Identification of the Novel Synthetic Opioid N‐Pyrrolidino Isotonitazene at an Australian Drug Checking Service

Blake Curtis; Douglas J Lawes; David Caldicott; Malcolm D McLeod

doi:10.1002/dta.3910

. 2025 May 19;17(10):1996–2004. doi: 10.1002/dta.3910

Identification of the Novel Synthetic Opioid N‐Pyrrolidino Isotonitazene at an Australian Drug Checking Service

Blake Curtis ^1,², Douglas J Lawes ¹, David Caldicott ^3,^4,⁵, Malcolm D McLeod ^1,^5,^✉

PMCID: PMC12489282 PMID: 40384477

ABSTRACT

2‐Benzylbenzimidazole opioids and related derivatives, also known as ‘nitazenes’, present a growing threat to public health. Emerging in Europe in 2019, the nitazene group of drugs is a recent addition to the novel synthetic opioid class and has been associated internationally with adverse effects in drug users, overdose clusters and significant mortality. The high potency of many nitazene derivatives, which can in many cases exceed that of fentanyl, poses a significant challenge to the public health and early warning systems used to detect and respond to the emergence of new high‐risk substances. This report describes close collaboration between an Australian drug checking service and a nearby university laboratory to identify and characterise the novel synthetic opioid N‐pyrrolidino isotonitazene in an expected oxycodone sample presented by a member of the public. Though no prior publications are available describing the presence of this nitazene in the drug market, previously reported in vitro evaluation of this compound reveals it to be among the most potent nitazene opioid agonists known. The study highlights the rapid response possible though engaging drug users with drug checking services as a market monitor and early warning system to alert health services and the broader community to the presence of unexpected, high‐risk substances. Integration of well‐resourced and supported drug checking services provides a powerful approach to tackle the public health threats associated with novel synthetic opioids and other drugs of concern.

Keywords: drug checking, harm reduction, isotonitazepyne, novel synthetic opioid, N‐pyrrolidino isotonitazene

Close collaboration between an Australian drug checking service and an affiliated university laboratory identifies and characterises the novel synthetic opioid N‐pyrrolidino isotonitazene in an expected oxycodone sample presented by a member of the public. The study highlights the rapid response possible using drug checking as a market monitor and early warning system to alert health services and the broader community to the presence of unexpected high‐risk substances.

graphic file with name DTA-17-1996-g004.jpg

1. Introduction

2‐Benzylbenzimidazole opioids and related derivatives, also known as ‘nitazenes’, present a growing threat to public health. Emerging in the recreational drug market in Europe in 2019 [1], the nitazene group of drugs is a recent addition to the novel synthetic opioid class characterised by variable structures and differing potencies that in many cases exceed that of fentanyl [2, 3, 4, 5, 6]. The variation of nitazene structure and potency poses a significant challenge to early warning systems that seek to protect public health by detecting and responding to the emergence of new high‐risk substances. Internationally, the adulteration or substitution of the opioid drug supply with nitazenes has been associated with adverse effects for drug users and significant mortality [6, 7, 8, 9, 10, 11]. Reports from late 2023 of significant overdose clusters in Dublin and Cork in Ireland associated with the nitazene N‐pyrrolidino protonitazene (NPP, protonitazepyne; Figure 1) highlight the challenges that attend the introduction of novel synthetic opioids into local drug markets [12, 13].

Structures of N‐pyrrolidino protonitazene (**NPP**) and N‐pyrrolidino isotonitazene (**NPI**).

In Sep 2024, a client of an Australian drug checking service (CanTEST Health and Drug Checking Service, CanTEST) [14, 15] presented unmarked, round, mottled yellow pills with expectation that they contained the morphinan opioid oxycodone, accompanied by reports of significant nonfatal drug‐related harm. Chemical analysis at the service failed to detect oxycodone, instead providing evidence indicating the presence of a nitazene drug related to but distinct from NPP, leading the client to discard the samples. Further analysis at an affiliated university laboratory (the Australian National University, ANU) the next day identified N‐pyrrolidino isotonitazene (NPI, isotonitazepyne; Figure 1) as a major component leading to notification of the detection to clinical first responders and the public. Given the limited information available in the primary literature associated with NPI, this report presents a full characterisation of this compound. This study highlights the prompt response possible though the integration of well‐resourced drug checking services within early warning systems designed to monitor drug markets for the emergence of high‐risk substances in the community.

