Quantitative estimation of urinary ortho-cresol by high pressure liquid chromatography for biomonitoring of toluene exposed population

Anupa Yadav; Amit Chakrabarti; Geoffrey Nengzapum

doi:10.4103/ed.ed_23_20

ORIGINAL ARTICLE

Year : 2020 | Volume : 5 | 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

Correspondence Address:
Ms. Anupa Yadav
Department of Industrial Hygiene, Regional Occupational Health Centre (Eastern), Block DP, Sector V, Salt Lake, Kolkata - 700 091, West Bengal
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ed.ed_23_20

Abstract

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 5];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.^[1],[2],[3] Inhalation is the main route of exposure to its vapors.^{[4],[5],[6],[7],[8],[9]} Chronic exposure to toluene in occupational settings or recreational activities reveals irreversible and anatomical change in brain, neurobehavioral impairment.^[8],[9] Also has genotoxic effects,^[10],[11] and neurological, cardiac, renal, and hepatic toxicity.^[12] After inhalation about 70% is absorbed in bloodstream, while a fraction of this excreted in the exhaled air without any change.^{[4],[5],[13],[14]} 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.^[15],[16] 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.^[17] 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.^[18] 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.^[19],[20] At present, there are many techniques for the determination of OC, includes gas and liquid chromatography with mass, flame ionization, and ultraviolet detectors.^{[6], 7, [21],[22],[23]} 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

Study population

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.

Experimental

Chemicals

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

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.

Method validation

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.^[24],[25] 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).^[26]

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.^[27] 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,^[28] 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

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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;^[29] here, results of urine samples are presented without creatinine correction.

Table 1: Recovery %, intra- and inter-day precision study of o-cresol

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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.^[19],[30]

Table 2: Evaluation of quantified o-cresol in urine of exposed and unexposed group

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Table 3: Urinary o-cresol, mean±standard deviation in exposed workers and unexposed subjects

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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.^[19] 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.^[30],[31] 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

Nil.

Conflicts of interest

There are no conflicts of interest.

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