1) Researchers developed a method to monitor personal exposure to benzyl violet 4B and direct blue 15 in workplace air.
2) The method involves sampling air through filters and extracting the chemicals using water and methanol, then analyzing by HPLC.
3) Validation tests showed good recovery (94-102% for BV and 94-99% for DB), limits of quantification of 0.250 μg for BV and 1.25 μg for DB, and reproducibility with variations of 0.6-4.1% for BV and 0.8-2.9% for DB.
4) The method enables monitoring of personal exposure to BV and DB at concentrations of 1-2,000
1) Researchers developed a method to monitor personal exposure to benzyl violet 4B and direct blue 15 in workplace air.
2) The method involves sampling air through filters and extracting the chemicals using water and methanol, then analyzing by HPLC.
3) Validation tests showed good recovery (94-102% for BV and 94-99% for DB), limits of quantification of 0.250 μg for BV and 1.25 μg for DB, and reproducibility with variations of 0.6-4.1% for BV and 0.8-2.9% for DB.
4) The method enables monitoring of personal exposure to BV and DB at concentrations of 1-2,000
1) Researchers developed a method to monitor personal exposure to benzyl violet 4B and direct blue 15 in workplace air.
2) The method involves sampling air through filters and extracting the chemicals using water and methanol, then analyzing by HPLC.
3) Validation tests showed good recovery (94-102% for BV and 94-99% for DB), limits of quantification of 0.250 μg for BV and 1.25 μg for DB, and reproducibility with variations of 0.6-4.1% for BV and 0.8-2.9% for DB.
4) The method enables monitoring of personal exposure to BV and DB at concentrations of 1-2,000
1) Researchers developed a method to monitor personal exposure to benzyl violet 4B and direct blue 15 in workplace air.
2) The method involves sampling air through filters and extracting the chemicals using water and methanol, then analyzing by HPLC.
3) Validation tests showed good recovery (94-102% for BV and 94-99% for DB), limits of quantification of 0.250 μg for BV and 1.25 μg for DB, and reproducibility with variations of 0.6-4.1% for BV and 0.8-2.9% for DB.
4) The method enables monitoring of personal exposure to BV and DB at concentrations of 1-2,000
1 Osaka Occupational Health Service Center, Japan Industrial Safety and Health Association, Japan, 2Kanto Regional Safety and Health Service Center, Japan Industrial Safety and Health Association, Japan, 3 National Institute of Occupational Safety and Health, Japan and 4Occupational Health Research and Development Center, Japan Industrial Safety and Health Association, Japan
Abstract: Objectives: The purpose of this research was performance liquid chromatography, Personal exposure to develop a method for monitoring personal exposure to monitoring benzyl violet 4B (BV) and direct blue 15 (DB) in work- place air for risk assessment. Methods: We evaluated the utility of the proposed method by examining the fol- Introduction lowing: recovery; method limit of quantification ; repro- ducibility; and storage stability of the samples. Results: Benzyl violet 4B (BV) and direct blue 15 (DB) were An air sampling cassette containing a glass fiber filter listed as target chemicals in a project on workplace risk was chosen as the sampler. BV and DB were extracted assessment carried out by the Ministry of Health, Labour from the sampler filters with a solution of water and and Welfare (MHLW) of Japan in 20151) because they methanol ( 7 : 3, v / v ) and then analyzed by a high- have been classified as Group 2B (possibly carcinogenic performance liquid chromatograph equipped with a to humans) compounds by the International Agency for photo-diode array detector. The overall recoveries from Research on Cancer2,3) and by the Japan Society for Occu- spiked samplers were 94-102% and 94-99% for BV and pational Health (JSOH) 4) . Occupational exposure limits DB, respectively. The recovery after seven days of stor- for BV and DB have not been proposed by the JSOH or age at 4℃ exceeded 95%. The method limits of quantifi- by the American Conference of Governmental Industrial cation were 0.250 and 1.25 μg/sample for BV and DB, Hygienists. respectively. The relative standard deviations, which rep- To our knowledge, a method for monitoring personal resent the overall reproducibility defined as precision, exposure to BV or DB in