Five cases of unintentional exposure to BZO-4en-POXIZID among nightclub attendees in New York City

Abstract

A new class of synthetic cannabinoids called OXIZIDs has emerged in recent years. This class consists of compounds with oxindole cores and hydrazide/hydrazone linker moieties and has often been described as being designed to circumvent a Chinese class-wide ban that was effective as of 1 July 2021. However, through hair testing of nightclub attendees in New York City—a high-risk population for recreational drug use—we have evidence suggesting exposures to an OXIZID called BZO-4en-POXIZID (4en-pentyl MDA-19) prior to the effective ban. Through analysis of 6 cm segmented hair samples from attendees collected in 2021, we detected five cases of exposure. Specifically, we detected a cluster of three cases based on hair samples collected on 20 June 2021, and then two additional cases from samples collected on 16 July 2021. Four of these hair samples were long enough to analyze two 6 cm hair segments (representing approximately two 6-month timeframes) and three of four of these cases tested positive for repeated exposure (for an estimated exposure over 6 months prior to hair collection). All cases included young adult females reporting past-year cannabis use but all tested negative for tetrahydrocannabinol exposure. Three cases also reported past-year use of cocaine, ecstasy, and/or ketamine, and four cases tested positive for exposure to cocaine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine and/or eutylone. These subjects were exposed to BZO-4en-POXIZID—likely as an adulterant in other drugs, and these cases are among the first documented cases which occurred approximately half a year before the Chinese legislative ban.

Introduction

A new class of synthetic cannabinoids called OXIZIDs has emerged in recent years. This class consists of compounds with oxindole cores, and the hydrazide/hydrazone linker moieties which have often been described as being designed to circumvent a Chinese class-wide synthetic cannabinoid (SC) ban (1–5). The ban was announced on 11 May 2021, and it has been effective as of 1 July 2021 (6). Since the ban targets all compounds containing one of seven general core structures, manufacturers appear to have moved towards the synthesis of SCs with alternative core components. These include SCs with oxindole cores and hydrazide/hydrazone linker moieties not banned by the recent Chinese legislation. These SCs are based on the substance MDA-19 (2) and include 4en-pentyl MDA-19 (BZO-4en-POXIZID).

A decade ago, SC use was highly prevalent in the USA. In 2011, after cannabis, this class of drugs was the most prevalent drug class used among 12th graders, although the prevalence of past-year use substantially decreased from 11.4% in 2011 to 3.2% in 2022 (7). The number of SC-related poisonings in the USA also substantially decreased from 6,968 reported poisonings in 2011 to 524 in 2022 (8). However, there have been notable outbreaks of SC intoxications in recent years, including New York City (NYC) (9). In addition, SCs included as adulterants in other drugs including cannabis products has occurred throughout parts of the world (10–12). There have also been clusters of overdoses in the USA in which purported ecstasy (3,4-methylenedioxymethamphetamine [MDMA]) was adulterated with SCs (13). As such, there has been increasing concern among international public health organizations about illicitly purchased drugs (including cannabis) containing SCs (14).

Since the Chinese class-wide legislation on SCs in 2021, few studies have investigated the distribution of OXIZIDs, or more specifically BZO-4en-POXIZID (4en-pentyl MDA-19) (1–5). This compound was first synthesized over a decade ago and reportedly has a high affinity for type 1 (CB₁) and type 2 (CB₂) cannabinoid receptors (15). It is not currently controlled in the USA, although a recent law was passed in North Dakota banning the substance (16). Furthermore, only a few documented submissions in the USA have contained BZO-4en-POXIZID. On 14 November 2021, US Customs and Border Protection inspected the first known batch of BZO-4en-POXIZID (17). Two parcels were shipped from China and inspected in Maryland, each containing ∼1 kg of powder, with one package testing positive for BZO-4en-POXIZID. The second submission received by NPS Discovery in Philadelphia, PA from Indianapolis, ID tested positive for BZO-4en-POXIZID (18). This compound was also identified by the US Drug Enforcement Administration in the first quarter of 2022 in the southern region of the country (19).

