Exploratory optimisation of a LC-HRMS based analytical method for untargeted metabolomic screening of Cannabis Sativa L. through Data Mining.

BACKGROUND: Recent increase in public acceptance of cannabis as a natural medical alternative for certain neurological pathologies has led to its approval in different regions of the world. However, due to its previous illegal background, little research has been conducted around its biochemical insights. Therefore, in the current framework, metabolomics may be a suitable approach for deepening the knowledge around this plant species. Nevertheless, experimental methods in metabolomics must be carefully handled, as slight modifications can lead to metabolomic coverage loss. Hence, the main objective of this work was to optimise an analytical method for appropriate untargeted metabolomic screening of cannabis. RESULTS: We present an empirically optimised experimental procedure through which the broadest metabolomic coverage was obtained, in which extraction solvents for metabolite isolation, chromatographic columns for LC-qOrbitrap analysis and plant-representative biological tissues were compared. By exploratory means, it was determined that the solvent combination composed of CHCl3:H2O:CH3OH (2:1:1, v/v) provided the highest number of features from diverse chemical classes, as it was a two-phase extractant. In addition, a reverse phase 2.6 µm C18 100 Å (150 × 3 mm) chromatographic column was determined as the appropriate choice for adequate separation and further detection of the diverse metabolite classes. Apart from that, overall chromatographic peak quality provided by each column was observed and the need for batch correction methods through quality control (QC) samples was confirmed. At last, leaf and flower tissues resulted to provide complementary metabolic information of the plant, to the detriment of stem tissue, which resulted to be negligible. SIGNIFICANCE: It was concluded that the optimised experimental procedure could significantly ease the path for future research works related to cannabis metabolomics by LC-HRMS means, as the work was based on previous plant metabolomics literature. Furthermore, it is crucial to highlight that an optimal analytical method can vary depending on the main objective of the research, as changes in the experimental factors can lead to different outcomes, regardless of whether the results are better or worse.

Target and suspect screening of substances liable to migrate from food contact paper and cardboard materials using liquid chromatography-high resolution tandem mass spectrometry.

This work describes the analysis of both target and non-target compounds in paper and cardboard materials together with the characterization of their migration to Tenax® by means of liquid chromatography coupled to both low (LC-QqQ) and high resolution tandem mass spectrometry (LC-q-Orbitrap), respectively. To this aim, an entire analytical procedure was fully developed and validated for both matrices: paper/cardboard and Tenax®. A total of 97 chemicals, including photoinitiators, phthalates, biocides, antioxidants, etc., listed by the European Regulation, were found in the materials under study together with other substances not included in this list. Moreover, the majority of annotated substances were present in the simulant, giving evidence of their migration capacity. Migration percentages of 10 photoinitiators, 4 phthalates, bisphenol A, bis-(2-ethylhexyl) adipate, acetyltributyl citrate and caprolactam to Tenax® were quantified. Despite not exceeding the established specific migration limit (SML) in any case, benzophenone, 4-phenylbenzophenone and bisphenol A concentrations in some paper and cardboard materials were very close to the SML values established by the EU legislation.

Calibration and field test of the Polar Organic Chemical Integrative Samplers for the determination of 15 endocrine disrupting compounds in wastewater and river water with special focus on performance reference compounds (PRC).

