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Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing
Abstract
Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1-250 ng/mL), inter- (2.4%-4.3%) and intra-day precision (0.7%-6.1%), accuracy (85%-105%), limit of detection (0.2-0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n = 100), in hospitalized patients receiving blood transfusion (n = 10), and in athletes (n = 468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.
... DEHP is rapidly hydrolyzed to MEHP and subsequently metabolized to form several secondary metabolites, including 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP, and 2cx-MMHP, which are excreted in urine after conjugation with glucuronic acid (29). Several of these metabolites have been demonstrated to be candidate biomarkers in urine for ABT for up to 48 h after reinfusion (6)(7)(8)30), even in supposedly plasticizer-free bags, although the urine concentration of the phthalates was significantly lower (8). Indeed, 5cx-MEPP and 2cx-MMHP seem to be the most sensitive metabolites for detection of blood reinfusion and provide the longest window for detection (8), possibly due to a longer half-life compared with other secondary DEHP metabolites (31), which for 5cx-MEPP is confirmed by the present findings. ...
... For instance, diet is a significant pathway for DEHP exposure (32), and CA-Glu exists in foods such as dairy products (33) and oils (34). Previously, samples from 4 of 127 (6) and 4 of 468 (30) athletes were found to have high levels of DEHP metabolites overlapping with the levels after a blood reinfusion, but whether the athletes performed a 1-10 d). The x indicates whether the marker is included. ...
... blood transfusion remains unknown. Importantly, in more than 180 control subjects, which are less likely to perform a blood transfusion compared with competing athletes, only one subject exceeded the secondary metabolites of DEHP levels observed after a blood reinfusion (6,8,30). Thus, the risk of a false-positive result exists but seems small, which is likely the reason for plasticizers apparently not being utilized in routine measures in antidoping laboratories and as an indirect proof of blood reinfusion. ...
Purpose:
Autologous blood transfusion is performance enhancing and prohibited in sport but remains difficult to detect. This study explored the hypothesis that an untargeted urine metabolomics analysis can reveal one or more novel metabolites with high sensitivity and specificity for detection of autologous blood transfusion.
Methods:
In a randomized, double-blinded, placebo-controlled, cross-over design, exercise-trained males (n=12) donated 900 ml blood or were sham phlebotomized. After four weeks, RBCs or saline were reinfused. Urine samples were collected before phlebotomy and 2 h, 1, 2, 3, 5 and 10 days after reinfusion and analyzed by UPLC-QTOF-MS. Models of unique metabolites reflecting autologous blood transfusion were attained by partial least squares discriminant analysis.
Results:
The strongest model was obtained 2 h after reinfusion with a misclassification error of 6.3% and 98.8% specificity. However, combining only a few of the strongest metabolites selected by this model provided a sensitivity of 100% at days 1 and 2 and 66% at day 3 with 100% specificity. Metabolite identification revealed the presence of secondary di-2-ethylhexyl phtalate metabolites and putatively identified the presence of (iso)caproic acid glucuronide as the strongest candidate biomarker.
Conclusion:
Untargeted urine metabolomics revealed several plasticizers as the strongest metabolic pattern for detection of autologous blood transfusion for up to 3 days. Importantly, no other metabolites in urine appear of value for anti-doping purposes.
... In contrast to flow cytometry, the LC-MS/MS-based method is cost-efficient and less time-consuming [23]. Furthermore, because plasticizers are exogenous compounds, the qualitative "dilute-and-shoot" screening approach can be applied and easily implemented in accredited laboratories [51][52][53]. Nevertheless, the main drawback of this detection method is that phthalates are ubiquitous and can be found in urine after occupational or dietary exposures [54]. ...
... Nevertheless, the main drawback of this detection method is that phthalates are ubiquitous and can be found in urine after occupational or dietary exposures [54]. Consequently, different studies have investigated normal daily exposure to plasticizers to define a threshold for each metabolite in athletes [50,51,55,56]. Using a control phase with saline infusion, Leuenberger et al [50] investigated the specificity of urinary DEHP quantification for the detection of blood transfusion. ...
Despite being prohibited by the World Anti-Doping Agency, blood doping through erythropoietin (EPO) injection or blood transfusion is frequently used by athletes to increase oxygen delivery to muscles and enhance performance. In contrast with allogeneic blood transfusion and erythropoietic stimulants, there is presently no direct method of detection for autologous blood transfusion (ABT) doping. Blood reinfusion is currently monitored with individual follow-up of hematological variables via the Athlete Biological Passport, which requires further improvement. Microdosage is undetectable and suspicious profiles in athletes are often attributed to exposure to altitude, heat stress, or illness. Additional indirect biomarkers may increase the sensitivity and specificity of the longitudinal approach. The emergence of ‘-omics’ strategies provides new opportunities to discover biomarkers for the indirect detection of ABT. With the development of direct quantitative methods, transcriptomics based on microRNA or messenger RNA expression is a promising approach. As blood donation and blood reinfusion alter iron metabolism, quantification of proteins involved in metal metabolism, such as hepcidin, may be applied in an “ironomics” strategy to improve the detection of ABT. As red blood cell (RBC) storage triggers changes in membrane proteins, proteomic methods have the potential to identify the presence of stored RBCs in blood. Alternatively, urine matrix can be used for the quantification of the plasticizer di(2-ethyhexyl) phthalate (DEHP) and its metabolites that originate from blood storage bags suggesting recent blood transfusion, and have an important degree of sensitivity and specificity. This review proposes that various indirect biomarkers should be applied in combination with mathematical approaches for longitudinal monitoring aimed at improving ABT detection.
... [32,35] Plasticizers in urine A promising method was developed to detect blood transfusion misuse, based on the measurement of the metabolites of the plasticizer di-ethylhexylphthalate (DEHP) in urine. [36][37][38] .The use of DEHP has been extended in medical devices, especially in the bags used to store blood products, which have been authorized for the last three decades. The good preservation conditions of blood and its components are the main benefits of using DEHP in the bags, [39][40][41][42] although there is a high exposition to this chemical during the transfusion process. ...
... These results were corroborated in other studies. Solymos et al. [37] measured the same DEHP metabolites (MEHP, MEHHP, MEOHP) in a control group, in hospitalized patients receiving blood transfusions and in athletes. The investigation also demonstrated that significantly increased levels of these DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion. ...
The use of blood doping is forbidden by the World Anti‐Doping Agency. Several practices, such as blood transfusions are used to increase oxygen delivery to muscles and all of them are highly pursued. In this regard, the development of accurate methodologies for detecting these prohibited practices is one of the current aims of the anti‐doping control laboratories. Flow cytometry methods are able to detect allogeneic blood transfusions but there is no official methodology available to detect autologous blood transfusions.
This paper reviews protocols, including the Athlete Biological Passport, that use indirect markers to detect misuse of blood transfusions, especially autologous blood transfusions. The methods of total haemoglobin mass measurements and the detection of metabolites of blood bags plasticizers in urine are reviewed. The latter seems to be an important step forward because it is a fast screening method and it is based on urine, a fluid widely available for doping control. Other innovative approaches to blood transfusion detection are also mentioned. A combination of the reported methodologies and the implementation of the Athlete Biological Passport is becoming a promising approach. Copyright © 2012 John Wiley & Sons, Ltd.
... Providing a longer shelf life for blood bags intended for reinfusion, di-(2-294 ethylhexyl)phthalate (DEHP) is a plasticizer commonly applied during blood storage to 295 preserve the flexibility of the plastic bag 107 . Therefore, an initial study investigated the 296 presence of DEHP metabolites using liquid chromatography-tandem mass spectrometry (performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) 300 quantification method 109,110 , these results were later confirmed in healthy moderately trained 301 individuals 111 , even with bags claimed to be plasticizer-free 112 , where 5cx-MEPP and 2cx-302 MMHP emerged as the most interesting markers because of their extended half-life 13 . In 303 addition, DEHP was also detected in blood samples using GC-MS, although the very short 304 detection window limits its interest 113 . ...
The detection of blood doping represents a current major issue in sports and an ongoing challenge for anti-doping research. Initially focusing on direct detection methods to identify a banned substance or its metabolites, the anti-doping effort has been progressively complemented by indirect approaches. The longitudinal and individual monitoring of specific biomarkers aims to identify non-physiological variations that may be related to doping practices. From this perspective, the identification of markers sensitive to erythropoiesis alteration is key in the screening of blood doping. The current Athlete Biological Passport (ABP) implemented since 2009 is composed of 14 variables (including two primary markers, i.e. hemoglobin concentration and OFF-score) for the hematological module to be used for indirect detection of blood doping. Nevertheless, research has continually proposed and investigated new markers sensitive to an alteration of the erythropoietic cascade, and specific to blood doping. If multiple early markers have been identified (at the transcriptomic level) or developed directly in a diagnostics' kit (at a proteomic level), other target variables at the end of the erythropoietic process (linked with the red blood cell functions) may strengthen the hematological module in the future. Therefore, this review aims to provide a global systematic overview of the biomarkers considered to date in the indirect investigation of blood doping.
... The diffusion property of DEHP is also used for sports drug screening. Therefore, detection of DEHP and its metabolites which derive from tube systems and blood bags can provide evidence of cumulative autologous (detection of extrinsic blood system antigens is not possible) and allogenic blood transfusions [9,10]. ...
... The method used in LAB 2 was previously described and it was different regarding sample preparation and instrumental analysis. 17 Concentrations used for the results presented in this paper were those of LAB 1. ...
... Dilution of urine samples is generally practiced to reduce the sample-to-sample matrix variation that can affect analyte recovery. Direct analysis without any extraction/cleanup, the so-called "dilute and shoot" methods, has been reported as a rapid screening tool in dope testing [14,15]. However, further cleanup/concentration steps are required for the removal of endogenous matrix, which otherwise cause clogging of HPLC column and influence analyte ionization in electrospray ionization (ESI) source. ...
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 suitable biomarkers of exposure to short- and long-chain phthalates, respectively. The determination of urinary phthalate metabolites significantly reduces the "phthalate blank problem," which arises due to the ubiquitous presence of this chemical compound in laboratory atmosphere. Sample preparation and analytical methodologies for the determination of urinary phthalate metabolites by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography- tandem mass spectrometry (LC-MS/MS) techniques were discussed. Issues on the validity of urinary phthalate metabolite data, such as intra- and interpersonal variations, variability in population subgroups, and variability due to time and type of urine sample collection, are discussed. Measures to minimize uncertainties associated with urinary phthalate metabolite concentration are also suggested.
... Most liquid chromatographic methods for MPAEs separation are carried out on phenyl-type stationary phases (SPs) [10][11][12][13][14][15][16][17][18][19][20][21], although octadecylsilane (ODS) SPs are sometimes used [21,22]. The employ of other types of SPs has been rather limited [23,24]. ...
