|Year : 2020 | Volume
| Issue : 3 | Page : 78-82
Quantitative estimation of urinary ortho-cresol by high pressure liquid chromatography for biomonitoring of toluene exposed population
Anupa Yadav, Amit Chakrabarti, Geoffrey Nengzapum
Department of Industrial Hygiene, Regional Occupational Health Centre (Eastern), Kolkata, West Bengal, India
|Date of Submission||28-Jul-2020|
|Date of Decision||30-Aug-2020|
|Date of Acceptance||08-Sep-2020|
|Date of Web Publication||30-Sep-2020|
Ms. Anupa Yadav
Department of Industrial Hygiene, Regional Occupational Health Centre (Eastern), Block DP, Sector V, Salt Lake, Kolkata - 700 091, West Bengal
Source of Support: None, Conflict of Interest: None
Aim: Quantification of urinary ortho-cresol (OC) by high pressure liquid chromatography coupled with photodiode array detector.
Materials and Methods: Includes acid hydrolysis of urine, liquid–liquid extraction, and chromatography quantification of extracted OC in urine.
Results: Limit of detection, limit of quantification, and coefficient of linearity (R2) were 0.18 μg/ml, 0.62 μg/ml, and 0.9998, respectively. Recovery % of method ranged from 92%, 97%, and 100%. For intraday and interday precision coefficient of variation was 0.41%, 0.64%, and 0.89%, 0.86% for urine samples spiked with OC standards final concentration of 0.25 μg/ml and 0.7 μg/ml, respectively. Results (mean ± standard deviation) of exposed and unexposed real urine samples analyzed for OC with this method were 0.92 ± 0.76 and 0.40 ± 0.20 μg/ml, respectively. Statistical analysis of results showed significant (P ≤ 0.001) difference between urinary OC among exposed and unexposed subjects.
Conclusion: The present work describes precise, easy, and less time consuming method for estimation of OC in urine of population exposed to toluene. It can be used as a promising tool for biomonitoring of population exposed to toluene.
Keywords: Biomonitoring, high pressure liquid chromatography, liquid–liquid extraction, ortho-cresol, toluene
|How to cite this article:|
Yadav A, Chakrabarti A, Nengzapum G. Quantitative estimation of urinary ortho-cresol by high pressure liquid chromatography for biomonitoring of toluene exposed population. Environ Dis 2020;5:78-82
|How to cite this URL:|
Yadav A, Chakrabarti A, Nengzapum G. Quantitative estimation of urinary ortho-cresol by high pressure liquid chromatography for biomonitoring of toluene exposed population. Environ Dis [serial online] 2020 [cited 2023 Jun 3];5:78-82. Available from: http://www.environmentmed.org/text.asp?2020/5/3/78/296805
| Introduction|| |
Toluene is widely used as solvent in industries of paint, rubber, cosmetic, printing, in adhesive used for shoes, sandals or bags, in many products as a synthetic intermediate and in fuel as additive to increase octane rating of gasoline.,, Inhalation is the main route of exposure to its vapors.,,,,, Chronic exposure to toluene in occupational settings or recreational activities reveals irreversible and anatomical change in brain, neurobehavioral impairment., Also has genotoxic effects,, and neurological, cardiac, renal, and hepatic toxicity. After inhalation about 70% is absorbed in bloodstream, while a fraction of this excreted in the exhaled air without any change.,,, Major part (60%–70%) of the absorbed toluene in liver is transformed to hippuric acid (HA) while only small portion (1%) of the absorbed dose is converted to ortho-cresol (OC); excreted in urine as glucuronide and sulfate conjugate., Literature reveals, if environmental air concentration of toluene is 50 ppm (i.e., threshold limit value [TLV]) only then HA is a significant biomarker of toluene. The American Conference of Governmental Industrial Hygienist (ACGIH) has suggested OC as a reliable exposure biomarker of toluene, mainly when toluene exposure level is low. As per the ACGIH (2017) recommended biological exposure indices (BEI) for urinary, OC is 0.3 mg/g creatinine. Studies found good correlation between urinary OC and ambient air toluene (10 times lower than TLV); OC is not commonly excreted in urine of unexposed person and its analysis is more sensitive and reliable tool for biomonitoring of exposure to toluene., At present, there are many techniques for the determination of OC, includes gas and liquid chromatography with mass, flame ionization, and ultraviolet detectors., 7, ,, The aim of the present work was to develop and optimize a simple, cost-effective, and less time consuming method for quantification of urinary OC by high pressure liquid chromatography (HPLC) coupled with photodiode array (PDA) detector. Method application is reported with real urine sample of subjects those were exposed/unexposed to toluene.
| Materials and Methods|| |
Urine samples were taken from workers engaged in making of footwear and exposed to toluene. Ethical clearance was obtained from institutional ethical committee. The study purpose was explained to the study subjects before their participation in study. Before collection of urine samples, written informed consent was taken from all the study subjects.
