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This study presents an overview of the analytical methods for the determination of biomarkers of exposure to phthalates in human urine samples. Phthalates are nonpersistent chemicals; hence, urine is the ideal matrix for biomonitoring besides being noninvasive and simple to collect. Phthalate monoesters and oxidative secondary metabolites are the s...
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... are rapidly hydrolyzed to monoesters and eliminated as free monoes- ter as well as glucuronide conjugates. Elimination half-life of short- chain phthalates is about 5-6 h [5]. By contrast, long-chain phthalates such as DEHP, DiNP, and DPHP metabolize to their monoesters, which are extensively transformed to secondary oxidized metabolites ( Fig. 2) [5]. For long-chain phthalates, the elimination occurs in at least two phases. In the first phase, monoesters are predomi- nantly eliminated, followed by the oxidative metabolites in a long second phase. For example, only 2-7% of the ingested DEHP is ex- creted as the monoester with an elimination half-life of about 12 h Fig. 1. ...
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Background:
In utero exposure to environmental chemicals can adversely impact pregnancy outcomes and childhood health, but minimal biomonitoring data exist on the majority of chemicals used in commerce.
Objectives:
We aimed to profile exposure to multiple environmental organic acids (EOAs) and identify novel chemicals that have not been previous...
Citations
... The ubiquitous presence of phthalates in the laboratory environment poses an analytical challenge known as the "phthalate blank problem" which is difficult to control [41]. Laboratory materials such as pipette tips, plastic containers, solvents, sorbents, analytical instrument, gloves, and plastic tubing contain PAEs. ...
... To ensure aseptic conditions and to minimize the error of quantification during the procedure, the blank should be free from the target analyte. Therefore, if a high contamination level is estimated, it is necessary to raise the number of procedural blanks [41][42][43]. Finally, the standard addition method is another measure required for the PAEs' quantification to standardize the analysis, reducing the matrix-effect artefacts. ...
... They consist in the process of transferring a dissolved substance from one liquid phase to another (immiscible or partially miscible) liquid phase in contact with it. However, there are a lot of drawbacks such as the long time of the analysis, large amounts of organic solvents, and analyte loss, which have promoted the use of more efficient extraction approaches [41]. SPE provides better selectivity and higher recovery, using a volume of solvent less than LLE. ...
Phthalate esters (PAEs) are a group of chemicals used to improve the flexibility and durability of plastics. The chemical properties and the resistance to high temperatures promote their degradation and release into the environment. Food and beverages can be contaminated by PAEs through the migration from packaging material because they are not covalently bound to plastic and also via different kinds of environmental sources or during processing. For instance, alcoholic drinks in plastic containers are a particular risk, since the ethanol contained provides a good solubility for PAEs. According to its role as an endocrine disruptor compound and its adverse effects on the liver, kidney, and reproductive and respiratory systems, the International Agency on Research Cancer (IARC) classified di-(2-ethylhexyl) phthalate (DEHP) as a possible human carcinogen. For this reason, to control human exposure to PAEs, many countries prohibited their use in food as non-food substances. For example, in Europe, the Commission Regulation (EU) 2018/2005 restricts the use of DEHP, dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), and diisobutyl phthalate (DiBP) to a concentration equal to or below 0.1 by weight in plasticizers in articles used by consumers or in indoor areas. There are reports from the US Food and Drug Administration (FDA) that some beverages (and food as well), particularly fruit juices, contain high levels of phthalates. In some cases, the deliberate adulteration of soft drinks with phthalate esters has been reported. This paper would like to show the difficulties of performing PAE analysis in beverage matrices, in particular alcoholic beverages, as well as the main solutions provided for quality control in the industrial branches.
