Toxic to the Heart: Target Organ Toxicity and NMR Spectroscopy of

Chapter 9

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Toxic to the Heart: Target Organ Toxicity and NMR Spectroscopy of NSAIDs Homar S. Barcena* Kingsborough Community College, Brooklyn, New York 11235, United States *E-mail: [email protected]

This chapter exploits the target organ toxicity of non-steroidal anti-inflammatory drugs (NSAIDs), their structure, and spectroscopic properties to engage student interest in STEM. Starting with the familiar medicine cabinet, the paradigm of chemical safety is challenged—all drugs are chemicals, and we are in essence, voluntarily ingesting materials with inherent toxicities. Pharmaceuticals are essential to society, but vigilance must be practiced to fully understand how they affect organisms and ecosystems. By introducing systemic toxicities of common drugs, students gain an appreciation of chemical toxicities in general. The NMR experiments described herein could serve as a gateway for educators to initiate discussions on toxicity as a motivation for green chemistry. It is further hoped that this account could inspire the upcycling of waste chemicals.

© 2016 American Chemical Society Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Introduction The development of green chemistry pedagogy highlights the commitment and creativity of educators to engage students not only in the science, but also in the broader impacts of the field. The application of the principles of green chemistry highlights the role of the chemist as a steward for health, safety, and for protecting the environment. Beyond academia, the green chemistry community continues to gain momentum and recruit partners in industry, politics, and the media, with the expressed goal of changing how chemical transformations are accomplished in laboratories and in industrial processes. Greater integration of green chemistry requires not only an expansion of the green chemistry toolbox, made up of reactions and alternatives that are safer and less wasteful, but also of chemists equipped to use them. The challenge then, is to attract, retain, and engage students so that they may be better prepared to tackle the challenges of redesigning established methods in chemistry. The paradigm shift for the next generation of chemists is how to make reactions and processes safer, cleaner, and more efficient, and not just a higher yield. A necessary step in accomplishing this is an awareness of chemical hazards and toxicity. Knowledge of toxicity is crucial in shifting the chemical safety model from risk response to one of risk prevention,yet there is a gap between chemistry and toxicology (1, 2) which has not been addressed in academia (3). Even as the Green Chemistry and Commerce Council, a network of 80 companies, elicit government support in incentivizing a green supply chain, leaders of the industry recognize that the education sector lags in the implementation of the discoveries and advancements of green science (4, 5). Ultimately, this could hurt the employability and competitiveness of chemistry graduates to enter the changing landscape of the chemical industry towards greener practices (6). Indeed, companies such as the signatories of the Green Chemistry and Commerce Council have made public commitments towards this transformation by employing a workforce that is knowledgeable about green strategies (7). The call from industry to transform academia, not just in chemistry, but also within engineering, science, and business curricula, will enable graduates to enter the workforce to (1) solve the challenges facing the chemical industry towards more sustainable practices, and (2) design and apply safer, more sustainable products and processes (7). The industry further urges the education of non-science majors in sustainability and green chemistry principles, to understand and support the emergence of clean technologies. Within this framework, implementation of curricula that engage students in the principles of green chemistry and sustainability is not only timely, but also necessary. This chapter reports on the use of proton nuclear magnetic resonance spectroscopy (1H NMR) to study over-the-counter (OTC) drugs to elicit student discussions on toxicity. These pharmaceuticals are widespread and readily available, thus an examination of their structures, properties, and identification make for palpable learning experiences in the laboratory. A short review on toxicity in chemistry pedagogy is provided, as well as a background on the use of commercial drugs as a means to engage student interest. Activities designed for 150 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

both introductory and advanced students illustrate the scope of green chemistry in instruction.


