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Table 2 Analytes, method code, and description of analytical methods

From: Harmful and potentially harmful constituents (HPHCs) in two novel nicotine pouch products in comparison with regular smokeless tobacco products and pharmaceutical nicotine replacement therapy products (NRTs)

Analyte

Method code

Description

Arsenic

Cadmium

Chromium

Lead

Nickel

Selenium

Beryllium

Eurofins: EN ISO 17294–2:2016/EN 13805:2014

Digestion was performed in a microwave oven with a mix of nitric acid, hydrochloric acid, and hydrogen peroxide, followed by detection and quantification on an inductively coupled plasma mass spectrometry (ICP-MS)

Mercury

Eurofins: EN 16277:2012

Digestion was performed according to Annex D of EN16277:2012 with a mix of nitric acid, hydrochloric acid and hydrogen peroxide, followed by detection and quantification using cold vapor atomic fluorescence spectroscopy

Uranium-235

Uranium-238

Swedish Match: FDA method: “CFSAN/ORS/DBC/CHCB April 25, 2011” (ICP-MS technique) (Modified)

The metals were released from a matrix through microwave digestion using Milli-Q water, nitric acid with a concentration of 67–69%, and hydrogen peroxide. The obtained solution was analyzed using an ICP-MS

Polonium-210

Labstat/Maxxam

Detected using alpha emission spectrometry

NAB (N-Nitrosoanabasine)

NAT (N-Nitrosoanabatine)

NNK (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone)

NNN (N-Nitrosonornicotine)

Eurofins: In-house LW0A0

Extracted with ethyl acetate in presence of d-labelled specific internal standards, followed by detection and quantification with high-performance liquid chromatography using a C18, 3 µm column and a tandem mass-spectrometry (HPLC–MS/MS), positive polarity

NDMA (N-Nitrosodimethylamine)

Eurofins: In-house LP061

Extracted with ethyl acetate in the presence of a specific internal standard, followed by detection and quantification with HPLC–MS/MS, positive polarity. An HSS T3, 1.8 µm APCI column was used

NDELA (N-Nitrosodiethanolamine)

Swedish Match: In-house, based on [30, 31]

Extracted with Milli-Q water with the addition of an internal standard (NDELA-d8). The water extract was cleaned up using two different SPE-columns following separation and quantification using ultra performance liquid chromatography-MS/MS (UPLC-MS/MS)

NMOR (N-Nitrosomorpholine)

NPIP (N-Nitrosopiperidine)

NPYR (N-Nitrosopyrrolidine)

NSAR (N-Nitrososarcosine)

Swedish Match: In-house, based on [32]

After addition of the internal standards (NSAR-d3, NMOR-d4, NPYR-d4, and NPIP-d4) the nitrosamines were extracted using 2% formic acid in acetonitrile. The extracts were diluted with 2% formic acid in water and filtered. Separation and quantification were performed using UPLC-MS/MS

Benz[a]antracene

Benzo[b,k]fluoranthene

Chrysene

Dibenz[a,h]anthracene

Indeno[1,2,3-cd]pyrene

Naphthalene

Eurofins: In-house SLF92

Extracted with acetone in presence of specific d-labelled internal standards. The extracts were transferred to a hexane solution, followed by detection and quantification with gas chromatography-mass spectrometry, GC–MS. A J&W DB-5 ms GC column 0.18 µm was used

B(a)P (Benzo[a]pyrene)

Eurofins: In-house LW0R7

Extraction was performed with methanol in presence of a d-labelled specific internal standard, followed by detection and quantification with HPLC-FLD

Nitrite

Eurofins: In-house LW 091

Extracted in Milli-Q water derivatized with sulfanilamide and naphtylethylendiamine hydrochloride and analyzed as a red complex at 540 nm

Acetaldehyde

Crotonaldehyde

Formaldehyde

Eurofins: CORESTA recommended method No. 86 [33]

Extraction and derivatization were performed in the presence of specific internal standards in a two-phase-system consisting of an aqueous buffer and isohexane using DNPH as a derivatization agent, followed by detection and quantification on UPLC-MS/MS

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

Eurofins: EN 14123 (mod)

Extracted using acetonitrile/methanol/water and transferred to a phosphatic buffer saline and cleaned using a monoclonal antibody affinity column. After elution from the column the aflatoxins were post-derivatized followed by detection and quantification using high performance liquid chromatography with fluorescence detection (HPLC-FLD)

Ochratoxin A

Eurofins: NMKL 143

Extracted with a mix of acetonitrile and water, followed by a concentration step on a preparative column based on monoclonal antibody technology. The eluate was subsequently analyzed by liquid chromatography with fluorescence detection

Coumarin

Eurofins: In-house method

Extracted in 50% ethanol in presence of a specific internal standard, followed by detection and quantification on UPLC-MS/MS, positive polarity. A BEH, 1.7 µm column was used

Ethyl carbamate

Eurofins: In-house method

Extracted with Milli-Q water in presence of a specific internal standard, followed by detection and quantification by UPLC-MS/MS, positive polarity. A HSS T3, 1.8 µm column was used

Ammonia

Eurofins: In-house LW0A3

Extracted with Milli-Q water and mixed with salicylate and dichlor-isocyanate in presence of sodium nitroprusside to form a blue complex detectable at 660 nm

Nicotine

Eurofins: Health Canada Official method T301 (Modified)

Extracted with alkaline methanol in presence of a specific d-labelled internal standard

Anabasine

Nornicotine

Swedish Match: CORESTA recommended method No. 62 [34] (Modified)

Mixed with sodium hydroxide and extracted using an extraction solution with methyl tert-butyl ether and quinoline as an internal standard. Separation and quantitation were performed using a gas chromatograph fitted with a capillary column and flame ionization detector

pH

Eurofins: CORESTA recommended method No. 69 [35]

Diluted with milli-q water 1:10, stirred for 5 min and measured with pH-meter

Unprotonated nicotine

Swedish Match

Calculated according to [36]