The retention behavior of nine MPAEs has been studied, using commercial LC columns with octadecylsilane (ODS), phenyl, and amide-type SPs. First, it was found that the use of methanol in the mobile phase is not advisable, because induce a transesterification reaction of MPAEs in the electrospray ion source, regardless of the SP used. On the other hand, different responses were observed when representing the logarithm of retention factors (k) vs. the volume fraction of ACN (φ) in the mobile phase, for the three SPs tested. A quite linear trend was obtained for ODS (at φ values below 0.80) and Phenyl columns. On the contrary, the Amide column shows a striking U-shape trend, typical of both hydrophobic and hydrophilic retention mechanisms. Therefore, the separation process was mainly hydrophobic in the ODS and phenyl SPs, but in the amide-type a dual retention mechanism was found, showing zones with predominant hydrophobic or hydrophilic interactions, depending on both the compound and the experimental conditions. A high content of acetonitrile (>75%) and low concentration of formic acid in the mobile phase promote the hydrophilic separation mechanism for MPAEs on the amide SP. So, this dual separation mechanism can be modulated modifying the pH and content of organic modifier in the mobile phase, allowing greater flexibility to develop improved methods. Taking advantage of this, a separation method was optimized in this amide column using a Box-Wilson Central Composite experimental design, which allows separating the studied MPAEs with a time-saving of around 40% comparing to the conventional phenyl SP.
... Since the presence of DEHP is not specific for the prior use of blood transfusion, it has been proposed that threshold levels of phthalate contamination could be used to provide supporting evidence against athletes suspected of using this banned practice [14]. Urinary concentration of phthalate metabolites showed considerable intra-individual variation yet were not comparable to the Table 1. ...
Background:
A method to detect the banned use of blood transfusions by athletes has proven elusive. In this article, we investigate the utility of analyzing contaminant levels in stored blood as a possible marker. The presence of the plasticizer di(2-ethylhexyl)phthalate (DEHP) was measured in red blood cells collected from transfused subjects.
Results:
GC-MS detected high levels of DEHP in stored red cells. However, values fell rapidly after the cells were reinfused, with only half of the samples exceeding a cut-off 4 SDs above the baseline 90 min postinfusion.
Conclusion:
It was demonstrated that intact DEHP could be measured in red blood cells. Owing to its short window of detection, this approach seems to have limited utility in the context of antidoping.
... A nondirect method, based on the identification of plasticizers (di-2-ethylhexylphthalate) metabolites excreted in the urine as a result of autologous blood transfusion, was recently proposed. The rationale for this strategy of detection is the presence of di-2ethylhexylphthalate in certain transfusion bags and, it is known to diffuse into the stored blood (Solymos et al., 2010). Nevertheless, this method has several drawbacks and is not currently implemented as a doping detection tool (Lombardi et al., 2012). ...
Blood doping has been defined as the misuse of substances or certain techniques to optimize oxygen delivery to muscles with the aim to increase performance in sports activities. It includes blood transfusion, administration of erythropoiesis-stimulating agents or blood substitutes, and gene manipulations. The main reasons for the widespread use of blood doping include: its availability for athletes (erythropoiesis-stimulating agents and blood transfusions), its efficiency in improving performance, and its difficult detection. This article reviews and discusses the blood doping substances and methods used for in sports, the adverse effects related to this practice, and current strategies for its detection.
... Phthalates are colorless and odorless oily liquids with high boiling points (280 -4008C), low volatility and limited water solubility [octanol -water partition coefficient (log Kow) ¼ 1.6 -8.1] (3,4). As pollutants in the environment, phthalates degrade very slowly through hydrolysis of an ester bond to the corresponding monoester, hydroxylation of the alkyl moiety, decarboxylation and finally, mineralization (5,6). Phthalates are used extensively as plasticizers to enhance the strength, softness and flexibility of plastic (7), and as solvents in various consumer products such as epoxy adhesives, cosmetics, fragrances, paints and printing inks (1,8). ...
A simple, accurate and reliable method was presented and validated for the simultaneous monitoring of six phthalates at trace level concentrations in seven different brands of commercial bottled mineral water from Jordan. Liquid-liquid extraction with a mixture of methylene chloride-petroleum ether (20:80, v/v) was used for isolation and enrichment of the phthalates and sample cleanup. This was followed by gas chromatography-mass spectrometry (GC-MS) for identification and high-performance liquid chromatography (HPLC) with ultraviolet detection for quantification. The linear range of the GC-MS calibration curve was 0.3-1.2 µg/L with a mean correlation coefficient (R(2)) of 0.9920 ± 0.0063, the detection limit was < 0.1 µg/L and the percentage recovery was >90%. For HPLC, the linear range was 0.5-10 mg/L with R(2) = 0.9985 ± 0.0012 and an average detection limit of 0.20 ± 0.15 µg/L. The results indicated that the Jordanian bottled water was contaminated with dibutyl-, di-2-ethylhexyl- and di-n-octyl-phthalate, with total phthalate concentrations between 8.1 and 19.8 µg/L. Increasing the storage temperature of the bottled water increased the content of leached phthalates in the water (total concentration of 23-29.2 µg/L).
... withdrawal and re-transfusions with one's own blood) are a prohibited method of the World Anti Doping Agency (WADA), there is no direct detection method for the procedure. Several non-direct methods have been proposed including the analysis of plastic residues (dioctyl phthalate) (Solymos et al., 2010) present in blood following storage in certain transfusion bags (but not all), gene expression changes (Pottgiesser et al., 2009), microR-NAs and proteomics (ongoing projects) and the concept of the ABP first introduced some 10 years ago (Cazzola, 2000;Malcovati et al., 2003), perhaps with the addition of nHb to the ABP (Mørkeberg et al., 2011). Of these, only the ABP seems realistically applicable at the present time and will be discussed following the section on Detection of rhEpo injections since the ABP is also designed to detect rhEpo misuse. ...
Blood doping practices in sports have been around for at least half a century and will likely remain for several years to come. The main reason for the various forms of blood doping to be common is that they are easy to perform, and the effects on exercise performance are gigantic. Yet another reason for blood doping to be a popular illicit practice is that detection is difficult. For autologous blood transfusions, for example, no direct test exists, and the direct testing of misuse with recombinant human erythropoietin (rhEpo) has proven very difficult despite a test exists. Future blood doping practice will likely include the stabilization of the transcription factor hypoxia-inducible factor which leads to an increased endogenous erythropoietin synthesis. It seems unrealistic to develop specific test against such drugs (and the copies hereof originating from illegal laboratories). In an attempt to detect and limit blood doping, the World Anti-Doping Agency (WADA) has launched the Athlete Biological Passport where indirect markers for all types of blood doping are evaluated on an individual level. The approach seemed promising, but a recent publication demonstrates the system to be incapable of detecting even a single subject as 'suspicious' while treated with rhEpo for 10-12 weeks. Sad to say, the hope that the 2012 London Olympics should be cleaner in regard to blood doping seems faint. We propose that WADA strengthens the quality and capacities of the National Anti-Doping Agencies and that they work more efficiently with the international sports federations in an attempt to limit blood doping.
... The method used in LAB 2 was previously described and it was different regarding sample preparation and instrumental analysis. 17 Concentrations used for the results presented in this paper were those of LAB 1. ...
Misuse of autologous blood transfusions in sports remains undetectable. The metabolites of the plasticizer di-(2-ethylhexyl)phthalate (DEHP) were recently proposed as markers of blood transfusion, based on high urinary concentrations of these compounds observed in patients subjected to blood transfusion. This study evaluates DEHP metabolites in urine for detecting autologous blood transfusion.
One blood bag was drawn from moderately trained subjects and the red blood cells (RBCs) were reinfused after different storage periods. Group 1 (12 subjects) was reinfused after 14 days, and Group 2 (13 subjects), after 28 days of storage. Urine samples were collected before and after reinfusion for determination of the concentrations of three DEHP metabolites, mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate, and mono-(2-ethyl-5-oxohexyl)phthalate.
Concentrations of DEHP metabolites on the days before reinfusion were in agreement with those described after common environmental exposure. A few hours after the reinfusion a significant increase was observed for all metabolites in all volunteers. Concentrations 1 day later were still higher (p < 0.05) than before reinfusion. Variations in urine dilution supported normalization by specific gravity. Concentrations of DEHP metabolites tended to be higher after longer storage times of RBCs.
Autologous transfusion with RBCs stored in plastic bags provokes an acute increase in the urinary concentrations of DEHP metabolites, allowing the detection of this doping malpractice. The window of detection is approximately 2 days. The method might be applied to urine samples submitted for antidoping testing.
Tri-(2-ethylhexyl) trimellitate (TOTM or TEHTM) is a substitute for the plasticizer di-(2-ethylhexyl) phthalate (DEHP). Here, a fast and robust HPLC method is presented for the first time enabling the simultaneous quantification of several TEHTM metabolites in urine. These are the three TEHTM monoester isomers 1-mono-(2-ethylhexyl) trimellitate (1-MEHTM), 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM), and 4-mono-(2-ethylhexyl) trimellitate (4-MEHTM) as well as several selected side chain oxidized monoesters of TEHTM, namely, 1-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-1-MEHTM), 2-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-2-MEHTM), 1-mono-(2-ethyl-5-oxohexyl) trimellitate (5oxo-1-MEHTM), 2-mono-(2-ethyl-5-oxohexyl) trimellitate (5oxo-2-MEHTM), 1-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-1-MEPTM), 2-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-2-MEPTM), 2-mono-(2-carboxymethylhexyl) trimellitate (2cx-2-MMHTM), and 1-mono-(2-carboxymethylhexyl) trimellitate (2cx-1-MMHTM). The method is characterized by a short sample preparation, for which the urine samples are enzymatically hydrolyzed and cleaned up by an online column arrangement. Separation of the analytes is enabled using liquid chromatography coupled with tandem mass spectrometry. Thus, in less than 30 min, 11 postulated metabolites of TEHTM can be selectively and sensitively quantified. The method is distinguished by its wide linear working range of up to 1800 μg/L with detection limits ranging from 0.3 μg/L (for 5oxo-1-MEHTM) to 1.5 μg/L (for 1-MEHTM). Precision and repeatability of the method were proven with determined relative standard deviations between 2.5 and 11.3%. The selection of the analytes of this method was based on a pilot study, by which several potential TEHTM metabolites were investigated in human urine of an orally exposed volunteer. Thus, the here presented method is a perfect tool for human biomonitoring of TEHTM exposure.
Screening and quantification of phthalate metabolites in biological matrices provides information on the phthalate exposure. The preferred tool for the determination of phthalate metabolites is liquid chromatography-mass spectrometry, typically preceded by a sample extraction step. Method development for the determination of phthalate metabolites by hyphenated techniques faces challenges due to the widespread occurrence of phthalates in the laboratory and sample collection materials which impairs their accurate quantification. Here the analytical methods that have been developed for the determination of biomarkers of phthalates in various matrices are presented, and limitations and challenges in these applications are discussed.
This research was mainly focused on the effects of food emulsifier on the bioavailability of six priority controlling phthalate acid esters (PAEs) for the further accurate assessment of their toxic effects, using the corresponding phthalate acid monoesters (PAMEs) in rats urine as biomarkers. Glycerin monostearate was chosen as typical food emulsifier. A method was established to determine PAMEs in urine from rats either in experimental group (integrated gavaged with glycerin monostearate and PAEs) or in control group (gavaged with PAEs only), by using solid-phase extraction (SPE) coupled with ultra performance liquid chromatography tandem mass spectrometry (SPE–UPLC–MS/MS). Extraction recoveries were more than 75% for all the PAMEs; the calibration curve was linear in the range of 1.0–1000.0 ng/mL with R2 > 0.995; the limits of detection (LOD) were 0.30 ng/mL–0.50 ng/mL. In addition, by analysing quality control (QC) urine samples in 3 days, it showed that the method was precise and accurate, for the intra-day and inter-day RSD within 16%, and the accuracy more than 82%. Internal exposure amount of all PAEs in experimental group was significantly higher than that in control group with p values of less than 0.05 except for butyl benzyl phthalates (BBP) (P = 0.07). The bioavailability of all PAEs ranged from 5.03% to 109.35% with the presence of food emulsifiers glycerin monostearate, observably higher than that without glycerin monostearate (1.12% to 54.39%). It indicated that food emulsifiers increased the bioavailability of PAEs and may lead to potential food safety risk, which should bring awareness and be further studied.