In this work, home-made Polar Organic Chemical Integrative Samplers (POCIS) were studied for passive sampling of 15 endocrine disrupting compounds (4 alkylphenols and steroid hormones) in influent and effluent samples of wastewater treatment plants (WWTPs) as well as up- and downstream of the receiving river water. POCIS calibration at laboratory conditions was carried out using a continuous-flow calibration system. The influence of the exposure position of the POCIS within the calibration device, horizontal or vertical, to the water flow direction was evaluated. While the sampling rates of most of the target substances were not affected by the sampler position, for cis-ADT, E1, E2 and E3, the vertical position provided the highest analyte accumulation. Hence, the POCIS samplers were preferably exposed vertical to the water flow in overall experiments. Using the continuous-flow calibration device, lab-based sampling rates were determined for all the target compounds (RSBPA = 0.0326 L/d; RScisADT = 0.0800 L/d, RSE1 = 0.0398 L/d, RSEQ = 0.0516 L/d, RSTT = 0.0745 L/d, RSE2 = 0.0585 L/d, RSEE2 = 0.0406 L/d, RSNT = 0.0846 L/d, RSPG = 0.0478 L/d and RSE3 = 0.1468 L/d), except for DES, MeEE2, 4tOP, 4OP, 4NPs, where the uptake after 14 days POCIS exposure was found to be insignificant or indicated a no linear behaviour. Recoveries from POCIS extractions were in the range between 71 and 152% for most of the target analytes except for DES and E3 with around 59%. Good precision of the sampling procedure up till 20% was observed and limits of detection were at ng/L level. Two deuterated compounds ([(2)H3]-E2 and [(2)H4]-EQ) were successfully tested as performance reference compounds (PRC, [Formula: see text] = 0.0507 L/d and [Formula: see text] = 0.0543 L/d)). Finally, the POCIS samplers were tested for monitoring EDCs at two wastewater treatment plants, in Halle and Leipzig (Germany). BPA, E1, EQ, E2, MeEE2, NT, EE2, PG and E3 were quantified and their time-weighted average concentrations calculated on the basis of the lab-derived sampling rates were compared with the results based on conventional grab samples. While the influent concentration of BPA, cisADT, E1, TT, PG, EE2 reached the µg/L level, the rest of the target analytes were determined at ng/L. The analyte concentrations in the effluent never exceed ng/L level except for BPA. The concentration determined by spot sampling was partially lower (BPA, E1, TT) or comparable (EQ, E2, EE2, PG, E3) to the concentration obtained by POCIS using performance reference compounds (PRC).

Solid-phase extraction combined with large volume injection-programmable temperature vaporization-gas chromatography-mass spectrometry for the multiresidue determination of priority and emerging organic pollutants in wastewater.

In the present work the simultaneous extraction for the multiresidue determination in wastewater samples of organic compounds such as polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), pesticides, polycyclic aromatic hydrocarbons (PAHs), phthalate esters (PEs), alkylphenols (APs), bisphenol A (BPA) or hormones included in different lists of priority and emerging pollutants because of their action as endocrine disrupting compounds (EDCs) was developed. Different solid phase extraction (SPE) variables such as the nature of the solid phase (Oasis-HLC, C18 and Lichrolut), the sample volume, the addition of MeOH and NaCl, the pH of the water phase and the volume of the eluent solvent were optimized in order to analyze simultaneously the priority and emerging families of pollutants mentioned above. Good recoveries were obtained for Milli-Q water (80-120%), however, since the use of deuterated analogues and dilution of the sample did not correct the matrix effect, additional SPE clean-up step using Florisil® cartridges was necessary to obtain good results for wastewater samples (80-125%). In order to improve the limits of detection (LODs), large volume injection (LVI) using programmable temperature vaporizer (PTV) coupled to gas chromatography-mass spectrometry (GC-MS) was also optimized. Since analytes losses in the case of the most volatile congeners occurred during the derivatization step and no separation of the derivatized and the non-derivatized analytes was possible during SPE elution, two different injections were optimized for each analyte group. LODs were in good agreement with those found in the literature and relative standard deviations (RSDs) were in the 10-25% range for Milli-Q and 12-30% for wastewater samples. The method was finally applied to the determination of target analytes in three different wastewater treatment plants (WWTPs, Bakio, Gernika and Galindo (Spain)) and in one water purification plant (WPP) in Zornotza (Spain).

Optimization of comprehensive two dimensional gas chromatography-flame ionization detection-quadrupole mass spectrometry for the separation of octyl- and nonylphenol isomers.

