The Determination of Diacetyl and Acetylpropionyl in Aerosols from Electronic Smoking Devices using Gas Chromatography
Triple Quad Mass Spectrometry
S.C. Moldoveanu, A.G. Hudson,A. Harrison
R.J. Reynolds Tobacco Co.
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Background
• 2,3-Butanedione (diacetyl) is used as a flavoring agent in food and it is a component of certain fragrances. Also the compound occurs naturally in certain alcoholic and non-alcoholic beverages and in several types of tobacco.
• Concerns have been raised about exposure to diacetyl, and it has been indicated that the compound may contribute or may even cause severe respiratory disorders in the workplace.
• Occupational Safety and Health Administration (OSHA) issued a guidance regarding the use of diacetyl as a food flavoring.
• National Institute for Occupational Safety and Health (NIOSH) also issued a number of reports regarding the exposure to diacetyl and 2,3-pentanedione (acetylpropionyl) in the working place.
• Analysis of diacetyl and acetylpropionyl in mainstream cigarette smoke (burn-down products) and in aerosol (both the particles and the suspending gas) from electronic smoking devices (e-cigarettes) is of considerable interest.
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Background (cont.)• Measurements for diacetyl were performed on various matrices such
as wine, beer, various types of food, urine, and air.• The analysis of diacetyl in e-liquids and tobacco products has been
previously reported and the procedures can be based on gas chromatography followed by mass spectrometry, or on HPLC.
• A CORESTA method for the analysis of aldehydes and ketones in smoke from cigarettes [1] has been modified to include the measurement of diacetyl and acetylpropionyl [2] at μg level.
• The evaluation of diacetyl and acetylpropionyl in e-vapor products has been previously presented at TSRC [3,4].
1. CORESTA Recommended Method No. 74 - March 2013. Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA).2. Pierce, J. S., A. Abelmann, L. J. Spicer, R. E. Adams, B. L. Finley: Crit. Rev. Toxicol, 44 (2014) 420–435.3. Huang. C. B., K. Wagner, J. W. Flora, 69th Tobacco Science Research Conference, Naples, Fl, Sept. 20-23, 2015, Paper 71.4. Fraley N., 70th Tobacco Science Research Conference, Palm Beach Gardens, Fl, Sept. 18-21, 2016, Poster 18.
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Analytical method summary• The aerosols from e-cigarettes were collected using a Cerulean SM
450 Linear Smoke Machine (Cerulean SM 450,Milton Keynes, UK) using a square wave profile.
• The collection from the e-cigarettes was performed on a Cambridge pad and one impinger containing 10 mL acetone.
• The e-liquids were obtained either directly from bottles or removed from cartridges by centrifugation.
• The analysis used a gas chromatographic separation on a Carbowaxtype column with the measurement of the analytes on a triplequadmass spectrometer (GC-MS/MS) working in positive MRM mode.
• The GC-MS/MS was 7890B-7000C from Agilent (Agilent Technologies Inc., Wilmington, DE, USA).
• The separation was performed on an Agilent J&W DB-WaxEtrchromatographic column, 30 m x 0.25 mm i.d. with 0.25 μm film.
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Analytical method (details on sample collection) • The smoking regimen for aerosol collection was 55 mL puff volume,
3 second puff duration, and 30 second puff interval.• Depending on the electronic smoking device, 50 puffs or 100 puffs
were collected at specific puff intervals such as 1-50, 51-100, etc.• The impingers were weighed before and after sample collection to
determine the remaining volume of acetone (some acetone evaporates during smoking).
• After the samples were collected, the particulate phase pads were placed in the impinger containing the corresponding vapor phase and extracted (to create combined whole aerosol samples).
• An aliquot of 1mL sample was taken and d7-quinoline is added.• The collection of smoke from 3R4F Kentucky reference cigarette
was performed under ISO or HCI protocols, using a Cambridge pad followed by two impingers each with 50 mL acetone and glass beads.
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Publication of the method
The present method has been published:
S. C. Moldoveanu, A. G. Hudson, W. A. Scott, A. Harrison, The Determination of Diacetyl and Acetylpropionyl in Aerosols from Electronic Smoking Devices using Gas Chromatography Triple Quad Mass Spectrometry, Beitraege Tabak. Intern., 27(7) (2017) 145-153.
