Portland State University Portland State University
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University Honors Theses University Honors College
3-1-2018
Evaluation of Synthetic Dyes and Food Additives in Evaluation of Synthetic Dyes and Food Additives in
Electronic Cigarette Liquids: Health and Policy Electronic Cigarette Liquids: Health and Policy
Implications Implications
Tetiana Korzun Portland State University
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Recommended Citation 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
by
Tetiana Korzun
An undergraduate honors thesis submitted in partial fulfillment of
the requirements for the degree of
Bachelor of Science
in
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
2
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
3
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
4
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.
5
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
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.
S NN
S
OHO
-OO
O
OO O-
Allura Red AC (FD&C Red No. 40)
NN
HO
SO-
OO
S-OO
O
Sunset Yellow FCF (FD&C Yellow No. 6)
NN
O
O
O-
NN
SO
O
O-
SO
OO-
Tartrazine (FD&C Yellow No. 5)
N+
NS
O-
O
OHO
SO-
SO-
OOOO
Fast Green FCF (FD&C Green No. 3)
N+NS-O O
O
S O-
O
O
SO-
O
O
Brilliant Blue FCF (FD&C Blue No. 1)
O
O
O
I
O-
I
I
-OI
Erythrosine (FD&C Red 3)
NH
NH
O
O
S
S
-OO
O
O-
O
O
Indigotine(FD&C Blue No. 2)
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.
8
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.
11
Table 1. Quantitative features of the HPLC-DAD method for the selected dye standards
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.
Dye Standard
Absorption Maxima (nm)
Regression (%)
LOD (µg/mL)
LOQ (µg/mL)
RSD (%)
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
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.
Standard Calculated Mass of Standards (m/z)
Observed Mass of Standards (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.
14
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.
Synthetic Dye Chemical Class European ADI (mg/kg/day)
ADI25
(mg/p/day)a
Per Capita Exposure25
(mg/p/day)a
Allura Red AC (FD&C Red No. 40) Azo 0-7 (2016) 26 420 17.91
Sunset Yellow FCF (FD&C Yellow No. 6) Azo 0-2.5 (1982) 13 225 10.74
Tartrazine (FD&C Yellow No. 5) Azo 0-10 (2016) 26 300 12.06 Brilliant Blue FCF
(FD&C Blue No. 1) Triarylmethane 0-12.5 (1969) 13 720 1.72 Fast Green FCF
(FD&C Green No. 3) Triarylmethane 0-25 (1986) 13 150 0.038 Erythrosine
(FD&C Red No. 3) Xanthene 0-0.1 (1990) 13 150 0.61 Indigotine
(FD&C Blue No. 2) Sulfonated Indigo 0-5 (1974) 13 150 1.95 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.
Sample Identified Dye
Absorption Maxima (nm)
Retention time (min)
Concentration (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).
16
Table 5. ESI-HRMS validation of unknown dyes in commercially available e-liquids.
E-liquid Sample Dye Identified Observed mass
(m/z) Calculated mass
(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.
Foods Tartrazine (µg/g)
Brilliant Blue FCF (µg/g)
Allura Red AC (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
sulfate (SO42-) ions in the collected aerosols indicates that sulfur dioxide (SO2) or/and sulfur
trioxide (SO3) are released from the sulfonated dyes (and oxidized to SO42- 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
e-liquid showed the highest level of SO42- 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
SO42- 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.
Mass concentration [SO4
2-] (µg/g)a,b
Mass concentration [Cl-] (µg/g)a
Replicate 1 2 3 1 2 3
Sam
ple Red >LOD >LOD >LOD 23.38 56.34 36.48
Blue >LOD 0.84 1.96 20.5 33.5 32.82 Green >LOD 2.72 2.81 17.27 218.51 184.19
a analyte yield (µg) per g of e-liquid consumed; b SO42- was calculated back to SO2
19
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 by-
products 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.
20
SUPPLEMENTARY INFORMATION
HPLC-DAD chromatograms
Figure S1. Additional chromatograms of dyes standards. Standards at 40 µg/mL concentrations.
