A determination of the aerosolization efficiency of drugs of abuse in a eutectic mixture with nicotine in electronic cigarettes

Abstract

Eutectic mixtures can be formed by adding drugs other than nicotine (DOTNs) to nicotine-based e-liquids in electronic cigarettes (e-cigarettes). Thus, the interaction between nicotine e-liquids and DOTNs must be evaluated. Presented is the change in e-cigarette aerosolization of nicotine and methadone alone versus a 1:1 nicotine: methadone mixture to evaluate the possible formation of a eutectic mixture that can result in an increase of drug delivery. E-liquids were prepared in-house using 1:1 propylene glycol (PG):vegetable glycerin (VG) as a base plus nicotine, methadone hydrochloride, or 1:1 nicotine:methadone hydrochloride. The e-liquids were aerosolized via an automated vaping machine using parameters adopted from the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) E-cigarette Task Force method. Drug recovery was determined by capturing the aerosol from 15 puffs generated by the e-cigarette. Concentrations of nicotine and methadone aerosolized were determined by gas chromatography–mass spectrometry using nicotine (n = 3), methadone (n = 3), and combined nicotine/methadone e-liquids (n = 3), each prepared in-house at 12 mg/ml. The concentration of nicotine and methadone in 15 puffs of the single drug e-liquids were determined to be 1.60 ± 0.20 and 2.67 ± 0.12 mg, respectively. The concentration of nicotine and methadone in 15 puffs of the multidrug e-liquid were determined to be 3.66 ± 0.49 and 3.65 ± 0.10 mg, respectively. The single nicotine and methadone e-liquids had recoveries of 70 ± 0.1% and 84 ± 0.1%, respectively. In the 1:1 mixture, the recovery of both drugs increased. The development of a eutectic mixture can promote aerosolization of the drug and deliver a greater dose to the user.

1 |. INTRODUCTION

Electronic cigarettes (e-cigarettes) gained popularity as the marketing narrative focused on the user experience and vaping as a lifestyle choice. The perpetuation of the persona of someone who vapes as trendy and cool has contributed to the rising trend in e-cigarette use.^1,2 From 2010 to 2018, the percentage of U.S. individuals who reported ever using an e-cigarette increased from 3.3% to 14.9%.^3,4 E-cigarettes are used for many reasons, including a user’s curiosity to experiment, socialization with peers who vape, the perception of the health benefits and harmlessness, the trendiness of the products, and for convenience and discreteness.^2,5–8 E-cigarettes are typically composed of a mouthpiece, a reservoir tank, a battery, and an atomizer, which volatilizes the e-cigarette liquid (e-liquid) formulation into a condensation aerosol for inhalation.⁹ E-liquids usually contain propylene glycol (PG) and vegetable glycerin (VG), an active drug such as nicotine, flavoring agents, and other compounds, which could include ethanol.^9,10 With the advancements of e-cigarette devices, the ability to vape e-liquids with drugs other than nicotine (DOTNs) has occurred.¹¹ Vaping of cannabis/cannabinoids, cocaine and other psychostimulants, hallucinogens, opioids such as heroin, methadone, fentanyl, and other drugs have been reported in the literature and Internet forum sites.^6,12–14 The convenience of adulterating e-liquids for recreational drug use is becoming increasingly common as drugs can be added into a commercially available e-liquid.¹¹ A report of 241 commercially available e-liquids identified nicotine e-liquids that also contained caffeine, ethanol, cannabidiol, and mitragynine.¹⁵ Synthetic cannabinoids have been reported in e-liquids, predominantly as an adulterant to phytocannabinoids.^16–20 Online recreational drug forums have discussed the impact of adding the drug of choice (usually an opioid) to a common drug like nicotine or caffeine,^21,22 which can complicate harm reduction treatment.²³

Several factors are reported that can impact drug recovery and the formation of volatile products in aerosols. In a study evaluating the aerosolization of a heroin/caffeine mixture, drug aerosolization was changed by increasing the temperature of the sample and the addition of a diluent.²⁴ The addition of a diluent has been extensively studied and found to affect the drug aerosolization efficiency. The presence of a diluent can either increase or decrease the recovery of the parent drug.^25,26 In a eutectic mixture, the diluent interacts with the active drug at a specific ratio, reducing the melting point to a temperature lower than the melting temperatures of the individual compounds, and it is at this point that aerosolization of the drugs occurs more efficiently.²⁷ The interaction reported between heroin and caffeine was shown to alter the thermal behavior of the individual compounds, reducing the melting temperature of the mixture.²⁸ The chemical form of drug(s) used is also reported to alter the recovery of the parent drug, and the pH of the solvent can also affect the amount of drug delivered.^26,29,30 Another study reported the flow rate of air through a smoking device was a factor in the volatilization of cocaine, whereas the temperature of the device increased the concentration of pyrolytic products.³¹ A high temperature during the drug aerosolization can facilitate the generation of pyrolytic products.^26,32 The formation of these pyrolytic products can impact the overall recovery of the parent drugs.

