Preparation of organic monolithic columns in polytetrafluoroethylene tubes for reversed-phase liquid chromatography.

In this work, a method for the preparation and anchoring of polymeric monoliths in a polytetrafluoroethylene (PTFE) tubing as a column housing for microbore HPLC is described. In order to assure a covalent attachment of the monolith to the inner wall of the PTFE tube, a two-step procedure was developed. Two surface etching reagents, a commercial sodium naphthalene solution (Fluoroetch®), or mixtures of H2O2 and H2SO4, were tried and compared. Then, the obtained hydroxyl groups on the PTFE surface were modified by methacryloylation. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM) confirmed the successful modification of the tubing wall and the stable anchorage of monolith to the wall, respectively. Special emphasis was also put on the reduction of the unwanted effects of shrinking of monolith during polymerization, by using an external proper mold and by selecting the adequate monomers in order to increase the flexibility of the polymer. Poly(glycidyl methacrylate-co-divinylbenzene) monoliths were in situ synthesized by thermal polymerization within the confines of surface-vinylized PTFE tubes. The modified PTFE tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 20 MPa. The column performance was also evaluated via the isocratic separation of a series of alkylbenzenes in the reversed-phase mode. The optimized monolithic columns gave plate heights ranged between 70 and 80 µm. The resulting monoliths were also satisfactorily applied to the separation of proteins.

Single-pump heart-cutting two-dimensional liquid chromatography applied to the determination of fatty alcohol ethoxylates.

A setup for heart-cutting bi-dimensional liquid chromatography (LC-LC), constructed with a chromatograph provided with a single pump, an auxiliary 6-port 2-position valve (V6/2) and a column selector valve (VCS), is described. The possible ways of connecting the two valves for LC-LC, namely with V6/2 first followed by VCS and vice versa, are compared. The possibility of using the setups for preconcentration followed by the backwards transfer of the preconcentrated solutes to the detector or to a second column is also shown. The V6/2-first configuration for LC-LC was applied to the characterization of industrial fatty alcohol ethoxylates (FAEs) using UV-vis detection. For this purpose, the phthalates of the FAE oligomers were first obtained. The hydrocarbon series were separated along the 1st dimension by MeOH/water gradient elution on a C8 column at 60°C. Selected segments of the eluate were transferred to the 2nd dimension, where the EO oligomers of the isolated series were resolved by gradient elution with a complementary ACN/water mobile phase on a C8 column at 25°C. In addition, an average response factor of the hydrocarbon series of FAEs was proposed. To apply the factors, the average EO number of the series is first established by chromatographing one of the series along the 2nd dimension. Then, the factors are used to correct the peak areas of the isolated series which are obtained along the 1st dimension chromatogram, thus allowing the fast and accurate determination of the series in industrial FAEs. The method is particularly useful to characterize FAEs having large average EO numbers or constituted by mixtures of even and odd series.

Determination of non-ionic and anionic surfactants in industrial products by separation on a weak ion-exchanger, derivatization and liquid chromatography.

A method for the determination of priority surfactants, including fatty alcohol ethoxylates (FAE), alkylether sulfates (AES) and linear alkylbenzene sulfonates (LAS) is described. The samples were diluted with 50% methanol at pH 4 prior to solid-phase extraction on a weak anionic exchanger (WAX). The AES and LAS surfactant classes were retained, whereas the non-ionic components, including most FAE oligomers were eluted. After washing the WAX cartridge to remove cations, the remaining hydrophobic FAE oligomers were eluted using hot 80% methanol at pH 4 (at ca. 50°C). These two eluates were combined to constitute the non-ionic fraction. Then, AES and LAS were eluted using 80% MeOH with 3M NH3 followed by 95% methanol with 0.75M NH3. The two eluates obtained in basic media were combined to constitute the anionic fraction. The solvents were evaporated, the residues were dissolved in 1,4-dioxane, and esterification of the alcohols and transesterification of AES with phthalic anhydride was performed. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid and 0.1M NaClO4. The chromatogram of the non-ionic fraction showed the peaks of the resolved FAE oligomers. The chromatogram of the anionic fraction showed the peaks of the LAS homologues well resolved from those of the AES oligomers. The method was applied to laundry and industrial cleaners, shampoos and a shower gel.