... The LOQs and LODs estimated in the present study (Table II) are appropriate for quantifying m-PAEs in human urine samples as demonstrated in human biomonitoring studies. 11,17,18,21,24,27,30,41,[45][46][47][48] To evaluate the proposed method's repeatability and accuracy, the precision (RSD%) and recovery (%) were assessed by extracting and measuring synthetic urine samples with three spiked concentrations (1.0, 10.0, and 20.0 ng mL −1 ) on one day (for intra-day precision) and three different days (n = 6) (for inter-day precision). The relative standard deviation values for intra-day and inter-day precision were in the ranges of 1-14% and 2-13%, respectively. ...
... ng mL -1 ), and di-(2-ethylhexyl)-phthalate monoesters(∑, mEOHP, mEHHP, mCMHP, and mECPP = 142 ng mL -1 ), when compared to existing data reported in the scientific literature. 7,11,13,20,26 The median concentrations of mIPrP and mPrP, metabolites of diisopropyl phthalate and di-n-propyl phthalate, were 0.45 and 0.89 ng mL −1 , respectively. ...
... However, this exposure has been observed only sporadically or not at all in studies conducted in different countries. 11,17,18,21,24,27,30,41,[45][46][47][48][49][50][51] Overall, the current findings are consistent with the results for other populations previously studied using traditional analytical methods. 11,17,18,21,24,27,30,41,[45][46][47][48] ...
In recent years, the number of epidemiological studies on phthalates that can inform and help update health risk assessments has grown rapidly. Developing reliable and rapid analytical methods for determining phthalate monoesters (m-PAEs) is an important biomonitoring tool for assessing exposure. In this study, a fast and sensitive method was developed to determine 15 m-PAEs in human urine samples as effective biomarkers for exposure assessment. Air-assisted dispersive liquid-liquid microextraction and liquid chromatography coupled to mass spectrometry were used. In order to determine the optimal conditions and model the variables influencing the extraction efficiency, a central composite rotatable design coupled with response surface methodology was used. Under the optimized conditions, the method achieved good linearities (R > 0.99), satisfactory intra- and inter-day accuracies (97–111%), and intra- and inter-day precision (RSD < 14%). The proposed procedure allowed the detection of the m-PAEs with limit of detection values between 0.02 and 0.10 ng mL-1, which makes the method sensitive and appropriate for assessing internal exposure to phthalates. The applicability of the proposed procedure was evaluated by screening fifty children’s urine from Brazil. High detection frequencies and urinary concentrations of several m-PAEs associated with using personal care products and diet were found.
... However, they are time-consuming and require rather large amounts of organic solvents, which are known to be hazardous to the environment and humans. Therefore, alternative procedures that aggregate speed, simplicity, reduced amounts of samples and reagents, and eliminate the matrix effects associated with analyte preconcentration are in high demand [10,[20][21][22][23]. In this way, microextraction techniques, such as solid-phase microextraction [24], hollow fiber liquid-phase microextraction [25,26], and dispersive liquid-liquid microextraction [27,28], have gained considerable popularity and are becoming alternatives in the analysis of mPAEs in human samples. ...
... Therefore, the pH adjustment is extremely necessary. It is important to highlight that all mPAEs are weak acids with typical pKa values around 3.5 [21,23]. Therefore, the urine samples were acidified with HCl to pH 2 to achieve complete protonation of the -COOH group for the next experiments. ...
... Over the past few Table 3 Analytical performance of the proposed AALLME-LC-MS/MS for determination of phthalate metabolites in human urine samples. [21]. A comparison of the proposed analytical procedure for sample extraction of mPAEs with previously published methods is summarized in Table 4. Compared to liquid-liquid extraction and solid-phase extraction, as classical methodologies, our developed method has the advantages of being simpler, less time-consuming, and using fewer solvent volumes. ...
