Ion chromatographic separation of alkylsulphonic acids with conductivity detection

Summary An ion chromatographic (IC) method has been developed for the separation of alkylsulphonic acid. Two different stationary phases, silica-based and polymer-based ion-exchange resins, were studied using pure ion exchange and/or hydrophobic interaction mechanisms. Correlations between analyte hydrophobicity and eluent polarity were made in order to investigate the possibility of changing the dominant separation mechanism by varying the eluent composition. The alkyl chain lengths of the sulphonic acids analysed ranged from C₁ to C₉. Detection limits in the submicromolar range were obtained by suppressed conductivity detection.     

Simultaneous assay of four stereoisomers of diltiazem hydrochloride. Application to in vitro chiral inversion studies

Summary The simultaneous stereospecific assay of four stereoisomers of diltiazem hydrochloride in bulk drug and aqueous solution was developed using HPLC on a Chiralcel OF column. The four isomers were quantitated with good precision by the internal standard method. The chiral inversion of (+)-cis-diltiazem hydrochloride in vitro, stability of its (2S, 3S) configuration in the solid and aqueous states was examined by HPLC. Chiral inversion of (+)-cis-diltiazem hydrochloride was not observed in the solid state, and its (2S, 3S) configuration was stable to heat, humidity and light. Chiral inversion of (+)-cis-diltiazem hydrochloride (2S, 3S) was observed in aqueous solution under UV, but not in aqueous solution stored at 80°C for 5h nor under visible light for 10 h. The (+)-cis-diltiazem hydrochloride (2S, 3S) epimerized to (+)-trans-diltiazem hydrochloride (2R, 3S) with a half-life of 5h in aqueous solution under UV but the reverse chiral inversion of (+)-trans-diltiazem hydrochloride (2R, 3S) to (+)-cis-diltiazem hydrochloride (2S, 3S) was not observed.     

Development and Validation of a Liquid Chromatographic Method for Determination of Efavirenz in Human Plasma

A simple, rapid, and sensitive high-performance liquid chromatographic method for estimation of efavirenz in human plasma has been developed and validated. Chromatography was performed with C₁₈ analytical column and 50:50 acetonitrile–phosphate buffer (pH 3.5) as mobile phase. Compounds were monitored by UV detection at 247 nm. The retention time for efavirenz was 6.45 min and that for the internal standard, nelfinavir, was 2.042 min. Response was a linear over the concentration range of 0.1 μg–10 μg mL⁻¹ in human plasma. The method was simple, specific, precise and accurate and was useful for bioequivalence and pharmacokinetic studies of efavirenz.     

Affinity Chromatography of Insulin with a Heptapeptide Ligand Selected from Phage Display Library

A heptapeptide phage display library was screened with insulin to find its ligands for affinity chromatography. The peptide was synthesized and coupled to EAH Sepharose 4B (5.4 mol mL^–1 bed). Then, insulin chromatography was carried out with mobile phases of different pH values and by the addition of urea and ethylene glycol. It was found that electrostatic interactions were predominant for the affinity binding, and hydrogen bonding might also contribute somewhat to the affinity. Finally, frontal analysis was performed and the dynamic binding capacity of the affinity column for insulin at 50% breakthrough was estimated at 60.6 mg mL^–1 bed, which was about two times higher than the theoretical binding capacity of the monomeric insulin. The result suggests that insulin was bound in dimer state in a stoichiometric relationship with the coupled peptide, indicating the high binding efficiency of the peptide ligand for insulin.     

Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.     

Capillary electrophoresis as an alternative to HPLC for determination of honey flavonoids

Summary The general objective is to provide an alternative methodology based on capillary electrophoresis (CE) to characterize flavonoids from honey and hence determine its botanical origin. The specific objective is to compare the separation of flavonoids by CE with those achieved by HPLC to assess CE as an alternative technique for the determination of honey flavonoids. Fourteen different flavonoids isolated from honey were analysed by MECC and compared to the HPLC separations. It was difficult to find specific experimental conditions to separate all the flavonoids from honey in a single MEKC run. Three chromatographic conditions are optimized and, depending on the flavonoid markers sought in honey, the appropriate detection method should be chosen. Compared to the HPLC results, it is clear that CE could be an alternative technique in honey flavonoids analysis and particularly in the study of its geographical and floral origin.     

