Evaluation of Synthetic Dyes and Food Additives in Electronic Cigarette Liquids: Health and Policy Implications

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3-1-2018 Evaluation of Synthetic Dyes and Food Additives in Electronic Cigarette Liquids: Health and Policy Implications

Tetiana Korzun Portland State University

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Recommended Citation Korzun, Tetiana, "Evaluation of Synthetic Dyes and Food Additives in Electronic Cigarette Liquids: Health and Policy Implications" (2018). University Honors Theses. Paper 499. https://doi.org/10.15760/honors.502

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Evaluation of synthetic dyes and food additives in electronic cigarette liquids:

Health and policy implications

Tetiana Korzun

An undergraduate honors thesis submitted in partial fulﬁllment of

the requirements for the degree of

Bachelor of Science

University Honors

and

Biology

Thesis Advisers

Professor Robert M. Strongin, PhD Dr. R. Paul Jensen, PhD

Portland State University

2018 1

TABLE OF CONTENTS

ABSTRACT ...... 4

INTRODUCTION ...... 5

METHODS AND INSTRUMENTS ...... 8

Materials ...... 8

Sample Preparation ...... 8

Samples for HPLC-DAD analysis: ...... 8

Samples for ESI-HRMS validation: ...... 9

Samples for IC analysis ...... 9

HPLC-DAD Analysis ...... 10

ESI-HRMS Validation ...... 12

Ion Chromatography ...... 12

RESULTS AND DISCUSSION ...... 14

CONCLUSION ...... 19

ACKNOWLEDGMENTS ...... 19

SUPPLEMENTARY INFORMATION ...... 20

HPLC-DAD chromatograms ...... 20

ESI-HRMS spectra of synthetic dye standards ...... 21

ESI-HRMS validation of synthetic dyes in e-liquid samples ...... 23

Ion chromatograms of vaporized samples ...... 25

REFERENCES ...... 26

INDEX OF TABLES

Table 1. Quantitative features of the HPLC-DAD method for the selected dye standards ...... 11 Table 2. Quantitative features of the ESI-HRMS method for the selected dye standards...... 12 Table 3. Seven synthetic dyes used in foods. Chemical classes and ingestion ADIs...... 14 Table 4. Identification and quantification of unknown dyes in the samples of commercially available e-liquids...... 15 Table 5. ESI-HRMS validation of unknown dyes in commercially available e-liquids...... 16 Table 6. Synthetic dye concentrations in selected foodstuffs...... 16 Table 7. Sulfate and chloride concentration in vaporized e-liquid samples...... 18

TABLE OF FIGURES

Figure 1. Structures of synthetic dyes...... 6 Figure 2. Representative chromatograms of synthetic dye standards ...... 11 Figure 3. Chromatograms of unknown dyes...... 15

SUPPLEMENTARY INFORMATION

Figure S1. Additional chromatograms of dyes standards ...... 20 Figure S2. ESI-HRMS spectrum of Allura Red AC Standard...... 21 Figure S3. ESI-HRMS spectrum of Erythrosine Standard...... 21 Figure S4. ESI-HRMS spectrum of Fast Green FCF Standard ...... 21 Figure S5. ESI-HRMS spectrum of Tartrazine Standard ...... 22 Figure S6. ESI-HRMS spectrum of Sunset Yellow FCF Standard ...... 22 Figure S7. ESI-HRMS spectrum of Brilliant Blue FCF Standard ...... 22 Figure S8. ESI-HRMS spectrum of Allura Red AC in Red E-liquid ...... 23 Figure S9. ESI-HRMS spectrum of Tartrazine in Green E-liquid ...... 23 Figure S10. ESI-HRMS spectrum of Brilliant Blue FCF Dye in Green E-liquid ...... 23 Figure S11. ESI-HRMS spectrum of Brilliant Blue FCF in Blue E-liquid...... 24 Figure S12. IC of vaporized Red E-liquid...... 25 Figure S13. IC of vaporized Blue E-liquid...... 25 Figure S14. IC of vaporized Green E-liquid ...... 25

To beloved chemists and the physicist

Зробити щось, лишити по собі, а ми, нічого, – пройдемо, як тіні, щоб тільки неба очі голубі цю землю завжди бачили в цвітінні. Щоб ці ліси не вимерли, як тур, щоб ці слова не вичахли, як руди. Життя іде і все без коректур, і як напишеш, так уже і буде. Ліна Костенко

ABSTRACT

Prior studies of e-liquid thermal degradants do not reflect many of the potential health hazards related to e-cigarettes. Although current studies have focused on solvents and flavoring additives in e-cigarette formulations, there have been no prior reports on the identity and levels of synthetic dye additives. Therefore, the purpose of this study was to identify and quantify these compounds to enhance understanding of the risks associated with the inhalation of colored e- liquids. Furthermore, e-liquids were subjected to thermal degradation under normal vaping conditions to quantify the sulfur oxides (SOx) content indicating dye decomposition. The dyes were analyzed by a combination of high-performance liquid chromatography and high resolution mass spectroscopy. The thermal decomposition of dyes in vaporized e-liquid samples was studied by ion chromatography. The findings of this investigation revealed that e-liquid manufacturers added synthetic dyes in concentrations comparable to those used in the food industry. In addition, SOx were present in the aerosolized e-liquids suggesting that dyes undergo thermal degradation. The aerosol samples contained a substantial amount of free chloride, which could be associated with a breakdown of the sucralose molecules, whose presence in the e- liquids was confirmed by nuclear magnetic resonance.

INTRODUCTION

Electronic cigarettes (e-cigarettes) may pose dangers to consumers, due to a lack of formal oversight regarding their regulation and manufacturing. In addition, the long-term effects of vaping on human health remain to be unknown. However, e-cigarettes are frequently lauded to be a healthier alternative to tobacco products. For decades, tobacco manufacturers have faced heavy advertising restrictions, in large part to avoid encouraging tobacco use among children. It is known that advertising has a positive correlation with youth cigarette smoking.1 The bright packaging designs of e-liquids, as well as their pleasant aromas (and potentially colors), are known to be enticing to young people.2,3 These attractive products, readily available on the largely unregulated market,4 are manufactured and advertised in a relaxed regulatory environment. As a result, in recent years, e-liquid poisoning amongst children has increased by

1500%.5 This includes child fatalities associated with e-liquid nicotine ingestion overdoses.6 If appealing color and flavoring additives in e-liquid formulations remain, there are substantive health risks to our nation’s youth.