Toxicity and the Curriculum In the sense that hazards are mere chemical properties like physical state or color (8), a descriptive approach for chemical toxicity is primary in subject matter delivery. Thus, it is not uncommon for instructors to require students to list chemical safety precautions found in safety data sheets (SDS). The physical, toxicological, and global hazards of chemicals have been listed, with categorical examples such as flammability, carcinogenicity, and environmental impacts, respectively (9). Although this information is easily accessible and is highly applicable to a chemical’s purpose in the teaching laboratory, there is still a disconnect between theory and practice. The mitigation of chemical toxicity with good laboratory practices that allow for hazards minimization would be facilitated by an understanding of what a chemical’s toxicity means. Treatment of toxicity in pedagogy literature includes discussions of risk as a resultant of hazard and degree of exposure. Practical experiments exploring toxicity also include assays with invertebrates such as brine shrimp (10–12), daphnia (13), and microbes (14–16). In biochemistry and ecotoxicology, fish embryo and zebra fish toxicity assays are instructive (17, 18). albeit more specialized. Toxicity may also be incorporated into the curriculum through discussions of molecular design such as functional group activation, structure-activity relationships, and mode of action of drugs and toxicants (9). Toxicants tend to induce systemic toxicity upon absorption and distribution throughout the organism’s blood stream (19). Toxicants may accumulate in certain organs, damage highly vascularized organs, accumulate in lipophilic tissues, or accrue due to pumping mechanisms within certain cells. Metabolites of toxicants may also collect within tissues containing high levels of enzymes that act on the toxicant, referred to as toxicological bioactivation. The target organ toxicity of chemicals can reveal their mode of action on the organism, and provides students a broader viewpoint. For example, while many students are aware of heavy metal poisoning, they may not know that these toxicants accumulate in the liver and kidneys, which are the organs responsible for excreting toxins from the body, resulting in hepatotoxicity and nephrotoxiticy. Moreover, there may be more than one susceptible organ for a particular chemical, and the manifestation of its effects may not be instantaneous. While target organ toxicity demystifies the physiological hazards of certain chemicals, anecdotal systemic toxicities are interesting trivia among students. For example, pyridine is believed to lower sperm count, whereas ethidium bromide is linked to female infertility. Listed in Table 1 are a few common chemicals encountered in the teaching laboratory and a summary of their target organ toxicities from Toxnet, which is searchable toxicology data network maintained by the U.S. National Library of Medicine (20). These examples ought not to scare students away from experiments, but rather encourage vigilance in the employment of safety measures, personal protective equipment, and waste 151 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

disposal. It can aid them in the choices they make in designing procedural and synthetic routes.


Table 1. Target organ toxicity of some chemicals (20). Chemicals

Target Organ

Acute Effects

Chronic Effects

carbon tetrachloride chloroform

liver

nausea, abdominal pain

jaundice, liver enlargement

ethylene glycol chloroform halogenated hydrocarbons

kidney

benzene toluene carbon disulfide

central nervous system

drowsiness, headache, nausea

carbon monoxide cyanides arsenic aniline

blood

cyanosis (lips, tongue turn blue), loss of consciousness

Silica asbestos Nitrogen dioxide Hydrogen sulfide

lungs

cough, shortness of breath, tightness in chest

lung disease, asthma

Ketones Phenol Alcohols Chlorinated compounds

skin

Defatting of skin, rashes, irritation

skin and mucous membrane damage

Methyl iodide Ethylene oxide formaldehyde

DNA

tumors, cancer

DMSO DMF Chloroform cyclohexanone

embryo

growth retardations, abortions

edema, proteinuria

tremors, speech, hearing, vision impairments

Pharmaceuticals as a Teaching Tool The discovery and efficacy of pharmaceuticals is among the drivers for the advancement of the chemical sciences. Rightly so, medicines provide the means to longer and healthier life, the alleviation of human suffering, and the promise of curing and eradicating diseases. The benefits of chemistry to human life are unquestionable in this sense, and many students recognize the relevance of pharmaceuticals not only for the benefits they confer, but also in the ubiquity of drugs in many careers. While students studying nursing, medicine, pharmacy, and allied health need to take chemistry to gain an appreciation of drugs, majors like forensics, environmental science, engineering, and other technical fields benefit from an understanding of molecular science. Discussions 152 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


