Metabolites and Endogenous Compounds

7 UNEXPECTED DRUG/ METABOLITES AND ENDOGENOUS COMPOUNDS Drugs undergo a variety of oxidation, reduction, hydrolytic, and conjugation reactions during the course of their metabolism and elimination from the body. These reactions result in alterations in the chemical structure leading to the production of multiple and/or unique metabolites. Many of these metabolites, in some cases, will be at low concentrations and not detected in the course of routine toxicological analyses. However, in the case of a large ingestion of the parent drug they may be readily identifiable. Some of these “metabolites” are pharmacologically active and will enhance the overall pharmacological activity and/or duration of effect of the ingested drug. There may also be situations in which a particular metabolite may increase the toxicity of the ingested drug. Some endogenous compounds are also exogenous drugs, that are already in the body either because the body produces them or they are from our environment, for example, food that is ingested. This too has the potential to lead to a misinterpretation of the cause of death and/or lead to additional studies, which may be unnecessary, and lead to additional investigation or testing, thus unnecessarily consuming resources. The awareness of these compounds may actually corroborate other analytical findings in the case or present as a compound that is initially unidentified, potentially calling into question the final determination of the cause of death. Whether or not endogenous ethyl alcohol is present in blood or tissue or is an artifact of the analyses has been raised and investigated as early as 1858 [1]. The early studies were challenged with the lack of specificity and sensitivity of the analytical techniques utilized to identify and quantitate ethyl alcohol. Lester [2] felt there were valid arguments for concluding “normal” ethyl alcohol concentrations were less than or equal to 10 mg/L (1 mg/dL or 0.001 g%) and some studies cited values Postmortem Toxicology. DOI: https://doi.org/10.1016/B978-0-12-815163-1.00007-1 © 2019 Elsevier Inc. All rights reserved.

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up to 39 mg/L (3.9 mg/dL or 0.039 g%), however, some evidence supported concentrations as high as 200 mg/L (20 mg/dL or 0.020 g%). A year later, Lester [3] conducted a series of experiments using a more specific and sensitivity gas chromatographic techniques for the identification and quantitation of normal ethyl alcohol concentrations. The result of his experiments lead to the conclusion that “normal” ethyl alcohol concentrations found in blood of man ranges up to 1.5 mg/L (0.15 mg/dL or 0.0015 g%). This “normal” or endogenous ethyl alcohol is constantly being formed from acetaldehyde which is a metabolic byproduct of pyruvate, threonine, deoxyribose-5phosphate, phosphoethanolamine, aniline, and possibly of substrates [4]. In addition to ethyl alcohol being formed through the metabolism of acetaldehyde, ethyl alcohol may also be formed in the intestinal tract in patients; the first reported case was in 1948 [5]. Patients who consume high carbohydrate meals and have an abnormal yeast proliferation, particularly Candida species would ferment the carbohydrate to ethyl alcohol and absorb the ethyl alcohol to concentrations that they would be clinically intoxicated [6,7]. These syndromes would also be referred to as Autobrewery syndrome or intragastrointestinal alcohol fermentation syndrome. The initial reported cases occurred in Japan and in Japanese the syndrome was called “Meitei-sho,” which translates to intragastrointestinal alcohol fermentation syndrome. Dahshan and Donovan [8] described a case in a 13-year-old girl. She had a history of longstanding short bowel syndrome secondary to jejunal atresia and necrotizing enterocolitis, and underwent surgical resection as a neonate. The young girl had recurring episodes of bizarre behavior, somnolence, disorientation, and a fruity odor to her breath. Her presentation was consistent with alcohol intoxication and subsequent testing revealed blood alcohol concentrations in the range of 250 350 mg/dL. She repeatedly denied alcohol ingestion and after repeated episodes of continued intoxication, she was placed on a closed alcohol rehabilitation facility. While in the facility she continued to exhibit alcohol intoxication with elevated blood levels. The family noted that the episodes of intoxication coincided with the ingestion of high carbohydrate meals or fructose containing drinks. A gastrointestinal endoscopy procedure obtained aspirates from her small intestine. These aspirates grew out two types of yeast: Candida glabrata and Saccharomyces cerevisiae. Fluconazole therapy was begun, her symptoms resolved, and there were no recurrence of the elevated ethyl alcohol level. The author investigated a case of a