The current lack of regulations allows manufacturers to add new ingredients that have no associated inhalation toxicological data. The yields of toxic aldehydes and related compounds that are produced via the thermally–induced degradation of propylene glycol and glycerol (the e- liquid solvents) are enhanced by the decomposition of additives that are often not listed as e- liquid ingredients and are considered to be the manufacturing secrets.7,8

Food additives influence the consumers’ perception of food flavor and flavor identity.9

Such additives are classified as generally recognized as safe (GRAS) for ingestion.10 However, their inhalation toxicity is unknown. Two categories of color additives used in food and drugs in the US include those certified and those exempt from certification. They are categorized based 6 on the US Food and Drug Administration (FDA) testing requirements.11,12 Certified food dyes are synthetic compounds that are widely used because of their uniform color and shelf-life stability, as well as their ability to encompass a full spectrum of colors when mixed, while not impacting or altering food taste.13 The synthetic dyes shown in Figure 1 are certified by the FDA for use in food, as well as in drugs and cosmetics (FD&C). Other FD&C colorants, not shown here, are dyes that are certified for usage only in specific foods (i.e. Orange B and Citrus Red

No. 2 are used to color the surfaces of sausages and oranges, respectively).14

HO O N+ O O N O - O- O S N I I O S O O S O O O- O -O O O- S O I I O- HO N O O- S Allura Red AC Erythrosine Fast Green FCF (FD&C Red No. 40) (FD&C Red 3) (FD&C Green No. 3) O

O - O N O O O- O S N S S N -O O- O N O N N+ O O O S O- O Tartrazine Brilliant Blue FCF O (FD&C Yellow No. 5) (FD&C Blue No. 1) S O- O

HO O O O- O N NH S -O S N O O O S NH -O O O S O O O- Sunset Yellow FCF Indigotine (FD&C Yellow No. 6) (FD&C Blue No. 2)

Figure 1. Structures of synthetic dyes.

Although disclosing the identity of synthetic dyes is mandatory on the labels of foodstuffs,12 this information is absent on the vast majority of e-liquid labels. Additionally, acceptable daily intake (ADI) values for dye inhalation are not provided by the FDA, as synthetic dyes had not been used or intended for inhalation previously. 7

Herein, the purpose of this study was to identify and quantify the specific coloring compounds in commercially available e-liquids in order to enhance our understanding of their potential risks associated with inhalation and to help raise awareness about the inhalation of substances originally intended for ingestion.

METHODS AND INSTRUMENTS

Materials

Three e-liquids were ordered from the manufacturer’s online shop. All HPLC-grade solvents (acetonitrile, methanol and water) were from Fisher Scientific (Fisher Scientific

Co., Chicago, IL, USA). Dibasic ammonium phosphate (ACS reagent, ≥98%) and potassium hydroxide (ACS reagent grade, >85%, pallets) were used for HPLC buffer preparation and were obtained from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA). Perchloric acid

(70 % aqueous solution; Acros Organics) and hydrogen peroxide (30 % aqueous solution;

Sigma Aldrich, St. Louis, MO, USA) were used for absorption solution preparation in ion chromatography experiments. Analytical standards of Allura Red AC (FD&C Red 40),

Sunset Yellow FCF (FD&C Yellow 6), Tartrazine (FD&C Yellow 5), Brilliant Blue FCF

(FD&C Blue 1), Fast Green FCF (FD&C Green 3), Erythrosine (FD&C Red 3) and Indigo

Carmine (FD&C Blue 2) were obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, MO,

USA). Water for sample preparation was purified using Thermo Scientific Barnstead

GenPure xCAD Plus UV–TOC Water Purification System (Thermo Scientific, Waltham,

MA, USA).

Sample Preparation

Samples for HPLC-DAD analysis: E-liquids were diluted with water (1:5 dilution) and sonicated for 20 minutes. The standard solutions of Allura Red AC, Fast Green FCF,

Sunset Yellow FCF, Erythrosine, Tartrazine, Brilliant Blue FCF were prepared in water by dissolving the solid powders to obtain a stock concentration of 1mg/mL. Four to eight calibration concentrations were prepared from stock solutions and used for calibration curves construction with curves forced through the origin. The concentration ranges for standard 9 solutions were 20-80 µg/mL for Allura Red AC, Fast Green FCF, Sunset Yellow FCF and

Erythrosine, and 2.5-80 µg/mL for Tartrazine and Brilliant Blue FCF. Each unknown and standard solution were filtered (PVDF, 0.22 µm pore size) and subjected to HPLC-DAD analysis.

Samples for ESI-HRMS validation: dye samples from three e-liquids were concentrated using SPE cartridges (Oasis HLB cartridges, Waters, Milford, MA, USA), connected to the vacuum manifold system (Waters, Milford, MA, USA) and the vacuum inlet.

Cartridges were conditioned using 3 ml of methanol and 5 ml of deionized water at a flow rate of about 1 drop per minute. E-liquid samples diluted in water were transferred to the SPE cartridges. The loaded cartridges were rinsed with 5 ml of water allowing e-liquid solvent separation at a flow rate of about 2 drops per minute. The cartridges were eluted with two 3-mL methanol rinses at the same flow rate. The eluent sample volumes containing dyes were reduced by rotary evaporation (R-210 Rotavap, Buchi, New Castle, De, USA). The residues were reconstituted in 1 ml of methanol. SPE of red and blue e-liquids yielded two samples containing red and blue dyes, respectively; SPE of the green e-liquid yielded a combination of two samples of yellow and blue dyes. The standard solutions of Allura Red AC, Fast Green FCF, Sunset

Yellow FCF, Erythrosine, Tartrazine, Brilliant Blue FCF dyes were prepared in water to give a final concentration of 20 Μm. Note, Indigotine was not used for ESI-HRMS analysis.

