Elisabeth Eckert

Efficient drug-delivery using magnetic nanoparticles — biodistribution and therapeutic effects in tumour bearing rabbits

UNLABELLED To treat tumours efficiently and spare normal tissues, targeted drug delivery is a promising alternative to conventional, systemic administered chemotherapy. Drug-carrying magnetic nanoparticles can be concentrated in tumours by external magnetic fields, preventing the nanomaterial from being cleared by metabolic burden before reaching the tumour. Therefore in Magnetic Drug Targeting (MDT) the favoured mode of application is believed to be intra-arterial. Here, we show that a simple yet versatile magnetic carrier-system (hydrodynamic particles diameter <200nm) accumulates the chemotherapeutic drug mitoxantrone efficiently in tumours. With MDT we observed the following drug accumulations relative to the recovery from all investigated tissues: tumour region: 57.2%, liver: 14.4%, kidneys: 15.2%. Systemic intra-venous application revealed different results: tumour region: 0.7%, liver: 14.4 % and kidneys: 77.8%. The therapeutic outcome was demonstrated by complete tumour remissions and a survival probability of 26.7% (P=0.0075). These results are confirming former pilot experiments and implying a milestone towards clinical studies. FROM THE CLINICAL EDITOR This team of investigators studied drug carrying nanoparticles for magnetic drug targeting (MDT), demonstrating the importance of intra-arterial administration resulting in improved clinical outcomes in the studied animal model compared with intra-venous.

Determination of six hydroxyalkyl mercapturic acids in human urine using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC–ESI-MS/MS) ☆

Mercapturic acids are increasingly used as biomarkers for exposure to certain carcinogenic substances. Glycidol, ethylene oxide, propylene oxide, acrolein and 1,3-butadiene are important intermediates of toxicological concern used in the industrial production of various chemicals. The main urinary metabolites of these alkylating substances are hydroxyalkyl mercapturic acids. Therefore, we developed and validated an analytical method for the simultaneous determination of six hydroxyalkyl mercapturic acids in human urine after solid phase extraction. The mercapturic acids were separated using hydrophilic interaction liquid chromatography (HILIC) and quantified by tandem mass spectrometry using isotopically labelled internal standards. The developed method enables for the first time the determination of 2,3-dihydroxypropyl mercapturic acid (DHPMA), a metabolite of glycidol, in human urine. Additionally, the mercapturic acids of ethylene oxide (hydroxyethyl mercapturic acid, HEMA), propylene oxide (2-hydroxypropyl mercapturic acid, 2-HPMA), acrolein (3-hydroxypropyl mercapturic acid, 3-HPMA) as well as of 1,3-butadiene(3,4-dihydroxybutyl mercapturic acid, DHBMA and monohydroxy-3-butenyl mercapturic acid, MHBMA) can be determined. The limits of detection range from 3.0 to 7.0 μg/L. Intra- and inter-day precision was determined to range from 1% to 9%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for the determination of occupational exposure to alkylating substances as well as for the determination of background concentrations of the respective mercapturic acids in the general population.

Mercapturic acids as metabolites of alkylating substances in urine samples of German inhabitants