In this report, we focus on five cases of detected BZO-4en-POXIZID exposure within participants surveyed and hair tested in NYC as part of an epidemiological study monitoring drug use and new psychoactive substance (NPS) exposure among nightclub attendees.

Materials and methods

Study details

In this study, adults (age ≥18 years) were surveyed before entering nightclubs and dance festivals in NYC. Participants were given an anonymous survey inquiring about past-year use of approximately 100 drugs, and participants were asked if they were willing to provide a hair sample for subsequent analysis. A total of 350 adults were surveyed in 2021 (from July through December); 100 participants provided a hair sample, and of these, 69 samples were adequate in size to be tested. Hair analysis was conducted in March 2023. All methods were approved by the first author’s institutional review board.

Collection of specimens

Using clean scissors, staff cut a small amount of hair from the vertex region, as close to the scalp as possible. All but one participant provided head hair, with one providing armpit hair. One sample was collected from each individual. Short hair (e.g., <2 cm) did not preclude hair submission eligibility. Hair samples were folded into a piece of tin foil and sealed in envelopes labeled with the participant’s study ID number. Hair samples were stored in a locked drawer at room temperature before being shipped to the toxicology lab for analysis.

Reagents and standards

All chemicals, including methanol, formic acid, and acetonitrile, were purchased from Sigma-Aldrich (Milan, Italy) (purity ≥ 99% for all) while ultra-pure water was obtained using a Milli-Q^® UF-Plus apparatus (Millipore, Bedford, MA, USA).

Sample preparation

All hair samples were treated with a procedure developed for the keratin matrix. Approximately 50 mg of hair was decontaminated by an initial wash with 1 mL dichloromethane followed by a second wash with 1 mL methanol, each performed under 3 min of stirring. After complete removal of the solvent wash, the hair was dried at room temperature overnight. The samples were then pulverized using six steel balls stirring in a Precellys^® homogenizer (Bertin Technologies, France). Half a milliliter of methanol and 2.5 µL of an internal standard mixture (0.01 ng/mg) were added to the pulverized samples, and the specimen tubes were sealed and incubated at 55°C ± 5°C for 15 h. The organic phase was collected, and an aliquot of 5 μL was directly injected into the ultra-high-performance liquid chromatography (UHPLC) system.

Instrumentation

Targeted screening for traditional drugs of abuse was performed using previously published methods (20, 21). A list of target analytes (n = 155) is presented in Supplementary Table S1. The untargeted approach was based on an existing method (22). All methods have been validated and have been routinely used in our laboratory. In particular, the selectivity was evaluated, as recently proposed (23).

UHPLC separation was performed on the SCIEX ExionLC™ AC system (Sciex, Darmstadt, Germany) using a Phenomenex Kinetex C18 column (100 × 2.1 mm, 1.7 μm) maintained at 45°C. The mobile phase was a mixture of water (A) and acetonitrile (B), both with 0.01% of formic acid. The LC flow rate was set at 0.5 mL/min. Mobile phase starting conditions were eluted under the following linear gradient conditions: starting conditions were 5% B hold for 0.5 min and increased to 95% over 7.5 min. Using a linear gradient, 95% B was maintained for 0.5 min, and a final re-equilibration was programmed for 1.5 min to the initial gradient conditions. The total run time was 10 min. All analyses were performed using a SCIEX X500R quadrupole time-of-flight mass spectrometer (Sciex, Darmstadt, Germany) equipped with a Turbo VTM source operated in positive-ion electrospray ionization mode. Data acquisition involved a preliminary TOF-MS high-resolution full scan followed by a Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH)™ acquisition protocol which used a variable window setup (18 windows covering mass range from m/z 100.0 to 600.0 at 0.025 resolving power), resulting in a final cycle time of 0.933 s. The variable windows technique allows the reduction of the size of the Q1 window in order to further improve the quality of the SWATH acquisition data, while maintaining a complete coverage of the mass range and optimal cycle times. In this case, it was decided to use 30 Da windows as they allowed an optimal acquisition of the peaks, improving the specificity and reducing interference from possible co-eluting analytes. The qualitative identification of the target analyte BZO-4en-POXIZID was based on the identification of precursor ion (m/z 334.1550) and characteristic fragment ion m/z values with mass error <5 ppm as identification parameters. Data were acquired using SCIEX OS 1.5 Software and raw data files were processed using the MarkerView^TM software from Sciex.

