Comparison of a non-aqueous capillary electrophoresis method with high performance liquid chromatography for the determination of herbicides and metabolites in water samples.

A method of capillary electrophoresis (CE) for the determination of triazine herbicides and some of their main metabolites in water samples has been developed. The proposed CE method includes an off-line solid-phase extraction (SPE) procedure with LiChrolut EN sorbent coupled to a non-aqueous capillary electrophoresis (NACE) separation with UV detection. The target compounds were the chloro-s-triazines simazine, atrazine, propazine; the methyltio-s-triazines ametryn and prometryn and three main derivatives from the atrazine degradation products; namely, deethylatrazine, deethylhydroxyatrazine and deisopropylhydroxyatrazine. The analytical characteristics of the CE method are reported. The repeatability of the method was studied considering the different steps of the method separately in order to determine the contributions of each step to the total variability of the method. The NACE-UV results are compared with those obtained with a high performance liquid chromatography with UV detection (HPLC-UV) method. The same off-line SPE procedure was applied to both techniques. The results obtained show that both methods afford the same results in the analysis of surface and drinking water samples, with a level of significance regarding the F- and t-tests greater than 0.05 in all the cases. The detection limits in surface water samples were in the 0.04-0.32 microg l(-1) and 0.11-1.2 microg l(-1) ranges for the NACE-UV and HPLC-UV methods, respectively. The recoveries (spiked/found) were significantly 100% in all cases.

Pressurized liquid extraction in the analysis of food and biological samples.

Originally, the use of the pressurized liquid extraction technique (PLE) was mainly focused on the extraction of environmental pollutants present in soil matrices, sediments, and sewage sludge. However, more recently the distinct advantages of this technique are being exploited in diverse areas, including biology, and the pharmaceutical and food industries. The aim of the present review is to explore recent analytical applications of this extraction technique (PLE) in the extraction of contaminant compounds and matrix components in food and biological samples, placing special emphasis on the strategies followed to obtain a rapid, selective, efficient and reliable extraction process.

Prediction of the behaviour of organic pollutants using cloud point extraction.

A preconcentration study based on the cloud point phenomenon was carried out for a set of triazine herbicides, three of them chloro-substituted and three of them methylthio-substituted. Concentration factors and recoveries were calculated as function of the percentage of the non-ionic surfactant Triton X-114 employed. From these values, obtained from a cloud point extraction (CPE) procedure, the distribution coefficient between the Triton X-114 micelles and water, Kc, prior to CPE was calculated for each triazine and related to the corresponding octanol-water partition coefficient, Kow. In order to confirm the results obtained with the triazine herbicides, two sets of data from chemically different organic pollutants--organophosporous and chlorophenols--obtained from the literature were assessed, concluding that they display a similar behaviour to that of the triazine herbicides. This can be used to predict the CPE behaviour of other organic pollutants from their octanol-water partition coefficients. The Kc values were compared with the analyte concentration ratio in the surfactant-rich phase and aqueous phase (Ksa) with a view to obtaining a link between the analyte behaviour prior to and after cloud point extraction procedures.

Determination of herbicides, including thermally labile phenylureas, by solid-phase microextraction and gas chromatography-mass spectrometry.

A method for the determination of 10 herbicides, including thermally unstable compounds, has been developed. The method uses solid-phase microextraction (SPME) with a polyacrylate fibre. Separation, identification and quantification were accomplished with gas chromatography-mass spectrometry. The herbicides chosen belong to different chemical groups and were alachlor, atrazine, chlorotoluron, diclofop, diflufenicam, ethofumesate, isoproturon, linuron, terbutryn and trifluralin. In the present work we studied the chromatographic behaviour of three phenylureas as a function of the medium and injection mode employed. The compounds generated as a function of the solvent used in direct injection of the phenylureas (ethyl acetate, methanol and methanol-water) and those obtained when injection was accomplished using the polyacrylate fibre were determined. The results allow us to propose a method for the determination of stable and thermally unstable herbicides as long as a preconcentration step involving SPME is carried out. In the proposed method, the limits of detection varied between 0.02 microg/l for ethofumesate and 0.11 microg/l for chlorotoluron. The method was applied to the determination of these herbicides in surface and ground water samples, performing quantification by standard addition calibration. The contents of chlorotoluron and atrazine found were significantly equal to those obtained using HPLC after a preconcentration stepwith styrene-divinylbenzene sorbents.

