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Application of 2, 4-Dinitro Phenyl Hydrazine as a Derivatizing Reagent for the Trace Level Quantification of Toxic Impurity Formaldehyde in Entecavir ? An Antiviral Drug for Treatment of Hepatitis B by High Performance Liquid Chromatography


Author(s): Deepali Gangrade, Rajesh Nema and Indrajeet Singhvi

The objective of present research work was to develop a suitable and sensitive analytical method for the quantification of trace level of toxic impurity Formaldehyde in the active pharmaceutical ingredient Entecavir. Formaldehyde was quantified through pre-column derivatization using 2,4-Dinitro Phenyl Hydrazine (2,4-DNPH) by gradient elution on HPLC. The analysis was accomplished on an Inertsil ODS C18 column (50mm x 4.6mm, 3µ) using 0.1% Triethyl amine in water and pH was adjusted to 3.0 with Trifluoro acetic acid as buffer and Acetonitrile as the organic modifier. The flow rate was set at 1.5 ml/minute with an analysis time of about 15 minutes. The method was validated for the analytical parameters such as System Suitability, Specificity, Linearity and Range, Accuracy, Limit of Detection (LOD), Limit of Quantification (LOQ), Precision, and Solution Stability. The limit of detection and limit of quantification of Formaldehyde was found to be 0.2µg/ml and 0.6µg/ml respectively with respect to Entecavir sample concentration (1mg/ml).

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