RATIONALEIn recent years, several ambient ionization techniques, where solid and/or liquid samples are brought directly into the ion source without any sample preparation and chromatographic separation, have been introduced for mass spectrometric (MS) analyses. Using the direct inlet probe–atmospheric pressure chemical ionization (DIP-APCI)-MS and DIP-APCI-MSn methods presented here, a non-destructive screening analysis for plasticizers directly from plastic articles can be performed.METHODS
The DIP-APCI ion source developed in our laboratory uses a temperature-programmed push rod to introduce solid or liquid samples into a homemade APCI ion source. The DIP-APCI ion source was coupled to an ion trap (IT) mass spectrometer and selected source parameters were optimized. To enable a screening analysis for plasticizers, standards substances of several phthalates and other plasticizers were analyzed and their fragmentation behavior during collision-induced dissociation (CID) was studied.RESULTSUsing DIP-APCI-ITMS, plasticizers can be detected directly from plastic articles and identification is possible through MSn experiments. For example, the isomeric phthalates di(2-ethylhexyl) phthalate and di-n-octyl phthalate can be differentiated according to their fragmentation behavior.CONCLUSIONS
There are several advantages of the DIP-APCI source in comparison to many other ambient desorption ion sources: (i) well-defined gas phase matrix, (ii) precisely adjustable reagent gases (e.g. O2 for negative APCI), (iii) well-defined probe temperature, and (iv) fully automated operation. Copyright © 2014 John Wiley & Sons, Ltd.
The collection of blood, its storage as red blood cell (RBC) concentrates and its reinjection is prohibited; until now, the practice cannot be reliably detected. A recent innovation-the haematological module of the athlete's biological passport-can provide authorities with indications towards autologous blood transfusion. In situations where a given athlete has been exposed to altitude, heat stress, sickness, etc, additional evidence may be needed to establish beyond any reasonable doubt that a blood transfusion may actually have occurred. Additional evidence may be obtained from at least three different approaches using parameters related to blood and urine matrices.Genomics applied to mRNA or miRNA is one of the most promising analytical tools. Proteomics of changes associated with RBC membranes may reveal the presence of cells stored for some time, as can an abnormal pattern of size distribution of aged cells. In urine, high concentrations of metabolites of plasticisers originating from the blood storing bags strongly suggest a recent blood transfusion. We emphasise the usefulness of simultaneously obtaining and then analysing blood and urine for complementary evidence of autologous blood transfusion ('blood doping').
During the last 30 years, the artificial increase of red blood cell volume ("blood doping") has changed the level of performance in all endurance sports. Many doping scandals have shown the extent of the problem. The detection of blood doping relies on two different approaches: the direct detection of exogenous manipulating substances (erythropoietic stimulants) or red cells (homologous transfusion) and the indirect detection, where not the doping substance or technique itself, but its effect on certain biomarkers is measured. Whereas direct detection using standard laboratory procedures such as isoelectric focusing can identify erythropoietic stimulants, homologous blood transfusion is identified through mismatches in minor blood group antigens by flow cytometry. Indirect methods such as the athlete biological passport are the only means to detect autologous transfusion and may also be used for the detection of erythropoietic stimulants or homologous transfusion. New techniques to unmask blood doping include the use of high-throughput 'omics' technologies (proteomics/metabolomics) and the combination of different biomarkers with the help of mathematical approaches. Future strategies should aim at improving the use of the available data and resources by applying pattern recognition algorithms to recognize suspicious athletes and, on the basis of these findings, use the appropriate testing method. Different types of information should be combined in the quest for a forensic approach to anti-doping.
Phthalates, which are ubiquitous in the environment, are readily metabolized in human bodies to their respective monoesters. These phthalate monoesters are non-persistent with short half-lives, which make them the ideal biomarkers of human exposure to phthalates. In this study a direct analysis method without preconcentration was developed and validated for the following phthalate ester metabolites in urine: mono-(2-ethylhexyl) phthalate, mono-(2-ethyl-5-hydroxyhexyl) phthalate, mono-(2-ethyl-5-oxohexyl)phthalate, monobenzyl phthalate, mono-isobutylphthalate, mono-n-butyl phthalate and monoethyl phthalate. The recovery of the phthalate ester metabolites varied between 97% and 104%. The intraday precision for the replicate analysis (n=10) of a urine sample did not exceed 5% for most of the compounds. The coefficient of variance amounted to 2-3%. The limit of quantification was set equal to 0.5μg/L for the majority of the compounds. A comparison between the direct analysis method and a foregoing solid phase extraction (SPE) of the urine sample was made. Finally, the applicability of the direct analysis method was tested in three interlaboratory comparisons.
Di-(2-ethylhexyl)phthalate (DEHP) is the most commonly used plasticizer for polyvinyl chloride, which is found in a large variety of products, including most of the bags used for blood storage because of its protective role on erythrocytes survival. DEHP metabolites have been recently proposed as markers of the misuse of blood transfusion in athletes. In this study, a method to quantify the main five DEHP metabolites in urine has been developed: mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP), and mono-(2-carboxymethylhexyl)phthalate (2cx-MMHP). The method involved an enzymatic hydrolysis with β-glucuronidase from Escherichia coli followed by an acidic extraction with ethyl acetate. The hydrolysed extracts were analysed by ultraperformance liquid chromatography tandem mass spectrometry. Isotope labelled MEHP, MEOHP and 5cx-MEPP were used as internal standards. Analysis of all the metabolites was achieved in a total run time of 10min, using a C(18) column and a mobile phase containing deionized water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6mLmin(-1). Detection of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive and negative ion modes. The method was validated for quantitative purposes. Extraction recoveries were greater than 90% and the limits of quantitation ranged from 1.2 to 2.6ngmL(-1). Intra-day precisions were better than 8% for all metabolites while inter-assay precisions were better than 12%. Concentrations of DEHP metabolites were measured in a control group (n=30, subjects reflecting the common environmental DEHP exposure), and in sportsmen (n=464), to evaluate population distribution exposure to DEHP. Additionally, threshold concentrations indicating outliers of common exposure for DEHP metabolites are proposed.
Prenylamine is a vasodilator of phenylalkylamine structure and was used for the treatment of angina pectoris, until reports of undesirable effects including ventricular tachycardia led to a decreasing use of the drug in the 1980s. Metabolic N-dealkylation of orally ingested prenylamine can liberate amphetamine in humans and cause positive findings for amphetamine in doping and forensic analysis. In 2010, the World Anti-Doping Agency (WADA) classified prenylamine as a non-specified stimulant according to the 2010 Prohibited List, thus banning its use in sports in-competition. Supporting the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based detection method, a post-administration urine sample following a single oral prenylamine ingestion (Segontin® 60 mg) was analyzed for urinary metabolites. The LC-separated analytes were ionized in positive electrospray ionization (ESI) mode and detected as protonated ions using an AB Sciex TripleTOF 5600 quadrupole-time-of-flight hybrid mass spectrometer. Over 40 phase I metabolites were detected, including previously unknown mono- bis-, tris- and tetra-hydroxylated prenylamine, several hydroxylated and methoxylated prenylamine metabolites and (hydroxylated) diphenylpropylamine. Investigation of the collision-induced dissociation behaviours of the metabolites by high resolution/high accuracy mass spectrometry allowed for the assignment of the nature and the site of observed metabolic transformations. The most abundant phase I metabolite was confirmed as p-hydroxy-prenlyamine by chemical synthesis and stable isotope labelling of reference material. An existing routine screening assay based on direct injection and LC-MS/MS analysis of urine was modified and validated according to common guidelines, in order to allow for the detection of p-hydroxy-prenylamine in sports drug testing. The assay demonstrated the ability to detect the target metabolite at 0.1 ng/ml at intra- and inter-day imprecisions below 10%. Copyright © 2012 John Wiley & Sons, Ltd.
Biomarker monitoring can be considered a new era in the effort against doping. Opposed to the old concept in doping control of direct detection of a prohibited substance in a biological sample such as urine or blood, the new paradigm allows a personalized longitudinal monitoring of biomarkers that indicate non-physiological responses independently of the used doping technique or substance, and may cause sanctioning of illicit practices. This review presents the development of biomarker monitoring in sports doping control and focuses on the implementation of the Athlete Biological Passport as the current concept of the World Anti Doping Agency for the detection of blood doping (hematological module). The scope of the article extends to the description of novel biomarkers and future concepts of application.
International anti-doping efforts are harmonized and regulated under the umbrella of the World Anti-Doping Code and the corresponding Prohibited List, issued annually by the World Anti-Doping Agency (WADA). The necessity for a frequent and timely update of the Prohibited List (as the result of a comprehensive consultation process and subsequent consensual agreement by expert panels regarding substances and methods of performance manipulation in sports) is due to the constantly growing market of emerging therapeutics and thus new options for cheating athletes to illicitly enhance performance. In addition, 'tailor-made' substances arguably designed to undermine sports drug testing procedures are considered and the potential of established drugs to represent a doping substance is revisited in light of recently generated information. The purpose of the annual banned substance review is to support doping controls by reporting emerging and advancing methods dedicated to the detection of known and recently outlawed substances. This review surveys new and/or enhanced procedures and techniques of doping analysis together with information relevant to doping controls that has been published in the literature between October 2010 and September 2011.
Recent developments in MS for the detection of small molecules in the context of doping control analysis are reviewed. Doping control analysis is evolving together with MS, which is the technique of choice in order to accomplish the analytical requirements in this field. Since these analytical requirements for the detection of a doping agent depend on the substance, in the first section we review the different scenarios. The commonly established approaches, together with their achievements and drawbacks are described. New developments in hyphenated MS techniques (both GC-MS/MS and LC-MS/MS) concerning interfaces and analyzers are mentioned. The use (or potential use) of these developments in order to minimize the limitations of the commonly established approaches in the doping control field is discussed. Finally, a brief discussion about trends and remaining limitations is presented.
To indicate homologous or autologous blood transfusion in sports drug testing, quantification of increased urinary concentrations of di(2‐ethylhexyl) phthalate (DEHP) metabolites presents a promising approach; however, the possible intra‐individual variation of the metabolite concentrations over time has not been well characterized.
The aim of this study was to explore the intra‐individual variability of urinary DEHP metabolites among seven volunteers without special occupational exposure to DEHP during one week (n = 253) in order to investigate the possibility of increased urinary concentrations of the metabolites caused by, for example, residential, dietary, or environmental exposure.
Quantification of three DEHP metabolites – mono(2‐ethylhexyl) phthalate, mono(2‐ethyl‐5‐oxohexyl) phthalate, and mono(2‐ethyl‐5‐hydroxyhexyl) phthalate – was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope‐dilution liquid chromatography‐tandem mass spectrometry.
Although urinary concentrations of DEHP metabolites showed considerable intra‐individual variation, no increased values were observed comparable to the concentrations measured in urine specimens collected after blood transfusion. Copyright © 2011 John Wiley & Sons, Ltd.
