Molecular Insights and Clinical Outcomes of Drugs of Abuse Adulteration: New Trends and New Psychoactive Substances

Adulteration is a well-known practice of drug manufacturers at different stages of drug production. The intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the number of available doses and the dealer’s profit. Adulterants found in different drugs change over time in response to different factors. A systematic literature search in PubMed and Scopus databases and official international organizations’ websites according to PRISMA guidelines was performed. A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as “non-biological” (n = 35) or “biological” (n = 35 case reports; n = 9 case series) according to the samples investigated. Although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin, the reported data disclosed new adulteration practices, such as the use of NPS as cutting agents for classic drugs of abuse and other NPS. For example, heroin adulterated with synthetic cannabinoids or cocaine adulterated with fentanyl/fentalogues raised particular concern. Notably, adulterants play a role in some adverse effects commonly associated with the primary drug, such as levamisole-adulterated cocaine that may induce vasculitis via an autoimmune process. It is essential to constantly monitor adulterants due to their changing availability that may threaten drug consumers’ health.


Introduction
In 2020, about 284 million people consumed drugs within the previous year, representing an increase of 26% compared to 2010. Cannabis products remain the most abused compounds all over the world, while the highest prevalence of drug-related deaths is attributed to opioids [1]. Over recent decades, the drug market continuously evolved in response to several factors, including recent laws banning substances and precursors, but also events involving the entire world such as the recent SARS-CoV-2 pandemic [2]. Between 2010 and 2020, an alarming increase in drug seizures was observed in Europe, reflecting a 477% increase in methamphetamine seizures, 278% increase in herbal cannabis seizures, 266% increase in cocaine seizures, and a decrease in heroin seizures [3]. Although seizures data reflect only partially the drug supply chain, it suggests that drug market dynamics are changing.
Besides the threat of illicit drug consumption, adulteration with other active compounds represents an additional risk for drug users, exposing them to unanticipated intake of a wide range of pharmacologically active substances. Adulteration is a well-known practice of drug manufacturers at different stages of drug production [4,5]. Different from drug dilution and drug counterfeiting, the intentional addition of active ingredients to adulterate the primary drug may enhance or mask pharmacological effects or may produce more potent drugs to increase the drug dealer's profit [6]. Furthermore, most of the adulterants are not internationally banned unless they are controlled under national health or food regulations [7]. Adulterants found in different drugs changed over time in response to different factors, such as controls implementation. In the last decades, the increased controls on methylenedioxymethamphetamine (MDMA) and its synthetic precursors affected the drug manufacturing process leading to poorer quality "ecstasy" tablets with increased adulterants [8]. On the other hand, some cutting agents frequently reoccur in the manufacturing process such as levamisole for cocaine or paracetamol and xylazine for heroin [4,5,7].
Recently, concerns were raised by the adulteration of low ∆-9-tetrahydrocannabinol (THC) cannabis products with more powerful synthetic cannabinoids [9]. Besides synthetic cannabinoids, other new psychoactive substances (NPS)-adulterated classical drugs of abuse have provoked a wave of fatal intoxications around the world. Fentanyl and its analogues were employed for decades as heroin adulterants; but recently "nitazenes", new synthetic benzimidazole opioids with high potencies, are found as common opioidcutting agents [10,11].
Here, we review the most recent reports of adulterants detected in seized drugs of abuse including NPS. Furthermore, we reviewed reported cases of adulterated drug intoxications published in the literature from 2017 to 2022, focusing on clinical signs, pharmacological peculiarities, reported intoxication symptoms, and toxicological analyses performed on different human specimens.

