A link between lead and cadmium kinetic speciation in seawater and accumulation by the green alga Ulva lactuca.

In this work, studies on the bioaccumulation of Cd and Pb by Ulva lactuca at different sites of Gulf San Jorge (Patagonia, Argentina) are presented. Higher values of bioaccumulated Cd were found in Punta Maqueda - a site believed to serve as a control - in comparison to those in Punta Borja, a place highly exposed to urban and industrial activities. Consequently; the labile fractions of Cd and Pb in seawater were determined with a flow injection-preconcentration manifold interfaced to a graphite furnace-atomic absorption spectrometer (FI-GFAAS). The results obtained by kinetic speciation showed that the variable that correctly explains heavy metals accumulation in the alga, is the labile metal fraction in seawater. We propose to use an enhancement ratio - on the basis of the kinetically labile metal fraction - for calculation of the metal accumulated by the alga relative to its environment.

Simultaneous determination of free and conjugated bile acids in serum by cyclodextrin-modified micellar electrokinetic chromatography.

A simultaneous determination of 15 free and most conjugated forms of bile acids (BA) in serum using capillary electrophoresis is described. The optimized and validated method proposed in this work is straightforward and rapid, employing affordable equipment. A background electrolyte of 5 mM beta-cyclodextrin, 5 mM 2-hydroxypropyl-beta-cyclodextrin, 50 mM SDS and sodium borate-dihydrogen phosphate pH 7.0 with 10% of acetonitrile was used. The complete separation of 15 BA, not easily achievable with other methods, is performed in less than 12 min using a UV detector with good precision and accuracy. BA were extracted from pretreated serum samples using a C(18)-solid-phase extraction and the recovery values ranged from 65 to 107.8%. Limits of quantitation were between 0.58 and 3.2 microM. This method proved to be suitable to determine individual BA profiles which are more useful than total serum bile acids as indicators of metabolic disorders and hepatobiliary diseases.

Retention of bile salts in micellar electrokinetic chromatography: relation of capacity factor to octanol-water partition coefficient and critical micellar concentration.

The capacity factors of 16 anionic cholates (from six bile salts, including their glyco- and tauro-conjugates) were determined in a micellar electrokinetic chromatography (MEKC) system consisting of buffer, pH 7.5 (phosphate-boric acid; 20 mmol/l) with 50 mmol/l sodium dodecyl sulfate (SDS) as micelle former and 10% acetonitrile as organic modifier. The capacity factors of the fully dissociated, negatively charged analytes (ranging between 0.2 and 60) were calculated from their mobilities, with a reference background electrolyte (BGE) without SDS representing "free" solution. For comparison, the capacity factors were derived for a second reference BGE where the SDS concentration (5 mmol/l) is close to the critical micellar concentration (CMC). The capacity factors are compared with the logarithm of the octanol-water partition coefficient, log Pow, as measure for lipophilicity. Clear disagreement between these two parameters is found especially for epimeric cholates with the hydroxy group in position 7. In contrast, fair relation between the capacity factor of the analytes and their CMC is observed both depending strongly on the orientation of the OH groups, and tauro-conjugation as well. In this respect the retention behaviour of the bile salts in MEKC seems to reflect their role as detergents in living systems, and might serve as model parameter beyond lipophilicity.

Determination of inorganic cations by capillary ion electrophoresis in Ilex paraguariensis (St. H.), a plant used to prepare tea in South America.

A practical and economical capillary ion electrophoresis method with indirect UV detection at 214 nm was developed for determination of inorganic cations in plants of Ilex paraguariensis(St. H.) and their infusion known as mate tea, a very popular beverage in South America. A microwave digestion procedure was used to prepare the herbal plants, but the infusion was only diluted. The background electrolyte contained 6mM imidazole and 10mM alpha-hydroxyisobutyric acid, pH 4.0. The running voltage was 20 kV and temperature was 25 degrees C. K, Na, Ca, Mg, and Mn ions were quantitated, and linearity was demonstrated between 0.6 and 120 ppm. The results were in good agreement with those obtained by flame atomic absorption and emission spectrometry. Accuracy of the method was verified by comparison with Beech leaves CRM 100, a standard reference material. The high content of minerals and several oligoelements, especially Mn in mate tea, is considered to be of nutritional interest.

