On-line Solid-phase Extraction-liquid Chromatography-tandem Mass Research Articles

Simultaneous determination of fourteen pharmaceuticals in sewage sludge using online solid-phase extraction-liquid chromatography-tandem mass spectrometry combined with accelerated solvent extraction.

An online solid-phase extraction (SPE) liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) combined with accelerated solvent extraction (ASE) was developed for simultaneous determination of 14 pharmaceuticals in sludge. In the online SPE procedures, ultrapure water with no additives was used as the loading solvent. In addition, low molecular weight targets such as atenolol were difficult to retain on SPE column after acetone was added to the washing solvent. The response signal of analytes can be greatly improved by adding 0.2% formic acid to the mobile phase. Under the optimized conditions, the recoveries of all the analytes ranged between 75.1 and 112%. Moreover, the limit of detections ranged from 1.8 to 7.9 ug/kg. The precision of analytical data was determined with relative standard deviation (RSD) ≤ 4.87%. This method was successfully applied to determine the concentration of pharmaceuticals in sludge.

Simultaneous online SPE-HPLC-MS/MS quantification of gefitinib, osimertinib and icotinib in dried plasma spots: Application to therapeutic drug monitoring in patients with non-small cell lung cancer

Gefitinib, osimertinib and icotinib are the most commonly used tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) with EGFR mutation. Therapeutic drug monitoring (TDM) for these TKIs has become a standard and essential procedure. Dried plasma spots (DPS) was choosen for microsampling strategies for TDM, allowing easy and cost-effective logistics in many settings. This study developd and validated an assay for the simultaneous quantitative determination of gefitinib, osimertinib and icotinib in DPS by online solid-phase extraction-liquid chromatography–tandem mass spectrometry (online SPE-LC-MS) system. The TKIs were extracted from DPS with methanol and enriched on a Welch Polar-RP SPE column (30 × 4.6 mm, 5 µm), followed by separation on Waters X Bridge C18 analytical column(4.6 × 100 mm, 3.5 µm). The method achieved LLOQ of 2 ng mL−1 for gefitinib and osimertinib (4 ng mL−1 for icotinib), respectively (r2 > 0.99). Precision (within-run 1.54–7.41 % RSD; between-run 3.03–12.84 % RSD), accuracy (range from 81.47 % to 105.08 %; between-run bias 87.87–104.13 %). Osimertinib and icotinib were stable in DPS stored at − 40 °C for 30 days, 4 °C, 42 °C and 60 °C for 5 days and well-sealed 37 °C,75 % humidity (except gefitinib). Lastly, the assay was applied to TDM of TKIs in 46 patients and the results were compared to SALLE assisted LC-MS analysis, it could be confirmed that the developed method achieves similarly good results as the already established one and no bias could be detected. It implies that this method capable of supporting clinical follow-up TDM of TKIs in DPS from poor medical environment.

Unraveling the dynamics of organic micropollutants in wastewater: Online LC-MS/MS analysis at high temporal resolution

Online monitoring of organic micropollutants (OMPs) in the aquatic environment at high temporal resolution is an upcoming technique that provides insights into their dynamics and has the potential to bring water research and management to a new level. An online monitoring setup was developed to quantify OMPs in wastewater treatment plant (WWTP) influent and effluent using automated and continuous sampling, sample preparation, online solid-phase extraction-liquid chromatography-tandem mass spectrometry analysis and data evaluation. This online monitoring setup provided high selectivity and sensitivity (limit of quantification down to 1 ng/L) as well as a stable performance during one week of constant operation whilst using a high sampling frequency of 10 min (>1000 samples). Custom automated data evaluation enabled quantification within seconds after each measurement and results were comparable to those from a commercial software. Additionally, an alarm tool was included in the evaluation application, which automatically notified the user in case a substance exceeded a predefined threshold. The online monitoring setup was applied to WWTP influent and effluent, where 57 substances were monitored over a period of one week and two days, respectively. High temporal resolution enabled the observation of periodic patterns of pharmaceuticals as well as pollution by OMPs originating from point and diffuse sources, while dynamics of OMPs in WWTP effluent were less pronounced. These new insights into the dynamics of OMPs in WWTP influent, which would not be observable using 24 h composite samples, will be a starting point for new stormwater and wastewater research and management strategies.

Automated sample preparation for the detection and confirmation of hypoxia-inducible factor stabilizers in urine.

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation.

Improved fully automated method for the determination of medium to highly polar pesticides in surface and groundwater and application in two distinct agriculture-impacted areas.

