the dry material into a weighing container. Some samples may be wet, or sticky, which complicates the process. Plant matter contains moisture, which impacts weight and thus legal consequences. As a result, it is vital that the laboratory ensures the material is dry and the weight recorded is dry weight. Figure 6.24 illustrates how much mari- juana weights can change after seizure \[58\]. The figure shows the drying process and how moisture levels off in about a week. The authors pointed out that the samples lost between 25% and 77% of the original weight under storage conditions of \~22°C and 49% relative humidity. In the past, this analytical scheme (microscopy, D--L test, and TLC) was usually considered sufficient for mari-juana analysis; this is no longer the case. In 2019, a regulation \[59\] was implemented in the US in response to legislation regarding hemp (fiber) production. **Hemp** differs from marijuana in THC concentration, which must be less than 0.3% dry weight. This is the standard set in the US regulation and which the European Parliament voted to accept in late 2020; 0.2% is still seen and cited. Besides, cannabidiol (CBD) has become known as a potential treatment for anxiety and seizures and is also used as a supplement and OTC drug. Thus, what used to be a simple qualitative analytical scheme has morphed into a quantitative one, emphasizing the concentrations of THC and CBD. As a result, analytical methods and schemes have been updated significantly in the past few years \[60--65\].Overview of Drug Analysis 249 **Table 6.4** Names, abbreviations, and structures of selected cannabinoids in marijuana and hemp. Compound (abbreviation) Structure Δ9-Tetrahydrocannabinol, generically referred to as tetrahydrocannabinol or THC. Dronabinol is the name used for the pharmaceutical form. Cannabidiol (CBD) Cannabinol (CBN) Δ8 THC Cannabigerol (CBG) Tetrahydrocannabivarin (THCV, Δ9) (*Continued*)250 Forensic Chemistry **Table 6.4 (*Continued*)** Names, abbreviations, and structures of selected cannabinoids in marijuana and hemp. Compound (abbreviation) Structure Tetrahydrocannabinolic Acid (THCA, Δ9) Cannabidiolic acid (CBDA) **EXAMPLE PROBLEM 6.2** Suppose you are drafting a formal report or paper for publication, and you need to show the structure of can-nabichromene, a cannabinoid found in marijuana. Summarize the steps and provide the structure, citing sources. *Answer:* Start with a PubChem search to find the compound. You are welcome to draw this structure by hand or recreate it atom by atom using a chemical drawing program, but why? Take advantage of the tools available. The search yields the structure in 2D and 3D format. You could copy and paste the 2D image but the quality is limited, and you cannot edit or add to the structure. An easy alternative is to exploit chemical structure drawing programs. We will use ChemDraw® here since it is readily available to students, but you can figure it out for other software once you see the process. As an example, PubChem currently supports a program called Sketcher that includes documentation and help files. It also supports SMILES cut and paste. In the PubChem results, navigate to the "Names and Identifiers" section. You will find headings such as "InChI" and "Canonical SMILES." These are systems used to convert a structure to a simple text descriptor. For this exam-ple, copy the SMILES string. In ChemDraw®, open a new document and navigate to Edit -- Paste Special. If the text string is in memory, there will be an option to paste SMILES. Do so, and the structure appears. You can edit how it appears and add text labels. You can also save it for future reference. It also may provide a framework for editing. This trick comes in handy with compound groups such as cannabinoids or novel psychoactive substances based on common core groups. The citation was obtained using the "Cite" button shown in Example Problem 6.1.Overview of Drug Analysis 251 The D--L test is designed to react with THC. Thus, one way to improve the analytical scheme is to add a screening test focused on THC and CBD. Knowing the relative amounts is useful in distinguishing hemp (THC \< 0.3%) from marijuana. A color test based on 4-aminophenol was evaluated by two forensic labs and described in a 2021 paper \[66\]. The test helps visualize the difference between the THC and CBD concentration in plant matter. The proposed mechanism is shown in Figure 6.25. Reagent B is used to convert the aminophenol to the unionized form, which is oxidized to the reactive form that produces colored species. Unlike the D--L test, the color provides information on the relative amounts of THC and CBD present. Based on how the test works and the colors that result, you can rationalize why. A marijuana sample will have more THC than CBD and thus produce a bluer color, while hemp in which CBD \> THC, the color should be pinkish. If the