Determination of bile acids in pharmaceutical formulations using micellar electrokinetic chromatography.

A micellar electrokinetic chromatography method (MEKC) has been developed and validated for the determination of bile acids (BA) such as ursodeoxycholic acid (UDCA), dehydrocholic acid (DHCA) and deoxycholic acid (DCA) in pharmaceuticals for quality control purpose. The background electrolyte consisted of 20 mM borate-phosphate buffer containing 50 mM sodium dodecylsulfate (SDS), and acetonitrile as additive. UV detection was set at 185 nm. Selectivity, linearity, range, repeatability, intermediate precision and accuracy showed good results. Comparison of the values obtained by MEKC and HPLC methods were in close agreement.

Analysis of cis-trans isomers and enantiomers of sertraline by cyclodextrin-modified micellar electrokinetic chromatography.

In this work development, optimization and validation of a cyclodextrin-modified micellar electrokinetic chromatography (CD-modified MEKC) method is proposed to resolve separation of the sertraline hydrochloride and synthesis-related substances. Sertraline hydrochloride, the cis-(1S,4S) enantiomer form, is used as an antidepressant therapeutic agent. A buffer concentration composed of 20 mM sodium borate, pH 9.0 with 50 mM sodium cholate, 15 mM sulfated beta-cyclodextrin and 5 mM hydroxypropyl-beta-cyclodextrin was found to be the most suitable background electrolyte. Quantitation of the impurities at levels of 0.1% in different samples of the bulk drug was determined. A comparison of the results with those obtained by HPLC methodology was also accomplished. The method proved appropriate for testing the purity of sertraline hydrochloride in bulk drug.

Fecal bile acid excretion profile in gallstone patients.

Epidemiological studies have shown a positive association between cholesterol gallstones and colonic cancer. These two diseases may be somehow related with bile acids metabolic alterations. The aim of this study was to evaluate the profiles of fecal bile acid in gallstone patients, in order to estimate the quality and amount of fecal bile acids. A fecal bile acid profile of ten gallstone patients and ten controls was compared using high performance liquid chromatography. Total fecal bile acid excretion was significantly increased in gallstone patients compared with controls (692.7 mg/day (302.5-846.2) vs 165.7 mg/day (138.7-221.3), p < 0.01) as was the excretion of secondary free bile acids 562.9 mg/day (253.3-704.9) vs 99.9 mg/day (88.9-154.2), p < 0.01). Lithocholic and glycodeoxycholic acid percentages have also been found to show differences with controls of 55.4 (47.4-73.9) vs 24.6 (22.1-38.4) (p < 0.01) and 29.4 (3.3-41.7) vs 2.8 (1.0-3.8) (p < 0.03), respectively but deoxycholic acid has not shown differences between the two groups. Moreover, the percentage of ursodeoxycholic acid diminished significantly in gallstone patients (1.5 (1.0-2.8) vs 8.6 (6.0-10.39) (p < 0.001), and the decrease of chenodeoxycholic acid was also significant (20.0 (11.4-23.6) vs 8.9 (3.1-10.9) (p < 0.03) along with a rise in the ratios lithocholic/deoxycholic acids (1.8 (1.4-6.4) vs 0.9 (0.6-1.6) (p < 0.05) and glycine/taurine of deoxycholic acid (7.3 (4.1-46.6) vs 0.2 (0.1-0.5) (p < 0.01). In conclusion, we have observed a significant increase of total and secondary fecal bile acid excretion as well as a rise of LCA and GDCA percentages and a rise in the ratios of LCA/DCA and glycinet/taurine of DCA.

Capillary electrophoretic method for quality control of phospholipid products.

