Determination of Benzoic Acid and Benzyl Alcohol in E-Liquids (JUULTM Pods) by Isotopic Dilution High-Performance Liquid Chromatography and Tandem Mass Spectrometry

Segment	Segment time (min)	Compound	Scan type	Ions monitored (m/z)	Collision energy (eV)
1	0.00 − 6.00	NA	Full Scan		NA
2	6.00 − 9.50	BOH_Q	SRM	91.0 → 65.0	20.0
		BOH_C1	SRM	91.0 → 91.0	10.0
		BOH_C2	SRM	91.0 → 39.1	34.0
		BOH_L	SRM	97.0 → 69.0	20.5
3	9.55 − 13.50	BCOOH_Q	SRM	123.0 → 79.0	10.2
		BCOOH_C1	SRM	123.0 → 77.0	22.1
		BCOOH_C2	SRM	123.0 → 105.0	13.6
		BCOOH_L	SRM	129.0 → 85.0	10.0
4	13.55 − 20.0	NA	Full Scan		NA

[i] BOH: benzyl alcohol; BCOOH: benzoic acid; _Q: quantitation; _C: confirmation; _L: Labeled; NA: Does not apply, SRM: selected reaction monitoring

Table 3

Limits of detection (LOD), accuracy, and precision of the method.

Analyte	LOD (ng/μL)	Accuracy (%)			% RSD intermediate precision
Analyte	LOD (ng/μL)	Low	Medium	High	QCL	QCH
Benzyl alcohol	0.11	96.6 ± 1.9	95.4 ± 1.4	98.9 ± 2.3	5.3	4.4
Benzoic acid	9.05	92.2 ± 1.7	91.7 ± 4.1	93.8 ± 2.5	4.8	4.6

[i] RSD: relative standard deviation; QCL: quality control samples at low concentration; QCH: quality control samples at high concentration

Representative chromatograms of benzyl alcohol (BOH) and benzoic acid (BCOOH) quantification transitions for (A) quality control sample low concentration, (B) HAUS Cool Ice ^TM, (C) JUUL Cool Mint ^TM, (D) labeled internal standard.

Table 4

Results and conditions for the determination of method robustness.

Test parameters	Analyte	Method settings		Low level settings			High level settings
Test parameters	Analyte	Method level	Amount (μg) ^a	Low level	Amount (μg) ^a	% Diff.	High level	Amount (μg) ^a	% Diff.
Column temperature	Benzyl alcohol	50 °C	3.76 ± 0.12	45 °C	3.69 ± 0.13	1.9	55 °C	3.66 ± 0.03	2.7
Column temperature	61.65 ± 0.08	50 °C	62.90 ± 1.34	45 °C	2.0	61.54 ± 0.82	55 °C	4.7
Benzoic acid	3279 ± 50	3241 ± 40	1.2	3171 ± 35	3.3
3158 ± 103	3038 ± 84	3.8	3053 ± 55	3.3
Sample dilution composition	Benzyl alcohol	MeOH:H₂O 50:50	2.95 ± 0.01	MeOH:H₂O 40:60	3.06 ± 0.06	3.7	MeOH:H₂O 60:40	2.96 ± 0.04	0.3
Sample dilution composition	52.12 ± 4.8	MeOH:H₂O 50:50	52.21 ± 0.59	MeOH:H₂O 40:60	0.2	52.42 ± 1.32	MeOH:H₂O 60:40	0.6
Benzoic acid	3344 ± 72	3324 ± 90	0.6	3415 ± 77	2.1
2711 ± 201	2831 ± 111	4.4	2813 ± 60	3.8
Injection volume	Benzyl alcohol	10 μL	3.00 ± 0.30	7 μL	3.11 ± 0.32	3.7	13 μL	3.01 ± 0.41	0.3
Injection volume	61.26 ± 6.95	10 μL	61.91 ± 7.09	7 μL	1.1	61.17 ± 6.76	13 μL	0.1
Benzoic acid	3332 ± 396	3419 ± 437	2.6	3434 ± 386	3.1
3077 ± 27	3137 ± 52	1.9	3147 ± 29	2.3
Sample weight	Benzyl alcohol	100 mg	3.03 ± 0.11	80 mg	3.09 ± 0.12	2.0	130 mg	3.05 ± 0.20	0.7
Sample weight	53.54 ± 4.25	100 mg	53.19 ± 3.05	80 mg	0.7	54.15 ± 2.25	130 mg	1.1
Benzoic acid	3360 ± 68	3484 ± 58	3.7	3308 ± 78	1.5
2806 ± 210	2909 ± 90	3.7	2856 ± 110	1.8
Extraction time	Benzyl alcohol	60 min	3.01 ± 0.07	42 min	2.98 ± 0.04	1.0	78 min	2.98 ± 0.06	1.0
Extraction time	51.64 ± 4.04	60 min	51.75 ± 1.49	42 min	0.2	52.12 ± 0.96	78 min	0.9
Benzoic acid	3345 ± 65	3293 ± 27	1.6	3327 ± 20	0.5
2773 ± 235	2714 ± 21	2.1	2721 ± 23	1.9