on pharmaceuticals capture student engagement since drugs are among the most widely used chemicals, yet they remain poorly understood. Drugs, for all the benefits they confer, are also toxicants. Focal to this chapter is the use of OTC NSAIDs, which are readily available and familiar to students. These are a class of analgesics that reduce fevers (antipyretic) and serve as antiinflammatory in higher doses. These are differentiated from narcotics, which also reduce pain but tend to have sedative and addictive side effects. The World Health Organization lists acetylsalicylic acid (aspirin), ibuprofen, and paracetamol (acetaminophen) as essential medicines (21). Some target organ toxicities for these drugs are familiar to most people. For example, aspirin is a known to damage the stomach and intestinal lining, which ultimately leads to ulcers and gastrointestinal bleeding. Aspirin also inhibits platelet aggregation, which is beneficial as a blood thinner in preventing strokes, but could cause prolonged bleeding when taken after a surgical operation. Paracetamol does not target the stomach nor thin the blood, but can cause serious liver damage when taken above the recommended amounts. To prevent such fatal overdoses in the United Kingdom, paracetamol may only be purchased in 16-tablet packs from shops. While target organ toxicity data serve as cautionary guides for drug use, they are also useful tools for instilling chemical safety in the laboratory. Students must note however, that these are just a part of the hazards assessment for any chemical. Toxic to the Heart: NSAIDs Recently, the Federal Drug Administration (FDA) issued a warning with a list of NSAIDs that can cause heart attacks or strokes (22). This was especially disconcerting to the general public since, although many of these drugs have been known to exacerbate heart disease, the recent findings implicate them as a cause of heart attacks. This announcement was made after an FDA advisory committee on drug safety and risk management drew findings from newly published papers, clinical trials, and observational studies in 2014. The stronger warning issued by the FDA in 2015 included some alarming statements for the drugs’ labels. For one, they noted that the risk of a heart attack or stroke could occur within the first weeks of using an NSAID, with an escalating risk for longer drug use. This increased risk is applicable even for patients without heart disease or who exhibit no risk factors of heart disease, although patients with risk factors are more susceptible. They also determined that individuals treated with NSAIDs after a first heart attack are more likely to die within a year, compared to heart attack patients not treated with NSAIDs. Thus, NSAIDs increase the risk of heart failure. It was also noted that although aspirin is an NSAID, it does not pose this risk (22). The cardiovascular toxicity of non-aspirin NSAIDs originates from their inhibition of cyclooxygenase (COX) enzymes. Common among these is COX-1, which performs a number of important regulatory functions and is produced by the cell under all physiological conditions, and COX-2, which is produced only in response to certain events. Both COX-1 and COX-2 are located in the blood vessels, stomach, and kidneys, with COX-2 production promoted under inflammation. Non-aspirin NSAIDs selectively inhibit COX-2, causing 153 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


homeostatic imbalance and exaggerating thrombotic response, increasing the likelihood of blood clot formation (23). Blood clots can inhibit blood flow, and when these occur in the heart muscle, a myocardial infarction or heart attack can occur. On the other hand, the anti-inflammatory effect of aspirin originates from its inhibition of both COX-1 and COX-2, so there is less chance of clot formation, but it poses higher risk of stomach bleeding. A recent study shows that paracetamol is a COX-2 inhibitor (24), although this drug has not been implicated in an increased stroke among hypertensive patients (25), and is not included in the FDA list. Table 2 summarizes the OTC drugs used in this activity, with the generic name and some brand name representatives, and their target organ toxicities (22).

Table 2. Some target organs for common NSAIDs (FDA = listed in the 2015 FDA Drug Safety Communication) Target Organ Toxicity

Heart

Kidney

Stomach

Liver

Aspirin (Bayer, Ecotrin) Ibuprofen (Advil, Motrin)

(FDA)

Paracetamol (Panadol, Tylenol) Naproxen (Aleve)

(FDA)

Toxicity is dose-dependent, and there are several live organism experiments that demonstrate this concept to students in literature (10–16). For this activity, students were instead provided the FDA Drug Safety Communication, which unequivocally states that the risk of heart attack from non-aspirin NSAIDs increases at higher doses (22). The communication further advises consumers to take the lowest effective dose for the shortest possible time. This is an effective demonstration of hazard minimization by limiting exposure to the toxicant. Before this activity, many students perceived these OTC drugs to be innocuous, and some even admitted to taking NSAIDs indiscriminately. This activity highlights that the scientific community’s understanding of toxicity, even for common substances, is still evolving, reflecting the challenge presented by the fourth principle of green chemistry, which is the design of safer chemicals. The FDA warning notes for instance, that treatment of patients with NSAIDs after the first heart attack results in higher fatalities, demonstrating that the timing of dosing, not merely the dose, is one of the future challenges in safer chemical design (26). That the toxicities of these indispensable and widely used drugs are still not fully understood demonstrates the need for greater collaboration between disciplines, particularly pharmacology, toxicology, and chemistry. 154 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Programmatic Elements