Chapter 7 UNEXPECTED DRUG/METABOLITES AND ENDOGENOUS COMPOUNDS

51-year-old female with Down’s syndrome who functioned in the severe range of mental retardation. She resided in a care home until her death. The medical examiner ruled the cause of death as “aspiration of gastric contents due to swallowing of a foreign object with obstruction of the pylorus.” Prior to her death, she made a couple of hospital visits where the clinicians found evidence of bowel stasis and noted a possible penny within her duodenum. This finding was confirmed at autopsy. Toxicology reflected a blood alcohol concentration of 0.07% (w/v) and based upon that the medical examiner opined that the decedent had been drinking alcohol shortly before death. However, investigation of the care home, where the patient resided, did not reveal a source of alcohol. The subsequent medical investigation determined that the most likely source of the alcohol was due to intragastrointestinal fermentation syndrome. Naturally occurring benzodiazepines were first reported to be found in bovine brain tissue in 1986 [9]. The benzodiazepine identified was N-desmethyldiazepam. The authors found benzodiazepine-like immunoreactivity in human brain they examined. Six of the brains had been stored in paraffin since 1940, 15 years prior to the synthesis of the first benzodiazepine drug. Thus, undisclosed drug therapy or use would not explain the findings. Medina et al. [10] was able to extract benzodiazepinelike compounds from bovine brain and milk, suggesting a possible dietary source for these compounds. In addition to N-desmethyldiazepam, Klotz [11] detected additional benzodiazepines; diazepam, desmethyldiazepam, delorazepam, deschlorodiazepam, delormetazepam, isodiazepam, lormetazepam, and oxazepam in different animal and human tissues. The concentrations were quite low, diazepam ranged from 0.005 to 1 ng/g of tissue and desmethyldiazepam ranging from 0.01 to 0.5 ng/g of tissue. It is unlikely that these low drug concentrations would exert any direct pharmacological effects. Pen˜a et al. [12] demonstrated benzodiazepine-like compounds in human milk and plasma; the concentrations as measured by radioimmunoassay ranged from approximately 2 3 ng/mL diazepam equivalents and approximately 2 ng/mL diazepam equivalents in milk. The authors were able to conclusively identify diazepam and at least three other benzodiazepine-like compounds. Duthel et al. [13] devolved a sensitive method (gas chromatography—selected ion mass spectrometry) to identify and quantitate diazepam, N-desmethyldiazepam, and oxazepam in human serum. They examined the serum of 20 volunteers for the presence of these

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benzodiazepines. Diazepam was found in all 20, ranging from 7.3 to 32.0 pg/mL, whereas N-desmethyldiazepam and oxazepam were found only in some subjects, with concentrations ranging from 1.0 to 7.6 pg/mL and 2.0 to 13.0 pg/mL, respectively. Wildmann et al. [14] presented evidence for a possible dietary source for these benzodiazepines. The authors extracted wheat and potato and were able to identify a number of benzodiazepines. In wheat they found diazepam, N-desmethyldiazepam, delorazepam, deschlorodiazepam, delormetazepam, lormetazepam, and isodiazepam, while in the potato they found diazepam, N-desmethyldiazepam, delorazepam, lorazepam, and delormetazepam. The concentrations found were in the low ppb range. It has been well-established that endogenous opiates, specifically morphine and codeine, are present in trace amounts in mammals [15,16] and man [17]. Kodaira et al. [18] isolated an intermediary of morphine biosynthesis, thebaine, in ovine (sheep) brains, along with codeine and morphine. The presence of thebaine provides evidence that codeine and morphine are endogenous in nature and arise from the biosynthesis of thebaine. Donnerer et al. [19] further substantiated this biosynthetic route by injecting thebaine into rats and then sacrificing them after 1 hour later. The analyses of the brain, intestine, liver, kidney, and blood showed an increased the concentration of both codeine and morphine. Laux-Biehlmann et al. [20] demonstrated that morphine is synthesized in mammalian cells from dopamine, an endogenous neurotransmitter. Endogenous morphine plays a role as an endogenous neural transmitter or modulator [21 23]. Although present, “Are codeine and morphine found at concentrations that are toxicologically significant and would show up in routine postmortem analyses?” The literature supports that the concentrations are very low and would not be pharmacologically or toxically significant. Cardinale et al. [24] examined the cerebrospinal fluid of 12 patients who had not received any opioid type medication and detected codeine and morphine in concentrations of 2 239 femtomole/mL; both mainly in the conjugated form. The preliminary evidence in this study suggested that the conjugated form of morphine was the sulfate. Fricchionee et al. [25] determined endogenous morphine, via morphine-specific radioimmunoassay technique, to be present at a concentration of 3.4 ng/g in heart tissue. Zhu et al. [26] utilizing a chromatographic technique found endogenous morphine and