Derivatization of hydroxyl functional groups for liquid chromatography and capillary electroseparation.

The derivatization reactions commonly used to enhance the analytical signal in the HPLC and CE determination of compounds with hydroxyl functional groups are revised. Focus is placed on the determination of compounds having aliphatic alcohols and phenols while lacking other reactive functional groups. The derivatization with acyl chlorides, organic anhydrides, isocyanates and a variety of other approaches, including oxidation of primary and secondary alcohols, sulfonation, esterification with carboxylic acids, and the use of azides, sulfonyl chlorides and other reagents having miscellaneous leaving groups, is covered. Reactions mainly addressed to introduce a chromophore or a fluorophore in the analyte molecule, or to introduce a charge to enhance sensitivity in MS detection, or to enable CE separation are included. Applications related to the industrial quality control of raw materials and manufactured products, and to the evaluation of their environmental impact are emphasized. The problem of the different response factors of the derivatives when complex mixtures of oligomers are derivatized, as occurs with non-ionic surfactants (mainly fatty alcohol ethoxylates) and soluble synthetic polymers, is discussed. Other applications related to the biochemical, biomedical, pharmaceutical, nutritional and toxicological fields are also reviewed. The reactions, the criteria to be applied to select the reagent, and the characteristics of the derivatives in relation to separation and detection, are discussed.

Determination of fatty alcohol ethoxylates and alkylether sulfates by anionic exchange separation, derivatization with a cyclic anhydride and liquid chromatography.

A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater.

Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products.

Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 µm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 µm or 3 µm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners.

Use of triacylglycerol profiles established by high performance liquid chromatography with ultraviolet-visible detection to predict the botanical origin of vegetable oils.

A method for the determination of triacylglycerols (TAGs) in vegetable oils from different botanical origins by HPLC with UV-vis detection has been developed. Using a core-shell particle packed column (C18, 2.6 µm), TAG separation was optimized in terms of mobile phase composition and column temperature. Using isocratic elution with acetonitrile/n-pentanol at 10 °C, excellent efficiency with good resolution between most of the TAG peak pairs, within a total analysis time of 15 min, was achieved. Using mass spectrometry detection, a total of 15 peaks, which were common to oils of six different botanical origins (corn, extra virgin olive, grapeseed, hazelnut, peanut and soybean) were identified. These peaks were used to construct linear discriminant analysis (LDA) models for botanical origin prediction. Ratios of the peak areas selected by pairs were used as predictors. All the oils were correctly classified with assignment probabilities higher than 95%.

Characterization and determination of poly(vinylpyrrolidone) by complexation with an anionic azo-dye and nonequilibrium capillary electrophoresis.

Using capillary zone electrophoresis in nonequilibrium conditions, the complexes of poly(vinylpyrrolidone) (PVP) with anionic azo-dyes dissociate following a first-order kinetics. Two peaks due to the remaining PVP-dye complexes and the equilibrium concentration of the free dye, plus an exponential region due to the dye liberated by the complexes during the electrophoretic run, are obtained. This behaviour was closely similar to that described in the literature for protein-probe and DNA-protein mixtures, upon application of the technique known as nonequilibrium capillary electrophoresis of equilibrium mixtures or NECEEM. Using Congo Red and Acid Blue 113, information about the maximal stoichiometry and average stability of the PVP-dye complexes was obtained. The procedure was also useful to predict the average molecular mass of PVP and to determine PVP in cleaning products and pharmaceutical preparations. By using an appropriate probe, the procedure should be also useful to characterize and determine many other synthetic or natural nonionic polymers, and to study polymer-probe interactions.

Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and linear discriminant analysis.

The enzymes present in raw materials of the cleaning industry (enzyme industrial concentrates) and in household cleaners were isolated by precipitation with acetone and hydrolyzed with HCl. The resulting amino acids were derivatized with o-phthaldialdehyde, and the derivatives were separated by HPLC. The peaks of 14 amino acids were observed using a C18 column and a multi-segmented gradient of acetonitrile-water in the presence of a 5 mM citric/citrate buffer of pH 6.5. Using either normalized peak areas (divided by the sum of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables, linear discriminant analysis models, capable of predicting the enzyme class, including proteases, lipases, amylases and cellulases, were constructed. For this purpose, both enzyme industrial concentrates and detergent bases spiked with them were included in the training set. In all cases, the enzymes of the evaluation set, including industrial concentrates, spiked detergent bases and commercial cleaners were correctly classified with assignment probabilities higher than 99%.

Determination of fatty alcohol ethoxylates with diphenic anhydride derivatization and liquid chromatography with spectrophotometric detection: a comparative study with other anhydrides.

A method for the determination of fatty alcohol ethoxylates (FAEs) using diphenic anhydride as derivatization reagent and RP-HPLC separation with UV-vis detection is presented and compared to derivatization with maleic, phthalic, and other cyclic anhydrides. With these anhydrides, the reaction rates increased when urea was added to the reaction medium, and the yields did not decrease when the samples contained moderate amounts of water. Gradient elution on a C8 column was performed with water/acetonitrile in the presence of 0.1% acetic acid. The use of diphenic anhydride was advantageous for both the chromatographic separation and the detection. Specifically, sensitivity at 200 and 220 nm was significantly better for the FAE diphenates, resulting in lower limits of detection at both wavelengths for the diphenates than for the maleates and phthalates (up to 30 and 4.3 times lower at 220 nm, respectively). Response factors for the diphenates decreased less than those of the phthalates when the number of ethylene oxide units, m, increased, reaching a constant value of ca. 0.62 when m>3. Peak symmetries and efficiencies were also better than those found for the other anhydrides. The optimized procedure was applied to the characterization and determination of FAEs in the effluent of a wastewater treatment plant and in sea water.

Characterization of the alcoholic fraction of vegetable oils by derivatization with diphenic anhydride followed by high-performance liquid chromatography with spectrophotometric and mass spectrometric detection.

Aliphatic and triterpene alcohols present in vegetable oils have been identified and determined by HPLC using UV-vis and MS detection after previous derivatization with diphenic anhydride. The alcoholic fraction was obtained by saponification, extraction and TLC (according to the European Union official procedure). Derivatization was performed in tetrahydrofuran in the presence of suspended grinded urea, which increases the reaction rate and yield. Derivatized extracts were chromatographed on a C8 column using gradient elution with acetonitrile/water mixtures containing 0.1% acetic acid, with UV-vis followed by negative-ion mode MS detection. Using linear discriminant analysis of the HPLC-MS data (extracted ion chromatograms), oil samples belonging to seven botanical origins (hazelnut, sunflower, corn, extra virgin olive, soybean, peanut and grapeseed) were correctly classified with excellent resolution among all the categories.

Determination of fatty alcohol ethoxylates by derivatization with phthalic anhydride followed by liquid chromatography with UV-vis detection.