Determination of fatty acids in the subcutaneous fat of Iberian breed swine by near infrared spectroscopy (NIRS) with a fibre-optic probe.

A near infrared spectrometer equipped with a standard 1210/210 bundle remote reflectance fibre-optic probe, with a 5×5 cm quartz window, was used for the determination of fatty acids in the subcutaneous fat of Iberian pigs. A comparative study was made of the determination of fatty acids (C14:0, C16:0, C18:0, C18:1, C18:2, C18:3, C20: 1, Σpolyunsaturated, Σmonounsaturated and Σsaturated) in samples of subcutaneous fat from Iberian pigs by direct application of the fibre-optic probe on samples of whole subcutaneous fat and with cam-lock cups, assessing extracts of total lipids with diethyl ether. The regression method employed was modified partial least squares (MPLS). Calibration of 157 samples, using the fibre optic probe, allowed determination of fatty acids in the following ranges: C14:0 (0.78-1.77), C16:0 (15.87-29.74), C18:0 (4.61-15.90), C18:1 (43.50-61.27), C18:2 (2.03-13.94), C18:3 (0.13-1.14), C20:1 (0.45-2.32), Σpolyunsaturated (2.31-14.82), Σmonounsaturated (47.37-65.62), Σsaturated (22.09-47.31), with corrected standard errors of prediction SEP(C) of 0.093, 0.56, 0.67, 0.94, 0.42, 0.10, 0.20, 0.46, 0.94, 0.83, respectively. The robustness of the method using the fibre-optic probe was tested in a slaughterhouse using 23 samples for external validation, giving multiple correlation coefficients (RSQ) for C14:0, C16:0, C18:0, C18:1, C18:2, C18:3 C20:1, Σpolyunsaturated, Σmonounsaturated, Σsaturated acids of 0.72, 0.94, 0.72, 0.79, 0.88, 0.55, 0.17, 0.88, 0.74, and 0.90, respectively, and a corrected standard error of prediction [SEP(C)] for these acids (%) of 0.11, 0.60, 0.84, 1.20, 0.77, 0.11, 0.30, 0.76, 1.21, and 1.18, respectively.

Analysis of vitamin E isomers in seeds and nuts with and without coupled hydrolysis by liquid chromatography and coulometric detection.

A method is described for the determination of vitamin E isomers [alpha-, (beta+gamma)- and delta-tocopherols] in seeds and nuts by reversed-phase HPLC with coulometric detection. Three methods of sample treatment were compared. The first method included alkaline hydrolysis, extraction of analytes from unsaponifiable and injection into the chromatographic system. In the second method, alkaline hydrolysis and later continuous membrane extraction of isomers were coupled with the HPLC system. The third method involved direct extraction of the analytes through a silicone membrane coupled on-line with the chromatographic system. The three methodologies used for the determination of vitamin E isomers in these samples afforded good results for alpha-tocopherol. However for (beta+gamma)- and delta-tocopherols the best results were obtained with the third method. The method without hydrolysis was the simplest one to carry out and analysis took no longer than 40 min from weighing of the samples. Accordingly, it is the method proposed.

Differentiation of dietary regimene of Iberian swine by means of isotopic analysis of carbon and sulphur in hepatic tissue.