In the present work, the separation of complex nonylphenol technical mixtures has been optimized using comprehensive two-dimensional gas chromatography coupled with a flame ionization detector and quadrupole mass spectrometer (GC×GC-qMS), using valve-based modulator. The optimization of GC×GC-qMS has been carried out using experimental designs and the optimal separation was obtained at the following conditions: 1st column flow: 1mL/min; 2nd column flow: 17.75 mL/min, oven temperature ramp: 1°C/min, modulation period: 1.5s and discharge time: 0.12s. These values have been used to determinate the previously synthesized 22OP, 33OP, 363NP and 22NP isomers in two different nonylphenol technical mixtures. Percentages obtained were as follows: 4.86% and 0.59% for 22OP, 4.91% and 2.82% for 33OP, 11.79% and 7.71% for 363NP and 2.28% and 1.98% for 22NP, in Fluka and Aldrich mixtures, respectively. The values obtained for NP isomers are in good agreement with the literature.

Optimization of large volume injection-programmable temperature vaporization-gas chromatography-mass spectrometry analysis for the determination of estrogenic compounds in environmental samples.

Large volume injection-programmable temperature vaporization-gas chromatography-mass spectrometry (LVI-PTV-GC-MS) was optimized for the determination of estrone (E1), 17ß-estradiol (E2), 17α-ethynyl estradiol (EE2), mestranol (MeEE2) and estriol (E3) for their determination in environmental samples (estuarine water, wastewater, fish bile and fish homogenate) after derivatization with 25 µL (BSTFA+1% TMCS) and 125 µL of pyridine. Experimental designs such as Plackett-Burman (PBD) and central composite designs (CCDs) were used to optimize the LVI-PTV variables (cryo-focusing temperature, vent time, vent flow, vent pressure, injection volume, purge flow to split vent, splitless time and injection speed). Optimized conditions were as follows: 45 µL of n-hexane extract are injected at 60°C and 6 µL/s with a vent flow and a vent pressure of 50 mL/min and 7.7 psi, respectively, during 5 min; then the split valve is closed for 1.5 min and afterwards the injector is cleaned at 100 mL/min before the next injection. The method was applied to the determination of estrogenic compounds in environmental samples such as estuarine water, wastewater, and fish homogenate and bile. Limits of detection (0.04-0.15 ng/L for water samples, 0.04-0.67 ng/g for fish bile and 0.1-7.5 ng for fish homogenate) obtained were approx. ten times lower than those obtained by means of a common split/splitless inlet.

Determination of alkylphenols and 17beta-estradiol in fish homogenate. Extraction and clean-up strategies.

The determination of target analytes such as nonyl- and octylphenols and 17beta-estradiol in fish homogenate require of solid-liquid extraction step. In this work microwave-assisted extraction (MAE) and focused-ultrasound liquid extraction (FUSLE) were studied as two different alternatives for extraction of the target compounds in zebrafish (Danio rerio) homogenate. In this work solid phase extraction (SPE) using 5-g and 10-g Florisil cartridges and gel permeation chromatography (GPC) were studied for the clean-up of the MAE and FUSLE extracts due to the non-selective extraction step. Although good recoveries were obtained both for SPE (106% and 126% range) and GPC (79% and 100% range) clean-up procedures, cleaner chromatograms were obtained after SPE and finally 5-g Florisil cartridges were tested since no improvement was observed when 10-g Florisil cartridges were used. Under optimized clean-up conditions, MAE and FUSLE provided comparable results for 4nOP and NP, while more accurate results were obtained for 4tOP and E2 after FUSLE. Finally, the method was applied to the determination of alkylphenols and 17beta-estradiol in zebrafish homogenate that had been exposed to known concentrations of the target analytes. In the case of alkylphenols two different isomers of nonyl- and octylphenol (4-(3',6'-dimethyl-3'-hepthyl)phenol, 363-NP, and 4-(3'-methyl-3'-hepthyl)phenol, 33-OP) were studied.

Stir-bar sorptive extraction: A view on method optimisation, novel applications, limitations and potential solutions.

Introduced in 1999 as a novel solventless sample preparation method, stir-bar sorptive extraction (SBSE) has become a popular analytical technique for the pre-concentration of organic compounds into a polydimethylsiloxane (PDMS)-coated stir-bar. In the last 10 years, hundreds of applications in the environmental, food and biomedical fields can be found in the literature. However, only PDMS-coated stir-bars are commercially available, which reduces the applicability of SBSE to the extraction of the non-polar compounds due to the poor extractability of more polar analytes. In this review, a view on method optimisation, limitations, potential solutions such as in-house coatings and derivatisation and novel applications in multi-residue analysis and passive sampling are revised.

