Chemical characterization of tobacco-free “modern” oral nicotine pouches and their position on the toxicant and risk continuums

Figures & data

Figure 1. An artist’s illustration of a typical nicotine pouch. (a) As sold in container with lid. (b) Individual pouch.

Table 1. Harmful and potentially harmful constituents analyzed and summary of analytical methods used for NPs, snus and NRTs.

Analyte	Method code	Brief description
Nicotine	Health Canada method: T-301^a	Nicotine was extracted from the products with alkaline methanol under ultrasonication. The liquid extract was then filtered and diluted prior to quantification by LC-MS. Quantification was performed against a 5-point standard curve and by using deuterated nicotine as internal standard.
Metals: Cadmium, chromium, nickel, arsenic, lead	EN ISO 17294-2:2016/EN 13805:2014	The products were digested in microwave oven with a mix of nitric acid, hydrochloric acid and hydrochloric peroxide, followed by detection and quantification of metals by ICP-MS.
Mercury	EN 16277:2012	Mercury was extracted by digestion, according to Annex D of EN16277:2012, with a mix of nitric acid, hydrochloric acid, hydrochloric peroxide. Detection and quantification were by CV-AFS.
TSNAs: NAB, NAT, NNK, NNN	In-house LW0A0	TSNAs were extracted from the products with ethylacetate in presence of d-labelled specific internal standards, followed by detection and quantification by HPLC-MS/MS.
Benzo(a)pyrene	In-house LW0R7	Benzo(a)pyrene extraction was performed with methanol, in presence of a d-labelled specific internal standard, followed by detection and quantification by HPLC-FLD.
NDMA	In-house LP061	NDMA was extracted with ethylacetate in presence of specific internal standard, followed by detection and quantification with HPLC-MS/MS.
Nitrite	In-house LW09I	Nitrite was extracted in water, derivatised with sulfanilamide and n-acetylethylendiaminehydrochloride and analyzed as a red complex at 540 nm.
Carbonyls: Formaldehyde, acetaldehyde, crotonaldehyde, acrolein	CORESTA CRM-86	Carbonyls were analyzed according to CORESTA CRM-86. Extraction and derivatisation occured in a two-phase system consisting of aqueous buffer and isohexane, using DNPH as the derivatising agent in the presence of specific internal standards, followed by detection and quantification on UPLC-MS/MS.
Aflatoxins: B1, B2, G1, G2	EN 14123 (mod)	Aflatoxins were extracted and transferred to a phosphate buffer saline and cleaned with monoclonal antibody affinity column. After elution from the column the aflatoxins were post-derivatised followed by detection and quantification on HPLC-FLD.
Ochratoxin A	NMKL 143	Ochratoxin was extracted with a mix of acetonitrile and water, followed by a concentration step on a preparative column based on monoclonal antibody technology. The eluate is subsequently analyzed by LC-FLD.
1,3-Butadiene and benzene	In-house HS-GC-MS	1,3-Butadiene and benzene were analyzed using headspace GC-MS.

^aDeviations to method T-301 include determination with LC-MS instead of GC-MS and addition of the internal standard during final dilution. Abbreviations: CORESTA: Cooperation Center for Scientific Research Relative to Tobacco; CRM: CORESTA recommended method; CV-AFS: cold vapor atomic fluorescence spectroscopy; DNPH: 2,4-Dinitrophenylhydrazine; FDA: US Food and Drug Administration; GC-MS: gas chromatography–mass spectrometry; HPLC-FLD: high-performance liquid chromatography with fluorescence detection; HPLC-MS/MS: HPLC with mass spectrometry; HS-GC-MS: Headspace GC-MS; ICP-MS: inductively coupled plasma MS; LC-FLD: liquid chromatography with fluorescence detection; NAB: N-nitrosoanabasine; NAT: N-nitrosoanatabine; NDMA: N-Nitrosodimethylamine; NNK: 4-(methylnitrosamino)-1–(3-pyridyl)-1-butanone; NNN: N-nitrosonornicotine; TSNA: tobacco-specific nitrosamines; UPLC-MS/MS: ultraperformance liquid chromatography–tandem MS.

Table 2. Toxicant content of NPs, snus and NRTs.

