On-line MSPD-SPE-HPLC/FLD analysis of polycyclic aromatic hydrocarbons in bovine tissues.

A fast method was optimized and validated for simultaneous trace determination of four polycyclic aromatic hydrocarbons: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in bovine tissues. The determination was performed by matrix solid-phase dispersion (MSPD) coupled on-line to solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with fluorescence detection (FLD). The sample was dispersed on C18 silica sorbent and then the on-line MSPD-SPE-HPLC/FLD method was applied. Several parameters were optimized: cleaning and elution sequences applied to the MSPD cartridge, the flow rate and dilution of extract used for SPE loading. The on-line method was validated over a concentration range of 0.1-0.6ngg-1 obtaining good linearity (r⩾0.998) and precision (RSD)⩽10%. Recovery ranged from 96 to 99% and the limits of detection were 0.012ngg-1. This methodology was applied to liver samples from unhealthy animals. The results demonstrate that MSDP-SPE-HPLC/FLD method provides reliable, sensitive, accurate and fast data to the food control.

Extraction and analysis of polycyclic aromatic hydrocarbons and benzo[a]pyrene metabolites in microalgae cultures by off-line/on-line methodology based on matrix solid-phase dispersion, solid-phase extraction and high-performance liquid chromatography.

This paper describes the development and validation of an analytical methodology to determine the presence of four PAHs: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in cultures of the green microalgae Selenastrum capricornutum. The metabolites of benzo[a]pyrene (BaP), 4,5-dihydrodiol benzo[a]pyrene, 9,10-dihydrodiol benzo[a]pyrene, 3-hydroxy benzo[a]pyrene and 9-hydroxy benzo[a]pyrene were also included. The methodology consisted of three parts: (1) separation of liquid media from biomass samples by centrifugation of pure cultures, (2) off-line extraction of analytes from biomass by a miniaturized matrix solid phase dispersion (MSPD) method and from liquid media by a solid phase extraction (SPE) method and (3) on-line SPE preconcentration and analysis of the MSPD and SPE extracts, separately, by high performance liquid chromatography with fluorescence detection (HPLC-FD). The off-line/on-line (MSPD/SPE-HPLC-FD) method was validated over a concentration range of 20-200 pg mg(-1) obtaining good linearity (r(2)>0.9912) and precision values measured as relative standard deviation (RSD)<5%, recovery values were in the range of (40-66%) and the limits of detection (LODs) ranged from 2 to 6.5 pg mg(-1). The off-line/on-line (SPE/SPE-HPLC-FD) method was validated over a concentration range of 5-120 pg mL(-1); r(2)>0.9913 and RSD<7.36%, recovery values were in the range of 38-74% and LODs ranged from 0.8 to 2.3 pg mL(-1). This methodology was applied to samples from cultures exposed to BaP at 5 ng mL(-1) with different exposure times (0.75, 1.5, 3, 6, 24 and 48 h). The analytical methodology was suitable for measuring the very low amounts of residual BaP and metabolites produced in bioassays. Results showed that some of the metabolites favored by the microalgae are the dihydrodiols. The microalgae cultures were able to decrease the BaP level in the liquid medium below the United States Environmental Protection Agency (USEPA) limit (<0.2 ng mL(-1)).

Determination of organophosphorus pesticides in bovine tissue by an on-line coupled matrix solid-phase dispersion-solid phase extraction-high performance liquid chromatography with diode array detection method.

A miniaturized method based on matrix solid-phase dispersion coupled to solid phase extraction and high performance liquid chromatography with diode array detection (MSPD-SPE-HPLC/DAD) was developed for the trace simultaneous determination of the following organophosphorus pesticides (OPPs) in bovine tissue: parathion-methyl, fenitrothion, parathion, chlorfenvinphos, diazinon, ethion, fenchlorphos, chlorpyrifos and carbophenothion. To perform the coupling between MSPD and SPE, 0.05 g of sample was dispersed with 0.2 g of C(18) silica sorbent and packed into a stainless steel cartridge containing 0.05 g of silica gel in the bottom. After a clean-up of high and medium polarity interferences with water and an acetonitrile:water mixture, the OPPs were desorbed from the MSPD cartridge with pure acetonitrile and directly transferred to a dynamic mixing chamber for dilution with water and preconcentration into an SPE 20 mm × 2.0 mm I.D. C(18) silica column. Subsequently, the OPPs were eluted on-line with the chromatographic mobile phase to the analytical column and the diode array detector for their separation and detection, respectively. The method was validated and yielded recovery values between 91% and 101% and precision values, expressed as relative standard deviations (RSD), which were less than or equal to 12%. Linearity was good and ranged from 0.5 to 10 µg g(-1), and the limits of detection of the OPPs were in the range of 0.04-0.25 µg g(-1). The method was satisfactorily applied to the analysis of real samples and is recommended for food control, research efforts when sample amounts are limited, and laboratories that have ordinary chromatographic instrumentation.