Standard, chemicals, and solvents used were OC (purity ≥99%), hydrochloric acid, dichloromethane, sodium hydroxide, methanol, phosphoric, acid, and water all of chromatography grade (Merck, Germany).
Standard solution and urine sample spiking
Stock solution of OC was prepared in HPLC grade distilled water (1 mg/ml). Working solutions were prepared in methanol-water (65:35, V/V) with final concentration of 10 μg/ml. Urine samples of unexposed healthy people was taken as blank sample and spiked with OC to final concentration ranges from 0.25 to 10 μg/ml and all solutions were stored at 4°C till analysis.
Chromatography involved HPLC (Shimadzu, Japan) coupled with system controller (Model: SLC-10 A), connected to an automated liquid sampler (Model: LC-10AT), Detector (Model: SPD-M 10A), column oven, and a monitor. Pump flow rate was 1.3 ml/min; chromatogram of OC was detected at specific λ max of 215 nm. The mobile phase used was methanol-water-orthophosphoric acid (65:35:0.1, V/V/V), sample injected was 10 μl, reverse phase column ODS-2 Hypersil (250 mm, 4.6 mm, and 5 μm) and oven temperature was 25°C. Retention time (RT) was at around 3.7 min.
Sample extraction process
Dichloromethane was used for extraction of OC in urine sample; blank and spiked urine aliquots (1 ml) were taken, acidified with concentrated hydrochloric acid (37%) and incubated at 95°C for 15 min in hot air oven (hydrolysis step). Then, samples were cooled at room temperature. Dichloromethane (4 ml) was added, mixed well, and centrifuged at 2,400 g for 10 min. Then, 2.5 ml from organic layer was taken and 1 ml of 0.01 M sodium hydroxide was added and vortex was done for 2 min, 0.3 ml of the aqueous layer was taken and brought to pH 7 with hydrochloric acid, 10 μl of this extract injected in HPLC.
Standard curve linearity
To obtain calibration curve, working standards of OC prepared in urine concentration ranged from 0.25, 0.5, 1, 2, 3, 5, and 10 μg/ml and run in HPLC-PDA at optimized chromatographic conditions to obtain linearity coefficient (R2) from calibration curve.
Limit of detection and limit of quantification
Limit of detection (LOD) is lowest concentration of analyte that can be differentiated from background level noise, while limit of quantification (LOQ) is lowest concentration of analyte that can be quantified with stated level of confidence., LOD and LOQ were determined as per guidelines of international conference on harmonization 2005; based on standard deviation (SD) (σ) and slope (S) of calibration curve (LOD = 3 × σ/S; LOQ = 10 × σ/S) (ICH, 1996).
Recovery and precision
Recovery % of OC was determined by spiking the urine with three concentration levels (0.25, 0.5, 0.7 μg/ml), in replicates of three. Extraction efficiency of the process was determined by comparison of detector response of spiked urine sample with same quantities of working standard solutions analyzed directly.
Recovery (%) = 100 × (C spiked − C sample)/C std.
Where C spiked is concentration of OC added to urine sample, C sample is concentration of OC in urine sample determined from calibration curve and C std is concentration of OC standard.
Coefficient of variation (%CV) was used to determine intraday and interday precision. Intraday precision measurement involved three times analysis of the sample within-day; while inter-day precision involved the estimation of run to run variation in analysis of same sample for the 3 consecutive days.
| Results|| |
Brega et al. used enzymatic hydrolysis of urine sample for the extraction of urinary OC and incubated the sample at 37°C for 12 h, but in our method, acidic hydrolysis was use for extraction of urinary OC, followed by 15 min incubation at 95°C in hot air oven. The present method has advantages (1) cost-effectiveness (can be used in any average research laboratory/institute) and (2) sample extraction is less time consuming.
Chromatogram obtained for standard solution of OC (5 μg/ml), blank urine and spiked urine, under optimized chromatographic conditions are presented in [Figure 1]. The calibration curve was prepared with OC concentration rages from 0.25, 0.5, 1, 2, 3, 5, and 10 μg/ml. This range was selected from the BEI recommended by the ACGIH (2017) at this selected range of OC concentrations the linearity coefficient (R2) for calibration curve was >0.998. The LOD and LOQ of the presented method were 0.18 and 0.62 μg/ml, respectively.