Chapter 7 UNEXPECTED DRUG/METABOLITES AND ENDOGENOUS COMPOUNDS

morphine-6-glucuronide at 106 and 48 ng/g, respectively, in human heart tissue. To place into perspective, Cravey and Reed [27] reported a brain concentration of morphine, by a morphine-specific radioimmunoassay technique, of 0.04 mg/kg. Rohrig and Hicks [28] reported a mean brain morphine concentration of 0.26 mg/kg, utilizing a gas chromatographic mass spectrometric assay, in decedents who died for a variety of reasons, some being heroin overdoses and other traumatic deaths, with overlapping of concentrations. Contrary to endogenous biosynthesis, Poeaknapo [17] suggests the most likely reason for the presence of “endogenous” morphine is due to exogenous or dietary sources. The consumption of various food products containing poppy seeds, which contain morphine and codeine, may lead to the detection of codeine and morphine in biological samples. Rohrig and Moore [29] detected morphine and codeine in urine samples and morphine in oral fluid samples from volunteers who ingested commercially available poppy seeds and/or poppy seed bagels. Following sample hydrolysis, maximal urine codeine concentrations were less than 40 ng/mL and maximal morphine concentrations in urine ranged from 314 to 603 ng/ mL. The highest morphine concentration measured in the oral samples was 205 ng/mL, codeine was not detected. Moeller et al. [30] measured the concentration of morphine found in blood and urine sample (free morphine in blood and total in urine) from five volunteers who consumed poppy seed products. These positive results have been substantiated by a number of other researchers, however, the measured concentrations were quite variable due to the poppy seed product consumed and the concentration of morphine and codeine in the ingested product. Pelders and Ros [31] analyzed poppy seeds samples from seven different regions; Australia, Czech Republic, Hungry, Spain, The Netherlands, and two areas of Turkey. The morphine concentration in the seed ranged from 2 to 251 µg/g of morphine and 0.4 to 57.1 µg/g of codeine. Volunteers who consumed products containing these variation poppy seeds from the different regions have variable amounts of opiates detected in their urine sample. Carbon monoxide is a common finding in many postmortem examinations and its presence at a significant concentration may support a cause of death. However, on occasion an elevated carbon monoxide concentration is unexplainable and the source of carbon monoxide is unknown. Low concentrations of carbon monoxide may adversely affect an individual’s ability to

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perform a complex task, extricate one from a hazardous situation and/or may aggravate preexisting cardiovascular disease. The toxicity of many halogenated hydrocarbons is due, in part, to their biotransformation products, such as stable toxic metabolites or reactive electrophilic metabolites [32]. In the 1970s, it was discovered that dichloromethane (methylene chloride) was metabolized to carbon monoxide. The metabolism of methylene chloride is via two routes; cytochrome P-450dependent route leading to carbon monoxide and a glutathione S-transferase route leading to formaldehyde and formic acid [32 35]. Guengerich et al. [36] demonstrated that cytochrome P-450 2E1 is the major isozyme responsible for the oxidation of methylene chloride to carbon monoxide. Stewart and Hake [37] report a case of a man, without a prior cardiac history, who was admitted to the coronary care unit with severe crushing retrosternal pain of 2 hours, which had radiated to his shoulder and left arm. The patient related a history of 6 hours prior to admission he had applied a commercial gel paint and varnish remover (80% methylene chloride by weight) to some furniture. He had worked on the furniture for 3 hours (exposure period) and an hour later become symptomatic. He had an uncomplicated hospital course and was discharged 2 weeks later. The patient again used the paint remover over a 3-hour period, and once again experienced chest pain and was readmitted to the hospital. During his hospital stay he developed cariogenic shock, dysrhythmia, and heat failure. The patient survives and was discharged. Six months after this last episode, he once again used the paint remover to complete his project and after 2 hours of exposure began to experience chest pain, collapsed, and died. The carboxyhemoglobin levels were not reported. They also reported on the observation of a local nonsmoker physician (RD Stewart), who had volunteered for a study evaluating the correlation of carboxyhemoglobin levels and air pollution. They noted that he had elevated carboxyhemoglobin levels (6% and 8%) 2 days in a row after utilizing a varnish remover the night before. To evaluate the potential for elevated carboxyhemoglobin following the inhalation of methylene chloride, they exposed the physician to low concentrations of methylene chloride in a controlled environment chamber for 1 hour. His carboxyhemoglobin rose from a preexposure level of 0.4% to 2.4%. To further investigate this observation, the authors exposed 11 male subjects to methylene chloride vapors in concentration ranges of 500 1000 ppm for 1 2 hours [38], they observed sustained elevation of carboxyhemoglobin levels in all subjects.