Simultaneous preconcentration of a wide variety of organic pollutants in water samples. Comparison of stir bar sorptive extraction and membrane-assisted solvent extraction.

Stir bar soptive extraction (SBSE) coupled to thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) and membrane-assisted solvent extraction (MASE) coupled to large volume injection-programmed temperature vaporisation-GC-MS (LVI-PTV-GC-MS) were optimised for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), nonylphenols (NPs), polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs) in water samples. In the case of SBSE-TD, variables affecting the extraction (extraction time, addition of sodium chloride or methanol and sample volume) and desorption (cryofocusing temperature, desorption time and temperature, vent pressure and desorption flow) were fitted for the simultaneous determination. The extraction solvent nature (n-hexane, cyclohexane, n-heptane, ethyl acetate, toluene, dichloromethane or cyclohexane:ethyl acetate mixtures), as well as the addition of methanol (0-30%) and sodium chloride (0-20%), the extraction temperature (30-60 degrees C), shaking speed (250-750 rpm) and extraction time (5-150 min) were studied for the simultaneous membrane-assisted preconcentration. Finally, PTV-LVI variables such as injection volume (100-600 microL), injection speed (10-40 microL s(-1)), vent pressure (0-12.7 psi), vent time (0.05-0.8 min), vent flow (30-80 mL min(-1)), cryofocusing temperature (20-70 degrees C), split flow (20-100 mL min(-1)) and split time (1-5 min) were optimised. The optimisation was carried out by means of experimental design approaches in most of the cases. Precision (approximately 3-19% for both SBSE-TD and MASE-LVI-PTV), accuracy (approximately 80-120% for both SBSE-TD and MASE-LVI-PTV), limits of detection (LoDs) (0.1-222 ng L(-1) for MASE-LVI-PTV and 0.03-20.4 ng L(-1) for SBSE-TD in dependence of substance) and linearity (from 25 ng L(-1) up to at least 500 ng L(-1) for both procedures) were established for both procedures. Finally, the developed methods were applied to the determination of the free concentrations of PAHs, PCBs, PEs, NPs, PBBs and PBDEs in natural water samples (estuarine water and sea water) from the Bilbao estuary (Northern Spain) and comparable results were obtained with both procedures.

Simultaneous extraction of several persistent organic pollutants in sediment using focused ultrasonic solid-liquid extraction.

Focused ultrasonic solid-liquid extraction (FUSLE) has been optimised for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), and nonylphenols (NPs) in sediment samples. Optimisation was performed using naturally polluted freeze-dried sediment samples. The variables studied during the optimisation process were: percentage of maximum power (10-60%), extraction time (10-300 s), number of cycles (1-9), composition of the extraction solvent (acetone-n-hexane, 10:90-90:10), and sample mass (0.1-1 g). The volume of the extractant was constant (10 mL) and the extraction was performed at 0 degrees C in an ice-bath during the optimisation process. All these variables were studied using an experimental design approach by means of The Unscrambler software. The extraction time and the operational variables (number of cycles and power) had no statistically significant effect in the extraction and they were held at 2 min, 20% power, and seven cycles, respectively. The mass and the addition of non-polar solvent (n-hexane) had a negative effect in the extraction yield and, thus, the mass was held at 0.5 g and pure acetone was used as extraction solvent. After those variables were optimised, the effect of the extraction temperature (0 degrees C or room temperature) was also studied. The validation of the extraction method was performed using NIST-1944 reference material in the case of PAHs and PCBs. Because no certified reference sediment is available for PEs and NPs, the results obtained for FUSLE were compared with those obtained for microwave-assisted extraction (MAE) under conditions optimised elsewhere. In all the cases the analysis were performed by gas chromatography-mass spectrometry (GC-MS). Good accuracy were achieved in all cases. The limits of detection (LODs) obtained were between 0.10 and 1.70 ng g(-1) for PAHs (except for naphthalene 5.33 ng g(-1)), 0.02 and 0.16 ng g(-1) for PCBs, 46 and 188 ng g(-1) for PEs, and 0.6 and 12.4 microg g(-1) for NPs. The precision was around 5-10% for most of the PAHs and PCBs and around 2-10% for most of the PEs and NPs.