The aim of this work was to differentiate the feed received by Iberian swine during fattening (acorns, feed) and their breed (Iberian or White) using analysis of the stable isotopes of carbon (δ(13)C) and sulphur (δ(34)S) in liver tissue samples. The results obtained in the determination of δ(34)S, using a procedure in which organic and inorganic sulphur are converted into BaSO(4) and the procedure that measures δ(34)S in samples of dried ground liver tissue were compared. Joint analysis of carbon (δ(13)C) and sulphur (δ(34)S) permits the differentiation of swine of different breeds receiving different diets (acorns or feed).

Automated analysis of vitamin E isomers in vegetable oils by continuous membrane extraction and liquid chromatography-electrochemical detection.

A rapid and automated method for the analysis of alpha-, gamma- and delta-tocopherols in vegetable oils is reported. Continuous extraction of vitamin E isomers from oil samples dissolved in Triton X-114 in the presence of methanol-hexane is achieved and coupled on-line with the chromatographic system. Using an acetic acid/sodium acetate buffer in a methanol-water (97:3) solution as the mobile phase, a C18 stationary phase and electrochemical detection in the coulometric mode, alpha-, gamma- and delta-tocopherol isomers can be successfully analyzed within 17 min. Thirteen commercially available oils of olive, sunflower, corn and seed mixtures were analyzed using 2,2,5,7,8-pentamethyl-6-chromanol as internal standard. The results obtained using three methodologies, one of them including classical sample treatment for liposoluble vitamin analysis, were in good agreement. To validate the proposed method, analysis of the only BCR Reference Material available, with a certified content of alpha-tocopherol (margarine CRM 122), was carried out using the automated methodology, the results found being in agreement with the certified value.

Detection of Chlamydia trachomatis in pregnant women by the Papanicolaou technique, enzyme immunoassay and polymerase chain reaction.

OBJECTIVE: To compare Papanicolaou staining, enzyme immunoassay (EIA) and the polymerase chain reaction (PCR) techniques for detecting Chlamydia trachomatis in pregnant women. STUDY DESIGN: Endocervical specimens were taken randomly from 125 pregnant women with or without symptoms. These women attended their first medical consultation at the Regional General Ignacio Zaragoza Hospital. Samples were analyzed for detection of C trachomatis. When results differed between tests, specimens were evaluated by direct immunofluorescence staining. RESULTS: The prevalence of chlamydial infection was 2.4%. The characteristics of patients positive for Chlamydia were: average age, 24 years; first sexual encounter at age 21 years, one partner and six to nine months of gestation. The sensitivity, specificity, accuracy, positive predictive values and negative predictive values were 100%, 99.18%, 99.20%, 75% and 100%, respectively, for Papanicolaou staining; 100%, 92.62%, 92%, 25% and 100% for EIA; and 100%, 100%, 100% and 100% for PCR. CONCLUSION: Both Papanicolaou staining and PCR were adequate for diagnosis of C trachomatis infection. EIA was not reliable and therefore is not recommended for use as a diagnostic technique in a pregnant population with low risk and low prevalence.

Micellar electrokinetic chromatography with bis(2-ethylhexyl)sodium sulfosuccinate vesicles determination of synthetic food antioxidants.