A practical, low cost capillary electrophoretic method for phospholipid products employed in pharmaceutical, cosmetic and food industries with quality control purposes is described. The phospholipid components of the samples were hydrolyzed prior to their electrophoretic separation. The carrier electrolyte to determine choline, ethanolamine and serine consisted of 5 mM imidazole, pH 3.0 using indirect UV detection at 214 nm. Inositol was quantified employing a buffer of 40 mM sodium borate, pH 10.0 at 40 degrees C and detection at 185 nm. Analytical parameters were evaluated and the results obtained of phospholipid composition in commercial samples from soybeans and animal sources showed acceptable values.

Monitoring the purity of a synthetic peptide by capillary electrophoresis: utilization of an on-line preconcentration method for improved separation and detection sensitivity.

To date, there has been a considerable amount of interest and success in the pharmaceutical industry in the discovery of drug targets and diagnostics utilizing peptides. The success of peptide pharmaceuticals has, however, been accompanied by some failures, both prior to entry and in the clinic. Progress has been made in various areas to improve the effectiveness of the final drug product. One major advance has been in the area of peptide synthesis and control of the purity of the peptide of interest. Recent advances in analytical instrumentation, including advances in capillary electrophoresis, have had a great impact on the ability to separate and detect low quantities of impurities and degradation products during the synthesis of a peptide drug. In this work, affinity capillary electrophoresis (ACE) was developed for the identification and characterization of a chemically synthesized peptide fragment of a snake toxin called fasciculin. The affinity capillary electrophoresis technology utilized in this study employed two powerful techniques coupled on-line for the direct and rapid determination of analytes in simple and complex matrices. The first technique, aimed for the nonselective extraction and concentration of one or more analytes of interest, utilizes a solid-phase analyte concentrator device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated target analyte(s), after elution from an analyte concentrator device. The on-line preconcentration step has proved valuable in terms of improving separation conditions as well as enhancing detection sensitivity values for the peptide fragment with a sensitivity increase ranging from 100- to 10,000-fold. Different types of analyte concentrator devices and a few binding-desorption conditions were tested. Bare and internal-wall-coated fused-silica capillaries were used. Comparative performance with HPLC in terms of selectivity and sensitivity is also discussed.

Fecal bile acid excretion profile in gallstone patients.

Epidemiological studies have shown a positive association between cholesterol gallstones and colonic cancer. These two diseases may be somehow related with bile acids metabolic alterations. The aim of this study was to evaluate the profiles of fecal bile acid in gallstone patients, in order to estimate the quality and amount of fecal bile acids. A fecal bile acid profile of ten gallstone patients and ten controls was compared using high performance liquid chromatography. Total fecal bile acid excretion was significantly increased in gallstone patients compared with controls (692.7 mg/day (302.5-846.2) vs 165.7 mg/day (138.7-221.3), p < 0.01) as was the excretion of secondary free bile acids 562.9 mg/day (253.3-704.9) vs 99.9 mg/day (88.9-154.2), p < 0.01). Lithocholic and glycodeoxycholic acid percentages have also been found to show differences with controls of 55.4 (47.4-73.9) vs 24.6 (22.1-38.4) (p < 0.01) and 29.4 (3.3-41.7) vs 2.8 (1.0-3.8) (p < 0.03), respectively but deoxycholic acid has not shown differences between the two groups. Moreover, the percentage of ursodeoxycholic acid diminished significantly in gallstone patients (1.5 (1.0-2.8) vs 8.6 (6.0-10.39) (p < 0.001), and the decrease of chenodeoxycholic acid was also significant (20.0 (11.4-23.6) vs 8.9 (3.1-10.9) (p < 0.03) along with a rise in the ratios lithocholic/deoxycholic acids (1.8 (1.4-6.4) vs 0.9 (0.6-1.6) (p < 0.05) and glycine/taurine of deoxycholic acid (7.3 (4.1-46.6) vs 0.2 (0.1-0.5) (p < 0.01). In conclusion, we have observed a significant increase of total and secondary fecal bile acid excretion as well as a rise of LCA and GDCA percentages and a rise in the ratios of LCA/DCA and glycinet/taurine of DCA.