Presented is the analysis of aerosols generated from 12 mg/ml nicotine, methadone and 1:1 nicotine:methadone e-liquids. Nicotine is an alkaloid and the main addictive component in traditional cigarettes and is often added to e-liquid formulations.^9,33 An evaluation of nicotine free-base concentrations in 27 commercially available e-liquids labeled with nicotine concentrations ranging from 6 to 18 mg/ml found the e-liquids contained 45–131% of the stated concentration.⁹ Nicotine salt concentrations in commercial e-liquids can be found up to 100 mg/ml; however, analysis demonstrated nicotine salt concentrations experimentally ranged from 20 to 89 mg/ml.³⁴ Methadone was used as a model for opioids for safety purposes. Methadone is a Schedule II synthetic opioid that is used to treat pain and opioid use disorder but has a high abuse liability.³⁵ Methadone can be taken by various routes of administration.³⁶ Methadone is commercially available in a 10 mg/ml liquid solution or 1, 5, 10, and 25 mg tablets for oral administration. The maximum therapeutic dose of methadone is suggested and restrictively distributed at 40 mg.^37,38 Both nicotine and methadone act on nicotinic acetylcholine receptors (nAChRs).^39,40 Methadone binds to the nAChR as an agonist and increases calcium levels, which can enhance neurotransmitter release.^40,41 Nicotine binds to the nAChR as a full agonist and increases dopamine release, reinforcing nicotine addiction.^39,42,43 It can also facilitate the release of endogenous opioids.⁴⁴ Nicotine can increase drug consumption and decrease the effects of opioid withdrawal whereas methadone can enhance the reinforcing effects of nicotine.^45–48 Oral administration of a drug like methadone undergoes first-pass metabolism, resulting in a majority of the drug being eliminated and a low bioavailability of the drug at the site of action.^49,50 Inhalation of drugs is a preferred route of administration as it results in a high bioavailability, a rapid absorption of the drug because of its avoidance of the metabolic pathway in the liver, and, subsequently, a rapid onset of drug effects.^51,52

Aerosols were generated and captured using a Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) E-cigarette Task Force method.⁵³ The captured aerosols were analyzed using a gas chromatography–mass spectrometry (GC-MS) method to determine the concentrations of each drug and to check for pyrolytic products. Changes in recoveries and concentrations of nicotine and methadone within the aerosols were used to determine if a eutectic mixture was formed.

2 |. EXPERIMENTAL

2.1 |. Materials

Methadone, methadone-d9, 2-ethylideine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and nicotine certified reference materials (CRMs) were purchased from Cerilliant (Round Rock, TX). Anhydroecgonine methyl ester (AEME) CRM was purchased from Supelco (Bellefonte, PA). Quinoline (purity 98%), nicotine liquid (purity ≥99%), and methadone hydrochloride powder (purity ≥98%) were purchased from Sigma-Aldrich (Saint Louis, MO). β-Nicotyrine CRM was purchased from Cayman Chemical (Ann Arbor, MI). Propylene glycol (USP grade) and vegetable glycerin (USP grade) were purchased from Wizard Labs (Altamonte Springs, FL). Methanol (HPLC grade) was purchased from Sigma-Aldrich and Thermo Fisher Scientific (Waltham, MA). Ethanol (200 proof) was purchased from Koptec (King of Prussia, PA). A KangerTech SUBOX^™ Mini-C e-cigarette, 0.5-Ω stainless steel organic cotton coils (SSOCC) with a nichrome wire, and SUBTANK^™ Mini-C tanks were purchased from Vapor Authority (San Diego, CA). The aerosol generating model (or automated vaping machine) and the filter holder were designed by Dr. Vinit Gholap and Phil Gunn Machine Co., Inc. (Richmond, VA), respectively. The syringe pump device and the stand for the SUBOX^™ e-cigarette were purchased from Gram Research (Oakland, CA). The Borgwaldt 44 mm Cambridge filters manufactured by Hauni Richmond, Inc. were purchased from Thermo Fisher Scientific. The aerosol capture proprietary software system was obtained from Gram Research.