Samples for IC analysis: the vapor was generated using the electronic cigarette consisted of Tesla Invader III battery unit (Teslacigs, Shenzhen, China) with two 18650

HG2 batteries (3.7 V, 3000 mAh) (LG Chem, Holland, MI) and KangerTech SubTank Mini atomizer with horizontal kenthal coil (1.2 Ω resistance) (KangerTech, Shenzhen, China). The e- cigarette was operated at 17 W. The aerosol was drawn into a -78 ºC cold trap (dry ice/acetone) 10 followed by the impinger containing acidified hydrogen peroxide absorption solution. The setup was connected to the SCSM-STEP single cigarette-smoking machine, simulating inhalation

(CH Technologies, Westwood, NJ). Aerosol was produced using CORESTA vaping mode (3- s puff with 1-s power button activation prior vaping, 30-s puff intervals, 55-mL puff volume).15

The sample from the cold trap was combined with the impinger solution. The samples were immediately subjected to analysis by ion chromatography. Triplicate experiments were performed using each e-liquid; each replicate represents the session of 40 puffs (20 puffs – 20 minutes cooling – 20 puffs).

HPLC-DAD Analysis

The samples of commercially available e-liquids were analyzed using adapted HPLC-

DAD method.16 The HPLC-DAD set-up included 1525 Binary HPLC pump, 2996 Photodiode

Array Detector and column heater (Waters, Milford, MA). The chromatographic separation was perfumed using Acclaim PA2 column with 3 µm particle size, 3×75 mm dimensions and injection volume of 5 µL (Thermo Scientific, Waltham, MA). The DAD detector was set to

210–650 nm spectral range. The column was kept at 30 °C. The mobile phase consisted of buffers A (20 mM (NH4)2HPO4, pH 8.8) and B (20 mM (NH4)2HPO4: CH3CN = 50:50, v/v).

The analysis of standard solutions was performed using the gradient of 12% B from 0.00 to 3 min, ramping to 100% from 3.00 to 3.50 min with hold for 1.0 min, and return to 12% B in

0.1 min (flow rate of 0.71 mL/min). The e-liquid samples followed the same gradient program and flow, except that the 100% B hold was extended to 29.50 min and returned to 12% in 1 min.

Table 1. Quantitative features of the HPLC-DAD method for the selected dye standards

Dye Absorption Maxima Regression LOD LOQ RSD Standard (nm) (%) (µg/mL) (µg/mL) (%) Allura Red AC 506 99.998 0.27 0.88 1.37 Fast Green FCF 619 99.988 0.97 3.23 2.39 Sunset Yellow FCF 485 99.972 1.60 5.35 0.36 Erythrosine 530 99.972 1.57 5.23 2.45 Tartrazine 426 99.991 0.57 1.91 0.81 Brilliant Blue FCF 630 99.973 0.10 0.34 1.49

Experiments and the construction of calibration curves were performed in triplicates.

Quantitative features of the HPLC-DAD method and chromatograms of the dye standards are presented in Table 1 and Figure 2 (additional chromatograms: Supplementary information).

Empower 2 Chromatography Data Software was used for data collection and processing.

Figure 2. Representative chromatograms of synthetic dye standards. Standards at 40 µg/mL concentrations. 12

ESI-HRMS Validation

Validation of identified analytes was performed using a high-resolution (30,000 resolution power) Thermo LTQ-Orbitrap Discovery hybrid mass spectrometry instrument equipped with Ion Max source with an electrospray ionization probe (Thermo Fisher Scientific,

San Jose, CA, USA). The ionization interface was operated in the negative mode using the following settings: source voltage, 4 kV; sheath and aux gas flow rates, 50 and 5 units, respectively; tube lens voltage, 90 V; capillary voltage, 49 V; and capillary temperature, 300 °C.

The Orbitrap mass analyzer was externally calibrated prior to analysis. ESI-HRMS spectra of standards (molecular ions: Allura Red AC [M]2-, Brilliant Blue FCF [M]2-, Tartrazine [M]3-,

Sunset Yellow FCF [M]2-, Erythrosine [M]2- and Fast Green FCF [M]2-) revealed the molecular ion masses with accuracy within ±7 ppm (Table 2 and Supplemental information).

Table 2. Quantitative features of the ESI-HRMS method for the selected dye standards.

Calculated Mass of Standards Observed Mass of Standards Standard (m/z) (m/z) Allura Red AC 225.00903 225.00924 Brilliant Blue FCF 373.07077 373.07067 Tartrazine 154.99315 154.99263 Sunset Yellow FCF 202.99592 202.99515 Erythrosine 834.64667 834.64394 Fast Green FCF 381.06823 381.06713

Ion Chromatography

Anion analysis of the vaped samples was conducted using an Dionex ICS-5000 ion chromatography system (Dionex, Sunnyvale, CA), outfitted with a conductivity detector cell and electrolytically regenerated suppressor (AERS 500, 4mm; Dionex, Sunnyvale, CA). 25 µL aliquots of the filtered (0.2 µm pore size) samples were injected onto the system for each run.

The separation was carried out on an IonPac-AS15 with an IonPac-AG15 guard columns 13

(Dionex, Sunnyvale, CA) and a flow of 0.75 mL/min. Gradient elution was used to obtain separation of the organic and inorganic peaks. The eluent concentrations were set as follows: 3 mM KOH for 36.5 minutes, 45 mM KOH for 16.5 minutes, and 3 mM KOH for 7 minutes.

Calibration curves were created using seven calibration standards with concentrations of chloride and sulfate ranging from 2.50 to 40.0 mg/L and 0.125 to 2.00 mg/L, respectively

(Supplementary Information). Data acquisition and analysis was performed using Chromeleon workstation. Linear calibration curves (without forcing the intercept through zero) were created based on peak height. R-squared values for all calibration curves were greater than 0.99; LOD values for chloride and sulfate were 0.063 and 0.079 mg/L, and LOQ values were 0.19 mg/L and

0.24 mg/L, respectively.