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Analytical method (details on GC separation)
Parameter Description Parameter DescriptionInitial oven temp. 35°C Inlet initial temp. 120°CInitial time 7.0 min Initial time 7 minOven ramp rate 2.5°C/min Heating rate 600 oC/minOven final first ramp 50°C Final inlet temp. 290 oCFinal time first ramp 0 min Injection volume 3.0 mLOven ramp rate 30°C/mm Carrier gas HeliumOven final second ramp 260oC Flow mode Constant flowFinal time 5.0 min Flow rate 1.1 mL/minTotal run time 25 min Nominal initial pressure 12.2 psiInlet type Multi Mode Average flow velocity 26.23 cm/secInlet mode Pulse splitless Total flow 19.1 mL/minPulse pressure 20 psi Holdup time 1.88 minPurge flow time 0.75 min Outlet pressure VacuumPurge flow 15 mL/min Transfer line heater 280°C
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Analytical method (details on MS/MS parameters)
Parameter DescriptionIon source EIAcquisition mode MRM (positive)Source temperature 230oC Collision cell He 2.25 mL/minN2 collision gas 1.5 mL/minGain factor 10Diacetyl transition 86.0 43.0CE energy for diacetyl 2 VAcetylpropionyl transition 100.0 57.0CE energy for acetylpropionyl 1 Vd7-Quinoline transition 136.0 108.0CE energy for d7-quinoline l 5 VMS1 resolution UnitMS2 resolution UnitDwell time for each compound 150 ms
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Typical chromatogram for a sample containing about 50 ng/mL of the analytes with I.S. at 50
ng/mL d7-quinoline
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Analytical method (various details) • Calibration standards in ng/mL.
StandardCompound Level 7 Level 6 Level 5 Level 4 Level 3 Level 2 Level 1Diacetyl 2100 525 105 52.5 21.0 5.25 1.05Acetylpropionyl 2460 615 123 62.1 24.6 6.15 1.23
• Coefficients for the calibration curves Y = a X2 + b X + c used in quantitation, Y = ng/mL, X = (analyte area)/(I.S. area)
• Internal standard (d7-quinolone) concentration in the analyzed samples was 50 ng/mL
Compound a b c R2
Diacetyl -1.13988 159.816 -0.70536 0.9996
Acetylpropionyl - 4.22341 348.6078 0.261438 1.0000
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Method validation• Limit of detection (LOD) and limit of quantitation (LOQ) in ng/mL.
• Limit of detection (LOD) and limit of quantitation (LOQ) in ng/puff for 50 puffs per pad extracted with 10 mL acetone.
Compound LOD (ng/mL) LOQ (ng/mL)Diacetyl 0.12 0.41Acetylpropionyl 0.06 0.21
Compound LOD (ng/puff) LOQ (ng/puff) Diacetyl 0.025 0.082Acetylpropionyl 0.013 0.042
• Limit of detection (LOD) and limit of quantitation (LOQ) in ng/g e-liquid for 0.5 g e-liquid in 10 mL acetone.
Compound LOD (ng/g) LOQ (ng/g) Diacetyl 2.46 8.2Acetylpropionyl 1.3 4.2
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Method validation
• Relative standard deviation (RSD%) for standard Level 1 (1.05 ng/mL diacetyl and 1.23 ng/mL acetylpropionyl), and standard Level 3 (21.0 ng/mL diacetyl and 24.6 ng/mL acetylpropionyl)
Compound Level 1 Level 3 Diacetyl 3.91% (n = 5) 4.89% (n = 10)Acetylpropionyl 1.48% (n = 5) 3.36% (n = 10)
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Method validation (cont.)• Results for the recovery study in ng/mL
• Results in ng/puff for diacetyl and acetylpropionyl from two different smoking devices and two impinger collection.
