Overview - ISO 21766:2021 (LC‑MS/MS method for TSNAs)
ISO 21766:2021 specifies a standardized laboratory method for the determination and quantification of four tobacco‑specific nitrosamines (TSNAs) in tobacco and tobacco products (cigarettes, cigars and smokeless tobacco) using reversed‑phase HPLC with tandem mass spectrometry (LC‑MS/MS). The analytes covered are NNN, NAT, NAB and NNK, and results are reported in ng/g (wet mass). The method uses deuterated internal standards, aqueous extraction and LC‑ESI‑MS/MS quantification to deliver trace‑level TSNA measurements for quality control, regulatory testing and research.
Key topics and technical requirements
- Analytes: N‑nitrosonornicotine (NNN), N‑nitrosoanatabine (NAT), N‑nitrosoanabasine (NAB), 4‑(methylnitrosamino)‑1‑(3‑pyridyl)‑1‑butanone (NNK).
- Technique: Reversed‑phase HPLC coupled to tandem mass spectrometry (LC‑MS/MS) with electrospray ionization (ESI).
- Internal standards: Deuterium‑labelled analogues (NNN‑d4, NAT‑d4, NAB‑d4, NNK‑d4) for accurate quantification.
- Sample prep: Addition of internal standards, extraction with aqueous buffer (100 mM ammonium acetate), filtration (PTFE recommended) and optional SPE clean‑up (described in Annex A).
- Chromatography: C18 reversed‑phase column (example: 2.1 × 50 mm, 2.5 μm) with mobile phases of water and 0.1% acetic acid in methanol.
- Quality control: System suitability tests, calibration with multiple standards, and procedures for calculation, repeatability and reproducibility (collaborative study data included).
- Reporting: Concentrations in ng/g (wet mass) and full test‑report requirements outlined.
Applications and users
- Tobacco industry laboratories: product testing, manufacturing quality control and product formulation assessment.
- Regulatory and compliance labs: monitoring TSNA levels for regulatory limits, market surveillance and enforcement.
- Research and public health: studies on TSNA formation, exposure assessment, harm‑reduction research and toxicology.
- Third‑party testing services: independent verification and contract testing for manufacturers and regulators.
Practical benefits include standardized comparability of results across laboratories, traceable quantification using deuterated internal standards, and validated repeatability/reproducibility data from international studies.
Related standards
- CORESTA Recommended Method No. 72 - LC‑MS/MS TSNA method (basis for this ISO document).
- ISO 21766:2018 - previous edition (this second edition cancels and replaces it).
Keywords: ISO 21766:2021, tobacco‑specific nitrosamines, TSNAs, LC‑MS/MS, HPLC‑MS/MS, NNN, NNK, NAT, NAB, smokeless tobacco testing, tobacco analysis, analytical method, deuterated internal standards.
Open preview (PDF)Tobacco and tobacco products — Determination of tobacco-specific nitrosamines in tobacco products — Method using LC-MS/MS
