Results from the INMUNOSUN-SOGUG trial: a prospective phase II study of sunitinib as a second-line therapy in patients with metastatic renal cell carcinoma after immune checkpoint-based combination therapy.

BACKGROUND: The INMUNOSUN trial had the objective of prospectively evaluating the efficacy and safety of sunitinib as a pure second-line treatment in patients with metastatic renal cell carcinoma (mRCC) who have progressed to first-line immune checkpoint inhibitor (ICI)-based therapies. PATIENTS AND METHODS: A multicenter, phase II, single-arm, open-label study was carried out in patients with a histologically confirmed diagnosis of mRCC with a clear-cell component who had progressed to a first-line regimen of ICI-based therapies. All patients received sunitinib 50 mg once daily orally for 4 weeks, followed by a 2-week rest period following package insert instructions. The primary outcome was the objective response rate. RESULTS: Twenty-one assessable patients were included in the efficacy and safety analyses. Four patients [19.0%, 95% confidence interval (CI) 2.3% to 35.8%] showed an objective response (OR), and all of them had partial responses. Additionally, 14 (67%) patients showed a stable response, leading to clinical benefit in 18 patients (85.7%, 95% CI 70.7% to 100%). Among the four assessable patients who showed an OR, the median duration of the response was 7.1 months (interquartile range 4.2-12.0 months). The median progression-free survival (PFS) was 5.6 months (95% CI 3.1-8.0 months). The median overall survival (OS) was 23.5 months (95% CI 6.3-40.7 months). Patients who had better antitumor response to first-line ICI-based treatment showed a longer PFS and OS with sunitinib. The most frequent treatment-emergent adverse events were diarrhea (n = 11, 52%), dysgeusia (n = 8, 38%), palmar-plantar erythrodysesthesia (n = 8, 38%), and hypertension (n = 8, 38%). There was 1 patient who exhibited grade 5 pancytopenia, and 11 patients experienced grade 3 adverse events. Eight (38%) patients had serious adverse events, four of which were considered to be related to sunitinib. CONCLUSION: Although the INMUNOSUN trial did not reach the pre-specified endpoint, it demonstrated that sunitinib is active and can be safely used as a second-line option in patients with mRCC who progress to new standard ICI-based regimens.

Study of antifungal agent caspofungin adsorption to laboratory materials.

Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.

Estimation of the post-mortem interval of human skeletal remains using Raman spectroscopy and chemometrics.

An important demand exists in the field of forensic analysis to objectively determine the post-mortem interval (PMI) when human skeletal remains are discovered. It is widely known that bones undergo different chemical and physical processes after death, mainly due to their interaction with the environment in which they are found, although it is not known exactly what these processes consist of. Multiple techniques have been used so far to follow up these and other post-mortem changes and thus establish the time elapsed since the individual's death, but they present important drawbacks in terms of reliability and accuracy. The aim of this research was to propose an analytical methodology capable of determining the PMI by using non-destructive Raman spectroscopy measurements of human skeletal remains. The recorded Raman spectra provided valuable and potentially useful information from which a multivariate study was performed by means of orthogonal partial least squares regression (OPLSR) in order to correlate the PMI with the detected spectral modifications. A collection of 53 real human skeletal remains with known PMI (15 years ≤ PMI ≤ 87 years) was analysed and used for building and validating the OPLS model. The PMI of 10 out of 14 validation samples could be determined with an accuracy error of less than 30%, demonstrating the adequate predictive performance of the OPLS model even in spite of the large inter-individual variability it handled. This opens up the possibility of applying the OPLS model in combination with non-destructive techniques to the determination of the PMI of human skeletal remains that have been buried in conditions similar or equal to those of cemetery niches and in a geographic location with a Mediterranean climate, which is an important achievement for forensic medicine and anthropology.

Determination of antifungal caspofungin in RPMI-1640 cell culture medium by column-switching HPLC-FLD.

