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An efficient analytical method for determination of S-phenylmercapturic acid in urine by HPLC fluorimetric detector to assessing benzene exposure
Journal of Chromatography B 1063 (2017) 136–140
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
An efficient analytical method for determination of S-phenylmercapturic acid in urine by HPLC fluorimetric detector to assessing benzene exposure
MARK
⁎
Michele P. Rocha Mendes , Josianne Nicácio Silveira, Leiliane Coelho Andre Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Brazil
A R T I C L E I N F O
A B S T R A C T
Key-words: Analytical validation Benzene S-phenylmercapturic acid
Benzene is an important occupational and environmental contaminant, naturally present in petroleum and as byproduct in the steel industry. Toxicological studies showed pronounced myelotoxic action, causing leukemic and others blood cells disorders. Assessing of benzene exposure is performed by biomarkers as trans, trans-muconic acid (AttM) and S-phenylmercapturic acid (S-PMA) in urine. Due to specificity of S-PMA, this biomarker has been proposed to asses lower levels of benzene in air. The aim of this study was to validate an analytical method for the quantification of S-PMA by High-Performance Liquid Chromatography with fluorometric detector. The development of an analytical method of S-PMA in urine was carried out by solid phase extraction (SPE) using C-18 phase. The eluated were submitted to water bath at 75 °C and nitrogen to analyte concentration, followed by alkaline hydrolysis and derivatization with monobromobimane. The chromatography conditions were reverse phase C-18 column (240 mm, 4 mm and 5 μm) at 35 °C; acetonitrile and 0.5% acetic acid (50:50) as mobile phase with a flow of 0.8 mL/min. The limits of detection and quantification were 0.22 μg/L and 0.68 μg/L, respectively. The linearity was verified by simple linear regression, and the method exhibited good linearity in the range of 10–100 μg/L. There was no matrix effect for S-PMA using concentrations of 40, 60, 80 and 100 μg/L. The intra- and interassay precision showed coefficient of variation of less than 10% and the recovery ranged from 83.4 to 102.8% with an average of 94.4%. The stability of S-PMA in urine stored at −20 °C was of seven weeks. The conclusion is that this method presents satisfactory results per their figures of merit. This proposed method for determining urinary S-PMA showed adequate sensitivity for assessment of occupational and environmental exposure to benzene using S-PMA as biomarker of exposure.
1. Introduction Benzene is an aromatic hydrocarbon, naturally present in the oil, in the steel industry as by-product, in the gasoline, in the cigarette and laboratory chemical synthesis. The International Agency for Research on Cancer (IARC) classifies benzene as carcinogenic to humans due to its pronounced myelotoxic action which is a highly causative agent of leukaemia disorders, lymphoproliferative, and quantitative disorders of blood cells; even at concentrations below 1 ppm [1–3]. Benzene is considered a ubiquitous chemical agent, and human exposure to this may occur by occupational or environmental sources. For this reason, the assessment of exposure to predict the risk is extremely important, whether in the context of occupational and environmental toxicology. Benzene research and/or its metabolites in inhaled air or body fluids such as urine make its exposure assessment prevailing important. Various benzene bio-transformation products have been proposed as exposure biomarkers, including – hydroquinone, phenol, catechol, and trans acid, trans-muconic (AttM) [4,5]. However, these acids did not ⁎
Corresponding author. E-mail address: [email protected] (M.P.R. Mendes).
http://dx.doi.org/10.1016/j.jchromb.2017.07.039 Received 18 September 2016; Received in revised form 17 July 2017; Accepted 21 July 2017 Available online 18 August 2017 1570-0232/ © 2017 Elsevier B.V. All rights reserved.
have the specificity and sensitivity required for being used as biomonitoring exposure to this chemical agent. The trans acid, trans urinary-muconic, despite being the biomarker adopted by Brazilian law, has limitations in its use since the excretion of this bio-transformation product is influenced by sorbic acid – a food additive, and by smoking habit since cigarette-smoke is a source of benzene [6–8]. As a result, other metabolites have been proposed, such as − Sphenylmercapturic acid (S-PMA) and benzene unchanged in urine and inhaled air [9–11]. S-PMA has high specificity, since exposure to benzene is the only authoritative source; moreover, this indicator the amount excreted in the urine is related to the concentration of benzene present in the air, especially at low concentrations [11,12]. So this has been employed especially for monitoring exposure to low concentrations of benzene in the air [10,13,14]. However, it is known that less than 1% of absorbed benzene is eliminated in the urine as S-phenylmercapturic acid (S-PMA) [15]. Thus, the determination of this metabolite in the urine requires sensitive analytical techniques, able to identify and quantify a biomarker to
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with 290 μL of buffer solution prepared from 90 μL of 5 mol/L phosphoric acid and 200 μL of 0.5 mol/L ammonium bicarbonate. After homogenization they were added 50 μL of derivatizing reagent – monobromobimane (MB) 2 mmol/L in acetonitrile and 100 μL of derivatized was injected into the chromatographic system.
a level capable to be traced. The biological exposure limit recommended by ACGIH [16] is 25 μg/g creatinine, which correspond to 0.5 ppm of benzene in the air. The most commonly used analytical technique for the determination of S-PMA is liquid chromatography high performance (HPLC), this technique associated with mass spectrometry (MS) analytical has shown great efficiency, regarding the high sensitivity of this technique. Several studies on the bio-monitoring of exposure to benzene have used HPLC/MS to quantify urinary S-PMA [13,16,18,19,14,20]. Despite all the advantages offered by this technique, it is expensive and is more susceptible to the matrix effect. Some studies have been reported a good analytical performance using the HPLC technique with fluorimetric detection [21–23]. The good analytical results set the HPLC technique with attractive fluorimetric detector for determining the S-PMA, in view of the analytical sensitivity and lower cost when compared to HPLC/MS. In this sense, the present study aimed to standardization and validation of analytical methodology for quantification of S-PMA in urine by high-performance liquid chromatography (HPLC) and fluorimetric detection and evaluation of this method in biomonitoring of occupational exposure to benzene.
