Cooperation Centre for Scientific Research

Relative to Tobacco

E-Vapour Sub-Group

CORESTA Recommended Method

No. 96

DETERMINATION OF FORMALDEHYDE AND

ACETALDEHYDE IN E-VAPOUR

PRODUCT AEROSOL

February 2021

CRM No. 96 – February 2021 Page 2/13

CORESTA RECOMMENDED METHOD Nº 96

Title:

DETERMINATION OF FORMALDEHYDE AND ACETALDEHYDE

IN E-VAPOUR PRODUCT AEROSOL

Status: Valid

Note:

Document history:

Date of review Information

February 2021 Version 1

CRM No. 96 – February 2021 Page 3/13

CORESTA RECOMMENDED METHOD Nº 96

DETERMINATION OF FORMALDEHYDE AND ACETALDEHYDE

IN E-VAPOUR PRODUCT AEROSOL

(February 2021)

1. SCOPE AND APPLICATION

The purpose of this document is to describe the procedures used for quantitation of

formaldehyde and acetaldehyde in electronic cigarette (e-cigarette) aerosols utilizing liquid

chromatography coupled with Ultraviolet or Diode Array Detector (LC-UV or LC-DAD).

Aerosols from vapour products are generated on a vaping machine conforming to the

requirements of ISO 20768. The trapping system that is used to trap carbonyls consists of a

pad holder containing a glass fibre filter pad in series with a fritted-tipped impinger containing

an acidified solution of 2,4-dinitrophenylhydrazine (DNPH) in 1:1 acetonitrile:water. Post-

vaping, the glass fibre pad is combined with the impinger solution and shaken mechanically for

30 minutes. An aliquot of the sample extract is subsequently neutralized with pyridine and

analysed using reversed phase liquid chromatography.

E-cigarette devices used in this method may be manual (push-button) or puff-activated. The

e-liquids used in the devices to generate aerosol may be unflavoured or flavoured and include

extracted tobacco derived flavours. This method may also be applied to e-liquid samples by

using a suitable reference device.

2. SUMMARY OF METHOD

Aerosols from e-cigarettes are collected using a vaping machine conforming to the requirements

of ISO 20768. The aerosol is passed through a sampling train consisting of a glass fibre filter

pad and a single fritted-tipped impinger containing a derivatization solution. The derivatization

solution generates a coloured adduct that can be visualized on an HPLC with a UV-Vis Diode

Array Detector (DAD) or Variable Wavelength Detector (VWD). Samples are collected using

a standard puffing regimen.

3. DEFINITIONS

3.1 Reagent Blank

An aliquot of the reagent (acetonitrile) used to prepare the standards and/or dilutions. The

reagent blank is analysed to ensure that no contamination is introduced by the acetonitrile.

3.2 Air Blank

A room air sample collected using the aerosol trapping system and the puffing systems.

This sample contains no vapour product aerosol and is carried through the same

collection, preparation and analysis steps as the samples.

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4. NORMATIVE REFERENCES

ISO 3308:2000, Routine analytical cigarette-smoking machine – Definitions and standard

conditions

ISO 20768, Vapour products — Routine analytical vaping machine — Definitions and standard

conditions

ISO 3696, Water for analytical laboratory use – Specification and test methods

5. SAFETY

5.1 All laboratory personnel handling chemicals used in this procedure should familiarize

themselves with the appropriate SDSs for health and safety information. Personnel

should wear appropriate personal protective equipment when handling hazardous

substances. This equipment should include, as a minimum, safety glasses, gloves, and

laboratory coat.

5.2 There are a number of hazardous chemicals used in this method, including the following:

5.2.1 Acetonitrile is toxic, highly flammable, causes severe eye irritation and may

cause damage to internal organs.

5.2.2 Formaldehyde-DNPH causes skin irritation, serious eye irritation and may cause

respiratory irritation.

5.2.3 Acetaldehyde-DNPH causes skin irritation, may cause an allergic skin reaction,

causes serious eye irritation and may cause respiratory irritation.

5.2.4 Dry dinitrophenylhydrazine (DNPH) is explosive, may cause liver damage, and

can be absorbed transdermally.

5.2.5 Pyridine is highly flammable, causes skin irritation and serious eye irritation.

5.2.6 Phosphoric acid is corrosive, acutely toxic and causes serious eye damage.

6. EQUIPMENT AND SUPPLIES

Note that equivalent supplies and equipment may be used provided that the equivalence is

demonstrated.