2.2 |. Generation and capture of the aerosols

E-liquids were made in-house using a 1:1 PG:VG mixture as the base. Drug free, 12 mg/ml methadone, 12 mg/ml nicotine, and 1:1 12 mg/ml nicotine:methadone e-liquids were prepared in-house using the PG:VG mixture. These e-liquids were aerosolized using a previously validated method with parameters adopted from the CORESTA method and an automated vaping machine.²⁹ The vaping machine was operated with an attached air flow sensor using the CORESTA guidelines to measure the puff duration, air flow, and puff volume (n = 3). In brief, an empty assembled e-cigarette was weighed, 2.5 ml of e-liquid were added to the tank, and the e-cigarette was weighed again to determine the pre-aerosolization weight. The e-cigarette was set aside for at least 1 min to allow the e-liquid to saturate the wick in the coil. A Cambridge filter was weighed before aerosolization and inserted into the filter holder. The voltage on the SUBOX^™ e-cigarette was set to 4.3 V, and then the trapping system and the e-cigarette were assembled and attached to the vaping machine, Figure 1. The aerosol capture parameters were programmed as described in Table 1 with the vaping profile optimized for a 3 s puff duration for 15 puffs. The puff volume was 60 ml rather than 55 ml as suggested by the CORESTA method. Total aerosol capture took approximately 7.5 min per run. After each run, the filter and the e-cigarette were weighed for the post-aerosolization weight. The filter holder and mouthpiece were washed with 50 ml of methanol, which was collected and sonicated for 10 min. Aliquots (n = 3) were collected for analysis. The nicotine aerosol samples were diluted 1:10 in methanol and the methadone and 1:1 mixture aerosol samples were diluted 1:20 in methanol. The theoretical amount of the drug aerosolized was calculated from the weight difference of the e-cigarette before and after aerosolization. The experimental amount of drug aerosolized and percent drug recovery were calculated from analysis of the filter pad using the presented methods.

FIGURE 1.

(a) Automated vaping machine (or aerosol generating model). (b) Schematic diagram of vaping machine (created with BioRender.com)

TABLE 1.

Aerosol capture parameters

Parameter	CORESTA conditions	Experimental conditions
Preheat, s	-	1
Puff volume, ml	55	60
Inhale, s	3	3
Exhale, s	-	10
Power, W	-	255
Number of puffs	-	15
Puff frequency, s	30	30

2.3 |. Standard preparation

Nicotine calibration standards were prepared at 500, 1000, 2000, 5000, 10,000, and 20,000 ng/ml in methanol using quinoline (100 μg/ml in methanol) as the internal standard. Methadone standards were prepared at 50, 100, 200, 500, 1000, 2000, and 5000 ng/ml in methanol using methadone-d9 (10 μg/ml in methanol) as the internal standard. Quality controls (QCs) for nicotine and methadone were prepared at 1500, 8000, and 16,000 ng/ml and 150, 1500, and 4000 ng/ml, respectively. EDDP and β-nicotyrine were prepared at concentrations of 100 μg/ml in methanol and 40 μg/ml in ethanol, respectively.

2.4 |. Determination of nicotine, methadone, and pyrolytic products using gas chromatography–mass spectrometry

Identification and quantitation of nicotine, methadone, and pyrolytic products were performed on a Shimadzu QP-2020 GC-MS (Kyoto, Japan) in electron ionization mode controlled by GCMS Real Time Analysis version 4 (Shimadzu Corp., Kyoto, Japan). Chromatographic separation was performed on a HP-5MS column (30 m × 0.25 mm id × 0.25 μm) (Agilent, Santa Clara, CA) using helium as the carrier gas at a linear velocity of 35 cm/s. The GC-MS was operated in splitless mode using 1 μl sample injections and an inlet temperature of 250°C with a split ratio of 6:1. The initial oven temperature was 100°C, followed by a ramp of 15°C/min to 170°C. The temperature was then ramped at 40°C/min to 300°C. The ion source was set to 250°C and the interface was set to 280°C. The qualifying and quantifying ions used for the analysis are listed in Table 2. For the identification of the analytes, a combined stock of 10 μg/ml nicotine, 100 μg/ml quinoline, 10 μg/ml methadone, 10 μg/ml methadone-d9, 40 μg/ml β-nicotyrine, and 100 μg/ml EDDP was analyzed to determine retention times and mass spectra.

TABLE 2.

