Introduction: The regulatory importance and health risks of primary aromatic amines
Primary aromatic amines (PAA) are organic compounds whose chemical structure contains a primary amino group (-NH₂) directly attached to one or more aromatic rings. This group of substances is of particular regulatory importance for the safety of packaging materials that come into contact with food. PAAs can arise from the breakdown of azo dyes used in packaging production or from incomplete curing reactions of polyurethane-based adhesives. The European Food Safety Authority (EFSA) and the International Agency for Research on Cancer (IARC) have classified many PAA compounds as genotoxic carcinogens. For this reason, Regulation (EU) No 10/2011 of the European Union on plastic materials strictly limits the migration of PAA from plastic materials that come into contact with food to an extremely low level. With the introduction of the PPWR, this control has been further tightened; PAA analyses have become an indispensable part of conformity assessments for packaging manufacturers. Nano-Test Laboratory Services performs PAA analyses using validated LC-MS/MS methods that fully comply with GLP principles; we offer our customers assessments of compliance with legal regulations as well as comprehensive technical consulting.Chemical structure, sources, and legal framework of PAAs
Primary aromatic amines are not a single chemical compound, but a broad family of substances that share a common structural feature. Within this family, certain compounds have been identified whose names are explicitly listed in Regulation (EU) No 10/2011 and whose migration is prohibited.Main sources of PAAs in packaging materials
The following table summarizes the reasons for the occurrence of PAAs in packaging materials, their formation mechanisms, and the types of packaging where these mechanisms are typically observed:| Mechanism of formation | Formation process | Typical packaging type / application | Explanation |
|---|---|---|---|
| Breakdown of azo dyes | Cleavage of the nitrogen-nitrogen double bond (N=N) | Printed plastic films, colored packaging, paper/cardboard packaging | Under reducing conditions (e.g., in the acidic or reducing environment of food), azo dyes can release PAAs by breaking the azo bond in their structure. |
| Incomplete curing of polyurethane adhesives | Conversion of aromatic isocyanates to PAA upon reaction with water | Multi-layer laminate packaging, flexible films, Bags | If polyurethane adhesives based on aromatic isocyanates are not fully cured, they can undergo hydrolysis when in contact with water and form PAAs (e.g., MDA – methylenedianiline). |
| Contaminations in raw materials | PAA residues that arise during the production process of monomers or additives | Polymer raw materials, masterbatches, technical additives | Some technical chemicals can contain small amounts of PAA impurities that originate from production processes. |
| Inks and coatings | Breakdown of pigments and binders used in ink recipes | Externally printed packaging, labels, coated cardboard | In particular, the migration of dyes (transfer of the print to the surface of the packaging that comes into contact with food) can lead to PAA contamination. |
Important PAA compounds subject to the regulation
Regulation (EU) No 10/2011 lists certain PAA compounds for which the migration from plastic materials that come into contact with food must not be detectable (limit: 0,01 mg/kg food or simulant). The following table shows the most important PAA compounds from this list along with their CAS numbers and typical sources:| Primary aromatic amine | Abbreviation / CAS No. | Toxicological classification | Typical source / Related azo dye |
|---|---|---|---|
| 4,4′-Methylenedianiline | MDA / 101-77-9 | Carcinogen (IARC Group 2B); Mutagen | Polyurethane adhesives; Isocyanate hydrolysis |
| Benzidine | BZD / 92-87-5 | Carcinogen (IARC Group 1); genotoxic | Azo dyes (based on benzidine); prohibited pigments |
| 2-Naphthylamin | 2-NA / 91-59-8 | Carcinogen (IARC Group 1); genotoxic | Azo dyes (based on 2-naphthylamine); prohibited pigments |
| 4-Aminobiphenyl | 4-ABP / 92-67-1 | Carcinogen (IARC Group 1); genotoxic | Azo dyes (based on 4-aminobiphenyl); prohibited pigments |
| o-Toluidine | 2-Methylaniline / 95-53-4 | Carcinogen (IARC Group 1); genotoxic | Azo dyes; printing inks |
| 4-Chloranilin | 4-CA / 106-47-8 | Suspected of carcinogenicity; Mutagen | Azo dyes; pigment impurities |
| Aniline | ANL / 62-53-3 | Carcinogen (IARC Group 2A); toxic | Azo dyes; a variety of pigments and additives |
| 2,4-Toluenediamine | 2.4-TDA / 95-80-7 | Suspected of carcinogenicity; mutagen | Polyurethane adhesives; Isocyanate hydrolysis |
PAA analysis methodology in the Nano-Test laboratory