2. Experimental

2.1. Materials

Fentanyl and nitazene test strips were obtained from BTNX (Ontario, Canada). All solvents and formic acid used throughout sample preparation and analysis were liquid chromatography grade from Thermo Fisher Scientific (Scoresby, Australia). Deuterated solvents were supplied by Cambridge Isotope Laboratories Inc. (MA, United States). Hydroquinone reference material for nuclear magnetic resonance (NMR) was supplied by the National Measurement Institute (Sydney, Australia). A sample containing NPP was acquired from a client submission to CanTEST, matching the previously reported ¹H NMR and gas chromatography‐electron ionisation‐mass spectrometry (GC‐EI‐MS) characterisation data [12].

2.2. Fourier Transform Infrared (FTIR) Analysis

The FTIR analysis was performed as previously described in Algar et al. [14]. One whole pill was homogenised by crushing and mixing in a mortar with pestle. Following cleaning and a background scan, approximately 1–2 mg of the sample powder was loaded onto the instrument for analysis.

2.3. Ultraperformance Liquid Chromatography‐Photo‐Diode Array (UPLC‐PDA) Analysis

The UPLC‐PDA analysis was conducted as previously described [14]. Chromatographic separation was achieved using an alternative gradient consisting of the following mobile phases: 0.1% formic acid in water and methanol at a flow rate of 0.5 mL/min where the percentage of organic solvent was linearly changed: 0 min, 3%; 0.01 min, 35%; 3.50 min, 55%; 3.80 min, 3%; 6.0 min, 3%.

2.4. Immunoassay Test Strip Analysis

Sample homogenate was prepared in 0.1% formic acid in water/methanol (97:3) in a 2‐mL glass sample vial to a final concentration of 1.00 mg/mL. BTNX Rapid Response fentanyl and nitazene test strips were applied to the solution for 15 s and then placed on a nonabsorbent surface to develop. After 60 s, the results were recorded as either positive, negative or invalid.

2.5. High Resolution Ultraperformance Liquid Chromatography–Electrospray Ionisation–Tandem Mass Spectrometry (UPLC‐ESI‐MS/MS) Analysis

A sample of homogenised pill (10.0 mg) was weighed into a 2‐mL glass vial, suspended in methanol (1.5 mL) and vortexed for 30 s. The suspension was allowed to settle, and the supernatant was transferred into a syringe and filtered through a PTFE 13‐mm, 0.2‐μm syringe filter (1× dilution). The resulting solution was diluted 100× in 0.1% formic acid in water/methanol (97:3), and dicyclohexylamine (0.0050 mg/mL final concentration) was added as internal standard. A sample containing NPP (approximately 0.001–0.002 mg/mL) and dicyclohexylamine (0.0050 mg/mL) was prepared and run alongside for comparison.

Analysis was conducted using a Dionex UltiMate RSLCnano liquid chromatograph coupled to a Thermo‐Fisher Orbitrap Fusion ETD mass spectrometer via a heated electrospray ionisation source, operated in positive mode with a collision energy of 40 eV. Acquisition was run without quadrupole mass selection but otherwise using conditions as previously described [14]. Fragment ion ratios were taken by normalising the peak intensity of each precursor and fragment ion to the most intense ion (m/z 98) in the product ion spectrum.

2.6. High‐Resolution GC‐EI‐MS Analysis

To the initial sample prepared for UPLC‐ESI‐MS/MS (1× dilution), dicyclohexylamine (0.05 mg/mL final concentration) was added as internal standard for GC‐EI‐MS analysis. A sample containing NPP (approximately 0.10–0.20 mg/mL) and dicyclohexylamine (0.05 mg/mL) was prepared and run alongside for comparison.

Analysis was conducted using an Agilent 8890 GC coupled to an Agilent 7250 quadrupole time‐of‐flight high‐resolution mass spectrometer equipped with a Gerstel MPS preparative autosampler. The specific conditions used were as previously described, with minor adjustments [14]. The GC inlet was operated in splitless mode, and the final temperature of 325°C was held for 10 min, for a total analysis time of 22 min. Fragment ion ratios were taken by normalising the area under the curve of extracted ion chromatograms for each precursor and fragment ion to that of the most abundant ion (m/z 84) in the spectra.

