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.

OPLS multivariate regression of FTIR-ATR spectra of acrylic paints for age estimation in contemporary artworks.

In recent years the interest and demand for artworks has been increasing as they are an interesting commercial investment due to their growing value in the market. This explains the increasing number of counterfeits dealing with artworks that has led to the development of new methodologies for their characterization. The material characterization of these types of works can provide relevant information for both authentication and conservation/restoration. Thus, in this study multivariate chemometric methods were applied to FTIR-ATR spectroscopic data for artwork dating purposes. To that end, ageing prediction models were developed for Liquitex® and Hyplar® brands. Paint samples containing the green synthetic organic pigment (PG7), were exposed to artificial ageing and analysed with FTIR-ATR and Py-GC/MS for characterization and monitorization of the main components (binding medium, pigment and additives). Although the OPLS ageing models were mainly characterized by the modifications suffered by both the binder and the surfactants, a universal model could not be developed due to differences in the modification trends of the different brands. The applicability of the OPLS modelling for artwork dating purposes was tested in artworks provided by internationally recognized contemporary Basque artists. For Liquitex® a significant correlation (p < 0.05) between natural and accelerated ageing could be established, in which approximately 50 h of accelerated ageing under the applied conditions were equivalent to one natural year. This correlation might have possible applications in the dating of artworks for up to at least 22 years. Thus, the study demonstrates that FTIR-ATR combined with chemometrics is a potential method for artwork dating and a valuable source of information about the chemical processes involved in paint ageing, which can be of great help in the conservation and restoration steps.

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.

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.

Quantitative determination of oxprenolol and timolol in urine by capillary zone electrophoresis.

A simple capillary zone electrophoretic method with UV detection has been developed for the quantitative determination of the beta-adrenoreceptor antagonists (beta-blockers) oxprenolol and timolol in human urine, preceded by a solid-phase extraction step. The electrophoretic separation was performed on a 78 cm x 75 microm I.D. fused-silica capillary (effective capillary length: 70 cm). The electrolyte consisted of a Na2B4O7-H3BO3 (50 mM), pH 9. The introduction of the sample was made hydrostatically for 20 s and the running voltage 25 kV at the injector end of the capillary. Photometric detection was used at a wavelength of 229 nm for oxprenolol and 280 nm for timolol. Under these conditions oxprenolol migrated at 4.76+/-0.05 min and timolol at 4.97+/-0.05 min. The solid-phase extraction methods were optimised for each beta-blocker and provided recoveries of 72.8% for timolol and 94.52% for oxprenolol. Good resolution from the endogenous compounds present in the urine matrix were achieved for both compounds. The method was applied to the determination of both beta-blockers in pharmaceutical formulations and urine samples obtained from hypertensive patients after the ingestion of a therapeutic dose (in a 24-h time interval after the ingestion). The quantitative results were compared with results previously obtained at our laboratories by HPLC and were found to be in good agreement. Good reproducibility, linearity, accuracy and quantitation limits (in urine) of 0.19 microg/ml for timolol and 0.20 microg/ml for oxprenolol were obtained, allowing the method to be applied to pharmacokinetic studies of these compounds.

k0-NAA, a valuable tool for reference-material producers.

The main concern of producers of certified reference materials (CRM) is the preparation of high-quality products with demonstrated homogeneity and stability, combined with a well established set of certified characteristics. CRM producers must, furthermore, comply with other constraints imposed by the ISO Guide 34: production processes, production control, and certification analyses should be performed by expert laboratories, using validated protocols documented in their respective quality assurance manuals; laboratory mean values and the corresponding "expanded" uncertainties, must be used for the determination of the certified values, as recommended by the ISO Guide to the Expression of Uncertainties in Measurements (GUM); and when possible, traceability of the certified value to the SI units, using appropriately validated and/or primary methods, must be ensured. k0-NAA, i.e. neutron activation analysis with k0 standardization, is one of the analytical techniques implemented at the Reference Material Unit of IRMM; it meets the first two requirements.

Determination of the pKa values of beta-blockers by automated potentiometric titrations.

