2018 Volume 60 Issue 2 Pages 192-195
Objectives: The purpose of this research was to develop a method for monitoring personal exposure to benzyl violet 4B (BV) and direct blue 15 (DB) in workplace air for risk assessment. Methods: We evaluated the utility of the proposed method by examining the following: recovery; method limit of quantification; reproducibility; and storage stability of the samples. Results: An air sampling cassette containing a glass fiber filter was chosen as the sampler. BV and DB were extracted from the sampler filters with a solution of water and methanol (7:3, v/v) and then analyzed by a high-performance liquid chromatograph equipped with a photo-diode array detector. The overall recoveries from spiked samplers were 94-102% and 94-99% for BV and DB, respectively. The recovery after seven days of storage at 4°C exceeded 95%. The method limits of quantification were 0.250 and 1.25 μg/sample for BV and DB, respectively. The relative standard deviations, which represent the overall reproducibility defined as precision, were 0.6-4.1% and 0.8-2.9% for BV and DB, respectively. Conclusions: The proposed method enables 4 h personal exposure monitoring of BV and DB at concentrations of 1-2,000 μg/m3 for BV and 5-2,000 μg/m3 for DB, with a 240 l sampling. Thus, the proposed method is useful for estimating worker exposure to BV and DB.
Benzyl violet 4B (BV) and direct blue 15 (DB) were listed as target chemicals in a project on workplace risk assessment carried out by the Ministry of Health, Labour and Welfare (MHLW) of Japan in 20151) because they have been classified as Group 2B (possibly carcinogenic to humans) compounds by the International Agency for Research on Cancer2,3) and by the Japan Society for Occupational Health (JSOH)4). Occupational exposure limits for BV and DB have not been proposed by the JSOH or by the American Conference of Governmental Industrial Hygienists.
To our knowledge, a method for monitoring personal exposure to BV or DB in workplace air has not been reported. The aim of the present study was to develop and validate a personal exposure monitoring method for BV and DB in workplace air with the sampling capacity and sensitivity required for the MHLW exposure survey.
BV and DB were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Acetonitrile, methanol, ammonium dihydrogen phosphate and disodium hydrogen phosphate were of analytical grade or better (for high-performance liquid chromatography). An air sampling cassette (Catalog No. 225-3LF, SKC Inc., Eighty Four, PA, USA) containing a glass fiber filter (37 mm in diameter, Catalog No. AP2004200, Merck Millipore Ltd., Cork, Ireland) was used as the sampler. Sampling was performed by drawing air through the sampler using a SKC Air Check 2000 (SKC Inc., Eighty Four, PA, USA). A mixture of water and methanol (7:3, v/v) was used as the extraction solution. Mixed standard working solutions of BV and DB were prepared in the extraction solution.
InstrumentsThe high-performance liquid chromatograph (HPLC) system used a Shimadzu (Kyoto, Japan) Prominence UFLC equipped with a SPD-M20A photo-diode array (PDA) detector. Separation was achieved using an InertSustain C18 (150 mm× 4.6 mm I.D., 5 μm; GL Sciences Inc., Tokyo, Japan) with a flow rate of 1.0 ml/min at 40°C. The mobile phase was composed of: (A) a 10 mM phosphate buffer containing 5 mM ammonium dihydrogen phosphate and 5 mM disodium hydrogen phosphate; and (B) acetonitrile. The gradient elution program was as follows: 0-20 min, 15-85% B; and 20-30 min, 15% B. The PDA acquisition wavelength was set in the range of 240-800 nm, and the detection wavelengths were 592 nm and 620 nm for BV and DB, respectively.
Sample preparationAfter sampling, the filter was placed in a glass test tube. Extraction solution (5 ml) was added and the tube was shaken for 5 min, followed by centrifugation at 3,000 rpm for 10 min. The supernatant was then filtered utilizing a DISMIC-13HP020AN (Advantec Toyo Kaisha, Ltd., Tokyo, Japan). A 10 μl aliquot of the sample solution was injected into the HPLC-PDA.