workplace air has not been re- were 0.6-4.1% and 0.8-2.9% for BV and DB, respec- ported. The aim of the present study was to develop and tively. Conclusions: The proposed method enables 4 h validate a personal exposure monitoring method for BV personal exposure monitoring of BV and DB at concen- and DB in workplace air with the sampling capacity and 3 3 trations of 1-2,000 μg/m for BV and 5-2,000 μg/m for sensitivity required for the MHLW exposure survey. DB, with a 240 l sampling. Thus, the proposed method is useful for estimating worker exposure to BV and DB. Materials and Methods (J Occup Health 2018; 60: 192-195) doi: 10.1539/joh.17-0190-BR Materials BV and DB were purchased from Tokyo Kasei Kogyo Key words : Benzyl violet 4 B, Direct blue 15, High- (Tokyo, Japan). Acetonitrile, methanol, ammonium dihy- drogen phosphate and disodium hydrogen phosphate were Received July 18, 2017; Accepted November 1, 2017 of analytical grade or better (for high-performance liquid Published online in J-STAGE December 19, 2017 chromatography). An air sampling cassette (Catalog No. Correspondence to : A. Takeuchi, Osaka Occupational Health Service 225-3LF, SKC Inc., Eighty Four, PA, USA) containing a Center, Japan Industrial Safety and Health Association, 2-3-8 Tosabori, glass fiber filter ( 37 mm in diameter, Catalog No. AP Nishi-ku, Osaka 550-0001, Japan (e-mail: [email protected]) 2004200, Merck Millipore Ltd., Cork, Ireland) was used Akito Takeuchi, et al.: Personal exposure monitoring method for BV and DB in air 193
as the sampler. Sampling was performed by drawing air
through the sampler using a SKC Air Check 2000 (SKC Results and Discussion Inc., Eighty Four, PA, USA). A mixture of water and methanol (7:3, v/v) was used as the extraction solution. Selection of sampler and extraction solution Mixed standard working solutions of BV and DB were We used a glass fiber filter as a sampler because it is prepared in the extraction solution. presumed that BV and DB are solids with very low vapor pressures at ambient temperature. Therefore, they exist as Instruments aerosols in the workplace air. We next explored a suitable The high-performance liquid chromatograph ( HPLC ) solvent to extract BV and DB from the glass fiber filter system used a Shimadzu ( Kyoto, Japan ) Prominence after sampling. Although BV and DB were predominantly UFLC equipped with a SPD-M 20 A photo-diode array insoluble in acetone, acetonitrile, methanol and ethanol, (PDA) detector. Separation was achieved using an Inert- they both were highly water soluble. However, the solu- Sustain C18 (150 mm × 4.6 mm I.D., 5 μm; GL Sciences tions that were dissolved in water were highly viscous Inc., Tokyo, Japan) with a flow rate of 1.0 ml / min at and contained numerous air bubbles that persisted in the 40℃. The mobile phase was composed of: (A) a 10 mM solution. Therefore, if water is used as an extraction solu- phosphate buffer containing 5 mM ammonium dihydro- tion, it may be difficult to obtain an accurate quantitative gen phosphate and 5 mM disodium hydrogen phosphate; analysis of BV and DB levels. To overcome this problem, and (B) acetonitrile. The gradient elution program was as we examined the utility of a mixture of water and metha- follows: 0-20 min, 15-85% B; and 20-30 min, 15% B. nol to be used as an extraction solution. A low viscosity The PDA acquisition wavelength was set in the range of solution lacking air bubbles was obtained by mixing 240-800 nm, and the detection wavelengths were 592 nm water and methanol at a ratio of 7:3 (v/v). Extraction effi- and 620 nm for BV and DB, respectively. ciencies obtained with this extraction solution from glass fiber filters spiked with BV and DB (each containing 500 Sample preparation μg/sample) were 100±0.9% (mean±standard deviation, n After sampling, the filter was placed in a glass test = 5) and 99±0.7%, respectively. Therefore, we utilized tube. Extraction solution (5 ml) was added and the tube this mixed solution as the extraction solution. was shaken for 5 min, followed by centrifugation at 3,000 rpm for 10 min. The supernatant was then filtered utiliz- Optimization of the HPLC analytical conditions ing a DISMIC-13 HP 020 AN ( Advantec Toyo Kaisha, The HPLC-PDA, which is commonly used in many Ltd., Tokyo, Japan). A 10 μl aliquot of the sample solu- laboratories, was used in this research because the tion was injected