Qualifying and quantifying ions

Compound	Ions
Methadone	72a, 223, 309
Methadone-d9	78a, 318
EDDP	262, 276, 277
Nicotine	84, 133a, 162
Quinoline	76, 102, 129a
β-Nicotyrine	116, 130, 158

^aIons for quantitation.

Method validation was performed by analyzing calibrators, QCs, and negative controls with and without internal standard for each drug in triplicate over 5 days. Linearity, limit of detection, bias, precision, carryover, and contamination were evaluated. A linear regression of the average of the ratio of the peak area counts of analyte and the corresponding deuterated internal standard versus concentration was used to construct the calibration curves. Bias was determined from the percent difference of the experimental concentration of the QCs and the theoretical concentration. Precision was determined from the standard deviation and mean experimental concentration of the QCs and analyzed by one-way ANOVA (α = 0.05) to determine the mean square between and within groups. Carryover was assessed by running blanks with internal standard after the highest calibrator, and contamination was assessed by injecting methanol blanks.

3 |. RESULTS

3.1 |. Aerosol capture

Nicotine, methadone, and their associated pyrolytic products in the various collected aerosol samples were identified using the presented GC-MS method. Figure 2 shows the chromatogram of the 1:1 mixture aerosol sample. The aerosol capture was performed four times for each e-liquid. The first trial was eliminated for analysis as the amount of e-liquid aerosolized was not consistent with the subsequent trials. This was hypothesized to be due to a characteristic of the e-cigarette manufacturing. In the single drug e-liquids (n = 3), 1.60 ± 0.20 mg of nicotine and 2.67 ± 0.12 mg of methadone were aerosolized, respectively. In the 1:1 nicotine:methadone mixture (n = 3), the amount of nicotine and methadone aerosolized increased to 3.66 ± 0.49 mg and 3.65 ± 0.10 mg, respectively (Figure 3). In the single drug e-liquids (n = 3), the nicotine recovery was 70 ± 0.1% and the methadone recovery was 84 ± 0.1% (Table 3). The aerosolizing of the 1:1 nicotine:methadone mixture resulted in recoveries of 97 ± 0.1% and 97 ± 0.1% for methadone and nicotine, respectively. In the 15 puffs of the nicotine and methadone e-liquids, 0.22 ± 0.02 g and 0.31 ± 0.01 g (n = 3) were aerosolized, respectively. In the 1:1 nicotine:methadone mixture, 0.37 ± 0.01 g (n = 3) of e-liquid was aerosolized (Table 3).

FIGURE 2.

Aerosol capture sample of 1:1 methadone:nicotine e-liquid. Parent drugs, internal standards, and pyrolytic products were identified.

FIGURE 3.

Drug dose of parent drugs in single- and mixed-drug e-liquids. Error bars indicate standard deviation. α = 0.05. For nicotine and methadone, p = 0.002 and 0.0004, respectively.

TABLE 3.

Percent recovery and amount of e-liquid aerosolized

	Recovery (%), n = 3	Amount e-liquid (g), n = 3
NIC	70 ± 0.1	0.22 ± 0.02
MTD	84 ± 0.1	0.31 ± 0.01
1:1 NIC:MTD	97 ± 0.1	0.37 ± 0.01

3.2 |. GC-MS method validation

The GC-MS method for the analysis of nicotine was determined to have a linear range of 500–20,000 ng/ml and coefficient of determination (r²) values for the five analytical runs had an average of 0.996. The standardized residual plots across the five runs showed that the residuals were within three standard deviations of the mean and no outliers were present. The limit of detection (LOD) was administratively set as the lowest calibrator, 500 ng/ml for nicotine, with a signal to noise ratio (S/N) > 10. Bias (n = 15), intraprecision, and interprecision for all controls were <−6%, 8%CV, and 6%CV, respectively. Methadone was determined to have a linear range of 50–5000 ng/ml and r² values had an average of 0.997 (n = 5). Residuals were within three standard deviations of the mean and no outliers were present. Bias (n = 15), intraprecision and interprecision for all controls were <−8%, 2%CV, and 4%CV, respectively (Table 4). The LOD was administratively set as the lowest calibrator, 50 ng/ml for methadone, with a S/N > 10. No carryover or contamination was detected; all carryover concentrations were below the respective LODs.

TABLE 4.

