Title:HPLC Stability Indicating Assay Method for Metformin Hydrochloride in Bulk Drug and Tablets and Cytotoxicity of Degradation Products
Volume: 8
Issue: 4
Author(s): Fernanda Cristina Stenger, Luciana Catia Block, Rogerio Correa, Rilton Alves de Freitas and Tania Mari Belle Bresolin
Affiliation:
Keywords:
Bulk Drug, Cytotoxicity, Degradation Products, HPLC-UV, Ion-pair liquid chromatography, Metformin hydrochloride,
Method validation, MTT assay, Stability-indicating assay method, Tablets
Abstract: The present work reports a stability-indicating reversed phase ion pair liquid chromatography method for quantitative
determination of metformin hydrochloride (MTF) (drug and tablets) and assessment of the cytotoxicity of degraded
MTF. Chromatographic separation was performed on a C18 (Phenomenex® Luna), 5.0 μm (250 mm x 4.6 mm)
column using isocratic elution. The optimized mobile phase consisted of 10 mmol L-1 heptane sulphonic acid (pH 3.0),
acetonitrile and methanol (75:8:17 v/v), flow 1.0 mL.min-1, at 30 ºC. The eluted compounds were monitored at 210 nm
wavelength using a DAD detector. The stability indicating capability of the developed method was established by analyzing
forced degradation samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of
MTF was ascertained, along with the separation of degradation products from the analyte peak. The cytotoxicity of MTF
and the degraded drug was analyzed in the L929 cell test by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. The 500 mg of commercial MTF tablets (Reference, Generic and Similar drugs) showed a similar chromatographic
profile, but lower intensity degradation in the same conditions, in relation to the bulk drug in almost forced
conditions. Neither the degraded drug samples, nor the tablet samples degraded in acid conditions, showed any cytotoxicity.
The developed stability-indicating ion-pair HPLC method was fully validated according to the International Conference
on Harmonization (ICH) guidelines. The degraded sample of MTF showed no affect on cell viability, compared with
the non-degraded drug.