-0.5
0
0.5
1A
505
time (min)
Erythrosine
-0.5
0
0.5
1
A61
8
time (min)
Fast Green FCF
-0.2
0
0.2
0.4
A48
2
time (min)
Sunset Yellow FCF
21
ESI-HRMS spectra of synthetic dye standards
Figure S2. ESI-HRMS spectrum of Allura Red AC Standard. [M]2- ion. Theoretical mass: 225.00903. Observed mass: 225.00924. Δ m/z: 0.93 ppm
Figure S3. ESI-HRMS spectrum of Erythrosine Standard. [M]2- ion. Theoretical mass: 834.64667. Observed mass: 834.64394. Δ m/z: 3.27 ppm
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-AR1 8/31/2017 9:46:51 AM 08-31-2017-AR
RT: 0.00 - 1.50
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 1.5Time (min)
0
50
1000
50
100
Rel
ativ
e Ab
unda
nce
0
50
1000.14
0.150.130.20
0.110.21
0.10 0.250.280.07 0.31 0.37 0.45 0.50 0.57 0.63 0.67 0.810.73 0.940.90 1.04 1.421.09 1.12 1.34 1.471.19 1.24
0.170.140.11
0.22
0.250.38 0.45 0.53 1.460.730.60 0.94 1.28 1.350.68 1.140.83 1.10 1.180.91 0.99 1.40
0.14
0.200.11
0.230.25
0.28 0.31 0.45 0.570.54 0.60 0.67 0.81 0.940.76 0.90 1.04 1.421.09 1.12 1.34 1.471.19 1.29
NL: 6.43E7TIC MS 08-31-2017-AR1
NL: 9.04E6TIC F: ITMS - p ESI Full ms [100.00-500.00] MS 08-31-2017-AR1
NL: 6.43E7TIC F: FTMS - p ESI Full ms [100.00-500.00] MS 08-31-2017-AR1
210 215 220 225 230 235 240 245 250 255 260 265 270 275 280m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
225.00924
226.50864
217.49825 234.99332
220.99128212.96994 252.30813 274.66849242.83986 264.71285
225.00903
228.50955
NL:2.40E708-31-2017-AR1#10-37 RT: 0.07-0.19 AV: 14 F: FTMS - p ESI Full ms [100.00-500.00]
NL:7.22E5
c18 h14 n2 o8 s 2: C18 H14 N2 O8 S2c (gss, s /p:40)(Val) Chrg 2R: 20000 Res .Pwr . @FWHM
K:\Strongin LAB\...\08-25-2017-ER 8/25/2017 7:22:22 PM 08-25-2017-ER
08-25-2017-ER #9-33 RT: 0.08-0.27 AV: 12 NL: 7.08E4F: FTMS - p ESI Full ms [200.00-1000.00]
300 400 500 600 700 800 900 1000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
834.64394
416.81803
248.95950 560.83080 856.62571316.94628 377.00322 708.74744628.82092 924.61278802.12402452.92050 520.90705 976.35045
825 830 835 840 845 850m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance
834.64394
835.64739
836.64996828.79191 843.22814823.27429 847.10615831.55430
833.78089
850.06508839.06697 854.81226834.64667
835.65003
836.65338 839.66098
NL:7.08E408-25-2017-ER#9-33 RT: 0.08-0.27 AV: 12 F: FTMS - p ESI Full ms [200.00-1000.00]
NL:7.96E5
c20 h6 i4 o5 +H: C20 H7 I4 O5pa Chrg 1
K:\Strongin LAB\...\08-25-2017-FG 8/25/2017 7:24:50 PM 08-25-2017-FG
08-25-2017-FG #9-33 RT: 0.07-0.28 AV: 25 NL: 1.12E3T: ITMS - p ESI Full ms [200.00-1000.00]
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
381.00001
785.00002690.50001296.00000238.83333 834.50002588.50001544.58334 964.83336416.75001 897.50002
377 378 379 380 381 382 383 384 385 386 387 388 389 390 391m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance
381.06713
381.56860
382.06364377.00330 384.93299380.67883378.91697 390.89010383.56740 387.94161
