Hsin-Chang Chen

Using liquid chromatography-ion trap mass spectrometry to determine pharmaceutical residues in Taiwanese rivers and wastewaters.

To establish their environmental concentrations and to support surface water protection programs, we have undertaken a preliminary study of the concentrations of selected acidic and neutral pharmaceutical residues (clofibric acid, ketoprofen, ibuprofen, diclofenac and carbamazepine) in Taiwanese river and wastewater samples. These pharmaceutical residues were extracted from the water samples through the Oasis HLB solid-phase extraction (SPE). The analytes were then identified and quantified using liquid chromatography-ion trap mass spectrometry with dual-polarity electrospray ionization in the product ion scan mode. The limits of quantification (LOQs) ranged between 0.5 and 20 ngl(-1) for 250 ml samples of water. We investigated the intra- and interbatch precision and accuracy at two levels of concentration. The selected analytes were detected at concentrations ranging from <0.5 to 960 ngl(-1) in wastewater treatment plant effluents and river water samples.

Determination of estrogenic compounds in wastewater using liquid chromatography-tandem mass spectrometry with electrospray and atmospheric pressure photoionization following desalting extraction.

Two complementary LC-MS ionization methods, electrospray (ESI) and atmospheric pressure photoionization (APPI), have been optimized to determine three natural estrogenic compounds (estrone, 17beta-estradiol and estriol) and two synthetic estrogenic compounds (17alpha-ethynylestradiol and diethylstilbestrol) in the influent and effluent of wastewater treatment plants (WWTPs). The wastewater samples were first subjected to solid-phase extraction coupled with desalting extraction to remove matrix interference. The analytes were then detected using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with ESI and dopant-assisted (DA) APPI to evaluate the ion suppression effect and to complement the detection and quantification of estrogenic compounds in complex wastewater samples. The average ion suppression factors for the extracts of the WWTP influent analyzed using ESI and APPI were 52+/-5% and 27+/-7%, respectively. The sensitivity and ionization efficiency of the LC-ESI-MS-MS system decreased dramatically when a complex matrix was present in the WWTP influent sample. Estrogenic compounds could be detected in the WWTP influent and effluent samples at concentrations below the parts-per-billion level. The lower detection limits obtained when using ESI and the higher matrix tolerance of the APPI method allowed the complete quantification of estrogenic compounds in very complex samples in a complementary manner.

Determination of volatile N-nitrosamines in meat products by microwave-assisted extraction coupled with dispersive micro solid-phase extraction and gas chromatography - Chemical ionisation mass spectrometry

A sensitive procedure, microwave-assisted extraction (MAE) coupled dispersive micro solid-phase extraction (D-μ-SPE), was developed to extract N-nitrosodimethylamine (NDMA) and other six volatile N-nitrosamines (NAms) from meat products. Parameters affecting the efficiency of MAE and D-μ-SPE were systematically investigated. For MAE, 5-g of a homogenised meat sample was extracted with 30 mL of a sodium hydroxide (0.025 M) solution at 100 °C for 10 min. The optimum D-μ-SPE conditions were immersing 100mg of Carboxen™ 1000 adsorbent in the MAE extract. After vigorously shaking for 30 min, the NAms were then desorbed by treatment with 200 μL of dichloromethane. A 10 μL aliquot was determined by gas chromatography with chemical ionisation mass spectrometry (GC-CI-MS) using the selected-ion-storage (SIS) mode. The limits of quantitation (LOQs) were 0.03-0.36 ng/g. Preliminary results revealed that NDMA was present in the highest concentration, ranging from 0.8 to 3.2 ng/g.

Determination of aqueous fullerene aggregates in water by ultrasound-assisted dispersive liquid–liquid microextraction with liquid chromatography–atmospheric pressure photoionization-tandem mass spectrometry

A simple and solvent-minimized method for the determination of three aqueous fullerene aggregates (nC₆₀, nC₇₀, and aqueous [6,6]-phenyl C₆₁ butyric acid methyl ester (nPCBM)) in water samples is described. The method involves the use of ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) coupled liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization (LC-APPI-MS/MS). The parameters affecting the extraction efficiency of the analytes from water samples were systematically investigated and the conditions optimized. The best extraction conditions involved the rapid injection of a mixture of 1.0 mL of 2-propanol (as a disperser solvent) and 10 μL of benzyl bromide (as an extraction solvent) into 10 mL of an aqueous solution (pH 10.0) containing 1% sodium chloride in a conical bottom glass tube. After ultrasonication for 1.0 min and centrifugation at 5000 pm (10 min), the sedimented phase 5.0 μL was directly injected into the LC-APPI-MS/MS system. The limits of quantification (LOQs) were 150, 60 and 8 ng L⁻¹ for nPCBM, nC₆₀ and nC₇₀, respectively. The precision for these analytes, as indicated by relative standard deviations (RSDs), were less than 12% for both intra- and inter-day analysis. Accuracy, expressed as the mean extraction recovery, was between 70 and 86%. A standard addition method was used to quantitate three aqueous fullerene aggregates, and the concentrations of these aqueous fullerene aggregates were determined to be in the range from n.d. to 130 ng L⁻¹ in various environmental samples including municipal influent and effluent samples, industrial wastewater samples, and surface water samples.