Chapter 7 UNEXPECTED DRUG/METABOLITES AND ENDOGENOUS COMPOUNDS

Langehennig et al. [39] reported on a limited study in which two nonsmoking subjects were exposed to methylene chloride from a furniture stripping product. The subjects were exposed for 6 hours in a large basement room, had a carboxyhemoglobin (COHb) level rise from baseline (not measured) to 26% and 40% saturation. In a multiple exposure experiment, over a short period of time; 2- or 3-hour exposure followed by a 24 hours break and reexposure for 2 or 3 hours, had COHb saturation of 14% and 33%, respectively. Interestingly, the subjects were reported to be asymptomatic during the exposure sessions. Fagan et al. [40] describes a case involving a 20-year-old female art student who was using methylene chloride in a poorly ventilated room. She began to develop nausea and vomiting, along with a severe headache. Approximately, an hour after leaving the room she continued feeling “unwell,” felt dizzy and shortly thereafter lost consciousness. She was transported to a local emergency department and upon admission she had regained consciousness, but was disorientated to time and space. Physical examination noted a cherry-red coloring of her skin and mucosa. Her admission carboxyhemoglobin was 50%. She was given oxygen therapy (60% at 4 L/min) and at 12 hours post admission, her carboxyhemoglobin saturation had dropped to 20%. The elimination kinetics of carbon monoxide did not follow the expected course; carbon monoxide in room air has a half-life of 3 4 hours and with 100% oxygen therapy is around 30 90 minutes. The extended elimination of the carbon monoxide was most likely due to the methylene chloride being sequestered into body tissues, slowly being released, and subsequently metabolized to carbon monoxide. She was discharged from the hospital 3 days later with no reported sequelae. Rioux and Myers [41] present an overview of the pathogenesis of methylene chloride, due to the hypoxic effects of the carbon monoxide formed via metabolism, along with 26 case studies; which were divided into four categories: accidental, abuse-related exposure, occupational exposure, and animal or organ studies. Another unusual source of carbon monoxide is the reaction of formic acid (methanoic acid) and a strong mineral acid. Toxic exposures to carbon monoxide have been described with the source originating from the combination of formic acid and sulfuric acid [42]. Lin and Dunn [43] described a case where a 26-year-old male was found in a car deceased. Located on the front passenger floor was a bucket containing an unidentified “boiling clear liquid.” The pH test of the liquid found a pH of 0. In the backseat of the car was an empty bottle of as sulfuric