Distribution and bioaccumulation of PAHs in the UNESCO protected natural reserve of Urdaibai, Bay of Biscay.

Along 10 campaigns, from June 2002 to September 2004, the concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were measured in sediments and oysters (Crassostrea sp.) taken from four sites in the Unesco protected natural reserve of Urdaibai (Basque Country, Bay of Biscay). Total PAH concentration ranged from 0.7 to 140 microg kg(-1) (dw) in the case of sediments, and from 300 to 1400 microg kg(-1) (dw) in the case of oysters. During this study, the coast of the Bay of Biscay was severely affected by the Prestige oil spill (November 2002). Presumably, as a consequence of this accident, both spatial and temporal variations of the PAHs, as well as the sources of the PAHs were affected by the oil spill, and this effect was observed in the total concentrations and, especially, in several diagnostic ratios and in multivariate data analysis. Finally, both BAF (bioaccumulation factor) and BSAF (biota-sediment accumulation factor) parameters were calculated to conclude that particulate matter seems to be the most favourable uptake pathway of PAHs in oysters from this estuary.

Thermodynamic and Raman spectroscopic speciation to define the operating conditions of an innovative cleaning treatment for carbonated stones based on the use of ion exchangers--a case study.

Thermodynamic and Raman spectroscopic speciation was used to define the operating conditions for an innovative cleaning treatment of the decayed carbonate-based stones using ion exchange technology. The conditioning process of the Lewatit OC1071 anion exchanger with EDTA (Y) was monitored by ionic chromatography. Characterisation of the initial (R-Cl) and the final (R2-H2Y) forms of the ion exchanger was carried out by dispersive Raman microprobe spectroscopy. The subtraction spectrum obtained between the chloride and the EDTA forms of the ion exchanger shows Raman bands at 1403, 1324, 1223, 1122, 932, 908, 591, 431 and 350 cm(-1), which agree with bands showed in the spectrum of the standard solution of the H2Y(2-) species. Therefore, this seems to be the EDTA species which takes part in the cleaning treatment performed by using resin poultices on black crusted limestone samples from an historical building.

Simultaneous speciation of methylmercury and butyltin species in environmental samples by headspace-stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry.

In this work, making use of experimental designs, headspace-stir bar sorptive extraction (HS-SBSE) followed by thermal desorption (TD) coupled to a gas chromatography-mass spectrometry (GC-MS) for the simultaneous determination of mercury and tin organometallic compounds present in surface water, sediment and biological tissue was optimized. All solid samples require a previous extraction typically done with diluted HCl or KOH/methanol solutions, and the derivatization, in all the cases, of the organometallic compounds with NaBEt(4). As a consequence, the preconcentration step was carried out in a 0.1 mol L(-1) HOAc/NaOAc buffer solution, with 0.1% (m/v) of NaBEt(4), without the addition of NaCl as a salting out reagent, and with the stir bar (20 mm x 1 mm) located in the headspace (HS). In addition, the desorption step required the following conditions: 250 degrees C (desorption temperature), 15 min (desorption time), 14.1 psi (approximately 97.2 kPa) (vent pressure) and 75 mL min(-1) (vent flow). Finally, to assure the extraction of all the analytes under equilibrium, 5h are required. Inorganic mercury (Hg(2+)) and tripropyltin (TPrT) were used as internal standards to correct for variations in the extraction, derivatization and detection steps. The resulting method provides precise (4-17%) and accurate (against four certified reference materials) results in the ng L(-1) and pg g(-1) range concentrations with recoveries within 80-120% for water samples. The proposed methodology is currently applied in the speciation analysis of specimens obtained in different estuarine sites of the Basque Coast.

Use of experimental design in the optimisation of stir bar sorptive extraction followed by thermal desorption for the determination of brominated flame retardants in water samples.