2.3. Equipment The high performance liquid chromatograph LC 1100 series equipped with isocratic pump, column oven, autosampler, fluorimetric detector 1200 and ChemStation for integration and processing of chromatograms − Agilent Technologies® was used. The chromatographic column used was Lichrocarth Hypersil ODS C18 column (250 mm × 4.0 mm × 5 μm, Merck ®) at 35 °C. The mobile phase used was acetonitrile-0.5% acetic acid (50:50 v/v) with a flow of 0.80 mL per minute. The wavelengths used for fluorimetric detector were 395 and 470 nm for excitation and emission respectively. 2.4. Quantification Pool urine samples spiked with standard S-PMA concentrations of 10, 20, 40, 60, 80 and 100 μg/L were used to construct calibration curves, where these solutions were prepared according to the procedure described in (2.2). To demonstrate the reliability of the method were determined by the following figures of merit − linearity, precision, recovery and matrix effect using standard samples of urine pool S-PMA added. The precision study was carried out by intra- and interassay analyses, using pooled urine added 20, 60 and 100 μg/L of S-PMA in triplicate for 5 consecutive days. The limits of detection and quantification were calculated according to the Eurachem Guide [28], which goes respectively to three and ten times the standard deviation of ten replicates extracted from urine pool and derivatized as described in item 2.2. The matrix effect was evaluated by comparing the slope and intercept calibration curve constructed with urine pooled samples spiked with standard S-PMA solution and the analyte calibration curve in water at concentrations of 40, 60, 80 and 100 μg/L. Recovery tests were performed using urine pool without adding standard S-PMA solution and addition of 10 concentrations; 20, 60 and 100 μg/L, prepared in triplicate.
2. Methods 2.1. Standards and reagents The standard S-phenylmercapturic acid (S-PMA) was purchased from Santa Cruz Biotechnology®. First of all standard-solutions were prepared and stocked in methanol at a concentration of 250 mg/L, which was used to prepare standard-solutions for using as matrix adjustment (matrix matching). The chromatographic grade solvents which were used: methanol, isopropanol and acetic acid obtained from SigmaAldrich®; and acetonitrile acquired from JT Backer®. The pure grade reagents for analysis were: ammonium bicarbonate and sodium hydroxide (Synth®), hydrochloric acid and phosphoric acid (Vetec®), ammonium acetate and monobromobimane (Sigma-Aldrich®). 2.2. Sample preparation The sample preparation procedure was based on the methodology described by Buratti et al. [21] and Einig and Dehnen [22]. Urine specimens, pooled, were used as template for optimization experiments and analytical validation. The pool was prepared by taking urine samples from both gender volunteers, not smoking, not occupationally exposed to benzene and who have not used drugs. The samples were collected in plastic containers, homogenized and filtered. The samples were then transferred to Falcon tubes with capacity of 50 mL, with lid and stored at −20 °C. The validated method was applied to urine samples from workers occupationally exposed to benzene. The samples preparation included five steps: acidification, solid phase extraction; analyte concentration at 75 °C under nitrogen flow; alkaline hydrolysis and derivatisation. Urine samples pooled from individuals occupationally exposed to benzene were acidified (pH = 1) to pre-hydrolysis and mercapturic acids extracted using SPE employing C18 cartridges (500 mg, Chromabond® 3 mL). The cartridges were conditioned with 3 mL of methanol and 6 mL of 1% acetic acid; 3 mL of urine was added to the cartridge and 1 min after contact with the stationary phase was carried out further washing step, using 2 mL of 1% acetic acid. For elution were used 2 mL buffer solution containing 20% ammonium acetate 0.1 mol/ L and 80% methanol (v/v), 2 mL eluate obtained from the extraction were concentrated as residual in a water bath at 75 °C under a nitrogen atmosphere. Then the residue was subjected to alkaline hydrolysis by adding 400 μL of 2 mol/L NaOH, followed by vigorous stirring at 2500 rpm until complete solubilisation. The residue solubilized cryovial was transferred to 2 mL screw cap and placed in a water bath at 95 °C for 25 min. After hydrolysis, the pH was adjusted (pH = 7.5 to 8.5)
3. Results and discussions The analytical conditions optimized in this study allowed good separation and quantification of urinary S-PMA. The S-PMA retention time was determined by comparison with peaks observed in the chromatograms of the standard solution S-PMA analysis mobile phase and urine pool added S-PMA standard solution. The observed retention time was 5.9 min with total time of 12 min of running. The results of the validation are shown in the Table 1. There was a linear relationship between the peak area of the S-PMA and their concentration in urine pool to the range of 10–100 μg/L, this result is similar to that reported by Einig and Dehnen [22] (1–200 μg/L) and Buratti et al. [21] (10–250 μg/L). The coefficients of variation (CV) intraassay ranged from 1.85 to 11.93; while interassay coefficients were between 4.3 and 6.2 as shown in Table 2; these results were considered acceptable. As Lin et al. [29], manual procedures for the preparation of samples influence the reproducibility of measurements, increasing the variability of results. The limits of detection (LD) and quantification (LQ) were respectively 0.22 μg/L and 0.68 μg/L. The methods developed by Einig and Dehnen [22] and Buratti et al. [21] for determining the S-PMA urine, using the same analytical technique used in this study also found high sensitivity; LD being reported by the authors respectively less than 1 μg/L and 1 μg/ L. These results demonstrate that despite the mass detector to be considered more sensitive, the use of fluorimetric detector has similar sensitivity. 137
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Table 1 − Results of the validation of the method for quantification of urinary SPMA.
