The importance of method validation in the determination of 2,3-butanedione (diacetyl) in e-liquids

Martin, A.(1) and Gillman, I.G.(2)

(1) Enthalpy Analytical Inc., Richmond VA 23228, USA

(2) Enthalpy Analytical Inc., Durham NC 27713, USA

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Outline

• Importance of Diacetyl and related Diketones

• What are they?

• Why are they important?

• Enthalpy Method Development

• Presentation will focus mainly on Diacetyl

• Analysis using methods not Fit-For-Purpose

• Examples

• Why is validation important?

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What are diketones and why are they important?

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Structures of Diketones

2,3-butanedione 2,3-pentanedione (diacetyl)

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Some Commonly Used Flavor Chemicals

Alcohols Aldehydes Esters Ketones

(3Z)-3-Hexen-1-ol Benzaldehyde 2-Methylbutylacetate Carvone

Benzyl alcohol Cinnamaldehyde Benzyl acetate Menthone

Ethyl maltol Ethyl vanillin Ethyl acetate γ-Undecalactone (peach)

Eugenol Piperonal Ethyl butyrate Acetylpyrazine

Maltol p-Tolualdehyde Ethyl hexanoate 2-Acetylpyridine

Menthol Vanillin Ethyl isovalerate Ionones

Linalool Isobutyraldehyde Hexyl acetate Damascenones

Rhodinol Methyl anthranilate 3-Methylcyclopentane-1,2-dione

(maple)

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So what is the concern?

• Flavors “generally recognized as safe” (GRAS) for use in food have not been shown to be safe when inhaled, only ingested.

• In fact, diacetyl has been implicated in the development of bronchiolitis obliterans, an irreversible respiratory disease also called “popcorn lung disease” because it was initially observed in workers at popcorn factories.

• Laboratory studies of diacetyl administered by inhalation show destruction of the lining of the respiratory system; unfortunately the risks associated with the inhalation of 2,3-pentanedione may be just as high.

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OSHA Methods 1012 and 1013

OSHA 1012 OSHA 1013

Extraction 2 mL EtOH:water 2 mL EtOH:water

Derivatization PFBHA none

Detection GC-ECD GC-FID

LOQ in sample 41.1 ng/sample 370 ng/sample

LOQ in extract 20.6 ng/mL 185 ng/mL

LOQ = Limit of Quantitation

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Method for determination of diacetyl and 2,3-pentanedione in e-liquids

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OSHA Methods 1012 and Enthalpy 227

OSHA 1012 Enthalpy 227

Extraction 2 mL EtOH:water 2 mL MeCN:water

Derivatization PFBHA PHBHA

Conditions Room Temp 36 hours 50°C for 20 hours

Extraction none 2 mL hexane

Detection GC-ECD GCMS-NCI

LOQ in extract 20.6 ng/mL 1.0 ng/mL

LOQ = Limit of Quantitation

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Calibration Standard @ 1 ng/mL

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Linearity and Range - Diacetyl

r2 – 0.999

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Accuracy – Diacetyl Recoveries

Recovery (%)

Day 1 Day 2 Day 3

Low Spike

Mean 102.2 101.3

SD 3.0 5.7

CV (%) 2.9 5.6

Mid Spike

Mean 105.4 105.4 102.8

SD 3.9 3.8 3.8

CV (%) 3.7 3.6 3.7

High Spike

Mean 107.9 102.4

SD 2.5 3.9

CV (%) 2.3 3.8

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Selectivity and Specificity

Standard solution containing diacetyl and 2,3-pentanedione at approximately 15 ng/mL (0.375

µg/g). Top chromatogram is full-scan mode. Bottom is selected ion monitoring (SIM).

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Selectivity and Specificity

Sample extract (“Peach”) containing diacetyl at approximately 4 ng/mL (0.1 µg/g). Top

chromatogram is full-scan mode. Bottom is selected ion monitoring (SIM).

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Use of methods not fit-for purpose

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Example 1: Milwaukee Journal Sentinel

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• “…the Milwaukee Journal Sentinel bought five locally made e-liquids from vape shops around Milwaukee and had them tested for diacetyl and 2,3-pentanedione by a team from Marquette University’s chemistry department. The samples were derivatized and then analyzed with gas chromatography-mass spectrometry. This method is considered to be highly sensitive and is designed to determine levels at 1 part per billion or greater.”

• “All five samples were found to have both chemicals, which are known to destroy lungs.“

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Results from Milwaukee Journal Sentinel Study

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Comparison of Liquid Test Results (all results in ppm)

Diacetyl

2,3-pentane-

dione Diacetyl

2,3-pentane-

dione Diacetyl

2,3-pentane-

dione

Foghorn: Randy 10 300 61.8 207.0 NA NA

Matik: Pomegranate 11 50 <1.13 ND <1.07 ND 0.10 0.11

Radioactive Vapes: Grape 4 13 <1.13 ND <1.07 ND 0.13 0.03

Marquette Enthalpy Method 1 Enthalpy Method 2

LLOQ ~ 0.02

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Example 2: Harvard Study

Allen JG, Flanigan SS, LeBlanc M, Vallarino J, MacNaughton P, Stewart JH, Christiani DC. 2016. Flavoring chemicals in e-cigarettes: diacetyl, 2,3-pentanedione, and acetoin in a sample of 51 products, including fruit-, candy-, and cocktail-

flavored e-cigarettes. Environ Health Perspect 124:733–739; http://dx.doi.org/10.1289/ehp.1510185

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Brief description

•Harvard Study used OSHA 1012.