2.7. NMR Analysis

A sample of the homogenised pill (100 mg) was weighed into a 20‐mL glass scintillation vial, suspended in methanol (1.8 mL) and vortexed for 30 s. The suspension was allowed to settle, and the supernatant (~1.5 mL) was transferred by micropipette to an Eppendorf tube. Following centrifugation for 30 s, the supernatant was again aspirated by micropipette, transferred to a 2‐mL glass vial and dried in a heating block at 60°C under a constant flow of nitrogen gas, resulting in a crude mixed yellow and white solid (4.1 mg).

One‐ and two‐dimensional ¹H and ¹³C NMR spectra were acquired in DMSO‐d ₆ at 298 K on a Bruker Avance III HD 800 spectrometer (800.13 MHz ¹H, 201.22 MHz ¹³C) equipped with 5‐mm TCI cryoprobe. Chemical shifts (δ) are reported in parts per million with ¹H shifts referenced to the residual solvent peaks (DMSO‐d ₅: ¹H δ 2.50) and ¹³C shifts referenced to the solvent peak (DMSO‐d₆: ¹³C δ 39.52). Coupling constants (J) are reported in Hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: m, multiplet; hept, heptet; q, quartet; t, triplet; d, doublet; and s, singlet. Analysis of these spectra was completed using the MestReNOVA (Version 14.2.1) and Bruker Topspin (Version 3.6.3) software packages.

Three individual pills were prepared as above for quantitative NMR (qNMR) analysis; the total recoverable mass of each crushed pill was weighed and then used in the extraction step. The volume of methanol for extraction was adjusted to reflect the larger sample mass (6.0 mL). All recoverable liquid was transferred, followed by one washing with methanol (1.0 mL). The extracted samples were dried and redissolved in DMSO‐d ₆ to a total volume of 1000 μL and transferred to 5‐mm NMR tubes; an external standard NMR sample was also prepared by dissolving 18.2 mg of hydroquinone (99.7 ± 0.3%) in DMSO‐d ₆ to a total volume of 1000 μL. ¹H NMR spectra were acquired at 298 K using qNMR conditions, of the three samples and the external standard; a blank DMSO‐d ₆ sample was also run to ensure that there were no underlying signals from the solvent contamination. The molar concentrations of NPI were determined by integrating the NPI proton at δ 8.485–8.450 (1H) and comparing this to the integral of the hydroquinone aromatic peak at δ 6.575–6.525 in the external standard, using the ERETIC 2 function in the Topspin 3.6.3 package. The total mass of NPI per original pill was then determined from the concentrations and volumes of the NMR samples, and the recoverable and original pill masses.

3. Results and Discussion

FTIR analysis (CanTEST) of the homogenised sample produced a relatively featureless spectrum (Figure S1.1) consistent with complex mixture of pill fillers/binders with no confident drug identification made using the OPUS Drug ID software.

UPLC‐PDA analysis (CanTEST) indicated a single component producing a UV spectrum with maximum absorptions at 239 and 309 nm, matching the spectrum of a previously identified NPP sample (Figure S1.2) when both spectra are manually overlayed. However, the retention times of the client sample (3.06 min) and NPP (3.56 min) differed (15% difference). The near identical UV spectra suggested the analyte contained a nitrobenzimidazole opioid of similar structure to NPP. No peak corresponding to the expected oxycodone was detected by UPLC‐PDA analysis.

Fentanyl test strip analysis (CanTEST) was recorded as negative indicating the likely absence of fentanyl or a fentanyl derivative for the client sample (Table S1.1). Nitazene test strip analysis (CanTEST) was recorded as positive indicating the likely presence of a nitazene derivative. The analytical findings obtained at the service were relayed to the client within minutes and led to the discard of the samples, which were transported to the nearby ANU laboratory for further analysis.

The GC‐EI‐MS analysis (ANU) of the client sample returned a peak (RT 19.4 min), differing from that of NPP (RT 20.6 min, 6% difference). The mass spectrum (Figures 2 and S2.1–S2.4 and Tables 1 and S2.1) revealed a molecular ion at m/z 408.2106 (C₂₃H₂₈O₃N₄ ^•+, +0.9 ppm) and fragments at m/z 84.0808 (C₅H₁₀N⁺, +0.0 ppm) consistent with an N‐methylene pyrrolidinium ion and m/z 107.0492 (C₇H₇O⁺, +0.9 ppm) [1, 16]. The NPP sample showed a similar mass spectrum [12]. The observation of different retention times but similar mass spectra suggested the presence of a constitutional isomer of NPP. Minor peaks (RT 9–12 min) observed in the GC‐EI‐MS of the samples were indicated as system related contaminants by inspection of solvent blanks, with no additional substances identified.