|Figure 1: Chromatogram for standard solution ortho-cresol of concentration 5 μg/ml (a), extracted blank urine (b) and extracted blank urine spiked with final concentration of 1 μg/ml of ortho-cresol (c) at optimized conditions, retention time 3.7 min. Note: Figure 1 (b) blank urine peak area is 6835 and for (c) urine spiked with 1 μg/ml of o-cresol peak area is 9678|
Click here to view
The study for recovery %, intraday and interday precision were carried out at three different concentration levels (0.25, 0.5, 0.7 μg/ml), precision was calculated by CV, presented in [Table 1]. Recovery % age was the mean of triplicate run for each concentration and ranged 100%, 92%, and 97% for the OC standards (0.25, 0.5, 0.7 μg/ml). For intraday day, precision triplicate of each standards were run within the same day, while for interday precision triplicate of each standards were run for the 3 consecutive days. The presented method has very good recovery % age and precision. It was also reflected from the results that precision was independent of the concentration of OC in sample. BEI of ACGIH (2009) is used to compare results of OC in exposed and unexposed group; here, results of urine samples are presented without creatinine correction.
Results obtained by analysis of real urine sample with present method are shown in [Table 2]. Out of total urine sample (n = 75), 73% (n = 55) had given chromatographic signal at the RT of OC but remaining 27% (n = 20) had no signal for OC. Results (mean ± SD) of quantified urine samples for exposed and unexposed were 0.92 ± 0.76 and 0.40 ± 0.20 μg/ml, respectively. These results were analyzed statistically using Student's t-test and found that reported results are statistically significant (P ≤ 0.001) presented in [Table 3]. For exposed subjects 45% of the results were above the BEI of ACGIH (2009) of 0.5 ppm, while 25% of the unexposed subjects also had urinary OC level higher than the BEI. It could be due to indirect exposure to toluene because unexposed subjects living in same community where exposed subject are living and working. Findings of the present work for exposed subjects are similar to the previous studies.,
|Table 2: Evaluation of quantified o-cresol in urine of exposed and unexposed group|
Click here to view
|Table 3: Urinary o-cresol, mean±standard deviation in exposed workers and unexposed subjects|
Click here to view
| Discussion|| |
Chemical compound after inhalation can be transformed into other forms (metabolites) by metabolic processes in human body. Accurate interpretation of these metabolites for biomarkers study is very important, because sometimes other molecules can be shared with metabolic pathway of target compound and results in false-positive results. OC is not usually excreted in the urine of unexposed person and can be used for monitoring of low level exposure to toluene. Presented method has excellent linearity, LOD, and LOQ, with very fine accuracy (92%–100%) and precision. Sample extraction is very simple, efficient, and run time is quite less. In this work, the results of urinary OC were presented without creatinine correction. To check the applicability of the method, real urine sample of exposed and unexposed subjects are analyzed and results obtained were statistically significant (P ≤ 0.001). Our results are supported by present literature., Exposed workers had higher level of OC in comparison to control subjects which make the presence of toluene exposure at work environment evident. Therefore, this method can be used for biomonitoring of low level exposure to toluene as an alternative tool of toluene air monitoring.
| Conclusion|| |
The present work describes precise, easy, and less time consuming method for estimation of OC in urine of population exposed to toluene. It can be used as a promising tool for biomonitoring of population exposed to toluene..
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Serap AA, Pembe O, Yusuf K, Huseyin K, Suheyla E. Medicolegal aspects of bloodurine toluene and urinary orthocresol concentration in toluene exposure. J Med Sci 2001;31:415-19.
Decharat S. Urinary hippuric acid and toluene levels in workers of printing factories in Thailand. Int J Occup Hyg 2016;2:85-92.
Murata K, Araki S, Vokoyama K, Yamashita K, Okajima F, Nakaaki K. Changes in autonomic function as determined by ECGR-R internal variability in sandal, shoe and leather workers exposed to n-hexane, xylene and toluene. Neurotox 1994;15:867-76.
Agency for Toxic Substances and Disease Registry, Toxicological Profile for Toluene. Atlanta: US Department of Health and Human Services; 2017.
U.S Environmental Protection Agency International Program on Chemical Safety. Washington, DC: National Center for Environmental Health, Office of Research and Development; 2017.
Thiesen FV, Noto AR, Barros HM. Laboratory diagnosis of toluene-based inhalants abuse. Clin Toxicol (Phila) 2007;45:557-62.
Perbellini L, Pasini F, Romani S, Princivalle A, Brugnone F. Analysis of benzene, toluene, ethylbenzene and m-xylene in biological samples from the general population. J Chromatogr B Analyt Technol Biomed Life Sci 2002;778:199-210.
Sullivan J, Vanert M. Aromatic Solvents in Clinical Environmental Health Toxic Exposures. Sullivan JB, Krieger GR, editors. Philadelphia, PA. Williams and Wilkins: Publisher ; 2001. p. 1146-51.
Kang SK, Rohlman DS, Lee MY, Lee HS, Chung SY, Anger WK. Neurobehavioral performance in workers exposed to toluene. Environ Toxicol Pharmacol 2005;19:645-50.