Method ValidationThe proposed method was validated in accordance with MHLW guidelines5). A 25 μl aliquot of the mixed standard solution was spiked onto the filter of a sampler. Simultaneously, room air (temperature, 18.6-22.9°C; relative humidity, 29-49%) was drawn through the samplers at a flow rate of 1 l/min for 240 min.
For the recovery test, the spiked amounts ranged from 0.250 to 500 μg for BV and from 1.25 to 500 μg for DB, in a sampling volume of 240 l; these amounts corresponded to air concentrations of approximately 1-2,000 μg/m3 for BV and 5-2,000 μg/m3 for DB. For storage stability tests, different amounts (0.250, 1.25, 25.0 and 500 μg for BV and 1.25, 25.0 and 500 μg for DB) were spiked onto the filter in a sampling volume of 240 l; these amounts corresponded to air concentrations of approximately 1, 5, 100 and 2,000 μg/m3 for BV and 5, 100 and 2,000 μg/m3 for DB. Air was drawn through the spiked samplers, which were then sealed and stored at 4°C for seven days.
We used a glass fiber filter as a sampler because it is presumed that BV and DB are solids with very low vapor pressures at ambient temperature. Therefore, they exist as aerosols in the workplace air. We next explored a suitable solvent to extract BV and DB from the glass fiber filter after sampling. Although BV and DB were predominantly insoluble in acetone, acetonitrile, methanol and ethanol, they both were highly water soluble. However, the solutions that were dissolved in water were highly viscous and contained numerous air bubbles that persisted in the solution. Therefore, if water is used as an extraction solution, it may be difficult to obtain an accurate quantitative analysis of BV and DB levels. To overcome this problem, we examined the utility of a mixture of water and methanol to be used as an extraction solution. A low viscosity solution lacking air bubbles was obtained by mixing water and methanol at a ratio of 7:3 (v/v). Extraction efficiencies obtained with this extraction solution from glass fiber filters spiked with BV and DB (each containing 500 μg/sample) were 100±0.9% (mean±standard deviation, n = 5) and 99±0.7%, respectively. Therefore, we utilized this mixed solution as the extraction solution.
Optimization of the HPLC analytical conditionsThe HPLC-PDA, which is commonly used in many laboratories, was used in this research because the MHLW exposure survey is conducted by several research institutes. To determine the optimum HPLC analytical conditions for BV and DB, we evaluated several reported HPLC analytical conditions for colorants6-9). We adapted the analytical conditions of the ISO 17234 method7,8) because it generated both efficient separation and enhanced sensitivity (Fig. 1). Under this analytical condition, no peaks were observed on chromatograms of a solution extracted from a blank glass fiber filter (Fig. 1[C] and [D]). In contrast, several peaks were observed on chromatograms of a solution extracted from a glass fiber filter spiked with a BV and DB mixed standard solution (Fig. 1[A] and [B]). These peaks were presumed to be derived from analogs of BV and DB since many analogs are generally contained in the commercially available grades of BV and DB. Therefore, the largest peak on the chromatogram from each standard solution of BV and DB was used as the quantification peak. Absorption spectra of the quantification peaks of BV and DB showed maximum absorption at approximately 592 nm and 620 nm, respectively. These absorption spectra agreed with those from the standard solutions. Therefore, 592 nm and 620 nm were used as the detection wavelengths for BV and DB, respectively.
Chromatograms of a solution extracted from a glass fiber filter spiked with a mixed standard solution containing 25.0 μg of benzyl violet 4B (BV) and 25.0 μg of direct blue 15 (DB) at (A) 592 nm and (B) 620 nm. Chromatograms of a solution extracted from a blank glass fiber filter at (C) 592 nm and (D) 620 nm.