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acid-based drain cleaner and another empty bottle (label unreadable), along with a receipt for a 950 mL bottle of formic acid. At autopsy, the decedent presented with a characteristic pink-red lividity, and an otherwise unremarkable autopsy. The carboxyhemoglobin saturation of the heart blood was 85%. Zeleny´ et al. [44] described a sophisticated apparatus for the timed and controlled release of the carbon monoxide generated from formic acid and sulfuric acid. The timer was set to release the carbon monoxide while the decedent was asleep. The carboxyhemoglobin saturation was 76.5%. Djafari and Ingemann-Hansen [45] describe an outdoor occupation exposure to carbon monoxide generated from the reaction of formic acid and sulfuric acid. The decedent was removing sulfuric acid which was reported to contain acetic acid from an industrial site. It was later determined that some of the collected liquid also contained formic acid. He was found dead at the top of his tanker near the lid. The decedent presented with the typical bright red lividity associated with carbon monoxide intoxication and laboratory studies confirmed carbon monoxide exposure with a carboxyhemoglobin saturation of 62%. Another short chain aliphatic hydrocarbon derivative, Gamma-hydroxybutyrate (GHB), is a drug commonly thought of as being abused and/or a drug utilized in drug facilitated crimes. In addition, GHB has/is used as an intravenous anesthetic agent, and has/is utilized in the treatment of various sleep disorders, opiate withdrawal syndrome, and in the treatment of alcohol withdrawal syndrome and dependence [46]. To further complicate the picture, GHB is a naturally present in mammalian tissues. It is thought to serve as a neurotransmitter or neuromodulator. GHB may also be formed de novo and/or in vitro depending on the container that the specimen is held in. Therefore, the question is—the GHB detected—was it from endogenous production or exogenous ingestion. This could have significant ramifications in the determination of the cause and/or manner of death, the circumstances surrounding the death being investigated, or could lead to a gross misinterpretation of these findings. Naturally occurring GHB is derived from the normal metabolism of gamma-aminobutyric acid (GABA); when the oxidation of succinate semialdehyde (SSA) to succinic acid is impeded (Fig. 7.1). Raknes et al. [47] reported that urine samples from pregnant women tend to have higher GHB and GBL concentrations than samples from nonpregnant females. However, the

Chapter 7 UNEXPECTED DRUG/METABOLITES AND ENDOGENOUS COMPOUNDS

Glutamate Glutamate decarboxylase

GABA GABA-transaminase

Succinate semialdehyde

GHB

Succinate semialdehyde dehydrogenase

Succinic acid

Krebs (TCA cycle)

Figure 7.1 GHB formation.

concentrations individually or combined did not exceed the generally utilized urinary administrative cut-off of 10 mg/L. Ando et al. [48] provide limited evidence that suggests patients with Huntington’s chorea will have elevated concentrations of GHB. Their data were limited to brain tissue concentrations; they did not have blood, urine, or cerebral spinal fluid available for analyses. Stahl and Swanson [49] suggest that the high levels of GHB found in Huntington patients may be related to the decrease in succinate:oxidoreductase (EC1.3.99.1) activity. Jakobs et al. [50] first described a rare genetic disorder in which there is a deficiency in SSA dehydrogenase. This alters the normal metabolism of GHB, leading to an accumulation of GHB in the body. Concentrations of GHB as high as 105 mg/L in serum and 260 mg/L in urine have been reported. SSA dehydrogenase deficiency has also been described as 4-hydroxybutyric aciduria [51]. This disorder was diagnosed, based upon urine GHB results. Wamelink et al. [52] describe two patients who were initially diagnosed with this disorder due to elevated levels of 4-hydroxybutyric acid in their urine. Upon further investigation it was found that a small amount of gamma-butyrolactone (GBL) was used in the manufacturing process for the catheters used and in an acidic environment would hydrolyze to GHB. There have been numerous studies that have looked at the “endogenous” antemortem concentrations of GHB (Table 7.1). The concentrations of GHB ranging from 0.00 to 6.63 mg/L in

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Table 7.1 Concentrations of Antemortem Endogenous GHB Study

Matrix

Concentration Range (mg/L)

LeBeau et al. [53] Kerrigan [54] Elian [55]

Urine Urine Urine (n 5 670) Blood (n 5 240) Urine Urine (n 5 207)

0.00 6.63 0 7a 0.34 5.75 0.17 1.51 0.9 3.5 0.00 2.70

Yeatman and Reid [56] LeBeau et al. [57] a

Stability study up to 5 mg/L after 244 days (NaF at 21˚C).