6.1 Balance, 4 to 5-place, 0,01 mg to 0,1 mg precision.

6.2 Class-A glassware: graduated cylinders, volumetric pipettes, and volumetric flasks.

6.3 Adjustable pipettes.

6.4 Amber vials with caps, 8 mL.

6.5 Glass autosampler vials with crimp or screw tops.

6.6 Bottle-top dispenser capable of dispensing 35 mL (±2 %).

6.7 Impinger: For example, bubbler Bottle, 24/40 outer joint, 215 mm, O.L. Hgt x 30 mm O.D.

NOTE: Alternate impinger designs may be used provided that equivalent trapping efficacy can

be demonstrated.

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6.8 Stem: For example, bubbler insert with course fritted tip, 24/40 inner o-ring and 12/5

socket/ball o-ring connections.

NOTE: Alternate impinger insert designs may be used provided that equivalent trapping

efficacy can be demonstrated.

6.9 HPLC columns:

6.9.1 Guard Column: Waters XTerra MS C18 20 mm × 3,9 mm × 5µm or equivalent.

6.9.2 Zorbax SB-C18, 4,6 mm x 15 cm, 1,8 µm, 600 bar or equivalent.

6.10 Instrument: HPLC system with UV detection capable of performing the method below.

7. REAGENTS AND STANDARDS

Note that equivalent reagents and reference materials may be used provided that the equivalence

is demonstrated.

7.1 Acetonitrile, ACN, HPLC grade.

7.2 Phosphoric acid, (H3PO4, 85 %, or 10 % (v/v) Aqueous Solution).

7.3 Water, Grade 1 (refer to ISO 3696).

7.4 2,4-Dinitrophenylhydrazine Hydrochloride (DNPH-HCL) min 98 % or equivalent.

7.5 Pyridine min. 99 %.

7.6 Formaldehyde-DNPH, min. 99 %.

7.7 Acetaldehyde-DNPH, min. 99 %.

NOTE: An ISO guide 34 solution may be used in place of neat standard materials.

7.8 Solution Preparation: Prepare appropriately proportioned amounts of the solutions listed

below. All solutions must be equilibrated to room temperature prior to use. Use

graduated cylinders and calibrated pipettes to combine components.

7.8.1 10 % H3PO4: Prepare by bringing 117,6 mL of 85 % H3PO4 to 1 L with water.

Vendor-prepared 10 % H3PO4 may be used instead.

7.8.2 DNPH Trapping Solution: Prepare trapping solution by dissolving 1.0 g

DNPH-HCL in 500 mL of ACN, combine with 40 mL of 10 % H3PO4 and bring

to 1 L with water. Store the solution in an amber glass bottle at room

temperature. Discard the solution when the Air Blank contains an analyte

concentration greater than that of the LOQ.

NOTE: The use of DNPH free base containing water may also be used with the

appropriate mass scaling to provide the same final concentration of DNPH solution.

7.8.3 Neutralized Trapping solution: Transfer 50 mL of DNPH trapping solution to

a suitable size glass bottle and add 2,5 mL of pyridine. Mix solution thoroughly.

CRM No. 96 – February 2021 Page 6/13

7.9 Preparation of Standards

NOTE: All solutions to be equilibrated to room temperature prior to use.

7.9.1 HPLC calibration standards and working solutions.

The calibration standards should cover the concentration range of interest.

Table 1 provides a suitable concentration range that can be used for the analysis.

However, it can be adjusted depending on the level of carbonyls detected in the

samples. The user shall ensure the low calibration standard has a sufficient

signal to noise ratio for accurate quantitation (≥10:1) and that the calibration

curve is linear.

7.9.2 Primary carbonyl standards

Weigh the hydrazones as described in Table 1.1 into individual 25 mL

volumetric flasks and dissolve in acetonitrile. Record the concentrations of the

free aldehyde equivalents in µg/mL.

7.9.3 Secondary carbonyl standards

Pipette predetermined volumes in Table 1.1 of each primary hydrazone standard

into a 25 mL volumetric flask and dilute to the mark with acetonitrile.

7.9.4 Carbonyl working standards

Take appropriate volumes (0,050 mL to 10 mL) of the secondary carbonyl

standard (6.9.3) and dilute to 10 mL with acetonitrile to prepare calibration

standards with approximate carbonyl concentrations listed in Table 1.2.

Table 1.1 — Stock standards

Primary standards Secondary standard a

Carbonyl hydrazone

Formula Wt

hydrazone

Formula Wt

carbonyl

Mass Purity Volume Stock

Volume of

primary stock

Dilute to

volume Stock

(mg) (%) (mL) (µg/m

L) (µl) (mL) (µg/mL)

Formaldehyde-DNPH

210,15 30,03 50,6 99,9 25 288,9 500 25 5,779

Acetaldehyde-DNPH

224,18 44,05 33,9 99,9 25 266,2 1 800 25 19,16

a In a single 25 mL volumetric flask, made to volume with acetonitrile.