Diacetyl AcetylpropionylElectronic device/flavor
Impinger 1 + Pad Impinger 2 Impinger 1 +
Pad Impinger 2
Device 1 ng/puff 32.42 0.3 22.66 0.8RSD% 0.89 0.4 0.68 12.0Device 2 ng/puff 9.00 0.0 4.19 0.2RSD% 14.76 - 13.38 5.2
Sample Added diacetyl
Diacetylng/mL
Recovery %
Added acetyl-
propionyl
Acetyl-propionyl
ng/mLRecovery%
E-Cig. X puffs 1-100 - 8.56 - - 7.83 -RSD% 0.1 - 4.86E-Cig. X puffs 1-100 21.8 31.31 104.37 19.2 26.90 99.32RSD% 4.24 1.59E-Cig. X puffs 1-100 54.5 63.88 101.51 48.0 59.55 107.74RSD% 0.68 2.11E-Cig. X puffs 101-200 - 6.65 - 4.19 -RSD% 1.56 5.08E-Cig. X puffs 101-200 21.8 29.66 105.55 19.2 24.71 106.88RSD% 7.28 4.49E-Cig. X puffs 101-200 54.5 60.64 99.06 48.0 53.33 102.38RSD% 1.67 4.22
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Method validation (cont.)
• Results in μg/cigarette for diacetyl in 3R4F cigarettes.
• Results in μg/cigarette for acetylpropionyl in 3R4F cigarettes.
• Literature reference [1] indicated an average of 107.5 μg/cig diacetyl and 15.4 μg/cig acetylpropionyl for ISO smoking.
• 1. Pierce, J. S., A. Abelmann, L. J. Spicer, R. E. Adams, B. L. Finley, Crit. Rev. Toxicol, 44 (2014) 420–435 (pag. 422)
Smoking protocol Pad Impinger 1 Impinger 2 TotalISO Average μg/cig 24.41 89.82 0.55 114.78RSD% 7.91 7.55 70.25HCI Average μg/cig 86.78 249.48 0.67 336.93RSD% 3.76 1.21 31.87
Smoking protocol Pad Impinger 1 Impinger 2 TotalISO Average μg/cig 9.07 19.75 0.70 29.52RSD% 17.64 9.11 23.31
HCI Average μg/cig 35.09 56.19 0.58 91.87RSD% 4.25 7.36 15.40
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Results for aerosols from various smoking devices expressed in ng/puff
Sample description Diacetyl Acetylpropionyl
Brand Puff range ng/puff RSD% ng/puff RSD%
E-cig. A 1-50 1.9 8.3 0.1 6.1
E-cig. A 101-150 2.5 26.4 1.2 58.1
E-cig. A 201-250 2.3 23.2 3.7 2.7
E-cig. A 401-450 2.9 8.9 0.6 5.2
E-cig. B 1-50 9.9 21.7 3.9 11.6
E-cig. B 101-150 13.0 24.1 4.7 13.4
E-cig. B 201-250 41.8 8.9 13.0 3.7
E-cig. B 401-450 43.6 9.0 14.1 15.3
E-cig. C 1-50 23.8 31.2 21.1 22.0
E-cig. C 101-150 34.2 25.1 12.7 22.6
E-cig. C 201-250 49.9 45.9 23.1 59.7
E-cig. C 401-450 49.7 9.5 22.9 8.1
E-cig. D 1-50 6.0 0.4 1.5 1.7
E-cig. D 101-150 5.1 42.2 1.2 69
E-cig. D 201-250 8.3 42.9 1.5 65.0
E-cig. D 401-450 9.1 42.5 1.6 71.5
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Results for aerosols from various smoking devices expressed in ng/puff (cont.)