RESULTS AND DISCUSSION

Three e-liquids were randomly chosen to represent the primary additive colors (red, green and blue). The manufacturers were contacted but declined the requests for the identity of ingredients, including the synthetic dyes used in the formulations.

Three dyes, Allura Red AC, Brilliant Blue FCF and Tartrazine, were identified in the e- liquid samples. According to their structures, Allura Red AC and Tartrazine are azo dyes, and

Brilliant Blue FCF is a triarylmethane (Table 3). Although these artificial food colorants are generally regarded as safe (GRAS) for human consumption, batches of Allura Red AC, Sunset

Yellow FCF and Tartrazine are known to contain the human carcinogen benzidene and other aromatic amines17-20, while Tartrazine is a known potent allergenic agent.21-24

Table 3. Seven synthetic dyes used in foods. Chemical classes and ingestion ADIs.

Per Capita European ADI ADI25 Synthetic Dye Chemical Class Exposure25 (mg/kg/day) (mg/p/day)a (mg/p/day)a Allura Red AC Azo 0-7 (2016) 26 420 17.91 (FD&C Red No. 40) Sunset Yellow FCF Azo 0-2.5 (1982) 13 225 10.74 (FD&C Yellow No. 6) Tartrazine Azo 0-10 (2016) 26 300 12.06 (FD&C Yellow No. 5) Brilliant Blue FCF Triarylmethane 0-12.5 (1969) 13 720 1.72 (FD&C Blue No. 1) Fast Green FCF Triarylmethane 0-25 (1986) 13 150 0.038 (FD&C Green No. 3) Erythrosine Xanthene 0-0.1 (1990) 13 150 0.61 (FD&C Red No. 3) Indigotine Sulfonated Indigo 0-5 (1974) 13 150 1.95 (FD&C Blue No. 2) a Per 60-kg person

The identification and quantification of the dyes in the samples was performed by high- performance liquid chromatography-diode array method (HPLC-DAD). The chromatograms of three e-liquid samples were compared to chromatograms of the standard solutions of Allura Red

AC, Sunset Yellow FCF, Tartrazine, Brilliant Blue FCF, Fast Green FCF, Erythrosine and 15

Indigotine (Figure 3). The green e-liquid contained two dyes, Brilliant Blue FCF and

Tartrazine, at concentrations and proportions consistent with those used in the food industry to afford neon green solutions.27 The blue and red e-liquids contained Brilliant Blue FCF and

Allura Red AC food colorants, respectively (Table 4).

Table 4. Identification and quantification of unknown dyes in the samples of commercially available e-liquids.

Identified Absorption Maxima Retention time Concentration Sample Dye (nm) (min) (µg/g)a

Red Allura Red AC 505 2.38 66.71 Blue Brilliant Blue FCF 630 3.01 51.42 Green Brilliant Blue FCF 630 3.00 1.85 Green Tartrazine 426 1.38 29.43 a µg of synthetic dye per g of e-liquid

Figure 3. Chromatograms of unknown dyes. Notice that the green sample is made of a combination of yellow and blue dyes. Detector wavelengths were set at 426 nm for Tartrazine, 505 nm for Allura Red AC and 630 nm for Brilliant Blue FCF. Follow-up validation by high resolution electrospray ionization mass spectroscopy (ESI-

HRMS) confirmed the results obtained by HPLC-DAD method. Allura Red AC, Brilliant Blue

FCF and Tartrazine were identified with mass accuracies within ±7 ppm (Table 5 and

Supplementary information).

Table 5. ESI-HRMS validation of unknown dyes in commercially available e-liquids.

E-liquid Observed mass Calculated mass Dye Identified Sample (m/z) (m/z) Red Allura Red AC 225.00829 225.00903 Blue Brilliant Blue FCF 373.06815 373.07077 Green Brilliant Blue FCF 373.07184 373.07077 Green Tartrazine 154.99319 154.99315

The overall data shows that manufacturers are using the three FDA certified dyes at concentrations similar to those used in the food industry (Table 6).

Table 6. Synthetic dye concentrations in selected foodstuffs.

Tartrazine Brilliant Blue FCF Allura Red AC Foods (µg/g) (µg/g) (µg/g) Soft drinks28 4.7-5.2 1.0-1.2 49.8 Confectioneries28 4.0-154.8 1.0-6.3 ND Water soluble foods29 0.5 0.5-4.8 18.1-27.8 Gummy candy30 1.7-80.4 0.7-9.6 1.0-47.8 Jellies30 2.2-20.2 ND ND Juices31 0.06-121.82 2.75-71.44 5.77-7.15 Cookies31 0.13-17.36 0.90-0.99 ND Fruit jam32 ND ND 17.9-33.4 Salted fish32 136.0-292.5 ND ND Soft drinks33 158 0.063-12.9 0.107-0.14 Juice and jelly powder34a 1.3-56.2 2.9-6.4 30.2-53.8 a Units for this study are µg/mL; ND = not disclosed or not discovered in specified foods.

The justification for colorant addition to e-liquids in the same proportions relative to foodstuffs is likely to enable a vaper’s identification of e-cigarette flavoring additives with corresponding foods. The color was shown to influence food sensory characteristics and acceptability of products due to possible learned associations of particular colors with foodstuffs.35,36 Other reasons for using artificial dyes in e-liquids could include the masking of unattractive natural colors of flavorings or other e-liquid constituents, or protecting e-liquids via the sunscreen effect13 to extend their shelf life. Although these reasons are valid for justifying 17 dye addition to foods, the benefits of e-liquid color enhancement may be undermined due to the unknown health risks associated with the inhalation of dyes and their potential degradation byproducts.

Several conundrums arise regarding the lack of (i) certified dye labeling on e-cigarette packaging and (ii) acceptable daily intake (ADI) values for their inhalation. In agreement with the requirements of CFR 21, §70.25, certified synthetic dye identities are required on the labels of not only food products, but also on drugs and cosmetics.12 Although e-liquids are intended for human consumption, the identities of any dyes used are not required to be disclosed.

