Martha P. García de Llasera

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.

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)).

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.

Speciation and transformation pathways of chlorophenols formed from chlorination of phenol at trace level concentration

Trace organic precursors remaining in water after primary treatment can originate a variety of toxic disinfection by-products during chlorination. Therefore, knowledge of conditions leading to their persistence or transformation in chlorinated media is crucial for human health protection. Using phenol as model compound at trace level (50 ppb), the short term formation and degradation of chlorophenols (CPs) in plain water and buffered water (pH 4.8, 7 and 9) treated with typical chlorine doses (1-5 ppm) was investigated. Total phenol consumption and quantitative degradation of formed CPs occurred in ⩽5h with 5 ppm chlorine in plain water and alkaline buffer, and with 1 ppm chlorine in phosphate buffer of pH 7. The enhanced reactivity in this buffer was attributed to high ionic strength (0.18 M). On the contrary, phenol was only slowly transformed to monochlorophenols (MCPs) in acidic media. Analysis of phenol and CPs concentration profiles indicated the coexistence of two competing reaction pathways in neutral and alkaline conditions: 1) successive ortho-para chlorination of aromatic ring up to 2,4,6-trichlorophenol followed by ring cleavage, 2) direct oxidation of MCPs to rapidly degradable oxygenated aromatics (dihydroxybenzenes, benzoquinones). Ionic strength and pH had some influence on preferred pathway but chlorine dose was determinant.

On-line SPE chromatography with spectrophotometric diode array detection as a simple and advantageous choice for the selective trace analysis of benzo(a)anthracene degradation products from microalgae

In this work, a methodology based on on-line solid phase extraction (SPE) chromatography with spectrophotometric diode array detection was optimized and validated for the trace analysis of benzo(a)anthracene dihydrodiol degradation products from microalgae cultures 5,6-dihydrodiol, 8,9-dihydrodiol and 10,11-dihydrodiol. The two on-line methods for the constituents of the culture, an SPE/on-line SPE chromatographic method for liquid medium and a matrix solid phase dispersion (MSPD)/on-line SPE chromatographic method for biomass presented good linearity in the ranges of 0.5-47ngmL-1 and 2-80ngmg-1 of samples, respectively, with correlation coefficients r>0.99. The percent relative standard deviation (RSD%) values were ≤4.9%. For the liquid medium and biomass methods, the global recoveries were between 84% and 90% and between 67% and 78%, and the limit of detection LODs were ≤0.3ngmL-1 and ≤0.8ngmg-1 respectively. The methodology was applied to exposure bioassays, and for the first time the three metabolites were detected and quantified individually by their appearance in the biomass and when they were excreted into the liquid medium. The metabolite formed in the greatest amount was 10,11-dihydrodiol, and the maximum production of all metabolites was at 6h of exposure. This work contributes to the study of the degradation route of BaA, which has not been elucidated for microalgae until now.

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.