Results
A total of 724 studies were initially screened, with 145 articles from PubMed and 462 from Scopus excluded according to the criteria described in the Method Section. The remaining 117 records were further assessed for eligibility to exclude articles without sufficient data. Finally, 79 studies were classified as "non-biological" (n = 35) or "biological" (n = 35 case reports; n = 9 case series) according to the samples investigated. All results are summarised in Tables 1-3
Levamisole and phenacetin were the most prevalent cocaine adulterants, followed by caffeine. The reported adulteration with local anaesthetics such as lidocaine, benzocaine, or procaine [12,13,16,[18][19][20][21][22][23][24][25] is a known practice to mimic the highly pure cocaine anaesthetic effect on the mucosa. Although fentanyl traces were detected in cocaine samples seized in the USA [26], other drugs of abuse or pharmaceuticals were rarely reported in cocaine street samples [12,13,16]. The investigated opioids were heroin, fentanyl, morphine, opium, and "crack heroin". The largest number of adulterants were found in fentanyl samples [27,28], primarily synthetic opioids, synthetic cathinones, caffeine, and acetaminophen. Paracetamol/acetaminophen and caffeine were confirmed as the most common opioids cutting agents according to Tittarelli et al. [5]. Fentanyl/fentalogues adulterated heroin was reported in 3 cases in Canada and in the USA [13,29,30], whereas heroin adulterated fentanyl was detected only in the USA and Canada [27]. Other psychotropic substances such as cocaine, methamphetamine, and benzodiazepines were detected in opioids seized samples. Lead was the most prevalent adulterant in opium samples, but it was detected also in heroin, methamphetamine, and ecstasy seized in Iran [15,31]. It was proposed that lead was added to heroin and other drugs during the first phases of the supply chain, to increase the weight of the batch and obtain more profit.

Non-Biological Samples
The literature search yielded 35 studies (Table 1) on seized cocaine (n = 20), opioids (n = 14), amphetamine-like stimulants (n = 8), NPS (n = 2), and THC (n = 2). Although most Amphetamine-type stimulants were adulterated with the most varied compounds, primarily other stimulants, most likely to boost the expected effect. Furthermore, the highest number of NPS (19 different substances in total) were detected in MDMA and methamphetamine seized in the USA and Canada [12,32].
Only 3 studies investigating NPS composition revealed that these adulteration practices also occurred with these compounds that were also used as adulterants of classic drugs [12,31,33] Although data are not sufficient to establish a trend, it seems that NPS are adulterated to enhance some desirable effects with substances producing these effects, such as the synthetic cannabinoids or NBOMe adulterated α-methyltryptamine [33].
Cannabis adulteration was the least investigated phenomenon since it was reported in only 2 studies. In this regard, Oomen et al. analysed a large number of samples recently seized in Europe, showing 24% of samples were adulterated with a potent synthetic cannabinoid, MDMB-4en PINACA, [34]. It was apparent that cannabis was adulterated more frequently in e-cigarette cartridges than in flowering tops or edibles.
The quality and quantity of adulterants identified in psychotropic drugs are affected by the analytical approach, as typical targeted methods do not reveal unexpected molecules [23,35]. Examples are on-site drug screening devices that contain antibodies that are specific for a class of drugs and do not react with adulterants. Conversely, the identification and structural characterization of adulterants was primarily conducted via two-step analysis by mass spectrometry and subsequently, nuclear magnetic resonance (NMR) [24,33,36]. Capillary electrophoresis and electrochemical methods are also available [14,23,27,36,37], but the gold standard are hyphenated techniques based on liquid or gas chromatography coupled to mass spectroscopy [12,15,17,20,22,[28][29][30][38][39][40][41][42][43][44].
The average age of the cocaine users that experienced levamisole toxicity due to adulteration was 44 years, including 11 males (average age: 41.8 years) and 7 females (average age: 45.3 years). Age and sex were factors related to levamisole and cocaine exposure in the different pathologies. CIMDL was associated with younger age and female sex (36.3 years, 60% women), older women experienced more vasculitis (46.5 years, 60% female) and older men more glomerulonephritis (42 years).
Adulterated opioids were found in 10 intoxications. The most frequent adulterated opioid was heroin laced with 5F-MDMB-PINACA (n = 8) [52], while methaqualone combined with oxazepam, ketazolam, nordiazepam, pinazepam, and alprazolam was detected in one heroin consumption case [55]. Lead was detected in one opium abuse case characterized by non-specific symptoms including nausea, acute/severe abdominal pain, and vomiting [49]. Patients with severe abdominal pain may undergo unnecessary emergency abdominal surgery, due to a diagnosis of occluded bowel rather than chronic lead intoxication. The average age of individuals experiencing heroin-adulterated intoxication was 30.4 years, with 8 male and two female heroin users reported as exposed to adulterants. Urine was analysed in 10 cases, while serum confirmation was conducted only in 3 cases. Urinalysis revealed the presence of other psychotropic substances combined with heroin including other opioids (n = 7), fentanyl (n = 5), and cocaine (n = 3) [49,52,55].