Analytical technique Linearity Interassay accuracy Intrassay accuracy Limit of detection Limits of quantification Recovery Analyte stability in urine
In this study
Buratti et al. [21]
Einig and Dehnen [22]
Wang et al. [20]
Sabatini et al. [30]
HPLC with fluorimetric detector 10–100 μg/L 1.85–11.93 4.3–6.2 0.22 μg/L 0.68 μg/L 89.81% 7 weeks (–20 °C)
HPLC with fluorimetric detector 10–250 μg/L < 13 <7 1 μg/ L – 90% 6 months (–18 °C)
HPLC with fluorimetric detector 1–200 μg/L < 10 <5 < 1 μg/ L – 103% –
HPLC/MS/MS
HPLC/MS/MS
0.2–200 μg/L 4.4–8 3.3–6.3 0.1 μg/L 0.2 μg/L 95.3–106.7% –
0.6–50 μg/L 2.8–6.2 3.9–9 0.3 μg/L 0.6 μg/L 82 ± 4.4% 2 months (–20 °C)
Fan Wang and She [13]
0.2–200 μg/L 3.12–9.37 – 0.10 μg/L – 88.7–107% 2 months (–20 °C)
Legend: − not specified. Table 2 Intra and interassay accuracy results for the determination of urinary S-PMA. Concentration nominal (μg.L -1)
Intraassay
Interassay
μg.L (n=3)
C.V
μg.L-1 (n=3)
C.V
21,37 62,86 102,03
11,93 3,53 1,85
20,0 60,4 101,9
6,2 5,0 4,3
-1
20 60 100
Table 3 Extraction Method recovery percentage obtained after analysis of urine samples added 10, 20, 60 and 100 μg.L−1 S-PMA. Nominal Concentration (μg.L −1)
Recovery percentage (n = 3)
C.V
10 20 60 100
76,13 102,82 83,45 96,87
13,02 0,43 1,68 6,09
Legend: n = number of replicates; C.V = coefficient of variation.
Legend: n = number of replicates; CV = coefficient of variation.
This study was not observed for the matrix effect S-PMA (Fig. 1). Work conducted by Wang et al. [20], and Sabatini et al. [30] also found no matrix effect on the elution of S-PMA. In contrast, recent work by Fan Wang and She [13] showed matrix effect during method validation for determination of catechol, AttM, S-PMA and benzyl-mercapturic acid in the urine. The average percentage recovery obtained was 89.81% (Table 3), a similar recovery was reported by other studies that also used the SPE technique for extracting the S-PMA urine through C18 cartridges [22,24]. Urine analyte stability was evaluated for a period of eight-week. The stability observed in this study was seven weeks, as shown in Fig.2, therefore, the recovery of stable below 25% was considered acceptable compared to the concentration of S-PMA determined on the first day. Lin et al. [29] and Fan Wang and She [13] reported 8 week stability for the S-PMA stored at −20 °C. The validated method was applied to urine samples from 10 workers from a fuel testing laboratory, who are occupationally exposed to benzene and agreed to participate in the study by signing the Free
Consent and Informed. These employees work for technical positions in chemistry. They are responsible for fuelling sample and carrying out laboratory tests; and administrative positions, responsible for managing, scheduling sampling, issuing reports, collection and others. All individuals work 8 h per day. The collection of urine samples was carried out on the fifth consecutive day of exposure, at the end of eight hours of the working day, as established by the Annex to Ordinance No. 34 of December 20, 2001 [6]. Seven out of ten employees were female and 3 males, aged 23–49 years (mean 43 years); 5 technicians and 5 employees in the administrative sector, none of them were smokers. Benzene in the air analyses were conducted by the Occupational Toxicology Laboratory of Federal University of Minas Gerais, Faculty of Pharmacy. The samples were collected from workers using samplers SKC® series 575, posted at the time of the respiratory region of each worker during the eight hours of work and on the day of collection of urine samples. The determination of the levels of benzene in air was conducted by using the Standard Method of the National Institute for Occupational Safety and Health − NIOSH [31], using gas chromatography with a flame ionization detector. The concentration of benzene Fig. 1. Matrix effect test result by comparison the slope and intercept calibration curve constructed with urine pooled samples spiked with standard S-PMA solution and the analyte calibration curve in water at concentrations of 40, 60, 80 and 100 μg/L. Legend: curve constructed using water as solvent; curve constructed using urine as solvent.
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Fig. 2. S-PMA stability during storage at −20 °C. Legend: C1 = 75% recovery; C2 = 100% recovery; C3 = 125% recovery.
for urinary creatinine present outside the limits recommended by the ACGIH [16]. The S-PMA was detected in only 2 urine samples from nine workers who participated in the study, being respectively 18.8 and 20.02 mg/g creatinine (Fig. 3). According to ACGIH, the S-PMA Biological Exposure Limit is 25 μg/g creatinine, which corresponds to 0.5 ppm benzene in air. Since the AttM was detected in all samples, its concentration ranged from 0.16 to 2.14 μg/g creatinine; and the average value AttM found for these individuals 0.82 mg/g creatinine. These results characterize a low occupational exposure to benzene by the group. Ordinance No. 34 of December 2001 of the Ministry of Labour and Employment recommends acid-trans, trans-muconic urinary as a biological indicator of exposure to assess exposure to benzene [6]. This legislation directs that the results of this biomarker should be used as the industrial hygiene monitoring tool and monitoring of worker health. According to the ordinance, the concentration of AttM in the urine of non-exposed individuals is less than 0.5 mg/g creatinine. Nevertheless, healthy individuals are not occupationally exposed to benzene, may also have the AttM in urine, the consumption of food, pharmaceutical and cosmetic preparations containing sorbic acid and its salts as preservatives for the biotransformation of sorbic acid and its salts may there be formation of AttM [26,27]. Thus, this indicator has limited specificity and can become a disrupting factor when its use in biomonitoring of exposure to benzene, mainly associated with exposure to low concentrations of the agent in the air. The combined interpretation of all results obtained in this study for the group of benzene occupationally exposed individuals are in agreement, and are consistent with exposure to low concentrations of benzene in the workplace.