Analyte	Units	Snus			NRT lozenge	NRT gum	Nicotine Pouch
		Granit Ice Blue White	Skruf Slim Fresh XStrong Mint	G3 Slim White XStrong Blue Mint	Nicorette peppermint 4 mg	Nicorette peppermint 4 mg	Lyft Freeze	Lyft Lime Strong	Lyft Berry Frost	Lyft Mint
Product mass	g	0.7	0.7	0.7	0.623	1.265	0.7	0.7	0.7	0.7
Replicates		2	2	2	3^a	3^a	3^a	3^a	3^a	3^a
pH		8.7	7.8	8.7	8.0	10.6	8.6	8.6	8.7	8.5
Moisture	%	47.7	34.7	47.3	1.1	3.3	49.5	43.9	50.4	48.2
Nicotine	%	1.2	2.2	1.5	n/a	n/a	1.7	1.4	0.58	0.89
Formaldehyde	µg/g	1.5	1.2	<1.0	<1.0	<1.0	<1.0	<1.0–1.0^b	1.13	1.10
Acetaldehyde	µg/g	7.1	9.2	8.4	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0
Acrolein	µg/g	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05
Crotonaldehyde	µg/g	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05
NAB	ng/g	n/a	n/a	n/a	<10	<10	<10	<10	<10	<10
NAT	ng/g	n/a	n/a	n/a	<10	<10	<10	<10	<10	<10
NNN	ng/g	610	560	640	<10	<10	<10	<10	<10	<10
NNK	ng/g	200	125	89	<10	<10	<10	<10	<10	<10
NDMA	ng/g	<0.2	0.23	0.37	<0.2	<0.2	<0.2	<0.2	<0.2	<0.2
Benzo(a)pyrene	ng/g	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0
1,3-Butadiene	ng/g	<20	<20	<20	<20	<20	<20	<20	<20	<20
Benzene	ng/g	<5.0	<5.0	<5.0	<5.0	<5.0	<5.0	<5.0	<5.0	<5.0
Cadmium	ng/g	285	460	245	<10	29	<10	<10	<10	<10
Chromium	ng/g	1550	1700	815	<50–52^c	743	<50–70^c	<50	<50–80^c	<50-51^c
Mercury	ng/g	<20	<20	<20	<20	<20	<20	<20	<20	<20
Nickel	ng/g	1400	2000	885	80	223	<50	<50	<50	<50
Arsenic	ng/g	92	130	94	<50	<50	<50	<50	<50	<50
Lead	ng/g	210	340	190	<20	55	<20	<20	<20	<20
Nitrite	µg/g	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0	<1.0
Aflatoxin B1	ng/g	<1.0	<1.0	<1.0	<0.1	<1.0	<1.0	<1.0	<1.0	<1.0
Aflatoxin B2	ng/g	<1.0	<1.0	<1.0	<0.1	<1.0	<1.0	<1.0	<1.0	<1.0
Aflatoxin G1	ng/g	<1.0	<1.0	<1.0	<0.1	<1.0	<1.0	<1.0	<1.0	<1.0
Aflatoxin G2	ng/g	<1.0	<1.0	<1.0	<0.1	<1.0	<1.0	<1.0	<1.0	<1.0
Ochratoxin A	ng/g	<0.5	1.6	1.1	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5

^aAflatoxins were measured with two replicates; ^btwo replicates were ≥ LOQ, ^cone replicate was ≥ LOQ. Abbreviations: n/a: not analyzed; NAB: N-nitrosoanabasine; NAT: N-nitrosoanatabine; NDMA: N-Nitrosodimethylamine; NNK: 4-(methylnitrosamino)-1–(3-pyridyl)-1-butanone; NNN: N-nitrosonornicotine; NRT: nicotine replacement therapy.

Table 3. Average daily consumption of traditional Swedish snus and NPs.

Survey	Traditional snus		Nicotine pouches
	ADC (pouches/day)	Sample size (n)	ADC (pouches/day)	Sample size (n)
Q1 2018	12.2	1345	8.7	39
Q2 2018	12.1	1261	8.2	20
Q3 2018	11.6	1242	9.0	36
Q4 2018	11.6	1244	8.4	43
Q1 2019	11.7	1202	8.8	35
Q2 2019	11.8	1197	7.7	62
Q3 2019	12.1	1194	10.4	68
Q4 2019	11.9	1192	8.4	99
Q1 2020	11.7	1102	7.8	190
Q2 2020	12.3	1104	8.0	196
Q3 2020	12.4	1092	9.5	228
Q4 2020	12.3	1123	8.6	238
Mean	12.0	1192	8.6	105

Abbreviations: ADC: average daily consumption.

Data availability statement

All data and materials are available upon request.