Case Series Reports
Among the 9 case series reports, cocaine and heroin were the main adulterated drug identified between 2017 and 2022 ( Table 3).
In 2019, Handley et. al. found higher levamisole concentration in urine than in plasma (565 ng/mL vs 10.6 ng/mL), due to its rapid metabolism (t 1/2 = 5.6 h). Interestingly, levamisole was more concentrated in brain tissue than in the other matrices (128 ng/mg) suggesting an accumulation in the parenchyma and facilitated passage through the bloodbrain barrier [66].

Discussion
Adulteration exposes drug consumers to unexpected threats, due not only to unintentional exposure to a potentially harmful substance but also due to pharmacological interactions between drugs and adulterants [75,76].
Similar to fluctuations in the availability of drugs of abuse, drug adulteration trends also may fluctuate in response to economic and political factors influencing the availability of certain substances [4].
The reported data disclosed new adulteration practices, although the seized samples analyses revealed the presence of well-established adulterants such as levamisole for cocaine or paracetamol/acetaminophen for heroin [5]. In 2022, quinine and quinidine were of particular concern in the USA, due to detection in more than 2,000 street drug samples. However, the reported data showed that quinine and quinidine were already used as cocaine adulterants in 2017 and 2018 [76]. Fentanyl was commonly detected as an adulterant in heroin samples in the USA and in Canada, presumably to enhance drug potency and reduce the amount of heroin needed for each dose [12,38,42,73].
NPS adulteration occurs not only with all classic drugs of abuse but also with other NPS. The wide use of these compounds as adulterants may be related to the ease of distribution due to uncontrolled status and ease of production since they are produced in "kitchen laboratories". However, it is not clear if certain NPS may be byproducts of the manufacturing process, such as fentanyl in butyrlfentanyl samples seized in Sweden between 2010-2014. Seized samples of kratom, a psychedelic plant containing mitragynine and 7-OH-mitragynine, were adulterated with caffeine and synthetic O-desmethyltramadol (ODT) and sold under the name "Krypton" [77]. ODT, a bioactive metabolite of tramadol, was added to mimic the sedative-narcotic effects of kratom [78,79]. Other adulterated kratom products contained high concentrations of 7-OH-mitragynine. Usually, 7-OHmitragynine is less than 2% of the mitragynine concentration in kratom; however, in this case, 7-OH-mitragynine was added as an adulterant to increase opioid-like effects [80]. Other synergistic effects were observed in the case of synthetic cannabinoids added to THC [34]. The addition of fentanyl to methamphetamine may lead to greater dependence on the mixture.
Although the reported data covered a limited time range, more recent evidence confirms the changing nature of adulteration practices, such as the xylazine-laced heroin seized in the USA [81,82].
Adulterants may play a role in the adverse effects commonly related to the primary drug, such as vasculitis induced by levamisole-adulterated cocaine [83]. P/c-ANCA positivity suggests that levamisole-induced vasculitis has an autoimmune mechanism. Immune pathologies require glucocorticoids, as was the case of a 29-year-old suffering from multifocal white matter lesions with brainstem and cerebellar involvement after cocaine/levamisole consumption, requiring immediate treatment with corticosteroids [84]. Noteworthy, p/c ANCA positivity was used as evidence of levamisole exposure in the presence of analytically confirmed cocaine positivity. Although, the unexpected negativity of p-c/ANCA despite vasculitis [5] in a case of certain cocaine consumption suggests that toxicological levamisole confirmation in biological matrices is necessary to prove consumption. The most challenging aspect of levamisole quantification in biological matrices may be its short half-life (5-6 h). In these cases, hair analysis may offer the best approach to document exposure to short half-life substances [45,59,67,68,85]. Interestingly, levamisole and cocaine quantification in post-mortem brains suggests they may have synergistic negative effects on the encephalic white matter [68]. Moreover, this synergistic effect was reported in living patients by diffusion tensor imaging and magnetic resonance imaging [86,87] revealing that levamisole increases the risk of white matter damage [87].
Although other adulterants were detected in seized samples, such as phenacetin, benzocaine and lidocaine, these substances were not reported in clinical cases, probably due to the lack of specific analyses of compounds other than principal drugs causing health threats. Noteworthy is the alarming increase in cocaine-related deaths due to fentanyladulterated cocaine [88]. Huhn et al. reported the case of a man who continued to test positive for fentanyl in urine for up 19 days [89]. Surprisingly, the new trend of heroin adulteration with synthetic cannabinoids and/or fentalogues was reported in a number of cases. In fact, 5F-MDMB-PINACA and fentanyl were reported in a case series in which the adulterants were confirmed by urine analysis [52] Apparently, lead adulteration of drugs such as opium, heroin and methamphetamine is a common practice in Iran [49]. In lead-adulterated drugs cases, the diagnosis is always difficult because of unspecific abdominal symptoms caused by chronic lead intoxication. The diagnosis of lead poisoning requires a clear clinical suspicion and sophisticated technological methods to detect it. It is important to highlight lead adulteration of opium because of its potential spread to Europe.
Rarely, cases of LSD-adulterated methamphetamine were reported, characterized by the inconsistency between the type of symptoms appearing in the acute intoxication phase and the substance reportedly taken by the patient. This inconsistency led to toxicological research to detect LSD and adulterants [48]. The state of well-being induced by MDMA including increased activation and emotional excitation is associated with a better response to LSD. The combination of MDMA/LSD enhances the psychedelic experience by inducing positive emotions. On the other hand, this has a minor enhancing effect relating to psychedelic experience. Club drugs, such as methamphetamine, MDMA, ketamine, cocaine, and GHB are often taken in combination with sildenafil, especially when sexual encounters are anticipated [50].