Table 4 − Benzene concentration in the breathing zone of workers. Sampler
Concentration (ppm)
1 2 3 4 5 6 7 8 9
0,03 ND ND 0,21 0,14 0,17 ND 0,41 0,01
Legend: ND = not detected.
in air was validated by calculated parameters to the Sampler SKC® and expressed in ppm. The results are shown in Table 4. In Brazil, the Department of Safety and Occupational Medicine established by Ordinance number 14 of December 20, 1995, the Technological Reference Value (TRV) of 1 ppm of benzene in the air for companies that produce, transport, store or handle benzene in their activities and 2.5 ppm for steel [25]. Benzene values in the air to which workers are exposed in the LEC, determined in this study were lower than the TRV and is therefore in accordance with the recommendations by those laws. The values of AttM and S-PMA obtained from these urine samples’ workers are presented in Table 5. The urine sample was not analysed
Table 5 PMA values and AttM in the urine of workers exposed to benzene.
4. Conclusion Sample identification
Function performed
S-PMA Concentration mg/g creatinine (n = 2)
Concentration AttM mg/g of creatinine
1 2 3 4 5 6 7 8 9 10
Technical Technical Administrative Administrative Technical Administrative Technical Administrative Technical Technical
ND ND ND ND NA ND ND ND 18,76 20,02
0,49 0,53 0,86 0,88 NA 2,14 1,03 1,09 0,21 0,16
The validated method can be implemented in health surveillance activities exposed to benzene worker, and in the evaluation of environmental exposure to this solvent. The analytical method developed using the solid and liquid phase technical extraction chromatography with fluorimetric detector showed excellent sensitivity with low limits of detection and quantification, and adequate precision and linearity for the analytical purpose. It demonstrates that the validated method can be implemented in health surveillance activities exposed to benzene worker, and in the evaluation of environmental exposure to this solvent. The S-phenylmercapturic acid has been proposed as biological indicator for biomonitoring of exposure to benzene, especially those associated with low concentrations of hydrocarbon. The results confirm
Legend: ND = not detected; NA = unanalyzed.
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Fig. 3. Chromatogram worker exposed to benzene.
[15] Suyoung. Kim, et al., Modeling human metabolism of benzene following occupational and environmental exposures, Cancer Epidemiol. Biomarkers Prevent. 15 (11) (2006) 2246. [16] AMERICAN Conference of Governmental Industrial Hygienists-ACGIH. 2016 TLVs and BEIs. Cincinnati, 2016. [18] C.B. Hymer, Validation of an HPLC-MS–MS method for the determination of urinary S-benzylmercapturic acid and s-phenylmercapturic acid, J. Chromatogr. Sci. 49 (7) (2011) 547–553. [19] L.V. Bi-Hua, et al., Urinary S-phenylmercapturic acid as a key biomarker for measuring occupational exposure to low concentrations of benzene in Chinese workers a pilot study, American college of, Occup. Environ. Med. 56 (March (3)) (2014). [20] W. Zhonghua, et al., A rapid and sensitive liquid chromatography–tandem mass spectrometry method for the quantitation of S-phenylmercapturic acid in human urine, Anal. Methods 5 (21) (2013) 6081–6085. [21] M. Buratti, et al., Determination of monobromobimane derivatives of phenylmercapturic and benzylmercapturic acids in urine by high-performance liquid chromatography and fluorimetry, J. Chromatogr. B: Biomed. Sci. Appl. 751 (2) (2001) 305–313. [22] T. Einig, W. Dehnen, Sensitive determination of the benzene metabolite S-phenylmercapturic acid in urine by high- performance liquid chromatography with fluorescence detection, J. Chromatogr. A 697 (1) (1995) 371–375. [23] H. Kivisto, et al., Biological monitoring of exposure to benzene in the production of benzene and in a cokery, Sci. Total Environ. 199 (1997) 49–63. [24] E. Paci, et al., Determination of free and total S-phenylmercapturic acid by HPLC/ MS/MS in the biological monitoring of benzene exposure, Biomarkers: Biochem. Indic.Exp.Response Suscept. Chem. 12 (2) (2007) 111. [25] BRAZIL, Ministério do Trabalho, Portaria n°14, De 20 De Dezembrode . Alteração Do Item Substâncias Cancerígenas Do Anexo 13. Brasília, (1995) Available: http:// portal.mte.gov.br/data/files/FF8080812C12AA70012C12C576E3005C/p_ 19951220_14. pdf. [26] G. Marrubini, et al., Effect of sorbic acid administration on urinary trans, transmuconic acid excretion in rats exposed to low levels of benzene, Food Chem. Toxicol. 40 (12) (2002) 1799–1806. [27] Negri Sara, et al., High-pressure liquid chromatographic-mass spectrometric determination of sorbic acid in urine: verification of formation of trans, trans-muconic acid, Chem. Biol. Interact. 153 (2005) 243–246. [28] EURACHEM, The Fitness for Purpose of Analytical Methods—A Laboratory Guide to Method Validation and Related Topics, 2nd ed., (2014) (ISBN 978-91-87461-59-0. Available:https://www.eurachem.org/images/stories/Guide/pdf/ MV_guide_2nd_ed_EN.pdf.). [29] L. Lin, et al., Validation of an online dual- loop cleanup device with an electrospray ionization tandem mass spectrometry-based system for simultaneous quantitative analysis of urinary benzene exposure biomarkers trans, trans-muconic acid and Sphenylmercapturic acid, Anal. Chim. Acta 555 (1) (2006) 34–40. [30] Sabatini Laura, et al., Validation of an HPLC-MS/MS method for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o- methylbenzyl mercapturic acid in urine as biomarkers of exposure to benzene, toluene and xylenes, J. Chromatogr. B 863 (1) (2008) 115–122. [31] Niosh -The National Institute for Occupational Safety and Health Centers for Disease Control and Prevention, Manual of Anayltical Methods. BENZENE by Portable GC (Available:), http://www.cdc.gov/niosh/docs/2003-154/pdfs/3700. pdf.