The esterification of fatty alcohol ethoxylates (FAEs) with phthalic anhydride in 1,4-dioxane was studied. At 110 degrees C and in the presence of urea, which increased the reaction rate, esterification was completed in 60 min. The reaction yield did not decrease when the sample contained up to 50% water. For the non-ethoxylated alcohols, the UV-vis response factors of the derivatives were not significantly different from each other (f=1 for C12E0 as reference). The response factors decreased when the number of ethylene oxide units, m, increased, reaching constant values of ca. 0.40 and 0.65 for the n=8 and 18 series, respectively, when m>or=3. Using a C8 column and gradient elution with acetonitrile/water plus 0.1% acetic acid, FAEs in industrial mixtures and cleaning products were characterized. At column temperatures of 25 and 35 degrees C, homologous series with even values of n from 8 up to 20, were resolved up to m=14 and 18, respectively, and at 25 degrees C, series with consecutive even and odd values of n were resolved up to m=7. Within the series, superior resolution of the ethoxymers with large values of m was achieved; however, the elution order was reversed for the m=1 and 0 ethoxymers, which produced overlapping of a few peaks within the series. A simple procedure for the accurate prediction of the concentrations of all the ethoxymers in industrial samples was described. After preconcentration with C18 solid-phase extraction cartridges, FAEs were also characterized and quantified in environmental samples (river and sea water). The LODs were ca. 2 microM (S/N=3) in the injected solutions.

Prediction of the genetic variety of Spanish extra virgin olive oils using fatty acid and phenolic compound profiles established by direct infusion mass spectrometry.

The genetic varieties of Spanish extra virgin olive oils (Arbequina, Hojiblanca and Picual) were predicted by direct infusion of the samples in the electrospray ionization source of a mass spectrometer, followed by linear discriminant analysis of the spectral data. The samples were 1:50 diluted (v/v) with an 85:15 propanol/methanol (v/v) mixture containing 40mM KOH and infused. The abundances of the [M-H](-) peaks of the free fatty acids (7 peaks) and 28 phenolic compounds (20 peaks) were measured. Ratios of pairs of peak abundances were used as predictors in the construction of the linear discriminant analysis models. An excellent resolution between the three genetic varieties was achieved.

Determination of fatty alcohol ethoxylates by derivatisation with maleic anhydride followed by liquid chromatography with UV-vis detection.

The esterification of fatty alcohol ethoxylates (FAEs) by melting with maleic anhydride has been studied in the presence of urea. The reaction rate and yield of non-ethoxylated and ethoxylated alcohols increase largely when grinded urea is suspended in the reaction medium, a 100% yield being achieved at 80 degrees C in 15 min. The procedure is tolerant to the presence of large amounts of water. The UV-vis response factors of the derivatives of individual FAE oligomers vary little with the number of carbon atoms in the alkyl chain, n, and the number of ethylene oxide units, m. Derivatised samples of industrial FAE mixtures, cleaning products and river and sea waters were chromatographed on a C8 column of the fused-core type using gradient elution with acetonitrile/water mixtures plus 0.1% acetic acid. The hydrocarbon series with even values of n from 8 up to 20, were well resolved using column temperatures in the 15-30 degrees C range. Superior resolution between the consecutive oligomers within the series was also obtained at large values of m; however, reversion of the elution order for the derivatives with m=1 and 0 hindered direct quantitation of a few oligomers at low m values. Full resolution of all the oligomers was achieved by deconvolution with a genetic algorithm assisted by the unconstrained local method of Powell. After preconcentration with C18 solid-phase extraction cartridges, both river and sea water samples also showed the signature of the FAE oligomers. The evaluation of total FAEs in these samples was demonstrated.

Separation and determination of homologues of linear alkylbenzenesulfonates by nonaqueous capillary zone electrophoresis using alkylammonium salts in ethanol.

The separation of linear alkylbenzene sulfonates (LAS) by nonaqueous capillary electrophoresis (NACE) using negative polarity, and a buffer containing acetic acid and an alkylamine in nonaqueous ethanol, has been investigated. Several primary, secondary, and tertiary alkylamines with alkyl chains of different length were compared. The solutes travelled against the electroosmotic flow (EOF), and at the same time were braked by association with the alkylamine molecules or with the alkylammonium ions. The best resolution between adjacent LAS homologues (R approximately 2.1), partial isomer resolution in two peaks, and at the same time an excellent repeatability, was obtained with a small dipentylamine excess over the acetic acid. When the buffer concentration increased, resolution between the homologues increased slightly (R approximately 2.4), and a different isomer group was partially separated. A background electrolyte (BGE) containing 10 mM acetic acid and 20 mM dipentylamine to separate and quantify the homologues within 25 min is recommended. The isomer peak profile with up to three peaks can be estimated using this buffer and another one with 80 mM acetic acid and 90 mM dipentylamine. The former BGE was used to determine LAS in liquid and powder laundry detergents. The detection limit for the determination of total LAS in these products was 2.5 microg mL(-1), and the peak area and migration time interday repeatabilities were below 4.3 and 2.8%, respectively.