GC‐EI‐MS (EI+, 70 eV) total ion chromatogram for (A) client sample (**NPI**, RT 19.4 min) and (B) **NPP** (RT 20.6 min). Dicyclohexylamine (RT 7.7 min) internal standard is indicated by an asterisk (*). Mass spectrum for (C) client sample (**NPI**) and (D) **NPP**.

TABLE 1.

High resolution GC‐EI‐MS and UPLC‐ESI‐MS/MS analysis of the client sample (NPI) and NPP.

Method	Sample	Retention time (min)	Observed ion (m/z)	Chemical formula	Error (ppm)	Relative abundance(%)
GC‐EI‐MS	Client sample (NPI)	19.4	84.0808	C₅H₁₀N⁺	+0.0	100 ^a
			107.0492	C₇H₇O⁺	+0.9	4.8 ^a
			408.2160	C₂₃H₂₈O₃N₄ ^•+	+0.9	0.5 ^a
	NPP	20.6	84.0807	C₅H₁₀N⁺	−1.2	100 ^a
			107.0490	C₇H₇O⁺	−0.9	4.8 ^a
			408.2155	C₂₃H₂₈O₃N₄ ^•+	−0.2	0.4 ^a
UPLC‐ESI‐MS/MS	Client sample (NPI)	7.37	98.0963	C₆H₁₂N⁺	−1.0	100 ^b
			107.0493	C₇H₇O⁺	+1.9	5.2 ^b
			409.2247	C₂₃H₂₉O₃N₄ ⁺	+3.2	0.2 ^b
	NPP	7.50	98.0964	C₆H₁₂N⁺	+0.0	100 ^b
			107.0494	C₇H₇O⁺	+2.8	4.3 ^b
			409.2243	C₂₃H₂₉O₃N₄ ⁺	+2.2	0.2 ^b

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^{^a}

Derived from scan MS extracted ion chromatogram peak areas.

^{^b}

Derived from product ion MS/MS spectrum peak heights.

During UPLC‐ESI‐MS/MS analysis (ANU), a peak was observed at a retention time of 7.37 min with m/z 409.2247, (C₂₃H₂₉O₃N₄ ⁺, +3.2 ppm) for the client sample, corresponding to the protonated molecule of NPP or an isomer (Figures S2.5 and S2.6 and Tables 1 and S2.2). Following collision induced dissociation, the client sample returned fragments consistent with an N‐ethylene pyrrolidine ion at m/z 98.0963 (C₆H₁₂N⁺, −1.0 ppm) [17] and an ion corresponding to that observed in GC‐EI‐MS at m/z 107.0493 (C₇H₇O⁺, +1.9 ppm) [1, 16]. Although the precursor and fragment ions were consistent between both the client sample and NPP (Table 1), the latter returned a different retention time of 7.50 min (3.8% difference) also consistent with an isomeric species.

The ¹H NMR analysis (ANU) of the client sample (Figures 3 and S2.7) closely resembled that for NPP (Figure S3.1) [12], producing broad peaks at 2.39 ppm (4H) and 1.36 ppm (4H) with observed ¹H → ¹H COSY and ¹H → ¹³C HMBC cross peaks between the two resonances, matching those expected for a pyrrolidine substituent. The presence of a heptet at 4.56 ppm (1H, J = 6.0 Hz) and doublet at 1.23 ppm (6H, J = 6.0 Hz) with expected two‐dimensional ¹H → ¹H COSY coupling indicated substitution by an isopropoxy group consistent with an isotonitazene rather than a protonitazene or other nitazene derivative. Due to overlap with impurities, likely resulting from pill binding agents or excipients in DMSO‐d ₆, integration of the observed doublet at 1.23 ppm was instead taken from a second spectrum taken using chloroform‐d (CDCl₃) as solvent, where the signal was better resolved (Figure S2.8). Two‐dimensional NMR spectra in DMSO‐d ₆ solvent were acquired, and a complete assignment for the client sample is presented in Table 2. Reference spectra can be found in the Supporting Information (Figures S2.7–2.12). Together, the analysis revealed the client sample to contain N‐pyrrolidino isotonitazene (NPI, isotonitazepyne), a constitutional isomer of NPP. The low‐resolution GC‐MS spectrum and a reference material for NPI are available from Cayman Chemical but were not obtained as part of this study due to a projected 3‐ to 4‐month delay for supply.