The actual scenario in the fight against fungal infections forces researchers to carry through with resistance studies to improve the therapies. These studies, which are performed in cell culture media, need accurate and sensitive analytical methodologies. That is why, in this work, an analytical method for caspofungin (CSF) concentration determination in RPMI-1640 cell culture medium with on-line sample treatment was developed and validated. CSF concentration was determined by HPLC-FLD using a column-switching procedure. The chromatographic analysis was carried out in less than 10 min using a C8 column (4 × 4 mm, 5 µm) as extraction stationary phase and a HSS T3 column (4.6 × 100 mm, 5 µm) as the analytical column. The used mobile phases were mixtures of phase A: pH 2 (adjusted with TFA) aqueous phase and phase B: ACN. For the extraction, the composition was (95:5, A:B v/v) and for the analysis (60:40, A:B v/v), both done in isocratic elution mode. These chromatographic conditions allowed reaching a limit of quantification of 10 µg/L, using 100 µL of sample with an injected volume of 40 µL. The proposed method was successfully validated in terms of selectivity, carryover, linear concentration range, accuracy and precision according to the criteria established by the Food and Drug Administration. Available amount of CSF in RPMI-1640 solution was found critical. CSF concentrations remained stable up to 2 h at room temperature. The developed method was applied for the direct analysis of CSF concentrations from in vitro experiments in presence of C. glabrata (CAGL18). The results highlight the decrease of cell proliferation even if the CSF amount decreases too, which asks question about the real value of the efficient concentration for CSF antifungal activity.

Graphene oxide electrodeposited electrode enhances start-up and selective enrichment of exoelectrogens in bioelectrochemical systems.

This study seeks to assess the impact that the anodic electrodeposition of graphene oxide (GO) has on the start-up process and on the development of microbial communities on the anode of BESs. The GO electrodeposited electrodes were characterised in abiotic conditions to verify the extent of the modification and were then transferred to a bioelectrochemical reactor. Results showed that the modified electrode allowed for a reduced start-up time compared to the control electrode. After three months, high throughput sequencing was performed, revealing that electrochemically reduced graphene oxide acts as a selective agent toward exoelectrogenic bacteria as Geobacter. Overall, this study shows that GO modified electrodes enhance biofilm build up in BES.

Quantitative determination of dobutamine in newborn pig plasma samples by HPLC-MS/MS.

A novel gradient reverse phase high performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) was performed as a method for the determination of dobutamine hydrochloride (DOB) in newborn pig plasma samples. It was developed and validated after optimization of sample treatment and various chromatographic and mass spectrometric conditions. Trimethoxydobutamine (TMD) was used as internal standard. Heptafluorobutyric acid (HFBA) and ethyl acetate were used for the treatment of plasma samples. The separation of dobutamine and internal standard was done using a Kinetex F5 (50×2.1mm, 2.6µm, 100Å) analytical column. The mobile phase was a mixture of acetonitrile and HCOOH 0.01%. The column oven temperature was optimized at 40° C and the flow rate was 0.25mL/min. DOB and TMD were detected by multiple reaction monitoring (MRM) mode in ESI+, using a cone voltage (CV) of 25V and a collision energy (CE) of 25eV. The weighted calibration curve (1/x2) was found to be linear over the concentration range of 1-100ng/mL (r2>0.999). The limit of quantification (LLOQ) of the method was 1ng/mL. The values of selectivity, carryover, LLOQ, linearity, accuracy, precision, matrix effect, stability and recovery obtained meet the acceptable range according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines. The method was efficiently applied to quantify DOB in plasma samples from a pharmacokinetic/pharmacodynamic study in a disease model of newborn piglet.

Effect of coating on the environmental applications of zero valent iron nanoparticles: the lindane case.