Urinary phthalate metabolite (mPAEs) analysis is a reliable tool for assessing human exposure to phthalates. With growing interest in urinary biomonitoring of these metabolites, there is a need for fast and sensitive analytical methods. Therefore, a simple, rapid procedure for simultaneous determination of fifteen phthalate metabolites in human urine samples by liquid chromatography-tandem mass spectrometry was developed. The novelty of the present procedure is based on the use of diethyl carbonate as a green biobased extraction solvent and air-assisted liquid-liquid microextraction (AALLME) as a sample preparation step. A Plackett-Burman design was used for screening the factors that influence the AALLME extraction efficiency of mPAEs. The effective factors were then optimized by response surface methodology using a central composite rotatable design. Under the optimized conditions, good linearity can be achieved in a concentration range of 1.0-20.0 ng mL-1 with correlation coefficients higher than 0.99. The repeatability and reproducibility precision were in the range of 2-12% and 1-10% respectively. Recoveries ranging from 90% to 110%. This, and the low limits of detection, ranging from 0.01 to 0.05 ng mL-1, make the proposed procedure sensitive and suitable for human biomonitoring of phthalate exposures. For proof-of-principle, the new method was used to measure the urinary concentrations of mPAEs in 20 urine samples from Brazilian women. The high frequency of detections and in part high concentrations of mPAEs indicate to widespread exposure to several phthalates among Brazilian women.
... PAEs are firstly esterolysed into monophthalic acid esters (MAEs) and phthalic acid (PA), and a small fraction of PA might be also metabolized into protocatechuic acid (PCA) or ultimately broken down into CO 2 and H 2 O (Lin et al., 2017;Mahajan et al., 2019;Ren et al., 2018). Alternatively, the hydrolyzed monoesters of PAEs could be eliminated rapidly from plants and animals through glucuronidation (Ramesh Kumar and Sivaperumal, 2016). The accumulation of DBP is approximately 15 mg/kg at the seeding time while the generation of monobutyl phthalate (MBP) is approximately 10 mg/kg at the jointing time in wheat under stress using 20 mg/kg DBP . ...
Phthalic acid esters (PAEs) are an important group of organic pollutants that are widely used as plasticizers in the environment. The PAEs in soil organisms are likely to be biotransformed into a variety of metabolites, and the combined toxicity of PAEs and their metabolites might be more serious than PAEs alone. However, there are only a few studies on PAE biotransformation by terrestrial animals, e.g. earthworms. Herein, the key biotransformation pathways of PAEs and their association with catalytic enzymes and intestinal symbionts in earthworms were studied using in vivo and in vitro incubation approaches. The widely distributed PAE in soil, dibutyl phthalate (DBP), was proven to be biotransformed rapidly together with apparent bioaccumulation in earthworms. The biotransformation of PAE congeners with medium or long side chains appeared to be faster compared with those with short side chains. DBP was biotransformed into butyl methyl phthalate (BMP), monobutyl phthalate (MBP), and phthalic acid (PA) through esterolysis and transesterification. Besides, the generation of small quantities of low-molecular weight metabolites via β-oxidation, decarboxylation or ring-cleavage, was also observed, especially when the appropriate proportion of NADPH coenzyme was applied to transfer electrons for oxidases. Interestingly, the esterolysis of PAEs was mainly regulated by the cytoplasmic carboxylesterase (CarE) in earthworms, with a Michaelis constant (Km) of 0.416 mM in the catalysis of DBP. The stronger esterolysis in non-intestinal tissues indicated that the CarE was primarily secreted by non–intestinal tissues of earthworms. Additionally, the intestinal symbiotic bacteria of earthworms could respond to PAE stress, leading to the changes in their diversity and composition. The enrichment of some genera e.g. Bacillus and Paracoccus, and the enhancement of metabolism function, e.g. amino acids, energy, lipids biosynthesis and oxidase secretion, indicated their important role in the degradation of PAEs.
... In a recent review, Wang et al. (2019aWang et al. ( , 2019bWang et al. ( , 2019c reported concentrations up to 2.54 μg/mL of major phthalate metabolites in urine, breast milk, serum, semen plasma and nails collected from human subjects in various countries. Kumar and Sivaperumal (2016) also reported high levels of phthalate metabolites in human urine samples, with average total concentrations up to 4.29 μg/mL. ...