Combining microwave-assisted extraction and liquid chromatography-ion-trap mass spectrometry for the analysis of hexabromocyclododecane diastereoisomers in marine sediments.

An efficient microwave-assisted extraction (MAE) procedure coupled with high performance liquid chromatography-electrospray-ion-trap mass spectrometry (HPLC-ESI-ITMS) has been evaluated to determine hexabromocyclododecane diastereoisomers (alpha-, beta- and gamma-HBCD) in marine sediments. The composition of the LC mobile phase (consisting of water, methanol and acetonitrile) and the parameters of electrospray ionization (ESI) were evaluated to obtain chromatographic baseline separation and high sensitivity for the detection of these diastereoisomers. The effects of various operating parameters on the quantitative extraction of the HBCDs through MAE were systematically investigated. The three diastereoisomers were then quantitated by HPLC-ITMS employing ESI operated in the negative ionization mode. The HBCDs were extracted from the sediments through MAE using 40mL of acetone/n-hexane (1/3, v/v) at 90 degrees C for 12min. The limits of quantitation (LOQ) ranged from 25 to 40pg/g (dry weight) in 5g of the sediment samples. The recoveries of the HBCDs in spiked sediment samples ranged from 68 to 91% (relative standard derivation: 2-11%). The extraction efficiency of the MAE technique was also compared with Soxhlet extraction and pressurized liquid extraction.

Determination of perchlorate in river by ion-pair hollow-fiber liquid-phase microextraction coupled with electrospray ionization tandem mass spectrometry.

This paper describes the analysis of perchlorate (ClO(4)(-)) in surface water samples by a rapid and reliable ion-pair hollow-fiber liquid-phase microextraction (HF-LPME) method coupled with flow-injection electrospray ionization tandem mass spectrometry (ESI-MS-MS) technique. The effects of the type and concentration of ion-pairing reagents, extraction time, temperature and pH value on the quantitative extraction of perchlorate by ion-pair HF-LPME were investigated and optimized. Di-n-hexyl ammonium acetate (DHAA) was employed to form an extractable ion-pair complex with aqueous perchlorate. The characteristic ions [ClO(4)-ClO(4)-DHA](-) at m/z 384.6 and 386.7 were observed in the ESI negative-ionization mode. The predominant product ions [ClO(4)](-) at m/z 99 and 101 were used for quantitation and to maximize the detection selectivity and sensitivity. The limit of detection (LOD) was 0.5 microg/L. The reliability and precision of the standard addition method of ion-pair HF-LPME for the determination of trace levels of perchlorate in surface water were demonstrated.

Analysis of naphthalenesulfonate compounds by cyclodextrin-mediated capillary electrophoresis with sample stacking

This study systematically investigates the optimal conditions for analyzing the positional isomers of multi-charged naphthalenesulfonate compounds by cyclodextrin-mediated capillary electrophoresis (CE). Specifically, this work employs large-volume sample injection with the electrode polarity switching technique. The most effective separation and sample stacking conditions were 15 mM borate buffer with a mixture of beta- and gamma-cyclodextrin (concentration ratio 3:7 mM) at pH 9.2, and the sample hydrodynamic injection of up to 60 s at 3 p.s.i. (around 1.8 microl, and 1 p.s.i. = 6.9 kPa). Significantly selective and sensitive improvements were observed and a more than 100-fold enrichment was achieved (based on peak area). The reproducibility of migration time and quantitative results of stacking CE can be improved by using an internal standard. The quantitation limits of these naphthalenesulfonate isomers, based on a signal-to-noise ratio above 10, can be about 4 microg/l with UV detection. This method was successfully applied to determine the trace amount of naphthalenesulfonate isomers in a spiked drinking water sample.

Determination of the maleic acid in rat urine and serum samples by isotope dilution-liquid chromatography-tandem mass spectrometry with on-line solid phase extraction.