Commercial stabilized slurry of zero-valent iron nanoparticles (nZVI) as well as laboratory-synthesized polymer-stabilized NZVI nanoparticles were used for lindane (γ-hexachlorocyclohexane) degradation studies in aqueous solution. In the present study, polymer-stabilized iron nanoparticles were stabilized using polyethylene glycol (PEG, Mn ~400 and ~950-1050) and polytetrahydrofuran (PTHF, Mn ~650). To study the effectiveness of the different nanoparticles, a quantitative monitorization of lindane degradation by using solid-phase extraction (SPE) and a qualitative measurement of generated volatile by-products by headspace-solid phase microextraction (HS-SPME) followed by GC/MS were carried out. The obtained data were compared and contrasted with the results obtained in previous work. Results showed that the nanoparticles studied in this work possess superior dechlorination performance compared with previous observations. The freshly prepared Fe(0)-PEG400, Fe(0)-PEG1050 and Fe(0)-PTHF exhibited high reactivity during the dechlorination process of lindane in a very short time. The results obtained with the synthesized nanoparticles were similar to those obtained with commercial nanoparticles. However, in all cases reactivity decreased at reaction's late stage. Degradation of lindane by the studied nanoparticles removed 99.9% of the lindane initial concentration after 72h, except for Fe(0)-PTHF nanoparticles, for which the reaction stopped after 5min. In all cases, the reaction followed a second order kinetics. Finally, comparing the results from this study with our previous work, where different nature polymers were considered (Fe(0)-CMC, Fe(0)-PAA and Fe(0)-PAP), more gradual degradation profile of lindane was observed for Fe(0)-PAA and Fe(0)-CMC. It should be noted that in the present case, the reaction of lindane was speeded up with commercial and Fe(0)-PEG nanoparticles. Nevertheless, in the later case, the composition of by-products was affected by the presence of partially degraded intermediates. Taking into account the current technologies, the high removal rates obtained and the acceptable degradation times required, the proposed technology is suitable for its aimed purpose.

DATINK pilot study: an effective methodology for ballpoint pen ink dating in questioned documents.

Establishing the approximate age of an ink entry from a questioned document is often a complicated task and a controversial issue in forensic sciences. Among the existing approaches, the analysis of solvents in ballpoint inks may be a useful parameter for determining the age of ink on paper. In recent years, several ink dating methods have been proposed. These methods have been based on the analysis of common ink solvents using gas chromatography/mass spectrometry (GC/MS) as the analytical platform. Despite these recent methods, several questions remain. The aim of this work was to develop an ink dating methodology (DATINK) for documents written by ballpoint pens based on the disappearance of volatile solvents from the ink entry. Multiple solid-phase microextraction (MHS-SPME) coupled to GC/MS was used to measure the solvents from ink entries made with four BIC(®) ballpoint pens. The ß parameter, the remaining fraction of the analyte in the system after one equilibration, corresponding to the successive extractions was considered for modelling a mathematical equation for later ink age dating. Preliminary tests of DATINK method showed that it was possible to detect the presence of ink solvents on documents up to the studied five years. The analyses of different real samples of known age were analyzed in terms of ß values, which provided a mean relative error of 21%. The proposed use of ß parameter for estimating the absolute age of ballpoint ink entries has shown promising results with a standard deviation of ß ranging from 0.002 to 0.004.

Quantitative determination of fentanyl in newborn pig plasma and cerebrospinal fluid samples by HPLC-MS/MS.

In this study, a selective and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method requiring low sample volume (≤100 µL) was developed and validated for the quantitative determination of the opioid drug fentanyl in plasma and cerebrospinal fluid (CSF). A protein precipitation extraction with acetonitrile was used for plasma samples whereas CSF samples were injected directly on the HPLC column. Fentanyl and (13) C6 -fentanyl (Internal Standard) were analyzed in an electrospray ionization source in positive mode, with multiple reaction monitoring (MRM) of the transitions m/z 337.0/188.0 and m/z 337.0/105.0 for quantification and confirmation of fentanyl, and m/z 343.0/188.0 for (13) C6 -fentanyl. The respective lowest limits of quantification for plasma and CSF were 0.2 and 0.25 ng/mL. Intra- and inter-assay precision and accuracy did not exceed 15%, in accordance with bioanalytical validation guidelines. The described analytical method was proven to be robust and was successfully applied to the determination of fentanyl in plasma and CSF samples from a pharmacokinetic and pharmacodynamic study in newborn piglets receiving intravenous fentanyl (5 µg/kg bolus immediately followed by a 90-min infusion of 3 µg/kg/h).

Analytical strategies based on multiple headspace extraction for the quantitative analysis of aroma components in mushrooms.

