Determination of gibberellic acid in fermentation broth and commercial products by micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC) was developed as a method for quantitative determination of gibberellic acid (GA3) in fermentation broth and commercial products, using 25 mM disodium tetraborate as a buffer at pH 9.2 and 100 mM sodium dodecyl sulfate as a micellar phase. The baseline resolution (Rs of GA3 from other compounds in fermentation broth was achieved with Rs > 2.5. The addition of methanol or acetonitrile in the MEKC buffer did not give a better resolution. Advantages of this MEKC method include high accuracy and precision and no sample preparation except for dilution and filtration.     

Determination of the fungicide validamycin A by capillary zone electrophoresis with indirect UV detection

Validamycin A, a main component of the antibiotic validamycin complex, which is widely used to control sheath blight disease of rice plants, can be determined by capillary zone electrophoresis with indirect UV detection. The influence of various separation conditions including background electrolyte and modifier concentration, pH, and voltage was investigated. By using 10 mM aminopyrine-2 mM ethylenediaminetetraacetic acid at pH 5.2 as the carrier electrolyte, high efficiency separation of validamycin A was achieved with the number of theoretical plates up to 350000 plates/m. The linear range was across 3 orders of magnitude. The relative standard deviations for migration times and peak areas were less than 0.5 and 3.0%, respectively. The limit of detection for validamycin A was 1.0 microg/mL. The average recoveries ranged from 103.0 to 104.8%. This method has many advantages as compared with high-performance liquid chromatography and micellar electrokinetic capillary chromatography in the determination of commercial formulations.     

Rapid analysis of essential and branched-chain amino acids in nutraceutical products by micellar electrokinetic capillary chromatography

A rapid method for the analysis of dansylated essential and branched-chain amino acids (BCAAs) by micellar electrokinetic capillary chromatography (MECC) is reported. Optimization of analytical conditions has been carried out, evaluating the influence on the performance of several parameters such as sodium dodecyl sulfate (SDS) concentration in the running electrolyte, temperature, and voltage. The effect of the addition of small amounts of isobutanol to the electrolyte has also been investigated. The best separation in the shortest time with a 37 cm capillary was obtained employing a 20 mM Borax buffer (pH 9.1) + 70 mM SDS at 25 degrees C and 20 kV. Under these conditions a mixture of nine essential amino acids was analyzed in 7 min, while separation of BCAAs occurred in less than 4 min. Using a shorter capillary (20 cm to the detector), the BCAA separation was performed in only 2.5 min. The method was applied to the quantitative analysis of amino acids in three commercial nutraceutical preparations. Assessment of analytical performance in terms of precision, linearity, and limit of detection has also been reported.     

Enantioseparation of isoxanthohumol in beer by hydroxypropyl-gamma-cyclodextrin-modified micellar electrokinetic chromatography

Chiral resolution of isoxanthohumol (IX) in beer samples was performed by hydroxypropyl-gamma-cyclodextrin-modified micellar electrokinetic chromatography. The optimum running conditions were found to be 20 mM phosphate buffer (pH 7.0) containing 45 mM hydroxypropyl-gamma-cyclodextrin and 100 mM sodium dodecyl sulfate with an effective voltage of +20 kV at 20 degrees C using direct detection at 210, 295, and 370 nm. IX was detected in 12 beer samples and the total levels of (+)- and (-)-IX ranged from 0.15 to 1.4 mg/L. But the amount of xanthohumol (XN) was below the detection limit (0.017 mg/L). Each level of (-)-IX was almost the same as that of (+)-IX, suggesting that IX was a racemic mixture in these beer samples. The racemization of IX in beer could be attributed to the production of a racemic mixture from XN during boiling and to the fact that IX enantiomers were easily interconverted.     

Fast determination of catechins and xanthines in tea beverages by micellar electrokinetic chromatography

Antioxidant properties and stimulating effects of green tea are related to its content of cathechins and xanthines; tea quality evaluation is based on organoleptic tests and on the presence of those components. In this work, by a MEKC method, eight cathechins and three xanthines were quantified in some tea-based beverages. The best separation was realized using a phosphate-borate running buffer, with sodium dodecyl sulfate as micellar agent. A 40 cm capillary, a temperature of 29 degrees C, a voltage of 30 kV, and UV detection at 200 nm were used. The method showed a very good sensitivity (limit of detection ranging from 0.0011 to 0.0051 microg/mL) and was applied to real tea samples to characterize their antioxidant content. Statistical studies were performed and showed a satisfactory reliability of the data.     