Capillary electrokinetic chromatography is suitable for the separation of mixtures of uncharged and charged solutes. In the present work the behavior of six synthetic food antioxidants--2[3]-tert.-butyl-4-hydroxyanisole, 2,6-di-tert.-butyl-p-cresol, tercbutylhydroquinone, 3,4,5-trihydroxybenzoic acid propyl ester, 3,4,5-trihydroxybenzoic acid octyl ester and 3,4,5-trihydroxybenzoic acid dodecyl ester--was studied in a capillary electrophoresis system using capillary electrokinetic chromatography with vesicles of the surfactant bis(2-ethylhexyl)sodium sulfosuccinate (AOT). Several studies aimed at calculating the critical aggregation concentration of the surfactant were conducted to check that under the conditions used the AOT was in a state of aggregation. Having checked the association shown by the surfactant, we then explored the greater or lesser capacity of the antioxidants to interact with this compound. We followed the evolution of the molecular absorption spectra of each of the antioxidants in the presence of the surfactant at different concentrations and the retention factors were calculated at different pH values. Additionally, in order to determine which species--anionic or neutral--was present at the pH of the buffer used (boric/borate), the pKa values in acetonitrile-water (20:80) were obtained. Resolution and quantification of the antioxidants demand optimization of the variables involved in the system, such as the percentage of acetonitrile, the concentration of AOT and boric/borate buffer, pH, voltage, etc. When this part of the study had been completed, calibrations were obtained for each of the antioxidants, obtaining good linear correlation coefficients in all cases. Finally, we propose a method that allows the resolution of the six most employed antioxidants in a capillary electrophoretic system in 15 min, using electrokinetic chromatography with AOT as the pseudostationary phase.

Analysis of pesticide residues in matrices with high lipid contents by membrane separation coupled on-line to a high-performance liquid chromatography system.

Separation through membranes coupled to an HPLC system was used as a technique for the analysis of pesticide multiresidues in samples with high lipid contents. As well as the usual procedure, in the proposed system it is possible to recirculate the sample through the membrane cell, which permits the extraction system to be applied to cases in which only a very small volume of sample is available. A procedure for pesticide multiresidue analysis in egg samples was developed as a representative example of the applicability of the proposed method. To accomplish this, the analytes (dichlorvos, dimethoate, propoxur, paraoxon, pirimicarb, atrazine, ametryne, terbutryne, azinphos-methyl, folpet) were subjected to prior extraction in a Soxhlet system, after which the extract was introduced into the membrane separation device coupled to the HPLC system. This procedure afforded clean chromatograms, hence considerably facilitating determination, and at the same time was efficient in removing macromolecular compounds. For egg samples, spiked at a concentration level of 0.750 mg/kg, recoveries ranged from 60 to 98%. The detection limits varied from 0.018 mg/kg for dichlorvos to 0.002 mg/kg for atrazine.

Determination of triazine herbicides in natural waters by solid-phase extraction and non-aqueous capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) in an organic medium was used to analyse triazines at sub-ppb concentration levels in natural waters after a preconcentration step using conventional C18 cartridges and new Oasis HLB devices. With both sorbents, satisfactory results were obtained on analysing deionized water. However, on analysing natural waters, both sorbents showed very different types of behaviour. The different variables affecting the elution of both sorbents were studied, resulting in the choice of Oasis HLB as the most suitable for later separation by CZE in non-aqueous medium. Combination of a preconcentration step with electrokinetic injection revealed that capillary electrophoresis with simple UV detection can also be used satisfactorily for the quantification of micropollutants in natural waters. The detection limits obtained varied between 0.01 and 0.05 microg l(-1), depending on the type of matrix analysed. The day-to-day precision varied between 0.9% and 2.3%, expressed as the relative standard deviation.

Cloud point extraction as a preconcentration step prior to capillary electrophoresis.

Cloud point extraction was applied as a preconcentration step prior to capillary electrophoresis. The behavior of a surfactant-rich micellar phase injected into a capillary electrophoresis system was studied using different separation modes: micellar electrokinetic capillary chromatography and capillary zone electrophoresis (CZE). A problem that appeared on introducing a surfactant-rich phase into a bare fused silica capillary was that the surfactant was adsorbed onto the wall of the capillary, leading to a marked loss of efficiency and reproducibility both in the migration times and in the areas of the electrophoretic peaks. The use of cetyltrimethylammonium bromide dynamically coated capillaries afforded reproducible results, although the half-life of the capillary was short. The most satisfactory results were obtained by using nonaqueous media in the CZE mode, thus avoiding surfactant adsorption. Other parameters related to the composition of the injection medium were also studied to optimize the electrophoretic behavior of the analytes and the sensitivity of the determination. The optimized procedure was applied to the determination of triazines in tap and river water samples.

