Validation and application of an HPLC method for determination of di (2-ethylhexyl) phthalate and mono (2-ethylhexyl) phthalate in liver samples

A reversed-phase gradient elution, UV detection method is developed for the simultaneous determination of mono (2-ethylhexyl) phthalate [MEHP] and di (2-ethylhexyl) phthalate [DEHP] in tissue samples. The method is validated with respect to extraction recovery, inter and intra-day precision, linearity of response, detect ability, and specificity. The validated method has been successfully applied to the study of DEHP and MEHP in liver, kidney, testis, brain, and plasma samples from rat.     

HPLC with diode-array detection for the simultaneous determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in seminal plasma

A high-performance liquid chromatographic method for the simultaneous determination of di(2-ethylhexyl)phthalate (DEHP) and its major metabolite mono(2-ethylhexyl)phthalate (MEHP) in seminal plasma was developed and validated. The method involves liquid-liquid extraction followed by isocratic reversed-phase chromatography with diode-array detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from the analysis of spiked seminal plasma samples. The effect of mobile-phase composition and pH on the retention of the target analytes was investigated. The limits of detection were 0.010 and 0.015 microg/mL, for DEHP and MEHP, respectively. This method was used to analyze real samples in support of clinical studies on these potential endocrine disruptors.     

Human Metabolism of Bis(2-ethylhexyl)phthalate

Abstract

To study the human metabolism of bis (2-ethylhexyl)-phthalate (DEHP) urine samples were analyzed from non-uremic psoriatic patients, uremic patients undergoing hemodialysis treatments and patients undergoing cardiac bypass surgery using High Performance Liquid Chromatography (HPLC). The urine of dialyzed non-uremic patients contained phthalic acid, mono (2-ethylhexyl) phthalate and bis (2-ethylhexyl) phthalate. Other compounds identified were p-hydroxy benzoic acid, m-hydroxy benzoic acid, o-hydroxy hippuric acid, o-hydroxy benzoic acid and benzoic acid, which may be either diet dependent normal urinary constituents or metabolites of bis (2-ethylhexyl) phthalate. The levels of phthalic acid and bis (2-ethylhexyl) phthalate found in the urine of patients who were on total body oxygenators containing a membrane during cardiac bypass surgery were comparable to levels obtained from non-uremic psoriatic patients. Significant levels of phthalic acid were detected in the urine of the uremic patients studied while mono (2-ethylhexyl) phthalate and bis (2-ethylhexyl)-phthalate were present only in small amounts or were completely absent. In general, the urinary phthalate content of uremic patients increased with urinary volume.     

邻苯二甲酸二(2-乙基)己酯和偏苯三酸三辛酯在临床不同药液中溶出量的比较

聚氯乙烯(PVC)材质的医疗器械产品中需要加入增塑剂以改善柔韧性,目前最常用的增塑剂是邻苯二甲酸二(2-乙基)己酯(DEHP)和偏苯三酸三辛酯(TOTM)。本文考察了PVC一次性使用输液器产品在脂溶性药液(紫杉醇注射液)、肠外营养液(脂肪乳)、酸性药液(左氧氟沙星,pH 3.0~5.0)和碱性药液(呋塞米,pH 8.0~9.0)中的DEHP和TOTM溶出量,并进行对比分析。先建立了一种高效液相色谱-紫外检测(HPLC-UV)方法测定增塑剂的溶出量,并利用该方法对增塑剂的溶出量进行了分析。实验结果表明,增塑剂在不同药液中均有一定的溶出情况,其中紫杉醇注射液对增塑剂的溶出量要高于脂肪乳,并远高于左氧氟沙星和呋塞米注射液。通过对比DEHP和TOTM的溶出量可以看出,在相同的浸提条件下,TOTM的溶出量远低于DEHP的溶出量。利用紫杉醇注射液浸提24 h,PVC输液器产品DEHP的溶出量为21.14 mg,而TOTM的溶出量仅为0.078 mg。DEHP的溶出量为TOTM溶出量的270倍。因此,TOTM具有的较好耐迁移性,是一种潜在的DEHP替代增塑剂。     

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

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.     

New contributions to the field of bead-injection spectroscopy—flow-injection analysis: determination of cobalt

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.     