The experiment described herein uses 1H NMR spectroscopy to determine the identity of unlabeled OTC tablets. This half-day activity was initially developed for a Science Technology Engineering and Mathematics (STEM) summer program at Kingsborough Community College, in order to recruit incoming freshmen into the STEM majors. The activity was later adapted for the Organic Chemistry II course, as a laboratory experiment on instrumentation.

Methods for Introductory STEM Program For the chemistry module of the summer STEM program, students were invited into the chemistry laboratory where they were given an introduction on NSAIDs and target organ toxicity, and were shown a news video on the FDA warning. They were then provided an introduction on drawing organic molecules and how to use an NMR correlation chart to predict 1H chemical shifts. After this, students were shown how to prepare an NMR sample from an NSAID tablet, and then tasked to prepare an unknown sample. A brief introduction on NMR was provided in the instrumentation room, and afterwards, students were asked to insert their sample, and the acquisition was performed by automation.

Drawing Organic Molecules Practically all students have seen line-angle drawings of organic molecules. In this tutorial, students were taught that each point is a carbon, and each carbon must have four bonds. Any missing or “invisible” bonds are connections to a hydrogen atom. They were also told that single bonds can rotate freely, and were shown a molecular model. In the example in Figure 1, the neurotransmitter dopamine is converted from a line-angle drawing into one where the hydrogen atoms are shown. Note that this is not a Lewis structure, which would have necessitated students to draw carbons and lone electron pairs. The expanded structure is also shown as a straight line, in order to facilitate a discussion on symmetry later on. Students were provided a worksheet with line-angle structures, which they then re-drew to include the missing hydrogen atoms. The worksheet is a valuable tool in introducing molecules of interest, such as amino acids, fragrance and flavor molecules, and even poisons.

Figure 1. Line-angle structure of dopamine, which students re-draw to show the missing H’s. 155 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Predicting 1H NMR Shifts The utility of the NMR instrument to discern different types of protons was introduced once students gained proficiency in drawing the missing hydrogens in a molecular structure. Students were provided an NMR correlation chart such as the one in Figure 2. Students were also told that the only signals observable for this experiment are hydrogens on carbon (C-H), whereas hydrogens bonded to heteroatoms (N-H, O-H) will not be detected. This holds true for the samples used for this experiment, since the solvent used is D2O. Thus, three sets of signals are predicted for dopamine, the aromatic protons (6.8-8 ppm), benzylic protons (2.1-2.8), and the protons next to the amine (2.5-3.2 ppm). It must be stressed that these are merely approximate chemical shifts.

Figure 2. Correlation chart for 1H NMR shift, showing the structural features of the functional groups bearing H’s.

For practice, students retrieved the previous worksheet and used the correlation chart (Figure 2) to predict the chemical shifts for each molecule they drew. This visual exercise promoted collaboration and discussions between students, and was the most challenging part of the activity, as it required students to scrutinize a molecule’s structural features. As a third exercise, students were provided another handout, which contained examples of 1H NMR spectra. The aim of the handout is to familiarize students with NMR spectra, including chemical shift terminology (ppm, downfield, upfield). For simplicity, the proton’s chemical environment is defined to shift when it is near a heteroatom (O, N) or a multiple bond. Students are referred to the NMR chart to explain the chemical shifts in simple molecules like ethanol, acetaldehyde, and acetic acid. In this sense, students were able to discern different types of hydrogens according to the functional groups they are immediately bonded to. 156 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Once students were able to identify different types of H’s, they were shown the spectrum and structure of ethylene glycol, which is an additive found in antifreeze and e-cigarettes. This introduces students to symmetry and chemical equivalence, since the symmetric molecule shows only one NMR signal. It was further noted that the signals may appear as a single peak or multiple peaks, and that the splitting further gives information on molecular structure. However, multiplicity is beyond the scope for an introductory cohort, and its discussion was reserved for the organic chemistry class. For this reason, illustrative spectra must have no overlapping peaks, except for the aromatic region where it is easily justifiable due to the similarity of the chemical shifts.