into the HPLC-PDA. MHLW exposure survey is conducted by several research institutes. To determine the optimum HPLC analytical Method Validation conditions for BV and DB, we evaluated several reported The proposed method was validated in accordance with HPLC analytical conditions for colorants 6-9) . We adapted MHLW guidelines 5) . A 25 μl aliquot of the mixed stan- the analytical conditions of the ISO 17234 method 7,8) be- dard solution was spiked onto the filter of a sampler. Si- cause it generated both efficient separation and enhanced multaneously, room air (temperature, 18.6-22.9℃; rela- sensitivity (Fig. 1). Under this analytical condition, no tive humidity, 29-49%) was drawn through the samplers peaks were observed on chromatograms of a solution ex- at a flow rate of 1 l/min for 240 min. tracted from a blank glass fiber filter (Fig. 1[C] and [D]). For the recovery test, the spiked amounts ranged from In contrast, several peaks were observed on chroma- 0.250 to 500 μg for BV and from 1.25 to 500 μg for DB, tograms of a solution extracted from a glass fiber filter in a sampling volume of 240 l ; these amounts corre- spiked with a BV and DB mixed standard solution (Fig. 1 sponded to air concentrations of approximately 1-2,000 [A] and [B]). These peaks were presumed to be derived μg/m3 for BV and 5-2,000 μg/m3 for DB. For storage sta- from analogs of BV and DB since many analogs are gen- bility tests, different amounts (0.250, 1.25, 25.0 and 500 erally contained in the commercially available grades of μg for BV and 1.25, 25.0 and 500 μg for DB) were spiked BV and DB. Therefore, the largest peak on the chroma- onto the filter in a sampling volume of 240 l ; these togram from each standard solution of BV and DB was amounts corresponded to air concentrations of approxi- used as the quantification peak. Absorption spectra of the mately 1, 5, 100 and 2,000 μg/m3 for BV and 5, 100 and quantification peaks of BV and DB showed maximum ab- 2,000 μg/m 3 for DB. Air was drawn through the spiked sorption at approximately 592 nm and 620 nm, respec- samplers, which were then sealed and stored at 4℃ for tively. These absorption spectra agreed with those from seven days. the standard solutions. Therefore, 592 nm and 620 nm were used as the detection wavelengths for BV and DB, respectively. 194 J Occup Health, Vol. 60, 2018
0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min
Fig. 1. 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 solu- tion extracted from a blank glass fiber filter at (C) 592 nm and (D) 620 nm.
Table 1. Recovery of BV and DB from spiked sampler Recovery of the glass fiber filter The minimum sampling capacity required for the Spiked BV DB MHLW exposure survey is 240 l (1 l/min, 240 min), and amount Recovery a RSD b Recovery a RSD b this was evaluated based on the results of the recovery (μg) (%) (%) (%) (%) test. The overall recoveries from spiked samplers were 0.250 94 ± 3.8 4.1 –±– – 94-102% and 94-99% for BV and DB, respectively (Ta- 1.25 97 ± 1.6 1.6 94 ± 2.8 2.9 ble 1). Therefore, the glass fiber filter is suitable as a sam- 5.00 100 ± 0.6 0.6 95 ± 0.8 0.9 pler for the MHLW exposure survey of BV and DB. 25.0 102 ± 0.7 0.7 99 ± 0.8 0.8 250 100 ± 0.9 0.9 94 ± 0.8 0.8 Storage stability of samples 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 A standard solution (25 μl) containing benzyl violet 4B the amounts of BV and DB in the samples analyzed im- (BV) and direct blue 15 (DB) diluted in a mixed solution mediately after preparation. After seven days storage, the of water and methanol (7:3, v/v) was spiked onto the glass fiber filter of a sampler. Simultaneously, room air was recoveries from all the spiked samplers exceeded 95%, drawn through the sampler at a flow rate of 1 l/min for 240 indicating that BV and DB on a glass fiber filter can be min. The spiked amounts correspond to air concentrations stored for at least seven days at 4℃. 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 Method limit of quantification and reproducibility not examined. The calibration curves for BV and DB exhibited linear- a Values are expressed as means ± SD (n = 5). ity in the ranges of 0.0500-100 μg/ml and 0.250-100 μg/ b Relative standard deviation ml, respectively, with correlation coefficients greater than 0.999. From the calibration curves, the instrumental limit of quantification, defined as 10 times the standard devia- tion (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 Akito Takeuchi, et al.: Personal exposure monitoring method for BV and DB in air 195