urine and 0.17 to 1.51 mg/L in blood samples of individuals not exposed to GHB. Seiler [58] demonstrated GABA formation from putrescine in visceral organs and in the CNS of invertebrates. Snead et al. [59] found GHB concentrations increased 80% 100% in rat brain after intracerebroventricular administration of putrescine. Elliott [60] and Marinetti et al. [61] have measured the presence of GHB in postmortem cases; n 5 40 and 26, respectively, without purported ingestion of the drug and found the following concentrations in various postmortem fluids (Table 7.2). Although the data are somewhat variable, interpretive ranges may be set. Table 7.3 lists commonly used administrative cutoffs for differentiate of endogenous versus exogenous GHB. However, in decomposed bodies the 50 mg/L cut-off may be exceeded [62]. Therefore, these ranges are useful for guidance but must be used with caution. The interpretation of low concentrations of GHB may be problematic given its endogenous nature, ability to rise from container artifact and/or production during the postmortem interval. One approach to sort out the endogenous versus exogenous nature of GHB is to examine physical evidence retrieved from the death scene. In a case of suspected suicide or rape/ homicide, the analysis of beverages and/or liquid food products may be utilized to determine a purported source of the compound. However, caution should be exercised since a variety of alcoholic, nonalcoholic beverages, and food products contain GHB and GBL as a natural component. Vose et al. [63] examined a number of wines and determined qualitatively that GBL

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89

Table 7.2 Concentrations of Postmortem Endogenous GHB Study

Matrix

Concentration Range

Elliott [60]

Urine

0 10 mg/L (NaF) 0 18 mg/L (unpreserved) 4 25 mg/L (NaF) 2 29 mg/L (unpreserved) 1 and 3 mg/La 0 (one case 7 mg/L) 0 (one case 7 mg/L) 0 13 mg/L (one outlier 119 mg/L) 0 23 mg/L (two outliers 90 and 97 mg/L)

Blood

Marinetti et al. [61]

Vitreous Urine Vitreous Blood (Heart) Blood (Femoral)

a

Unknown if preservative was used.

Table 7.3 Interpretive Cut-Offs for GHB Specimen

Concentration (mg/L)

Urine/vitreous Blood (antemortem)a Blood (postmortem)

$ 10 $ 10 $ 50

a

Blood samples in citrate tubes use postmortem blood administrative cut-off.

is a naturally occurring substance in the wines. The authors did estimate the concentration in a sample of red merlot examined contained approximately 5 mg/L of GBL. They did not test for GHB. Elliott and Burgess [64] examined various alcoholic and nonalcoholic beverages and found naturally occurring GHB and GBL in various beverages involving the fermentation of white and red grapes. The concentrations ranged from ,3 to 21.4 mg/L. Collison et al. [65] examined alcoholic, nonalcoholic, and liquid food products for the presence of GHB. Their results were consistent with the values reported by Elliott and Burgess [64] and also detected GHB in three different kinds of soy sauce (2.79 18.10 mg/L). They concluded that the concentrations of

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GHB were very low, and one would have to consume a large volume of liquid (e.g., 210 L) to achieve a pharmacological significant amount. However, if one was not aware of the natural occurring presence of GHB/GBL in these products, this could lead to the misinterpretation that the alleged “adulterated” drink was the source of the low level of GHB/GBL detected in the decedent. Another “endogenous source” of GHB is derived from 1,4-butandiol which is formed from the breakdown of some fatty acids. The 1,4-butandiol is then further oxidized to GHB. There are a variety of analytical methods for the identification and quantitation of GHB in biological fluids and tissues. Many of the methods involve the derivatization of GHB with trimethylsilane (TMS derivative) [66]. A potential pitfall, if not controlled for, is that the di-TMS derivative of GHB and the naturally occurring urea share common ions and have similar chromatographic characteristics, thus creating the potential for a false-positive result if SIM analyses are used. Some forensic laboratories will use a urea and urea plus GHB control to verify separation and preclude the misidentification of GHB. Ingestion of amphetamine salt preparations may result in trace amounts of methamphetamine being detected due to its presence as an impurity. This could lead to drawing a wrong conclusion that the decedent was using methamphetamine in addition to his or hers prescribed amphetamine salt medication. Fleming et al. [67] evaluated the 297 and 67 urines of patients prescribed Adderall (amphetamine salts) and Vyvanse (lisdexamfetamine dimesylate), respectively for the presence of methamphetamine. In a number of these specimens from both the Adderall and Vyvanse patients, methamphetamine was detected; albeit, the methamphetamine concentration in the Vyvanse patients was quite low as compared to the other group. The authors did test pharmaceutical preparations of Adderall and Vyvanse, in the Adderall preparations they confirmed the presence of methamphetamine, however, were unable to detect methamphetamine in the Vyvanse pharmaceutical. They concluded that a number of these positive methamphetamine urine were due to an impurity (methamphetamine) in the pharmaceutical preparation. Jemionek et al. [68] analyzed 22 urines from a military drug testing program and corroborated the findings of Fleming. The methamphetamine concentrations ranged from 4.2 to 275 ng/mL. Famprofazone is an analgesic, antiinflammatory, and antipyretic medication that is metabolized to methamphetamine and amphetamine. Shin et al. [69] identified a number of urinary