% Diff.: Percentage of difference between the final method settings and the new settings used for the robustness study.

a Average amount in sample with standard deviation. All samples were prepared in triplicates.

Table 5

Levels of benzyl alcohol (n = 21), benzoic acid (n = 21), and nicotine (n = 7) in JUUL^TM liquids and two commercially available e-liquids.

Sample type	Benzyl alcohol Total (mg/g)			Benzoic acid (BA) Total (mg/g)			Benzoic acid Total (%,W/W)	Nicotine (N) Total (mg/g)			Molar ratio
Sample type	Average	STD	CV	Average	STD	CVs	Average	Average	STD	CV	N:BA
QCL	0.0340	0.0009	2.5	13.03	0.67	5.2	1.30	NP	—	—	NA
JUUL^TM - Fruit Medley	ND	NA	NA	32.58	2.06	6.3	3.26	49.4	0.84	1.7	1.14
JUUL^TM - Mango	0.0283	0.0045	16.0	33.64	3.74	11.1	3.36	50.6	0.92	1.8	1.13
JUUL^TM - Cool Mint	0.6420	0.0485	7.6	33.18	2.84	8.5	3.32	49.3	0.75	1.5	1.12
JUUL^TM - Virginia Tobacco	ND	NA	NA	32.52	2.54	7.8	3.25	50.3	0.88	1.7	1.16
JUUL^TM - Creme Brulee	ND	NA	NA	32.20	2.62	8.2	3.22	50.2	1.08	2.2	1.17
HAUS^TM - Cool Ice	0.0487	0.0021	4.3	ND	NA	NA	ND	NP	—	—	NA
Pacha Mama - Mango	0.0972	0.0065	6.7	ND	NA	NA	ND	NP	—	—	NA
QCH	0.3317	0.0118	3.6	29.87	1.27	4.3	2.99	NP	—	—	NA

[i] ND: Non-detectable; NA: Does not apply; % CV: Coefficient of variation; STD: Standard deviation; NP: Non-performed; QCL: quality control sample-low concentration; QCH: quality control sample-high concentration; N: nicotine; BA: benzoic acid

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DOI: https://doi.org/10.2478/cttr-2021-0016 | Journal eISSN: 2719-9509 (formerly 1612-9237)

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Language: English, French, German

Page range: 212 - 220

Submitted on: Mar 30, 2021

Accepted on: Oct 14, 2021

Published on: Jan 2, 2022

Published by: Beiträge zur Tabakforschung GmbH

In partnership with: Paradigm Publishing Services

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© 2022 Roberto Bravo Cardenas, Clifford Watson, Liza Valentin-Blasini, published by Beiträge zur Tabakforschung GmbH
This work is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License.

Volume 30 (2021): Issue 4 (November 2021)

Determination of Benzoic Acid and Benzyl Alcohol in E-Liquids (JUULTM Pods) by Isotopic Dilution High-Performance Liquid Chromatography and Tandem Mass Spectrometry

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