Headspace (HS) and headspace solid phase microextraction (HS-SPME) analysis by gas chromatography-mass spectrometry (GC/MS) have been found to be suitable methods for the analysis of volatile organic compounds. The objectives of this paper are to study the possibilities of multiple headspace extraction (MHE) for the quantitative determination of volatile compounds in mushroom samples and to compare the results obtained using three different sample treatment techniques. For this purpose, HS with two different injection techniques (pressure-loop system and gas-tight syringe autosampling system) and HS-SPME have been studied. Three processes were optimized for the analysis of 20 volatile compounds by experimental design technique based on Central Composite Design (CCD) and Full Factorial Design depending on the used methodology. Once the designs were finished, a trade off among optimum conditions for each compound analyzed was reached. At optimum conditions, appropriate extraction time and sample amount for the three techniques used were established. Finally, the methods were validated in terms of linearity, detection and quantitation limits and repeatability. The most suitable method was then applied to the quantitative analysis of seven mushroom samples. A detailed comparison of the analytical performance characteristics of HS and HS-SPME as sample treatment techniques for final GC/MS determination is given. In addition, MHE has been proved to be an adequate technique to avoid matrix effects in complex samples quantitation. Its applicability to the determination of volatile mushroom components, along with its limitations, is discussed in this work.

Relevance study of bare and coated zero valent iron nanoparticles for lindane degradation from its by-product monitorization.

Zero-valent iron nanoparticles (NZVI) as well as polymer-stabilized nanoparticles were synthesized and used for lindane (γ-hexachlorocyclohexane) degradation in aqueous solution. To study the effectiveness of the different coated nanoparticles, simple and rapid analytical methods have been developed to measure and to detect lindane and its by-products. For the monitorization of lindane degradation solid-phase extraction (SPE) was used, while volatile by-products formation measurement was carried out by headspace-solid phase microextraction (HS-SPME) followed by GC/MS. The SPE-GC/MS method provides low detection limits (0.2 µg L(-1)), high recovery (above 95%) and it is a valuable tool for kinetic studies of the degradation process for each polymer used, while HS-SPME-GC/MS has proved to be an effective tool for the extraction and evaluation of volatile degradation by-products.

Hollow fibre-based liquid-phase microextraction technique combined with gas chromatography-mass spectrometry for the determination of pyrethroid insecticides in water samples.

A simple, easy-to-use, efficient and environmentally friendly method has been developed for the simultaneous analysis of nine pirethroid pesticides in water samples by the combination of hollow fibre-based liquid-phase microextraction (HF-LPME) and gas chromatography-mass spectrometry (GC/MS). For the developed method, nine pirethroid pesticides (esbiothrin, prallethrin, bifenthrin, tetramethrin, phenothrin, permethrin, cyfluthrin, cypermethrin and deltamethrin) were concentrated and well separated under optimal conditions. Several factors that influence the efficiency of HF-LPME were investigated and optimized by means of experimental design. The proposed method has good linearity in the concentration range of 10-400 µg L(-1) with correlation coefficients between 0.995 and 0.999. Overall enrichment factors for the optimized method ranged from 139 to 255 times except for cypermethrin and deltamethrin which ranged from 35 to 128. Detection and quantitation limits of the chromatographic method were in the range of 0.002-0.012 µg L(-1) and 0.003-0.026 µg L(-1) respectively, with RSD values between 4.2% and 18.4%. The recoveries varied in the range of 69.4%-122.7% except for cypermethrin and deltamethrin (17.5%-64.1%) with relative standard deviations between 1.0% and 24.0% for intra and inter-day experiments at different concentrations (0.1 µg L(-1), 0.5 µg L(-1), 1 µg L(-1)). The HF-LPME method optimized was applied to the analysis of three spiked real water samples with good results.

Micellar electrokinetic chromatography method for the determination of several natural red dyestuff and lake pigments used in art work.

