Romeo Pomponio

Analysis of phenolic acids by micellar electrokinetic chromatography: application to Echinacea purpurea plant extracts.

A micellar electrokinetic chromatographic (MEKC) method was developed for the separation of ten phenolic acids including cichoric acid and caftaric acids, specific marker phytochemicals of Echinacea purpurea. The MEKC method involved the use of 70 mM sodium deoxycholate (SDC) in 40 mM borate (pH 9.2) buffer and UV detection at 300 nm. The bile acid was used as biosurfactant able to provided a micellar system with different and more selective properties than sodium dodecyl sulfate. The effects of SDC and borate concentration and buffer pH on the analyte resolution were evaluated. The validated method was applied to the determination of cichoric acid and related compounds in E. purpurea root extracts, and in commercial E. purpurea based dried extracts and tablets.

Analysis of catechins in extracts of Cistus species by microemulsion electrokinetic chromatography.

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.

Determination of 5-aminosalicylic acid related impurities by micellar electrokinetic chromatography with an ion-pair reagent.

A micellar electrokinetic chromatographic (MEKC) method was developed for the quantification of mesalazine or 5-aminosalicylic acid (5-ASA) and its major impurities 3-aminosalicylic acid, salicylic acid, sulfanilic acid and 4-aminophenol. The optimisation of the experimental conditions was carried out considering some important requirements: resolution, reproducibility, detection limits of 0.1% (m/m) or less, short total analysis time. Preliminary investigations employing sodium dodecyl sulfate (SDS) as surfactant did not lead to the necessary resolution of the studied compounds; the addition of tetrabutylammonium bromide (TBAB) to the SDS micellar system resulted in the complete separation of all the compounds. The effects on the separation by several parameters such as TBAB concentration, SDS concentration, background electrolyte pH and concentration, were evaluated. Using a fused-silica capillary (8.5 cm effective length) complete analysis was obtained in a very short time. Under the optimised final conditions [120 mM 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer, pH 10.20, 65 mM SDS in the presence of 55 mM TBAB and 5% methanol] the method was validated for specificity, precision, linearity, limits of detection and quantitation, and robustness: the 5-ASA related impurities can be quantified at least at the 0.1% (m/m) level.

Photostability studies on nicardipine-cyclodextrin complexes by capillary electrophoresis.

Nicardipine (NC)-cyclodextrin solid systems were prepared in equimolar ratios and their photostability in aqueous solution under exposure to UV(A)-UV(B) radiations was evaluated. The photodegradation process was monitored by a capillary electrophoresis (CE) method able to provide the enantioresolution of the rac-nicardipine. Enantioresolution was achieved using the mixture 3.0% sulfate-beta-cyclodextrin (SbetaCD) and 2.0% heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) as chiral selector in 20mM triethanolammonium phosphate solution (pH 3.0). The photostability studies were carried out on inclusion complexes of rac-nicardipine with alpha-cyclodextrin (alphaCD), beta-cyclodextrin (betaCD), gamma-cyclodextrin (gammaCD), hydroxypropyl-alpha-cyclodextrin (HPalphaCD), hydroxypropyl-beta-cyclodextrin (HPbetaCD), hydroxypropyl-gamma-cyclodextrin (HPgammaCD), (2-hydroxyethyl)-beta-cyclodextrin (HEbetaCD) and methyl-beta-cyclodextrin (MbetaCD). A photoprotective effect was observed by betaCD, HPalphaCD, HEbetaCD, whereas gammaCD, MbetaCD, HPbetaCD and HPgammaCD did not affect the nicardipine photostability. Conversely, alphaCD was found to favour the drug photodegradation. Evidences for CDs-mediated stereoselective photodegradation of rac-nicardipine were observed only for the beta-CD complex. In this case, two distinct photodegradation profiles, with two different kinetic constants (k), were observed for the nicardipine enantiomers.

Analysis and enantioresolution of donepezil by capillary electrophoresis.

The analysis of donepezil, a centrally acting acetylcholine esterase inhibitor, is described by a CZE method suitable for applications in pharmaceutical field. A rapid migration of the analyte was obtained under acidic conditions (pH 3.0); with detection wavelength of 320 nm a LOD of 0.8 x 10(-3) mg/ml was provided. Applications on real sample (pharmaceuticals) were carried out using two different instruments with comparable results in terms of reproducibility and accuracy. The use of chiral selectors in the running buffer allowed the enantioseparation of donepezil; charged cyclodextrins (carboxymethyl-beta-cyclodextrin and sulfated-beta-cyclodextrin) were suitable for the chiral resolution of the analyte. Interesting results were also obtained using human serum albumin. The protein-based CE enantioseparation was carried out at pH 7.4 avoiding the partial filling technique due to the good absorptivity of donepezil at 320 nm. Interestingly, the use of bicine as BGE provided a significative improvement in the enantioresolution compared to that obtained by phosphate buffer.

Analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis

The ability of capillary zone electrophoresis in the development of analytical methods devoted to the quality control of the thiol drug penicillamine is shown. Using 50 mM phosphate running buffer (pH 2.5), good quantitations of underivatized penicillamine and its disulfide were achieved; detection at 200 nm allowed checking the presence of the disulfide impurity in pharmaceuticals. The use of 1,1-[ethenylidenebis(sulfonyl)]bis-benzene as a thiol specific reagent resulted in an increased sensitivity for the quantitation of D-penicillamine (limit of detection at 200 nm wavelength was 1.5 microM). Introducing beta-cyclodextrin as chiral selector in the running buffer, enantioseparation of D-L-penicillamine was obtained; for this purpose (+)-camphor-10-sulfonic acid, a chiral ion-pairing reagent, was found to be an essential additive in obtaining a baseline separation. The resulting enantioseparative system was validated in order to evaluate the presence of the toxic L-penicillamine enantiomer in pharmaceutical samples.