Sample description Diacetyl Acetylpropionyl
Brand Puff range ng/puff RSD% ng/puff RSD%
E-cig. E 1-100 6.24 21.21 4.55 19.67E-cig. E 101-200 3.85 23.75 3.16 20.59E-cig. F 1-100 0.72 16.94 0.51 4.88E-cig. F 101-200 0.50 7.26 0.23 15.16E-cig. G 1-100 7.16 20.81 5.57 10.89E-cig. G 101-200 7.96 11.44 6.35 7.93E-cig. H 1-100 3.44 38.78 3.27 32.57E-cig. H 101-200 4.90 15.17 4.33 22.04E-cig. I 1-100 2.18 49.84 0.23 53.82E-cig. J 1-100 0.34 14.58 0.66 10.26E-cig. K 1-100 1.28 12.66 0.92 93.21E-cig. K 101-200 2.52 85.86 0.76 42.12E-cig. L 1-100 0.96 52.72 0.25 72.92E-cig. L 101-200 1.96 56.17 0.40 54.12
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Results for diacetyl in the aerosols from various smoking devices expressed in ng/puff 20
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Results for acetylpropionyl in the aerosols from various smoking devices expressed in ng/puff 20
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Results for various e-liquids expressed in ng/g
Sample description Diacetyl Acetylpropionylng/g RSD% ng/g RSD%
1 E-liquid 1 64.8 35.7 32 13.1
2 E-liquid 2 73.1 25.2 35.1 3.2
3 E-liquid 3 85.3 27.9 387.4 3.9
4 E-liquid 4 101.7 4.3 602.5 0.6
5 E-liquid 5 109.3 19.2 518.8 2.9
6 E-liquid 6 208.1 71 203.9 26.9
7 E-liquid 7 219.8 82.8 97.4 16.6
8 E-liquid 8 317.1 98.6 368.3 23.1
9 E-liquid 9 502.9 2.3 111 9.6
10 E-liquid 10 546.5 6.2 419.3 3.6
11 E-liquid 11 566.1 110.5 548.7 2.9
12 E-liquid 12 597 9.5 26.4 14.2
13 E-liquid 13 677.4 115.1 575.6 3.1
14 E-liquid 14 773.8 3.8 416.5 4.8
15 E-liquid 15 786.1 1.7 490 2.5
16 E-liquid 16 1042.5 129.6 897.7 2.6
17 E-liquid 17 1648.3 1.8 429.7 3
18 E-liquid 18 2078.1 3.3 465.1 2.8
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Results for various e-liquids expressed in ng/g (cont.)
Sample description Diacetyl Acetylpropionylng/g RSD% ng/g RSD%
19 E-liquid 19 2084.3 3.9 248.4 220 E-liquid 20 2824.8 6.5 8132.8 1.921 E-liquid 21 3157.3 1.8 687.5 1.222 E-liquid 22 3717.4 5.1 229.7 14.923 E-liquid 23 6164.8 5 725.3 1.224 E-liquid 24 6682.1 1.5 1540.8 1.725 E-liquid 25 11504.6 0.7 20982.6 226 E-liquid 26 16886.7 2.4 234.7 11.327 E-liquid 27 17532.4 3.3 151856.9* 2.528 E-liquid 28 18639.9 3.4 543.1 42.929 E-liquid 29 25226.6 0.4 8737.2 230 E-liquid 30 26113.5 2.5 303.7 4.931 E-liquid 31 27433 1 6846.3 0.732 E-liquid 32 39509.2 1.9 3419.2 1.833 E-liquid 33 54965.1* 4.5 21514.2 3.834 E-liquid 34 73196.7* 1.9 10778 0.535 E-liquid 35 74256.3* 3.6 156356.4* 336 E-liquid 36 105498.9* 2.2 1022.9 1.737 E-liquid 37 452567.0* 2.4 261.5 8.9
*Note: These values were outside the calibration range and were calculated based on a linear dependence Y (ng/ml) =144.13 X +8.6368 (for diacetyl) and Y = 315.47 X + 10.466 (for acetylpropionyl).
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Results for aerosols from various smoking devices expressed in ng/puff 20
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Results for acetylpropionyl in various e-liquids expressed in ng/g 20
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Conclusions• A new method for the quantitation of diacetyl and
acetylpropionyl in the aerosols of electronic smoking devices (e-cigarettes) has been developed.
• The method also has been applied for the measurements of these two analytes in the liquids used in e-cigarettes (e-liquids).
• The method uses a GC-MS/MS instrument with separation on a Carbowax type column and detection in MRM positive mode.
• The method has been validated.• The limit of quantitation (LOQ) for the method was determined
to be 0.41 ng/mL for diacetyl and 0.21 ng/mL for acetylpropionyl as measured for standards.
• The accuracy of the method has been verified on a 3R4F Kentucky reference cigarette.
• The results for different e-cigarette brands and e-liquids indicated a wide range of levels of the two analytes.
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