Analysis of reducing carbohydrates by reductive tryptamine derivatization prior to micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography method for determination of low molecular weight carbohydrates (dp 1-2) with an unbound carbonyl group as in aldoses or other reducing carbohydrates has been developed. Reductive amination of aldoses on the carbonyl group using tryptamine introduced a chromophor system to the carbohydrates enabling their sensitive UV detection at 220 nm and identification based on the indole group using diode array detection. Twelve carbohydrates including pentoses (d-ribose, l-arabinose, and d-xylose), hexoses (d-glucose, d-mannose, and d-galactose), deoxy sugars (l-rhamnose and l-fucose), uronic acids (d-glucuronic acid and d-galacturonic acid), and disaccharides (cellobiose and melibiose) are included in the study, using d-thyminose (2-deoxy-d-ribose) as the internal standard. Detection of all 12 carbohydrates is performed within 30 min. Linearity with correlation coefficients from 0.9864 to 0.9992 was found in the concentration range of 25-2500 micromol/L for all carbohydrates; the relative standard deviation on the migration times was between 0.27 and 0.80 min, and limits of quantification and limits of determination were in the picomole range.     

Determination of monoterpene hydrocarbons and alcohols in Majorana hortensis Moench by micellar electrokinetic capillary chromatographic

Micellar electrokinetic capillary chromatography was used to determine the essential oils obtained by steam distillation of different samples of marjoram (Majorana hortensis Moench) dried leaves and flowers. The electrophoretic method consisted of a running buffer of 10 mM NaH2PO4, 6 mM Na2B4O7, 50 mM SDS, 7 mM gamma-cyclodextrin, and 10% acetonitrile, adjusted to pH 8.0 by the addition of 0.1 M H3PO4. The following monoterpene hydrocarbons and alcohol compounds were extracted from real samples and determined by the method proposed: alpha-pinene, gamma-terpinene, alpha-terpinene, terpinolene, p-cymene, linalool, alpha-terpineol, and terpinen-4-ol. The most prominent component of dried leaves, flowers, and commercial samples was terpinen-4-ol in four of the samples analyzed; only in one sample was alpha-terpineol present as the major compound.     

Determination of tetracycline and streptomycin in mixed fungicide products by capillary zone electrophoresis

A method of capillary zone electrophoresis (CZE) was used to determine tetracycline and streptomycin content in commercial agriculture products. The results indicated that this method was capable of analyzing the mixed fungicide in formulated products with instrument detection limit (IDL) of 0.50 microg/mL and a method detection limit (MDL) of 0.52 microg/mL for tetracycline, and IDL of 1.00 microg/mL and MDL of 1.22 microg/mL for streptomycin. Precision expressed by relative standard deviation (RSD) ranged from 1.44 to 4.37% of tetracycline and 1.00 to 4.20% of streptomycin. Recoveries were in the region of 98.2-102.5% for tetracycline and 95.3--103.0% for streptomycin. The low detection limit, the low RSD values, and the high percentage of recovery confirmed that the CZE technique is a sensitive and selective method. And the CZE method can analyze both tetracycline and streptomycin at the same time without complicated extraction and further derivative reaction.     

Determination of nabam fungicide in crops by liquid chromatography with postcolumn reaction detection

The ethylenebisdithiocarbamate (EBDC) fungicide, nabam, was determined in several crop matrixes using liquid chromatography with postcolumn reaction detection. After separation by micellar liquid chromatography, nabam (EBDC sodium salt) was acid hydrolyzed to ethylenediamine and fluorigenically labeled with o-phthalaldehyde-mercaptoethanol (OPA-MERC). Standard curves were linear from the detection limit of ca 1 ng to 1000 ng. Nabam was recovered in high yield (89 plus or minus 7.7%) over a range of concentrations (0.1 to 20 ppm) from fortified samples of papaya, lettuce, cucumber, spinach, and applesauce using a simple extraction method. Efforts to convert the more popular EBDC fungicides, maneb and mancozeb, to nabam are discussed.     

