title: Development and validation of an HPLC method for tetracycline-related USP monographs.
abstract: A novel reversed-phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4-epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H3 PO4 as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80-120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50-150% of the acceptance criteria specified in the individual USP monographs for 4-epianhydrotetracycline hydrochloride. The limit of quantification for 4-epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust.
authors.affiliation: Visiting Scientist, Research and Development Laboratories, US Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, MD, 20852-1790, USA.
authors.forename: Emad M
authors.initials: EM
authors.lastname: Hussien
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D004340:Drug Contamination; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013752:Tetracycline / Q000032:analysis* / Q000737:chemistry / Q000592:standards*
chemical_list: D013752:Tetracycline
keywords: US Pharmacopeia monographs; impurities; method development; reversed-phase HPLC; tetracycline hydrochloride