A new multi‐target approach based on liquid chromatography – electrospray ionization tandem mass spectrometry (LC‐(ESI)‐MS/MS) is presented to screen for various classes of prohibited substances using direct injection of urine specimens. With a highly sensitive new generation hybrid mass spectrometer classic groups of drugs – for example, diuretics, beta2‐agonists – stimulants and narcotics are detectable at concentration levels far below the required limits.
Additionally, more challenging and various new target compounds could be implemented. Model compounds of stimulant conjugates were studied to investigate a possible screening without complex sample preparation. As a main achievement, the integration of the plasma volume expanders dextran and hydroxyethyl starch (HES), commonly analyzed in time‐consuming, stand‐alone procedures, is accomplished. To screen for relatively new prohibited compounds, a common metabolite of the selective androgen receptor modulator (SARMs) andarine, a metabolite of growth hormone releasing peptide (GHRP‐2), and 5‐amino‐4‐imidazolecarboxyamide ribonucleoside (AICAR) are analyzed. Following a completely new approach, conjugates of di(2‐ethylhexyl) phthalate (DEHP) metabolites are monitored to detect abnormally high levels of plasticizers indicating for illicit blood transfusion.
The assay was fully validated for qualitative purposes considering the parameters specificity, intra‐ (3.2–16.6%) and inter‐day precision (0.4–19.9%) at low, medium and high concentration, robustness, limit of detection (1–70 ng/ml, dextran: 30 µg/ml, HES: 10 µg/ml) and ion suppression/enhancement effects.
The analyses of post‐administration and routine doping control samples demonstrates the applicability of the method for sports drug testing. This straightforward and reliable approach accomplishes the combination of different screening procedures resulting in a high‐throughput method that increases the efficiency of the labs daily work. Copyright © 2011 John Wiley & Sons, Ltd.
Autologous blood transfusions (ABTs) has been used by athletes for approximately 4 decades to enhance their performance. Although the method was prohibited by the International Olympic Committee in the mid 1980s, no direct detection method has yet been developed and implemented by the World Anti-Doping Agency (WADA). Several indirect methods have been proposed with the majority relying on changes in erythropoiesis-sensitive blood markers. Compared with the first methods developed in 1987, the sensitivity of subsequent tests has not improved the detection of blood doping. Nevertheless, the use of sophisticated statistical algorithms has assured a higher level of specificity in subsequent detection models, which is a crucial aspect of antidoping testing particularly to avoid "false positives." Today, the testing markers with the best sensitivity/specificity ratio are the Hbmr model (an algorithm based on the total amount of circulating hemoglobin level [hemoglobin level mass] and percentage of reticulocytes, 4.51·ln(Hbmass)-√%ret) and the OFF-hr model (algorithm based on hemoglobin level concentration and percentage of reticulocytes, Hb(g/L)-60·√%ret). Only the OFF-hr model is currently approved by WADA. Recently, alternative indirect strategies for detecting blood doping have been proposed. One method is based upon a transfusion-induced immune-response resulting in specific changes in gene expression related to leukocytes such as T lymphocytes. Another method relies on detecting increased plasticizer metabolite levels in the urine caused by the leakage of plasticizers from the blood bags used during the blood storage. These methods need further development and validation across different types of transfusion regimes before they can be implemented. In addition, several research projects have been funded by WADA in recent years and are now under development including "Detection of Autologous Blood Transfusions Using Activated Red Blood Cells (the red blood cells eNOS system)" and "Detection of Autologous Blood Transfusion by Proteomic: Screening to find Unique Biomarkers, Detecting Blood Manipulation from Total Hemoglobin Mass using 15-nitric Oxide as a Tracer Gas, Storage Contamination as a Potential Diagnostic Test for Autologous Blood Transfusion and Test for Blood Transfusion (Autologous/Homologous) based on Changes of Erythrocyte Membrane Protome" (WADA, WADA Funded Research Projects. http://www.wada-ama.org/en/Science-Medicine/Research/Funded-Research-Projects/. 2010). Although strategies to detect autologous blood transfusion have improved, a highly sensitive test to detect small volumes of transfused autologous blood has not yet been implemented.
Aerobic sport performance may be strongly influenced by the number of red blood cells available for transport and delivery of oxygen from lungs to muscles. Often, athletes search for an acute increase in red blood cells by means of blood transfusions. This paper reviews the possibilities for detecting such prohibited practice. Flow cytometry methods are able to detect a double population of red blood cell membrane surface antigens, thus revealing an allogeneic transfusion. Other ingenious approaches for total hemoglobin mass measurements or to test for the metabolites of blood bag plasticizers in urine are new trends for facing the detection of autologous transfusions. Steady increase of red blood cell number may be obtained also by erythropoietic stimulant agents such as erythropoietin, analogs and mimetics. The challenge of detecting those substances has stimulated the development of indirect markers of altered erythropoiesis, leading to the consequent development of the hematological blood passport approach, which is gaining legal acceptance.
During the past decade OMICS-methods not only continued to have their impact on research strategies in life sciences and in particular molecular biology, but also started to be used for anti-doping control purposes. Research activities were mainly reasoned by the fact that several substances and methods, which were prohibited by the World Anti-Doping Agency (WADA), were or still are difficult to detect by direct methods. Transcriptomics, proteomics, and metabolomics in theory offer ideal platforms for the discovery of biomarkers for the indirect detection of the abuse of these substances and methods. Traditionally, the main focus of transcriptomics and proteomics projects has been on the prolonged detection of the misuse of human growth hormone (hGH), recombinant erythropoietin (rhEpo), and autologous blood transfusion. An additional benefit of the indirect or marker approach would also be that similarly acting substances might then be detected by a single method, without being forced to develop new direct detection methods for new but comparable prohibited substances (as has been the case, e.g. for the various forms of Epo analogs and biosimilars). While several non-OMICS-derived parameters for the indirect detection of doping are currently in use, for example the blood parameters of the hematological module of the athlete's biological passport, the outcome of most non-targeted OMICS-projects led to no direct application in routine doping control so far. The main reason is the inherent complexity of human transcriptomes, proteomes, and metabolomes and their inter-individual variability. The article reviews previous and recent research projects and their results and discusses future strategies for a more efficient application of OMICS-methods in doping control.
Human metabolism of di(2-ethylhexyl)phthalate (DEHP) was studied after a single oral dose of 48.1mg to a male volunteer. To avoid interference by background exposure the D4-ring-labelled DEHP analogue was dosed. Excretion of three metabolites, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP), was monitored for 44h in urine and for 8h in serum. Peak concentrations of all metabolites were found in serum after 2h and in urine after 2h (MEHP) and after 4h (5OH-MEHP and 5oxo-MEHP). While the major metabolite in serum was MEHP, the major metabolite in urine was 5OH-MEHP, followed by 5oxo-MEHP and MEHP. Excretion in urine followed a multi-phase elimination model. After an absorption and distribution phase of 4 to 8h, half-life times of excretion in the first elimination phase were approximately 2h with slightly higher half-life times for 5OH- and 5oxo-MEHP. Half-life times in the second phase—beginning 14 to 18h post dose—were 5h for MEHP and 10h for 5OH-MEHP and 5oxo-MEHP. In the time window 36 to 44h, no decrease in excreted concentrations of 5OH- and 5oxo-MEHP was observed. In the first elimination phase (8 to 14h post dose), mean excretion ratios of MEHP to 5oxo-MEHP and MEHP to 5OH-MEHP were 1 to 1.8 and 1 to 3.1. In the second elimination phase up to 24h post dose mean excretion ratios of MEHP to 5oxo-MEHP to 5OH-MEHP were 1 to 5.0 to 9.3. The excretion ratio of 5OH-MEHP to 5oxo-MEHP remained constant through time at 1.7 in the mean. After 44h, 47% of the DEHP dose was excreted in urine, comprising MEHP (7.3%), 5OH-MEHP (24.7%) and 5oxo-MEHP (14.9%).
Phthalates are metabolized and eliminated in urine within hours after exposure. Several reports suggest that concentrations of phthalate metabolites in a spot urine sample can provide a reliable estimation of exposure to phthalates for up to several months.
We examined inter- and intraperson and inter- and intraday variability in the concentrations of monoethyl phthalate (MEP), the major metabolite of diethyl phthalate, commonly used in personal care products, and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), a metabolite of di(2-ethylhexyl) phthalate (DEHP), a polyvinyl chloride plasticizer of which diet is the principal exposure source, among eight adults who collected all urine voids (average, 7.6 samples/person/day) for 1 week.
We analyzed the urine samples using online solid-phase extraction coupled to isotope dilution-high-performance liquid chromatography-tandem mass spectrometry.
Regardless of the type of void (spot, first morning, 24-hr collection), for MEP, interperson variability in concentrations accounted for > 75% of the total variance. By contrast, for MEHHP, within-person variability was the main contributor (69-83%) of the total variance. Furthermore, we observed considerable intraday variability in the concentrations of spot samples for MEHHP (51%) and MEP (21%).
MEP and MEHHP urinary concentrations varied considerably during 1 week, but the main contributors to the total variance differed (interday variability, MEHHP; interperson variability, MEP) regardless of the sampling strategy (spot, first morning, 24-hr collection). The nature of the exposure (diet vs. other lifestyle factors) and timing of urine sampling to evaluate exposure to phthalates should be considered. For DEHP and phthalates to which people are mostly exposed through diet, collecting 24-hr voids for only 1 day may not be advantageous compared with multiple spot collections. When collecting multiple spot urine samples, changing the time of collection may provide the most complete approach to assess exposure to diverse phthalates.
In most DEHP exposure assessment studies, single spot urine sample was used. It could not compare the exposure level among studies. Therefore, we are going to represent the necessity of selection of proper sampling time of spot urine for assessing the environmental DEHP exposure, and the association urinary DEHP metabolites with steroid hormones.
We collected urine and plasma from 25 men. The urine sampling times were at the end of the shift (post-shift) and the next morning before the beginning of the shift (pre-shift). Three metabolites of DEHP {mono(2-ethylhexyl) phthalate [MEHP], mono-(2-ethyl-5-hydroxyhexyl)phthalate [MEHHP], and mono(2-ethyl-5-oxohexyl)phthalate [MEOHP]} in urine were analyzed by HPLC/MS/MS. Plasma luteinzing hormone, follicle stimulating hormone, testosterone, and 17ß-estradiol were measured at pre-shift using a ELISA kit. A log-transformed creatinine-adjusted urinary MEHP, MEHHP, and MEOHP concentration were compared between the post- and pre-shift. The Pearson's correlation was calculated to assess the relationships between log-transformed urinary MEHP concentrations in pre-shift urine and hormone levels.
The three urinary metabolite concentrations at post-shift were significantly higher than the concentrations in the pre-shift (p<0.0001). The plasma hormones were not significantly correlated with log-transformed creatinine - adjusted DEHP metabolites.
To assess the environmental DEHP exposure, it is necessary to select the urine sampling time according to the study object. There were no correlation between the concentration of urinary DEHP metabolites and serum hormone levels.
