New method for low molecular weight heparin quantification in tablets by suppressed conductivity detection and cryptand column.

Unfractioned Heparins (UFH) and Low Molecular Weight Heparins (LMWHs) are non-chromophoric, high charged sulphated molecules which are difficult to elute and detect with conventional liquid chromatography methods. Moreover, the detection of LMWHs at low concentration level, like in samples coming from dissolution test of tablets formulated with Heparins is problematic due to the weak response by UV or refractive index detection. According to the European Pharmacopoeia requirements, the assay of LMWHs is based on the anti-Xa activity and anti-IIa activity factors, which are biological parameters related to the antithrombotic activity of this compound family. The same assay method could also be applied to determine the dissolution rate of LMWHs contained in tablets, but it is time consuming and expensive test. The challenge was to develop a simpler and faster alternative method based on Ion Chromatography coupled with Suppressed Conductivity detection, but the particular polycharge interaction of LMWHs with anions exchanger makes the analyte with standard eluents and columns difficult to elute . The analytical problem could be solved by using a particular chromatographic phase with 2,2,2 cryptand sites which gives variable capacity in anion exchange depending on the used eluent. Such phase was very effective for the elution of strongly retained polyanions using simple inorganic eluent phase suitable for suppressed conductivity detection as well. The developed method allows the elution of a unique peak, which represents the whole polymeric distribution, and therefore it makes easy to quantify quickly and selectively the active ingredient in case of multiple analysis like when a dissolution test is required to characterize and discriminate between different tablets formulations. The method was tested and developed in Cosmo Pharmaceuticals QC labs and the validation results are reported in the present paper. Besides, a comparison between biological and chromatographic methods was carried out as well.

Electrochemical detection of tobramycin or gentamicin according to the European Pharmacopoeia analytical method.

Tobramycin and gentamicin are two aminoglycosidic antibiotics used in lung infection, ophthalmic treatments as well as in skin infections. Pharmaceutical companies which produce remedies containing tobramycin and gentamicin need an analytical method for their internal quality control. For several years a simple chromatographic method based on anion exchange separation coupled with amperometric detection was proposed for aminoglycosides. This analytical approach was partially used in the last edition of the European Pharmacopoeia (EP) for tobramycin and gentamicin analysis. In fact they use integrated pulsed amperometric detection (IPAD) on a gold electrode while the separation is obtained on a polymeric wide pore reversed phase instead of anion exchange in alkaline conditions. Such coupling seems to be cumbersome and not so easy to realize and to reproduce from one laboratory to another. Besides, the described method lacks some of the details as important as the waveform steps duration. Unfortunately the quality control (QC) laboratories have to use exactly the method described in the EP, so they complained about the troubles. Therefore, the EP authors published recently a paper regarding the guidelines for good practice in the method application, but the suggestion was not yet resolutive. In our work we evaluated the eluent composition and the kind of amperometric cell, work electrode diameter and cell volume. Mainly we optimized the amperometric waveform. In addition, for tobramycin analysis another chromatographic phase was explored in order to achieve better efficiency and to separate all the impurities confirming the effectiveness of the detection. The conditions described in the paper seem to allow the analyst to operate in conformity with the EP method.

Aliphatic long chain quaternary ammonium compounds analysis by ion-pair chromatography coupled with suppressed conductivity and UV detection in lysing reagents for blood cell analysers.

A method has been developed which allows simultaneous determination of three linear alkyl trimethylammonium salts. Dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride are widely used as main active ingredients of lysing reagents for blood cell analyzers which perform white blood cells differential determination into two or more sub-populations by impedance analysis. The ion-pair on styrene-divinyl benzene chromatographic phase looks like a suitable, reliable and long term stable tool for separation of such quaternary compounds. The detection based on suppressed conductivity was chosen because of the lack of significance chromophores. A micromembrane suppressor device compatible with high solvent concentration (up to 80%) was used in order to minimize the conductivity background before the detection. In the present work we show how the chemical post column derivatization makes the alkyl chain detectable also by UV direct detection at 210 nm.

Determination of biogenic amines in fish tissues by ion-exchange chromatography with conductivity detection.

Some biogenic amines, such as putrescine, cadaverine, spermidine and histamine, have been found to be useful as quality indices for the decomposition of fish, so research on the simultaneous analysis of various biogenic amines in food is of interest and importance. The intake of histamine may cause an allergic intoxication known as "scombroid poisoning" while secondary biogenic amines can potentiate the toxicity of histamine and in addition can react with nitrite to form carcinogenic nitrosamines. A new method for the simultaneous determination of underivatized biogenic amines based on ion-exchange chromatography with conductivity detector has been developed. The proposed method offers a number of advantages over previous pulsed amperometric detection and integrated pulsed amperometric detection methods such as simpler extraction procedure and sharp peaks. Separations were performed on a new low hydrophobic weak cation-exchange analytical column. This technique is simple, rapid and useful for routine checks in repetitive analyses.