Determination of alkylphenol ethoxylates by micellar electrokinetic chromatography with bile salts.

Octyl- and nonylphenol ethoxylates (OPEs and NPEs) with different numbers of ethoxy units (average values: n = 10 and N = 40 for OPEs, and n = 10 for NPEs) were separated by micellar electrokinetic chromatography under positive polarity using an 80 mM borate buffer of pH 8.5 containing sodium deoxycholate (SDC) or sodium cholate (SC). When sodium dodecyl sulfate (SDS) was added to the background electrolyte (BGE) in the absence of the bile salt, a single peak at a migration time longer than that of the EOF was obtained. Substituting the SDS by a bile salt, the homologues were resolved. At the same bile salt concentration, resolution between the homologues was higher with SDC than using SC. Optimum resolution between consecutive homologues was obtained with 50 mM SDC. In the presence of low or moderate amounts of acetonitrile or n-propanol, the background line improved significantly, whereas resolution may increase or decrease slightly. We propose a procedure for the determination of OPEs and NPEs with optimum resolution between the homologues as well as a modified procedure with improved selectivity for the single-run determination of other absorbing nonionic, cationic, and anionic (such as linear alkylbenzene sulfonates) surfactants in industrial and household cleaning products and its application to a variety of samples. The detection limit was ca. 28 microg x mL(-1) of total NPE (n = 10), and peak area repeatabilities at 50 microg x mL(-1) were 1.7% (intraday) and 5.6% (interday).

Determination of cationic surfactants by capillary zone electrophoresis and micellar electrokinetic chromatography with deoxycholate micelles in the presence of large organic solvent concentrations.

Mixtures of the cationic surfactants benzalkonium chloride (BKC) and cetylpyridinium chloride (CPC) were quickly resolved and reproducibly and reliably determined by using background electrolytes (BGEs) containing 80 mM borate, pH 8.5, bile salts and large concentrations of an organic solvent. When the bile salt is present, the separation mechanism changes from capillary zone electrophoresis (CZE) to a mixed micellar electrokinetic chromatography (MEKC)-CZE, with predominant MEKC interactions, which lead to an excellent resolution of all the solutes, including the C12-C18 homologues of BKC and CPC. A BGE containing 50 mM sodium deoxycholate and 30% ethanol for an extreme resolution, or 20% tetrahydrofuran for an adequate resolution within a much shorter analysis time, is recommended. The procedure was applied to the determination of the surfactants in industrial and household formulations, with excellent resolution between the homologues, detection limits of a few microg ml(-1) and reproducibilities below 2%.

Determination of cow's milk in non-bovine and mixed cheeses by capillary electrophoresis of whey proteins in acidic isoelectric buffers.