The reproducibility of urinary phthalate metabolite concentrations has not been well characterized in non-pregnant women of reproductive age. Our primary study objectives were to describe the distribution of urinary phthalate metabolites concentrations among a population of Hmong women of reproductive age, and to evaluate intra- and inter-individual variability of phthalate metabolite concentrations. Ten phthalate metabolites were measured in first-morning urine samples collected from 45 women and 20 of their spouses, who were members of the Fox River Environment and Diet Study cohort in Green Bay, Wisconsin. Repeated first-morning urine samples were collected and analyzed from 25 women, who provided up to three samples over approximately 1 month. Measurement variability was assessed using intraclass correlations (ICCs) and surrogate category analysis. Linear mixed models were used to evaluate the associations between participant characteristics and phthalate metabolite concentrations. Nine of the 10 phthalate metabolites were detected in >80% of all analyzed samples, of which seven were detected in all samples. As a measure of reliability, ICCs were strongest for monobenzyl phthalate (0.64) and weakest for the metabolites of di(2-ethylhexyl)phthalate (DEHP) (ranging from 0.13 to 0.22). Similarly, surrogate category analysis suggested that a single urine sample characterized an average 1-month exposure with reasonable accuracy across low, medium and high tertiles for all metabolites, except the DEHP metabolites. Geometric mean concentrations of monoethyl phthalate increased with age, but patterns by education, income, body mass index, environmental tobacco smoke or season were not observed when measures were adjusted for urinary dilution. Our results suggest that the participant characteristics assessed in this study have limited influence on inter-individual variability of phthalate metabolite concentrations. With regard to intra-individual variability, our results suggest that urinary concentrations of some phthalate metabolites are more reproducible over time and are less subjected to exposure misclassification than others (e.g., metabolites of DEHP).
Using a novel and highly selective technique, we measured monoester metabolites of seven commonly used phthalates in urine samples from a reference population of 289 adult humans. This analytical approach allowed us to directly measure the individual phthalate metabolites responsible for the animal reproductive and developmental toxicity while avoiding contamination from the ubiquitous parent compounds. The monoesters with the highest urinary levels found were monoethyl phthalate (95th percentile, 3,750 ppb, 2,610 microg/g creatinine), monobutyl phthalate (95th percentile, 294 ppb, 162 microg/g creatinine), and monobenzyl phthalate (95th percentile, 137 ppb, 92 microg/g creatinine), reflecting exposure to diethyl phthalate, dibutyl phthalate, and benzyl butyl phthalate. Women of reproductive age (20-40 years) were found to have significantly higher levels of monobutyl phthalate, a reproductive and developmental toxicant in rodents, than other age/gender groups (p < 0.005). Current scientific and regulatory attention on phthalates has focused almost exclusively on health risks from exposure to only two phthalates, di-(2-ethylhexyl) phthalate and di-isononyl phthalate. Our findings strongly suggest that health-risk assessments for phthalate exposure in humans should include diethyl, dibutyl, and benzyl butyl phthalates.
Phthalates are a group of industrial chemicals with many commercial uses, such as solvents, additives, and plasticizers. For example, di-(2-ethylhexyl) phthalate (DEHP) is added in varying amounts to certain plastics, such as polyvinyl chloride, to increase their flexibility. In humans, phthalates are metabolized to their respective monoesters, conjugated, and eliminated. However, despite the high production and use of DEHP, we have recently found that the urinary levels of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in 2,541 persons in the United States were lower than we anticipated, especially when compared with urinary metabolite levels of other commonly used phthalates. This finding raised questions about the sensitivity of this biomarker for assessing DEHP exposure. We explored the utility of two other DEHP metabolites, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), as additional DEHP biomarkers. These metabolites are formed by oxidative metabolism of MEHP. In urine from 62 people, both the range and the mean urinary levels of MEOHP and MEHHP were on average 4-fold higher than those of MEHP; the mean of the individual ratios of MEHHP/MEOHP, MEHHP/MEHP, and MEOHP/MEHP were 1.4, 8.2, and 5.9, respectively. These data suggest that MEOHP and MEHHP are more sensitive biomarkers of exposure to DEHP than is MEHP. These findings also suggest a predominant human metabolic route for DEHP hydrolysis to MEHP followed by oxidation of MEHP; they also imply that a similar mechanism may be relevant for other high-molecular-weight phthalates, such as di-n-octyl, di-isononyl, and di-isodecyl phthalates.
We measured the urinary monoester metabolites of seven commonly used phthalates in approximately 2,540 samples collected from participants of the National Health and Nutrition Examination Survey (NHANES), 1999-2000, who were greater than or equal to 6 years of age. We found detectable levels of metabolites monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), and mono-(2-ethylhexyl) phthalate (MEHP) in > 75% of the samples, suggesting widespread exposure in the United States to diethyl phthalate, dibutyl phthalate or diisobutylphthalate, benzylbutyl phthalate, and di-(2-ethylhexyl) phthalate, respectively. We infrequently detected monoisononyl phthalate, mono-cyclohexyl phthalate, and mono-n-octyl phthalate, suggesting that human exposures to di-isononyl phthalate, dioctylphthalate, and dicyclohexyl phthalate, respectively, are lower than those listed above, or the pathways, routes of exposure, or pharmacokinetic factors such as absorption, distribution, metabolism, and elimination are different. Non-Hispanic blacks had significantly higher concentrations of MEP than did Mexican Americans and non-Hispanic whites. Compared with adolescents and adults, children had significantly higher levels of MBP, MBzP, and MEHP but had significantly lower concentrations of MEP. Females had significantly higher concentrations of MEP and MBzP than did males, but similar MEHP levels. Of particular interest, females of all ages had significantly higher concentrations of the reproductive toxicant MBP than did males of all ages; however, women of reproductive age (i.e., 20-39 years of age) had concentrations similar to adolescent girls and women 40 years of age. These population data on exposure to phthalates will serve an important role in public health by helping to set research priorities and by establishing a nationally representative baseline of exposure with which population levels can be compared.
Exposure to di-(2-ethylhexyl) phthalate (DEHP) is prevalent based on the measurement of its hydrolytic metabolite mono-(2-ethylhexyl) phthalate (MEHP) in the urine of 78% of the general U.S. population studied in the 1999-2000 National Health and Nutrition Examination Survey (NHANES). However, despite the high level of production and use of DEHP, the urinary MEHP levels in the NHANES samples were lower than the monoester metabolites of phthalates less commonly used than DEHP, suggesting metabolic differences between phthalates. We measured MEHP and two oxidative DEHP metabolites, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) to verify whether these other metabolites account for a greater proportion of DEHP metabolic products in 127 paired human urine and serum samples. We found that the urinary levels of MEHHP and MEOHP were 10-fold higher than levels of MEHP; concentrations of urinary MEOHP and MEHHP were strongly correlated (r = 0.928). We also found that the serum levels of MEOHP and MEHHP were comparatively lower than those in urine. Furthermore, the glucuronide-bound conjugates of the oxidative metabolites were the predominant form in both urine and serum. MEOHP and MEHHP cannot be formed by serum enzymes from the hydrolysis of any contamination from DEHP potentially introduced during blood collection and storage. Therefore, concentrations of MEHHP and MEOHP in serum may be a more selective measure of DEHP exposure than is MEHP. However, additional data on the absorption, distribution, metabolism, and elimination of these oxidative metabolites are needed to completely understand the extent of DEHP exposure from the serum concentrations of oxidative DEHP metabolites.
A guide to using S environments to perform statistical analyses providing both an introduction to the use of S and a course in modern statistical methods. The emphasis is on presenting practical problems and full analyses of real data sets.
High-performance liquid chromatography (HPLC) was used for the routine monitoring of the plasticizers di(2-ethylhexyl) phthalate
(DEHP) and tri(2-ethylhexyl) trimellitate (TOTM), in blood products. It allows easy sample clean-up, solvent extraction using
Celite 545 sorbent, good recoveries and opportunity to inject large number of samples without effect on column performance.
The plasticizer levels were investigated in two types of poly(vinyl chloride) (PVC) bags containing whole blood plasma, platelet
concentrates (PCs) during blood taking and storage.
In a retrospective human biomonitoring study we analyzed 24h urine samples taken from the German Environmental Specimen Bank for Human Tissues (ESBHum), which were collected from 634 subjects (predominantly students, age range 20-29 years, 326 females, 308 males) in 9 years between 1988 and 2003 (each n >or= 60), for the concentrations of primary and/or secondary metabolites of di-n-butyl phthalate (DnBP), di-iso-butyl phthalate (DiBP), butylbenzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP). Based on the urinary metabolite excretion we estimated daily intakes of the parent phthalates and investigated the chronological course of the phthalate exposure. In over 98% of the urine samples metabolites of all five phthalates were detectable indicating a ubiquitous exposure of the German population to all five phthalates throughout the last 20 years. The median daily intakes in the subsets between 1988 and 1993 were quite constant for DnBP (approx. 7 microg/kg bw/d) and DEHP (approx. 4 microg/kg bw/d). However, from 1996 the median levels of both phthalates decreased continuously until 2003 (DnBP 1.9 microg/kg bw/d; DEHP 2.4 microg/kg bw/d). By contrast, the daily intake values for DiBP were slightly increasing over the whole time frame investigated (median 1988: 1.1 microg/kg bw/d; median 2003: 1.4 microg/kg bw/d), approximating the levels for DnBP and DEHP. For BBzP we observed slightly decreasing values, even though the medians as of 1998 levelled off at around 0.2 microg/kg bw/d. Regarding daily DiNP exposure we found continuously increasing values, with the lowest median being 0.20 microg/kg bw/d for the subset of 1988 and the highest median for 2003 being twice as high. The trends observed in phthalate exposure may be associated with a change in production and usage pattern. Female subjects exhibited significantly higher daily intakes for the dibutyl phthalates (DnBP p=0.013; DiBP p=0.004). Compared to data from US National Health and Nutrition Examination Surveys (NHANES) exposure levels of the dibutyl phthalates were generally higher in our German study population, while levels of BBzP were somewhat lower. Overall, for a considerable 14% of the subjects we observed daily DnBP intakes above the tolerable daily intake (TDI) value deduced by the European Food Safety Authority (EFSA) (10 microg/kg bw/d). However, the frequency of exceedance decreased during the years and was beneath 2% in the 2003 subset. Even though transgressions of the exposure limit values of the EFSA and the US Environmental Protection Agency (US EPA) occurred only in a relatively small share of the subjects, one has to take into account the cumulative exposure to all phthalates investigated and possible dose-additive endocrine effects of these phthalates.
Subjects submitted to intravenous (IV) blood transfusions for medical reasons or blood doping to increase athletic performance are potentially exposed to the plasticizer di-(2-ethylhexyl)phthalate (DEHP) found in IV bags. Exposure to DEHP has been evaluated by measuring DEHP metabolites in selected groups of subjects.
Urinary DEHP metabolites, mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), and mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP) were measured in a control group with no explicit known exposure to DEHP (n = 30), hospitalized patients receiving blood transfusions (n = 25), nontransfused hospitalized patients receiving other medical care involving plastic materials (n = 39), and athletes (n = 127). Patients were tested in the periods 0 to 24 and 24 to 48 hours after exposition.
Urinary concentrations of all three DEHP metabolites were significantly higher in patients receiving blood transfusion than in nontransfused patients and the control group, except for MEHHP and MEOHP in the period 24 to 48 hours. Samples from four athletes showed increased concentrations of DEHP metabolites comparable to urinary concentrations of patients receiving blood transfusion.
Elevated concentrations of urinary DEHP metabolites represent increased exposure to DEHP. High concentrations of DEHP metabolites present in urine collected from athletes may suggest illegal blood transfusion and can be used as a qualitative screening measure for blood doping.