A rapid and simple on-line solid-phase extraction coupled with isotope dilution-liquid chromatography-tandem mass spectrometry (SPE-ID-LC-MS/MS) method was developed to quantitate maleic acid in serum and urine of SpragueDawley (SD) rats. The aforementioned biological samples were spiked with (13)C2-maleic acid, vigorously vortexed, added with acetonitrile to precipitate proteins, and then injected into the on-line SPE-LC-MS/MS system for quantification. Upon validation, this method demonstrated excellent feasibility and sensitivity: calibration curves for maleic acid in serum and urine display excellent linearity with the coefficient of determination (R(2)) greater than 0.999; the limits of detection and quantitation (LOD and LOQ) for maleic acid were determined at 0.2 and 0.5μg L(-1), respectively. Additionally, intra-day accuracy for maleic acid in serum and urine samples ranged from 94.0% to 100.2% and 101.3% to 104.4%, respectively. Furthermore, inter-day accuracy ranged from 93.6% to 101.0% and from 102.3% to 111.4% in serum and urine samples, respectively. Intra-day precision %RSD of maleic acid in serum and urine samples was 13.8% or less, whereas the inter-day precision was 6.1% or less. The matrix effects were not found to be statistically significant (p=0.9145 and p=0.5378, correspondingly) based on the calculations of recovery functions. The collected serum and urine samples were analyzed using SPE-ID-LC-MS/MS. Our results reveal trace levels of maleic acid in the control rats, demonstrating that this method is capable of analyzing background levels of contaminants in biofluids with excellent sensitivity and specificity at part-per-billion levels concentrations in complex matrices.

Study of urinary 2-{[2-(acetylamino-2-carboxyethyl]sulfanyl}butanedioic acid, a mercapturic acid of rats treated with maleic acid

Maleic anhydride was reported illegally adulterated into starch to prepare traditional foods for decades in Taiwan. Maleic acid (MA), hydrolyzed from maleic anhydride, could cause kidney damages to animals. The potential health effects due to long-term MA exposures through food consumption have been of great concerns. Assessment of the dietary MA exposures could be very difficult and complicated. One of the alternatives is to analyze an MA-specific biomarker to assess the daily total MA intake. Therefore, this paper aimed to study the mercapturic acid of MA, 2-{[2-(acetylamino)-2-carboxyethyl]sulfanyl}butanedioic acid (MAMA), with our newly-developed isotope-dilution online solid-phase extraction liquid chromatography tandem mass spectrometry (ID-SPE-LC-MS/MS) method. MAMA was first synthesized, purified, and characterized with NMR to reveal two diastereomers and used for developing the analytical method. The method was validated to reveal excellent sensitivity with a LOD at 16.3ng/mL and a LOQ at 20.6ng/mL and used to analyze MAMA in urine samples collected from Sprague-Dawley rats treated with a single dose of 0mg/kg, 6mg/kg, and 60mg/kg (n=5) of MA through gavage. Our results show dose-dependent increases in urinary MAMA contents, and 70% MAMA was excreted within 12h with no gender differences (p>0.05). A half life of urinary MAMA was estimated at 6.8h for rat. The formation of urinary MAMA validates it as a chemically-specific biomarker for current MA exposure. Future study of MA metabolism in vivo will elucidate mechanisms of MAMA formation, and analysis of this marker in epidemiology studies could help to shed light on the causal effects of MA on human.

The interactions among organophosphate pesticide exposure, oxidative stress, and genetic polymorphisms of dopamine receptor D4 increase the risk of attention deficit/hyperactivity disorder in children

Objective The aim of this study was to clarify the association between organophosphate pesticides (OPs) and attention‐deficit/hyperactivity disorder (ADHD) related to oxidative stress and genetic polymorphisms. Methods This case‐control study enrolled 93 children with ADHD and 112 control children in north Taiwan. Six dialkyl phosphate (DAP) metabolites of OPs and oxidative stress biomarkers were analyzed. Polymorphisms of the dopamine receptor D4 gene (DRD4) were identified. Results Children with ADHD had significantly higher dimethylphosphate (DMP, 236.69 nmol/g cre. vs. 186.84 nmol/g cre., p value = 0.01) and 4‐hydroxy‐2‐nonenal‐mercapturic acid (HNE‐MA, 28.95 &mgr;g/g cre. vs. 16.55 &mgr;g/g cre., p value<0.01) concentrations than control children. Children who carried DRD4 GA/AA genotypes (rs752306) were less likely than those who carried the DRD4 GG genotype to have ADHD (odds ratio [OR]: 0.45, 95% CI: 0.24–0.84). The estimated value of the AP (attributable proportion due to interaction) was 0.59 (95% CI: 0.13–1.05), indicating that 59% of ADHD cases in DMP‐exposed children with the DRD4 GG genotype were due to the gene‐environment interaction. After adjustment for other covariates, children who carried the DRD4 GG genotype, had been exposed to high DMP levels (more than the median), and had high HNE‐MA levels had a significantly increased risk for developing ADHD (OR = 11.74, 95% CI: 2.12–65.04). Conclusion This study indicated a gene‐environment interaction in the risk of ADHD in children. The association between DMP and ADHD in children might relate to the mechanism of lipid peroxidation. Dose‐response relationships and the combined effects of OPs, oxidative stress, and genetic polymorphism on ADHD should not be neglected. HighlightsChildren with ADHD had high dimethylphosphate (DMP) and lipid peroxidation levels.An additive interaction between DRD4 polymorphism and DMP levels for ADHD existed.The combined effects of DRD4, DMP, and HNE‐MA increased the risk of developing ADHD.