An improved method for the determination of cow's milk in non-bovine cheese is reported: electrophoresis of whey proteins in acidic, isoelectric buffers. Two background electrolytes (BGEs) have been tested: (i) 50 mM iminodiacetic acid (pH=isoelectric point=2.30 at 25 degrees C), 0.5% hydroxyethylcellulose, 0.1% Tween 20 and 6 M urea (apparent pH 3.1), E=300 V/cm, for the separation of alpha-lactalbumins (alpha-LAs); (ii) a BGE with the same composition, but supplemented with 10% Tween 20, E=450 V/cm, for the fractionation of beta-lactoglobulins (beta-LGs). Surfactants have a discriminating effect on the retention behaviour of the bovine alpha-LA and beta-LG proteins, owing to the different strength of the protein-surfactant association complexes, and are needed for separating these two proteins from small peaks in the electropherograms generated by degradation of casein during cheese ripening. Novel equations are given for deriving the ratio of the area (or height) of bovine alpha-LA, or beta-LG, to the area (or height) of ovine or caprine alpha-LA or beta-LG (such ratios being typically used to determine the percentage of cow's milk in dairy products), since previous equations had marked drawbacks, such as non-linearity of the plots with increasing slopes at high cow's milk percentages, and too broad confidence limits at high cow's milk contents, where the peak area (or height) ratio tends asymptotically to infinite. With the novel procedures reported, contents of cow's milk as low as 1% can be quantified in goat's and ewe's cheeses. The present protocols give lower detection limits, are cheaper and more rapid than any other methodology reported in the literature, and can be easily applied to the routine quality control of binary and ternary cheeses.

Determination of cow's milk and ripening time in nonbovine cheese by capillary electrophoresis of the ethanol-water protein fraction.

A novel method is reported for analyzing adulteration of goat and ewe cheeses with cow's milk: capillary zone electrophoresis (CZE) in isoelectric, acidic buffers (50 mM imino diacetic acid, IDA, pH = pI 2.3). The cheese samples were extracted with a 20:80 v/v ethanol-water mixture in presence of 3 M urea and 1% beta-mercaptoethanol for 1 h. After centrifugation and lipid extraction, the samples were dissolved in 50 mM IDA, 6 M urea and 0.5% hydroxyethyl cellulose and analyzed by CZE at 700 V/cm. A total of 18 characteristic peaks were resolved among the three types of cheeses and 18 variables were defined as their respective areas. There was excellent similarity among the electrophoretic patterns obtained with cheeses of a given type of milk, while cheeses made with different types of milk were easily distinguishable. Most peaks were common to all cheeses, but the profile differed depending on the type of milk used. Principal component analysis, linear discriminant analysis, and partial least squares regression (PLS) were used for statistical analysis of the data obtained by CZE. In particular, by using PLS multivariate regression, the contents of cow's milk in presumably pure goat and ewe cheeses, as well as in binary and ternary mixtures, could be predicted with relative standard deviations of ca. 6-7%. In addition, the ripening time in goat and ewe cheeses could also be predicted.

Determination of thyreostatics in animal feed by micellar electrokinetic chromatography.

The determination of the thyreostatics 2-thiouracil, its derivatives (4-methyl-2-thiouracil, 4-propyl-2-thiouracil and 4-phenyl-2-thiouracil) and methimazole in manufactured dried animal feed by micellar electrokinetic chromatography (MEKC) is described. A 99 +/- 5% extraction yield at the 20 micrograms g-1 level (n = 8) was achieved by shaking the milled fodder with methanol-1 M NaOH (80 + 20). Aliquots of the supernatant were injected in a 75 microns x 33.5 cm uncoated silica capillary using pressure; separation was performed at 23 degrees C with 15 kV (positive polarity) in a background electrolyte (BGE) containing 40 mM sodium dihydrogenphosphate, 50 mM sodium dodecyl sulfate and 15 mM Tween 20 at pH 9. When the surfactants were added to the BGE, all the thyreostatics were well resolved and the fodder extracts showed lower backgrounds. The peaks appeared within the 2.25-5.2 min range with efficiencies in the 2.5 x 10(4)-8 x 10(4) range; methimazole appeared in the vicinity of the electroosmotic migration time. Calibration curves were linear within the studied range (20-200 micrograms ml-1, r2 > 0.998). Limits of detection in the extracts of spiked fodder samples ranged from 0.25 to 0.4 microgram ml-1, which corresponded to 0.6-1.0 microgram of drug per gram of fodder. Peak area repeatabilities were about 4% at the 20 micrograms ml-1 level.