Hantzsch pre-column derivatization for simultaneous determination of alendronate sodium and its pharmacopoeial related impurity: Comparative study with synchronous fluorometry using fluorescamine.

ABSTRACT

High performance liquid chromatographic (HPLC) method with a pre-column derivatization based on Hantzsch condensation reaction was applied for simultaneous determination of alendronate sodium (ALN) and its main related impurity, 4-Aminobutanoic acid (ABA) at its pharmacopeial limit. The separation of colored condensation products of ALN and ABA were achieved on Agilent Zobrax Eclipse SB-C18 analytical column (250 × 4.6 mm, 5 µm) using a mobile phase composed of acetonitrile-0.1 M acetate buffer, pH 5.0 (1585, v/v). The flow rate was 1 mL min-1. The detection was carried out at 340 nm using photo-diode array detector. Peak areas were used for the linear regression line in the range of 10-500 and 0.2-40 µg mL-1 for ALN and ABA, respectively. Different conditions for the optimization of the derivatization reactions as well as for the HPLC measurement were studied. The proposed method was validated for linearity, precision, accuracy, specificity and robustness. This method was used to check the purity of ALN in the presence of ABA (related impurity) at the pharmacopeial limit (0.5%). For comparison purpose, another method was proposed which involves synchronous fluorescence measurement after ALN reaction with fluorescamine. In this method, the third derivative synchronous spectra were estimated as peak to peak measurement from 339 to 370 nm for ALN determination with LOD and LOQ of 24 and 73 ng mL-1, respectively, showing very high sensitivity. Both methods have been applied for determination of the alendronate sodium (ALN) in bulk and pharmaceutical preparations without interference of additives in tablets or oral solution.