On-line Solid-phase Extraction Liquid Chromatography Research Articles

Simultaneous, rapid, and sensitive quantification of 8-hydroxy-2'-deoxyguanosine and cotinine in human urine by on-line solid-phase extraction LC-MS/MS: correlation with tobacco exposure biomarkers NNAL.

Cigarette smoke can increase oxidative DNA damage. The main component in cigarette smoke is nicotine. Nicotine is metabolized to cotinine, which can be regarded as a biomarker for measuring exposure to tobacco smoke. A sensitive, simple, and robust method based on on-line solid-phase extraction liquid chromatography with tandem mass spectrometry (on-line SPE LC-MS/MS) has been developed and validated for the simultaneous determination of 8-OHdG and cotinine. The matrix effects of 8-OHdG and cotinine were measured at 97.1 and 91.7%, with values for CV at 4.4 and 4.2%, respectively. The limits of detection of 8-OHdG and cotinine were 10.0 and 5.5pgmL(-1), and the limits of quantification were 40.0 and 20.0pgmL(-1), respectively. The total run time was 12min. We quantified 8-OHdG and cotinine in the urine of 80 male subjects. The results showed the levels of 8-OHdG and cotinine in smokers were significantly higher than that in non-smokers. Furthermore, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide conjugate (defined as total NNAL) are the nitrosation metabolites of nicotine. In this study, urinary levels of 8-OHdG and cotinine were well correlated with urinary levels of total NNAL. This is also the first study to focus on the future risk of oxidative stress from exposure to cigarette smoke based on the relationship between 8-OHdG levels, cotinine levels, and total NNAL concentrations in the urine of humans. Graphical Abstract On-line SPE LC-MS/MS for the simultaneous determination of 8-OHdG and cotinine in human urine.

Application of LC-MS/MS MRM to Determine Staphylococcal Enterotoxins (SEB and SEA) in Milk.

Staphylococcus aureus is one of the important aetiological agents of food intoxications in Europe and can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Due to their stability and ease of production and dissemination, some SEs have also been studied as potential agents for bioterrorism. Therefore, specific and accurate analytical tools are required to detect and quantify SEs. Online solid-phase extraction liquid chromatography electrospray ionization tandem mass spectrometry (online SPE-LC-ESI-MS/MS) based on multiple reaction monitoring (MRM) was used to detect and quantify two types of SE (A and B) spiked in milk and buffer solution. SE extraction and concentration was performed according to the European Screening Method developed by the European Reference Laboratory for Coagulase Positive Staphylococci. Trypsin digests were screened for the presence of SEs using selected proteotypic heavy-labeled peptides as internal standards. SEA and SEB were successfully detected in milk samples using LC-MS/MS in MRM mode. The selected SE peptides were proteotypic for each toxin, allowing the discrimination of SEA and SEB in a single run. The detection limit of SEA and SEB was approximately 8 and 4 ng/g, respectively.

Development and validation of a fully automated online-SPE–ESI–LC–MS/MS multi-residue method for the determination of different classes of pesticides in drinking, ground and surface water

ABSTRACTThe present work describes a fully automated method based on online solid phase extraction–liquid chromatography–tandem mass spectrometry for the determination of different classes of pesticides, including acidic and polar pesticides and six thiamethoxam metabolites. Sample preconcentration was performed by extracting 4 mL of the sample with a single styrene-divinylbenzene polymer. Elution of the compounds was done within the high performance liquid chromatography gradient and tandem mass spectrometry determination was performed in the selected reaction monitoring mode, by recording 1–3 transitions per compound. The overall pretreatment and analysis time per sample was less than 15 min. Method validation was performed in drinking, ground and surface water. For nearly all compounds a recovery between 70% and 120% could be achieved. The limit of detection ranges from 1.2 to 18 ng/L in drinking water and 3.0 to 23 ng/L in ground and surface water. The correlation coefficients for a calibration range of 0.05–2 µg/L are between 0.9915 and 0.9999. The limit of quantification (LOQ) for all compounds lies below the required limit of 0.1 µg/L, to fulfil the Council Directive 98/83/EC. Most of the compounds easily reach an LOQ below 0.05 µg/L.

Analysis of lignans in Magnoliae Flos by turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry.

In this study, a method coupling turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry was developed for analyzing the lignans in Magnoliae Flos. By the online pretreatment of turbulent flow chromatography solid-phase extraction, the impurities removal and analytes concentration were automatically processed, and the lignans were separated rapidly and well. Seven lignans of Magnoliae Flos including epieudesmin, magnolin, 1-irioresinol-B-dimethyl ether, epi-magnolin, fargesin aschantin, and demethoxyaschantin were identified by comparing their retention behavior, UV spectra, and mass spectra with those of reference substances or literature data. The developed method was validated, and the good results showed that the method was not only automatic and rapid, but also accurate and reliable. The turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry method holds a high potential to become an effective method for the quality control of lignans in Magnoliae Flos and a useful tool for the analysis of other complex mixtures.