Chapter 7 UNEXPECTED DRUG/METABOLITES AND ENDOGENOUS COMPOUNDS

metabolites; with methamphetamine representing approximately 15% 20% of the administered dose. There were several metabolites in low concentrations; amphetamine, norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, p-hydroxyamphetamine, p-hydroxymethamphetamine, and p-hydroxydemethylfamprofazone. Chan et al. [70] dosed six subjects with 25 mg of famprofazone and collected their urine over a 48-hour period. Analysis of the urine samples by gas chromatography mass spectrometry found urine concentrations ranging from 901 to 2670 ng/mL and 208 to 711 ng/mL for methamphetamine and amphetamine, respectively. Fenethylline (Captagon) has been used as a therapeutic agent in Germany for the treatment of attention deficit hyperactivity disorder, narcolepsy, epileptic absences syndrome, and depression. Fenethylline has recently has appeared in the clandestine market [71]. It is amphetamine conjugated with theophylline. Metabolically it is cleaved into amphetamine and theophylline; 24.5% and 13.7% of an oral dose, respectively and amphetamine is detected in the urine. Selegiline is a medication used in the treatment of early stage Parkinson’s disease, depression, and dementia. Metabolism studies have identified three main metabolites; l-desmethylselegiline, l-methamphetamine, and l-amphetamine [72]. Unless a chiral analysis is performed to differentiate the stereoisomers, a wrong conclusion of [d-] methamphetamine abuse may occur. Levamisole is a veterinary deworming agent commonly found as a diluent/adulterant in cocaine. In 2009, it was reported that horses could metabolize Levamisole to the amphetamine-like compound aminorex and in the following year human metabolism was reported [73]. This amphetamine like compound will induce hallucinogens and also possesses a significant pulmonary hypertension risk (Fig. 7.2). Bertol et al. [73] dosed eight (four males and four females) subjects with B50 mg of Levamisole and collected urines at 3 and 6 hours. The concentrations of Levamisole and aminorex were determined (Table 7.4).

Figure 7.2 In vivo metabolism of levamisole.

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Table 7.4 Urinary Concentrations of Levamisole and Aminorex Compound

Time Zero

3h

6h

Levamisole Aminorex

ND ND

40.63 ng/mL (30.05 53.22 6 8.62) 30.63 ng/mL (22.52 38.12 6 5.48)

20.63 ng/mL (12.45 28.34 6 6.56) 28.32 ng/mL (20.08 43.6 6 7.95)

ND, not detected.

Unique metabolites are formed when certain drugs are coingestion with alcohol. Two particularly common metabolites detected are cocaethylene and ethylphenidate. Cocaethylene (ethylbenzoylecgonine) is a unique cocaine metabolite found in individuals using/abusing cocaine and ethyl alcohol concurrently. Dean et al. [74] identified a nonspecific carboxylesterase that catalyzed the ethyl transesterification of cocaine in the presence of ethyl alcohol. Boyer and Petersen [75] and others [76,77] further characterized the microsomal carboxylesterases involved in the transesterification reactions. Cocaethylene has similar pharmacological activity to cocaine, however, a longer half-life (B3.5 5.5 hours) and a greater toxicity as compared to cocaine [78 81]. Xu et al. [82] suggests the greater cardiac toxicity of cocaethylene is due to its effect upon cardiac sodium channels. Ethylphenidate was first reported in blood and liver tissues from two suicide victims [83]. The history had indicated coingestion of methylphenidate and ethyl alcohol. The concentration of ethylphenidate in these two cases was low relative to the concentration of methylphenidate; 8 and 1 ng/mL, respectively. The formation appears to be mediated by a carboxylesterasedependent transesterification process [84]; similar to that seen with the formation of cocaethylene. Markowitz et al. [85] studied six individuals who consumed a single 20 mg dose of methylphenidate and coingested a moderate amount of alcohol (0.66 mL/kg; 95%). Ethylphenidate was detected in blood and urine of all subjects. The reported half-life is significantly lower than that of methylphenidate, contrary to what is seen with cocaethylene. However, the shorter measured half-life may be due to sampling frequency and/or the limit of detection of the analytical methodology. In the presence of higher dosing