Application of micellar electrokinetic capillary chromatography to the analysis of uncharged pesticides of environmental impact

A test mixture of five pesticides and metabolites (naphthalene acetamide, carbaryl, 1-naphthol, thiabendazole, and carbendazime) has been investigated by capillary electrophoresis with an ultraviolet diode array detector. These compounds were separated in <10 min by micellar electrokinetic capillary chromatography (MEKC). MEKC was performed in 30 mM ammonium chloride/ammonia buffer (pH 9.0) containing 15 mM sodium dodecyl sulfate. The lowest detection limit was obtained for the insecticide carbaryl (0.22 microg mL(-)(1)) and the highest for its metabolite 1-naphthol (1.13 microg mL(-)(1)). This method was applied to the analysis of the pesticides in cultivated vegetables such as cucumbers, which were extracted with a liquid-liquid extraction procedure, obtaining recovery percentages ranging from 90.1 to 110.2%.     

Analysis of antioxidants from orange juice obtained by countercurrent supercritical fluid extraction,using micellar electrokinetic chromatography and reverse-phase liquid chromatography

Antioxidants from orange juice were determined by the combined use of countercurrent supercritical fluid extraction (CC-SFE) prior to reverse-phase liquic chromatography (RP-LC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published LC procedure, both using diode array detection. The characterization of the different antioxidants was further done by LC-mass spectrometry. Advantages and drawbacks of LC and MEKC for analyzing the antioxidants found in the different orange extracts are discussed. Although LC yields higher peak area and slightly better reproducibility than MEKC, the latter technique provides information about the CC-SFE extracts in analysis times 7 times faster than by LC. This analysis advantage can be used for the quick adjustment of CC-SFE conditions, thus providing a fast way to obtain orange fractions of specific composition.     

Fast determination of Sudan dyes in chilli tomato sauces using partial filling micellar electrokinetic chromatography

A new method based on partial filling micellar electrokinetic chromatography (MEKC) for the quantitative determination of Sudan dyes (I, II, III, and IV) in chilli sauces is presented. The separation is achieved filling 25% of the capillary with a MEKC buffer composed of 40 mM NH(4)HCO(3), 25 mM sodium dodecyl sulfate, and 32.5% (v/v) acetonitrile (ACN). The rest of the capillary is filled using a capillary zone electrophoresis (CZE) buffer composed of 40 mM NH(4)HCO(3) and 32.5% (v/v) ACN. Under optimized conditions, the azo dyes are baseline separated in less than 8 min with limits of detection ranging from 0.57 to 0.71 μg mL(-1) (S/N > 3). Using an internal standard, the repeatability of the quantitative determination is improved almost four times. The applicability of the method for rapid screening and determination of Sudan dyes is corroborated by analyzing spiked chilli sauce samples with recoveries from 85 to 99%. The reported conditions are demonstrated to be compatible with mass spectrometry detection.     

Separation of food grade antioxidants (synthetic and natural) using mixed micellar electrokinetic capillary chromatography.

A capillary electrophoretic method, for the determination of antioxidants present in food, has been developed using mixed micellar electrokinetic capillary chromatography. The buffer consists of sodium cholate (40 mM), sodium dodecyl sulfate (15 mM), 10% methanol, and 10 mM borate at pH 9.3. A separation was obtained for nine antioxidants (synthetic and natural) commonly found in food. High-performance liquid chromatography and capillary electrophoresis were applied to the analysis of sesame oil and wine. Ascorbic acid was identified in wine.     

Determination of oxytetracycline in premixes and veterinary products by liquid chromatography

A liquid chromatographic method for the assay of oxytetracycline in premixes and veterinary products is described. Premix samples are extracted with acidified methanol, diluted with mobile phase, and filtered before chromatography on a C-8, reverse phase column. The assay method separates oxytetracycline from epioxytetracycline, tetracycline, and chlortetracycline. Total elution time for oxytetracycline is less than 5 min at 1.5 mL/min. Five spiked premix samples each of 2 and 50 g/lb had a coefficient of variation of 3.5 and 4.5% and a mean recovery of 99 and 104%, respectively. The results of premixes and veterinary products assayed by this method compared closely with those of the same assayed by the official AOAC microbiological method.     