Determination of related impurities of bile acids in bulk drugs by cyclodextrin-modified micellar electrokinetic chromatography.

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) method has been developed and validated for purity determination of two bile acids, ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA). Quantitation of related impurities such as lithocholic acid (LCA), chenodeoxycholic acid (CDCA), cholic acid (CA), and DCA in UDCA and CA in DCA was performed. A running buffer containing 20 mM borate-phosphate, 50 mM sodium dodecyl sulfate (SDS), 2.0 mM beta-cyclodextrin, and acetonitrile was used. Modifiers were added to improve resolution and selectivity. The applied voltage was 25 kV and detection was performed at 185 nm. Validation parameters such as selectivity, linearity, repeatability, intermediate precision, limit of detection, limit of quantitation, and robustness were evaluated. The method was simple and proved to be useful for the purity testing of bile acids in bulk drugs. Good results were obtained for related impurities at concentration levels from 0.05 to 1.5% with respect to the main component, according to international requirements.

Development and validation of capillary electrophoresis methods for pharmaceutical dissolution assays.

The determination of active compounds in samples of dissolution tests of oral solid dosage forms based on the USP 23 methods was performed by capillary electrophoresis after the use of solid-phase extraction disks for the preconcentration of drugs. Enrichment factors of 20:1 allowed the determination of betamethasone and ergotamine tartrate at levels of 0.33 microgram/mL and 1.0 microgram/mL, respectively. CE analysis was performed using fused-silica capillaries (35 or 60 cm length x 75 microns i.d.) and the operating conditions consisted of 15 kV applied voltage and UV detection at 254 nm. The background electrolyte was 20-mM phosphate borate buffer, pH 9.0, containing 50 mM of sodium cholate for the separation of betamethasone and 25 mM phosphate buffer, pH 3.0, for ergotamine tartrate. Validation of the methods was also performed. Accuracy and precision of the intraday and interday assays showed comparable results with those obtained by HPLC.

Determination of suramin by micellar electrokinetic chromatography with direct serum injection.

A simple and reproducible micellar electrokinetic chromatography (MEKC) method has been developed for the quantitation of suramin serum levels to be used for its therapeutic drug monitoring (TDM). A running buffer solution of 20 mM sodium borate, 75 mM sodium dodecyl sulfate (SDS), and 4 M urea at pH 9.2 were employed and samples were introduced directly into the capillary. The voltage applied for sample separation was 25 kV and UV detection was at 254 nm. Linearity was proved over the range 47.6 micrograms/mL-523.6 micrograms/mL of suramin (r: 0.9996). Orange G was used as internal standard and no interferences of common drugs simultaneously administered to patients were observed. Recovery values of the intraday and interday assays were between 92.5 and 97.2%.

Determination of noscapine, hexylresorcinol and anethole in cough lozenges by liquid chromatography.

A liquid chromatographic method was developed for the simultaneous separation and determination of noscapine hydrochloride, hexylresorcinol and anethole in cough lozenges. Analysis was performed on a phenyl column with phosphate buffer- acetonitrile as mobile phase and the separated components were detected at 282 mm. Recoveries obtained for the analytes were of 94.6% for noscapine hydrochloride, 99.1% for hexylresorcinol and 96.3% for anethole. The values of the relative standard deviation were 0.8% for noscapine hydrochloride, 1.5% for hexylresorcinol and 1.1% for anethole. The analytical method was validated and a system suitability test was accomplished for the chromatographic method.

Application of extraction disks in dissolution tests of clenbuterol and levothyroxine tablets by capillary electrophoresis.

