Pesticides in sediments of the Ebro River Delta cultivated area (NE Spain): Occurrence and risk assessment for aquatic organisms.

Intense agricultural activities are performed in the Ebro River Delta (NE Spain) with extensive use of pesticides. Medium to highly polar pesticides have not been studied intensively in sediments despite its larger use in the recent years. This work aimed at assessing the occurrence of 69 pesticides, including medium to highly polar compounds, in sediments collected from drainage and irrigation channels of the Ebro River Delta during the main rice growing season. In addition, an environmental risk assessment was performed to evaluate the potential adverse effects to sediment-dwelling organisms with the risk quotient approach. A total of 24 pesticides were detected in sediments with bentazone and cypermethrin exhibiting high detection frequencies (79%) as well as high mean concentration levels (61.9 and 81.8 ng g-1 dw, respectively). Overall, the Alfacs bay, in the South of the delta, presented higher pesticide contamination than the Fangar bay, in the North. A similar pesticide distribution profile was observed in both bays, with oxadiazoles, organochlorines, pyrethroids, benzothiazinones and organophosphates as major, predominant classes. The presence of oxadiazon, pendimethalin and thifensulfuron methyl in the sediments may pose a moderate risk to sediment-dwelling organisms while bentazone, chlorpyrifos, and cypermethrin exhibited a potential high risk. Thus, the importance of the inclusion of medium to highly polar pesticides in the analysis of sediments is emphasized since some polar pesticides such as bentazone, imidacloprid, and thifensulfuron-methyl have been detected at concentrations that may pose a risk to aquatic organisms. Moreover, the co-occurrence of pesticides may potentially pose a high risk to sediment-dwelling organisms in 13 out of the 14 investigated locations. Finally, it could be concluded that the risk derived from the presence of pesticides in sediments must be assessed since some pesticides not detected at concerning levels in water, may pose a moderate/high risk in the sediments.

Fast analysis of relevant contaminants mixture in commercial shellfish.

One of the major challenges currently faced is to develop systematic ways of addressing chemical mixtures in environmental assessment. With this purpose, a simple, rapid, and sensitive method for the detection and quantification of a mixture of relevant contaminants in molluscs has been developed. The method is based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS). It includes a mixture of 23 compounds formed by pesticides, endocrine disruptors and pharmaceuticals (metolachlor, simazine, desethylatrazine, atrazine, thiabendazole, diazinon, malathion, bentazone, MCPA, propanil, acetamiprid, imidacloprid, caffeine, bisphenol A, triclosan, triclocarban, methylparaben, ethylparaben, propylparaben, 1H-benzotriazole, sulfamethoxazole, venlafaxine and carbamazepine). The method was developed and validated in 4 different types of shellfish of high commercial interest such as mussel (Mytilus galloprovincialis), oyster (Crassostrea gigas), cockle (Cerastoderma edule) and razor shell (Solen marginatus). The mean percentage of recoveries obtained for all the compounds in each mollusc type (intra-specie) ranged from 96% to 107% showing the good performance of the method developed. The relative standard deviation was under 10% for the intra-day and 17% inter-day analyses. Method detection limits and method quantification limits were below 10 ng/g dry weight for all the species and compounds targeted. Finally, the method was applied to aquaculture samples, oysters and cockles, from Ebro Delta (Spain), after some episodes of mortality occurred in 2017. A high level of bisphenol A was detected in C. edule which may explain the mortality suffered by this organism. C. gigas presented low levels of metolachlor, bentazone, acetamiprid, and methylparaben.

Validation of micellar LC-based methods applied to analyze foodstuffs.