Case details

The five cases were surveyed (with hair samples collected) on 20 June 2021 (n = 3) and on 16 July 2021 (n = 2), with 2–3 participants surveyed in June completing the survey at about the same time, suggesting they might have arrived at the venue together. As shown in Table I, all cases were female, and four provided a hair sample ≥12 cm in length. This permitted segmental analysis—with four cases each having two analyzable (i.e., sufficient quantity) hair segments, corresponding roughly to 0–6 months and 6–12 months before collection, respectively. All cases reported past-year cannabis use, and none reported past-year SC use. Cases 1, 2 and 4 reported having vaped in the past year, but none reported vaping of cannabis or tetrahydrocannabinol (THC) products. Case 1 also reported use of cocaine, ecstasy, amphetamine (nonmedical use), ketamine and 2C-I; Case 2 also reported use of ecstasy; and Case 5 also reported use of cocaine and ecstasy.

Table I.

Case Details

Case	Date of collection	Sex	Age	Past-year reported drug use	Toxicology results (ng/mg)
1	20 June 2021	F	27	Cannabis	Segment 1:
				Ecstasy	BZO-4en-POXIZID
				Cocaine	Cocaine 0.64
				Amphetamine	BZE 0.18
				Ketamine	Amphetamine 1.0
				2C-I	Ketamine 0.21
					Segment 2:
					BZO-4en-POXIZID
					Cocaine 0.87
					BZE 0.36
					Ketamine 0.19
2	20 June 2021	F	23	Cannabis	Segment 1:
				Ecstasy	BZO-4en-POXIZID
					Cocaine 0.16
					Amphetamine 0.87
3	20 June 2021	F	36	Cannabis	Segment 1:
					BZO-4en-POXIZID
					Segment 2:
					BZO-4en-POXIZID
4	16 July 2021	F	25	Cannabis	Segment 1:
					BZO-4en-POXIZID
					Methamphetamine 0.17
					Segment 2:
					BZO-4en-POXIZID
5	16 July 2021	F	23	Cannabis	Segment 1:
				Ecstasy	BZO-4en-POXIZID
				Cocaine	Cocaine 0.06
					BZE 0.23
					MDMA 2.60
					MDA 1.0
					Eutylone 0.28
					Segment 2:
					Cocaine 0.08
					BZE 0.62
					MDMA 0.93
					ketamine 0.14
					MDA 0.17
					Eutylone 0.03

M = male, F = female

All five cases tested positive for BZO-4en-POXIZID exposure. Three of the four cases which provided enough hair for segmental analysis tested positive in two segments, with these segments corresponding roughly to the past 6 months and the previous six months. In addition, three cases also tested positive for exposure to cocaine, MDMA, amphetamine, ketamine and/or eutylone, and these three cases all reported past-year use of various party drugs. Cases 3 and 4, however, only reported cannabis use, with one testing positive for exposure to methamphetamine and the other not testing positive for exposure to any other drugs.

Results and discussion

BZO-4en-POXIZID identification

Because of the continuous fluctuation of the NPS landscape, targeted analysis based on a well-defined panel of analytes may miss the identification of molecules which are present in analyzed samples. Taking advantage of untargeted high resolution-mass spectrometry (HRMS)-based screening, a qualitative identification of NPS (https://www.cfsre.org/nps-discovery/scope-recommendations) was performed. The candidate BZO-4en-POXIZID was singled out from the chromatographic profile of the full-scan analysis by checking the exact mass of the corresponding protonated molecular ion. Then, the elemental composition of the relative fragment ions and the rationality of its fragmentation pattern were checked in the MS-HRMS spectra to confirm the presumptive molecule identification (HRMS fragmentation patterns are presented in Figure 1). The fragmentation spectrum was then compared to results from a recently published study (2).

Figure 1.

HRMS fragmentation pattern of BZO-4en-POXIZID detected in the samples.

