Abstract
Objectives: The purpose of this research was to develop a method for the simultaneous determination of p-phenylazoaniline (also called 4-aminoazobenzene, AAB) and 2-methyl-4-(2-tolylazo)aniline (also called o-aminoazotoluene, AAT) in workplace air for risk assessment. Methods: The characteristics of the proposed method, such as recovery, limit of quantitation, reproducibility and storage stability of the samples were examined. Results: An air sampling cassette containing two sulfuric acid-treated glass fiber filters was chosen as the sampler. The AAB and AAT were extracted from the sampler filters by methanol and then analyzed by a high-performance liquid chromatograph equipped with a photo-diode array detector. The overall recoveries from spiked samplers were 77–98 and 85–98% for AAB and AAT, respectively. The recovery after 5 days of storage in a refrigerator exceeded 96%. The overall limits of quantitation were 5.00 and 2.50 μg/sample for AAB and AAT, respectively. The relative standard deviations, which represent the overall reproducibility defined as precision, were 0.6–1.8 and 0.5–2.2% for AAB and AAT, respectively. Conclusions: The proposed method enables 4-h personal exposure monitoring of AAB and AAT at concentrations of 21 to 2,000 μg/m3 for AAB and 10 to 2,000 μg/m3 for AAT, respectively. The proposed method is useful for estimating worker exposure to AAB and AAT.
Introduction
p-Phenylazoaniline (also called 4-aminoazobenzene, AAB) and 2-methyl-4-(2-tolylazo)aniline (also called o-aminoazotoluene, AAT) were listed as the target chemicals in a project on workplace risk assessment carried out by the Ministry of Health, Labour and Welfare (MHLW) of Japan in 20111) because they have been classified as Group 2B (possibly carcinogenic to humans) compounds by the International Agency for Research on Cancer (IARC)2) and by the Japan Society for Occupational Health (JOH)3). Occupational exposure limits for AAB and AAT have not been proposed by the American Conference of Governmental Industrial Hygienists (ACGIH) or by the JOH.
To our knowledge, a determination method for AAB or AAT in workplace air has not been reported. In this paper, we report on the development and validation of a high-performance liquid chromatograph (HPLC) method for the simultaneous determination of AAB and AAT in workplace air.
Materials and Methods
Materials
AAB and AAT were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Acetonitrile, formic acid, ammonium acetate, methanol and sulfuric acid were of analytical grade or better (for high-performance liquid chromatography or super special grade). An air sampling cassette (Catalog No. 225-3LF, SKC Inc., Eighty Four, PA, USA) containing two sulfuric acid-treated glass fiber filters (Catalog No. 303, GASTEC Corporation, Kanagawa, Japan) was used as the sampler. Sampling was performed by drawing air through the sampler using an SKC Air Check 2000 (SKC Inc., Eighty Four, PA, USA). For calibration curves, working mixed standard solutions of AAB and AAT were prepared in methanol.
Instruments
The HPLC system used a Shimadzu (Kyoto, Japan) Prominence UFLC equipped with a SPD-M20A photo-diode array (PDA) detector. Separation was achieved using a InertSustain C18 HP (100 mm × 3.0 mm I.D., 3 μm; GL Sciences Inc., Tokyo, Japan) at a flow rate of 1.0 ml/min and at 40°C. The mobile phase was composed of (A) 10 mM ammonium acetate in 0.1% formic acid and (B) acetonitrile. The gradient elution program was as follows: 0.00–5.50 minutes, 20–90% B; 5.50–7.00 minutes, 90% B; 7.00–7.01 minutes, 90–20% B; and 7.01–10.00 minutes, 20% B. The PDA detector was set to scan from 240 to 800 nm, and 385 nm was used as the detection wavelength.
Sample preparation
After sampling, the front and back sample filters were placed in separate glass test tubes. Methanol (5 ml) was added to each tube, the tubes were shaken for 5 minutes, and they were then centrifuged at 3,000 rpm for 10 minutes. An aliquot of 4 μl of the samples was injected into the HPLC.
Preparation of spiked samplers for retention efficiency and storage stability tests
Mixed standard solutions of AAB and AAT were dissolved in methanol and then diluted with 0.125 M H2SO4 in methanol solution. A 25 μl of mixed standard solution was spiked onto the front filter of a sampler. Simultaneously, room air (temperature, 21–26°C; relative humidity, 22–32%) was drawn through the samplers at a flow rate of 1 l/min for 240 minutes.
For the retention efficiency test, the spiked amounts were varied from 2.5 to 500 μg for each of AAB and AAT for a sampling volume of 240 l; these correspond to air concentrations of approximately 10–2,000 μg/m3. For storage stability tests, three different amounts (5.00, 25.0 and 250 μg for each of AAB and AAT) were spiked onto the filter for a sampling volume of 240 l; these correspond to air concentrations of approximately 20, 100 and 1,000 μg/m3. Air was drawn through the spiked samplers, and the samplers were then sealed and stored in a refrigerator (4°C) for 5 days.