the specificity and applicability of this indicator for this purpose, and validated method appeared as an economically viable alternative to costly analytical methods. Acknowledgements The authors acknowledge Coordination for the Improvement of Higher Education Personnel (Capes) and Foundation for Research Support of the State of Minas Gerais (FAPEMIG) for funding. References [1] Fustinoni Silvia, et al., Methodological issues in the biological monitoring of urinary benzene and S-phenylmercapturic acid at low exposure levels, J. Chromatogr. B 878 (27) (2010) 2534–2540. [2] International agency for research on cancer - IARC, Benzene. Monografia 100F. IARC Monogr Evaluation Carcinogenic Risks to Human, (2012) Disponível em: http://monographs.iarc.fr/ENG/Monographs/vol100F/mono100F-24. pdf. [3] Stephen M. Rappaport, et al., Human benzene metabolism following occupational and environmental exposures, Chem. Biol. Interact. 184 (1) (2010) 189–195. [4] Scott M. Arnold, et al., The use of biomonitoring data in exposure and human health risk assessment: benzene case study, Crit. Rev. Toxicol. 43 (2) (2013) 119–153. [5] Q.U. Qingshan, et al., Validation of biomarkers in humans exposed to benzene: urine metabolites, Am. J. Ind. Med. 37 (5) (2000) 522–531. [6] BRAZIL, Ministério do Trabalho e Emprego. Portaria. N°. 34, de 20 de dezembrode . Determina procedimentos para a utilização de indicador biológico de exposição ao benzeno, (2001) Available: http://www.trabalho.gov.br. [7] Maico Menezes, et al., Influência do hábito de fumar na excreção urinária do ácido trans, trans-mucônico, Revista Brasileira de Ciências Farmacêuticas 44 (July (3)) (2008). [8] Negri Sara, et al., High-pressure liquid chromatographic-mass spectrometric determination of sorbic acid in urine: verification of formation of trans, trans-muconic acid, Chem. Biol. Interact. 153 (2005) 243–246. [9] Fustinoni Silvia, et al., Environmental and lifestyle factors affect benzene uptake biomonitoring of residents near a petrochemical plant, Environ. Int. 39 (1) (2012) 2–7. [10] Lovreglio Piero, et al., Biomarkers of internal dose for the assessment of environmental exposure to benzene, J. Environ. Monit. 13 (10) (2011) 2921–2928. [11] Clifford P. Weisel, Benzene exposure: an overview of monitoring methods and their findings, Chem. Biol. Interact. 184 (1–2) (2010) 58–66. [12] Piero Lovreglio, et al., Il monitoraggio dell’esposizione occupazionale ed ambientale a basse dosi di benzene, G Ital Med Lav Erg 35 (4) (2013) 251–255. [13] Ruifang Fan, Dongli Wang, Jianwen She, Method development for the simultaneous analysis of trans, trans-muconic acid, 1,2-dihydroxybenzene, S−phenylmercapturic acid and S−benzylmercapturic acid in human urine by liquid chromatography/ tandem mass spectrometry, Anal. Methods 7 (2) (2015) 573–580. [14] J. Tuakuila, S-phenylmercapturic acid (S-PMA) levels in urine as an indicator of exposure to benzene in the Kinshasa population, Int. J. Hyg. Environ. Health 216 (4) (2013) 494–498.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
An efficient analytical method for determination of S-phenylmercapturic acid in urine by HPLC fluorimetric detector to assessing benzene exposure
MARK
⁎
Michele P. Rocha Mendes , Josianne Nicácio Silveira, Leiliane Coelho Andre Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Brazil
A R T I C L E I N F O
A B S T R A C T
Key-words: Analytical validation Benzene S-phenylmercapturic acid
Benzene is an important occupational and environmental contaminant, naturally present in petroleum and as byproduct in the steel industry. Toxicological studies showed pronounced myelotoxic action, causing leukemic and others blood cells disorders. Assessing of benzene exposure is performed by biomarkers as trans, trans-muconic acid (AttM) and S-phenylmercapturic acid (S-PMA) in urine. Due to specificity of S-PMA, this biomarker has been proposed to asses lower levels of benzene in air. The aim of this study was to validate an analytical method for the quantification of S-PMA by High-Performance Liquid Chromatography with fluorometric detector. The development of an analytical method of S-PMA in urine was carried out by solid phase extraction (SPE) using C-18 phase. The eluated were submitted to water bath at 75 °C and nitrogen to analyte concentration, followed by alkaline hydrolysis and derivatization with monobromobimane. The chromatography conditions were reverse phase C-18 column (240 mm, 4 mm and 5 μm) at 35 °C; acetonitrile and 0.5% acetic acid (50:50) as mobile phase with a flow of 0.8 mL/min. The limits of detection and quantification were 0.22 μg/L and 0.68 μg/L, respectively. The linearity was verified by simple linear regression, and the method exhibited good linearity in the range of 10–100 μg/L. There was no matrix effect for S-PMA using concentrations of 40, 60, 80 and 100 μg/L. The intra- and interassay precision showed coefficient of variation of less than 10% and the recovery ranged from 83.4 to 102.8% with an average of 94.4%. The stability of S-PMA in urine stored at −20 °C was of seven weeks. The conclusion is that this method presents satisfactory results per their figures of merit. This proposed method for determining urinary S-PMA showed adequate sensitivity for assessment of occupational and environmental exposure to benzene using S-PMA as biomarker of exposure.