Table 1.2 — Carbonyl working standards

Carbonyl

Volume of secondary standard (mL)a

10,0 7,0 4,0 2,0 0,80 0,40 0,20 0,05

(µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL)

Formaldehyde 5,779 4,045 2,311 1,156 0,462 3 0,231 1 0,115 6 0,028 9

Acetaldehyde 19,16 13,42 7,666 3,833 1,533 0,766 6 0,383 3 0,095 8

a In a single 10 mL volumetric flask, made to volume with acetonitrile.

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8. SAMPLE PRESERVATION, STORAGE, AND HANDLING

8.1 Analyse the e-cigarette aerosol solutions within 2 days, if stored at room temperature; or

analyse within 7 days when stored at 4 °C (± 2 °C).

9. CALIBRATION

9.1 Initial Calibration:

9.1.1 Analyse the initial calibration consisting of at least 5 consecutive standard

concentrations per analyte. The regression is linear and should have a coefficient

of determination (r2) of at least 0,995. It is recommended that curve type is

linear, origin is ignored with the regression fitted with linear weighting. The initial

calibration standards are acceptable if they are within 15 % of their assigned

values for the LOQ (low standard) and within 10 % for all other standards.

9.1.2 If the calibration does not meet criteria in section 9.1.1, check the instrument for

problems; analyse fresh standard aliquots and/or fresh standards prepared from

stock solutions.

10. PROCEDURE

10.1 Sample Preparation:

10.1.1 Label all tubes and vials needed for sample preparation. Labels will be generated

by a computer or hand-written on labels that are resistant to smearing by solvent

etc.

10.1.2 Using the bottle top dispenser, add 35 mL of trapping solution to a separate

impinger with a fritted stem insert for each sample to be collected.

10.1.3 Assemble trapping system of the vaping machine (see Figure 1) as follows:

preweighed glass filter pad/holder → impinger → backup filter pad → vaping

machine. Aerosol collection is carried out as specified in the study protocol.

10.1.4 After aerosol collection is complete, weigh the glass filter pad/holder for

determination of collected aerosol mass.

10.1.5 Transfer the glass filter pad to a 60 mL flask.

10.1.6 Mix the contents of the impinger. Using a clean disposable pipette, rinse the

inside of the impinger stem five (5) times being sure to wet the inside.

10.1.7 Transfer the impinger solution to the 60 mL flask containing the glass filter pad.

10.1.8 Extract the filter pad by mechanical shaking. Mechanical shakers are not

standardized and suitable extraction conditions should be determined prior to

use.

10.1.9 Transfer 5 mL of impinger solution into an 8 mL glass vial containing 0,25 mL

of pyridine. This solution may become cloudy and can be filtered into an auto-

sampler vial using a 0,45 µm PTFE syringe filter and appropriate disposable

syringe.

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10.1.10 Transfer an aliquot to an auto-sampler vial and close the vial with crimp-cap.

The samples are now ready for analysis.

10.1.11 The neutralized samples have been shown to be stable when stored at

approximately 4° C. Stability and storage time should be evaluated by the

laboratory.

10.1.12 Samples may be diluted in neutralized trapping solution if necessary. Document

the dilution factor and dilution preparation.

10.1.13 Place impingers and stems in hot soapy water after use.

Figure 1 — Aerosol Collection setup for Carbonyls Analysis

NOTE: Since there is no standard impinger design, trapping efficiency shall be verified

when validating this method. Ensure the end of the bubbler tube is completely submerged

in the trapping solution and that the impinger can adequately hold 35 mL of trapping

solution. The trapping system should effectively trap 95 % of the analytes of interest. To

check the trapping efficiency of the method, add an additional impinger after the impinger

and analyse the contents separately according to the method. If less than 5 % of the

compounds are detected in the backup impinger then only one impinger is required to trap

all the carbonyls effectively. Breakthrough or poor trapping efficiency can be due to the

size of the impinger or bubbler tube or due to products with high carbonyl yields.

NOTE: A volume of trapping solution may be modified provided that sufficient trapping

efficiency is demonstrated prior to use.

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10.2 Instrument conditions using the Zorbax SB-C18 column.