Hydroxyalkyl mercapturic acids (HAMA) are the main urinary metabolites of several alkylating substances that possess a carcinogenic potential, like acrolein, 1,3-butadiene, ethylene oxide, propylene oxide and glycidol. These alkylating substances are used extensively in industrial processes, but they do also occur environmentally, e.g. in tobacco smoke. The aim of this study was the determination of six HAMA, as biomarkers of exposure, in human urine of smokers and non-smokers. We applied a sensitive analytical method, using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) for the determination of 2-hydroxyethyl mercapturic acid (HEMA, biomarker for ethylene oxide), 2-hydroxypropyl mercapturic acid (2-HPMA, biomarker for propylene oxide), 3-hydroxypropyl mercapturic acid (3-HPMA, biomarker for acrolein), 2,3-dihydroxypropyl mercapturic acid (DHPMA, biomarker for glycidol) as well as 3,4-dihydroxybutyl mercapturic acid and 3-monohydroxybutenyl mercapturic acids (DHBMA and MHBMA, biomarkers for 1,3-butadiene). Background concentrations of four HAMA were detected in each urine sample we analyzed. The mercapturic acids HEMA and MHBMA were detected in 55% and 10% of the samples, respectively. In the urine of non-smokers (n = 54) we observed median levels of 206, 1.6, 12.1, 146, 159, and <5.0 μg/g creatinine for DHPMA, HEMA, 2-HPMA, 3-HPMA, DHBMA and MHBMA, respectively. Among smokers (n = 40) median levels of DHPMA, HEMA, 2-HPMA, 3-HPMA, DHBMA and MHBMA were determined to be 217, 4.9, 46.2, 884, 211 and <5.0 μg/g creatinine, respectively. The excretion rate of the biomarkers HEMA, 2-HPMA and 3-HPMA was distinctly higher in smokers than in non-smokers. Furthermore, our study revealed a comparatively high background level of DHPMA in urine of smokers and non-smokers whose origin is still unknown. The presented data may contribute to the evaluation of reference values for urinary HAMA levels in the general population.

Comparative study on the migration of di-2-ethylhexyl phthalate (DEHP) and tri-2-ethylhexyl trimellitate (TOTM) into blood from PVC tubing material of a heart-lung machine.

Medical devices like blood tubing often consist of PVC material that requires the addition of plasticizers. These plasticizers may migrate into the blood leading to an exposure of the patients. In this study the migration behavior of three different blood tubing sets (PVC material with two different plasticizers and silicone as control material) applied on a heart-lung machine standardly used for cardiopulmonary bypass (CPB) in children was studied. We analyzed the total plasticizer migration by analysis of both, the parent compounds as well as their primary degradation products in blood. Additionally, the total mass loss of the tubing over perfusion time was examined. The PVC tubing plasticized with DEHP (di-2-ethylhexyl phthalate) was found to have the highest mass loss over time and showed a high plasticizer migration rate. In comparison, the migration of TOTM (tri-2-ethylhexyl trimellitate) and its primary degradation products was found to be distinctly lower (by a factor of approx. 350). Moreover, it was observed that the storage time of the tubing affects the plasticizer migration rates. In conclusion, the DEHP substitute TOTM promises to be an effective alternative plasticizer for PVC medical devices particularly regarding the decreased migration rate during medical procedures.

Simultaneous determination of polyvinylchloride plasticizers di(2-ethylhexyl) phthalate and tri(2-ethylhexyl) trimellitate and its degradation products in blood by liquid chromatography-tandem mass spectrometry

Di(2-ethylhexyl) phthalate (DEHP) and tri(2-ethylhexyl) trimellitate (TEHTM or TOTM) are common plasticizers that are also largely used for PVC medical devices, e.g. bags and tubing for blood transfusions and infusions. The leachability of medical devices is a well-known situation of increasing toxicological concern. To assess the migration of plasticizers from PVC medical devices into human blood we developed and validated an analytical method for the determination of DEHP and TOTM in combination with the determination of their primary degradation products mono(2-ethylhexyl) phthalate (MEHP), 1,2-di(2-ethylhexyl) trimellitate (1,2-DEHTM) and 2-mono(2-ethylhexyl) trimellitate (2-MEHTM). The presented method involves liquid-liquid extraction of the analytes from the blood followed by the subsequent analytical separation and detection using LC-MS/MS analysis. The validation of the procedure showed a good precision in the range of 1.8 to 5.3%. Mean accuracy ranged from 86% for 1,2-DEHTM to 109% for MEHP. LOQ was found to be 2 to 5μg/L for each of the analytes. Additionally, the method is characterised by its wide linear range up to 2mg/L each for the degradation products of TOTM to 100mg/L for the parent plasticizer DEHP. The presented method promises to be of major advantage for further studies as it allows for the first time the simultaneous determination of DEHP and TOTM in human blood in combination with the analysis of their degradation products that render possible to investigate the leachability of a broad range of PVC medical devices in human blood using only one analytical method.