The identification of organic colorants used in artistic paintings is an important information source for reconstructing the working techniques found in a particular work and for defining a programme for the restoration and conservation of the painting. In this work, sodium dodecyl sulfate (SDS) was used as a surfactant in micellar electrokinetic chromatography (MEKC) for separating a broad range of red organic pigments, based on their colouring matters: madder (colouring matters: alizarin, quinizarin and purpurin), cochineal (colouring matter: carminic acid), red sandalwood (colouring matter: santalin), brazilwood (colouring matter: brazilin), lac dye (colouring matter: laccaic acid) and dragon's blood (colouring matter: dracorhodin). The running electrolyte used was 20 mM borax (pH 9), containing 20 mM SDS and 10% acetonitrile as organic modifier. Separation was carried out by applying a +20 kV voltage at the injection end, 25 degrees C and 214 nm/254 nm as detection wavelengths. All colorants were separated within less than 13 min with a good baseline resolution. The method was applied to the analysis of paint samples obtained from the Diocesan Museum of Holy Art of Bilbao.

Development of a solid phase extraction procedure for HPLC-DAD determination of several angiotensin II receptor antagonists in human urine using mixture design.

The optimisation of a solid phase extraction procedure involves several variables whose influence has been widely studied. However, in most cases, only process variables are taken into account. In this work, the influence of those process variables together with the fact of using mixtures of solvents during the elution step of the solid phase extraction of four angiotensin II receptor antagonist drugs has been studied. Since the influence on the extraction efficiency of several process variables were simultaneously tested, a D-optimal design was constructed. The composition of the elution solvent (a mixture of methanol, acetonitrile, ethanol and acetone at different proportions from 0 to 100% each solvent), the percentage and pH of the buffer solution added to the urine samples at the beginning of the extraction procedure; the percentage of the organic component and the volume of the washing solution, the drying time and the volume of the elution solvent were the studied variables. The chromatographic separation was carried out by gradient elution mode with 0.026% trifluoroacetic acid (TFA) in the organic phase and 0.031% TFA in the aqueous phase using an Atlantis dC18, 100mmx3.9mm I.D. chromatographic column at a flow rate of 1mL/min and a column temperature of 35+/-0.2 degrees C. For detection a diode array detector set at 232nm was used. The extraction procedure for spiked human urine samples was developed using C8 cartridges, phosphate buffer pH 6.8 as conditioning agent, a drying step of 10min, a washing step with methanol-phosphate buffer (20:80, v/v) and methanol as eluent. Recovery percentages obtained: 84% for eprosartan, 74% for telmisartan, 74% for irbesartan and 89% for valsartan allow the determination of these drugs concentration levels in urine.

Prevalencia de síndrome disfórico premenstrual en población general / Prevalence of premenstrual dysphoric syndrome in general population

OBJETIVOS. Medir la prevalencia de síndrome disfórico premenstrual mediante encuesta heteroadministrada con los criterios diagnósticos de la Asociación Americana de Psiquiatría presentes en el DSM-IV. MÉTODOS. Se diseña un estudio observacional, descriptivo y transversal, con emplazamiento en la Zona Básica de Salud de Porzuna (Ciudad Real). Ámbito: Atención Primaria de salud; medio rural. Población diana: todas las mujeres de entre 18 y 50 años de edad de la Zona Básica de Salud (948); se seleccionaron 226 por muestreo aleatorio simple, con reemplazo automático y consecutivo de aquellas que rehusaron participar (8), no fueron localizables (21) o reunían criterios de exclusión: menopausia, gestación, déficit mental (11), participando finalmente en el estudio 186 mujeres. A dichas mujeres se les aplicó una encuesta heteroadministrada recabando información referente a los criterios de la APA para el diagnóstico del síndrome disfórico premenstrual durante el último año, así como datos sociodemográficos, de comorbilidad y sobre tratamiento farmacológico. RESULTADOS. El 84,33% de las encuestadas presentaba algún tipo de molestia premenstrual, siendo moderada (al menos un síntoma durante todos o la mayoría de los ciclos del último año) en el 69,73% e intensa (5 o más síntomas la mayoría de los ciclos pero sin afectar a la vida laboral, social o familiar) en el 8,11%. La prevalencia encontrada de síndrome disfórico premenstrual según criterios de la Asociación Americana de Psiquiatría es del 6,49% (n = 12; IC 95%: 2,95-10,03%). CONCLUSIONES. La prevalencia encontrada de síndrome disfórico premenstrual en nuestra población concuerda con los datos publicados en estudios internacionales (2,5-14%) y se aleja de las prevalencias publicadas en nuestro país (20,6-28,6%) con encuestas autoadministradas

