The 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.

Background:The present work is focused on developing a simple stability indicating method for the estimation of the assay of benzyl alcohol and glycopyrrolate in glycopyrrolate injection using reverse high performance liquid chromatography. Methods: A simple, selective, rapid, precise, and gradient reversed-phase high-pressure liquid chromatography procedure has been proposed to estimate the assay of benzyl alcohol and glycopyrrolate for glycopyrrolate injection. It was developed on a Kromasil 100-5, C 8 250 mm x 4.6 mm, 5 µm column using mobile phase-A, containing pH 2.5 potassium dihydrogen phosphate buffer and methanol in a ratio of 80:20 v/v. and mobile phase-B, containing pH 2.5 potassium dihydrogen phosphate buffer and methanol in a ratio of 10:90 v/v at a flow rate of 1.5 mL/min. The detection of glycopyrrolate and benzyl alcohol was carried out at 222 nm and 254 nm. A sharp peak was obtained for glycopyrrolate and benzyl alcohol at a retention time of about 8.4 and 4.8 min. Results:The method was validated for specificity (blank, system suitability solution, standard, and sample and stress samples for any interference), precision (six replicate sample preparations), robustness (by slightly varying critical method parameters), and solution stability, and the results were found satisfactory. Conclusion: The method is specific, precise, accurate, and robust for estimation of the assay of benzyl alcohol of glycopyrrolate in glycopyrrolate injection.