Antibiotics in Austrian drinking water resources, survey 2014

Summary We surveyed antibiotics in drinking water in Austria. Testing for 37 antimicrobials was performed by online solid-phase extraction–liquid chromatography–high-resolution mass spectroscopy method. Selection of sample sites for drinking water resources focused on areas considered susceptible to contamination and on geographic population distribution. Sulfamethoxazole was detected in 10 (5%) of 200 samples collected in 2014. Five samples showed concentrations above the limit of quantification (2.5 ng/l). Concentrations measured were ≤8.9 ng/l, making toxic effects highly unlikely. However, even low-level concentrations of antibiotics could increase bacterial resistance. The presence of antibiotics is presently not regulated. We assume anthropogenic pollution (not animal husbandry) as source and ask for a maximum permissible value of antibiotics in drinking water.

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.

Determination of Morphine in Pharmaceutical Products by On-Line Solid-Phase Extraction and High-Performance Liquid Chromatography

ABSTRACTA method for the rapid determination of morphine in Chinese medicines was developed using on-line solid-phase extraction combined with double-ternary gradient liquid chromatography. Licorice tablets and camphor tincture were used as representative examples of traditional Chinese medicine. The results indicated that morphine provided a linear response from 0.0239 to 0.239 m g mL−1. The average recovery was as high as 99% with a relative standard deviation of 0.9%. Moreover, compared with conventional off-line solid-phase extraction and liquid chromatography, this approach offered a significant reduction in the analysis time. This method may provide for more efficient determination of morphine in traditional Chinese medicine.

Development of On-Line Solid-Phase Extraction-Liquid Chromatography Coupled with Tandem Mass Spectrometry Method to Quantify Pharmaceutical, Glucuronide Conjugates and Metabolites in Water

The present work describes the development of an analytical method, based on automated on-line solid phase extraction followed by ultra high-performance liquid chromatography coupled with tandem mass spectrometry (SPELC- MS/ MS) for the quantification of 37 pharmaceutical residues, covering various therapeutic classes, and some of their main metabolites, in surface and drinking water. A special attention was given to some glucuronide conjuguates and metabolites of active subtances. Multiple Reaction Monitoring (MRM) was chosen and two transitions per compound are monitored (quantification and confirmation transitions). Quantification is performed by standard addition approach to correct matrix effect. The method provides limit of quantification inferior to 20 ng.L-1 for all compounds. The methodology was successfully applied to the analysis of surface water and drinking water of 8 drinking water treatment plant in west of France. The highest drug concentrations in surface water and drinking water were reported for ketoprofen, hydroxyibuprofen, acetaminophen, caffeine and danofloxacin.

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.

An on-line solid phase extraction–liquid chromatography tandem mass spectrometry method for the determination of perfluoroalkyl substances in the Antarctic ice core samples

An on-line solid phase extraction–high performance liquid chromatography–tandem mass spectrometry method for the analysis of perfluoroalkyl substances (PFASs) in water samples was developed. The optimal analytical conditions were obtained through the optimization of the extraction efficiency of on-line solid phase extraction column, sample loading rate and loading volume, and the concentration of ammonium acetate in mobile phase. Under the optimal condition, the analytical method displayed good linearity (r2 > 0.99) for 12 PFASs (C5–C14 perfluoroalkyl carboxylic acids and C6/C8 perfluoroalkyl sulfonic acids) over a concentration range of 0.5–100 ng/L. The limits of quantitation for samples were between 0.025 ng/L and 0.5 ng/L and the relative standard deviations (RSD) of five consecutive analyses were less than 10% for 1 ng/L standard solution. Satisfactory results were obtained using this analytical method for the analysis of perfluoroalkyl substances in Antarctic ice core samples. The recoveries of all perfluoroalkyl substances were in a range of 73%–117% when the samples were spiked with standards at the concentrations of 2.5 ng/L and 25 ng/L.

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.

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.