OBJECTIVES. Measure the prevalence of premenstrual dysphoric syndrome (PMDS) by heteroadministered survey with diagnostic criteria of the American Association of Psychiatry (AAP) present in the DSM-IV. METHODS. An observational, descriptive and cross-sectional study was designed with location in the Basic Health Area (BHA) of Porzuna (Ciudad Real). Scope: primary health care; rural setting. Target population: all women between 18 and 50 years of age from the BHA (948). A total of 226 were selected by simple random sampling, with automatic and consecutive replacement of those who refused to participate (8). Of these, 21 could not be located or fulfilled exclusion criteria: menopause, gestation, mental retardation (11), 186 women finally participating in the study. These women were administered a heteroadministered survey, collecting information regarding the AAP criteria for the diagnosis of PMDS during the last year and sociodemographic, comorbidity and pharmacological treatment data. RESULTS. A total of 84.33% of those surveyed had some type of premenstrual discomfort, these being moderate (at least one symptom during all or most of the cycles of the last year) in 69.73% and intense (5 or more symptoms in most of the cycles but without affecting work, social or family life) in 8.11% Prevalence of PMDS found according to AAP criteria is 6.49% (n = 12; 95% CI: 2.95 - 10.03%). CONCLUSIONS. Prevalence of PMDS found in our population agrees with the data published in international studies (2.5 - 14%) and moves away from the prevalences published in our country (20.6 - 28.6%) with self-applied surveys

Validation of a solid phase extraction-high performance liquid chromatographic method for the determination of eprosartan in human plasma.

In this work, a solid phase extraction-reversed phase high performance liquid chromatographic (SPE-RP-HPLC) method with photometric detection for monitoring the antihypertensive drug eprosartan has been validated in order to assure good quantitation of eprosartan in plasma samples obtained from patients under cardiovascular treatment. This analytical method was developed by using experimental design and quantitation was accomplished with the internal standard method. No interferences were observed from endogenous compounds of plasma and other drugs which are commonly co-administered in elderly patients. The recoveries of eprosartan from plasma samples, measured at three levels of the linear concentration range (150-4000 ng/mL) were found to be between 93.4 and 102.8%. The intraday and interday precision and accuracy (measured by relative standard deviation, RSD, and relative error, RE, respectively) were always lower than 13% (RSD) and 4% (RE). Stability studies showed that eprosartan stock solutions are stable for at least 3 months when stored at 8 degrees C and plasma samples containing the drug were stable at least during the whole analytical method.

MultiSimplex and experimental design as chemometric tools to optimize a SPE-HPLC-UV method for the determination of eprosartan in human plasma samples.

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug eprosartan from human plasma samples. MultiSimplex program was used to optimize the HPLC-UV method due to the number of experimental and response variables to be studied. The measured responses were the corrected area, the separation of eprosartan chromatographic peak from plasma interferences peaks and the retention time of the analyte. The use of an Atlantis dC18, 100mmx3.9mm i.d. chromatographic column with a 0.026% trifluoroacetic acid (TFA) in the organic phase and 0.031% TFA in the aqueous phase, an initial composition of 80% aqueous phase in the mobile phase, a stepness of acetonitrile of 3% during the gradient elution mode with a flow rate of 1.25mL/min and a column temperature of 35+/-0.2 degrees C allowed the separation of eprosartan and irbesartan used as internal standard from plasma endogenous compounds. In the solid phase extraction procedure, experimental design was used in order to achieve a maximum recovery percentage. Firstly, the significant variables were chosen by way of fractional factorial design; then, a central composite design was run to obtain the more adequate values of the significant variables. Thus, the extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer pH 2 as conditioning agent, a drying step of 10min, a washing step with methanol-phosphate buffer (20:80, v/v) and methanol as eluent liquid. The SPE-HPLC-UV developed method allowed the separation and quantitation of eprosartan from human plasma samples with an adequate resolution and a total analysis time of 1h.