The validation of several micellar LC-based analytical methodologies was described. These methods were able to quantify quinolones in fish from fisheries, hydroxytyrosol in olive extracts and biogenic amines in anchovy sauce. The validation was performed following the requirements of official guides to provide more reliability. Two guides suggested by renowned institution are described: US FDA Guidance for Industry and EU Regulation 2002/657/EC Decision. The appropriate guide was used for each method, depending of the analyte, the matrix and the scope of sample. The calculated validation parameters were those proposed by the guide: selectivity, calibration range, linearity, LOD and LOQ, inter- and intra-day accuracy and precision, limit of decision, detection capability, robustness, recovery and stability. The methodologies were successfully validated by the selected guideline, indicating their suitability to be applied to analysis of real samples, proven to be useful to its intended purpose.

Monitoring of HAART regime antiretrovirals in serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography.

A methodology based on micellar liquid chromatography to monitor five antiretroviral drugs (lamivudine, stavudine, tenofovir, zidovudine and efavirenz) was proposed. Antiretrovirals were studied in sets of three, corresponding to each highly active antiretroviral therapy (HAART) regime, prescribed to acquired immunodeficiency syndrome (AIDS)-infected patients. Four aqueous micellar mobile phases buffered at pH 7 were optimized to separate these compounds, using sodium dodecyl sulfate as the tensioactive, and 1-propanol or 1-pentanol as the organic modifier. The composition of each mobile phase was optimized for each antiretroviral. The common separation conditions were: C18 apolar column (125 × 4.6 mm, 5 µm particle size), UV detection set at 214 nm, and mobile phase running at 1 mL min(-1) without controlling the temperature. The finally suggested method was validated for five analysed antiretroviral drugs following the US Food and Drug Administration guidelines in terms of: linearity between 0.5 and 50 ppm (r(2) > 0.9995), sensitivity (LOD lower than 0.25 ppm), intra- and inter-day precision (<7.1 and <5.2%, respectively) and accuracy (recovery 88.5-105.3% and 93.5-101.3%, respectively), as well as robustness (<6.5%). The proposed method was used to monitor the level of antiretrovirals in the serum of AIDS patients. The suggested methodology was found to be useful in the routine analysis of antiretrovirals in serum samples.

Validation of an analytical methodology to quantify melamine in body fluids using micellar liquid chromatography.

Measurement of urine and plasma melamine-concentration is helpful in confirming melamine-associated renal diseases. A chromatographic procedure using a C18 column and a micellar mobile phase of sodium dodecyl sulphate (0.2M), buffered at pH 3 and detection set at 210 nm, was reported for the resolution and quantification of melamine in plasma and urine. In this work, direct injection was used, thus avoiding long extraction and experimental procedures. Melamine was eluted in nearly 6.3 min without overlapping the protein band or other endogenous compounds. The optimal mobile phase composition was taken by studying the influence of each chromatographic parameter. Validation was satisfactorily performed following the US Food and Drug Administration (FDA), in terms of: linearity (0.25-25 ppm; r2>0.9995 in both cases), sensitivity, limit of detection (50 ppb), limit of quantification (250 ppb), intra- and inter-day precision (R.S.D. 0.7-10.2% and 1.0-9.1%, respectively) and recovery, calculated as accuracy (85.7-103.8% and 94.8-103.6%, respectively) and robustness (R.S.D.<7.1%). The suggested methodology has been applied to the analysis of real samples of volunteers, and no melamine was found in any of them.

Analytical determination of hydroxytyrosol in olive extract samples by micellar liquid chromatography.

Hydroxytyrosol is a well-known natural phenolic component obtained from olive extract samples with antioxidant effects. A micellar liquid chromatography method to detect and quantify hydroxytyrosol in olive extract samples is described. Matrix resolution was performed using a Kromasil C18 column and a micellar mobile phase of sodium dodecyl sulphate (SDS) 0.05M and 4% methanol buffered at pH 7. Detection was set by absorbance at 280nm. Samples were diluted with 0.05M SDS at pH 3 and directly injected, thus avoiding long tedious extractions. Hydroxytyrosol was eluted in 3.5min without overlapping other matrix compounds. Validation was performed following the US FDA guideline. The main analytical parameters studied were: linearity (0.03-250µgmL-1; r2=0.999), limit of detection and quantification (3 and 30ngmL-1, respectively), intra- and inter-day precision (RSD, % <1.4 and <8.2, respectively), and robustness (RSD, %<6.6). Recoveries were in the 88.5-98.9% range.