Identification of NSAID Tablets by NMR For practice, students were provided the line-angle structure for aspirin, paracetamol, and naproxen, as well as their unlabeled 1H NMR spectra (Figure 3). Student teams successfully assigned the spectra to the structure, using the correlation chart, after drawing the missing H’s in the structure. Students found the identification of naproxen the easiest, since it has more than one aliphatic proton, and has the most abundant signals in the aromatic region. Detailed NMR assignments may be discussed, including the overlap between the methoxy and benzylic protons. Aspirin and paracetamol both show only one aliphatic proton signal, but differ markedly in the aromatic region. Students within their groups determined that paracetamol is symmetrical, and must have equivalent protons, giving the simplest NMR spectrum. It is helpful to remind students that single bonds can freely rotate, so the amide in paracetamol can be re-drawn facing down.

Methods for Organic Chemistry Students The activity was further re-designed to coincide with the lectures in NMR spectroscopy for organic chemistry students. These students are more advanced, and in addition to predicting the NMR shifts, were tasked to predict multiplicity and integration. A more advanced worksheet was distributed for practice, which they completed while taking turns to individually acquire their own spectra.

157 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 3. Students assign the structures to the 1H spectra (D2O, 4.8 ppm) for naproxen, paracetamol, and aspirin using an NMR chart.