Humans are exposed to phthalates due to the ubiquitous use of these chemicals in consumer products. In the body, phthalates metabolize quickly to form hydrolytic and oxidative monoesters which, in turn, can be glucuronidated before urinary excretion. Exposure assessment studies typically report the total urinary concentrations of phthalate metabolites (i.e., free plus glucuronidated species). Nevertheless, because conjugation may potentially reduce the bioactivity of the metabolites by reducing their bioavailability, measuring the concentrations of free species may be of interest. An accurate, quantitative measurement of phthalate monoesters and their conjugated species requires data on the stability of these species in urine after sample collection and before analysis. We studied the stability of eight phthalate metabolites and their glucuronide conjugates at 25, 4, and -70 degrees C. Interestingly, the total concentrations of phthalate metabolites decreased over time at 25 and 4 degrees C, but not at -70 degrees C for up to 1 year and despite several freeze-thaw cycles. We further observed a considerable decrease in the concentrations of the glucuronides of some phthalate metabolites 1 day and 3 days after collection when the samples were stored at 25 and 4 degrees C, respectively. By contrast, the concentrations of the glucuronide conjugates at -70 degrees C remained unchanged for the whole duration of the study (1 year). Based on these findings, we recommend transferring urine specimens to a cooler or a refrigerator immediately after collection followed by permanent storage at subfreezing temperatures within hours of sample collection.
A method using gas chromatography--chemical-ionization mass spectrometry has been developed for determination of the plasticizer di(2-ethylhexyl) phthalate (DEHP) in blood plasma. With selective monitoring of the protonated molecular ion, DEHP concentrations down to 75 ng per 500 microliters of human plasma can be measured. Methods using electron-impact mass spectrometry with single-ion monitoring have also been developed for determination of mono(2-ethylhexyl) phthalate (MEHP) in human blood plasma, and of MEHP and other DEHP-derived metabolites in rat plasma. After extraction and derivatization with pentafluoropropanol--pentafluoropropionic anhydride, the metabolites are monitored at m/z 281. The precision and sensitivity of these methods indicate that they will be valuable in studies of the pharmacokinetics of DEHP and its metabolites.
The metabolites appearing in the urine of rats fed di(2-ethylhexyl)phthalate have been isolated by two procedures, thin-layer chromatography of the free metabolites, or thin-layer and gas-liquid chromatography after treatment with diazomethane. The metabolites were characterized by infrared spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry. The only metabolites found were those to be expected from ω- and (ω—1)-oxidation of mono(2-ethylhexyl) phthalate without attack on the aromatic ring. Conjugates were apparently not formed, and free phthalic acid amounted to less than 3% of the urinary metabolites.
Because of the ubiquity of phthalates and their potential role in increasing risk for cancer and reproductive dysfunction, the need for human exposure assessment studies is urgent. In response to this need, we developed a high-throughput, robust, sensitive, accurate, and precise assay for simultaneous measurement of trace levels of eight phthalate metabolites in human urine by HPLC-MS/MS. Human urine samples were processed using enzymatic deconjugation of the glucuronides followed by solid-phase extraction. The eluate was concentrated, and the phthalate metabolites were chromatographically resolved by reversed-phase HPLC, detected by APCI-tandem mass spectrometry, and quantified by isotope dilution. This selective analytical method permits rapid detection (7.7 min total run time) of eight urinary metabolites of the most commonly used phthalates with detection limits in the low nanagram per milliliter range. Assay precision was improved by incorporating 13C4-labeled internal standards for each of the eight analytes, as well as a conjugated internal standard to monitor deconjugation efficiency. This selective, sensitive, and rapid method will help elucidate potential associations (if any) between human exposure to phthalates and adverse health effects.
In 1999, a Blue Ribbon Panel convened to examine the health effects of two commonly used plasticizers present in medical devices and toys. Of particular interest were the plasticizers used in medical tubing. Hospitalized patients can be exposed to a high dose of these chemicals while receiving respiratory therapy or during hemodialysis, and are more likely to be vulnerable to potentially ill effects than healthy individuals. After extensive review of existing research, the Panel concluded that there was not enough evidence of harmful health effects to remove di-(2-ethylhexyl) phthalate (DEHP) and other plasticizers from use in medical tubing. The Panel recognized the importance of plasticizers in enabling tubing such as PVC for respiratory support or in dialysis to be flexible enough for use in a variety of clinical applications that might not be possible with rigid (plasticizer-free) tubing. Nevertheless, we reviewed the literature regarding the health effects for five of the most common compounds, plasticizers and antioxidants found in medical tubing, and suggest that further clinical studies be conducted. We concur with the Panel's recommendation that alternative materials be developed and studied.
Blood doping has the explicit goal of increasing the level of circulating hemoglobin in the bloodstream to enable greater oxygen transport during exercise and therefore to improve endurance performance. Tests have been developed that are capable of detecting athletes using either recombinant human erythropoietins or hemoglobin-based oxygen carriers to boost hemoglobin levels, but to date there are no standard methods for the detection of blood transfusion in an athlete intent on achieving increased oxygen-carrying capacity through the traditional form of blood doping.
In 2001, the U.S. Food and Drug Administration (FDA) convened to conduct a safety assessment of a plasticizer, di(2-ethylhexyl) phthalate (DEHP), released from polyvinyl chloride (PVC) medical devices. Hospitalized patients may be exposed to high concentrations of plasticizers, antioxidants, and chemical contaminants in PVC medical devices during blood transfusion or hemodialysis, thus, making them vulnerable to more potentially adverse effects of these chemicals than healthy people. At the same time, recently, the effect of endocrine-disrupting chemicals on hospitalized patients has attracted a great deal of attention. This study aims to investigate the harmful effects of estrogenic compounds in medical PVC tubing by two approaches of gas chromatography-mass spectrometry (GC-MS) and estrogen receptor (ER) binding assay.
Residual plasticizers, antioxidants, and chemical contaminants in PVC tubing were subjected to GC-MS in the full-scan mode with an original library for plastic additives. Such residual compounds in PVC tubing were screened at very low concentrations (10(-2)-10(5) nmol/l) to determine whether they competed with fluorescein-labeled estradiol for ER (alpha).
DEHP, 2-ethylhexanol, butylated hydroxytoluene, and 4-nonylphenol (NP) were detected in medical PVC tubing. In addition, only NP in PVC tubing was found to bind with ER.
The current study proves that the main residual chemical for estrogenic effect was NP in medical PVC tubing.
We developed a new and fast multidimensional on-line HPLC-method for the quantitative determination of the secondary, chain oxidized monoester metabolites of diethylhexylphthalate (DEHP), 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) in urine samples from the general population. Also included in the method were the simple monoester metabolites of DEHP, dioctylphthalate (DOP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Except for enzymatic hydrolysis for deconjugation of the metabolites no further sample pre-treatment step is necessary. The phthalate metabolites are stripped from urinary matrix by on-line extraction on a restricted access material (LiChrospher((R)) ADS-8) precolumn, transferred in backflush-mode and chromatographically resolved by reversed-phase HPLC. Eluting metabolites are detected by ESI-tandem mass spectrometry in negative ionization mode and quantified by isotope dilution. Within a total run time of 25 min we can selectively and sensitively quantify seven urinary metabolites of six commonly occurring phthalate diesters including the controversial di(2-ethylhexyl)phthalate (DEHP). The detection limits for all analytes are in the low ppb range (0.5-2.0 microgram/l urine). First results on a small non-exposed group (n=8) ranged for 5OH-MEHP from 0.59 to 124 microgram/l, for 5oxo-MEHP from <LOQ to 73.0 microgram/l, and for MEHP from <LOQ to 41.1 microgram/l. The other short chain monoester metabolites were detectable in every sample with mean concentrations for MnBuP of 36.5 microgram/l, for MBzP of 7.19 microgram/l and MEP of 1.0 mg/l. With this rapid and economic method we can determine the internal exposure of the general population to DEHP and other phthalates as well as the body burden of occupationally and medically exposed subjects. The results can help to rank the risks of phthalates in the areas of carcinogenesis, peroxisome proliferation and endocrine disruption. Since secondary, functionalized metabolites of DEHP are included in the method an enduring problem of the past is excluded: sample contamination in the pre-analytical and analytical phase by both di- and monoesters.
A novel method based on column-switching high-performance liquid chromatography-electrospray mass spectrometry (LC-MS) coupled with an on-line extraction column containing conjugated avidin has been developed for direct injection analysis of di(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono(2-ethylhexyl) phthalate (MEHP), in blood samples. The sample preparation for on-line extraction involved the mixing of blood samples with internal standards, DEHP-d(4) and MEHP-d(4), in LC glass vials. A linear response was found for column-switching LC-MS when tests were conducted within the validated range of 25 to 1000 ng mL(-1) for DEHP and 5 to 1000 ng mL(-1) for MEHP, with correlation coefficients (r) greater than 0.999. In addition, the recoveries of DEHP and MEHP from human plasma were calculated by using this method with on-line extraction, yielding recoveries of up to 91.2% (RSD<5%). We measured the background levels of DEHP and MEHP in six human plasma samples from healthy volunteers and three fetal bovine serum samples for cell-line culture. DEHP and MEHP were not detected in all human plasma samples (N.D. is <25 ng mL(-1) for DEHP, and N.D. is <5.0 ng mL(-1) for MEHP). In contrast, high DEHP contamination of commercially available fetal bovine serum samples was found by this method.
We analyzed 85 urine samples of the general German population for human specific metabolites of phthalates. By that we avoided contamination with the parent phthalates being omnipresent in the environment and for the first time could deduce each individual's internal exposure to phthalates without contamination. Determined were the secondary metabolites mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) of di(2-ethylhexyl)phthalate (DEHP) and the primary monoester metabolites of DEHP, di-noctylphthalate (DnOP), di-n-butylphthalate (DnBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Based on these internal exposure values we calculated the daily intake of the parent phthalates using urinary metabolite excretion factors. For DEHP we determined a median intake of 13.8 micrograms/kg body weight/day and an intake at the 95th percentile of 52.1 micrograms/kg body weight/day. The tolerable daily intake (TDI) value settled by the EU Scientific Committee for Toxicity, Ecotoxicity and the Environment (CSTEE) is 37 micrograms/kg body weight/day. Twelve percent of the subjects (10 out of 85 samples) within our collective of the general population are exceeding this value. Thirty-one percent of the subjects (26 out of 85 samples) had values higher than the reference dose (RfD) of 20 micrograms/kg body weight/day of the U.S. Environmental Protection Agency (EPA). For DnBP, BBzP, DEP and DnOP intake values at the 95th percentile were 16.2, 2.5, 22.1 and 0.42 micrograms/kg body weight/day respectively. Our results unequivocally prove that the general German population is exposed to DEHP to a much higher extent than previously believed. This is of greatest importance for public health since DEHP is not only the most important phthalate with respect to its production, use, occurrence and omnipresence but also the phthalate with the greatest endocrine disrupting potency. DEHP is strongly suspected to be a developmental and reproductive toxicant. We are not aware of any other environmental contaminant for which the TDI and RfD are exceeded to such an extent within the general population. The transgressions of the TDI and RfD for DEHP are accompanied by considerable ubiquitous exposures to DnBP and BbzP, two phthalates under scrutiny for similar toxicological mechanisms.