Method of determination of nitrosamines in sausages by CO2 supercritical fluid extraction (SFE) and micellar electrokinetic chromatography (MEKC)

In this paper, the use of supercritical fluid extraction (SFE) and micellar electrokinetic capillary chromatography (MEKC) is proposed for the complete analysis of volatile nitrosamines in sausages. The extraction fluid used was CO2 and variables such as density, temperature of thimbles, extraction time, modifier, fluid flow, and kind of traps were investigated. Several experiments were carried out to obtain the most favorable conditions for analysis of volatile nitrosamines in sausages. The recoveries ranged from 21 to 82% for the five nitrosamines studied. The optimal condition of extraction was 0.2 g of sample fortified with 10 mg/kg, using dynamic extraction during 20 min and with adsorbent Florisil in the trap. The solvent selected for the elution of the analytes was methanol.     

Microbiological determination of neomycin in feeds and formulated products

An AOAC modified method is described for the microbiological assay of neomycin, which has been adapted to include complete feeds, supplements, premixes, liquids, oil suspensions, boluses, and antibiotic-impregnated paper. The method features a more sensitive standard response line with a monolayer plating system. The use of a buffered plating medium in place of the water-prepared medium results in a curve with less degree of slope, which allows for more accurate interpretation of the standard response. The feed extract diluent used for standard response line dilution, which is prepared from exposure of the feed extract fluid to pH changes, heat, and sodium hypochlorite, has been eliminated. The constant salt concentration diluent used for the preparation of standards is the same as the salt concentration of the sample extract solution to be tested. Results for 50 commercial complete feeds and 50 commercial premixes received over the last 5 years produced an overall mean recovery of 101% with a mean percent recovery range of 80-112%. A statistical analysis of these 100 commercial, complete feeds and premixes, ranging in concentration from 47 g/ton to 70 g/lb, indicates the assay has little, if any, concentration-related bias. Precision and accuracy of the method was supported by laboratory studies of 20 assays that produced a mean recovery of 101% and standard deviation of 3.     

Determination of tetracycline antibiotic residues in edible swine tissues by liquid chromatography with spectrofluorometric detection and confirmation by mass spectrometry

A sensitive and specific method is described for the simultaneous determination of oxytetracycline, tetracycline (TC), and chlortetracycline residues in edible swine tissues, by combining liquid chromatography with spectrofluorometric and mass spectrometry detection. The procedure involved a preliminary extraction with EDTA-McIlvaine buffer acidified at pH 4.0, followed by solid-phase extraction cleanup using a polymeric sorbent. The liquid chromatography analysis was performed with spectrofluorometric detection after postcolumn derivatization with magnesium ions. The limits of quantification were 50 microg/kg for muscle and 100 microg/kg for kidney tissues. The recovery values were greater than 77.8% for muscle and 65.1% for kidney. The method has been successfully used for the quantification of tetracyclines in swine tissues samples. The selective liquid chromatography mass spectrometric analysis for confirmation of oxytetracycline in one positive swine muscle sample was made by atmospheric pressure chemical ionization (APCI). The APCI mass spectra of the TCs gave the protonated molecular ion and two typical fragment ions, required for their confirmation in single ion monitoring scan mode in animal tissues.     

Combined use of supercritical fluid extraction, micellar electrokinetic chromatography, and reverse phase high performance liquid chromatography for the analysis of antioxidants from rosemary (Rosmarinus officinalis L.)

Antioxidants from rosemary were determined by the combined use of supercritical fluid extraction (SFE) prior to reverse-phase high-performance liquid chromatography (RP-HPLC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published HPLC procedure, both with diode array detection. The characterization of the different antioxidants was further done by HPLC-mass spectrometry. Advantages and drawbacks of HPLC and MEKC for analyzing the antioxidants found in the different extracts are discussed. From the results it is concluded that HPLC renders higher peak area and is better in its reproducibility than MEKC; both techniques provide similar analysis time reproducibility. The main advantage of MEKC is its much higher separation speed, which is demonstrated to be useful for the quick adjustment of SFE conditions, allowing rosemary fractions of higher antioxidative power to be obtained quickly. Moreover, the possibilities of this approach for following the degradation of antioxidants are discussed.     

Determination of potassium guaiacolsulfonate by ion-pairing partition chromatography.

Potassium guaiacolsulfonate, a highly polar, acidic substance, is readily eluted by chloroform from a pH 4.5 Celite column in the form of its ion-pair with trihexylamine, thereby effecting facile separation from other pharmaceuticals. The sulfonic acid is then back-extracted into aqueous alkali and determined spectrophotometrically. Assay of standard solutions by this procedre averaged 100.44 plus or minus 0.81%. The method was applied successfully to 4 commercial cough preparations containing a variety of other drugs.