concentrations are equal, the color is a combination color with a purplish tint. Figure 6.26 shows several tests conducted on plant material and the resulting colors. The range of colors blue-purple-pink is evident in the image, which shows results from the DEA lab. The tests were also conducted by a Virginia Department of Forensic Science (DFS) laboratory with the same results except for sample \#46 (upper left). The DFS was inconclusive, while DEA reported the results as pink. All these samples had been analyzed quantitatively, so the concentrations of both cannabinoids were known. Sample 46 contained 0.11% THC and 0.64% CBD. Sample 60 contained 0.38%/0.10% respectively and was blue (middle frame, third from left, top). Sample 81 contained 0.43%/13.64% and was pinkish (same frame, top, right). While color alone is not quantitative here, it does provide useful information regarding the relative concentrations of each and is an improvement in that regard over the D--L test. Not surprisingly, improvements in TLC methods have been proposed that address the same two substances. The marijuana TLC shown in Chapter 3 is typical of traditional TLC, but improvements in coatings have led to improved high-performance TLC (HPTLC). A recent publication \[67\] explored different mobile phases used in conjunction with HPTLC plates to characterize cannabinoids. An example run is shown in Figure 6.27. **Figure 6.24** Plots of moisture loss from marijuana based on time elapsed from the day of the seizure (top) and one day after sei-zure (bottom). (Reproduced with permission from 1. Warne, M. L., et al., Comparative Analysis of Freshly Harvested Cannabis Plant Weight and Dried Cannabis Plant Weight, *Forensic Chemistry* 3 (2017) 52--57. Copyright Elsevier.)252 Forensic Chemistry **Figure 6.25** Proposed mechanism for color formation with THC and CBD and the 4-aminophenol test. (Reproduced with permission from 1. Lewis, K., et al., Validation of the 4-Aminophenol Color Test for the Differentiation of Marijuana-Type and Hemp-Type Cannabis, Journal of Forensic Sciences 66 (1) (2021) 285-294. Copyright Elsevier.) **Figure 6.26** Results of testing on plant matter at the DEA Special Testing and Research laboratory. (Reproduced with permis-sion from 1. Lewis, K., et al*.*, Validation of the 4-Aminophenol Color Test for the Differentiation of Marijuana-Type and Hemp-Type Cannabis, Journal of Forensic Sciences 66 (1) (2021) 285--294. Copyright Elsevier.)Overview of Drug Analysis 253 The methods were validated with figures of merit produced for repeatability, reproducibility, and linearity. The fac-tors validated were the **retardation factor** (Rf) and quantitation using densitometry. Densitometry measures the intensity of each band's color and thus an objective response value for quantitation. The retardation factor is a measure of how far the spot travels up the plate and is analogous to GC and LC retention time: R spot location application location Solventmax position application position f = − − (6.2) with positions in mm. Calculated Rfs are labeled in the figure. The application point is the baseline, and the solvent maximum position refers to how far the solvent front traveled up the plate. The Rf represents how far the compound traveled relative to the solvent front. The authors established the repeatability of the Rf as \< 5% RSD and the repro-ducibility as \>5%. As shown in the figure, lanes 1 and 15 were methanol blanks (negative controls), lanes 2 and 14 standard mixtures, lane 3 was an internal standard, and the remaining lanes case samples. Frame a (top) and frame b show plant matter samples, while frame c shows e-cigarette liquids and edibles. The hemp samples shown in the top frame (lanes 4 and 5) have significant CBD concentrations with less THC than in the plant matter samples. Figure 6.28 compares marijuana with hemp using two of the solvent systems evaluated by the authors. The THC/ CBD ratios are different for the two plant matter samples. This figure also demonstrates the importance of the mobile phase composition. Although densitometry provided quantitative capability in the HPTLC example, it is insufficient for distinguish-ing hemp from marijuana. GC methods are frequently used for quantitation using FID and MS detectors \[63,64\]. **Figure 6.27** Samples characterized using HPTLC. Lane 1 is a blank and lane 2 contains standards at the indicated concentrations. The colors were developed using FBB. (Reproduced with permission from 1. Liu, Y. F., et al., High Performance Thin-Layer Chromatography (HPTLC) Analysis of Cannabinoids in Cannabis Extracts, Forensic Chemistry 19 (2020). Copyright Elsevier.)254 Forensic Chemistry Other methods employed include LC-UV (or PDA), and LC-MSn \[61\]. It will be interesting to see how these analytical schemes change in the next few years, given the continuing evolution in marijuana regulation and hemp production.