Simultaneous analysis of the lipophilic and hydrophilic markers of Echinacea plant extracts by capillary electrophoresis

The phytochemical profile of Echinacea plant extracts has been obtained by a CD-MEKC method using sodium dodecyl sulfate (SDS) as surfactant and hydroxypropyl-β-cyclodextrin (HP-β-CD). The separation of the major lipophilic markers of E. purpurea (alkyl isobutylamides and alkyl methylbutylamides; designated as alkamides) was affected by the nature and concentration of both SDS and CD, while the nature, concentration, and pH of running buffer exerted little influence on the migration of alkamides. On the other hand, the separation of hydrophilic compounds of the different Echinacea species (caffeoyl conjugates, namely: echinacoside, cynarin, chlorogenic acid, caffeic acid, caftaric acid, and cichoric acid) were strongly influenced by the nature, concentration, and pH of running buffer. Thus a step-by-step optimization procedure was carried out in order to choose the best BGE able to provide simultaneous separation of both alkamides and phenolic compounds. By using a SDS/HP-β-CD mixture of concentration 110 mM/100 mM in a BGE consisting of Britton-Robinson buffer (10 mM, pH 8.0), complete separation of all the principal phytochemical markers of Echinacea species was achieved in less than 10 min. The proposed conditions were applied to the evaluation of the phytochemical profile of Echinacea purpurea plant extract; quantitative determination was performed on the hydrophilic caffeoyl conjugates in commercially available phytopharmaceutical products.

Linear, neutral polysaccharides as chiral selectors in enantioresolution of basic drug racemates by capillary electrophoresis

SummaryA comparison of the enantiorecognition ability of linear, neutral polysaccharides was performed on a series of basic drug racemates in acidic running buffer (pH 3.0). Dextrin 20, Dextran 70 and Pullulan were chosen as chiral selectors for their different characteristics. Dextrin 20, high-dextrose equivalent maltodextrin, showed good enantioresolution for a limited number of racemic drugs. In contrast, Dextran 70, a low-equivalent dextrose polysaccharide, exhibited poorer enantioresolution but had wider applicability allowing nine basic racemates to be resolved; in particular, at high concentrations enantioseparation of amphetamine and congeners was achieved in relatively short time. The results obtained appear to support different mechanisms of enantiorecognition for the polysaccharides studied.

Analysis of prostaglandin E1 and related impurities by mixed aqueous-organic capillary electrophoresis

A capillary electrophoretic method was developed for the separation and quantification of prostaglandin E 1 (PGE 1 , alprostadil) and its major impurities prostaglandin E 2 (PGE 2 , dinoprostone), prostaglandin A 1 (PGA 1 ), and prostaglandin B 1 (PGB 1 ). The documented good stability of the studied analytes in organic solvents led us to try CE separation in non-aqueous media. In mixed solvents (methanol-acetonitrile), good analyte peak shapes were obtained using bile acid salts as buffer electrolytes; in particular, sodium cholate and sodium deoxycholate were found to be suitable additives when used at sub-micellar concentrations. Interestingly, the addition of water to the organic running buffer permitted simultaneous improvement of the separation selectivity and the sample compatibility with the CE system. Optimisation of the experimental conditions was carried out considering the requirements for a method devoted to analysis of impurities: precision, selectivity, and sensitivity (quantitation limits of 0.1%). In a fused-silica extended path capillary of 32.5 cm total length, the complete prostaglandin separation was obtained in about 10 min, using 15 mM sodium deoxycholate in a mixed aqueous-organic solvent. The best separation was attained in the presence of 20% water and 80% of methanol-acetonitrile 75:25 (v/v). The method was then validated for linearity, LOD and LOQ, precision, selectivity, accuracy, and robustness; finally, it was applied to real samples, demonstrating its ability to quantify prostaglandin-related impurities.

Analysis of guaifenesin-based cough syrups by micellar electrokinetic chromatography and GC-MS

Capillary electrophoretic methods, under MEKC conditions, were developed for thesimultaneous rapid analysis of neutral (guaifenesin) and basic (salbutamol and dex-tromethorphan) active ingredients of commercially available cough syrups. The run-ning buffer nature played a key role on the migration of the studied analytes: using0.04 M sodium dodecyl sulfate (SDS) as surfactant, sodium tetraborate and 3-(cyclo-hexylamino)-2-hydroxy-1-propane-sulfonic acid sodium salt (CAPSO) were chosenas running buffer allowing the separation of active ingredients and excipients of differ-ent cough syrups. Qualitative and quantitative information on the composition of theanalyzed formulations were simultaneously obtained for both active ingredients andexcipients by the proposed MEKC methods; when syrups containing volatile UV-non-absorbing components (flavors) were analyzed a GC-MS approach was chosen. Theaqueous matrices of the formulations allowed direct sample injection in CE, whileextraction procedures were applied in order to perform GC analyses. The obtaineddata support the suitability of MEKC and GC-MS as effective, complementary techni-ques for the selective and sensitive quality control of cough syrups.Key Words: Cough syrups; Capillary electrophoresis; GC-MS; Guaifenesin; Salbuta-mol;DextromethorphanMs received: January 10, 2001; revised Ms received: February 16, 2001; accepted:February17,2001