Stability indicating validated HPLC method for quantification of levothyroxine with eight degradation peaks in the presence of excipients.
A simple, sensitive, accurate, and robust stability indicating analytical method is presented for identification, separation, and quantitation of l-thyroxine and eight degradation impurities with an internal standard. The method was used in the presence of commonly used formulation excipients such as butylated hydroxyanisole, povidone, crospovidone, croscarmellose sodium, mannitol, sucrose, acacia, lactose monohydrate, confectionary sugar, microcrystalline cellulose, sodium laurel sulfate, magnesium stearate, talc, and silicon dioxide. The two active thyroid hormones: 3,3',5,5'-tetra-iodo-l-thyronine (l-thyroxine-T4) and 3,3',5-tri-iodo-l-thyronine (T3) and degradation products including di-iodothyronine (T2), thyronine (T0), tyrosine (Tyr), di-iodotyrosine (DIT), mono-iodotyrosine (MIT), 3,3',5,5'-tetra-iodothyroacetic acid (T4AA) and 3,3',5-tri-iodothyroacetic acid (T3AA) were assayed by the current method. The separation of l-thyroxine and eight metabolites along with theophylline (internal standard) was achieved using a C18 column (25 degrees C) with a mobile phase of trifluoroacetic acid (0.1%, v/v, pH 3)-acetonitrile in gradient elution at 0.8 ml/min at 223 nm. The sample diluent was 0.01 M methanolic NaOH. Method was validated according to FDA, USP, and ICH guidelines for inter-day accuracy, precision, and robustness after checking performance with system suitability. Tyr (4.97 min), theophylline (9.09 min), MIT (9.55 min), DIT (11.37 min), T0 (11.63 min), T2 (14.47 min), T3 (16.29 min), T4 (17.60 min), T3AA (22.71 min), and T4AA (24.83 min) separated in a single chromatographic run. Linear relationship (r2>0.99) was observed between the peak area ratio and the concentrations for all of the compounds within the range of 2-20 microg/ml. The total time for analysis, equilibration and recovery was 40 min. The method was shown to separate well from commonly employed formulation excipients. Accuracy ranged from 95 to 105% for T4 and 90 to 110% for all other compounds. Precision was <2% for all the compounds. The method was found to be robust with minor changes in injection volume, flow rate, column temperature, and gradient ratio. Validation results indicated that the method shows satisfactory linearity, precision, accuracy, and ruggedness and also stress degradation studies indicated that the method can be used as stability indicating method for l-thyroxine in the presence of excipients.
Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Sciences, Center for Drug Evaluation and Research, Food and Drug Administration, USA.
R B
RB
Shah
A
A
Bryant
J
J
Collier
M J
MJ
Habib
M A
MA
Khan
D002138:Calibration; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D004340:Drug Contamination; D004355:Drug Stability; D005079:Excipients; D006868:Hydrolysis; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D013974:Thyroxine / Q000008:administration & dosage / Q000032:analysis*
D005079:Excipients; D013974:Thyroxine