Molecularly imprinted microspheres and nanospheres for di(2-ethylhexyl)phthalate prepared by precipitation polymerization

Molecularly imprinted microspheres (MIMs, >3 μm) and nanospheres (MINs, ≈450 nm) for the environmental endocrine disruptor di(2-ethylhexyl)phthalate (DEHP) were prepared by a precipitation polymerization (PP) procedure. The effect of the dispersive solvents acetonitrile (ACN) and cyclohexane (CH), the cross-linkers ethylene glycol dimethacrylate (EDMA) and trimethylpropane trimethacrylate (TRIM), and the template on particle size and morphology of polymers was investigated in detail by scanning electron microscopy (SEM) and BET adsorption isotherm determination. When used as HPLC stationary phase, the microspheres exhibited strong affinity for the template DEHP with an imprint factor (IF) higher than 8.0 in ACN/water (60:40, v/v) as mobile phase. Furthermore, baseline separation of DEHP from benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) could be achieved. In contrast, no or only poor separation could be observed with non-imprinted polymeric polymers (NIPs) or imprinted bulk polymers (MIB), respectively. Similarly, the obtained MINs exhibited an imprinting effect in pure ACN, i.e. the bond amount of DEHP was significantly higher compared to NIPs, as was shown in rebinding experiments. Besides their use as an HPLC stationary phase, MIMs might further be applicable for SPE sample cleanup, while MINs could be used as a recognition layer on sensor surfaces. Figure Molecularly imprinting of di(2-ethylhexyl)phthalate (DEHP)     

Rapid determination by high performance liquid chromatography of di-2-ethylhexyl phthalate in plasma stored in plastic bags

A rapid sensitive technique was developed for the analysis of di-2-ethylhexyl phthalate (DEHP) in plasma stored in plastic bags by using high performance liquid chromatography (HPLC) with UV detection and a Hypersil ODS column. The compound was easily and efficiently extracted with a mixture of sodium hydroxide and acetonitrile, which allowed the deproteinization of plasma samples. The recovery was greater than 95% and the intra- and inter-assay coefficients of variation were better than 6.5%. The results obtained showed that the amount of DEHP accumulated in plasma varied according to different parameters and depended on the storage conditions (time, temperature and shaking) and also on the lipid content of the stored plasma and the sterilization process of the PVC bags.     

Fast Chromatographic Separation of Plasticizers on Thin Layers of an Inorganic Ion-Exchanger: Quantitative Determination of Di(2-ethylhexyl)phthalate

Fast and selective separation of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, di(2-ethylhexyl)phthalate (DEHP), benzyl butyl phthalate, diisodecyl phthalate, dimethyl adipate, diethyl adipate, di(2-ethylhexyl)adipate, triethyl citrate, tributyl citrate, tributyl acetyl citrate and n-butyl stearate have been developed on thin layers of inorganic ion-exchanger stannic silicate using a mixture of toluene + ethyl acetate (10:1, v/v) as mobile phase. The development distance and time were 12 cm and 25 min, respectively. Quantitative determination of DEHP was made at wavelength 280 nm by Camag TLC Scanner-3. Limit of quantitation for DEHP was 0.50 μg per zone while its limit of detection was 0.05 μg per zone.     

High-performance liquid chromatographic procedure for the determination of di-(2-ethylhexyl)phthalate in human blood specimens. Problems of variable-extraction yield and the use of standard addition for calibration

A high-performance liquid chromatographic procedure was developed for the determination of di-(2-ethylhexyl)phthalate (DEHP) concentrations in human whole blood samples. The solvent extraction of DEHP was found to be highly variable between samples obtained from different subjects (coefficient of variation of 30.4%). The recovery of DEHP following extraction with ethyl acetate was negatively correlated with serum lipid content, as expressed by the sum of serum cholesterol and triglyceride concentrations (r = -0.864). The technique of standard addition of DEHP allowed a single-point calibration of DEHP extractability in individual blood samples, and provided an accurate estimation of DEHP concentration (coefficient of variation of approximately 6% in replicate samples). The potential for intersample variability in the solvent extraction of other highly lipid-soluble compounds should be considered.     

Synthesis of bis(2-ethylhexyl) phthalate over methane sulfonic acid catalyst. Kinetic investigations

The kinetic model for the synthesis of bis(2-ethylhexyl) phthalate from phthalic anhydride and 2-ethylhexanol in the presence of methane sulfonic acid as a catalyst has been derived, based on the investigation carried out in an isothermal, semibatch reactor. The first step, the formation of mono(2-ethylhexyl) phthalate, is very fast and irreversible. The second step, the esterification of monoester with 2-ethylhexanol, is relatively slow and needs a catalyst. The second reaction appears to be of the first order with respect to mono(2-ethylhexyl) phthalate and does not depend on the concentration of alcohol.     