Phthalates are widely used as industrial solvents and plasticizers, with global use exceeding four million tons per year. We improved our previously developed high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC-APCI-MS/MS) method to measure urinary phthalate metabolites by increasing the selectivity and the sensitivity by better resolving them from the solvent front, adding three more phthalate metabolites, monomethyl phthalate (mMP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP); increasing the sample throughput; and reducing the solvent usage. Furthermore, this improved method enabled us to analyze free un-conjugated mono-2-ethylhexyl phthalate (mEHP) by eliminating interferences derived from coelution of the glucuronide-bound, or conjugated form, of the mEHP on measurements of the free mEHP. This method for measuring phthalate metabolites in urine involves solid-phase extraction followed by reversed-phase HPLC-APCI-MS/MS using isotope dilution with (13)C(4) internal standards. We further evaluated the ruggedness and the reliability of the method by comparing measurements made by multiple analysts at different extraction settings on multiple instruments. We observed mMP, monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mEHP, mEHHP and mEOHP in the majority of urine specimens analyzed with DEHP-metabolites mEHHP and mEOHP present in significantly higher amounts than mEHP.
Mass spectrometry (MS) is being introduced into a large number of clinical laboratories. It provides specificity because of its ability to monitor selected mass ions, sensitivity because of the enhanced signal-to-noise ratio, and speed because it can help avoid the need for intensive sample cleanup and long analysis times. However, MS is not without problems related to interference, especially through ion suppression effects. Ion suppression results from the presence of less volatile compounds that can change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector.
This review discusses materials shown to cause ion suppression, including salts, ion-pairing agents, endogenous compounds, drugs, metabolites, and proteins. Experimental protocols for examining ion suppression, which should include, at a minimum, signal recovery studies using specimen extracts with added analyte, are also discussed, and a more comprehensive approach is presented that uses postcolumn infusion of the analyte to evaluate protracted ionization effects. Finally, this review presents options for minimizing or correcting ion suppression, which include enhanced specimen cleanup, chromatographic changes, reagent modifications, and effective internal standardization.
Whenever mass spectrometric assays are developed, ion suppression studies should be performed using expected physiologic concentrations of the analyte under investigation.
A number of phthalates and their metabolites are suspected of having teratogenic and endocrine disrupting effects. Especially the developmental and reproductive effects of di(2-ethylhexyl)phthalate (DEHP) are under scrutiny. In this study we determined the concentrations of the secondary, chain oxidized monoester metabolites of DEHP, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) in urine samples from the general population. The utilization of the secondary metabolites minimized any risk of contamination by the ubiquitously present phthalate parent compounds. Included in the method were also the simple monoester metabolites of DEHP, dioctylphthalate (DOP), di-n-butylphthalate (DnBuP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Automated sample preparation was performed applying a column switching liquid chromatography system enabling online extraction of the urine on a restricted access material (RAM) and separation on a reversed phase analytical column. Detection was performed by negative ESI-tandem mass spectrometry in multiple reaction monitoring mode and quantification by isotope dilution. The excretion of DEHP and the other phthalates was studied by analyzing first morning urine samples from 53 women and 32 men aged 7-64 years (median: 34.2 years) living in northern Bavaria (Germany) who were not occupationally exposed to phthalates. Phthalate metabolites, secondary and primary ones, were detected in all specimens. Concentrations were found to vary strongly from phthalate to phthalate and subject to subject with differences spanning more than three orders of magnitude. Median concentrations for excretion of DEHP metabolites were 46.8 microg/L for 5OH-MEHP (range 0.5-818 microg/L), 36.5 microg/L for 5oxo-MEHP (range 0.5-544 microg/L), and 10.3 microg/L for MEHP (range:<0.5 (limit of quantification, LOQ) to 177 microg/L). A strong correlation was found between the excretion of 5OH-MEHP and 5oxo-MEHP with a correlation coefficient of r=0.991, indicating close metabolic proximity of those two parameters but also the absence of any contaminating interference. Median concentrations for the other monoester metabolites were for mono-n-butylphthalate (MnBuP) 181 microg/L, for monobenzylphthalate (MBzP) 21.0 microg/L, for monoethylphthalate (MEP) 90.2 microg/L and for mono-n-octylphthalate (MOP)<1.0 microg/L (LOQ). These results will help to perform health risk assessments for the phthalate exposure of the general population.
Metabolism of most diesters of phthalic acid in humans occurs by an initial phase I biotransformation in which phthalate monoesters are formed, followed by a phase II biotransformation in which phthalate monoesters react with glucuronic acid to form their respective glucuronide conjugates. The phase II conjugation increases water solubility and facilitates urinary excretion of phthalate, and reduces the potential biological activity because the putative biologically active species is the monoester metabolite. In this study, we report percentages of glucuronidation of four common phthalate monoesters, monoethyl (mEP), monobutyl (mBP), monobenzyl (mBzP), and mono-2-ethylhexyl phthalate (mEHP) in a subset of urine (mEP n=262, mBP n=283, mBzP n=328, mEHP n=119) and serum (mEP n=93, mBP n=149, mEHP n=141) samples from the general US population. The percentages of free and conjugated monoester excreted in urine differed for the various phthalates. For the more lipophilic monoesters (i.e., mBP, mBzP, and mEHP), the geometric mean of free monoester excretion ranged from 6 to 16%. The contrary was true for the most hydrophilic monoester, mEP, for which about 71% was excreted in urine as its free monoester. Furthermore, percentages of free and conjugated monoesters were similar for mEP, mBP and mEHP among serum and urine samples. Serum mBzP was largely below the method limit of detection. Interestingly, the serum mEP and mBP levels were less than 3% and 47%, respectively, of their urinary levels, whereas the level of mEHP was similar both in urine and serum.
Concentrations of mono(2-ethylhexyl)phthalate (MEHP), and di(2-ethylhexyl)phthalate (DEHP), in serum of healthy volunteers were determined by high performance liquid chromatography (HPLC) with tandem mass spectrometry (LC/MS/MS). The serum was extracted with acetone, followed by hexane extraction under acidic conditions, and then applied to the LC/MS/MS. Recoveries of 20 ng/ml of MEHP and DEHP were 101+/-5.7 (n=6) and 102+/-6.5% (n=6), respectively. The limits of quantification (LOQ) of MEHP and DEHP in the method were 5.0 and 14.0 ng/ml, respectively. The concentration of MEHP in the serum was at or less than the LOQ. The concentration of DEHP in the serum was less than the LOQ. Contaminations of MEHP and DEHP from experimental reagents, apparatus and air during the procedure were less than the LOQ and were estimated to be <1.0 and 2.2+/-0.6 ng/ml, respectively. After subtraction of the contamination, the net concentrations of MEHP and DEHP in the serum were estimated at or <5 and <2 ng/ml, respectively. To decrease contamination by DEHP, the cleanup steps and the apparatus and solvent usage were minimized in the sample preparation procedures. The high selectivity of LC/MS/MS is the key for obtaining reliable experimental data from in the matrix-rich analytical samples and for maintaining a low level contamination of MEHP and DEHP in this experimental system. This method would be a useful tool for the detection of MEHP and DEHP in serum.
We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.
While the demonstrated benefits associated with breastfeeding are well recognized, breast milk is one possible route of exposure to environmental chemicals, including phthalates, by breastfeeding infants. Because of the potential health impact of phthalates to nursing children, determining whether phthalates are present in breast milk is important. We developed a sensitive method for measuring 13 phthalate metabolites in breast milk using automated solid phase extraction (SPE) coupled to isotope dilution-high-performance liquid chromatography (HPLC)-negative ion electrospray ionization-tandem mass spectrometry. We used D(4)-phthalate diesters to unequivocally establish the presence in human breast milk of enzymes capable of hydrolyzing the ubiquitous phthalate diesters to their respective monoesters. The analytical method involves acid-denaturation of the enzymes after collection of the milk to avoid hydrolysis of contaminant phthalate diesters introduced during sampling, storage, and analysis. The method shows good reproducibility (average coefficient of variations range between 4 and 27%) and accuracy (spiked recoveries are approximately 100%). The detection limits are in the low ng/ml range in 1ml of breast milk. We detected several phthalate metabolites in pooled human breast milk samples, suggesting that phthalates can be incorporated into breast milk and transferred to the nursing child.
Urine samples from the 2001/2002 pilot study for the German Environmental Survey on children (GerES IV) were analysed for concentrations of the primary DEHP metabolite MEHP (mono(2-ethylhexyl)phthalate) and two secondary DEHP metabolites SOH-MEHP (2-ethyl-5-hydroxy-hexylphthalate) and 5oxo-MEHP (2-ethyl-5-oxo-hexylphthalate). Urine samples had been taken from 254 children aged 3 to 14. In addition, DEHP was analysed in house dust samples. These samples had been collected with vacuum cleaners in the homes of the children. The geometric mean (GM) was 7.9 microg/l for MEHP in urine, and the GMs for the secondary metabolites 5OH-MEHP and 5oxo-MEHP were 52.1 microg/l and 39.9 microg/l. 5OH-MEHP and 5oxo-MEHP concentrations were highly correlated (r = 0.98). The correlations of 5OH-MEHP and 5oxo-MEHP with MEHP were also high (r = 0.72 and r = 0.70). The concentrations of 5OH-MEHP and 5oxo-MEHP were 8.0-fold and 6.2-fold higher than the concentrations of MEHP. The ratios 5OH-MEHP/Soxo-MEHP and 5oxo-MEHP/MEHP decreased with increasing age. Boys showed higher concentrations than girls for all three metabolites of DEHP in urine. Children aged 13-14 had the lowest mean concentrations of the secondary metabolites in urine. The house dust analyses revealed DEHP contamination of all samples. The GM was 508 mg/kg dust. No correlation could be observed between the levels of any of the urinary DEHP metabolites and those of DEHP in house dust.
We present a fast and reliable on-line clean-up HPLC-method for the simultaneous determination of the five major urinary metabolites of di-(2-ethylhexyl)phthalate (DEHP) namely mono-(2-ethyl-5-carboxypentyl)phthalate (5carboxy-MEPP), mono-[2-(carboxymethyl)hexyl]phthalate (2carboxy-MMHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono-(2-ethylhexyl)phthalate (MEHP). These metabolites represent about 70% of an oral DEHP dose. We for the first time succeeded to reliably quantify 5carboxy-MEPP and to identify 2carboxy-MMHP as major metabolites in native urines of the general population. The analytical procedure consists of an enzymatic hydrolysis, on-line extraction of the analytes from urinary matrix by a restricted access material column (RAM), back-flush transfer onto the analytical column (betasil phenylhexyl), detection by ESI-tandem mass spectrometry and quantification by isotope dilution (limit of detection (LOD) 0.25 microg/l). Median concentrations of a small collective taken from the general population (n=19) were 85.5 microg/l (5carboxy-MEPP), 47.5 microg/l (5OH-MEHP), 39.7 microg/l (5oxo-MEHP), 9.8 microg/l (MEHP) and about 37 microg/l (2carboxy-MMHP). The presented method can provide insights into the actual internal burden of the general population and certain risk groups. It will help to further explore the human metabolism of DEHP-an occupational and environmental toxicant of great concern.
We developed an on-line solid-phase extraction (SPE) method, coupled with isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and with automated sample preparation, to simultaneously quantify 16 phthalate metabolites in human urine. The method requires a silica-based monolithic column for the initial preconcentration of the phthalate metabolites from the urine and a silica-based conventional analytical column for the chromatographic separation of the analytes of interest. It uses small amounts of urine (100 microL), is sensitive (limits of detection range from 0.11 to 0.90 ng/mL), accurate (spiked recoveries are approximately 100%), and precise (the inter- and intraday coefficients of variation are <10%). The method is not labor intensive, and, because pretreatment of the urine samples was performed automatically using an HPLC autosampler, involves minimal sample handling, thus minimizing exposure to hazardous chemicals. The method was validated on spiked, pooled urine samples and on urine samples from 43 adults with no known exposure to phthalates. The high sensitivity and high throughput (HPLC run time, including the preconcentration step, is 27 min) of this analytical method combined with the ease of use and effective automated sample preparation procedure make it suitable for large epidemiological studies to evaluate the prevalence of human exposure to phthalates.