Among the 69 analyzable samples, we detected five cases of exposure to BZO-4en-POXIZID which appear to have occurred prior to the Chinese ban on SCs. In fact, in three of four cases providing hair long enough for segmental analysis, repeated exposure was detected suggesting additional exposure six or more months prior to hair collection. This provides evidence that BZO-4en-POXIZID was available on the recreational drug market (possibly in the NYC area) months before such compounds were thought to originally have emerged. To our knowledge, the first cases of BZO-4en-POXIZID discovered prior to our study were in November 2021 and January 2022, respectively (17, 18).

None of the five cases testing positive for exposure reported past-year SC use, suggesting unintentional exposure. While inaccurate self-report (e.g., underreporting of drug use) is possible, the items on the survey that assess past-year drug use have been shown to have strong or almost perfect test–retest reliability (κ = 0.88–1.00) (24). People in this high-risk population also tend to use common party drugs such as ecstasy, but SC use is relatively rare (25). Further, at the end of the survey, all five cases answered that they responded to all questions honestly. This is important because it has been found that those who report answering less than all questions honestly on this survey are more likely to provide discordant responses regarding drug use (24). As such, what is more likely was unintentional exposure to BZO-4en-POXIZID as an adulterant in other drugs used.

Previous studies have noted that BZO-4en-POXIZID (and pentyl MDA-19) has tended to be detected in the form of yellow powder (1, 3, 26) and one lab detected such compounds in e-liquid for vaping (1). If in powder form, it is possible that the drug was mixed into drugs purported to be ecstasy, cocaine, or ketamine, although if BZO-4en-POXIZID powder was yellow in nature, it is possible that the mixture could have been visible to consumers ingesting the substances. Although three cases reported past-year vaping (likely of nicotine), none reported vaping cannabis or THC products. As such, it is unknown whether exposure through vape cartridges was likely. Most cases reported using and/or tested positive for a variety of party drugs, but despite all cases reporting cannabis use, none tested positive for THC exposure. However, THC exposure can be difficult to detect in hair particularly when use is infrequent (27, 28). Historically, of the party drugs commonly used in this population, ecstasy in particular has been known to contain a wide variety of adulterants (29, 30), and extensive unintentional exposure to synthetic cathinones and methamphetamine in particular have been detected in hair samples submitted by people in this NYC population who use ecstasy (31). While to our knowledge, studies have not detected SCs as adulterants in purported ecstasy, in 2015, a cluster of 11 individuals at a university in Connecticut overdosed on a drug purported to be “Molly” (powder ecstasy) which was later found to contain the SC AB FUBINACA (13). Two students were in critical condition after the exposure, and one was revived after heart failure.

It is important to keep in mind that standard drug tests (e.g., dip sticks) cannot detect the presence of SCs so the prevalence of related poisonings from BZO-4en-POXIZID or SCs is unknown. Relatedly, many studies focusing on SC use are limited to poisoning cases, which limits ability to estimate the prevalence of use—regardless of whether a poisoning occurs. We are not aware whether these cases experienced adverse effects due to exposure, but we believe the strength of this epidemiology study is that we were able to detect potential clusters of repeated unknown exposure.

Conclusions

We detected five cases of unreported exposure to BZO-4en-POXIZID—likely as an adulterant in other drugs. We believe these findings are unique—first, because these were detected in a largescale epidemiology survey study rather than in poisonings; and second, we believe these are among the first detected cases of exposure—which appear to have occurred many months prior to the Chinese SC ban. We recommend that more studies monitor potential exposure to novel compounds without relying on poisonings.

Supplementary data

Supplementary data is available at Journal of Analytical Toxicology online.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Funding

This work was supported by the National Institute on Drug Abuse of the National Institutes of Health [R01DA044207 to J.P. and R01DA057289 to J.P.]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. A.S. acknowledges support from the Project CH4.0 under the MUR program “Dipartimenti di Eccellenza 2023-2027” (CUP: D13C22003520001) and funding from ‘Implementazione dell’identificazione e studio degli effetti delle NPS: Sviluppo di una multicentrica di ricerca per potenziare la base dati dell’Osservatorio Nazionale Tossicodipendenze e del Sistema di Allerta Precoce’ (CUP: I55E22000320001).