A method for the determination of polybrominated diphenyl ethers (PBDEs) and polybrominated biphenyls (PBBs) in water samples is proposed. The method involving stir bar sorptive extraction (SBSE) and thermal desorption followed by gas chromatography coupled with mass spectrometry was optimised using statistical design of experiments. In the first place, the influence of different polydimethylsiloxane stir bars was studied. A Plackett-Burman design was chosen to estimate the influence of five factors on the efficiency of the SBSE process: desorption time (5-10 min), desorption temperature (250-300 degrees C), desorption flow (50-100 mL min(-1)), cryofocusing temperature (-130 to 40 degrees C) and vent pressure (0-12.8 psi). Afterwards, two central composite designs were used to find the optimal process settings that were applied to the optimisation of both desorption and extraction efficiency. In the case of the desorption parameters, long desorption times (10 min) and desorption flows lower than 70 mL min(-1) yielded the best signals for the majority of compounds. However, different behaviour among the analytes was observed for the vent pressure and we decided to fix it at an intermediate value (7 psi). In the case of extraction parameters, the sample volume and the addition of NaCl did not have a significant effect, while the addition of methanol yielded better extraction responses. Remarkable recovery (82-106%) and repeatability (less than 18%) were attained. Furthermore, excellent regression coefficients (r2 = 0.991-0.999) and low detection limits (1.1-6.0 ng L(-1)) were also achieved for the congeners studied. The proposed method was applied to the analyses of PBDEs and PBBs in waters from the Basque Country, Spain.

Optimisation of the on-fibre derivatisation of volatile fatty acids in the simultaneous determination together with phenols and indoles in cow slurries.

The on-fibre derivatisation of volatile fatty acids (VFAs) using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was optimised in the simultaneous determination of VFAs together with phenols and indoles by headspace solid-phase microextraction (SPME)-gas chromatography-mass spectrometry. Firstly, the nature of the SPME fibre was optimised and four different fibres were studied (100 microm polydimethylsiloxane, 85 microm Carboxen/polydimethylsiloxane, 5/30 microm divinylbenzene/Carboxen/polydimethylsiloxane and 85 microm polyacrylate). The optimum fibre (50/30 microm divinylbenzene/Carboxen/polydimethylsiloxane) was used to study the exposure time of the fibre to the derivatisation agent and the desorption time and temperature. Firstly, a factorial design was built but since the three variables had a significant effect, a central composite design was used to build the response surfaces. The best signals were obtained after the exposure of the fibre in the headspace of the MTBSTFA derivatisation reagent for 1 h and desorption at 300 degrees C for 9 min. The determination of underivatised phenols and indoles was not affected by the presence of the derivatisation reagent in the fibre.

Development of a stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry method for the simultaneous determination of several persistent organic pollutants in water samples.

Stir bar sorptive extraction (SBSE) and thermal desorption followed by capillary gas chromatography coupled to mass spectrometry (SBSE-TD-GC-MS) was applied to the simultaneous determination of ultra-traces of 16 polycyclic aromatic hydrocarbons (PAHs), 12 polychlorinated biphenyls (PCBs), 6 phthalate esters (PEs) and 3 nonylphenols (NPs) in water samples. The parameters that could affect the sorption-desorption efficiency were studied. A Plackett-Burman design was used for the screening of the main effects of the experimental parameters related to the desorption step (desorption time, desorption temperature, desorption flow, cryo-focusing temperature and vent pressure). Afterwards, two central composite designs were used to find the optimal process settings for the extraction and desorption steps. The best analytical compromise conditions for the simultaneous determination of analytes from spiked water samples were found to be: sample volume (20 mL), sodium chloride addition (30%), methanol addition (20%), desorption time (10 min), desorption temperature (300 degrees C), desorption flow (23 mL min(-1)), cryo-focusing temperature (-50 degrees C) and vent pressure (7 psi). Remarkable recovery, repeatability and reproducibility were attained. Furthermore, excellent linearities (r(2) = 0.959-0.999) and low detection limits (0.1-10 ng L(-1)) were also achieved for the congeners studied. The proposed methodology was applied for the simultaneous determination of PAHs, PCBs, PEs and NPs in sea and estuarine waters. The influence of humic acids on the recovery was also studied.

Spectroscopic characterisation of moonmilk deposits in Pozalagua tourist cave (Karrantza, Basque Country, North of Spain).