Use of isotope analysis to characterize meat fromIberian-breed swine.

We describe a method based on isotope analysis (δ(13)C) for characterization and differentiation of Iberian pork meat as a function of the diet of the animal. Using adipose tissue, it is possible to classify unknown samples in group of animals designated "acorn-fed", "recebo=mixed-fed" and "feed raised" according the δ(13)C value obtained, from the calibration straight line of y=-22.12-0.35x, with a correlation coefficient r=0.982 and s=0.1‰; where y=δ(13)C and x=arrobas of acorn and range forage received by the animals during the fattening period. Stress should be placed on the economic and industrial importance of Iberian-breed swine because the market prices of Iberian swine products depends on the classification of the animals according to the type of feeding regimen which they are subjected to.

Capillary zone electrophoresis in nonaqueous solvents in the presence of ionic additives.

Capillary zone electrophoresis (CZE) in nonaqueous media and in the presence of ionic additives has been successfully applied to the determination of compounds that differ only slightly in their electrophoretic mobilities. Triazine herbicides of environmental interest were chosen as test compounds because they behave as very weak bases. CZE separation of these analytes (especially chlorotriazines) in aqueous solution is difficult due to the low pH required for their conversion into protonated cationic form (HA(+)). However, in mixed nonaqueous solvents, 50% (v/v) acetonitrile-methanol, the acid-base characteristics of these compounds are modified, yielding the protonated ionic species that is susceptible to migration when subjected to an electric field. A noteworthy increase in separation selectivity and resolution can be achieved by using ionic additives. Thus, in this mode of capillary zone electrophoresis, separation is based on ionic interactions between the charged analytes and the ionic additive present in the separation medium. These interactions contribute to enhancing mobility differences and to improving analyte separation. For the separation of chloro- and methylthiotriazines, 10 mM perchloric acid in 50% (v/v) acetonitrile-methanol and 20 mM SDS proved to be satisfactory, providing high resolution in short analysis times. The selectivity achieved was found to depend on the degree of association of the analyte with the ionic additive in the nonaqueous medium. This permits manipulation of the selectivity of the electrophoretic separations as a function of the type and concentration of the ionic additive and of the nature of the nonaqueous medium employed.

Determination of fat-soluble vitamins in yogurt by HPLC with electrochemical detection.

A method employing HPLC with electrochemical detection for the rapid and simultaneous determination of vitamins A, D(3) and E is described. The method uses a C-18 reverse phase column and 2.5 mM HAcO-NaAcO in methanol-water (99:1, v/v) solution as the mobile phase. The compounds are quantified using amperometric detection with a glassy carbon electrode at a potential of + 1300 mV (vs. Ag/AgCl) and the results are compared with those obtained using UV detection at a wavelength of 280 nm. The method was successfully applied to the analysis of vitamins A, D(3) and E in yogurt samples. After saponification, fat-soluble vitamins were extracted and the methanolic solution of the extracts was injected directly into the chromatographic system, avoiding the clean-up step which is necessary when no electrochemical detection is used. Good recovery percentages were obtained.

Determination of vitamins A, E and K1 in milk by high-performance liquid chromatography with dual amperometric detection.

A high-performance liquid chromatographic method with dual-electrochemical detection (HPLC-DEC) for the simultaneous determination of vitamins A, E and K1 is described. Separation was carried out using a C18 reversed-phase column and 2.5 mmol l-1 acetic acid-sodium acetate in methanol-water (99 + 1, v/v) as the mobile phase. The compounds eluted with good resolution in the above mentioned order within about 20 min and were quantified by dual-amperometric detection with a glassy carbon electrode at -1100 mV (E1) and + 700 mV (E2) (versus Ag/AgCl). This reductive-oxidative detection mode gave reproducible results and the detection limits were of the order of 0.06, 0.19 and 3.1 ng for vitamins A, E and K1, respectively. The HPLC-DEC method was successfully applied to the analysis of vitamins A, E and K1 in liquid cows' milk and infant-formula powdered-milk samples after applying alkaline hydrolysis of the fatty material and extraction of the vitamins with hexane for the analysis of vitamins A and E. In the case of vitamin K1 it was necessary to carry out enzymic hydrolysis, since this vitamin is degraded in basic medium, followed by cleaning with a Sep-Pak silica cartridge to isolate it from the other vitamins. Good recoveries (between 81 and 106%) were obtained.