Evaporative loss kinetics of di(2-ethylhexyl)phthalate (DEHP) from pristine DEHP and plasticized PVC

The migration of di(2-ethylhexyl)phthalate (DEHP) from poly(vinyl chloride) (PVC) to a surrounding gas phase at temperatures below 120 °C kinetically is controlled by evaporation. The effects on the DEHP loss rate of nitrogen flow rate, relative humidity and degradation of the plasticizer at 100 °C was assessed. The sample mass decreased linearly with time for both pristine DEHP and plasticized PVC at comparable rates, suggesting that a thin film of DEHP was present on the jacketing insulation during desorption. The latter hypothesis was supported by infrared spectroscopy and by the fact that DEHP is an amphiphilic molecule that will tend to aggregate at the surface with the hydrophobic 2-ethylhexyl units at the air interface. The effect on the migration rate of moisture present in the gas phase was negligible. The DEHP loss rate increased in a retarding non-linear fashion with increasing gas flow rate. In one of the experiments, DEHP was accidently degraded as revealed by discoloration, the presence of low molar mass degradation products (liquid chromatography) containing additional carbonyl groups (infrared spectroscopy) and an increase in the evaporation rate at temperatures between 100 and 130 °C.     

氧化石墨烯-邻苯二甲酸二(2-乙基己)酯表面分子印迹吸附剂的合成及应用

合成了以纳米材料氧化石墨烯为载体的表面分子印迹固相萃取材料，建立了分子印迹萃取联用高效液相色谱法检测牛奶塑料包装袋中的塑化剂邻苯二甲酸二（2-乙基己）酯（DEHP）残留的方法。以氧化石墨烯为基质、DEHP为模板分子、甲基丙烯酸为功能单体、N，N-二甲基甲酰胺为溶剂，通过沉淀聚合法合成表面分子印迹材料，优化了合成条件并对产品进行红外光谱、透射电镜表征。对产品的吸附性能（包括选择性、吸附平衡时间、吸附容量、重复使用率等）进行测定。在最优萃取条件下对牛奶包装袋提取液中DEHP进行选择性富集，通过高效液相色谱-紫外法检测，线性范围为0.5~50 mg/L，检出限为0.03 mg/L，定量限为0.1 mg/L。3种加标浓度下回收率为81.6%~92.4%，相对标准偏差（RSDs）小于7%。结果表明，该方法能够应用于实际样品中DEHP分析。     

Determination of DEHP in blood products stored in plastic bags by HPLC

Summary 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.     

Determination of bis(2-ethylhexyl) phthalate in water by high-performance liquid chromatography with direct on-column preconcentration

A technique for the direct preconcentration of bis(2-ethylhexyl) phthalate (DEHP) on a reversedphase analytical column was proposed for the analysis of water samples by high-performance liquid chromatography (HPLC). A procedure for determining DEHP in surface-water and atmospheric precipitations in the laboratory and in the field was developed (the limit of detection is 0.1 Μg/L, the limit of determination is 0.3 Μg/L, and the relative standard deviation is 20 or 6% at a DEHP concentration of 0.3 or 10 Μg/L, respectively). The concentration levels of DEHP as a chemical tracer for the transfer and migration of air and water masses were examined in Lake Baikal water and in the snow cover of the Baikal region.     

液相色谱-串联质谱法测定饮料中邻苯二甲酸二(2-乙基)己酯和邻苯二甲酸二异壬酯

建立了饮料中邻苯二甲酸二(2-乙基)己酯(DEHP)和邻苯二甲酸二异壬酯(DINP)残留的液相色谱-串联质谱(LC-MS/MS)检测方法。2.0 g样品经8 mL甲醇振荡提取、定容、离心,取上清液过滤,采用LC-MS/MS电喷雾电离,多反应监测(MRM)模式对样品进行分析。DEHP在浓度范围为2～200μg/L,DINP在10～1000μg/L内线性良好,相关系数均大于0.998。实验表明:样品无明显的基质效应。样品中添加0.01～5 mg/kg的DEHP和DINP,其回收率为86.2%～111.6%;相对标准偏差(n=6)小于11%;DEHP检出限为0.008 mg/kg,定量限为0.01 mg/kg;DINP检出限为0.01 mg/kg,定量限为0.05 mg/kg。本方法提取效果好,具有良好的灵敏度、回收率和重复性,被成功用于实际饮料样品中DEHP和DINP的测定。     