More importantly, there are no inhalation ADI values for these dyes, as inhalation is just emerging as an intended method of their consumption. The European ADI values for dye ingestion are defined by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the

European Food Safety Authority (EFSA). In the US, the ADI levels are regulated by the FDA and are listed in terms of dye intake in mg per 60-kg person (Table 3). To achieve an ADI upper limit of Brilliant Blue FCF (ADI: 720 mg/person/day), a 60-kg person would have to inhale 14 kg of the blue e-liquid per day. In order to achieve an ADI value for Allura Red AC (ADI: 420 mg/person/day), one would have to inhale 6 kg of red e-liquid. For Tartrazine (ADI: 300 mg/person/day), the value would be 1 kg of green e-liquid. To reach average per-capita exposure levels listed by FDA (Table 3), a person would have to consume 34, 268 and 410 g of blue, red and green e-liquids, respectively (per-capita exposure levels for Brilliant Blue FCF, Allura Red

AC and Tartrazine are 1.72, 17.91 and 12.06 mg/60-kg person/day (Table 3).

In addition to the dilemma of inhaling these dyes, food colorants in e-liquids may produce toxic byproducts via thermal degradation. Some of the potential degradants can include dye precursors and aromatic amines that are considered to have carcinogenic potential.37 18

In vaped samples in the investigation herein, dye desulfonation was observed at a moderate operational power of the e-cigarette (Table 7 and Supplementary Information). The presence of

2- sulfate (SO4 ) ions in the collected aerosols indicates that sulfur dioxide (SO2) or/and sulfur

2- trioxide (SO3) are released from the sulfonated dyes (and oxidized to SO4 according to the method described by Makkonen et al. for analysis).38 This suggests that the dyes start to degrade at temperatures as low as those needed to form e-liquid aerosols.7 The loss of sulfonate groups via thermal destruction in sulfonated dyes has been previously reported.39,40 Rehorek showed that the extent of desulfonation is a function of temperature due to the different positions of sulfonic group in the molecules (i.e., ortho, meta or para to the bonds adjoining the aryl rings), with para- sulfonated compounds having the lowest optimal pyrolysis temperature.40 Accordingly, the green

2- e-liquid showed the highest level of SO4 in the vaporized samples, as the yellow dye, tartrazine, has two para-sulfonyl groups.

In addition, the samples contained relatively high levels of free chloride (Cl-) (Table 7 and Supplementary Information). This could be derived from the breakdown of sucralose. The presence of sucralose in the e-liquids was confirmed by nuclear magnetic resonance.41 Cl- or

2- SO4 were not observed in the air samples, nor in the negative (absorption solution) or positive controls (e-liquid samples diluted in the absorption solution).

Table 7. Sulfate and chloride concentration in vaporized e-liquid samples.

2- - Mass concentration [SO4 ] Mass concentration [Cl ] (µg/g)a,b (µg/g)a Replicate 1 2 3 1 2 3

Red >LOD >LOD >LOD 23.38 56.34 36.48 Blue >LOD 0.84 1.96 20.5 33.5 32.82

Sample Green >LOD 2.72 2.81 17.27 218.51 184.19 a b 2- analyte yield (µg) per g of e-liquid consumed; SO4 was calculated back to SO2

While the in-depth investigation of sucralose decomposition was not an objective of the current study, finding chloride in the absorption solution suggested the fragmentation of sucralose molecules. The loss of hydrogen chloride (HCl) from sucralose is a known initial step in its breakdown, and can ultimately lead to the release and/or formation of potentially toxic byproducts such as chloropropanols.42 Recent studies demonstrate that sucralose decomposition, including HCl release and the formation of chlorinated derivatives, occurs at relatively mild temperatures.43-44 The formation of chlorinated compounds from sucralose in the presence of glycerol (one of the two common e-liquid solvents) and metal oxides is well-precedented.45,46

CONCLUSION

The data presented herein suggests that the infusion of e-cigarette formulations with food additives, including synthetic dyes and sweeteners, creates potential health risks since their inhalation toxicity as well as the toxicity of their byproducts is largely unknown. We hope the results of this study will increase awareness and inform decisions about the regulation of e- cigarettes, whether as inhalable foodstuffs, or as nicotine delivery matrices primarily intended for smoking cessation.

ACKNOWLEDGMENTS

I am deeply grateful to the Strongin Research Group for the immense support, practical guidance and the enormous contribution to this work. We thank the NIH and the FDA for their support via award R01ES025257. The content is solely the responsibility of the authors and does not necessarily represent the views of the NIH or the FDA. Support from the National Science

Foundation (Grant number 0741993) for purchase of the LTQ-Orbitrap Discovery is gratefully acknowledged.

SUPPLEMENTARY INFORMATION

HPLC-DAD chromatograms

1 Erythrosine 0.5 505

A 0

-0.5

time (min)

1 Fast Green FCF 0.5 618

A 0

-0.5

time (min)

0.4 Sunset Yellow FCF 0.2 482

A 0

-0.2

time (min)

Figure S1. Additional chromatograms of dyes standards. Standards at 40 µg/mL concentrations.

K:\Strongin LAB\...\08-31-2017-AR1 8/31/2017 9:46:51 AM 08-31-2017-AR

RT: 0.00 - 1.50 0.14 NL: 6.43E7 100 0.13 0.15 TIC MS 0.20 08-31-2017-AR1 0.11 0.21 50 0.10 0.25 0.07 0.28 0.31 0.37 0.45 0.50 0.57 0.63 0.67 0.73 0.81 0.90 0.94 1.04 1.09 1.12 1.19 1.24 1.34 1.42 1.47 0 NL: 9.04E6 0.14 0.17 100 TIC F: ITMS - p ESI 0.11 Full ms 0.22 [100.00-500.00] MS 50 08-31-2017-AR1 0.25 0.38 0.45 0.53 0.60 0.68 0.73 0.83 0.91 0.94 0.99 1.10 1.14 1.18 1.28 1.35 1.40 1.46 0 RelativeAbundance 0.14 NL: 6.43E7 100 TIC F: FTMS - p ESI 0.20 Full ms 0.11 [100.00-500.00] MS 21 50 0.23 08-31-2017-AR1 0.25 0.28 0.31 0.45 0.54 0.57 0.60 0.67 0.76 0.81 0.90 0.94 1.04 1.09 1.12 1.19 1.29 1.34 1.42 1.47 0 ESI-HRMS0.0 0.1spectra0.2 of0.3 synthetic0.4 0.5 dye0.6 standards0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 Time (min)