Bias and precision

	Intra-run, n = 3	Inter-run, n = 15
Concentration (ng/ml)	%CV	Bias (%)	%CV
Nicotine
1500	8	−1	6
8000	1	−6	3
16,000	4	4	4
Methadone
150	2	−3	3
1500	2	−1	2
4000	1	−8	4

4 |. DISCUSSION

The presented analytical methods were successfully validated according to ANSI/ASB Standard Practices for Method Validation in Forensic Toxicology and used to evaluate the prepared e-liquids and corresponding aerosol samples.⁵⁴ Calculated concentrations of nicotine and methadone standards were within ±20% of the theoretical concentration. The amount of nicotine and methadone in the single-drug e-liquids increased in the mixed-drug e-liquid. Employing a two-tailed t-test with α = 0.05, there is statistically significant evidence that the amount of nicotine (p = 0.002, n = 3, |μ₁ − μ2| = 2.06) and methadone (p = 0.0004, n = 3, |μ₁ − μ₂| = 0.984) aerosolized is significantly different between the single drug e-liquids and the mixed-drug e-liquid. The amount of e-liquid aerosolized in the single-drug e-liquids increased in the mixed-drug e-liquid, supporting the formation of a eutectic mixture. The experimental design was chosen to mitigate issues that arise with prepared e-liquids aggregating water with hygroscopic PG and VG while an experiment with another e-liquid tank is in process. Therefore, three replicates of each condition could be completed in a single e-liquid preparation. Statistical techniques produced statistically significant findings with enough power to detect this difference while still controlling for Type I error rates, even though the sample size and power may not be sufficient to detect other differences.

The formation of the eutectic mixture and the subsequent increase in recovery and aerosolization of nicotine and methadone in this experiment was similar to other reports. At 25%, 50%, and 75% w/w of heroin base and caffeine, recoveries of heroin and caffeine were also reported to increase compared to the recoveries of the single drugs.²⁴ In varying ratios with caffeine and barbital, the recovery of heroin hydrochloride and heroin base were reported to increase.²⁶ Cocaine base in 1:1 mixtures with benzocaine and procaine were reported to increase the recovery of cocaine.²⁵

The presence of the pyrolytic products in the aerosol samples collected in this experiment did not affect the aerosolization of the parent drug in the eutectic mixture. Uchiyama et al.⁵⁵ reported the generation of pyrolytic products as a result of the aerosolization of e-liquids in e-cigarettes. Contrary to what has been reported, we did not identify any pyrolytic products generated by the e-cigarette. β-Nicotyrine is a product formed from thermal degradation or oxidation of nicotine that can interfere with the metabolism of nicotine, resulting in increased levels of nicotine in the body.^56–58 EDDP is a major metabolite of methadone and is the primary biomarker used to indicate the presence of methadone in biological fluids.⁵⁹ The pyrolytic products β-nicotyrine from nicotine and EDDP from methadone were identified in the aerosol samples, but they were also present in the standards and e-liquids. Their presence could indicate they were generated as an artifact of high temperature in the GC injection port and not formed due to the aerosolization event in the e-cigarette. Although the pyrolytic products were not observed solely as a result of aerosolization in this experiment, their production in the e-cigarette could raise issues about the hazards associated with inhaling other byproducts and about their pharmacological interactions with the active drug.^60,61

The adapted CORESTA method for aerosol collection successfully captured approximately 100% recovery of drug from each e-liquid. The GC-MS method was successfully validated and able to identify the analytes and resolve the pyrolytic products from their respective parent drugs. Both the aerosolized nicotine and methadone dose increased in the eutectic mixture. The pyrolytic products were not observed solely as a result of aerosolization as they were also detected in the standards, e-liquids, and aerosol samples, possibly due to volatilization in the GC injection port. No current literature has been published on the toxicological impacts of the increased aerosolization of multi-drug combinations via an e-cigarette. With this increase in dose for both the active drug and the adulterant, users must be cautious of unintended effects from inhalation of adulterated e-liquids. Thus, a greater understanding on how DOTNs interact with the e-liquid constituents is necessary.

Online recreational drug forums discuss the advent of combining drugs to vape with benefits that range from easier-to-manipulate to enhanced experiences. Pharmacologically, the potentiation of a drug is fundamentally problematic since the development of tolerance could become more advanced, faster. It has been reported that studies on the simultaneous use of drugs has increased significantly in the last 5 years.²³ The description of co-use drug seeking behaviors and rituals is an imperative for identifying acute risk and intervention strategies for immediate life-saving mediation and long-term substance use disorder treatment.

5 |. CONCLUSIONS

Changes in e-liquid formulations, such as the addition of a second drug, can result in the creation of a eutectic mixture. The eutectic mixture can allow for an increase in the amount of e-liquid and drug being aerosolized. This increase can heighten the risk of adverse effects.