Method
A systematic literature search was performed in PubMed and Scopus databases and official international organizations' websites; according to PRISMA guidelines (12). Keywords "drug of abuse"; "new psychoactive substances"; "fentanyl analogues"; "heroin"; "cocaine"; "amphetamine"; "THC"; "cannabis"; "stimulants"; "synthetic cathinones"; "synthetic opioids"; "opioids"; "opiates"; "phenethylamine"; "synthetic cannabinoids"; were combined with "adulteration"; "adulterants"; and "contaminated". A total of 3604 scientific articles (1250 from PubMed and 2354 from Scopus) published from 2017 to 2021 were initially screened for eligibility. Two scientists individually evaluated each entry from one database; considering for full text reading only titles and abstracts mentioning analytical assessment of drugs and adulterants in seized materials and/or in drug-related intoxications. Further screening excluded studies according to the following criteria: (1) Articles not written in English (2) Commentary; editorial letters; and surveys (3) Duplicates were removed (4) Irrelevant studies Articles were classified by matrix investigated as "biological specimens" or "non biological specimens". Finally, articles were excluded for insufficient data.

Conclusions
Drug of abuse adulteration is a common practice involving a wide range of different active substances added to the primary drugs for pharmacological or profit purposes. The data showed that adulterants are widely added to all the classes of drugs of abuse, including NPS. Furthermore, NPS may be used as adulterants to potentiate the primary drug effect or obtain cocktails exerting unexpected effects on the abusers. However, they represent an additional threat to the users' health since a synergistic effect is observed in the comparison of certain intoxication symptoms. Moreover, the unexpected presence of certain psychotropic drugs may lead to incorrect treatment of intoxication cases. For these reasons, adulteration practices must be monitored to assess changes in the drug supply and adulterants should be analytically assessed in intoxication cases. Funding: The review paper was partially funded from the Project "Implementation of the identification and study of the effects of NPS: Development of a multicenter research to enhance the database of the National drug Addiction Observatory and the Early Warning System" and by the project: "National Early Warning System on Drugs (SNAP)" by the Italian Department of Antidrug policies.