A method for the simultaneous determination of mercapturic acids as biomarkers of exposure to 2-chloroprene and epichlorohydrin in human urine

We developed and validated an analytical method for the simultaneous determination of several chlorine and non-chlorine containing mercapturic acids in urine as specific metabolites of the hazardous chemicals 2-chloroprene and epichlorohydrin. The method involves an online column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using LC-MS/MS. The developed method enables for the first time the determination of Cl-MA-I (4-chloro-3-oxobutyl mercapturic acid), Cl-MA-II (4-chloro-3-hydroxybutyl mercapturic acid), Cl-MA-III (3-chloro-2-hydroxy-3-butenyl mercapturic acid) and HOBMA (4-hydroxy-3-oxobutyl mercapturic acid) as potential biomarkers of 2-chloroprene in urine. Additionally, CHPMA (3-chloro-2-hydroxypropyl mercapturic acid) as a specific metabolite of epichlorohydrin in urine and DHBMA (3,4-dihydroxybutyl mercapturic acid) can be determined. The analytical method proved to be both sensitive and reliable with detection limits ranging from 1.4 μg/L (for Cl-MA-III) to 4.2 μg/L (for HOBMA). Intra- and interday imprecision was determined to range from 4.7 to 11.8%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for application in biomonitoring studies in order to determine occupational exposure to 2-chloroprene and epichlorohydrin.

Rapid determination of four short-chain alkyl mercapturic acids in human urine by column-switching liquid chromatography–tandem mass spectrometry

We developed and validated an analytical method for the simultaneous determination of methyl mercapturic acid (MeMA), ethyl mercapturic acid (EtMA), n-propyl mercapturic acid (PrMA) and iso-propyl mercapturic acid (iPrMA) in human urine. These alkyl mercapturic acids are known or presumed biomarkers of exposure to several alkylating agents including methyl bromide, dimethyl sulfate, ethyl bromide, 1-bromopropane and 2-bromopropane. The method involves a column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using liquid chromatography and tandem mass spectrometry. Within day and day-to-day imprecision was determined to range from 4.5 to 12.2%. The analytical method is distinguished by its wide linear working range of up to 2,500 μg/L with detection limits ranging from 2.0 μg/L (for PrMA) to 5.1 μg/L (for MeMA) that render possible the application in various biomonitoring studies regarding exposure to alkylating agents. The results of a pilot study on urine samples of 30 individuals occupationally non-exposed to alkylating agents using the new procedure confirmed the background excretion of MeMA (<5.1-35.6 μg/L) and PrMA (<2.0-95.7 μg/L).

Isomeric separation and quantitation of di-(2-ethylhexyl) trimellitates and mono-(2-ethylhexyl) trimellitates in blood by LC–MS/MS

A new and fast HPLC-method for the simultaneous determination of tri-(2-ethylhexyl) trimellitate (TOTM or TEHTM), its diesters 2,4-di-(2-ethylhexyl) trimellitate (2,4-DEHTM), 1,4-di-(2-ethylhexyl) trimellitate (1,4-DEHTM), 1,2-di-(2-ethylhexyl) trimellitate (1,2-DEHTM) and monoesters 1-mono-(2-ethylhexyl) trimellitate (1-MEHTM), 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM) and 4-mono-(2-ethylhexyl) trimellitate (4-MEHTM) together with di-(2-ethylhexyl) phthalate (DEHP) and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) in blood was developed and validated. The analytes are extracted from blood using liquid-liquid extraction and are chromatographically separated by reversed-phase HPLC using core shell material. Quantitative assessment was performed by ESI-tandem mass spectrometry in negative ionization mode using stable isotope dilution. In less than 30min six postulated primary metabolites of TOTM along with the DEHP metabolite MEHP can be selectively and sensitively quantified. Additionally, the method enables the determination of the parent plasticizers TOTM and DEHP. The detection limits in blood were found to range between 0.7-5.5μg/L for all TOTM analytes. Precision and repeatability of the method were proven by relative standard deviations between 0.9% and 8.7%. TOTM, an alternative plasticizer to DEHP, is already increasingly used for medical devices. Nevertheless, data about the human metabolism of TOTM are still limited. The presented method is the first one enabling the simultaneous determination of the parent plasticizers TOTM and DEHP together with their primary degradation products (DEHTM, MEHTM, MEHP) and can thus be applied manifold including the investigation of the human metabolism of TOTM.