Heuser VD, Erdtmann B, Kvitko K, Rohr P, da Silva J. Evaluation of genetic damage in Brazilian footwear-workers: Biomarkers of exposure, effect, and susceptibility. Toxicology 2007;232:235-47.
Roma-Torres J, Teixeira JP, Silva S, Laffon B, Cunha LM, Méndez J, et al
. Evaluation of genotoxicity in a group of workers from a petroleum refinery aromatics plant. Mutat Res 2006;604:19-27.
National Institute of Occupational Safety and Health. Criteria document for toluene. Atlanta, GA; Department of human health and services US; 2005.
Dou JL, Rosemberg J, Coney J, Katz E. Solvents in Diagnosis and Treatment in Occupational and Environmental Medicine. 3rd
ed. The Modern Manual; 2005. p. 543-78.
Lenzken S, Diaz M, Olmos V, Merini L, Panzuto R, Schkolnik L, et al
. Reference value of hippuric acid in urine from a population not exposed to toluene at work. Argent Toxicol Act 2010;11:45-50.
Pierce CH, Chen Y, Dills RL, Kalman DA, Morgan MS. Toluene metabolites as biological indicators of exposure. Toxicol Lett 2002;129:65-76.
Fustinoni S, Buratti M, Giampiccolo R, Brambilla G, Foà V, Colombi A. Comparison between blood and urinary toluene as biomarkers of exposure to toluene. Int Arch Occup Environ Health 2000;73:389-96.
Bahrami A, Jonidi-Jafari A, Folladi B, Mahjub H, Sadri Q, Zadeh M. Comparison of urinary o-Cresol and hippuric acid in drivers, gasoline station workers and painters exposed to toluene in West of Iran. Pak J Biol Sci 2005;8:1001-5.
American Conference of Governmental Industrial Hygienists. Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices. Cincinnati, Ohio, USA: American Conference of Governmental Industrial Hygienists; 2017.
Nakajima T, Wang RS, Murayama N. Immunochemical assessment of the influence of nutritional, physiological and environmental factors on the metabolism of toluene. Int Arch Occup Environ Health 1993;65:S127-30.
Truchon G, Tardif R, Brodeur J. Gas chromatographic determination of urinary o-cresol for the monitoring of toluene exposure. J Anal Toxicol 1996;20:309-12.
Yadav A, Basu A, Chakarbarti A. Method for estimation of hippuric acid as a biomarker of toluene exposure in urine by high-performance liquid chromatography after extraction with ethyl acetate. Environ Dis 2019;4:17-22. [Full text]
Mukherjee AK, Chattopadhyay BP, Roy SK, Das S, Mazumdar D, Roy M, et al
. Work-exposure to PM10 and aromatic volatile organic compounds, excretion of urinary biomarkers and effect on the pulmonary function and heme-metabolism: A study of petrol pump workers and traffic police personnel in Kolkata City, India. J Environ Sci Health A Tox Hazard Subst Environ Eng 2016;51:135-49.
Sabatini L, Barbieri A, Indiveri P, Mattioli S, Violante FS. 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 Analyt Technol Biomed Life Sci 2008;863:115-22.
Quattrocchi OA, Andrizzi DS, Laba RF. Introduction to HPLC: Application and practice. Ferro Graphics Arts Farri, Buenos Aire, Argentiana, 1992; 301-28.
Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, et al
. Analytical methods validation: Bioavailability, bioequivalence and pharmacokinetic studies. Conference report. Eur J Drug Metab Pharmacokinet 1991;16:249-55.
International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human use, Validation of Analytical Procedures: Text and Methodology Q2 (R1) (1996); 2005.
Hasegawa K, Shiojima S, Koizumi A, Ikeda M. Hippuric acid and o-cresol in the urine of workers exposed to toluene. Int Arch Occup Environ Health 1983;52:197-208.
Brega A, Prandini P, Amaglio C, Pafumi E. Determination of phenol, m-, o- and p-cresol, p-aminophenol and p-nitrophenol in urine by high-performance liquid chromatography. J Chromatogr 1990;535:311-6.
American Conference of Governmental Industrial hygienists. Threshold Limit Values for Chemicals Substances and Physical Agents, Biological Exposure Indices. Cincinnati, Ohio, USA: American Conference of Governmental Industrial hygienists; 2009.
Navoni J, Ridolf A, Olivera M, Álvarez G, Lepori EV. Quantitative analysis of urinary o-cresol by gas chromatography - Flame ionization detection for the monitoring of population exposed to toluene. SM Anal Bioanal Technique 2018;3:1018.
Fustinoni S, Mercadante R, Campo L, Scibetta L, Valla C, Consonni D, et al
. Comparison between urinary o-cresol and toluene as biomarkers of toluene exposure. J Occup Environ Hyg 2007;4:1-9.
[Table 1], [Table 2], [Table 3]