1. Introduction Benzene is an aromatic hydrocarbon, naturally present in the oil, in the steel industry as by-product, in the gasoline, in the cigarette and laboratory chemical synthesis. The International Agency for Research on Cancer (IARC) classifies benzene as carcinogenic to humans due to its pronounced myelotoxic action which is a highly causative agent of leukaemia disorders, lymphoproliferative, and quantitative disorders of blood cells; even at concentrations below 1 ppm [1–3]. Benzene is considered a ubiquitous chemical agent, and human exposure to this may occur by occupational or environmental sources. For this reason, the assessment of exposure to predict the risk is extremely important, whether in the context of occupational and environmental toxicology. Benzene research and/or its metabolites in inhaled air or body fluids such as urine make its exposure assessment prevailing important. Various benzene bio-transformation products have been proposed as exposure biomarkers, including – hydroquinone, phenol, catechol, and trans acid, trans-muconic (AttM) [4,5]. However, these acids did not ⁎
Corresponding author. E-mail address: [email protected] (M.P.R. Mendes).
http://dx.doi.org/10.1016/j.jchromb.2017.07.039 Received 18 September 2016; Received in revised form 17 July 2017; Accepted 21 July 2017 Available online 18 August 2017 1570-0232/ © 2017 Elsevier B.V. All rights reserved.
have the specificity and sensitivity required for being used as biomonitoring exposure to this chemical agent. The trans acid, trans urinary-muconic, despite being the biomarker adopted by Brazilian law, has limitations in its use since the excretion of this bio-transformation product is influenced by sorbic acid – a food additive, and by smoking habit since cigarette-smoke is a source of benzene [6–8]. As a result, other metabolites have been proposed, such as − Sphenylmercapturic acid (S-PMA) and benzene unchanged in urine and inhaled air [9–11]. S-PMA has high specificity, since exposure to benzene is the only authoritative source; moreover, this indicator the amount excreted in the urine is related to the concentration of benzene present in the air, especially at low concentrations [11,12]. So this has been employed especially for monitoring exposure to low concentrations of benzene in the air [10,13,14]. However, it is known that less than 1% of absorbed benzene is eliminated in the urine as S-phenylmercapturic acid (S-PMA) [15]. Thus, the determination of this metabolite in the urine requires sensitive analytical techniques, able to identify and quantify a biomarker to
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with 290 μL of buffer solution prepared from 90 μL of 5 mol/L phosphoric acid and 200 μL of 0.5 mol/L ammonium bicarbonate. After homogenization they were added 50 μL of derivatizing reagent – monobromobimane (MB) 2 mmol/L in acetonitrile and 100 μL of derivatized was injected into the chromatographic system.
a level capable to be traced. The biological exposure limit recommended by ACGIH [16] is 25 μg/g creatinine, which correspond to 0.5 ppm of benzene in the air. The most commonly used analytical technique for the determination of S-PMA is liquid chromatography high performance (HPLC), this technique associated with mass spectrometry (MS) analytical has shown great efficiency, regarding the high sensitivity of this technique. Several studies on the bio-monitoring of exposure to benzene have used HPLC/MS to quantify urinary S-PMA [13,16,18,19,14,20]. Despite all the advantages offered by this technique, it is expensive and is more susceptible to the matrix effect. Some studies have been reported a good analytical performance using the HPLC technique with fluorimetric detection [21–23]. The good analytical results set the HPLC technique with attractive fluorimetric detector for determining the S-PMA, in view of the analytical sensitivity and lower cost when compared to HPLC/MS. In this sense, the present study aimed to standardization and validation of analytical methodology for quantification of S-PMA in urine by high-performance liquid chromatography (HPLC) and fluorimetric detection and evaluation of this method in biomonitoring of occupational exposure to benzene.
2.3. Equipment The high performance liquid chromatograph LC 1100 series equipped with isocratic pump, column oven, autosampler, fluorimetric detector 1200 and ChemStation for integration and processing of chromatograms − Agilent Technologies® was used. The chromatographic column used was Lichrocarth Hypersil ODS C18 column (250 mm × 4.0 mm × 5 μm, Merck ®) at 35 °C. The mobile phase used was acetonitrile-0.5% acetic acid (50:50 v/v) with a flow of 0.80 mL per minute. The wavelengths used for fluorimetric detector were 395 and 470 nm for excitation and emission respectively. 2.4. Quantification Pool urine samples spiked with standard S-PMA concentrations of 10, 20, 40, 60, 80 and 100 μg/L were used to construct calibration curves, where these solutions were prepared according to the procedure described in (2.2). To demonstrate the reliability of the method were determined by the following figures of merit − linearity, precision, recovery and matrix effect using standard samples of urine pool S-PMA added. The precision study was carried out by intra- and interassay analyses, using pooled urine added 20, 60 and 100 μg/L of S-PMA in triplicate for 5 consecutive days. The limits of detection and quantification were calculated according to the Eurachem Guide [28], which goes respectively to three and ten times the standard deviation of ten replicates extracted from urine pool and derivatized as described in item 2.2. The matrix effect was evaluated by comparing the slope and intercept calibration curve constructed with urine pooled samples spiked with standard S-PMA solution and the analyte calibration curve in water at concentrations of 40, 60, 80 and 100 μg/L. Recovery tests were performed using urine pool without adding standard S-PMA solution and addition of 10 concentrations; 20, 60 and 100 μg/L, prepared in triplicate.