Direct separation of the enantiomers of cetirizine and related compounds by reversed-phase chiral HPLC

Summary Direct separations of the enantiomers of cetirizine and related compounds have been achieved by reversed-phase HPLC on the Chiralcel OD-R, a polysaccharide-derived chiral stationary phase; the mobile phase was usually perchlorate solution supplemented with acetonitrile. Resolution of the enantiomers of cetirizine and related compounds was good. The effect of the acetonitrile content of the mobile phase was investigated, and the effect of the structure of the chiral compounds on their behavior on the Chiralcel OD-R column is discussed.     

Luminarin 9: A new fluorescent reagent for the precolumn derivatizatioin of main volatile and non-volatile N-nitrosamines in RP-HPLC

Summary A sensitive precolumn fluorescence derivatization method for low level detection of the, volatile (N-nitrosodimethylamine and N-nitrosopyrrolidine) and non-volatile N-nitrosamines (N-nitrosoproline and N-nitrosodiethanolamine) an high-performance liquid chromatography was developed. This method is based on the denitrosation of the compounds of interest by a mixture of hydrobromic acid and acetic acid to produce the corresponding secondary amines. These are, then, able to react with, a quinolizinocoumarin derivative (luminarin 9^®) to form highly fluorescent labelled N-nitrosamine derivatives. The structural elucidation of the luminarin 9^® derivatives of N-nitrosoproline and N-nitrosodimethylamine by way of example, were established by liquid chromatography-mass spectrometry (LC-MS) and by direct chemical ionization-mass spectrometry (CI-MS). The separation, derivatization and detection conditioins were optimized for all the studied compounds. The detection limits (signal to noise ratio=3) were between 0.4 and 1.0 pmol injected depending on the compound. The calibration graphs were linear for derivatized amounts in the range of 0.5–40 nmol for N-nitrosodimethylamine and N-nitrosopyrrolidine, 0.4–2- nmol for N-nitrosoproline and 1.0–40 nmol for N-nitrosodiethanolamine. The repeatability (RSD less than 3.5%, n=6) and reproducibility (RSD less than 4.8%, n-6) were satisfactory.     

Enantiomeric separation of amphetamine,methamphetamine and ring substituted amphetamines by means of a β-cyclodextrin-chiral stationary phase

Summary The enantioseparation of amphetamine, methamphetamine and various ring-substituted amphetamines by use of a chiral stationary phase carrying immobilized native -cyclodextrin (-CyD) selectors is reported. The system is evaluated for resolving the specified compounds directly without any derivatization and after derivatization with phenyl isothiocyanate (PITC), naphthyl isothiocyanate (NITC) and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). This direct enantioseparation is compared with the features of indirect separation of diasteromeric derivatives after reaction with the optically pure Marfey's reagent employing a simple non-chiral alkyl-silica (RP-8) column. A selection of those methods best suited for each single amphetamine is given.Seventeen different samples of amphetamine, confiscated by the Swedisch police, were analyzed with respect to their enantiomeric composition. Within this set of samples synthesized by the same method no significant deviation from a racemic ratio could be observed.     

Simultaneous Determination of Pioglitazone and Glimepiride by High-Performance Liquid Chromatography

A rapid and accurate HPLC method has been developed for simultaneous determination of pioglitazone and glimepiride. Chromatographic separation of the two pharmaceuticals was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm, 5 m) with a 45:35:20 (v/v) mixture of 0.01 m triammonium citrate (pH adjusted to 6.95 with orthophosphoric acid), acetonitrile, and methanol as mobile phase, at a flow rate of 1.0 mL min^–1, and detection at 228 nm. Separation was complete in less than 10 min. The method was validated for linearity, accuracy, precision, limit of quantitation, and robustness [1, 2]. Linearity, accuracy, and precision were found to be acceptable over the ranges 2.50–30.00 g mL^–1 for pioglitazone and 0.10–10.00 g mL^–1 for glimepiride.