Water is an essential resource for all living organisms. The continuous and increasing use of pesticides in agricultural and urban activities results in the pollution of water resources and represents an environmental risk. To control and reduce pesticide pollution, reliable multi-residue methods for the detection of these compounds in water are needed. In this context, the present work aimed at providing an analytical method for the simultaneous determination of trace levels of 51 target pesticides in water and applying it to the investigation of the target pesticides in two agriculture-impacted areas of interest. The method developed, based on an isotopic dilution approach and on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry, is fast, simple, and to a large extent automated, and allows the analysis of most of the target compounds in compliance with European regulations. Application of the method to the analysis of selected water samples collected at the lowest stretches of the two largest river basins of Catalonia (NE Spain), Llobregat and Ter, revealed the presence of a wide suite of pesticides in the Llobregat basin, some of them at concentrations above the water quality standards (irgarol and dichlorvos) or the acceptable method detection limits (methiocarb, imidacloprid, and thiacloprid), and much cleaner waters in the Ter River basin. Risk assessment of the pesticide concentrations measured in the Llobregat River indicated high risk due to the presence of irgarol, dichlorvos, methiocarb, azinphos ethyl, imidacloprid, and diflufenican (hazard quotient (HQ) values>10), and moderate potential risk in the Ter River, associated to the occurrence of bentazone and irgarol (HQ > 1).

Determination of glyphosate, AMPA, and glufosinate in honey by online solid-phase extraction-liquid chromatography-tandem mass spectrometry

ABSTRACTA simple method was developed for the simultaneous determination of glyphosate, its main degradation product (aminomethylphosphonic acid), and glufosinate in honey. Aqueous honey solutions were derivatised offline prior to direct analysis of the target analytes using online solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry. Using the developed procedure, accuracies ranging from 95.2% to 105.3% were observed for all analytes at fortification levels of 5, 50, and 150 μg kg−1 with intra-day precisions ranging from 1.6% to 7.2%. The limit of quantitation (LOQ) was 1 μg kg−1 for each analyte. Two hundred honey samples were analysed for the three analytes with AMPA and glyphosate being most frequently detected (99.0% and 98.5% of samples tested, respectively). The concentrations of glyphosate were found to range from <1 to 49.8 μg kg−1 while those of its degradation product ranged from <1 to 50.1 μg kg−1. The ratio of glyphosate to AMPA was found to vary significantly amongst the samples where both analytes were present above the LOQ. Glufosinate was detected in 125 of 200 samples up to a maximum concentration of 33.0 μg kg−1.

Development and validation of an analytical method based on liquid chromatography-tandem mass spectrometry detection for the simultaneous determination of 13 relevant wastewater-derived contaminants in lettuce.

We present an analytical method developed and validated to study the potential uptake of 13 selected drugs (ten pharmaceuticals, one illicit drug, and two transformation products) into lettuce plants from contaminated water and soil. Some of the selected drugs (i.e., cocaine, methadone, cis-diltiazem, valsartan, and valsartan acid), which are commonly present in treated wastewater, were investigated for the first time in plant tissues. The method is based on ultrasonic solvent extraction with acetonitrile-methanol (1:1, v/v) and subsequent automated extract cleanup and analysis by means of online solid-phase extraction-liquid chromatography-tandem mass spectrometry. Optimum extraction conditions were selected after evaluation of analyte recoveries with four different extraction techniques (ultrasonic solvent extraction, solid-liquid extraction, pressurized liquid extraction, and a "quick, easy, cheap, effective, rugged, and safe" based method) and six different solvent mixtures. Furthermore, two different solid-phase extraction cleanup sorbents were evaluated. The method developed has high sensitivity (with limits of detection between 0.1 and 12.6 ng per gram dry weight and limits of quantification between 0.5 and 42.0 ng per gram dry weight), satisfactory accuracy (with analyte relative recoveries above 80% for all analytes but acridone and oxcarbazepine), and good repeatability (with relative standard deviations below 9% for all analytes). As part of the validation procedure, the analytical method was applied to the analysis of lettuce plants irrigated with water fortified with the selected compounds for the entire growing period. The results obtained evidenced the transfer of all the investigated drugs into lettuce leaves. Graphical Abstract ᅟ.