¹H NMR expanded region of the client sample (**NPI**) in DMSO‐d ₆; 3.24 ppm = H₂O; 2.50 ppm DMSO‐d ₅. An unidentified impurity coextracted from the client sample is labelled (imp.); the isopropoxy substituent doublet at 1.23 ppm is obscured by the impurity.

TABLE 2.

NMR data of client sample (NPI) in DMSO‐d ₆. Assignment for proton‐coupled ¹³C signals were determined using HSQC and HMBC ¹H → ¹³C cross‐peak analysis.

Structure	Label	Chemical shifts (ppm)		Multiple bond correlations
		¹H	¹³C	COSY	HMBC
		¹H	¹³C	¹H → ¹H cross‐peak	¹H → ¹³C cross‐peak
Open in a new tab	a	8.46	114.7	b (weak)	c, n, p, o
	b	8.13	117.6	c	a, n, o
	c	7.73	110.9	b	n, p
	d	4.34	43.1	e	e, o
	e	2.53	54.3	d	g
	f	11.95
	g	2.39	53.8	h	h, g′
	h	1.62	23.2	g	g, h′
	i	4.31	32.3		j, q, r
	j	7.18	129.9	j	i, k, s, j′
	k	6.90–6.84	115.8	k	s, r, j, k′
	l	4.56	69.1	m	m, s
	m	1.23	21.8	l	l, m′
	n		142.6
	o		139.9
	p		141.4
	q		158.4
	r		127.8
	s		156.3

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To establish an approximate quantity of NPI per pill, an extraction followed by quantitative‐NMR (qNMR) was undertaken on three whole individual pills. The quantity of NPI in each pill ranged from 3.5 to 3.3 mg per pill (3.4 ± 0.1 mg, mean ± SD; Table S2.3 and Figures S2.13–S2.17).

First described in the illicit drug market 2019 [1], nitazenes were originally developed as experimental analgesic agents in the 1950s but were never brought to market [6, 18]. In September 2024, the nitazene opioid NPI was detected at an Australian drug checking service in an expected oxycodone sample. Although this nitazene analogue is available as a reference material from Cayman Chemical and has been investigated as a predicted novel synthetic opioid by several laboratories [5, 6], this is the first reported detection of this analogue within the illicit drug market.

Of particular concern in this case was the reported misrepresentation of the product purchased online as containing a pharmaceutical agent oxycodone that is commonly diverted in Australia. The unmarked, round mottled yellow appearance suggested the sample was not prepared to closely mimic an authentic pharmaceutical but may have been produced for sale by anonymous online vendors or cryptomarkets as an alternative to street level drug diversion. Recent reports have highlighted the prevalence of cryptomarkets that offer a wide range drugs, including nonheroin opioids like nitazenes, internationally and to the Australian market [19, 20]. Research on the phenomenon of cryptomarkets is nascent, with commentary suggesting the potential for higher quality of drug supply due to features such as aggregated marketplaces and client reviews of drug quality [21]. The current example highlights the potential for concerning substitutions in products sourced from online vendors. More contemporary drug alerts suggest this product remains in circulation several months after the notification associated with this study was released [22, 23].

Analysis by FTIR proved incapable of identifying any drug component within the presented pill, likely due to the low abundance of NPI (< 2% w/w by qNMR) in the homogenised sample, a known limitation of this analytical technique [24]. Nitazene immunoassay test strips provided an initial indication of the presence of a nitazene, with reported limits of detection between 1000–9000 ng/mL for a range of common nitazene derivatives [25, 26]. However, the inability of test strips to differentiate between nitazenes remains a limitation given the wide variation in reported in vitro potency reported for members of this class [2, 3, 4, 5, 6].