225.00924 NL: 100 2.40E7 08-31-2017-AR1#10-37 RT: K:\Strongin80 LAB\...\08-25-2017-ER 8/25/2017 7:22:22 PM 08-25-2017-ER 0.07-0.19 AV: 14 F: FTMS - p 60 ESI Full ms [100.00-500.00] 08-25-2017-ER #9-33 RT: 0.08-0.27 AV: 12 NL: 7.08E4 40F: FTMS - p ESI Full ms [200.00-1000.00] 834.64394 100 226.50864 20 220.99128 RelativeAbundance 90212.96994 217.49825 234.99332 242.83986 252.30813 264.71285 274.66849 0 416.81803 225.00903 NL: 100 80 7.22E5

c18 h 14 n 2 o 8 s 2: 80 70 C 18 H 14 N 2 O8 S2 c (gss, s /p:40)(Val) Chrg 2 60 60 R: 20000 Res .Pwr . @FWHM 40 50

20 40 228.50955

Relative Abundance 0 30 210 215 220 225 230 235 240 245 250 255 260 265 270 275 280 20 m/z

10 248.95950 560.83080 316.94628 377.00322 856.62571 Figure S2. ESI-HRMS spectrum of Allura452.92050 Red520.90705 AC Standard.628.82092 708.74744 802.12402 924.61278 976.35045 2- 0 [M] ion. Theoretical mass:300 225.00903.400 Observed500 mass: 225.00924.600 Δ700 m/z: 0.93 800ppm 900 1000 m/z

NL: K:\Strongin LAB\...\08-25-2017-FG 834.64394 8/25/2017 7:24:50 PM 08-25-2017-FG 100 7.08E4 08-25-2017-ER#9-33 80 08-25-2017-FG #9-33 RT: 0.07-0.28 AV: 25 NL: 1.12E3 RT: 0.08-0.27 AV: 12 T: ITMS - p ESI Full ms [200.00-1000.00] 60 833.78089 F: FTMS - p ESI Full 381.00001 ms [200.00-1000.00] 100 40 90 835.64739 20 Relative Abundance 80 823.27429 828.79191 831.55430 836.64996 839.06697 843.22814 847.10615 850.06508 854.81226 0 834.64667 NL: 10070 7.96E5

8060 c 20 h6 i4 o5 +H: C20 H7 I4 O5 6050 pa Chrg 1

40 835.65003 Relative Abundance 2030 836.65338 839.66098 200 825 830 835 840 845 850 10 m/z 785.00002 238.83333 296.00000 416.75001 544.58334 588.50001 690.50001 834.50002 897.50002 964.83336 Figure S3. ESI0 -HRMS spectrum of Erythrosine Standard. 2- 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 [M] ion. Theoretical mass: 834.64667. Observed mass: 834.64394.m/z Δ m/z: 3.27 ppm

381.06713 NL: 100 2.18E5 08-25-2017-FG#9-33 80 RT: 0.08-0.27 AV: 12 60 F: FTMS - p ESI Full ms [200.00-1000.00] 381.56860 40

Relative Abundance 382.06364 377.00330 378.91697 380.67883 383.56740 384.93299 387.94161 390.89010 0 381.06823 NL: 100 5.54E5

80 c 37 h34 n2 o10 s 3: C37 H34 N2 O10 S 3 60 pa Chrg 2 381.56991 40

20 382.06613 383.06948 384.56906 385.57118 0 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 m/z Figure S4. ESI-HRMS spectrum of Fast Green FCF Standard [M]2- ion. Theoretical mass: 381.06823. Observed mass: 381.06713. Δ m/z: 2.89 ppm

K:\Strongin LAB\...\08-31-2017-TA-NP 8/31/2017 4:47:20 PM 08-31-2017-TA-NP

RT: 0.00 - 1.50 0.15 NL: 7.74E7 100 TIC MS 0.14 0.16 0.11 0.19 08-31-2017-TA-NP 50 0.22 0.24 0.09 0.26 0.32 0.40 0.47 0.56 0.60 0.73 0.79 0.83 0.94 0.97 1.05 1.09 1.17 1.23 1.32 1.41 0 0.17 NL: 1.13E7 100 0.13 TIC F: ITMS - p ESI 0.11 Full ms 0.20 50 [100.00-500.00] MS 08-31-2017-TA-NP 0.24 0.36 0.46 0.56 0.61 0.71 0.75 0.82 0.89 0.93 1.02 1.11 1.21 1.31 1.41 1.46 0

RelativeAbundance 0.15 NL: 7.74E7 100 TIC F: FTMS - p ESI 0.16 0.11 0.19 Full ms 50 [100.00-500.00] MS 08-31-2017-TA-NP 0.24 0.26 0.40 0.43 0.56 0.60 0.73 0.79 0.86 0.94 1.02 1.05 1.09 1.17 1.23 1.32 1.41 0 22 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Time (min)

K:\Strongin LAB\...\08-31-2017-SY-NP154.99263 8/31/2017 4:49:57 PM 08-31-2017-SY-NP NL: 100 8.78E6 08-31-2017-TA-NP#10-43 RT:800.00 - 1.50 0.12 210.99789 RT: 0.08-0.23NL: 1.13E8 AV: 17 F: FTMS 100 0.16 0.20 - p ESI Full ms 60 TIC MS 0.11 [100.00-500.00] 197.98478 08-31-2017-SY-NP 40 0.21 50 0.10 0.22 20 0.24 232.99244 RelativeAbundance 0.09140.32963 170.99774 0.31 0.36 0.45 0.50274.732910.57 0.62303.754960.70 0.76343.292170.86 376.548120.96 1.01 1.07434.126921.19 1.24 488.975681.34 1.40 1.45 00 0.15 NL: NL: 1.75E7 100100 1.71E4 0.18 TIC F: ITMS - p ESI 0.12 Full ms 80 c16 h 9 n 4 o 9 s 2: 0.19 C 16 H 9 N[100.00-500.00]4 O9 S2 MS 50 0.10 60 p (gss, s08-31-2017-SY-NP/p:40) Chrg 3 0.23 R: 20000 Res .Pwr . @FWHM 0.29 0.35 0.43 0.53 0.58 0.66 0.69 0.79 0.84 0.95 1.02 1.07 1.17 1.23 1.28 1.38 1.43 400