Multiresidue trace analysis of pharmaceuticals, their human metabolites and transformation products by fully automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry

A novel, fully automated analytical methodology based on dual column liquid chromatography coupled to tandem mass spectrometry (LC-LC-MS2) has been developed and validated for the analysis of 12 pharmaceuticals and 20 metabolites and transformation products in different types of water (influent and effluent wastewaters and surface water). Two LC columns were used – one for pre-concentration of the sample and the second for separation and analysis – so that water samples were injected directly in the chromatographic system. Besides the many advantages of the methodology, such as minimization of the sample volume required and its manipulation, both compounds ionized in positive and negative mode could be analyzed simultaneously without compromising the sensitivity. A comparative study of different mobile phases, gradients and LC pre-concentration columns was carried out to obtain the best analytical performance. Limits of detection (MLODs) achieved were in the low ngL−1 range for all the compounds. The method was successfully applied to study the presence of the target analytes in different wastewater and surface water samples collected near the city of Girona (Catalonia, Spain). Data on the environmental presence and fate of pharmaceutical metabolites and TPs is still scarce, highlighting the relevance of the developed methodology.

Ecological risk assessment associated to the removal of endocrine-disrupting parabens and benzophenone-4 in wastewater treatment

The occurrence of four widely used and endocrine disrupting parabens (PBs) (methylparaben, propylparaben, butylparaben and benzylparaben) and a polar UV filter (benzophenone-4) were determined in influent and effluent wastewater from the 19 major wastewater treatment plants (WWTPs) of Catalonia, Spain. For their analysis an on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry (SPE-HPLC–MS/MS) method was developed and validated. Laboratory analysis revealed high levels for both PBs and BP4, with maximum concentrations of 5700ngL−1 and 1806ngL−1, respectively, in influent samples, and 137ngL−1 and 1080ngL−1, respectively in effluent wastewaters. Removal rates (RE%) for the target compounds in each WWTPs were calculated. RE% for parabens were almost 100%, whereas for BP4 values where in the range 5–91%. The half-life time (t1/2), hydraulic retention time (HRT), and annual mass load (ML) for each facility was estimated. Results indicated that there was no clear influence of HRT on the RE% of BP4. MLs for BP4 were in the range 0.9–110.1kgy−1, with the highest values in the most populated areas. Finally, a risk assessment, estimated in terms of hazard quotients (HQs), was carried out for aquatic biota. HQs for the target compounds in effluent wastewaters indicated a negligible effect, whereas for some influent wastewaters’ HQs pointed out that some species are at risk.

Fully automated analysis of four tobacco-specific N-nitrosamines in mainstream cigarette smoke using two-dimensional online solid phase extraction combined with liquid chromatography–tandem mass spectrometry

A fully automated method for the detection of four tobacco-specific nitrosamines (TSNAs) in mainstream cigarette smoke (MSS) has been developed. The new developed method is based on two-dimensional online solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE/LC–MS/MS). The two dimensional SPE was performed in the method utilizing two cartridges with different extraction mechanisms to cleanup disturbances of different polarity to minimize sample matrix effects on each analyte. Chromatographic separation was achieved using a UPLC C18 reversed phase analytical column. Under the optimum online SPE/LC–MS/MS conditions, N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were baseline separated with good peak shapes. This method appears to be the most sensitive method yet reported for determination of TSNAs in mainstream cigarette smoke. The limits of quantification for NNN, NNK, NAT and NAB reached the levels of 6.0, 1.0, 3.0 and 0.6pg/cig, respectively, which were well below the lowest levels of TSNAs in MSS of current commercial cigarettes. The accuracy of the measurement of four TSNAs was from 92.8 to 107.3%. The relative standard deviations of intra-and inter-day analysis were less than 5.4% and 7.5%, respectively. The main advantages of the method developed are fairly high sensitivity, selectivity and accuracy of results, minimum sample pre-treatment, full automation, and high throughput. As a part of the validation procedure, the developed method was applied to evaluate TSNAs yields for 27 top-selling commercial cigarettes in China.

Detection of human exposure to saxitoxin and neosaxitoxin in urine by online-solid phase extraction-liquid chromatography-tandem mass spectrometry.