Chromatographic analysis indicated the presence of a ‘nitazene like’ component (UPLC‐PDA, CanTEST) and gave structural information in the form of the molecular formula and the production of several characteristic fragment ions (GC‐EI‐MS and UPLC‐ESI‐MS/MS, ANU). The spectroscopic and spectrometric features of the two constitutional isomers NPP and NPI were essentially identical across the three chromatographic analytical techniques, but the compounds differed in their retention times. Such scenario provides obvious challenges to methods of analysis like direct MS that operate without chromatographic resolution of isomeric species [27, 28, 29, 30].

Access to an NPI reference material would conventionally be required to match chromatographic retention times. However, in the current context, the lengthy 3‐ to 4‐month delay to acquisition of these materials, and the need for a rapid response, made this approach impractical. NMR facilitated the rapid structural elucidation of NPI and clearly differentiated this from the previously observed constitutional isomer NPP. Furthermore, qNMR analysis found there to be a significant dosage of NPI in a single pill (3.4 ± 0.1 mg, mean ± SD), raising concerns regarding the risk to public health of this presentation.

The potency of nitazenes is often reported relative to morphine or fentanyl. Both NPP and NPI are powerful activators of the μ‐opioid receptor, as demonstrated by their in vitro EC₅₀ values for stimulation of [³⁵S]GTPγS binding to activated G‐proteins [5], β‐arrestin 2 recruitment (MOR‐βarr2) or inhibition of cAMP accumulation [6]. One study reported NPP and NPI have MOR‐βarr2 agonist potencies approximately 350 and 1100 times that of morphine (Table S4.1) and 30 and 90 times that or fentanyl, respectively [6]. Though complicated by a range of factors, including drug bioavailability and metabolism, studies of nitazene drugs indicate that this high in vitro potency translates into in vivo activity in animal subjects [4, 6, 31, 32, 33].

The adulteration or substitution of the opioid drug supply with nitazenes internationally has been associated with adverse effects for drug users and significant mortality [6, 7, 8, 9, 10, 11]. Recent reports of overdose clusters in Dublin and Cork in Ireland associated with NPP highlight the public health threat that accompanies the introduction of novel synthetic opioids into the drug market [12, 13]. In the Australian context, a range of nitazenes have been detected in the drug supply and subject to alerts from public health authorities [11, 34, 35]. These detections have not only been associated with opioid supply but also, concerningly, the adulteration or substitution of other drugs such as benzodiazepines, cocaine, ketamine and MDMA [11, 34, 35]. The data for such alerts typically arise from the analysis of seized drugs or the toxicology samples obtained in association with emergency department presentations [11, 35, 36]. Wastewater analysis, which has limited coverage of NPS and targets only representative members of the nitazene family [37], has reported on the presence of nitazenes in the United states and recently Australia [38, 39].

Drug checking provides an alternative pathway to monitor the illicit drug market and respond to the introduction of high‐risk substances such as novel synthetic opioids. Working directly with members of the public, drug checking offers the potential to immediately respond to client reports of unexpected effects or drug related harm and, where appropriate, undertake public health messaging associated with high‐risk detections. The detection of NPI in an expected oxycodone sample reported here was achieved in a rapid time frame. Within 24 h of sample presentation at the drug checking service, an alert was issued to local clinical first responders to communicate the detection of a nitazene and provide information on the clinical management of nitazene harms. In the following 48 h, a community notice was released by the CanTEST service [22], distributed on social media and covered by the local media. Similarly rapid responses attended earlier detections of nitazene drugs at the service, including the detection of metonitazene in December 2022 and NPP in July 2024 (Table S4.1). Together, this series of detections highlights the unpredictability and rapid change within drug markets, with a local trend towards increasing potency and potential for harm for the nitazene drugs identified.

In addition to drug market monitoring and the issue of drug alerts, drug checking services form part of a broader response to the introduction high‐risk substances like nitazenes. Drug checking services provide avenues for science communication and public education. The provision of analytical test results is accompanied by health and alcohol and other drug (AOD) interventions that can be tailored to individual circumstances. This could include advice on safer using practices or dissemination of information regarding fluctuations or concerning detections in the local illicit drug market. The service also provides access to and training in the use of the opioid reversal agent naloxone, one effective measure to address nitazene‐related overdose [11, 35, 40].