Directly coupled sample treatment-high-performance liquid chromatography for on-line automatic determination of liposoluble vitamins in milk.

We developed an on-line system for the determination of liposoluble vitamins A, D3 and E in milk, both liquid and in powder form, using an automated sample treatment system coupled to chromatographic determination. For this, C18 cartridges were used because of the strong capacity of this material for the extraction and preconcentration of such vitamins, its ease in handling and the possibility of coupling it with automatic analysis systems. Alkaline hydrolysis of the samples was performed in an on-line system comprising two confluent channels through which the sample solution and alcoholic sodium hydroxide plus ascorbic acid flowed for a given period of time. A third channel merged with the other two to neutralize the solution before it arrived at the C18 cartridge. The latter, inserted into a loop with a six-port injection valve, retained the soluble vitamins. The vitamins were eluted with a stream of methanol and the eluate was automatically injected into the chromatographic system. Variables affecting the on-line system were optimized: sample size, flow-rate, preconcentration, washing and elution times, etc. The recoveries for powdered and liquid milk for the three vitamins assayed ranged between 80 and 105% (n = 10). Additionally, a day-to-day precision (10 days) of the method of 4.5% was obtained for the different vitamins.

Determination and speciation of arsenic in human urine by ion-exchange chromatography/flow injection analysis with hydride generation/atomic absorption spectroscopy.

A flow injection-hydride generation/atomic absorption spectroscopic method for the measurement of total urinary arsenic and for the selective determination of inorganic arsenic, monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) was developed. Destruction of the organic matrix is necessary to measure total arsenic content of urine samples. Digestion of this matrix with HNO3-H2SO4-H2O2 is described. The separation of inorganic, monomethylated, and dimethylated arsenic compounds in urine was performed with ion-exchange chromatography on AG 50 W-X8 resin. Detection limits of 2 ppb for each arsenic form and of 3 ppb for total arsenic in urine analyzed after mineralization were found. Recoveries in triplicate urine samples spiked with 10 ppb inorganic arsenic, 20 ppb MMAA, and 40 ppb DMAA were 93, 91, and 85%, respectively. The precision (relative standard deviation) of the method obtained in 10 replicate analyses of urine samples spiked with arsenic metabolites varied from 3.2 to 4.6%. This method is applicable to urine samples in studies relating to arsenic exposure and its monitoring.

High-performance liquid chromatography with electrochemical detection for the simultaneous determination of vitamin A, D3 and E in milk.

A high-performance liquid chromatographic electrochemical detection for the rapid and simultaneous determination of the vitamin A, D3 and E is described. The separation is carried out by using a C18 reversed-phase column and 0.1 M LiClO4 in methanol-water (99:1, v/v) as the mobile phase. The compounds are eluted with good resolution in the above order within about 15 min and are determined by amperometric detection with a glassy carbon electrode at +1050 mV (vs. Ag/AgCl). The method gave reproducible results and the detection limits were of the order of 0.07, 4 and 0.2 ng of vitamin A, D3 and E, respectively. The method was successfully applied to the determination of vitamin A, D3 and E in liquid cow milk and milk powder samples. After saponification, fat-soluble vitamins were extracted with hexane and a methanolic solution of the dried extract was injected directly into the chromatographic system, avoiding the clean-up step that is necessary for vitamin D3 when electrochemical detection is not used. Good recoveries were obtained.