A method for the analysis of organophosphorus pesticide residues in Mexican axolotl.

A method based on matrix solid-phase dispersion (MSPD) was developed for quantitative extraction of three organophosphorus pesticides (OPPs) from the Mexican axolotl, Ambystoma mexicanum. The determination was carried out using high- performance liquid chromatography (HPLC) with diode array spectrophotometric UV detection (DAD). The MSPD extraction with octadecylsilyl (C18) sorbent combined with a silica gel clean-up and acetonitrile elution was optimised for chlorpyrifos, fenthion and methyl parathion. The method was validated, yielding recovery values higher than 90%. The precision, expressed as the relative standard deviation (RSD), was less than or equal to 6% in muscle samples at spiking levels of 10 and 5 ppm. Linearity was studied from 15 to 60 ppm for chlorpyrifos and fenthion, and from 7.5 to 30 ppm for methyl parathion. The limits of detection (LODs) were found to be less than or equal to 0.5 ppm. This method was applied to the analysis of samples from a chlorpyrifos-exposed axolotl, demonstrating its use as an analytical tool for toxicological studies.

Binding characteristics of bovine serum albumin encapsulated in sol-gel glasses: an alternative for protein interaction studies.

Silica glasses doped with 500-700 microg of bovine serum albumin were prepared by the sol-gel method; two pH conditions (pH 5 and 7) were assayed for protein encapsulation. Both biomaterials showed a highly porous structure, with pore sizes in the range 5-28 nm. Columns packed with the ground biogels were on-line coupled to a C18 HPLC column for evaluation of the entrapped protein binding properties using propranolol. Binding capacities (at saturation) were approximately 3.7 and 7.1 microg of propranolol (drug-protein molar ratios 1.4 and 2.7) for the biogels prepared at pH 5 and 7, respectively. The significant difference indicates increased albumin denaturation upon encapsulation at pH 5. A frontal analysis study was then performed in cartridges packed with biogel prepared at pH 7 to evaluate the protein interaction with naproxen at low concentrations (

Relative influence of the dissolved humic material on the solid-phase extraction efficiency of pesticides from environmental water.

The effect of organic matter on the solid-phase extraction (SPE) efficiency for pesticides belonging to different chemical groups (urea-derivatives, carbamates and triazines) and having different polarities, was simultaneously studied for the first time in pure and simulated water samples. SPE was carried out in precolumns packed with C18 silica or styrene-divinylbenzene copolymer PLRP-S phases on-line coupled to high performance liquid chromatography (HPLC) analysis. Retention factors in water (k'(W)) were estimated for 25 compounds and used for the calculation of the theoretical breakthrough volume (Vb(T)) in pure water. Experimental breakthrough volumes (Vb(E)) were first determined using purified and deionized water as the matrix for selected compounds having Vb(T) < 500 mL; then, the same water with an added humic acid sodium salt (HA) at 0.4-5.6 mg/L of dissolved organic carbon (DOC) content, was used as the matrix for compounds having VbE < 500 mL in pure water. Several polar pesticides showed negative linear or logarithmic Vb(E) curves depending on HA content; their recoveries were also determined in environmental samples having low dissolved organic carbon values, between 0.5-6.4 mg/L. A similar behavior was observed for these compounds in simulated and natural water samples, where DOC concentration and the percolated volume (Vp) mainly determine the solute recoveries values. However, the variation of recoveries as a function of DOC content could be negative or null depending on the two examined conditions (Vp lower or larger than Vb(E) in pure water). Results demonstrated that breakthrough volume must always be considered to correctly interpret the participation of dissolved humic material on the SPE efficiency of organic micropollutants in water.