The acid-base equilibrium constants of the beta-blockers atenolol, oxprenolol, timolol and labetalol were determined by automated potentiometric titrations. The pKa values were obtained in water-rich or water methanol medium (20% MeOH) to obviate the solubility problems associated with the compounds. The initial estimates of pKa values were obtained from Gran's method and then, were refined by the NYTIT and ZETA versions of the LETAGROP computer program. The resultant values were 9.4 (I = 0.1 M KCl, 20% methanol) for atenolol, 9.6 (I = 0.1 M KCl) for oxprenolol, 9.4 (I = 0.1 M KCl, 20% methanol) for timolol and 7.4 and 9.4 (I = 0.1 M KCl) for labetalol. The potentiometric method was found to be accurate and easily applicable. The operational criteria for applying the methodology are indicated.

Simultaneous determination of the beta-blocker atenolol and several complementary antihypertensive agents in pharmaceutical formulations and urine by capillary zone electrophoresis.

A simple capillary zone electrophoresis method is developed for the quantitation of the beta-blocker atenolol and the complementary antihypertensive agents bendroflumethiazide, amiloride, and hydrochlorothiazide in human urine samples. The electrophoretic separation is performed using a 78-cm x 75-micron-i.d. (70-cm effective length) fused-silica capillary. A borate buffer (pH 9) is used as running electrolyte. The sample is hydrostatically introduced for 20 s, and the running voltage is 25 kV at the injector end of the capillary. The analysis of urine samples requires the optimization of solid-phase extraction methods, achieving recoveries > or = 61% for all the drugs and good separation from the urine matrix. The method is successfully applied to the determination of these compounds in pharmaceutical formulations and in urine samples collected after the intake of Neatenol Diu (100 mg atenolol-5 mg bendroflumethiazide) and Kalten (50 mg atenolol-25 mg hydrochlorothiazide-2.5 mg amiloride). The method is validated in terms of reproducibility, linearity, and accuracy.

Quantitative determination of the beta-blocker labetalol in pharmaceuticals and human urine by high-performance liquid chromatography with amperometric detection.

A rapid and simple high-performance liquid chromatographic (HPLC) method with amperometric detection has been developed for the quantitation of labetalol in urine. The chromatography was performed at 30 degrees C using a reversed-phase column with a base deactivated silica stationary support and an alkylamide bonded phase (Supelcosil ABZ+Plus). A 5 mM acetate buffer (pH 4.5)-acetonitrile (70:30, v/v) mixture was employed as the mobile phase, pumped at a flow-rate of 1 ml/min. Sample preparation was carried out using a simple solid-phase extraction (SPE) procedure, and recoveries higher than 85% were achieved. The method was found to be accurate, precise (R.S.D lower than 8%), and sensitive enough (experimental quantitation limit of 20 ng/ml, detection limit 10 ng/ml) to be applied to doping analysis and pharmacokinetic studies in human urine. The method was applied to the determination of labetalol in pharmaceutical formulations and urine samples obtained from a healthy volunteer after the ingestion of a therapeutic dose of the drug, and the results obtained were in agreement with the pharmacokinetic data.

High-performance liquid chromatography with amperometric detection applied to the screening of beta-blockers in human urine.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of six beta-blockers: atenolol, nadolol, timolol, metoprolol, oxprenolol, and alprenolol. The chromatographic separation was performed using a mu Bondapack C18 column, a mobile phase of acetonitrile-water (40:60), containing 5 mM KH2PO4/K2HPO4 proved to be optimal at a 1.3 ml/min flow-rate, and a pH of 6.5. The temperature was optimized at 30 +/- 0.2 degrees C. The amperometric detector, equipped with a glassy carbon electrode, was operated at 1300 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/ml), obtaining relative standard deviations lower than 5% at ppm levels and lower than 10% at ppb levels, and quantitation limits ranging from 15 ppb to 500 ppb. The method was applied to the screening of beta-blockers in spiked urine samples, with a total elution time lower than 12 min, obtaining the best recoveries for timolol and metoprolol (never greater than 93%). These recoveries together with the low limits of quantitation achieved, allows its application to doping analysis in human urine.