RelativeAbundance 0.12 NL: 1.13E8 100 154.993150.16 0.20 20 TIC F: FTMS - p ESI Full ms 0 50 [100.00-500.00] MS 100 150 200 250 300 350 400 450 500 08-31-2017-SY-NP m/z 0.40 0.44 0.57 0.65 0.70 0.76 0.86 0.96 1.01 1.07 1.19 1.24 1.34 1.40 1.45 Figure S0 5. ESI-HRMS spectrum of Tartrazine Standard 3- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 [M] ion. Theoretical mass: 154.99315. Observed mass:Time (min) 154.99263. Δ m/z: 3.35 ppm

202.99515 NL: 100 3.26E7 08-31-2017-SY-NP#6-47 RT: 80 0.04-0.24 AV: 21 F: FTMS - p 60 ESI Full ms [100.00-500.00]

40K:\Strongin LAB\...\08-25-2017-BB 8/25/2017 7:19:54 PM 08-25-2017-BB 203.99338 20 201.01408 RelativeAbundance 08-25-2017-BB193.08564 #6-21 RT: 0.05-0.17199.27224AV: 8 NL: 4.42E5 206.99679 211.09484 213.98532 218.51252 221.99647 228.53435 0 F: FTMS - p ESI Full ms [200.00-1000.00] 202.99592 NL: 100 373.07067 100 1.74E4 80 c16 h 10 n 2 o 7 s 2: 90 C 16 H 10 N 2 O7 S2 60 p (gss, s /p:40) Chrg 2 80 R: 20000 Res .Pwr . @FWHM 40 70 203.99507 20 60 205.49560 0 50 190 195 200 384.93403 205 210 215 220 225 m/z 40

Relative Abundance 30 Figure S6. ESI-HRMS spectrum of Sunset Yellow FCF Standard. 20 [M]2- ion. Theoretical mass: 202.99592. Observed mass: 202.99515. Δ m/z: 3.79 ppm 10 422.05928 769.13041 225.00958 333.09229 481.15675 561.11336 616.84228 700.85510 837.11770 889.08438 961.23393 0 300 400 500 600 700 800 900 1000 m/z

373.07067 NL: 100 4.42E5 08-25-2017-BB#6-21 80 RT: 0.05-0.17 AV: 8 60 F: FTMS - p ESI Full 373.57197 ms [200.00-1000.00] 40

20 374.06995 Relative Abundance 368.88981 370.35115 372.69431 375.07189 377.00351 378.91762 380.83104 384.93403 0 373.07077 NL: 100 5.56E5

80 c 37 h34 n2 o9 s 3: C37 H34 N2 O9 S 3 60 pa Chrg 2 373.57245 40

20 374.06867 375.07203 376.57160 378.06994 0 368 370 372 374 376 378 380 382 384 m/z

Figure S7. ESI-HRMS spectrum of Brilliant Blue FCF Standard. [M]2- ion. Theoretical mass: 373.07077. Observed mass: 373.07067. Δ m/z: 0.27 ppm

K:\Strongin LAB\...\AR 9/13/2017 7:04:25 PM AR+pnitrophenol

RT: 0.00 - 1.50 0.22 0.23 NL: 1.12E7 100 0.28 TIC MS AR 0.07 0.21 0.35 0.39 0.10 0.48 0.51 0.78 0.61 0.72 0.84 0.89 1.21 50 0.12 0.90 1.05 1.08 1.27 1.37 1.43

0 NL: 2.80E6 0.20 0.27 100 0.08 TIC F: ITMS - p ESI Full ms 0.36 0.39 0.43 [100.00-500.00] 50 0.48 0.57 0.64 0.74 MS AR 0.78 0.88 0.97 1.00 1.07 1.13 1.22 1.27 1.38 1.41

0 NL: 1.12E7 RelativeAbundance 0.22 100 0.28 TIC F: FTMS - p 0.07 0.35 0.39 0.10 0.41 ESI Full ms 0.51 0.57 0.72 0.78 [100.00-500.00] 0.61 0.84 0.89 1.00 1.05 1.21 1.27 50 1.08 1.37 1.43 MS AR 23

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 Time (min) ESI-HRMS validation of synthetic dyes in e-liquid samples

225.00829 NL: 100 1.38E6 AR#32 RT: 0.18 AV: 1 F: K:\Strongin80 LAB\...\GREEN+TA 9/13/2017 7:06:54 PM greenTA+pnitrophenol FTMS - p ESI Full ms 60 [100.00-500.00] RT: 0.00 - 1.50 0.25 0.30 NL: 8.20E7 10040 0.22 0.08 0.33 TIC MS 0.35 225.50980 20 0.13 0.44 0.49 GREEN+TA