Many classes of compounds are known or suspected to disrupt the endocrine system of vertebrate and invertebrate organisms. This review of the sources and fate of selected endocrine disrupting chemicals (EDCs) in the environment includes classes of compounds that are “legacy” contaminants, as well as contaminants of emerging concern. EDCs included for discussion are organochlorine compounds, halogenated aromatic hydrocarbons, brominated flame retardants, per- and polyfluoroalkyl substances, alkylphenols, phthalates, bisphenol A and analogues, pharmaceuticals, drugs of abuse and steroid hormones, personal care products, and organotins. An exhaustive survey of the fate of these contaminants in all environmental media (e.g., air, water, soil, biota, foods and beverages) is beyond the scope of this review, so the priority is to highlight the fate of EDCs in environmental media for which there is a clear link between exposure and endocrine effects in humans or in biota from other taxa. Where appropriate, linkages are also made between the fate of EDCs and regulatory limits such as environmental quality guidelines for water and sediments and total daily intake values for humans.
... primary phthalate metabolites) were ubiquitously detected at all sampling locations. This may be attributed to the metabolic transformation (and excretion) from aquatic species (Hu et al., 2016) or human excretion (Ramesh Kumar and Sivaperumal, 2016). Furthermore, despite its extensive use in products and applications and previous reports on its widespread occurrence in human biofluids (Fox et al., 2011), Bisphenol A was mainly not detected (below detection limits). ...
... lung, heart, brain, skin, adipose, muscles, bone, bone marrow, stomach and intestines (lumped), liver, and kidney) and calculates parent compound and one or more metabolite concentrations in organs over time, as well as the amount excreted in urine. This allows for the study of phthalates as they are rapidly metabolised (Frederiksen et al., 2007) and the concentration of some of their metabolites are an adequate indicator of exposure to the parent compound (Ramesh Kumar and Sivaperumal, 2016). By default, the model assumes physiological and anatomical parameters of a 70 kg reference human; however, several different subjects (e.g. ...
... Furthermore, conversion of a parent compound to one or more metabolites may be modelled in parallel. This is particularly relevant for phthalates as they are rapidly metabolised (Frederiksen, Skakkebaek and Andersson, 2007) and the concentration of some of their metabolites are an adequate indicator of exposure to the parent compound (Ramesh Kumar and Sivaperumal, 2016). A steady-state concentration was only reached for BPA whereas the venous blood concentration of all other substances continues to steadily increase, even when the simulation is performed for a period of 100-300 days. ...
Human biomonitoring (HBM) data can provide insight into co-exposure patterns resulting from exposure to multiple chemicals from various sources and over time. Therefore, such data are particularly valuable for assessing potential risks from combined exposure to multiple chemicals.
One way to interpret HBM data is establishing safe levels in blood or urine, called Biomonitoring Equivalents (BE) or HBM health based guidance values (HBM-HBGV). These can be derived by converting established external reference values, such as tolerable daily intake (TDI) values. HBM-HBGV or BE values are so far agreed only for a very limited number of chemicals. These values can be established using physiologically based kinetic (PBK) modelling, usually requiring substance specific models and the collection of many input parameters which are often not available or difficult to find in the literature.
The aim of this study was to investigate the suitability and limitations of generic PBK models in deriving BE values for several compounds with a view to facilitating the use of HBM data in the assessment of chemical mixtures at a screening level. The focus was on testing the methodology with two generic models, the IndusChemFate tool and High-Throughput Toxicokinetics package, for two different classes of compounds, phenols and phthalates. HBM data on Danish children and on Norwegian mothers and children were used to evaluate the quality of the predictions and to illustrate, by means of a case study, the overall approach of applying PBK models to chemical classes with HBM data in the context of chemical mixture risk assessment.