4. Conclusion

Close collaboration between an Australian drug checking service and an affiliated university laboratory has identified and characterised the novel synthetic opioid NPI in an expected oxycodone sample, presented by a member of the public. Previously reported in vitro evaluations of this compound reveals it to be among the most potent nitazene opioid agonists known [5, 6]. The study highlights the rapid response possible though engaging drug users with drug checking services as a market monitor and early warning system to alert health services and the broader community to the presence of unexpected, high‐risk substances. Integration of well‐resourced and supported drug checking services provides a powerful approach to tackle the public health threats associated with novel synthetic opioids and other drugs of concern.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1.1 FTIR spectrum of client sample pill homogenate.

Figure S1.2 UV‐spectra of A) NPI (RT 3.06 min) and NPP (RT 3.56 min).

Table S1.1 Results from immunoassay test strip analysis.

Figure S2.1 GC‐EI‐MS, total ion chromatogram (TIC) and selected extracted ion chromatograms (EIC, m/z 84, 107, 408) for client sample (NPI).

Figure S2.2 GC‐EI‐MS, total ion chromatogram (TIC) and selected extracted ion chromatograms (EIC, m/z 84, 107, 408) for NPP.

Table S2.1 Integration of GC‐EI‐MS EIC peak areas for m/z 84, 107 and 408 for client sample (NPI) and NPP.

Figure S2.3 GC‐EI‐MS spectrum of client sample (NPI) at RT 19.4 min.

Figure S2.4 GC‐EI‐MS spectrum of NPP at RT 20.6 min.

Figure S2.5 Extracted ion chromatograms (EIC) (m/z 182.1903 and 409.2235 ± 5 ppm) of client sample (NPI, blue), internal standard in client sample (green), NPP (black) and internal standard in NPP sample (red).

Figure S2.6 +ESI MS spectra of A) client sample (NPI), B) NPP, and MS/MS (HCD @ 40 eV) spectra of C) client sample (NPI), D) NPP.

Table S2.2 Ion intensity for major fragments and precursor ion from UPLC‐ESI‐MS/MS product ion spectrum.

Figure S2.7 ¹H NMR spectrum of client sample (NPI) in DMSO‐d ₆.

Figure S2.8 ¹H NMR spectrum of client sample (NPI) in CDCl₃.

Figure S2.9 ¹³C NMR spectrum of client sample (NPI) in DMSO‐d ₆.

Figure S2.10 COSY of client sample (NPI) in DMSO‐d ₆.

Figure S2.11 HSQC of client sample (NPI) in DMSO‐d ₆.

Figure S2.12 HMBC of client sample (NPI) in DMSO‐d ₆.

Table S2.3 Values used for qNMR in the calculation of the mass of NPI per pill, using the ERETIC 2 function in Bruker TopSpin 3.6.3. The ERETIC 2 function applies a method reported in the primary literature.1.

Figure S2.13 qNMR, ¹H NMR spectrum for hydroquinone external standard in DMSO‐d ₆.

Figure S2.14 qNMR, ¹H NMR spectrum of pill extract 1 in DMSO‐d ₆.

Figure S2.15 qNMR, ¹H NMR spectrum of pill extract 2 in DMSO‐d ₆.

Figure S2.16 qNMR, ¹H NMR spectrum of pill extract 3 in DMSO‐d ₆.

Figure S2.17 qNMR, ¹H NMR blank spectrum DMSO‐d ₆.

Figure S3.1 ¹H NMR spectrum of sample containing N‐pyrrolidino protonitazene (NPP), with benzoic acid impurity CDCl₃.

Table S4.1 Substitutions of various nitazenes, their in vitro² and in vivo ³ potencies relative to morphine, and date detected at CanTEST. ^aNot detected at CanTEST.

DTA-17-1996-s001.pdf^{(1.6MB, pdf)}

Acknowledgements

The CanTEST Health and Drug Checking Service is operated by Directions Health Services with funding from ACT Health and support from Pill Testing Australia and the Canberra Alliance for Harm Minimisation and Advocacy. We thank Mr Joeseph Boileau and Ms Anitha Jeyasingham and Dr Adam Carroll for helpful discussions on mass spectrometry. Open access publishing facilitated by Australian National University, as part of the Wiley ‐ Australian National University agreement via the Council of Australian University Librarians.

Curtis B., Lawes D., Caldicott D., and McLeod M., “Identification of the Novel Synthetic Opioid N‐Pyrrolidino Isotonitazene at an Australian Drug Checking Service,” Drug Testing and Analysis 17, no. 10 (2025): 1996–2004, 10.1002/dta.3910.

Funding: This study was supported by the ACT Health.

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information.

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