The US FDA and The Ministry of Health, Labor and Welfare of Japan have indicated that the risk assessment of di(2-ethylhexyl) phthalate (DEHP) released from polyvinyl chloride (PVC) medical devices requires immediate attention. In particular, the analysis of the exposure to DEHP from blood bags is very important for medical treatment. However, human exposure to DEHP via blood transfusion remains poorly understood. We evaluated DEHP and mono(2-ethylhexyl) phthalate (MEHP) levels, migration patterns, and metabolism in blood products for the detailed assessment of exposure to DEHP.
A method that is based on column-switching liquid chromatography-electrospray mass spectrometry (LC-MS) coupled with on-line extraction was used for the direct analysis of DEHP and MEHP in the blood products. From the Japanese Red Cross Society, 78 blood products (red blood cell concentrate: n=18, irradiated red blood cell concentrate: n=18, whole blood: n=18, blood platelet: n=18, and frozen plasma: n=6) were sampled in January 2003 for use in this study.
The detection levels of DEHP and MEHP ranged from 1.8 to 83.2 microg/ml and from 0.1 to 9.7 microg/ml, respectively. The levels of MEHP and DEHP in the blood products were increased with increasing storage time. In addition, whole blood products in PVC bags had the highest DEHP levels compared to the other blood products. Our results indicate that the maximum level of human exposure to DEHP released from blood bags is 0.7 mg/kg weight/time.
This first quantitative evidence may be useful for the risk assessment of DEHP released from blood bags.
Daily exposure of humans to phthalates may be a health risk because animal experiments have shown these compounds can affect the differentiation and function of the reproductive system. Because milk is the main source of nutrition for infants, knowledge of phthalate levels is important for exposure and risk assessment. Here we describe the development and validation of a quantitative analytical procedure for determination of phthalate metabolites in human milk. The phthalate monoesters investigated were: monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mono-(2-ethylhexyl) phthalate (mEHP), and monoisononyl phthalate (mNP). The method is based on liquid extraction with a mixture of ethyl acetate and cyclohexane (95:5) followed by two-step solid-phase extraction (SPE). Detection and quantification of the phthalate monoesters were accomplished by high-pressure liquid chromatography using a Betasil phenyl column (100 mmx2.1 mmx3 microm) and triple tandem mass spectrometry (LC-MS-MS). Detection limits were in the range 0.01 to 0.5 microg L(-1) and method variation was from 5 to 15%. Analysis of 36 milk samples showed that all these phthalates were present, albeit at different concentrations. Median values (microg L(-1)) obtained were 0.11 (mMP), 0.95 (mEP), 3.5 (mBP), 0.8 (mBzP), 9.5 (mEHP), and 101 (mNP). We also analysed seven samples of consumer milk and ten samples of infant formula. Only mBP and mEHP were detected in these samples, in the ranges 0.6-3.9 microg L(-1) (mBP) and 5.6-9.9 microg L(-1) (mEHP).
An on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (on-line SPE-HPLC-MS/MS) method was developed for the analysis of metabolites of three phthalate esters in human urine at the low nanogram per milliliter level. The recoveries were above 84.3% and relative standard deviations varied from 0.8 to 4.8%. The compounds along with their deuterated internal standards were detected in the negative ion mode by selective reaction monitoring and the accuracy of the method was improved by isotope dilution. Monobutyl phthalate was detected with median level of 22.5 ng/ml. The median levels for monobenzyl phthalate and monoethylhexyl phthalate were less than the limit of quantitation (LOQ). The on-line SPE-HPLC-MS/MS method allowed the possibility of determining these metabolites within a short time, with increased sensitivity and by using decreased amounts of sample and solvent.
We developed an analytical method using off-line solid-phase extraction (SPE) coupled with on-line SPE and isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of phthalate metabolites in human meconium and in semen. First, we used off-line SPE to remove interfering proteins and other biomolecules from the samples. Then, we preconcentrated the phthalate metabolites in the extract using on-line SPE before measuring them by HPLC-MS/MS. For most of the analytes, the limits of detection ranged between 0.2 and 0.7 ng/g for meconium and between 0.3 and 0.7 ng/mL for semen. The recovery after off-line SPE varied for most analytes between 65 and 99% at concentrations ranging from 3.0 to 30.0 ng/mL in semen and between 67 and 103% at concentrations ranging from 2.0 to 10.0 ng/mL in meconium. Precision measured by the relative standard deviation ranged from 3.2 to 19.1% for intraday and from 3.9 to 18.6% for interday. We validated this novel approach--which is applicable to other biological matrixes, including serum and breast milk--on spiked samples and on five meconium samples and one pooled semen sample from people with no known occupational exposure to phthalates.
Phthalates like di-(2-ethylhexyl) phthalate (DEHP) are commonly used as plasticizers and their metabolites are suspect of especially reproductive toxicity. The aim of our study was to assess phthalate exposure in adults by measuring urinary phthalate metabolite levels and to explore individual temporal variability. Urine samples were collected by 27 women and 23 men aged 14-60 years during 8 consecutive days. We quantified four monoesters, four oxidative DEHP metabolites, and two secondary metabolites of di-isononyl phthalate (DiNP) by a LC/LC-MS/MS method. If we analyzed all 399 available samples independent of classification, the highest median values of primary metabolites in this study were found for mono-n-butyl phthalate (MnBP: 49.6 microg/l), followed by mono-isobutyl phthalate (MiBP: 44.9 microg/l), mono-benzyl phthalate (MBzP: 7.2 microg/l), and mono-2-ethylhexyl phthalate (MEHP: 4.9 microg/l). The median concentrations of the oxidized metabolites of DEHP were 8.3 microg/l for mono-(2-carboxymethylhexyl) phthalate (2cx-MMHP), 19.2 microg/l for mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), 14.7 microg/l for mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), and 26.2 microg/l for mono-(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP). The concentrations of the two DiNP secondary metabolites mono (oxoisononyl) phthalate (oxo-MiNP) and mono(hydroxyisononyl) phthalate (OH-MiNP) ranged from <LOD to 304 microg/l (median: 3.0 microg/l, 2.9 microg/g creatinine) and <LOD to 698 microg/l (median: 5.5 microg/l, 5.2 microg/g creatinine), respectively. Phthalate metabolite levels did not consistently differ by sex or age. There was substantial day-to-day variation of urinary levels with considerable within-subject variability. Intraclass correlation coefficients adjusted for sex and age ranged between 0.21 and 0.48 for unadjusted metabolite levels and between 0.20 and 0.57 for creatinine-adjusted levels. The secondary metabolites of DiNP were detectable in nearly all samples and were therefore sensitive biomarkers of DiNP exposure. Our results of within-subject variability suggest that exposure assessment should not be based on a single urine measurement.
The aim of the present study was to improve and validate a flow cytometric method for the detection of homologous blood transfusion in doping control analysis. A panel of eight different primary antibodies and two different phycoerythrin-conjugated secondary antibodies was used for the detection of different blood populations. The flow cytometer used in this study was the BD FACSArray instrument. Mixed red blood cell populations were prepared from phenotype known donors. Linearity, specificity, recovery, precision, robustness and interday-precision were tested for every primary antibody used in the presented assay. The technique of signal amplification was utilized for an improved separation of antigens with weak or heterozygous expression to improve the interpretation of histograms. The resulting method allowed to clearly identify mixed red blood cell populations in homologous blood transfusion samples containing 0.3 - 2.0 % of donor blood.
An increase of hemoglobin (Hb) mass is the key target of blood doping practices to enhance performance as it is a main determinant of maximal oxygen uptake. Although detection methods exist for doping with recombinant EPO and homologous blood transfusions, autologous transfusions remain virtually undetectable. In this context, the most sensitive parameter would be a determination of Hb mass itself. The purpose therefore was to establish whether Hb mass measurements by the optimized CO-rebreathing method allow screening for the withdrawal and reinfusion of autologous red blood cells.
The optimized CO-rebreathing method was used for evaluation of Hb mass in two groups at three time points (duplicate measurements: 1) baseline, 2) after donation, and 3) after reinfusion). Group I (N = 6) was to donate and receive 1 unit of packed red cells (PRC) in contrast to two PRC in group II (N = 4). The time span between withdrawal and reinfusion was 2 d.
The mean Hb content of the blood units was 59.0 +/- 3.9 g (group I) and 108.3 +/- 1.3 g (group II). Hb mass decreased significantly after blood withdrawal (-89 +/- 16 g in group I and -120 +/- 14 g in group II) and increased significantly after reinfusion (group I: 70 +/- 16 g; group II: 90 +/- 9 g) but was lower than at baseline (group I: -19 +/- 17 g; group II: -30 +/- 14 g). The total error of measurements for the duplicate measures ranged between 0.8 and 3.1% (Hb mass: 6.4-22.1 g).
Hb mass determination with the optimized CO-rebreathing method has sufficient precision to detect the absolute differences in Hb mass induced by blood withdrawal and autologous reinfusion. Thus, it may be suited to screen for artificially induced alterations in Hb mass.
Athletes may undergo blood transfusion to increase their red cell mass and the oxygen carrying capacity of their blood in order to confer a competitive advantage. Allogeneic transfusions are normally mismatched at one or more minor blood group antigens. The most sensitive and accurate method known to detect this form of blood doping is flow cytometry. Low percentages of antigen-positive and antigen-negative red blood cells (RBCs) can be quantitated using suitable specific alloantibodies and careful analysis. By testing blood samples taken at various times, a reduction in the percentage of a minor population of RBCs will indicate transfusion has occurred.
Since the introduction in 2001 of a urine-based detection method for recombinant erythropoietin (rHuEPO), transfusion-doping practices have regained interest. To address this problem, an efficient antidoping test designed to obtain direct proof of allogeneic blood transfusion was developed and validated. This test, based on flow cytometry analysis of red blood cell (RBCs) phenotypes, was used to determine the absence or the presence of numerous RBCs populations in a blood sample. A such, it may constitute a direct proof of an abnormal blood population resulting from homologous transfusion. Single-blind and single-site studies were carried out to validate this method as a forensic quality standard analysis and to allow objective interpretation of real cases. The analysis of 140 blood samples containing different percentages (0-5%) of a minor RBCs population were carried on by four independent analysts. Robustness, sensitivity, specificity, precision and stability were assessed. ISO-accredited controls samples were used to demonstrate that the method was robust, stable and precise. No false positive results were observed, resulting in a 100% specificity of the method. Most samples containing a 1.5% minor RBCs population were unambiguously detected, yielding a 78.1% sensitivity. These samples mimicked blood collected from an athlete 3 months after a homologous blood transfusion event where 10% of the total RBCs present in the recipient originated in the donor. The observed false negative results could be explained by differences in antigen expression between the donor and the recipient. False negatives were more numerous with smaller minor RBCs populations. The method described here fulfils the ISO-17025 accreditation and validation requirements. The controls and the methodology are solid enough to determine with certainty whether a sample contains one or more RBCs populations. This variable is currently the best indicator for homologous blood transfusion doping.
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