Quinolones control in milk and eggs samples by liquid chromatography using a surfactant-mediated mobile phase.

Four quinolones (danofloxacin, difloxacin, flumequine and marbofloxacin) were determined in milk and egg samples by a simplified high-performance liquid chromatographic procedure using a micellar mobile phase. No extraction was needed to precipitate the proteins from the matrices since they were solubilised in micelles. The only pretreatment steps required were homogenisation, dilution and filtration before injecting the sample into the chromatographic system. An adequate resolution of the quinolones was achieved by a chemometrics approach where retention was modelled as a first step using the retention factors in only five mobile phases. Afterwards, an optimisation criterion was applied to consider the position and shape of the chromatographic peaks. Analytical separation involved a C18 reversed-phase column, a hybrid micellar mobile phase of 0.05 M sodium dodecyl sulphate, 10% (v/v) butanol and 0.5% (v/v) triethylamine buffered at pH 3 and fluorimetric detection. Quinolones were eluted in less than 15 min without the protein band or other endogenous compounds from the food matrices interfering. The calculated relevant validation parameters, e.g., decision limit (CC(α)), detection capability (CC(ß)), repeatability, within-laboratory reproducibility, recoveries and robustness, were acceptable and complied with European Commission Decision 2002/657/EC. Finally, the proposed method was successfully employed in quantifying the four quinolones in spiked egg and milk samples.

Determination of trazodone in urine and pharmaceuticals using micellar liquid chromatography with fluorescence detection.

A simple and reliable liquid chromatographic procedure is described for the determination of trazodone in pharmaceutical formulations and urine samples. The optimized procedure uses fluorimetric detection, a C18 column and a micellar mobile phase of sodium dodecyl sulfate (SDS) and 1-butanol. The mobile phase selected for use was 0.2M SDS and 8% 1-butanol fixed at pH 3 with phosphate buffer. The total analysis time was 10 min. For the analysis of urine samples, one great advantage of the method is that no extraction step is required. The quantification limit was 9.5 ng mL(-1), ensuring the analysis of the drug in biological fluids. The procedure shows good accuracy, repeatability and selectivity. Repeatability and intermediate precision were tested for several concentrations of the drug. Good claim percentages were obtained in the analysis of pharmaceutical formulations. Calibration repeatability in urine matrix was also studied in the 0.06-22.4 microg mL(-1) range. Good recoveries were obtained from spiked urine samples. No interferences from common additives frequently administered with trazodone or from endogenous compounds in urine samples were found. The results show that the procedure is suitable for routine analysis of the drug.

Direct determination of verapamil in urine and serum samples by micellar liquid chromatography and fluorescence detection.

Verapamil, a calcium channel antagonist, is one of the most commonly prescribed drugs in the treatment of hypertension. In this work, it was determined in serum and urine samples by a sensitive and precise chromatographic procedure without any pre-treatment step in a C18 column using a micellar mobile phase of 0.15M sodium dodecyl sulfate and 5% pentanol at pH 7. Fluorescence detection set at 230 nm (excitation) and 312 nm (emission) was used. Verapamil is eluted at 12.5 min with no interference by the protein band or endogenous compounds. Linearities (r > 0.998), as well as intra- and inter-day precision, were studied in the validation of the method. LODs were also calculated to be 11.0, 18.5 and 20.2 ng/mL in micellar solution, serum and urine, respectively. Recoveries in the biological matrices were in the 97-99% range. Drug excretion in urine was studied in a volunteer receiving treatment for hypertension, and verapamil, as an unchanged drug, was separated from other metabolites. The procedure developed can be useful in the field of toxicology and clinical analysis.