Online solid-phase extraction–liquid chromatography–mass spectrometry to determine free sterols in human serum

An automated method for analyzing free non-cholesterol sterols in human serum using online solid phase extraction–liquid chromatography–mass spectrometry is proposed herein. The method allows the determination of three phytosterols (sitosterol, stigmasterol and campesterol) and two cholesterol precursors (desmosterol and lanosterol). The analysis of sterols in human serum is critical in the study of cholesterol-related disorders, such as inherited familial hypercholesterolemias. Special effort was made to isolate the analytes from the serum lipoproteins, their natural conveyance through the bloodstream. The sample treatment consisted of a Bligh–Dyer extraction followed by dilution of the extract. This treatment allowed the sample to be injected into the online system and ensured the correct detection of the analytes, while avoiding the matrix effects commonly related to serum samples.The analytical performance showed linear ranges that covered two orders of magnitude, with correlation coefficients above 0.99. Limits of detection and quantification ranged from 0.2ng/mL to 13ng/mL and from 1.0ng/mL to 43ng/mL, respectively. Recovery when spiking serum with a half or a tenth of the average concentration reported in human serum ranged from 99% to 111% and from 102% to 120%, respectively. Intra-day precision and inter-day precision were below 20%.

A novel method for the measurement of plasma metanephrines using online solid phase extraction-liquid chromatography tandem mass spectrometry.

Measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine is useful in the diagnosis of phaeochromocytomas, but many assays require a large volume of plasma due to poor assay sensitivity, and often require lengthy sample preparation. Our aim was to develop a method for measurement of plasma metanephrines using a small sample volume with minimal hands-on preparation. Samples were deproteinised using 10 K spin filters prior to online solid phase extraction using a Waters Acquity UPLC Online SPE Manager (Waters, Manchester, UK) coupled to a Waters Xevo TQ-S mass spectrometer (Waters, Manchester, UK). The assay was validated and results compared to a previously published method. We achieved a limit of quantification of 37.5 pmol/L for metanephrine and 3-methoxytyramine and 75 pmol/L for normetanephrine using only 150 µL of sample. The assay was linear up to 30,000 pmol/L for all analytes and in a method comparison study results showed good agreement with a previously published LC-MS/MS assay. We have developed a simple method for measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine using only 150 µL of sample. There is minimal hands-on sample preparation required and the assay is suitable for routine use in a clinical laboratory.

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.

Short term variability in urinary bisphenol A in Australian children

Used frequently in food contact materials, bisphenol A (BPA) has been studied extensively in recent years, and ubiquitous exposure in the general population has been demonstrated worldwide. Characterizing within- and between-individual variability of BPA concentrations is important for characterizing exposure in biomonitoring studies, and this has been investigated previously in adults, but not in children. The aim of this study was to characterize the short-term variability of BPA in spot urine samples in young children. Children aged ≥2–<4years (n=25) were recruited from an existing cohort in Queensland, Australia, and donated four spot urine samples each over a two day period. Samples were analysed for total BPA using isotope dilution online solid phase extraction–liquid chromatography–tandem mass spectrometry, and concentrations ranged from 0.53 to 74.5ng/ml, with geometric mean and standard deviation of 2.70ng/ml and 2.94ng/ml, respectively. Sex and time of sample collection were not significant predictors of BPA concentration. The between-individual variability was approximately equal to the within-individual variability (ICC=0.51), and this ICC is somewhat higher than previously reported literature values. This may be the result of physiological or behavioural differences between children and adults or of the relatively short exposure window assessed. Using a bootstrapping methodology, a single sample resulted in correct tertile classification approximately 70% of the time. This study suggests that single spot samples obtained from young children provide a reliable characterization of absolute and relative exposure over the short time window studied, but this may not hold true over longer timeframes.

Trace determination of primary nerve agent degradation products in aqueous soil extracts by on-line solid phase extraction–liquid chromatography–mass spectrometry using ZrO2 for enrichment

A method for determination of the primary nerve agent degradation products ethyl-, isopropyl-, isobutyl-, cyclohexyl- and pinacolyl methylphosphonic acid in aqueous soil extracts has been developed utilizing on-line solid phase extraction-liquid chromatography and mass spectrometry (SPE–LC–MS). Four different stationary phases (ZrO2, TiO2, polymeric mixed mode anion exchange and porous graphitic carbon) were investigated for their suitability as SPE materials in the on-line SPE–LC–MS setup. Zirconium dioxide was chosen due to its high affinity for the alkyl methylphosphonic acids (AMPAs), and its compatibility with LC–MS. Aqueous soil extracts were acidified with 0.1% acetic acid and aliquots of 300μL were injected on a 2mm×10mm ZrO2 column. Separation of the analytes was performed on a reversed phase column with acetonitrile/water gradient and 15mM ammonium acetate. Method validation was performed with the analytes added to an aqueous extract of a loam soil, and the AMPAs could be determined at concentrations as low as 0.05–0.5μgL−1. The method was linear (R2>0.995) from the limit of quantification (LOQ) to 100×LOQ, and the within assay repeatability was below 10% and 5% relative standard deviation at LOQ and 50×LOQ, respectively. The developed method was employed for determination of the AMPAs which had been added to the aqueous extracts of five different soil types from cultivated and uncultivated areas. The obtained recoveries showed that the analytes could be determined at the sensitivities achieved in the method validation in four of the extracts. For the first time, we have demonstrated a method capable of detecting primary nerve agent degradation products at sub ppb levels in the aqueous extracts of various soils. The method requires no sample preparation after soil extraction other than pH adjustment of the aqueous extract.