NMR Experiment The instructor demo for this module was modified to include a discussion on solubility. The preparation of the NMR sample was performed for the students using ibuprofen. This organic acid exhibits low solubility in water, and the 1H NMR spectrum for the tablet will initially show only peaks from dissolved excipients. Upon addition of a small amount of potassium carbonate, the acquired spectrum will show the expected peaks, dwarfing the excipient peaks, owing to the increased water solubility of the deprotonated carboxylic acid. Depending on the set-up, students may be tasked to lock and shim the instrument. Students may also be tasked to analyze their spectra for integration, multiplicities, and coupling constants, especially if there is site license for NMR software. As a demonstration, the spectrum for ibuprofen further provides a good example for multiplicity, since one would expect a septet splitting pattern. For integration, the equivalent methyl groups will give a doublet with 6 H. Practical Considerations and Student Impact Worksheets may be prepared using 1H NMR spectra generated using the web-based prediction software from nmrdb.org, which gives crisp spectra (27). This software is free to use, and may be incorporated as a module for students to draw structure and predict spectra, especially for institutions with limited NMR access. For colleges that have an NMR instrument dedicated for teaching, the NMR analyses of NSAIDs is an inexpensive activity to engage students in STEM. Minimal sample preparation is required, however batch testing for each type of tablet is recommended prior to the experiment, to ensure that only the protons coming from the molecule of interest show up in the spectrum. Chemical hazards are minimized by utilizing OTC drugs and D2O for analyses. For clean-up, it is recommended that a laboratory technician wash the NMR tubes after use by inexperienced students, to minimize breakage of expensive tubes. Organic chemistry students may clean their NMR tubes with soapy water, followed by an acetone wash. In its entirety, the activity takes 4-5 hours, but may be broken down to shorter modules if needed. The NMR is an impressive facility for any science department, and it is used not only as an analytical instrument, but also as a recruitment tool. For students interested in medicine and allied health, the similarities between the NMR and MRI are compelling reasons to understand chemistry. Students interested in engineering and physics are more curious about how the instrument works, such as the superconducting magnet that is being kept cold by cryogenic gases. The activity educates students on the target organ toxicity of NSAIDs, and directly impacts their decisions when they take analgesics. The challenge for the instructor is to relate target organ toxicity to chemicals that are not ingested, but are used in the laboratory. Students may be referred to the National Institutes of Health’s Toxnet, a comprehensive database on toxicology, hazards, and environmental health (20). Moreover, the activity highlights our need to understand toxicity better. For example, ibuprofen has been extensively used for decades, yet it has taken a long time to fully understand how it impacts the heart. 159 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Green Chemistry and Upcycling The green synthesis of ibuprofen is one of the successes of green chemistry, which gained a Presidential Green Chemistry Challenge Award (28). This synthesis may be incorporated in discussions of NSAIDs chemistry, particularly in an organic chemistry class. Procedures for the green synthesis of aspirin (29) and paracetamol (30) for the teaching laboratory have been reported, and may be incorporated in the activity. However, what remains a challenge in green chemistry education is the integration of toxicology in the curriculum. While the FDA advisory on NSAIDs highlights to students that dose is the most important determinant of toxicity, the activity also highlights that toxins may not produce general effects but are specific to a few target organs. Toxicity is found in two tenets of green chemistry—Principle 3, “Wherever practicable, synthetic methods should be designed to use and generate substances that possess little or no toxicity to human health and the environment,” and Principle 4, “Chemical products should be designed to affect their desired function while minimizing their toxicity” (8). The choice of solvent (D2O over CDCl3) for this experiment illustrates adherence to Principle 3, as chlorinated solvents have a greater environmental impact. Providing the students a historical perspective of the drugs used in this experiment further highlights how chemists can develop the means to reduce the inherent toxicity of chemicals. For example, in 1897 Felix Hoffman discovered that by transforming salicylic acid to acetylsalicylic acid, he created a product that is more tolerable to the gastrointestinal tract, resulting in its marketability as a pain reliever (31). Interestingly, it was later discovered that aspirin’s ability to prevent myocardial infarction was also due to the acetyl group, as its antithrombotic property is absent in salicylic acid (31). Paracetamol has a more intricate pharmacopeia record, largely due to a misunderstanding of its toxicity (32). Paracetamol was first synthesized as a precursor for phenacetin, a commercial pain reliever. While the analgesic properties of paracetamol were known since the late 1800s, it was not until the 1950s that paracetamol was understood to be safe, and was entered to the market. Subsequently, it was discovered that phenacetin is metabolized to paracetamol in the body, lending it its therapeutic effect. It was also discovered that phenacetin is metabolized to phenetidine, which results in methemoglobinemia (32). Phenacetin was ultimately banned by the FDA in 1983, owing to the discoveries that it is reasonably anticipated to be a human carcinogen that targets the renal system. The similarity of the structures of these drugs in Figure 4 highlights molecular design as a means of attenuating toxicity (Principle 4), and students may be tasked to identify the functional groups and predict how to differentiate the molecules by NMR. Students interested in medicinal chemistry and pharmacy may also be engaged in discussions on how stereochemistry could play a role in drug efficacy and even toxicity. The fate of chiral drugs in biological systems is complex, as enantiomers may have different metabolism and elimination pathways, receptor interactions, and even racemize in vivo, which could present deleterious effects (33). Among the drugs used in this activity, naproxen has an asymmetric carbon, 160 Fahey and Maelia; Green Chemistry Experiments in Undergraduate Laboratories ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


and is marketed as a single enantiomer drug. Thus, (S)-naproxen is a drug, whereas the R-enantiomer has no analgesic effect and causes liver damage. From a green chemistry standpoint, the environmental fate of naproxen has attracted attention since it is one of the most widely used drugs and has been found to undergo enantiomeric inversion during wastewater treatment (34).

Figure 4. Paracetamol, phenacetin, phenetidine. Furthermore, while the described experiment does not generate a new analytical method, it follows the footsteps of green analytical chemistry (35). The materials required are non-toxic when used as described, and no sample derivatization nor treatment is needed. For some NMR instruments, the insoluble solids may need to be filtered, which can be easily accomplished using a short plug to minimize solvent waste. Each sample requires only ~400 μL of D2O, which may be discarded as aqueous waste. Tablets past their expiration date may be used for this activity. This is especially helpful not only to minimize costs and generation of chemical wastes, but also allows for upcycling. The FDA recommends household disposal of unused drugs in the trash after mixing with an unpalatable substance like kitty litter. However, there is rising concern that the presence of xenobiotics such as NSAIDs in ecosystems are toxic to organisms (36), and that they threaten the drinking water supply (37). Instead of discarding expired medicines, whose environmental fates are yet undefined, this activity repurposes them as chemicals for student inquiry.

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