E. De Lorenzi

Validation of a RP-LC method for the simultaneous determination of isoniazid, pyrazinamide and rifampicin in a pharmaceutical formulation.

A simple and accurate liquid chromatographic method was developed and validated for estimation of isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF) in combined dosage forms. Drugs were chromatographed on a reverse phase C18 column using a mobile phase gradient and monitored at the corresponding maximum of each compounds. Peaks were identified with retention time as compared with standards and confirmed with characteristic spectra using diode-array detector. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method does not require any specific sample preparation except the use of a guard column. The method is linear (r(2)>0.999), precise (RSD%: 0.50% for ISN, 0.12% for PYR and 0.98% for RIF), accurate (overall average recovery yields: 98.55% for ISN, 98.51 for PYR and 98.56% for RIF) and selective. Due to its simplicity and accuracy the method is suitable for routine quality control analysis of antitubercolosis combination dosage form.

Egg yolk riboflavin binding protein as a new chiral stationary phase in high-performance liquid chromatography

A chiral stationary phase for high-performance liquid chromatography based on hen egg yolk riboflavin binding protein is introduced. The purified protein was immobilized on activated 5NH2 Nucleosil silica. Chiral acidic, basic and uncharged drugs were chromatographed and the influence of the mobile phase parameters on the retention times and enantioselectivity was studied. Thirteen out of the twenty compounds tested were partially or baseline resolved. These encouraging preliminary results suggest that this chiral stationary phase may be applicable to a wide range of drug enantiomers in the reversed-phase mode.

Evaluation of stereoselective dissolution of verapamil hydrochloride from matrix tablets press-coated with chiral excipients

Abstract In most cases the modulation of the drug delivery rate from modified-release formulations is achieved with polymers also used as chiral stationary phases in liquid chromatography. It is therefore hypothesized that the interaction of the enantiomers with the excipient may lead to differentiated delivery rates from the devices for each enantiomer. This study evaluates the stereoselective dissolution of (±)-verapamil, a model racemic drug and, for this purpose, different matrix compositions, a commercial product and a particular delivery device have been considered. The delivery device, recently proposed for the delayed release of drugs, consists of an active core containing the drug, coated by compression with different types of chiral polymeric materials. The quantitative determination of verapamil enantiomers released by these systems was carried out using a stereospecific HPLC method. Hydroxypropylmethylcellulose, β-cyclodextrin, hydroxypropyl-β-cyclodextrin and cross-linked amylose did not show any stereoselective dissolution properties while pectin, galactomannan and scleroglucan seemed to give a slightly higher dissolution rate of the R, compared with the S enantiomer. It is, however, to be verified whether these small differences in the release rate of the two enantiomers detected ‘in vitro’ could lead to real ‘in vivo’ effects.

Evaluation of quail egg white riboflavin binding protein as a chiral selector in high-performance liquid chromatography and capillary electrophoresis

A new chiral stationary phase for high-performance liquid chromatography of quail egg white riboflavin binding protein is presented. Several chiral acidic, basic and uncharged drugs were analysed and the influence of the mobile phases parameters on the retention times and enantioselectivity was evaluated. On the basis of the results obtained, the same protein was studied as a background electrolyte additive in free solution capillary electrophoresis, in order to evaluate if capillary electrophoresis (CE) could be used as a rapid scouting technique for screening the enantioselectivity of novel proteins without immobilisation on a solid support. To investigate if it is possible to directly compare the results obtained by each technique, the CE experiments were planned on the basis of both the findings and ideas originated in liquid chromatography.

Synthesis and chromatographic evaluation of molecularly imprinted polymers prepared by the substructure approach for the class-selective recognition of glucuronides

Two series of molecularly imprinted polymers (MIPs) for the class-selective recognition of glucuronides have been prepared by using lipophilic substructures of the target analyte as template molecule and potent host monomers against oxyanions, that are expected to establish a strong stoichiometric interaction with the single carboxylic group of the template. The polymers were tested as stationary phases in liquid chromatography for specific recognition. A preliminary investigation of the imprinting properties of eleven MIPs was carried out, by comparing the retention time of the template and of structurally related compounds on the MIP column with that on the corresponding non-imprinted polymer (NIP). The two polymers showing the best performance were selected to further test cotinine, mycophenolic acid, testosterone and their respective glucuronides as model compounds. The high specificity obtained against glucuronides and the different chemical structure of the parent drug make the two MIPs class-selective imprinted receptors, also suitable for SPE application.

