Identification Of Related Substances Research Articles

Green HPLC quantification method of lamivudine, zidovudine and nevirapine with identification of related substances in tablets

ABSTRACT A green HPLC method for lamivudine (3TC), zidovudine (AZT) and nevirapine (NVP) determination in fixed-dose combination tablets was developed using ethanol as both one of the mobile phases and the solvent for sample preparation. This method was validated according to ICH Q2(R1) guideline. Additionally, the method was adapted to complete the analysis of five related substances described in the International Pharmacopoeia (Ph. Int.), five other known related substances and two excipients. The separation was obtained with a C18 column (ARV4 5 µm 250 × 3.0 mm, Interchim) using a gradient mode with 0.1M ammonium acetate buffer (pH 4.5) and ethanol as mobile phase at 35°C, a flow rate of 0.4 mL/min, at 270 nm and an injection volume of 10 µL. The combination of ethanol (biodegradable and low-cost alternative solvent) with the use of a column diameter of 3 mm instead of 4.6 mm as used in pharmacopoeias, makes the wastes of the analysis more environmentally friendly and allows the use of a conventional HPLC pump (<400 bar) for easy implementation in quality control in countries with limited resources. Assessment of the method greenness was evaluated using three analytical tools: Analytical Eco-scale, AGREE metrics and Analytical Method Greenness Score (AMGS).

Dummy Molecularly Imprinted Polymers Using DNP as a Template Molecule for Explosive Sensing and Nitroaromatic Compound Discrimination

This work reports a rapid, simple and low-cost voltammetric sensor based on a dummy molecularly imprinted polymer (MIP) that uses 2,4-dinitrophenol (DNP) as a template for the quantification of 2,4,6-trinitrotoluene (TNT) and DNP, and the identification of related substances. Once the polymer was synthesised by thermal precipitation polymerisation, it was integrated onto a graphite epoxy composite (GEC) electrode via sol–gel immobilisation. Scanning electron microscopy (SEM) was performed in order to characterise the polymer and the sensor surface. Responses towards DNP and TNT were evaluated, displaying a linear response range of 1.5 to 8.0 µmol L−1 for DNP and 1.3 to 6.5 µmol L−1 for TNT; the estimated limits of detection were 0.59 µmol L−1 and 0.29 µmol L−1, for DNP and TNT, respectively. Chemometric tools, in particular principal component analysis (PCA), demonstrated the possibilities of the MIP-modified electrodes in nitroaromatic and potential interfering species discrimination with multiple potential applications in the environmental field.

Determination of diminazene aceturate in pharmaceutical formulations by HPLC and identification of related substances by LC/MS

A validated, reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous assay of diminazene aceturate, antipyrine (excipient) and diminazene impurities in pharmaceutical formulations is described. The chromatographic system consisted of a Lichrospher-60 RP-select B column with a mobile phase composition of acetonitrile–methanol–ammonium formate (pH 4.0, 20 mM) (10:10: 80 v/v/v) and UV detection at 254 nm. The method is specific, precise and accurate for the determination of diminazene in the presence of its manufacturing and degradation impurities with a limit of detection and quantification of 50 ng/ml and 10 μg/ml (RSD<3.0%), respectively. The major manufacturing impurity [1-(4 amidino phenyl)3-(4 carbamoyl phenyl)-triazene] and a degradant ( p-aminobenzamidine) of diminazene aceturate have been resolved and identified by liquid chromatography/electrospray ionization-mass spectrometry operated in a positive ion mode.

The identification of related substances in triamcinolone acetonide by means of high-performance liquid chromatography with diode array detector and mass spectrometry.

A study of an HPLC method for the analysis of related substances in triamcinolone acetonide is described. Several systems of solvents and samples of different lots and preparative origins were examined and a rapid-scanning diode array UV detector (DAD) was particularly useful. With the proposed technique it was possible to identify 9 alpha-bromo desonide as a principal impurity, which was present in all examined samples of triamcinolone acetonide. This identification was rendered possible by the investigation of the second derivative of the UV spectra and by means of study of the mass spectrum. Furthermore, it was possible, primarily on the basis of the spectrophotometric data, to formulate reliable hypotheses on the possible identification of 9 beta, 11 beta-epoxide of the desonide which was present at very low levels and to exclude the presence of 11-deoxy-9(11)-unsaturated desonide. The presence of the above-mentioned related substances was explained considering the scheme of synthesis described in the literature. The spectrophotometric characteristics of the studied compounds and the limits of applicability of the present procedure are discussed.

The identification of related substances in 9α-fluoroprednisolone-21 acetate by means of high-performance liquid chromatography with diode array detector and mass spectrometry

A method for the analysis and identification of the principal related substances in 9α-fluoroprednisolone acetate is described. This compound has been chosen as a model for the investigation of related substances which can be originated in the general procedure for introducing a fluorine substituent at position 9 of a corticosteroid molecule. HPLC procedures, both in reversed and in normal phase were used; a rapid scanning UV detector which allows direct spectrophotometric data to be obtained on chromatographic peaks, proved to be a tool of great importance. Thus, after reversed-phase chromatographic separation and observation of the UV spectra and their respective second derivatives, it was possible to characterize some of the principal effective and potential related substances such as 9α-fluorohydrocortisone acetate, 9α-bromoprednisolone acetate, 9β, 11β-epoxyprednisolone acetate and 9(11)-dehydroprednisolone acetate, emerging as chromatographic peaks. Identification of 9α-bromoprednisolone acetate and of 9α-fluorohydrocortisone acetate which proved to be the most significant impurities, was confirmed by means of an exhaustive study of the mass spectra of these substances conveniently isolated by normal-phase HPLC. The chromatographic, spectrophotometric and mass-spectrometric characteristics of the studied compounds are reported.

Absorbance Ratioing as a Screen for Related Substances of Pharmaceutical Products

The identification of related substances in pharmaceutical products is generally incomplete during the initial stages of chromatographic method development. Thus one often makes the tacit assumption of constant molar absorptivity and relies on peak normalization for related substance quantitation. Absorbance ratioing, by simultaneously monitoring the column effluent at two wavelengths and ratioing the responses, provides a powerful screen for the selection of those related substances with significant changes in spectral characteristics. Several model compounds are examined to identify important considerations for effective application of this technique. A number of observations have been made from these examples concerning the selection of instrumental parameters such as the optimization of the two wavelengths under consideration, selection of absorbance thresholds, and interpretation of the resultant ratiograms. Finally, application of this technique to method validation strategies for related substances methods is described.