The minimum sampling capacity required for the MHLW exposure survey is 240 l (1 l/min, 240 min), and this was evaluated based on the results of the recovery test. The overall recoveries from spiked samplers were 94-102% and 94-99% for BV and DB, respectively (Table 1). Therefore, the glass fiber filter is suitable as a sampler for the MHLW exposure survey of BV and DB.
| Spiked amount (μg) | BV | DB | |||
|---|---|---|---|---|---|
| Recovery a (%) | RSD b (%) | Recovery a (%) | RSD b (%) | ||
| A standard solution (25 μl) containing benzyl violet 4B (BV) and direct blue 15 (DB) diluted in a mixed solution of water and methanol (7:3, v/v) was spiked onto the glass fiber filter of a sampler. Simultaneously, room air was drawn through the sampler at a flow rate of 1 l/min for 240 min. The spiked amounts correspond to air concentrations of approximately 1-2000 μg/m3 for BV and 5-2000 μg/m3 for DB. The recovery obtained with 0.250 μg of DB was not examined. a Values are expressed as means ± SD (n = 5). b Relative standard deviation |
|||||
| 0.250 | 94 ± 3.8 | 4.1 | – ± – | – | |
| 1.25 | 97 ± 1.6 | 1.6 | 94 ± 2.8 | 2.9 | |
| 5.00 | 100 ± 0.6 | 0.6 | 95 ± 0.8 | 0.9 | |
| 25.0 | 102 ± 0.7 | 0.7 | 99 ± 0.8 | 0.8 | |
| 250 | 100 ± 0.9 | 0.9 | 94 ± 0.8 | 0.8 | |
| 500 | 99 ± 1.3 | 1.3 | 97 ± 1.3 | 1.4 | |
Storage stabilities were evaluated by comparing the amounts of BV and DB remaining in stored samples with the amounts of BV and DB in the samples analyzed immediately after preparation. After seven days storage, the recoveries from all the spiked samplers exceeded 95%, indicating that BV and DB on a glass fiber filter can be stored for at least seven days at 4°C.
Method limit of quantification and reproducibilityThe calibration curves for BV and DB exhibited linearity in the ranges of 0.0500-100 μg/ml and 0.250-100 μg/ml, respectively, with correlation coefficients greater than 0.999. From the calibration curves, the instrumental limit of quantification, defined as 10 times the standard deviation (n = 5) of the peak area of the lowest standard, was 0.153 μg/sample for BV and 0.798 μg/sample for DB. From the results of the recovery test, the method limit of quantification, defined as the smallest amounts of BV and DB spiked on a sampler filter that resulted in a recovery of more than 90%, was 0.250 μg/sample for BV and 1.25 μg/sample for DB. Therefore, the measurable air concentration ranges for the proposed method are 1-2,000 μg/m3 for BV and 5-2,000 μg/m3 for DB, with a 4 h sample. The relative standard deviations (RSD) of the overall reproducibility of the proposed method, including sampling and analysis, were 0.6-4.1% and 0.8-2.9% for BV and DB, respectively (Table 1). This range of RSD values indicates that the proposed method has good reproducibility.
The proposed method enables 4 h personal exposure monitoring of BV and DB at concentration ranges of 1-2,000 μg/m3 for BV and 5-2,000 μg/m3 for DB. Thus, this proposed method will be useful for estimating worker exposure to BV and DB. To our knowledge, to date there is no report on the personal exposure levels of workers. The results of the MHLW exposure survey in the future may suggest the necessity for development of a new method with higher sensitivity than this proposed method.
Acknowledgments: This study was commissioned by the Ministry of Health, Labour and Welfare of Japan. We are deeply grateful to Prof. Kimiaki Sumino (former Director of the Osaka Occupational Health Service Center, Japan Industrial Safety and Health Association) who offered continuing support and constant encouragement. Advice and comments provided by Mr. Akira Jukurogi (Technical Application Department, GL Sciences Inc., Japan) have been extremely helpful for this study.
Conflicts of interest: None declared.
