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.

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.