quantification, defined as the smallest amounts of BV and Japanese)
DB spiked on a sampler filter that resulted in a recovery 2) International Agency for Research on Cancer (IARC). Benzyl of more than 90%, was 0.250 μg/sample for BV and 1.25 violet 4B. IARC monographs on the evaluation of carcino- μg/sample for DB. Therefore, the measurable air concen- genic risks to humans. Overall evaluations of carcinogenicity: tration ranges for the proposed method are 1-2,000 μg/m3 An updating of IARC monographs volumes 1 to 42, Supple- for BV and 5-2,000 μg/m3 for DB, with a 4 h sample. The ment 7. Lyon (France): IARC; 1987. p. 58. relative standard deviations (RSD) of the overall repro- 3) International Agency for Research on Cancer (IARC). CI Di- ducibility of the proposed method, including sampling rect blue 15. IARC monographs on the evaluation of carcino- and analysis, were 0.6-4.1% and 0.8-2.9% for BV and genic risks to humans, Volume 57, Occupational exposures of DB, respectively (Table 1). This range of RSD values in- hairdressers and barbers and personal use of hair colourants; dicates that the proposed method has good reproducibil- some hair dyes, cosmetic colourants, industrial dyestuffs and ity. aromatic amines. Lyon (France): IARC; 1993. p. 235-245. 4) Japan Society for Occupational Health. Recommendation of Conclusions occupational exposure limits (2016-2017). Journal of Occupa- tional Health 2016; 58: 489-518. The proposed method enables 4 h personal exposure 5) Ministry of Health, Labour and Welfare ( MHLW ) , Japan. monitoring of BV and DB at concentration ranges of 1- Guidelines for exposure assessment of workers to hazardous 2,000 μg/m3 for BV and 5-2,000 μg/m3 for DB. Thus, this substances. [ Online ] . 2009 [ cited 2017 Jul. 18 ] ; Available proposed method will be useful for estimating worker ex- from: URL: http://www.mhlw.go.jp/shingi/2010/01/s0115-4.ht posure to BV and DB. To our knowledge, to date there is ml (in Japanese) no report on the personal exposure levels of workers. The 6) Takeuchi A, Jukurogi A, Kaifuku Y, et al. Determination results of the MHLW exposure survey in the future may method for p-Phenylazoaniline and 2-methyl-4- ( 2-tolylazo ) suggest the necessity for development of a new method aniline in workplace air by high-performance liquid chroma- with higher sensitivity than this proposed method. tography. Journal of Occupational Health 2013; 55(1): 43-46. 7) International Organization for Standardization (ISO). Leather - Acknowledgments : This study was commissioned by Chemical tests for the determination of certain azo colorants in the Ministry of Health, Labour and Welfare of Japan. We dyed leathers - , Part 1 : Determination of certain aromatic are deeply grateful to Prof. Kimiaki Sumino (former Di- amines derived from azo colorants (ISO 17234-1, IULTCS/ rector of the Osaka Occupational Health Service Center, IUC 20-1). Geneva (Switzerland): ISO; 2010. Japan Industrial Safety and Health Association) who of- 8) International Organization for Standardization (ISO). Leather - fered continuing support and constant encouragement. Chemical tests for the determination of certain azo colorants in Advice and comments provided by Mr. Akira Jukurogi dyed leathers -, Part 2: Determination of 4-aminoazobenzene (Technical Application Department, GL Sciences Inc., Ja- (ISO 17234-2, IULTCS/IUC 20-2) . Geneva ( Switzerland ) : pan) have been extremely helpful for this study. ISO; 2011. 9) British Standards Institution (BSI). Safety of toys - Part 11: Conflicts of interest: None declared. Organic chemical compounds - Methods of analysis (BS EN 71-11: 2005). London (UK): BSI; 2005. References 1) Ministry of Health, Labour and Welfare (MHLW), Japan. In- Journal of Occupational Health is an Open Access article distributed under vestigative commission on risk assessment of chemical sub- the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Interna- stances. [Online]. 2016[cited 2017 Jul. 18]; Available from: tional License. To view the details of this license, please visit (https://creative-
URL: http://www.mhlw.go.jp/stf/shingi2/0000130880.html (in commons.org/licenses/by-nc-sa/4.0/).
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