2. Methods 2.1. Standards and reagents The standard S-phenylmercapturic acid (S-PMA) was purchased from Santa Cruz Biotechnology®. First of all standard-solutions were prepared and stocked in methanol at a concentration of 250 mg/L, which was used to prepare standard-solutions for using as matrix adjustment (matrix matching). The chromatographic grade solvents which were used: methanol, isopropanol and acetic acid obtained from SigmaAldrich®; and acetonitrile acquired from JT Backer®. The pure grade reagents for analysis were: ammonium bicarbonate and sodium hydroxide (Synth®), hydrochloric acid and phosphoric acid (Vetec®), ammonium acetate and monobromobimane (Sigma-Aldrich®). 2.2. Sample preparation The sample preparation procedure was based on the methodology described by Buratti et al. [21] and Einig and Dehnen [22]. Urine specimens, pooled, were used as template for optimization experiments and analytical validation. The pool was prepared by taking urine samples from both gender volunteers, not smoking, not occupationally exposed to benzene and who have not used drugs. The samples were collected in plastic containers, homogenized and filtered. The samples were then transferred to Falcon tubes with capacity of 50 mL, with lid and stored at −20 °C. The validated method was applied to urine samples from workers occupationally exposed to benzene. The samples preparation included five steps: acidification, solid phase extraction; analyte concentration at 75 °C under nitrogen flow; alkaline hydrolysis and derivatisation. Urine samples pooled from individuals occupationally exposed to benzene were acidified (pH = 1) to pre-hydrolysis and mercapturic acids extracted using SPE employing C18 cartridges (500 mg, Chromabond® 3 mL). The cartridges were conditioned with 3 mL of methanol and 6 mL of 1% acetic acid; 3 mL of urine was added to the cartridge and 1 min after contact with the stationary phase was carried out further washing step, using 2 mL of 1% acetic acid. For elution were used 2 mL buffer solution containing 20% ammonium acetate 0.1 mol/ L and 80% methanol (v/v), 2 mL eluate obtained from the extraction were concentrated as residual in a water bath at 75 °C under a nitrogen atmosphere. Then the residue was subjected to alkaline hydrolysis by adding 400 μL of 2 mol/L NaOH, followed by vigorous stirring at 2500 rpm until complete solubilisation. The residue solubilized cryovial was transferred to 2 mL screw cap and placed in a water bath at 95 °C for 25 min. After hydrolysis, the pH was adjusted (pH = 7.5 to 8.5)
3. Results and discussions The analytical conditions optimized in this study allowed good separation and quantification of urinary S-PMA. The S-PMA retention time was determined by comparison with peaks observed in the chromatograms of the standard solution S-PMA analysis mobile phase and urine pool added S-PMA standard solution. The observed retention time was 5.9 min with total time of 12 min of running. The results of the validation are shown in the Table 1. There was a linear relationship between the peak area of the S-PMA and their concentration in urine pool to the range of 10–100 μg/L, this result is similar to that reported by Einig and Dehnen [22] (1–200 μg/L) and Buratti et al. [21] (10–250 μg/L). The coefficients of variation (CV) intraassay ranged from 1.85 to 11.93; while interassay coefficients were between 4.3 and 6.2 as shown in Table 2; these results were considered acceptable. As Lin et al. [29], manual procedures for the preparation of samples influence the reproducibility of measurements, increasing the variability of results. The limits of detection (LD) and quantification (LQ) were respectively 0.22 μg/L and 0.68 μg/L. The methods developed by Einig and Dehnen [22] and Buratti et al. [21] for determining the S-PMA urine, using the same analytical technique used in this study also found high sensitivity; LD being reported by the authors respectively less than 1 μg/L and 1 μg/ L. These results demonstrate that despite the mass detector to be considered more sensitive, the use of fluorimetric detector has similar sensitivity. 137
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Table 1 − Results of the validation of the method for quantification of urinary SPMA.
Analytical technique Linearity Interassay accuracy Intrassay accuracy Limit of detection Limits of quantification Recovery Analyte stability in urine
In this study
Buratti et al. [21]
Einig and Dehnen [22]
Wang et al. [20]
Sabatini et al. [30]
HPLC with fluorimetric detector 10–100 μg/L 1.85–11.93 4.3–6.2 0.22 μg/L 0.68 μg/L 89.81% 7 weeks (–20 °C)
HPLC with fluorimetric detector 10–250 μg/L < 13 <7 1 μg/ L – 90% 6 months (–18 °C)
HPLC with fluorimetric detector 1–200 μg/L < 10 <5 < 1 μg/ L – 103% –
HPLC/MS/MS
HPLC/MS/MS
0.2–200 μg/L 4.4–8 3.3–6.3 0.1 μg/L 0.2 μg/L 95.3–106.7% –
0.6–50 μg/L 2.8–6.2 3.9–9 0.3 μg/L 0.6 μg/L 82 ± 4.4% 2 months (–20 °C)
Fan Wang and She [13]
0.2–200 μg/L 3.12–9.37 – 0.10 μg/L – 88.7–107% 2 months (–20 °C)
Legend: − not specified. Table 2 Intra and interassay accuracy results for the determination of urinary S-PMA. Concentration nominal (μg.L -1)
Intraassay
Interassay
μg.L (n=3)
C.V
μg.L-1 (n=3)
C.V
21,37 62,86 102,03
11,93 3,53 1,85
20,0 60,4 101,9
6,2 5,0 4,3
-1
20 60 100
Table 3 Extraction Method recovery percentage obtained after analysis of urine samples added 10, 20, 60 and 100 μg.L−1 S-PMA. Nominal Concentration (μg.L −1)
Recovery percentage (n = 3)
C.V
10 20 60 100
76,13 102,82 83,45 96,87
13,02 0,43 1,68 6,09
Legend: n = number of replicates; C.V = coefficient of variation.
Legend: n = number of replicates; CV = coefficient of variation.