Development of a liquid-liquid extraction procedure for five 1,4-dihydropyridines calcium channel antagonists from human plasma using experimental design.

A liquid-liquid extraction method using diethyl ether as organic solvent was optimized simultaneously for five 1,4-dihydropyridines (amlodipine, nitrendipine, felodipine, lacidipine and lercanidipine) belonging to the group of calcium channel blockers. Some experimental tools such as a full factorial design, a central composite design and the Multisimplex program were used to optimise the concentration of NaOH, volume of organic solvent and shaking time as main factors that influence the liquid-liquid extraction procedure. Following the extraction, the quantitation of the 1,4-dihydropyridines concentrations were performed by high-performance liquid chromatography with diode-array detector. Therefore, the studied compounds were separated quantitatively on a Supelcosil ABZ+Plus, 25cmx4.6mm i.d., 5mum column which was set at 30 degrees C, using as mobile phase, a mixture of acetonitrile-water (70:30, v/v) containing 10mM acetate buffer (pH 5) and setting the detector at a wavelength value of 360nm. It was concluded that the main factors that influence in the extraction process were the volume of organic solvent and the shaking time. The Multisimplex program suggested as optimal conditions an average of 6ml of organic solvent and 23min of shaking time. For these values, the optimised liquid-liquid extraction method showed good values of recoveries (80% for amlodipine and higher than 90% for the rest of the compounds) and low values of R.S.D. (<10%) in the reproducibility of the extraction what makes it reliable for the quantification of all the studied compounds in human plasma.

Capillary electrophoresis as a useful tool for the analysis of chemical tracers applied to hydrological systems.

A capillary electrophoretic method was optimised for the separation and determination of iodide used as artificial tracer in hydrology. The influence of the buffer concentration and pH, electroosmotic flow modifier concentration (cetyltrimethylammonium bromide (CTAB)), the injection time and voltage applied, on the electrophoretic separation was studied. A running buffer of 20 mM phosphate (pH 8) containing 1 mM CTAB was found to provide the optimum separation of iodide with respect to resolution, migration time and selectivity. The water samples were injected hydrostatically at 10 cm for 110 s, the voltage applied was -20 kV and a detection wavelength of 214 nm. The influence of the sulphite added to water samples in order to prevent the oxidation of iodide to iodate was also studied. This method can be applied to the determination of iodide free of sulphite interference up to at least a ratio of 1:1000 (I(-):SO3(2-)). The other inorganic anions, which are present in the water samples (mainly chloride, sulphate, nitrate, carbonate), do not interfere with the determination of iodide. This method allows the simultaneous determination of bromide, nitrite, and nitrate together with iodide. The electrophoretic method showed to be linear from 0.5 to 5 mg l(-1) of iodide (the migration time was 2.6 min) with a quantitation limit of 0.45 mg l(-1) and a intraday repeatability lower than 4% of R.S.D. at different concentration levels.

Experimental design optimization of a capillary zone electrophoresis method for the screening of several diuretics and ACE inhibitors.

Experimental design methodologies are applied to the development of a capillary zone electrophoretic method for the separation of the angiotensin-converting enzyme inhibitor enalapril and its derivative enalaprilat and the diuretics xipamide and hydrochlorothiazide. The effects of pH, buffer concentration, proportion of boric acid in the mixed boric acid-potassium dihydrogen phosphate background electrolyte, temperature, applied voltage, and percentage of organic modifier are studied. Critical factors are identified in a screening design (a 2(6-2) fractional factorial design), and afterwards, optimal conditions for the separation are reached by means of an optimization design (a 2(2) + 2 x 2 + k central composite design). The studied response is the resolution between peaks. The four studied compounds can be separated in less than 3.5 min using an electrolyte of 20mM boric acid-potassium dihydrogen phosphate (75:25, v/v) with 5% MeOH adjusted to pH 8.0 with KOH, at a potential of 30 kV. The detection wavelength and temperature are 206 nm and 35 degrees C, respectively.