Application of PBK models provides a better understanding and interpretation of HBM data. However, the study shows that establishing safety threshold levels in urine is a difficult and complex task. The approach might be more straightforward for more persistent chemicals that are analysed as parent compounds in blood but high uncertainties have to be considered around simulated metabolite concentrations in urine. Refining the models may reduce these uncertainties and improve predictions. Based on the experience gained with this study, the performance of the models for other chemicals could be investigated, to improve the accuracy of the simulations.
... However, the sample preparation step is the main challenge when it comes to biological matrices. Therefore, an extraction process is necessary to remove endogenous compounds and to the preconcentration of analytes [38]. ...
Bisphenol A and phthalates are endocrine disruptors widely used as chemical additives mainly in plastic products, including materials for dentistry procedures. Besides, many plasticizers have been associated with important diseases requiring performed methods for their quantification. In the present study, an alternative method for the determination of bisphenol A (BPA) and phthalate metabolites in saliva was developed and validated using hollow fiber liquid phase microextraction (HF-LPME) for sample preparation and gas chromatography coupled to ion trap mass spectrometry (GC/MS) for analysis. A mixture of octanol and ethyl octanoate (1:1 v/v) was used as an acceptor phase in hollow fiber to extract the analytes from saliva samples. A Doehlert design was performed to optimize the variable sample agitation and extraction time. The HF-LPME-GC/MS method developed for saliva analysis showed good selectivity, linearity (R2 > 0.900), and precision (CV = 0.86–18.68%). Limits of detection and quantification ranged from 0.03 to 0.53 μg L−1 and 0.09 to 1.78 μg L−1, respectively. A high concentration of BPA in the oral cavity and oropharyngeal space is a warning of the possible association with the main cancer of the mouth. The method developed and validated was applied to patients with oral squamous cell carcinoma (study group, n = 16) and patients who did not present any oral lesion (control group, n = 16). A principal component analysis was performed and showed a tendency for the association between oral squamous cell carcinoma (OSCC) and plasticizers.
Graphical abstract
... However, gas chromatography (GC) can be used to separate phthalate metabolites, although a derivatization step is needed, which makes it slightly more time-consuming than LC analytics. The GC method nevertheless offers some advantages over LC, especially in the separation of secondary metabolites of high molecular weight phthalates (Gries et al., 2012;Kumar and Sivaperumal, 2016). For example, Gries et al. (2012) presented a GC-high-resolution mass spectrometry (MS) method, which allows the specific determination of DPHP metabolites in the presence of DiDP metabolites. ...
... Typically, the LOD of the reported analytical methods ranges from sub-μg/L to <5 μg/L for the different metabolites in human urine samples (Kumar and Sivaperumal, 2016). This is also the case with most of the occupational biomonitoring studies (Table 2). ...
... The use of validated, sensitive, and accurate analytical methods to measure trace concentrations of phthalate metabolites in humans is also essential for assessing exposure to phthalates. The analysis of phthalate metabolites follows a typical procedure for organic contaminants, namely sample pretreatment, extraction and cleanup, concentration and re-constitution in a suitable solvent, separation by chromatography, and detection by mass spectrometry (Kumar and Sivaperumal, 2016). The LOD (and LOQ) of phthalate metabolites used in the occupational HBM studies should be low enough to compare results across different population subgroups and countries and between occupational HBM studies and national HBM studies in the general population (SpFrance, 2019; CDC, 2019; Koch et al., 2017;Health Canada, 2013). ...
Introduction
Phthalates, a group of ubiquitous industrial chemicals, have been widely used in occupational settings, mainly as plasticizers in a variety of applications. Occupational exposure to different phthalates has been studied in several occupational settings using human biomonitoring (HBM).
Aim
To provide a comprehensive review of the available literature on occupational exposure to phthalates assessed using HBM and to determine future data needs on the topic as part of the HBM4EU project.