• Collected aerosol from e-cigarettes, splitting the flow to obtain an appropriate flow through the sample collection tubes.

• Collected until the e-cigarette cartridge was exhausted.

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OSHA Method 1012– applied to e-cigarette aerosol

Concerns:

• E-cigarette aerosol is not air. The aerosol contains humectants, nicotine and many, many other flavors.

• A large number of the flavors are ketones and aldehydes which all have a carbonyl group. Therefore they will all be derivatized by PFBHA and,

• ECD is a selective detector but will detect all compounds with a halogen attached (not specifically the compound of interest).

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Comparison of published results to Enthalpy results (µg/g of e-liquid)

Flavor Diacetyl 2,3-pentanedione Diacetyl 2,3-pentanedione

Bold 11.8 <LOD 0.3 <LOQ

Gold 1.2 <LOD 0.2 <LOQ

Menthol 9.8 <LOQ 0.2 <LOQ

Pomegranate <LOD 0.4

Pomegranate 13.8 <LOD

Cherry 8.4 <LOD 0.1 <LOQ

Double Apple Hookah 42.2 4.6 21.0 0.03

Franks Lemon Lime 8.4 2.2 0.8 0.06

Grape Hookah 3.0 3.2 0.6 0.67

Orange Mint 2.2 3.0 0.1 <LOQ

Peach 16.6 <LOD 0.1 <LOQ

Pina Colada 23.2 1.4 0.5 0.05

Watermelon 26.6 2.8

Watermelon 14.8 <LOD

0.4 <LOQ

Enthalpy ResultsHarvard Study Results

0.3 <LOQ

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Why is there so much variability in results?

• The Marquette U. method uses derivatization and GCMS. The selectivity and specificity of the method are unknown.

• The OSHA methods are not validated for the matrix we are interested in – e-cigarette liquid and aerosol are vastly more complex than room air and neither of the OSHA methods are selective or specific enough.

• The Durham method is selective and specific but was designed to detect very high levels.

• The Richmond method is selective and specific but was designed to only detect very low levels.

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Selectivity and Specificity

Sample extract (“Pomegranate”) containing diacetyl at approximately 18 ng/mL (0.4 µg/g).

Top chromatogram is full-scan mode. Bottom is selected ion monitoring (SIM).

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Why is it important to use the right method?

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Information is quickly disseminated… (from medicinenet.com)

“Do e-cigarettes cause popcorn lung?

It is not only microwave popcorn that contains dangerous chemical flavorings such as diacetyl. A study published in 2015 in the journal Environmental Health Perspectives showed that harmful chemicals associated with "popcorn lung" are present in many types of flavored e-cigarettes, particularly those with flavors like fruit and candy that may appeal to young smokers. Of the 51 flavored e-cigarettes tested, flavoring chemicals were found in 47 and diacetyl specifically in 39 samples. This suggests a potentially dangerous level of exposure via e-cigarettes to chemicals that can cause severe lung damage.”

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(from lung.org)

“Even though we know that diacetyl causes popcorn lung, this chemical is found in many e-cigarette flavors. It is added to "e-juice" liquid by some e-cigarette companies to complement flavorings such as vanilla, maple, coconut and more. So while diacetyl was swiftly removed from popcorn products since it could cause this devastating disease among factory workers, e-cigarette users are now directly inhaling this harmful chemical into their lungs. In fact, researchers at Harvard found that 39 of 51 e-cigarette brands contained diacetyl. The study also found two similarly harmful chemicals—2,3 pentanedione and acetoin—present in 23 and 46 of the 51 flavors it tested. And roughly 92 percent of the e-cigarettes had one of the three chemicals present

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(from vapingex.com)

• “A study published in the 2015 journal of Environmental health Perspectives showed that the chemical Diacetyl was found in many types of e-cigarette juice flavors especial in candy and fruit flavors. The researchers at Harvard tested the chemical components of 51 types of e-cigarettes and liquid and found that 39 of these contained diacetyl. This suggests that exposure to the chemicals in e-cigarettes can be harmful to your health, and cause lung damage.”

However, they go on to say:

• “…the level of diacetyl exposure from vaping compared with smoking differs by ”orders of magnitude.””

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(from rollingstone.com)

While studies like Harvard's are critical to fully understanding e-cigs, they too often have the opposite effect. Tobacco cigarettes, for instance, have also long been known to contain diacetyl — at levels over 100 times those found in electronic cigarettes — yet earlier tobacco studies found that even these levels were not enough to cause popcorn lung in smokers.

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De-bunking the myth? (from snopes.com)

CLAIM: A study proved e-cigarette use (or vaping) leads to an incurable condition known as "popcorn lung."

MOSTLY FALSE

WHAT'S TRUE: A December 2015 Harvard study concluded diacetyl is present in e-cigarettes.

WHAT'S FALSE: That presence of diacetyl was well-known prior to the publication of the research, e-cigarettes don't contain more diacetyl than cigarettes, and research has not deemed e-cigarettes a "popcorn lung risk."

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Conclusions…

• There is some evidence that diacetyl and related diketones are toxic when inhaled.

• These compounds are just a few of many flavors “generally recognized as safe” in food but are being used in e-liquids. Their safety when inhaled may not be established.

• In order to accurately report the amount of these substances in e-liquids and aerosols, the analytical method must be validated for the matrix to be tested, or fit-for-use.

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…or More Questions?

• Is there significant amount of diacetyl in e-liquids and e-vapor?

• What if published results are wrong?

• Will regulatory decisions be made based on erroneous or selective data?

• Is this risk any greater than from smoking cigarettes?

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Vielen Dank

Thank you

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O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine hydrochloride

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