Saxitoxin (STX) and neosaxitoxin (NEO) are potent neurotoxins that cause paralytic shellfish poisoning (PSP). PSP typically occurs through the ingestion of bivalve shellfish that have consumed toxin producing dinoflagellates. Due to initial presentation of symptoms being nonspecific, a clinical measurement is needed to confirm exposure to these toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of STX and NEO in human urine with tandem mass spectrometry. A unique feature of this online method is the incorporation of a new synthetic (15)N4-STX labeled internal standard used for quantitation. Manual sample preparation time was reduced by approximately 70% for 98 urine samples as compared to a previously reported method. The lowest reportable limit for STX was improved from 5.0ng/mL to 1.01ng/mL and from 10.0ng/mL to 2.62ng/mL for NEO. Three analysts validated the method with 20 calibration curves total over 30 days with precision and accuracy within ±15% for all QCs. This new online method rapidly identifies STX and NEO exposure with improved sensitivity, which can facilitate the work of public health authorities to confirm the cases of PSP, complementing the many shellfish monitoring programs worldwide.

Degradation of the cytostatic etoposide in chlorinated water by liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry: Identification and quantification of by-products in real water samples

Once discharged into the sewage system, many pharmaceuticals may undergo degradation reactions in the presence of chemical disinfectants, generating by-products that may possess enhanced toxicity relative to the parent compounds. For this reason, the stability of the widely used cytostatic etoposide in chlorinated water has been investigated for the first time in the present work. Taking advantage of the high-resolution/accurate-mass capabilities of the hybrid quadrupole-Orbitrap mass spectrometer Q Exactive, two new oxidation by-products of etoposide were reliably identified. The time course of etoposide and its by-products was followed at different pH values, free chlorine concentrations and water matrices. Finally, the occurrence of etoposide and its major identified by-product (3′-O-desmethyl etoposide) was investigated in real water samples by on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry using a 4000QTRAP hybrid quadrupole-linear ion trap mass spectrometer. The etoposide by-product was found in various river and wastewater samples at levels between 14 and 33ngL−1, whereas etoposide was not detected in any sample.

Removal of polar UV stabilizers in biological wastewater treatments and ecotoxicological implications

The present study describes the development, validation and application of a fully automated analytical method based on on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (on line SPE–HPLC–MS/MS) to assess the removal efficiency in water works and the ecotoxicological implications derived of the two most used benzotriazole-class UV stabilizers (BZTs), namely 1H-benzotriazole (BZT) and 5-methyl-1H-benzotriazole (MeBZT). Influent and effluent wastewater samples from 20 wastewater treatment plants (WWTPs) were analyzed. Removal rates (RE%) and half-lives (t1/2) for each BZTs were calculated and correlated to the hydraulic retention time (HRT) of each plant. Both BZTs were detected in all influent and effluent samples (concentrations in the range 26.7ngL−1–42.9μgL−1), with the highest concentrations corresponding to MeBZT. Results indicated that both compounds were recalcitrant (RE% in the range 11.8–94.7%) and that no clear influence of HRT on removals could be drawn.Finally, the potential environmental risk posed by the levels of BZTs detected was evaluated calculating the hazard quotients (HQs) MeBZT was the only BZTs posing a risk to Vibrio fischeri, Daphnia galeata and Pimephales promelas.

On-line solid phase extraction–liquid chromatography–tandem mass spectrometry for the determination of 17 cytostatics and metabolites in waste, surface and ground water samples

A fully automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) method has been developed for the determination of 13 cytostatics and 4 metabolites in aqueous matrices, including groundwater, surface water, and raw and treated wastewater. On-line SPE is performed by loading 5mL of water sample at pH 2 through a PLRP-s cartridge. MS/MS is performed with an electrospray (ESI) interface operating in the positive ion mode and registering two selected reaction monitoring (SRM) transitions per compound. Quantification is carried out by the isotope dilution method using 15 different isotope-labelled compounds, specific for the target analytes, as internal standards (IS). The main advantages of the method are high sensitivity, with limits of determination in groundwater, surface water, and raw and treated wastewater below 5ngL−1 for all compounds except for gemcitabine (6.9–9.3ngL−1), temozolomide (26–50ngL−1), imatinib (80–180ngL−1) and etoposide (38–65ngL−1), repeatability, with relative standard deviations (RSDs) in most cases below 15%, and selectivity and reliability of results. The method is also fairly simple and fast, with an analysis time per sample (excluding the manual steps, i.e., sample filtration, pH adjustment, and addition of IS) of 40min. Application of the method to influent wastewater samples collected daily during eight consecutive days from a wastewater treatment plant (WWTP) from Catalonia showed the presence of methotrexate, ifosfamide, capecitabine, tamoxifen and 6(α)-hydroxypaclitaxel but at fairly low concentrations (up to 43ngL−1).