381.06823
381.56991
382.06613383.06948 384.56906 385.57118
NL:2.18E508-25-2017-FG#9-33 RT: 0.08-0.27 AV: 12 F: FTMS - p ESI Full ms [200.00-1000.00]
NL:5.54E5
c37 h34 n2 o10 s3: C37 H34 N2 O10 S3pa Chrg 2
22
Figure S5. ESI-HRMS spectrum of Tartrazine Standard [M]3- ion. Theoretical mass: 154.99315. Observed mass: 154.99263. Δ m/z: 3.35 ppm
Figure S6. ESI-HRMS spectrum of Sunset Yellow FCF Standard. [M]2- ion. Theoretical mass: 202.99592. Observed mass: 202.99515. Δ m/z: 3.79 ppm
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\...\08-31-2017-TA-NP 8/31/2017 4:47:20 PM 08-31-2017-TA-NP
RT: 0.00 - 1.50
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 1.4Time (min)
0
50
1000
50
100
Rel
ativ
e Ab
unda
nce 0
50
1000.15
0.14 0.160.11 0.19
0.220.24 0.260.09 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
0.170.13
0.110.20
0.240.36 0.46 0.56 0.61 0.750.71 0.82 0.89 1.02 1.21 1.311.110.93 1.41 1.46
0.15
0.160.11 0.19
0.24 0.260.40 0.43 0.56 0.60 0.73 0.79 0.86 0.94 1.05 1.09 1.171.02 1.23 1.32 1.41
NL: 7.74E7TIC MS 08-31-2017-TA-NP
NL: 1.13E7TIC F: ITMS - p ESI Full ms [100.00-500.00] MS 08-31-2017-TA-NP
NL: 7.74E7TIC F: FTMS - p ESI Full ms [100.00-500.00] MS 08-31-2017-TA-NP
100 150 200 250 300 350 400 450 500m/z
0
20
40
60
80
1000
20
40
60
80
100R
elat
ive
Abun
danc
e154.99263
210.99789
197.98478
232.99244170.99774140.32963343.29217303.75496 488.97568434.12692376.54812274.73291
154.99315
NL:8.78E608-31-2017-TA-NP#10-43 RT: 0.08-0.23 AV: 17 F: FTMS - p ESI Full ms [100.00-500.00]
NL:1.71E4
c16 h9 n4 o9 s 2: C16 H9 N4 O9 S2p (gss, s /p:40) Chrg 3R: 20000 Res .Pwr . @FWHM
K:\Strongin LAB\...\08-31-2017-SY-NP 8/31/2017 4:49:57 PM 08-31-2017-SY-NP
RT: 0.00 - 1.50
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 1.4Time (min)
0
50
1000
50
100
Rel
ativ
e Ab
unda
nce 0
50
1000.12 0.16 0.20
0.110.21
0.10 0.220.240.09 0.31 0.36 0.45 0.50 0.57 0.62 0.70 0.76 0.86 1.071.010.96 1.241.19 1.34 1.451.40
0.150.18
0.120.19
0.100.23
0.29 0.35 0.43 0.53 0.58 0.66 0.69 1.171.020.79 0.84 0.95 1.281.231.07 1.431.380.12 0.16 0.20
0.40 0.44 0.57 0.65 0.70 0.76 0.86 1.071.010.96 1.241.19 1.34 1.451.40
NL: 1.13E8TIC MS 08-31-2017-SY-NP
NL: 1.75E7TIC F: ITMS - p ESI Full ms [100.00-500.00] MS 08-31-2017-SY-NP
NL: 1.13E8TIC F: FTMS - p ESI Full ms [100.00-500.00] MS 08-31-2017-SY-NP
190 195 200 205 210 215 220 225m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
202.99515
203.99338
206.99679 213.98532199.27224 211.09484
201.01408
221.99647193.08564 228.53435218.51252202.99592
203.99507
205.49560
NL:3.26E708-31-2017-SY-NP#6-47 RT: 0.04-0.24 AV: 21 F: FTMS - p ESI Full ms [100.00-500.00]
NL:1.74E4
c16 h10 n2 o7 s 2: C16 H10 N2 O7 S2p (gss, s /p:40) Chrg 2R: 20000 Res .Pwr . @FWHM
K:\Strongin LAB\...\08-25-2017-BB 8/25/2017 7:19:54 PM 08-25-2017-BB
08-25-2017-BB #6-21 RT: 0.05-0.17 AV: 8 NL: 4.42E5F: FTMS - p ESI Full ms [200.00-1000.00]