Determination of Phthalate Plasticisers in Palm Oil Using Online Solid Phase Extraction-Liquid Chromatography (SPE-LC)

Contamination of phthalates plasticisers to food has raised concern as some of the phthalates are suspected to be endocrine disruptors. The phthalates have high affinity with oily environment and analysing these chemicals in such matrices is difficult because of the trace amount of the analyte and interference from matrix. An online solid phase extraction (SPE) technique using a large volume (3.5 mL) injection was developed for the analysis of 6 common plasticisers in palm oil. A simple sample preparation involving alumina as a fat retainer and methanol : acetonitrile (1 : 1) as the extraction solvent was performed prior to the usage of online SPE-LC system. This system consists of two columns, C16for the solid phase extraction (SPE) and C18as the analytical column, and a photo diode array detector. The calibration curves were linear from 5 to 1000 μg L−1, with correlation coefficients above 0.99. The instrumental limit of detection was 3 μg L−1and satisfactory recovery was obtained. A screening on a few samples in the retail market revealed the presence of dibutyl phthalate (DBP) and butylbenzylphthalate (BBP) in the palm oil, with concentration less than 1 mg L−1.

A snapshot of illicit drug use in Sweden acquired through sewage water analysis

Analytical measurements of sewage water have been used many times to estimate the consumption of specific drugs in an area. This study measured a large number of illicit drugs and metabolites (>30) at a large number of sewage treatment plants (STPs) distributed across Sweden. Twenty-four illicit and prescription drugs, classified as narcotic substances in Sweden, and seven selected metabolites were included in the study. A 24hour composite sample of incoming sewage water was collected from 33 different municipalities at various geographic locations across Sweden. Species were analyzed using an on-line solid-phase extraction–liquid chromatography electrospray tandem mass spectrometry method. The method proved to be rapid with minimum need for sample work up and was able to detect 13 compounds above their respective limits of quantification. The results for all compounds were presented as per capita loads. Multivariate data analysis was used to relate drug consumption to geographical location and/or population of cities. The results showed that geographical differences in drug consumption were apparent across the country. For the narcotic pharmaceuticals, the geographical differences suggested by the multivariate model were supported by prescription statistics.

Four-year advanced monitoring program of polar pesticides in groundwater of Catalonia (NE-Spain)

Pesticide contamination of groundwater is of paramount importance because it is the most sensitive and the largest body of freshwater in the European Union. In this paper, an isotopic dilution method based on on-line solid phase extraction–liquid chromatography (electrospray)–tandem mass spectrometry (SPE–LC(ESI)–MS/MS) was used for the analysis of 22 pesticides in groundwater. Results were evaluated from monitoring 112 wells and piezometers coming from 29 different aquifers located in 18 ground water bodies (GWBs), from Catalonia, Spain, for 4years as part of the surveillance and operational monitoring programs conducted by the Catalan Water Agency. The analytical method developed allows the determination of the target pesticides (6 triazines, 4 phenylureas, 4 organophosphorous, 1 anilide, 2 chloroacetanilides, 1 thiocarbamate, and 4 acid herbicides) in groundwater with good sensitivity (limits of detection <5ng/L), accuracy (relative recoveries between 85 and 116%, except for molinate), and repeatability (RSD<23%), and in a fully automated way. The most ubiquitous compounds were simazine, atrazine, desethylatrazine and diuron. Direct relation between frequency of detection of each target compound and Groundwater Ubiquity Score index (GUS index) is observed. Desethylatrazine and deisopropylatrazine, metabolites of atrazine and simazine, respectively, presented the highest mean concentrations. Compounds detected in less than 5% of the samples were cyanazine, molinate, fenitrothion and mecoprop. According to the Directive 2006/118/EC, 13 pesticides have individual values above the requested limits (desethylatrazine, atrazine and terbuthylazine lead the list) and 14 samples have total pesticide levels above 500ng/L. The GWB with the highest levels of total pesticides is located in Lleida (NE-Spain), with 9 samples showing total pesticide levels above 500ng/L. Several factors such as regulation of the use of pesticides, type of activities in the area, and irrigation were discussed in relation to the observed levels of pesticides.