Rapid determination of 33 primary aromatic amines in kitchen utensils, textiles dyes and food packaging materials
Xiaojie Sun1，Haiyan Cheng1, Lijun Li1，Jianru Stahl-Zeng2, Ashley Sage3, and Wenhai Jin1
1SCIEX, China；2SCIEX, Germany; 3SCIEX, UK
Introduction
Primary Aromatic Amines (PAAs) are a class of compound of which the simplest form is aniline. PAAs are substances that are used, for example, in the production of certain colorants, so-called azo pigments, notably in the color range yellow-orange-red. Whereas a large number of PAAs are safe for human health, some PAAs are known human carcinogens. For kitchenware, paper napkins, baker’s bags with colorful print and other printed items that come in contact with food, some PAAs may pose a health risk, if they are transferred to the food. PAAs are small, basic compounds, which are ionized with low PH. As a result of their basic properties and the 3% acetic acidic sample solvent, some PAAs don’t focus well on the head of the column, resulting in poor peak shape or loss of retention. 33 PAAs including 4 groups isomer which increase the difficulty of separation.
Because of the potential health risk, specific migration limits are put in place. According to the regulation on plastics EU/2011, plastic materials and articles shall not release PAAs in a detectable quantity into food or food simulant. The LOD of 0.01 mg of substance per kg of food or food simulant is applied to the sum of PAAs released. Recently, the Federal Institute for Risk Assessment in Germany released an opinion and suggested that the limit value of not detectable with an LOD of 0.01 mg kg-1 food for PAAs indicated in Annex Ⅱ of Regulation (EU) No 10/2011 should be applied to PAAs in kitchen utensils, textiles dyes and food packaging.
Here a targeted method has been established for the selective and sensitive quantitation of 33 primary aromatic amines.
Figure 1. Extraction ion chromatograms of 33 primary aromatic amines. (Top) Separation of all 33 PAAs on the Kinetex® F5 column. (Bottom) Separation of the 4 isomers.
Key advantage of the targeted PAA method
- Good chromatography was obtained using the Kinetex F5 column (Phenomenex) with good retention and peak shape, including the resolution of 4 isomers (Figure 1)
- Fast separation method of 12 mins
- Very good recoveries were obtained for the method, between 82.1% to 105.7% with the RSD% (n=6) of all PAAs were less than 9%
- Matrix effects were largely eliminated using two simulant solvents. There were almost no matrix effects for all PAAs when using the simulant C of 20% ethanol, but then over 50% matrix effects were observed for PAA 30 and PAA 32, and there were no matrix effects for the other compounds when using the simulant B of 3% acetic acidic. This may because of the formation of the ionic state of the two compounds with acetic acidic, which could cause the ionization efficiency becomes worse.
Methods
Sample preparation: All samples were extracted with a 3% acetic acid solution and 20% ethanol solution according to the procedure described in the EU 10/2011 guidelines. Briefly, samples were cut into pieces approximately 1-2 cm2 in size (Table 5). A total of 10 g of samples were weighted into a 200 mL conical flask. 20% ethanol was used as simulated solvent in the food packaging migration experiment, which was immersed at 40°C for 7 days. 3% acetic acid was used as simulated solvent in the kitchen utensils, textiles dyes migration experiment, which was immersed at 100°C for 2 hours.
Chromatography: Compound separation was performed using the ExionLC™ AD System with a Phenomenex Kinetex F5 column (2.6 μm F5 100 Å, 100 X 3.0 mm). The flow rate was 0.3 mL/min, column temperature was 40 ºC and the injection volume was 5 μL. Gradient conditions are shown in Table 1.
Table 1. LC separation gradient.
Mass Spectrometry: Analysis was performed using a SCIEX Triple Quad™ 5500 System with the Turbo V™ Source operating in positive ion mode, source conditions are shown in Table 2. At least two MRM transitions were optimized for each analyte as outlined in Supplementary infomration,2 final data was collected using Scheduled MRM™ Algorithm using Analyst® Software 1.6.3.
Data Processing: All data was processed using MultiQuant™ Software 3.0.2.
Table 2. Source parameters for the SCIEX Triple Quad 5500 System.
Figure 2. Extracted 25 PAAs excluding 4 groups of isomers.