300 400 500 600 700 800 900 1000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
373.07067
769.13041225.00958 333.09229 837.11770561.11336481.15675
384.93403
889.08438422.05928
961.23393616.84228 700.85510
368 370 372 374 376 378 380 382 384m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance
373.07067
373.57197
374.06995375.07189 377.00351372.69431 384.93403368.88981 378.91762 380.83104370.35115
373.07077
373.57245
374.06867375.07203 376.57160 378.06994
NL:4.42E508-25-2017-BB#6-21 RT: 0.05-0.17 AV: 8 F: FTMS - p ESI Full ms [200.00-1000.00]
NL:5.56E5
c37 h34 n2 o9 s3: C37 H34 N2 O9 S3pa Chrg 2
23
ESI-HRMS validation of synthetic dyes in e-liquid samples
Figure S8. ESI-HRMS spectrum of Allura Red AC in Red E-liquid. Theoretical mass: 225.00903. Observed mass: 225.00829. Δ m/z: 3.29 ppm
Figure S9. ESI-HRMS spectrum of Tartrazine in Green E-liquid. Theoretical mass: 154.99315. Observed mass: 154.99319. Δ m/z: 0.26 ppm
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\...\AR 9/13/2017 7:04:25 PM AR+pnitrophenol
RT: 0.00 - 1.50
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 1.5Time (min)
0
50
1000
50
100
Rel
ativ
e Ab
unda
nce 0
50
1000.22 0.23 0.28
0.21 0.35 0.390.07 0.10 0.48 0.780.51 0.72 0.84 0.890.61 1.21 1.270.90 1.050.12 1.08 1.37 1.43
0.20 0.270.08
0.36 0.39 0.43 0.48 0.57 0.740.64 0.88 1.000.78 1.130.97 1.411.22 1.271.07 1.38
0.22 0.280.35 0.390.07 0.10 0.41 0.780.51 0.57 0.72 0.84 0.890.61 1.21 1.271.051.00 1.08 1.37 1.43
NL: 1.12E7TIC MS AR
NL: 2.80E6TIC F: ITMS - p ESI Full ms [100.00-500.00] MS AR
NL: 1.12E7TIC F: FTMS - p ESI Full ms [100.00-500.00] MS AR
224.5 225.0 225.5 226.0 226.5 227.0 227.5m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
225.00829
225.50980226.00616
226.50769224.83192 225.18503 227.05444225.00903
225.51048226.00837
226.50937 227.00828 227.50897
NL:1.38E6AR#32 RT: 0.18 AV: 1 F: FTMS - p ESI Full ms [100.00-500.00]
NL:1.69E4
c18 h14 n2 o8 s 2: C18 H14 N2 O8 S2p (gss, s /p:40) Chrg 2R: 20000 Res .Pwr . @FWHM
K:\Strongin LAB\...\GREEN+TA 9/13/2017 7:06:54 PM greenTA+pnitrophenol
RT: 0.00 - 1.50
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 1.4Time (min)
0
50
1000
50
100
Rel
ativ
e Ab
unda
nce 0
50
1000.300.250.22 0.330.08
0.350.440.13 0.49 0.580.07 0.730.63 0.840.76 1.020.90 1.360.94 1.421.11 1.271.17 1.49
0.390.08 0.37 0.44 0.530.300.25 0.58 0.64 0.810.19 0.73 1.450.88 1.08 1.211.06 1.240.93 1.281.16 1.40
0.300.250.22 0.330.080.38 0.440.13 0.49 0.58 0.730.63 0.840.76 1.020.90 1.360.94 1.421.11 1.271.17 1.49
NL: 8.20E7TIC MS GREEN+TA
NL: 8.98E6TIC F: ITMS - p ESI Full ms [100.00-500.00] MS GREEN+TA
NL: 8.20E7TIC F: FTMS - p ESI Full ms [100.00-500.00] MS GREEN+TA
153.5 154.0 154.5 155.0 155.5 156.0 156.5 157.0 157.5 158.0m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
154.99319
154.03465157.12236
155.32773155.65848
154.99315
155.32736155.65932
156.32589 156.65970 157.32673 157.66028
NL:1.20E5GREEN+TA#178 RT: 0.99 AV: 1 F: FTMS - p ESI Full ms [100.00-500.00]