Methods
A systematic search was carried out in the databases of Pubmed, Scopus, and Web of Science for articles published between 2000 and September 4, 2019 using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 22 studies on the occupational HBM of phthalates was considered suitable for review.
Results and discussion
Among the reviewed studies, 19 (86%) focused on DEHP, an old phthalate that is now subject to authorization and planned to be restricted in the EU. Concentrations of MEHHP, one of its metabolites, varied up to 13-fold between studies and across sectors when comparing extreme geometric means, ranging from 11.6 (similar to the general populations) to 151 μg/g creatinine. Only 2 studies focused on newer phthalates such as DiNP and DPHP. Concerning the geographical distribution, 10 studies were performed in Europe (including 6 in Slovakia), 8 in Asia, and 4 in North America, but this distribution is not a good reflection of phthalate production and usage levels worldwide. Most HBM studies were performed in the context of PVC product manufacturing. Future studies should focus on: i) a more uniform approach to sampling timing to facilitate comparisons between studies; ii) newer phthalates; and iii) old phthalates in waste management or recycling.
Conclusion
Our findings highlight the lack of recent occupational HBM studies on both old and new phthalate exposure in European countries and the need for a harmonized approach. Considering the important policy actions taken in Europe regarding phthalates, it seems relevant to evaluate the impact of these actions on exposure levels and health risks for workers.
... Indeed, phthalates are not biologically persistent and, therefore, are excreted from the body in a short period through a metabolic pathway that involves hydrolysis and subsequent oxidation reactions. The corresponding phthalate monoesters and oxidation products can combine with glucuronic acid and, therefore, be eliminated as free as well as glucuronyl conjugated forms [1,6]. In general, polar shortchain phthalates do not require the addition of glucuronyl moiety as hydrophilic group to increase their water solubility. ...
... The considerable attention for human risk to phthalates has led to the development of several analytical methods for the assay of phthalates metabolites in urine, mainly based on chromatographic separation [6,9]. The typical analytical approach relies on HPLC with mass spectrometric detection (MS) [7,[10][11][12][13][14][15][16][17] as well as GC with MS [14,[18][19][20][21][22][23] in selected ion monitoring (SIM) acquisition mode. ...
... These protocols require multiple tedious and time-consuming steps for sample purification, they work well only with water-free extracts and, therefore, are error-prone. In these cases, the derivatization contributes significantly to extend the analysis time and, as a result, makes GC-MS methods less attractive for large-scale biomonitoring studies [6]. In this regard, an important advancement was the introduction of alkyl chloroformate as derivatizing agents because they allow for the reliable and simple esterification of acidic moiety directly in the aqueous phase without the need for heating the solution [24]. ...
In the following work, a new method for the analysis of the phthalate monoesters in human urine was reported. Phthalate monoesters are metabolites generated as a result of phthalate exposure. In compliance with the dictates of Green Analytical Chemistry, a rapid and simple protocol was developed and optimized for the quantification of phthalate monoesters (i.e., monoethyl phthalate, monoisobutyl phthalate, mono‐n‐butyl phthalate, mono‐(2‐ethylhexyl) phthalate, mono‐n‐octyl phthalate, monocyclohexyl phthalate, mono‐isononyl phthalate) in human urine, which entails preceding derivatization with methyl chloroformate combined with the use of commercial solid phase microextraction and the analysis by gas chromatography‐triple quadrupole mass spectrometry (GC‐QqQ‐MS). The affinity of the derivatized analytes toward five commercial coatings was evaluated, and in terms of analyte extraction, the best results were reached with the use of the divinylbenzene/carboxen/polydimethylsiloxane fiber. The multivariate approach of experimental design was used to seek for the best working conditions of the derivatization reaction and the solid phase microextraction extraction, thus obtaining the optimum response values. The proposed method was validated according to the guidelines issued by the Food and Drug Administration achieving satisfactory values in terms of linearity, sensitivity, matrix effect, intra‐ and inter‐day accuracy, and precision.
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