10.2.1 Mobile Phase A – 100 % DIUF H2O

10.2.2 Mobile Phase B – 100 % Acetonitrile

10.2.3 Flow Rate: 1,0 mL/min

10.2.4 Run Time: 12 min with a 2 min post time

10.2.5 Pump Timetable:

Time (min) % Mobile Phase A % Mobile Phase B

0,00 35 65

1,49 35 65

1,50 45 55

3,00 45 55

3,01 35 65

3,56 35 65

7,50 25 75

8,00 0 100

9,50 0 100

9,55 35 65

12,00 35 65

10.2.6 Injection Volume: 5µL

10.2.7 Column Compartment Temperature: 32 ºC

10.2.8 DAD:

Signal A: 360 nm, 16 nm slit, reference 510 nm, 100 nm slit

UV and Vis lamp is required

Pre-run and post-run balancing

Margin for negative absorbance: 100 mAU

10.2.9 VWD:

Signal wavelength: 360 nm

Signal peakwidth: > 0,1 min

Prerun balancing: Yes

Margin for negative absorbance: 100 mAU

Signal Polarity: Positive

Enable analysis when lamp is off: No

NOTE: Instrument conditions are provided as examples. Alternative columns or

instrument settings may be used provided that the equivalence is demonstrated.

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10.3 Sample Analysis:

10.3.1 Sample concentrations shall be within the instrument’s calibration range; if

sample concentrations exceed the range, dilute the impinger sample using

neutralized trapping solution.

NOTE: Neutralized Trapping Solution may be prepared by the addition of 2,5 mL of

pyridine to 50 mL of impinger solution (7.8.3).

11. DATA ANALYSIS AND CALCULATIONS

11.1 Acetaldehyde may elute as two peaks because its corresponding hydrazone exists in two

isomers. The peak areas should be integrated consistently in both the standards and

samples. This may be accomplished by drawing a single baseline tangent across both

peaks.

11.2 Samples are neutralized with pyridine after collection. This “dilution” must be accounted

for in sample calculations. When method volumes are followed, multiply the calculated

carbonyl concentration by total sample volume to determine yield in µg/mL as follows:

[𝑆 − 𝐵] ∗ [𝑡𝑜𝑡𝑎𝑙 𝑖𝑚𝑝𝑖𝑛𝑔𝑒𝑟 𝑣𝑜𝑙𝑢𝑚𝑒] ∗ [1,05] = 𝐴𝑠 (µg/mL)

Where

- S is the sample concentration and B is the blank concentration.

NOTE: A dilution factor of 1,05 is applied to the determined value to account for dilution

of the sample with pyridine e.g. 5,25 mL/5,00 mL.

NOTE: When determining the amount of carbonyls present in the samples a background

subtraction may be required if the DNPH has inherent levels of carbonyls.

Carbonyl concentrations in the vapour product emissions are typically reported as

micrograms per puff (µg/puff), microgram per mg of aerosol mass (µg/mg), or microgram

per mg of e-liquid vaporised mass (µg/mg), based on the following equations:

𝑀𝑝𝑢𝑓𝑓 = [𝐴𝑠] 𝑑 𝑉

# 𝑜𝑓 𝑃𝑢𝑓𝑓𝑠 (1)

MACM = [As] d V

ACM (2)

𝑀𝐸𝑉𝑀 = [𝐴𝑠] 𝑑 𝑉

𝐸𝑉𝑀 (3)

Where

- M is the concentration of the analyte;

- AS is the concentration of the analyte, in micrograms per millilitre, from the linear

regression reported by the software;

- d is the dilution factor (final volume/aliquot volume);

- V is the impinger volume;

- ACM is Aerosol Collected Mass (mg);

- EVM is E-liquid Vapourised Mass (mg).

CRM No. 96 – February 2021 Page 11/13

12. REPEATABILITY AND REPRODUCIBILITY

An international collaborative study involving 11 laboratories that used the specified LC-UV

method was conducted by the CORESTA EVAP Sub-Group in 2019. This collaborative study

included the determination of carbonyl compounds in e-vapour product aerosol generated under

ISO 20768 puffing conditions. The target compounds for this study included formaldehyde and

acetaldehyde, which were determined following a proposed recommended method for the

determination of carbonyl compounds in e-vapour product aerosol. Formaldehyde and

acetaldehyde yields were corrected based on the device ACM yield and reported in units of

g per gram of ACM.

Results were analysed in basic conformance with ISO 5725-2:1994 and ISO/TR 22971:2005.

The mean values, %r, and %R for formaldehyde and acetaldehyde are presented in Tables 2

and 3. The value of ‘N’ is the number of laboratories used to determine the statistics after the

removal of outliers. To reduce overall variability, this study included e-liquids that were

fortified with formaldehyde and acetaldehyde. The fortification amount is listed in Tables 2

and 3 in the column “added g/g”.