NL:1.71E4
c16 h9 n4 o9 s 2: C16 H9 N4 O9 S2p (gss, s /p:40) Chrg 3R: 20000 Res .Pwr . @FWHM
K:\Strongin LAB\...\BB+TA_171029212949 10/29/2017 9:29:49 PM BB+TA
RT: 0.00 - 1.50
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 1.4Time (min)
0
50
1000
50
100
Rel
ativ
e Ab
unda
nce 0
50
1000.120.10 0.13
0.440.16 0.270.26 0.29 0.40 0.540.500.320.24 0.550.07 0.61 0.62 0.65 1.120.76 1.020.90 1.110.79 0.980.87 0.94 1.17 1.18 1.27 1.391.300.02 1.41 1.47
0.090.13 0.15 0.29 0.41 0.520.430.18 0.38 0.54
0.630.58 0.67 1.120.770.74 1.031.000.89 1.150.94 1.060.81 1.17 1.401.24 1.421.280.03 1.36
0.120.100.440.27 0.40 0.540.50
0.200.07 0.61 0.64 1.120.760.72 1.020.90 0.980.87 1.171.07 1.23 1.27 1.391.30 1.47
NL: 2.39E7TIC MS BB+TA_171029212949
NL: 1.13E6TIC F: ITMS - p ESI Full ms [300.00-400.00] MS BB+TA_171029212949
NL: 2.39E7TIC F: FTMS - p ESI Full ms [300.00-400.00] MS BB+TA_171029212949
372.5 373.0 373.5 374.0 374.5 375.0 375.5 376.0 376.5 377.0 377.5 378.0 378.5m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
373.07184
373.57358
374.57083
373.07077
373.57231
374.07094
374.57151 375.07090 375.57109 376.07096 376.57112 377.07130 377.57160 378.07188
NL:2.24E5BB+TA_171029212949#94 RT: 0.52 AV: 1 F: FTMS - p ESI Full ms [300.00-400.00]
NL:1.30E4c 37 h 34 n 2 o 9 s 3: C 37 H34 N2 O 9 S3p (gss, s /p:40) Chrg 2R: 20000 Res .Pwr . @FWHM
24
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
K:\Strongin LAB\...\bb_180105094228 1/5/2018 9:42:28 AM BB
RT: 0.00 - 1.49
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 1.4Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
0.230.210.24
0.200.25 0.28
0.17
0.300.06
0.090.32
0.350.370.40 0.43
0.47 0.510.56
0.64 0.69 0.77 0.84 0.92 1.03 1.12 1.16 1.23 1.31 1.36 1.48
NL:2.02E7TIC MS bb_180105094228
373 374 375 376 377m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance
373.07220
373.57342
374.07160374.57127 377.10868375.07295372.69606 375.67752 376.27824 377.60504
373.07077
373.57231
374.07094
374.57151 375.07090 376.07096 376.57112 377.57160
NL:9.04E5bb_180105094228#1-180 RT: 0.00-1.49 AV: 180 T: FTMS - p ESI Full ms [300.00-400.00]
NL:1.30E4
c37 h34 n2 o9 s3: C37 H34 N2 O9 S3p (gss, s /p:40) Chrg 2R: 20000 Res .Pwr . @FWHM
25
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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27
(18) Lancaster, F. E.; Lawrence, J. F. Determination of benzidine in the food colours tartrazine and sunset yellow FCF, by reduction and derivatization followed by high-performance liquid chromatography. Food Addit. Contam. 1999, 16, 381-90.
(19) Lancaster, F. E.; Lawrence, J. F. Determination of total non-sulphonated aromatic amines in tartrazine, sunset yellow FCF and allura red by reduction and derivatization followed by high-performance liquid chromatography. Food Addit. Contam. 1991, 8, 249-263.
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