This study was not observed for the matrix effect S-PMA (Fig. 1). Work conducted by Wang et al. [20], and Sabatini et al. [30] also found no matrix effect on the elution of S-PMA. In contrast, recent work by Fan Wang and She [13] showed matrix effect during method validation for determination of catechol, AttM, S-PMA and benzyl-mercapturic acid in the urine. The average percentage recovery obtained was 89.81% (Table 3), a similar recovery was reported by other studies that also used the SPE technique for extracting the S-PMA urine through C18 cartridges [22,24]. Urine analyte stability was evaluated for a period of eight-week. The stability observed in this study was seven weeks, as shown in Fig.2, therefore, the recovery of stable below 25% was considered acceptable compared to the concentration of S-PMA determined on the first day. Lin et al. [29] and Fan Wang and She [13] reported 8 week stability for the S-PMA stored at −20 °C. The validated method was applied to urine samples from 10 workers from a fuel testing laboratory, who are occupationally exposed to benzene and agreed to participate in the study by signing the Free
Consent and Informed. These employees work for technical positions in chemistry. They are responsible for fuelling sample and carrying out laboratory tests; and administrative positions, responsible for managing, scheduling sampling, issuing reports, collection and others. All individuals work 8 h per day. The collection of urine samples was carried out on the fifth consecutive day of exposure, at the end of eight hours of the working day, as established by the Annex to Ordinance No. 34 of December 20, 2001 [6]. Seven out of ten employees were female and 3 males, aged 23–49 years (mean 43 years); 5 technicians and 5 employees in the administrative sector, none of them were smokers. Benzene in the air analyses were conducted by the Occupational Toxicology Laboratory of Federal University of Minas Gerais, Faculty of Pharmacy. The samples were collected from workers using samplers SKC® series 575, posted at the time of the respiratory region of each worker during the eight hours of work and on the day of collection of urine samples. The determination of the levels of benzene in air was conducted by using the Standard Method of the National Institute for Occupational Safety and Health − NIOSH [31], using gas chromatography with a flame ionization detector. The concentration of benzene Fig. 1. Matrix effect test result by comparison the slope and intercept calibration curve constructed with urine pooled samples spiked with standard S-PMA solution and the analyte calibration curve in water at concentrations of 40, 60, 80 and 100 μg/L. Legend: curve constructed using water as solvent; curve constructed using urine as solvent.
138
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Fig. 2. S-PMA stability during storage at −20 °C. Legend: C1 = 75% recovery; C2 = 100% recovery; C3 = 125% recovery.
for urinary creatinine present outside the limits recommended by the ACGIH [16]. The S-PMA was detected in only 2 urine samples from nine workers who participated in the study, being respectively 18.8 and 20.02 mg/g creatinine (Fig. 3). According to ACGIH, the S-PMA Biological Exposure Limit is 25 μg/g creatinine, which corresponds to 0.5 ppm benzene in air. Since the AttM was detected in all samples, its concentration ranged from 0.16 to 2.14 μg/g creatinine; and the average value AttM found for these individuals 0.82 mg/g creatinine. These results characterize a low occupational exposure to benzene by the group. Ordinance No. 34 of December 2001 of the Ministry of Labour and Employment recommends acid-trans, trans-muconic urinary as a biological indicator of exposure to assess exposure to benzene [6]. This legislation directs that the results of this biomarker should be used as the industrial hygiene monitoring tool and monitoring of worker health. According to the ordinance, the concentration of AttM in the urine of non-exposed individuals is less than 0.5 mg/g creatinine. Nevertheless, healthy individuals are not occupationally exposed to benzene, may also have the AttM in urine, the consumption of food, pharmaceutical and cosmetic preparations containing sorbic acid and its salts as preservatives for the biotransformation of sorbic acid and its salts may there be formation of AttM [26,27]. Thus, this indicator has limited specificity and can become a disrupting factor when its use in biomonitoring of exposure to benzene, mainly associated with exposure to low concentrations of the agent in the air. The combined interpretation of all results obtained in this study for the group of benzene occupationally exposed individuals are in agreement, and are consistent with exposure to low concentrations of benzene in the workplace.
Table 4 − Benzene concentration in the breathing zone of workers. Sampler
Concentration (ppm)
1 2 3 4 5 6 7 8 9
0,03 ND ND 0,21 0,14 0,17 ND 0,41 0,01
Legend: ND = not detected.
in air was validated by calculated parameters to the Sampler SKC® and expressed in ppm. The results are shown in Table 4. In Brazil, the Department of Safety and Occupational Medicine established by Ordinance number 14 of December 20, 1995, the Technological Reference Value (TRV) of 1 ppm of benzene in the air for companies that produce, transport, store or handle benzene in their activities and 2.5 ppm for steel [25]. Benzene values in the air to which workers are exposed in the LEC, determined in this study were lower than the TRV and is therefore in accordance with the recommendations by those laws. The values of AttM and S-PMA obtained from these urine samples’ workers are presented in Table 5. The urine sample was not analysed
Table 5 PMA values and AttM in the urine of workers exposed to benzene.
4. Conclusion Sample identification
Function performed
S-PMA Concentration mg/g creatinine (n = 2)
Concentration AttM mg/g of creatinine
1 2 3 4 5 6 7 8 9 10
Technical Technical Administrative Administrative Technical Administrative Technical Administrative Technical Technical
ND ND ND ND NA ND ND ND 18,76 20,02
0,49 0,53 0,86 0,88 NA 2,14 1,03 1,09 0,21 0,16
The validated method can be implemented in health surveillance activities exposed to benzene worker, and in the evaluation of environmental exposure to this solvent. The analytical method developed using the solid and liquid phase technical extraction chromatography with fluorimetric detector showed excellent sensitivity with low limits of detection and quantification, and adequate precision and linearity for the analytical purpose. It demonstrates that the validated method can be implemented in health surveillance activities exposed to benzene worker, and in the evaluation of environmental exposure to this solvent. The S-phenylmercapturic acid has been proposed as biological indicator for biomonitoring of exposure to benzene, especially those associated with low concentrations of hydrocarbon. The results confirm
Legend: ND = not detected; NA = unanalyzed.
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Fig. 3. Chromatogram worker exposed to benzene.
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