Excretion of mercapturic acids in human urine after occupational exposure to 2-chloroprene

A pilot study was conducted for human biomonitoring of the suspected carcinogen 2-chloroprene. For this purpose, urine samples of 14 individuals occupationally exposed to 2-chloroprene (exposed group) and of 30 individuals without occupational exposure to alkylating substances (control group) were analysed for six potential mercapturic acids of 2-chloroprene: 4-chloro-3-oxobutyl mercapturic acid (Cl-MA-I), 4-chloro-3-hydroxybutyl mercapturic acid (Cl-MA-II), 3-chloro-2-hydroxy-3-butenyl mercapturic acid (Cl-MA-III), 4-hydroxy-3-oxobutyl mercapturic acid (HOBMA), 3,4-dihydroxybutyl mercapturic acid (DHBMA) and 2-hydroxy-3-butenyl mercapturic acid (MHBMA). In direct comparison with the control group, elevated levels of the mercapturic acids Cl-MA-III, MHBMA, HOBMA and DHBMA were found in the urine samples of the exposed group. Cl-MA-I and Cl-MA-II were not detected in any of the samples, whereas HOBMA and DHBMA were found in all analysed urine samples. Thus, for the first time, it was possible to detect HOBMA and Cl-MA-III in human urine. The mercapturic acid Cl-MA-III could be confirmed as a specific metabolite of 2-chloroprene in humans providing evidence for the intermediate formation of a reactive epoxide during biotransformation. The main metabolite, however, was found to be DHBMA showing a distinct and significant correlation with the urinary Cl-MA-III levels in the exposed group. The obtained results give new scientific insight into the course of biotransformation of 2-chloroprene in humans.

Regioselective ester cleavage of di-(2-ethylhexyl) trimellitates by porcine liver esterase

In a comparative study the ester hydrolysis of the plasticizers di-(2-ethylhexyl) phthalate (DEHP) and tri-(2-ethylhexyl) trimellitate (TEHTM) as well as of the diester isomers 1,2-di-(2-ethylhexyl) trimellitate (1,2-DEHTM), 1,4-di-(2-ethylhexyl) trimellitate (1,4-DEHTM) and 2,4-di-(2-ethylhexyl) trimellitate (2,4-DEHTM) was investigated by a newly developed in vitro experimental design using porcine liver esterase (PLE). The substrates were incubated with PLE for 48h at 25°C in borate buffer and samples were taken at predetermined intervals during the experiment. The samples were processed using liquid-liquid extraction and analyzed using LC-MS/MS. The results demonstrated a rapid and extensive hydrolysis of the diester DEHP to the monoester mono-(2-ethylhexyl) phthalate (MEHP) during the incubation with PLE. The isomers of DEHTM were also hydrolyzed by PLE to a high extent, whereas TEHTM showed a high stability against enzymatic hydrolysis. The regioselective analysis revealed that the monoester isomers 1-MEHTM and 2-MEHTM were predominantly produced during the degradation of DEHTM isomers, indicating a preferred hydrolysis at the para-position. These findings are eminent for planning further investigations on the human TEHTM metabolism, as the extent, rate and route of metabolism are of crucial importance for a toxicological assessment.