The analysis of primary aromatic amines is an analytical procedure that requires high sensitivity and selectivity due to the low migration limits of these compounds (0.01 mg/kg). In the Nano-Test laboratory, PAA analyses are performed using LC-MS/MS methods that are optimized and fully validated for these requirements.Analytical workflow: From migration testing to quantitative determination
The following table describes the entire workflow for PAA analyses in the Nano-Test laboratory:| Step | Procedure | Technical Details |
|---|---|---|
| 1. Experimental planning | Definition of simulants and test conditions | An appropriate food simulant is selected according to Annexes III and V of Regulation (EU) No 10/2011. The simulant most frequently used for PAA analyses is Simulant B (3 % acetic acid), which represents acidic foods and best simulates the risk of reduction of azo dyes. Simulant A (10 % ethanol) and Simulant C (20 % ethanol) can also be used. |
| 2. Migration test | Controlled contact | The packaging sample is brought into contact with the selected simulant using standard contact cells or by means of the immersion method at the specified temperature and duration. The test conditions are chosen to reflect the worst-case application scenario. |
| 3. Sample preparation | Extraction and purification | The simulant extract is purified by solid-phase extraction (SPE) or liquid-liquid extraction after appropriate pH adjustment. To ensure the stability of PAA compounds, an antioxidant is added. Matrix effects are corrected by adding isotopically labeled internal standards (e.g., aniline-d5, MDA-d4). |
| 4. LC-MS/MS analysis | Quantitative Determination | After separation on a reverse-phase LC column, the purified extract is analyzed using ESI ionization in a triple quadrupole mass spectrometer. For each PAA compound, two MRM transitions are monitored. The LOQ value was validated at ≤ 0.005 mg/kg to enable a reliable assessment of the SML limit of 0.01 mg/kg. |
| 5. Evaluation and Reporting | Comparison with the SML and declaration of conformity | The quantitative results obtained are compared with the total PAA limit of 0.01 mg/kg. For compounds not detected, the result is reported as “< LOQ”. A comprehensive, GLP-compliant analysis report is generated. |
Parameters for quality control and method validation
The following table summarizes the key quality control parameters and acceptance criteria applied for PAA analyses at Nano-Test Laboratory:| Quality Control Parameters | Frequency of Application | Acceptance criteria |
|---|---|---|
| Procedure blank | At least 1 per analysis run | All target PAA compounds < LOQ |
| Spike recovery in matrix | Per run for each simulant type | 80 % – 120 % recovery |
| Recovery Rate of Internal Standard | Added to each sample | 60 % – 140 % recovery |
| Control standard (mixed PAA solution) | At least 1 per analysis run | Within ±20 % of Expected Concentration |
| Duplicate analysis | 1 per 10 samples | Relative percent deviation (RPD) ≤ 20 % |
| Accuracy (recovery) | At 3 different concentration levels during validation | Between 80 % and 120 % |
| Limit of quantification (LOQ) | Determined during validation | ≤ 0.005 mg/kg (SML limit below 0.01 mg/kg) |
The integrated service model of Nano-Test for PAA analyses
As Nano-Test Laboratory Services, we do not offer PAA analyses as standalone tests but as an integrated part of the compliance process according to PPWR and 10/2011/EU. Our service model consists of the components summarized in the following table:| Service column | Scope | Benefits for the customer |
|---|---|---|
| Consultation on regulatory requirements | Identification of PAA compounds to be analyzed based on the product composition as well as the adhesives and inks used; selection of suitable simulants and test conditions | Cost optimization through the right test strategy; avoidance of incomplete or unnecessary analyses |
| Analytical Testing Services | GLP-compliant, validated LC-MS/MS method for the simultaneous quantitative determination of more than 20 PAA compounds | Analysis data accepted by the regulatory authorities, are well-founded and traceable |
| Conformity assessment | Issuance of a declaration of conformity/non-conformity by comparing the results with the SML limit of 0.01 mg/kg total PAA, resulting in an explanation of conformity or non-conformity | A clear and binding assessment of the legal status of the product |
| Support with technical documentation | Provision of a comprehensive analysis report and a declaration of conformity (DOC) | Support for the customer in the complete and correct creation of their own declaration of conformity |
| Consulting in Case of Non-Conformity | Determination of the cause of the problem in the case of exceeding the limit value (glue, ink, pigment or raw material) and recommendations for corrective measures | Permanently eliminating the problem and avoiding recurring non-conformities |