Chapter 7 UNEXPECTED DRUG/METABOLITES AND ENDOGENOUS COMPOUNDS

(abuse) of methylphenidate, when coingested with alcohol, the resulting formation of ethylphenidate will most likely contribute to the overall pharmacological effect of the drug. The analytical process may lead to the formation of unexpected compounds which may alter the analytical result or interpretation of the result. Juhascik et al. [86] reports the formation of the isopropyl ester of benzoylecgonine (BE) when isopropyl alcohol is used in the extraction of this compound. A reported result of isopropylbenzoylecgonine (BEIE) could suggest the possibility of antemortem ingestion of isopropyl alcohol. If the laboratory utilized BEIE as an internal standard for BE quantitation, this would result in an inaccurate quantitation of BE. There is an isolated reference that suggests the formation of isopropylbenzoylecgonine in a patient who ingested both ethanol and isopropanol along with cocaine [87]. Bailey [88] did report the in vitro transesterification of cocaine with n-propanol in human liver homogenates. An expected compound may be formed and/or detected due to an impurity in a reagent used in the analytical process or an impurity formed during the manufacturing of the illicit compound. Ethyl chloroformate is an impurity sometimes found in chloroform. Chloroform has been used in the extraction or as a final solvent in the analyses of many substances. In the determination and quantitation of meperidine and its metabolite normeperidine, Siek et al. [89] noted the formation of the ethyl carbamate derivative of normeperidine. The reaction was quite efficient noting up to a 90% loss of normeperidine, converting to the carbamate derivative. A similar observation was made with the extraction of desipramine, with the desipramine ethyl carbamate derivative being identified. Illicit cocaine is a source of several compounds that are structurally related to cocaine; cis- and trans-cinnamoylcocaine and n-formyl cocaine. A wide variety of cutting agents for cocaine may also be the source of unexpected compounds, such as lidocaine, benzocaine, and levamisole as previously described. Crude cocaine is extracted from the coca leaves, with the resulting coca paste containing a mixture of nitrogenous bases; which include cis- and trans-cinnamoylcocaine, tropacocaine, tropine, ecgonine, ecgonine methyl ester, and BE [90,91]. Cinnamoylcocaine isomers are one of the major impurities found in coca paste. A “clean-up” step in some processes involves the oxidation of the mixture to remove the cis- and trans-cinnamoylcocaine (oxidized to ecgonine) with potassium

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Figure 7.3 Oxidation of cinnamoylcocaine to ecgonine.

permanganate (Fig. 7.3). The resulting ecgonine is water soluble and easily removed via precipitation or solvent extraction. N-Formyl cocaine has also been found as an impurity in cocaine resulting from a clandestine manufacturing process involving permanganate oxidation [92]. The systematic toxicological analyses of specimens associated with cases involving illicit drugs may reveal a wide variety of other compounds used as cutting agents, diluents, and or impurities (starting material and/or process-derived). Methoxyflurane anesthesia use has been associated with renal dysfunction in some patients [93]. This has been manifested by postoperative azotemia, diuresis, and oxalosis. Two metabolites of methoxyflurane metabolism are nephrotoxic; fluoride, and oxalate. Frascino et al. [94] examined renal biopsies from seven patients after various procedures using methoxyflurane as the anesthesia that developed renal failure. The authors noted significant oxalate precipitation in the renal tissue. In six of the patients, urine oxalate levels were elevated; 96 480 mg in a 24-hour urine. The deposition of oxalate crystals is generally associated with the ingestion of ethylene glycol or oxalic acid; this represents a unique source of oxalate.

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