RelativeAbundance 226.00616 0.07 0.58 50 224.83192 225.18503 0.63 0.73 0.84226.50769 227.05444 0 0.76 0.90 0.94 1.02 1.11 1.17 1.27 1.36 1.42 1.49 225.00903 NL: 100 1.69E4 0 80 0.39 c18 h 14 n 2 oNL:8 s 2 8.98E6: 100 0.08 0.37 0.44 C 18 H 14 N 2 OTIC8 S F:2 ITMS - p ESI 0.25 0.30 0.53 60 0.58 p (gss, s /pFull:40) msChrg 2 0.19 0.64 0.73 0.81 0.88 1.08 1.21 R: 200001.45 Res[100.00-500.00].Pwr . @FWHM 50 0.93 1.06 1.16 1.24 1.28 1.40 40 MS GREEN+TA 225.51048 20 226.00837 0 226.50937 RelativeAbundance 0.25 0.30 227.00828 227.50897 NL: 8.20E7 1000 0.22 0.08 0.33 TIC F: FTMS - p 224.5 225.0 0.38 225.5 226.0 226.5 227.0 227.5 0.13 0.44 ESI Full ms 0.49 m/z 0.58 0.63 0.73 0.84 [100.00-500.00] 50 0.76 0.90 0.94 1.02 1.36 Figure S8. ESI-HRMS spectrum of Allura Red AC in Red E-liquid. 1.11 1.17 1.27 1.42 1.49 MS GREEN+TA

Theoretical0 mass: 225.00903. Observed mass: 225.00829. Δ m/z: 3.29 ppm 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Time (min)

154.99319 NL: 100 1.20E5 GREEN+TA#178 RT: 0.99 80 AV: 1 F: FTMS - p ESI Full ms 60 [100.00-500.00]

40 154.03465 157.12236 20 155.32773

K:\StronginRelativeAbundance LAB\...\BB+TA_171029212949 10/29/2017 9:29:49 PM 155.65848BB+TA 0 154.99315 NL: RT: 0.00 - 1001.50 0.10 0.12 1.71E4 NL: 2.39E7 100 0.13 0.44 TIC MS 0.16 0.26 0.27 0.29 0.40 0.54 80 0.32 0.50 c16 h 9 n 4 o 9 s 2: BB+ 0.24 0.55 0.07 C 16 H 9 N 4 O9 S2 TA_171029212949 50 0.61 0.62 0.65 0.76 1.12 60 0.79 0.87 0.90 0.94 0.98 1.02 1.11 p (gss, s /p:40) Chrg 3 0.02 1.17 1.18 1.27 1.30 1.39 1.41 R: 20000 Res1.47.Pwr . @FWHM 0 40 0.09 NL: 1.13E6 100 0.13 155.32736 TIC F: ITMS - p ESI 0.15 0.29 20 0.18 0.38 0.41 0.43 0.52 155.65932 Full ms 0.54 [300.00-400.00] MS 50 0.58 0.63 0.67 156.32589 156.65970 157.32673 157.66028 BB+ 0 0.74 0.77 0.89 1.00 1.03 1.12 1.15 0.03 0.81 0.94 1.06 1.17 1.24 1.28 1.36 1.40 1.42 TA_171029212949 153.5 154.0 154.5 155.0 155.5 156.0 156.5 157.0 157.5 158.0 0 Relative Abundance 0.10 0.12 NL: 2.39E7 100 m/z 0.44 TIC F: FTMS - p ESI 0.27 0.40 0.54 0.50 Full ms 0.20 Figure S0.079. ESI-HRMS spectrum of Tartrazine0.61 in Green E-liquid. [300.00-400.00] MS 50 0.64 0.72 0.76 0.90 1.02 1.12 BB+ 0.87 0.98 1.07 1.17 Theoretical mass: 154.99315. Observed mass: 154.99319. Δ m/z: 0.26 ppm 1.23 1.27 1.30 1.39 1.47 TA_171029212949 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Time (min) 373.07184 NL: 100 2.24E5 BB+TA_171029212949#94 80 RT: 0.52 AV: 1 F: FTMS - p ESI Full ms [300.00-400.00] 60

40 373.57358 20

Relative Abundance 374.57083 0 373.07077 NL: 100 1.30E4 80 c 37 h 34 n 2 o 9 s 3: C 37 H34 N2 O 9 S 3 60 p (gss, s /p:40) Chrg 2 373.57231 R: 20000 Res .Pwr . @FWHM 40 374.07094 20 374.57151 375.07090 375.57109 376.07096 376.57112 377.07130 377.57160 378.07188 0 372.5 373.0 373.5 374.0 374.5 375.0 375.5 376.0 376.5 377.0 377.5 378.0 378.5 m/z Figure S10. ESI-HRMS spectrum of Brilliant Blue FCF Dye in Green E-liquid. Theoretical: 373.07077. Observed: 373.07184. Δ m/z: 2.87 ppm

K:\Strongin LAB\...\bb_180105094228 1/5/2018 9:42:28 AM BB

RT: 0.00 - 1.49 0.21 0.23 NL: 100 0.24 2.02E7 TIC MS 90 0.20 bb_1801050 0.25 0.28 94228 80

70 0.17

60 0.06 0.30

50 0.09 0.32 40 0.35 Relative Abundance 30 0.37 0.40 0.43 20 0.47 0.51 0.56 10 0.64 0.69 0.77 0.84 0.92 1.03 1.12 1.16 1.23 1.31 1.36 1.48 0 24 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Time (min)

373.07220 NL: 100 9.04E5 bb_180105094228#1-180 RT: 80 0.00-1.49 AV: 180 T: FTMS - 60 p ESI Full ms [300.00-400.00] 373.57342 40 374.07160 20 Relative Abundance 374.57127 372.69606 375.07295 375.67752 376.27824 377.10868 377.60504 0 373.07077 NL: 100 1.30E4

80 c 37 h34 n2 o9 s 3: C37 H34 N2 O9 S 3 60 p (gss, s /p:40) Chrg 2 373.57231 R: 20000 Res .Pwr . @FWHM 40 374.07094 20 374.57151 375.07090 376.07096 376.57112 377.57160 0 373 374 375 376 377 m/z Figure S11. ESI-HRMS spectrum of Brilliant Blue FCF in Blue E-liquid. Theoretical mass: 373.07077. Observed mass: 373.07220. Δ m/z: 3.83 ppm

Ion chromatograms of vaporized samples

Figure S12. IC of vaporized Red E-liquid. Bottom to top: replicates 1 – 3 and the spiked sample.

Figure S13. IC of vaporized Blue E-liquid. Bottom to top: replicates 1 – 3 and the spiked sample.

Figure S14. IC of vaporized Green E-liquid. Bottom to top: replicates 1 – 3 and the spiked sample. 26

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