Properties of a stationary phase based on immobilised chicken liver basic fatty acid-binding protein

The fatty acid-binding proteins (FABPs) are a class of low-molecular-mass proteins that bind fatty acids and are thought to be involved in their intracellular transport. FABPs have been isolated and studied from several tissues, but their precise function and mechanism of action are still not clear. Chicken liver (basic) fatty acid-binding protein (bFABP) was immobilised on aminopropyl silica and the developed stationary phase was used to examine the enantioselective properties of this protein and to study the binding of drugs to bFABP. The retention and enantioselectivity of the new column for a large number of chiral drugs was investigated. The enantiomers of basic and neutral compounds were poorly retained and not resolved by the bFABP column. On the contrary the resolution of the enantiomers of some acidic compounds was obtained. Therefore the influence of the mobile phase pH and organic modifier on the chromatographic performance of acidic compounds was studied. In order to clarify the retention mechanism, competitive displacement studies were also carried out by adding short-chain fatty acids to the mobile phase as displacing agents and preliminary quantitative structure-retention relationship correlations were developed to describe the nature of the interactions between the chemical structures of the analytes and the observed chromatographic results.

Evaluation of β-lactoglobulin as a stationary phase in high-performance liquid chromatography and as a buffer additive in capillary electrophoresis : observation of a surprising lack of stereoselectivity

Previous studies have reported that alpha1-acid glycoprotein is quite similar in amino acid sequence and disulfide bond arrangements to members of a group of proteins which include beta-lactoglobulin (BLG). Since generally homologous proteins retain some similarity in function at the molecular level, we decided to evaluate the enantioselective properties of BLG as an high-performance liquid chromatographic chiral stationary phase (HPLC-CSP), and as an additive in capillary electrophoresis (CE). Two columns with differences in internal diameter and method of immobilisation on epoxide silica were prepared. Chiral acidic, basic and uncharged drugs were chromatographed and mobile phase parameters, namely pH and type of organic modifier, were varied in order to test the column performance. The CE approach has some advantages in that there is no need for immobilisation and only a small amount of protein is required. BLG was therefore tested as a CE buffer additive, using the same analytes as in the HPLC study. Although one would expect that a protein would display some enantioselectivity, BLG did not show any enantioselectivity whatsoever in either system; the protein has fairly weak interaction with the majority of the test solutes, as indicated by both techniques.

High-performance liquid chromatography post-column derivatization with fluorescence detection to study the influence of ambroxol on dipalmitoylphosphatidylcholine levels in rabbit eustachian tube washings

In this work an appropriate high-performance liquid chromatography method was set up to guarantee specificity, sensitivity, precision and accuracy in analyzing dipalmitoylphosphatidylcholine (DPPC) in rabbit eustachian tube washings, as well as to determine its varying levels after administration of ambroxol chloride. The procedure is based on a post-column derivatization with fluorescence detection using 1,6-diphenyl-1,3,5-hexatriene which exhibits increased fluorescence in a lipid environment. DPPC was chromatographed on a Hypersil C18. The mobile phase for the isocratic elution consisted of 40 mmol/l choline chloride in methanol-tetrahydrofuran (97:3). Ambroxol was given to a group of New Zealand white rabbits at a dose of 30 mg/kg. A second group receiving vehicle only acted as controls.

Determination of 5-aminosalicylic acid and related compounds in raw materials and pharmaceutical dosage forms by high-performance liquid chromatography

Abstract A high-performance liquid chromatographic method using a μBondapak C 18 column and a UV detector at 230 nm was developed for the simultaneous determination of 5-aminosalicylic acid (5-ASA) and related compounds (4-aminophenol, 3-aminosalicylic acid and 3-aminobenzoic acid). A method for the determination of 5-ASA in pharmaceutical preparations (tablets, granules and rectal suspension) is also described. Calibration graphs for both 5-ASA and related compounds are reported. The standard deviation is ± 0.623 ( n = 10).

Chromatographic investigation on the binding site characteristics of quail egg-white riboflavin binding protein as a chiral stationary phase.

Recently we described the use of riboflavin binding protein extracted from quail egg-white, as a new HPLC chiral stationary phase. In this study we show the further results obtained with the use of high-performance affinity chromatography to provide a better understanding of the chiral recognition mechanism for the observed enantioselectivity and to gain a deeper knowledge into the binding site that has been recently characterised by X-ray crystallography for chicken egg-white. High-performance affinity chromatography provides information on the potential protein structural changes occurring upon its immobilisation and enables competitive binding studies as well as the assessment of binding constants through frontal analysis experiments.