Results and Discussion
Choice of sampler and optimization of the HPLC analytical conditions
We adopted a sulfuric acid-treated glass fiber filter as a sampler for two reasons. One reason is that it is presumed that AAB and AAT exist as aerosol in the workplace air considering their physical and chemical properties. The other reason is that AAB and AAT are transformed to the corresponding stable sulfate salts on this filter; this was supported by our preliminary experimental results showing that other (a non-treated glass fiber filter and a solid-phase extraction disk) showed low recoveries.
Several HPLC analytical methods have been reported for the determination of AAB and AAT in products (such as leather and toys)4–6). We adopted the method of BS EN 71-116) because the HPLC analytical condition of this method could separate 16 colorants (including AAB and AAT) that could potentially coexist in the workplace. However, this method requires a run time of 45 minutes. Therefore, we modified it to shorten the analysis time. The HPLC analytical conditions (the choice of column, the composition of the mobile phase, the gradient elution program and the detection wavelength) were optimized to ensure a satisfactory separation for fast analysis. Absorption spectra of AAB and AAT showed maximum absorption at around 385 nm. Therefore, the detection wavelength was set at 385 nm. Although overlap of the peaks of AAT and Disperse Blue 124 was observed in the max plot [Fig. 1 (A)], this interference was not observed at 385 nm [Fig. 1 (B)]. Therefore, at least these colorants will not interfere with the determination of AAB and AAT. The run time of the proposed method was reduced to less than a quarter of that of the original method [Fig. 1 (C)].
The sampling capacity required for the MHLW exposure survey was 240 l (1 l/min, 240 minutes), and this was evaluated based on the results of the recovery test. No AAB or AAT was detected on the back filter of any of the spiked samplers. Therefore, the acid-treated filter is suitable as a sampler for the MHLW exposure survey of AAB and AAT. The overall recoveries from spiked samplers were 77–98 and 85–98% for AAB and AAT, respectively (Table 1). We consider these results to be satisfactory.
Table 1.
Recovery from spiked sampler
Spiked
amount
(μg) |
AAB |
AAT |
Recoverya
(%) |
RSDb
(%) |
Recoverya
(%) |
RSDb
(%) |
| 2.50 |
77 ± 1.4 |
1.8 |
85 ± 1.7 |
2.0 |
| 5.00 |
82 ± 0.5 |
0.6 |
87 ± 2.0 |
2.2 |
| 12.5 |
88 ± 1.0 |
1.2 |
92 ± 0.9 |
1.0 |
| 25.0 |
97 ± 0.7 |
0.7 |
98 ± 0.6 |
0.6 |
| 250 |
96 ± 0.5 |
0.6 |
96 ± 0.5 |
0.5 |
| 500 |
98 ± 0.6 |
0.6 |
98 ± 0.7 |
0.7 |
A 25 μl of p-phenylazoaniline (also called 4-aminoazobenzene, AAB) and 2-methyl-4-(2-tolylazo)aniline (also called o-aminoazotoluene, AAT) mixed standard solution diluted with 0.125 M H2SO4 in methanol solution was spiked onto the front filter of a sampler. Simultaneously, room air was drawn through the sampler at a flow rate of 1 l/min for 240 minutes. The spiked amounts correspond to air concentrations of approximately 10–2,000 μg/m3.
a Values are expressed as means ± SD (n=5).
b Relative standard deviation.
Storage stabilities were evaluated by comparing the amounts of AAB and AAT remaining in storage samples with the amounts of AAB and AAT in the samples analyzed immediately after preparation. After 5 days of storage, the recoveries from all the spiked samplers exceeded 96%, indicating that AAB and AAT on an acid-treated filter can be stored for at least 5 days in a refrigerator.
Limit of quantitation and reproducibility
The calibration curves for AAB and AAT exhibited linearity in the ranges of 0.0500–100 μg/ml, respectively, with correlation coefficients of above 0.999. From the calibration curves, the instrumental limit of quantitation (LOQ), defined as 10 times the standard deviation (n=5) of the peak area of the lowest standard, was 0.166 μg/sample for AAB and 0.251 μg/sample for AAT. From the results of the recovery test, the overall LOQs were found to be 5.00 and 2.50 μg/sample for AAB and AAT, respectively. These values were the smallest amounts of AAB and AAT spiked on a sampler filter that resulted in a recovery of more than 80%. Therefore, the measurable air concentration ranges for the proposed method are 21 to 2,000 μg/m3 for AAB and 10 to 2,000 μg/m3 for AAT, respectively, with a 4-h sample. The relative standard deviations (RSD) of the overall reproducibility of the proposed method, including sampling and analysis, were 0.6–1.8 and 0.5–2.2% for AAB and AAT, respectively (Table 1). This range of RSD values indicates that the proposed method has good reproducibility.
Conclusions
The proposed method enables 4-h personal exposure monitoring of AAB and AAT at concentrations of 21 to 2,000 μg/m3 for AAB and 10 to 2,000 μg/m3 for AAT, respectively, and will be useful for estimating worker exposures to AAB and AAT.
Acknowledgments
This study was commissioned by the Ministry of Health, Labour and Welfare of Japan.
References
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- 4) International Organization for Standardization (ISO). Leather–Chemical tests for the determination of certain azo colorants in dyed leathers–, Part 1: Determination of certain aromatic amines derived from azo colorants (ISO 17234-1, IULTCS/IUC 20-1). Geneva (Switzerland): ISO; 2010.
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