Development and Validation of Highly Selective LC-MS/MS Method for the Simultaneous Estimation of Pioglitazone, Hydroxy Pioglitazone and Glimepiride in in Rat Plasma
Kalyani Lingamaneni* and Mukthinuthalapati Mathrusri Annapurna
Department of Pharmaceutical Analysis and Quality Assurance, Gandhi Institute of Technology and Management (Deemed to be University), GITAM Institute of Pharmacy, Visakhapatnam, Andhra Pradesh, India
*Corresponding Author: Kalyani Lingamaneni, Department of Pharmaceutical Analysis and Quality Assurance, Gandhi Institute of Technology and Management (Deemed to be University), GITAM Institute of Pharmacy, Visakhapatnam, Andhra Pradesh, India.
July 30, 2021; Published: August 04, 2021
The rapid, sensitive, selective and precise liquid chromatography-electrospray ionization-tandem mass spectrometry method (LC-ESI-MS/MS) for the simultaneous estimation and pharmacokinetic investigation of Pioglitazone, Hydroxy pioglitazone (M-IV), a key metabolite of pioglitazone; and glimepiride in rat plasma has been developed and fully validated. Pioglitazone and Glimepiride exert synergistic effects on blood glucose control, were investigated in rat plasma using Rosiglitazone as internal standards (IS). Solid phase extraction was carried out on 0.32 mL of rat plasma using Strata-X 33 mm polymeric sorbent cartridges, and chromatographic separation was performed on an Agilent Zorbax SB C18 column (50 mm x 4.6 mm, 3.5 μm) using a mobile phase consisting of an 80:20 (v/v) mixture of acetonitrile and 10mM ammonium formate (pH: 3.0 + 0.05) at a flow rate of 0.8 mL/min. To quantify Pioglitazone, Hydroxy pioglitazone, Glimepiride and IS multiple reaction monitoring (MRM) transitions of m/z 357.0 → 134.1, m/z 373.0 → 150.1, m/z 491.2 → 352.2 and m/z 358.3 → 135.2 respectively were performed in positive mode. The total run time was 3.5 min and the elution time was about 2.8 min. The method exhibited good separation of analytes, without interference from endogenous substances. The method has been validated according to the USFDA guidelines for bioanalytical method validation. A linear response was observed over the range of 2 - 4100 ng/mL for Pioglitazone 1 - 1800 ng/mL for hydroxy pioglitazone and 3 - 6400 ng/ml for Glimepiride with satisfactory precision and accuracy. The lower limit of quantification (LLOQ) was 2, 1 and 3 ng/Ml for Pioglitazone, hydroxy pioglitazone and Glimepiride respectively. The percent relative error (%RE) of accuracy studies at three quality control levels was in the range of -3.33 to 3.60% for PIO, 0.35 to 0.55% for OH-PIO and -0.83 to 0.53% for GLM at Intra-day and inter-day precision studies respectively. The precision %RSD values were less than 4.01% for PIO, 5.23% for OH-PIO and 1.78% for GLM at overall comparison of both intra-day and inter-day studies. The method was reproducible and sensitive enough to quantitative Pioglitazone, hydroxy pioglitazone and Glimepiride in rat plasma samples of a preclinical pharmacokinetic study. Due to the potential scope of pioglitazone-Glimepiride combination to be therapeutically explored, this method is expected to have significant usefulness in future.
Keywords: Pioglitazone; Glimepiride; Hydroxy Pioglitazone; Method Validation; LC-MS/MS; Rat Plasma; Pharmacokinetic Applicability
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