A stability‐indicating, reversed‐phase HPLC method for quantification of assay and organic impurities in doxycycline hyclate bulk and parenteral dosage forms

Abstract

AbstractThe aim of this study is to develop a stability‐indicating, reversed‐phase HPLC method for the quantification of assay and organic impurities (process and degradation) of doxycycline hyclate in a doxycycline injectable formulation. Both the active and dosage forms are officially present 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, considering the quality control requirements and user‐friendliness. A simple stability‐indicating HPLC method with a shorter run time was developed for the simultaneous quantification of assay and impurity. The method was developed using HPLC with a gradient program and a reversed‐phase Waters XBridge BEH C8 column (150 × 4.6 mm, 3.5 μm i.d.). Mobile phase A consisted of phosphate buffer (pH 8.5, 25 mM potassium phosphate, 2 mM ethylenediaminetetraacetic acid, and 0.5 ml of triethylamine). Mobile phase B consisted of methanol with a flow rate of 1.7 ml/min, a column temperature of 55°C, a UV wavelength of 270 nm, and an injection volume of 25 μl. Modern research represents a concomitant method for quantifying assay and organic impurities of doxycycline hyclate (active form) and doxycycline for injection (dosage form). The assay and impurity method were validated per United States Pharmacopeia (USP) 1225 and International Conference on Harmonization (ICH) guidelines. The retention time of doxycycline and degradation impurity, 4‐epidoxycycline, was about 9.8 and 6.4 min, respectively. The linearity range of doxycycline and 4‐epidoxycycline was 0.5–150 and 0.5–18 μg/ml, respectively. The percentage of recovery of doxycycline and 4‐epidoxycycline was 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 estimating the assay, known and degraded impurities of doxycycline, and doxycycline for injection.