Method optimization
During verification of the method, the primary focus was on achieving stable peak shapes and retention times for all analytes. Initial conditions, gradient and pH of the mobile phase had very significant effects, so the final optimized method should be fixed, and fresh mobile phases prepared regularly. The content of formic acid, the content of initial methanol and the selection of the column was optimized to improve the sensitivity and the separation for all PAAs. Compared with 0.0% formic acid, 0.01% formic acid, 0.05% formic acid, 0.1% formic acid, and 0.2% formic acid in water, the best sensitivity may be observed when added 0.05% formic acid.
Also, the initial percent of methanol was optimized for separation and sensitivity. A higher initial percent of methanol provided the best of sensitivity for all PAAs, but the worse of separation for isomers. Initial percent of methanol of 30% provided the best compromise between sensitivity and separation (Figure 1, bottom).
Multiple columns were compared for performance including the Phenomenex Omega, Phenomenex F5, Waters HSS T3. The Kinetex F5 column provided the best separation results (Figure 1, top).
Figure 2 shows the individual chromatograms of 25 PAAs.
Source parameters were optimized for the best sensitivity. During the optimization, it was found that the IonSpray Voltage optimized at 1200 V for the best sensitivity of all PAAs (Figure 3).
Figure 3. Optimization of ionization voltage. The ISV setting was tuned for each compound and 1200 V was selected for the method.
Recovery analysis
For the determination of the recovery, samples which had previously been determined to not contain any PAAs were used at matrix. They were spiked with three levels of a solution containing a mix of PAAs ( 0.1, 5, 20 ng/mL) in duplicate and the spiked samples were extracted and analysed as described above. The recovery was calculated for each PAA from the difference of results for the spiked samples and the simulants. Under the concentrations tested, the recoveries of the method were from 82.1% to 105.7% (Table 4). Only 2 PAA showed recoveries less than 80%.
Analytical performance
Repeatability of the analysis was calculated on injections of 3% acetic acid and 20% ethanol solutions spiked with 0.1, 5, 20 ng/mL of each PAA and it ranged from 1.9% to 8.9%. Within-laboratory reproducibility (intermediate precision) was also calculated by repeating the procedure described for repeatability at three different times and it ranged from 2.3% to 9.1%.
The linearity of instrument response evaluated in a concentration range between 0.05 and 50 ng/mL showed very good regression coefficients for all the PAAs (Table 3). LODs were in the range of 0.001-0.029 ng/mL for 33 PAAs. LODs were lower than the limit of total PAA of 10 ng/mL in the EU plastic FCM Regulation (EU) No 10/2011.
Sample analysis
A set of samples were obtained from the market and prepared as specified above. Using the established method and the external calibration curves created, the PAA levels in the samples were measured (Table 4).
Some PAAs are detected in some of the samples, but total concentrations of PAAs in all samples were lower than 10 ng/mL, not exceeding the limit of total PAA in the EU plastic FCM Regulation (EU) No 10/2011. Figure 4 shows the chromatograms of some detected PAAs.
Regulation (EU) No 10/2011. Figure 4 shows the chromatograms of some detected PAAs.
Table 3. Main Analytical Parameters. a
Figure 4. XICs for PAAs detected in some of the food contact materials tested.
Table 4. Some detected PAAs in samples. Samples were purchased from the market and consisted of kitchen utensils (5 plastic spoons, 5 plastic forks, 2 paper cups), textile dyes (5 colored paper napkins) and food packaging materials (5 plastic cookie packages).
Conclusions
This study developed a multi-analyte method based on UHPLC-MS/MS quantification for the analysis of PAAs content in kitchen utensils, textiles dyes and food packaging from the market. A sensitive method for 33 PAAs has been established with very easy sample preparation. Linearity was observed over a large dynamic range and up to 50 ng/mL. The established lower limits of detections of the method were all below the levels required by the regulations. The total PAAs content for all tested samples was below the 0.01 mg/kg as stipulated in the regulations EU 10/2011.
References
- Commission regulation (EU) No 10/2011 of 14 January 2011 on plastic materials and articles intended to come into contact with food. Official Journal of the European Union L 12, 15.1.2011.
- Supplementary method information.