Composition of moonmilk deposits located in different zones of the tourist Dolomite Cave of Pozalagua (Karrantza, Basque Country, North of Spain) was established by Raman spectroscopy. The deposits were located in column bases and detached rocks near a gour full of water or a dripping zone. Hydromagnesite (Mg(5)(CO(3))(4)(OH)(2).4H(2)O) with a strong Raman band at 1116 cm(-1) and weaker ones at 1522vw, 1487vw, 1452vw, 756w, 727w, 466w, 434w, 371w, 327m, 291w, 258w, 247vw and 230 m cm(-1) was found as the main component of the moonmilk. Aragonite is the unique calcium carbonate compound that sometimes composes the moonmilk but always together with the hydromagnesite. Among non-carbonate minerals, some nitrates (nitrocalcite, niter, nitromagnesite, nitratine and gwihabaite) and sulphates (arkanite) were also identified as minor compounds. Most of the deposits were matt white and pasty, but occasionally some samples appear greyish on the surface. In these samples, carbon particles were also found, apart from the above, and Raman shift changes were observed in the hydromagnesite spectra. Apart from the elements involved in the mentioned minerals, Si, Fe, Mn, Zn and Ti were identified by X-ray microfluorescence as trace elements and the results were correlated with mineral compositions found by Raman measurements. Furthermore, quantification of the soluble salts of moonmilk deposits was carried out by ionic chromatography and the results were chemometrically treated to find correlations among soluble ions and the composition of the mineral phases spectroscopically characterised.

Simultaneous microwave-assisted extraction of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters and nonylphenols in sediments.

A new method was developed for the simultaneous extraction of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), nonylphenols (NPs) and nonylphenol mono- and diethoxylates (NP1EOs and NP2EOs, respectively) in sediment samples by means of a closed microwave system. The extractions were carried out at 21 psi and 80% of microwave power and 15 ml of acetone were used as the common extraction solvent. The filtered extract was further fractionated in two groups using Florisil cartridges: PAHs and PCBs were eluted with n-hexane:toluene (4:1) and the PEs, NPs and ethoxylates were eluted with ethyl acetate. All the compounds were analysed by gas chromatography-mass spectrometry (GC-MS). In case of PAHs and PCBs, the developed method was validated by comparison of the results obtained for the certified reference material NIST 1944 with the certified values. In the absence of a reference material for phthalate esters and nonylphenols, one sediment sample was extracted twice under the optimal conditions in order to check than an exhaustive extraction of the analytes occurred. This method is currently used in the study of the distribution of those organic contaminants in the estuaries of the Bay of Biscay (Spain).

Automation of a procedure to find the polynomial which best fits (kappa, c1, c2, T) data of electrolyte solutions by non-linear regression analysis using MATHEMATICA software.

A MATHEMATICA package, 'CONDU.M', has been developed to find the polynomial in concentration and temperature which best fits conductimetric data of the type (kappa, c, T) or (kappa, c1, c2, T) of electrolyte solutions (kappa: specific conductivity; ci: concentration of component i; T: temperature). In addition, an interface, 'TKONDU', has been written in the TCL/Tk language to facilitate the use of CONDU.M by an operator not familiarised with MATHEMATICA. All this software is available on line (UPV/EHU, 2001). 'CONDU.M' has been programmed to: (i) select the optimum grade in c1 and/or c2; (ii) compare models with linear or quadratic terms in temperature; (iii) calculate the set of adjustable parameters which best fits data; (iv) simplify the model by elimination of 'a priori' included adjustable parameters which after the regression analysis result in low statistical significance; (v) facilitate the location of outlier data by graphical analysis of the residuals; and (vi) provide quantitative statistical information on the quality of the fit, allowing a critical comparison among different models. Due to the multiple options offered the software allows testing different conductivity models in a short time, even if a large set of conductivity data is being considered simultaneously. Then, the user can choose the best model making use of the graphical and statistical information provided in the output file. Although the program has been initially designed to treat conductimetric data, it can be also applied for processing data with similar structure, e.g. (P, c, T) or (P, c1, c2, T), being P any appropriate transport, physical or thermodynamic property.