Studies on the thermal decomposition of phthalate esters: IX. Thermal decomposition of bis(2-ethylhexyl) phthalate

A flow apparatus equipped with a spray nozzle was developed for investigating the thermal decomposition of esters with high boiling points and viscosities. The thermal decomposition of bis(2-ethylhexyl) phthalate (BEHP), which is a typical plasticizer for poly(vinyl chloride) with a high boiling point and viscosity, was carried out by the use of the flow apparatus. The thermal decomposition products were analysed by gas chromatography and the kinetic parameters were calculated. The kinetic parameter of bis(2-ethylhexyl) phthalate was k_BEHP (s^?1)=4.59·10¹¹ exp(?40600/RT). From a comparison with the values for phthalic esters, it is proposed that a cis-elimination reaction takes place with a two-step mechanism in which the rate-determining step is α-carbon cleavage.     

Micro-organic ion-associate phase extraction via in situ fresh phase formation for the preconcentration and determination of di(2-ethylhexyl)phthalate in river water by HPLC.

A high-enrichment method was proposed for the HPLC determination of trace di(2-ethylhexyl)phthalate (DEHP) in environmental water. A micro-organic ion-associate phase (IAP) was formed in situ from an aqueous sample by adding 4-trifluoromethylanilinium ion and dodecylbenzenesulfonate ions. Centrifugation of the solution led to the isolation of a liquid organic phase containing DEHP at the bottom of the centrifuge tube. The volume of the phase formed was less than 30 microL. DEHP was extracted into the IAP quantitatively during phase formation. After discarding the aqueous phase, the ion associate was dissolved with 50 microL of 2-methoxyethanol, and DEHP in the concentrate was determined by HPLC with an ultra-violet (UV) diode-array detector. DEHP in the concentrations range from 0.8 to 78 microg L(-1) was determined with good precision. The recovery tests for DEHP added to some river water were satisfactory. The detection limit of DEHP, defined as 3-times the standard deviation of the blank signals, was 0.07 microg L(-1) (n = 3). The present method is very simple, and was applied to the determination of DEHP in the river water samples collected around Toyama City, Japan.     

One-step in-syringe dispersive liquid–liquid microextraction and GC-FID determination of trace amounts of di(2-ethylhexyl) phthalate and its metabolite in human urine samples

Di(2-ethylhexyl) phthalate (DEHP) is used as plasticizer in polyvinylchloride (PVC) plastics. Its metabolites and the parent phthalates are considered toxic. As the DEHP plasticizers are not chemically bound to PVC, they can migrate, evaporate or be leached into indoor air and atmosphere, foodstuff, and other materials. We have reported a novel, easy and available analytical method for the determination of DEHP and its metabolite, mono(2-ethylhexyl) phthalate (MEHP) in human urine samples by the in-syringe dispersive liquid–liquid microextraction method coupled with gas chromatography with flame ionization detector. The limits of detection and precision (RSD) were 2.5 μg/L and 1.4% for DEHP and 1.1 μg/L and 3.0% for MEHP, respectively. This method could be utilized for routine monitoring of the trace DEHP and MEHP in urine of human exposure to plasticizers.     

多壁碳纳米管表面邻苯二甲酸二(2-乙基)己酯印迹聚合物的制备及其在固相萃取中的应用

以苯基修饰的多壁碳纳米管为载体,邻苯二甲酸二(2-乙基)己酯为模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,在碳纳米管表面接枝一层塑化剂邻苯二甲酸二(2-乙基)己酯印迹聚合层.采用红外光谱和扫描电镜对聚合物进行表征和分析.结果表明,在碳纳米管表面成功接枝一层20～30 nm厚的印迹聚合层.采用高效液相色谱研究该印迹聚合物的吸附性能,结果表明,碳纳米管分子印迹聚合物对邻苯二甲酸二(2-乙基)己酯最大吸附量为69.1 μmol/g,达到吸附平衡时间约为60 min.选择性吸附实验表明,与其它结构类似物相比,该印迹复合材料对邻苯二甲酸二(2-乙基)己酯有良好的识别能力.作为固相萃取材料装填于固相萃取柱中,该印迹聚合物能对芒果汁样品中塑化剂进行有效的分离和富集.