Ecotoxicity evaluation and removal of sulfonamides and their acetylated metabolites during conventional wastewater treatment

The present study describes the evaluation of the risk posed by the occurrence of sulfonamides (SAs) in wastewaters. A fully automated analytical method based on on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry (on line SPE-LC–MS/MS) was developed, validated and applied for the analysis of sixteen SAs and, for the first time in wastewaters and sewage sludge, five of their acetylated metabolites. Influent and effluent samples from twenty two different wastewater treatment plants (WWTPs) in Catalonia (Spain) and sewage sludge from fifteen of them were analyzed; removal rates (RE%) and half-lives (t1/2) for each SA were calculated. The estimated correlations between RE% and to the hydraulic retention time (HRT) of the different plants indicated no clear influence of HRT on removals. Sulfamethoxazole (SMX), sulfapyridine (SPY) and their corresponding acetylated metabolites were detected with the highest frequencies of detection and at the highest concentrations. The ecotoxicity of both SPY and AcSPY was evaluated for the first time through bioluminescent inhibition assays, resulting in a higher toxicity being attributed to the metabolite. Finally, the potential environmental risk posed by the levels of SAs detected was evaluated calculating the hazard quotients (HQ) to different non-target aquatic organisms in treated wastewaters. SMX was the only SA posing a risk to algae, with an HQ>10.

Targeting metabolomics analysis of the sunscreen agent 2-ethylhexyl 4-( N, N-dimethylamino)benzoate in human urine by automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry with liquid chromatography–time-of-flight/mass spectrometry confirmation

The in vivo metabolism of the xenobiotic agent 2-ethylhexyl 4-( N, N-dimethylamino)benzoate (EDP), a UV filter commonly used in sunscreen cosmetic products, was studied by targeting metabolomics analysis in human urine. The metabolomic study involved the use of urine from male and female volunteers before and after application of an EDP-containing sunscreen cosmetic. The metabolism of EDP in urine was studied by using the triple quadrupole detector in a combination of Precursor Ion Scanning and Neutral Loss Scanning modes, with and without enzymatic hydrolysis. Detected metabolites were subsequently confirmed as glucuronide conjugates of 4-( N, N-dimethylamino)benzoic acid and 4-( N-methylamino)benzoic acid by liquid chromatography–time-of-flight/mass spectrometry (LC–TOF/MS) in the accurate mass mode. In this way, the existence of phase II metabolism in the detoxification of EDP by effects of the lipophilic character of this sunscreen agent was confirmed. Hence, to study the in vivo metabolism of EDP, a fully automated method using a solid-phase extraction (SPE) workstation connected on-line to a liquid chromatograph and a triple quadrupole mass analyzer (LC–MS/MS) was developed. The ensuing hyphenated method is very simple and requires minimal human intervention. Following thorough optimization of the SPE and LC–MS/MS conditions, the analytical procedure was validated and standard addition calibration used for the quantitative correction of matrix effects. The proposed method was applied to determine the phase I metabolites of EDP in urine samples and afforded limits of detection from 0.1 to 1.1 ng and accuracy of 91–107% with relative standard deviations in the range 1.5–8.7% (sample volume: 100 μL). Based on the results of in vivo percutaneous absorption of a single application of the sunscreen, about 0.5% of the amount of the applied EDP is excreted in urine.

An ultra-sensitive online SPE-LC-MS/MS method for the quantification of levonorgestrel released from intrauterine devices

A selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of very low levonorgestrel (D-(-)-norgestrel) serum levels such as those found in patients using levonorgestrel-releasing intrauterine devices (IUDs) was developed. To achieve the sub-nanomolar sensitivity needed to measure such serum levels, a diethyl ether extraction sample preparation protocol was applied prior to the online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) assay. Analyte quantification from the selected reaction monitoring experiments relied on the use of sixfold deuterated norgestrel as internal standard. The final method was linear up to 1.50 ng/ml with a lower limit of quantification (LLOQ) of 0.05 ng/ml. It was found to be precise and accurate with imprecision <8% and bias <6% assessed at three control levels. Total analyte recovery measured in patient pools at three concentration levels was found to exceed 92%. Matrix interferences were excluded by post-column analyte infusion experiments. As a proof of concept, a set of IUD patient serum samples was screened for their levonorgestrel content. A total of 97.5% (n = 94) of the samples did show serum levels exceeding the LLOQ, proving the applicability of the assay in relevant clinical cohorts. This method must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the in vitro diagnostic directive (98/79/EG) of the European community.