Table 2: Repeatability (r) and Reproducibility (R) Limits for Acetaldehyde (μg/g)

Under ISO 20768 Conditions

Product Added

(μg/g) N Labs* Average r r % R R %

Menthol/Tobacco 0 8 2,33 1,32 56,7 % 4,80 206 %

Tobacco 0 9 2,43 1,36 55,9 % 5,06 208 %

Unflavoured 0 9 4,03 2,42 60,1 % 6,98 173 %

Menthol/Tobacco 15 8 9,06 3,86 42,6 % 10,50 116 %

Tobacco 15 9 11,45 3,51 30,6 % 15,34 134 %

Unflavoured 15 9 13,23 4,15 31,4 % 17,34 131 %

Menthol/Tobacco 25 7 15,79 5,31 33,6 % 18,00 114 %

Tobacco 25 9 19,24 9,65 50,1 % 21,19 110 %

Unflavoured 25 8 17,97 5,45 30,3 % 19,32 108 %

Menthol/Tobacco 35 8 23,20 7,86 33,9 % 28,72 124 %

Tobacco 35 9 24,96 12,97 52,0 % 33,36 134 %

Unflavoured 35 9 26,59 13,28 49,9 % 35,64 134 %

* The number of laboratory data sets after removal of outliers.

CRM No. 96 – February 2021 Page 12/13

Table 3: Repeatability (r) and Reproducibility (R) Limits for Formaldehyde (μg/g)

Under ISO 20768 Conditions

Product Added

(μg/g) N Labs* Average r r % R R %

Menthol/Tobacco 0 7 8,46 3,16 37,4 % 12,51 148 %

Tobacco 0 8 8,29 4,33 52,2 % 11,48 139 %

Unflavoured 0 8 11,57 3,95 34,1 % 14,41 124 %

Menthol/Tobacco 15 7 17,26 3,27 19,0 % 17,02 99 %

Tobacco 15 8 16,72 3,52 21,1 % 13,67 82 %

Unflavoured 15 8 20,93 2,68 12,8 % 12,70 61 %

Menthol/Tobacco 25 7 27,98 12,41 44,3 % 18,56 66 %

Tobacco 25 8 25,78 6,19 24,0 % 13,28 52 %

Unflavoured 25 8 28,55 5,90 20,7 % 17,31 61 %

Menthol/Tobacco 35 7 32,64 10,21 31,3 % 17,20 53 %

Tobacco 35 8 30,40 7,39 24,3 % 19,10 63 %

Unflavoured 35 8 33,63 6,98 20,7 % 13,25 39 %

* The number of laboratory data sets after removal of outliers.

13. REFERENCES

[1] Determination of Selected Carbonyls in Mainstream Tobacco Smoke, Health Canada,

Tobacco Control Programme

[2] UK Smoke Constituents Study: Part 2 Method: Determination of Eight Carbonyl Yields

in Cigarette Smoke by High Performance Liquid Chromatography

[3] Determination of Carbonyl Compounds by Reversed-Phase-High-Performance Liquid

Chromatography – Dionex Application Note 97 ISO 3308:1991 Sections 4.2 – 4.4

[4] Intorp M, Purkis S, Whittaker M, Wright W. Determination of “Hoffmann Analytes” in

cigarette mainstream smoke. The CORESTA 2006 Joint Experiment. Contributions to

Tobacco Research 2009;23-4:161–202,

https://content.sciendo.com/view/journals/cttr/23/4/cttr.23.issue-4.xml

[5] Jason W. Flora, Celeste T. Wilkinson, James W. Wilkinson, Peter J. Lipowicz, James A.

Skapars, Adam Anderson, John H. Miller, Method for the Determination of Carbonyl

Compounds in E-Cigarette Aerosols, Journal of Chromatographic Science, Volume 55,

Issue 2, 1 February 2017, Pages 142-148, https://doi.org/10.1093/chromsci/bmw157

[6] CORESTA E-Vapour Sub-Group Technical Report, 2019 Collaborative Study for the

Determination of Formaldehyde and Acetaldehyde in E-Vapour Product Aerosol, available

at https://www.coresta.org/2019-collaborative-study-determination-formaldehyde-and-

acetaldehyde-e-vapour-product-aerosol-34082

CRM No. 96 – February 2021 Page 13/13

14. TABLES, DIAGRAMS AND FLOW CHARTS

14.1 Appendix A – Example Chromatogram – Agilent Zorbax SB-C18 column

Example Standard Chromatogram – Agilent Zorbax SB-C18