Sample preparation procedures using octadecyl (C18) extraction disks were developed to obtain accurate and reproducible results for determinations of clenbuterol(20 micrograms per dose) and levothyroxine (100 micrograms per dose) in dissolution media of solid oral dosage forms. Preconcentration of samples allowed final concentrations of 1.1 micrograms/ml of clenbuterol and 4.0 micrograms/ml of levothyroxine to be reached prior to CE analysis. The results obtained by CE were in good agreement with those of HPLC. The precision of the migration time, peak area, peak height and accuracy were determined in both intra-day (n = 6) and inter-day (n = 18) assays. Linearity was demonstrated over the ranges 0.5-80.0 micrograms/ml of clenbuterol and 1.0-30.0 micrograms/ml of levothyroxine. The mean recoveries were higher than 94.0%, ranging from 50 to 125% levels with respect to dose potencies. The proposed methodology may be generally applied to determine drugs at ng/ml concentrations.

A survey of some trace elements in seaweeds from Patagonia, Argentina.

Cadmium and lead concentrations were determined in some common algal species living in the southernmost coasts of Argentina. Two different sampling areas were chosen: Gulf Nuevo, a locality being influenced by a developing industrial city, and Bay Camarones, a traditional harvest area for seaweed exploitation. Selected species of the genera Lessonia, Macrocystis and Gigartina, all of commercial interest, were collected from the harvest area, and analyses showed low levels of the metals in these species. Accumulation of Pb and Cd was also evident in other common brown seaweeds from the industrial site. Analysis of Al was included in this study due to an aluminum works near one of the sampling sites. High values of this metal ranging between 300 and 3000 mg Al/kg (dry basis) were recorded in the industrialized area. Amongst all of the species studied, Colpomenia sinuosa from Gulf Nuevo exhibited the highest values of aluminum. This preliminary survey showed that, except for Al, the levels of Cd and Pb were lower than those reported in same species of seaweeds from the polluted marine waters of the rest of the world.

Morphologic changes in livers of hamsters treated with high doses of ursodeoxycholic acid: correlation with bile acids in bile.

The effects of high doses of ursodeoxycholic acid on bile acid composition and the liver morphology was examined in 60 male Syrian golden hamsters. The animals were allocated to five groups: I, control; II and IV received 0.5 g and 1 g of ursodeoxycholic acid per 100 g of standard diet respectively over 30 days and III and V received 0.5 g and 1 g of ursodeoxycholic acid per 100 g of standard diet respectively over 60 days. Bile acids were determined by high performance liquid chromatography. In all treated groups there was a significant increase in chenodeoxycholic and lithocholic acid in the bile. The mean glyco/tauro ratio was significantly higher than in the control group, reaching values > 1 for individual bile acids, except for lithocholic acid values which remained < 1. Under light microscopy, the livers of the hamsters showed damage which was dose/time related, namely portal inflammatory infiltrate, bile duct proliferation, cholestasis, fat infiltration and necrosis. Electron microscopy revealed pronounced changes starting with microvilli edema and extending to canalicular membrane destruction and necrosis. The changes observed in the relation glyco/tauro lithocholic acids, may be due to defence mechanisms to avoid hepatotoxicity. The hepatotoxicity resulting from ursodeoxycholic acid administration is presumed to be due primarily to lithocholic acid or some lithocholic acid metabolite.

Relationship between serum concentrations of flecainide and fluoride in humans.

The biotransformation of flecainide to serum fluoride after the oral administration of 100 mg to six healthy subjects was studied. Fluoride, flecainide acetate, calcium and alkaline phosphatase serum levels were determined at 0, 3, 4.5 and 6 hours after administration. Higher mean serum concentrations for fluoride and alkaline phosphatase (P less than 0.01, P less than 0.005) and lower mean calcium levels (P less than 0.05) were reached at 4.5 hours. The flecainide acetate serum levels ranged from 108 to 261 ng/ml. The results were analyzed for statistical significance by single factor analysis of variance with repeated measures. The Spearman rank correlation coefficient between flecainide acetate and fluoride serum increases was calculated. Our results would suggest that fluorine-containing flecainide could be biotransformed to yield some ionic fluoride which contribute to the daily fluoride intake.