An online solid-phase extraction–liquid chromatography–tandem mass spectrometry method to study the presence of thyronamines in plasma and tissue and their putative conversion from 13C6-thyroxine

Thyronamines are exciting new players at the crossroads of thyroidology and metabolism. Here, we report the development of a method to measure 3-iodothyronamine (T(1)AM) and thyronamine (T(0)AM) in plasma and tissue samples. The detection limit of the method was 0.25 nmol/l in plasma and 0.30 pmol/g in tissue both for T(1)AM and for T(0)AM. Using this method, we were able to demonstrate T(1)AM and T(0)AM in plasma and liver from rats treated with synthetic thyronamines. Although we demonstrated the in vivo conversion of (13)C(6)-thyroxine ((13)C(6)-T(4)) to (13)C(6)-3,5,3'-triiodothyronine, we did not detect (13)C(6)-T(1)AM in plasma or brain samples of rats treated with (13)C(6)-T(4). Surprisingly, our method did not detect any endogenous T(1)AM or T(0)AM in plasma from vehicle-treated rats, nor in human plasma or thyroid tissue. Although we are cautious to draw general conclusions from these negative findings and in spite of the fact that insufficient sensitivity of the method related to extractability and stability of T(0)AM cannot be completely excluded at this point, our findings raise questions on the biosynthetic pathways and concentrations of endogenous T(1)AM and T(0)AM.

Integrated ecotoxicological and chemical approach for the assessment of pesticide pollution in the Ebro River delta (Spain)

Application of pesticides in the Ebro River delta (NE Spain) during the rice growing season is suspected to be one of the major causes behind the shellfish mortality episodes that occur yearly in this area at spring time. In an attempt to shed light on this suspicion, a monitoring study combining ecotoxicity measurements in water using three different bioassays and pesticides analysis in both water and shellfish has been carried out in this area in April–June 2008. Water and shellfish samples have been collected at six selected sites, two of them located in the bays where seafood (mussels and oysters) are grown, and four located in the main draining channels discharging the output water from the rice fields into the bays. Toxicity of the water samples has been evaluated using three standardized bioassays: 24–48 h immobilization of Daphnia magna, growth inhibition of Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) and bioluminescence inhibition of Vibrio fischeri. Analysis of pesticides (six triazines, four phenylureas, four organophosphorous, one anilide, two chloroacetanilides, one thiocarbamate and four acid herbicides) in water has been carried out by on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry (SPE-LC–MS/MS). Analysis of pesticides in shellfish has been performed by pressurized liquid extraction (ASE), followed by SPE clean-up and analysis by gas chromatography–mass spectrometry (GC/MS). Results have shown individual pesticides concentrations in water above 100 ng/L for about 50% of the compounds investigated, and total pesticides levels above 5 μg/L in the draining channels some days. The most ubiquitous compounds have been bentazone and MCPA and the highest levels have been observed for malathion (up to 5825 ng/L) and MCPA (up to 4197 ng/L). In shellfish, malathion has shown the highest concentration (53 mg/kg). A reasonable coherence has been observed between pesticide concentration (in water and shellfish), toxicity, and mortality episodes of shellfish, for the different locations studied. Taking into consideration the pesticides concentrations found, the toxicity values for the individual compounds, and the toxicity data measured on real samples, the pesticides suspected to be the main contributors to the total ecotoxicity are malathion, and to a lesser extent diazinon and molinate.

Determination of 19 sulfonamides in environmental water samples by automated on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry (SPE-LC–MS/MS)

The present study describes the development, validation and a practical application of a fully automated analytical method based on on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for the simultaneous determination of 19 sulfonamides, including one acetylated metabolite, in different water matrices. MS/MS detection was carried out in a quadrupole-linear ion trap (QqLIT) mass analyzer. Target compounds were identified in the selected reaction monitoring (SRM) mode, recording two transitions between precursor ions and the two most abundant product ions. The method developed was applied to evaluate the occurrence of the target antibiotics in different water samples: influent and effluent water from waste water treatment plants (WWTP), ground water and surface water. Under optimal conditions, the method detection limits achieved were in the range 0.05-7.84 ng/L for WWTP influent water, 0.01-6.90 ng/L for WWTP effluent water, 0.02-5.13 ng/L for ground water and 0.02-4.52 ng/L for surface water samples. The instrumental repeatability, expressed as RSD, was usually below 10% for the different water matrices. Results showed the wide presence of sulfonamides in the four types of water, including one acetylated metabolite, with maximum concentrations up to 855 ng/L corresponding to sulfapyridine in an influent waste water sample near a densely populated urban area.