A Stability Indicating RP-HPLC Method for Quantification of Assay and Organic Impurities in Doxycycline Hyclate Bulk and Parenteral Dosage Form.

The Scope of this study is to develop a reverse phase- HPLC stability-indicating method for the estimation of assay and organic impurities (process and degradation) of Doxycycline Hyclate in a Doxycycline injectable formulation. Both the active and dosage forms are official in the USP monograph and assay and impurity methods are provided by separate UPLC techniques which are highly sensitive to the flow rate and temperature by considering the quality control requirements and the user-friendly technique. A simple HPLC simultaneous stability indicating method was developed for the quantification of assay and impurity with a shorter run time. The method was developed by using HPLC with a gradient program and a reverse phase column of Waters XBridge BEH C8, 150 mm 4.6 mm, i.d 3.5μm particle size with a mobile phase A contains pH 8.5 Phosphate buffer (25mM potassium phosphate, 2mM EDTA, and 0.5mL of triethylamine). Mobile phase B contains methanol with a 1.7 mL/minute flow rate, 55 °C of column temperature with a 270 nm UV wavelength, and an injection volume of 25 μL, the modern research represents a concomitant method for developing assay and organic impurities of doxycycline hyclate and doxycycline for injection. The assay and impurity method were validated as per USP <1225>, and per ICH guidelines [28,29]. The retention time of doxycycline and degradation impurity 4-epi doxycycline (Figure 1B) is about 9.8 minutes and 6.4 minutes respectively. the linearity range of doxycycline and 4-epi doxycycline is 0.5-150μg per mL and 0.5-18μg per mL respectively. Recovery of doxycycline and 4-epi doxycycline are 98.7%-100.6% and 88.0%- 112.0%. Validation of the analytical method demonstrated that the method is suitable, specific, linear, accurate, precise, rugged, and stability indicating for the estimating of the assay, known and degraded impurities of Doxycycline active and Doxycycline for Injection.