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Bioequilalence Studies Decoded

06 Apr 2023

Simpler, Swifter & Smarter In-vitro methods for Bioequivalence Studies, DECODED

A novel analytical assay of in-vitro bile acid binding studies to Colesevelam Hydrochloride tablets:An ultra-high performance liquid chromatography tandem mass spectrometric method

Colesevelam hydrochloride is a novel non-absorbed, lipid lowering polymer that binds bile acids in the intestine, obstructing their reabsorption and it is largely used for the treatment of elevated LDL cholesterol in patients with primary hyperlipidemia as well as to improve glycemic control in patients with type 2 diabetes. Colesevelam is a water insoluble polymer that is not hydrolyzed by any digestive enzymes and forms an insoluble complex with bile acids which is not absorbed in intestines and is excreted entirely through the gastrointestinal system. As drug is not absorbed into the systemic circulation, in vivo measurement is not possible and hence pharmacokinetic information is not available.

An in-vivo BE study in its traditional way is not a practical approach to exhibit the efficacy of Colesevelam tablets. A draft guidance has already been developed by FDA to offer direction to industry for such kind of drugs and suggested to perform in-vitro equilibrium binding study and kinetic binding study. Analytes to measure (in appropriate biological fluid): Unbound bile salts [glycocholic acid (GCA), glycochenodeoxycholic acid (GCDA) and taurodeoxycholic acid (TDCA)] in filtrate (to calculate bile salts bound to resin)

Bio-analytical method:
A simple, precise and accurate, reverse-phase, ultra-performance liquid chromatography tandem mass spectrometric method was developed for the quantitative determination of bile acids [glycocholic acid (GCA), glycochenodeoxycholic acid (GCDA) and taurodeoxycholic acid (TDCA)] in in-vitro bile acid-binding study of Colesevelam HCl tablets. The separation was achieved using Poroshell EC C18 2.7µm 4.6 x 100mm column with mobile phase containing solvent A consists Methanol and solvent B consists 2mM Ammonium formate buffer (80:20) with total run time of 6.0 minutes. The detection was performed in negative ion mode and quantitation was performed by multiple reaction monitoring mode having m/z 464.5 > 74.0 for GCA, 448.4 > 74.0 for GCDA and 498.5 > 80.1 for TDCA with dwell time 300 msec per transition.

Design of In-vitro bioequivalence study for Colesevelam Hydrochloride tablets


As per USFDA guidance this in-vitro study was carried out in 2 parts, i.e. Equilibrium Binding Study and Kinetic Binding Study

No biological matrix and internal standard was used in the estimation of bile acids. Calibration curves were found to be linear in equilibrium binding study (GCA + GCDA + TDCA) 0.050-30.000 mM [GCA (0.022 - 12.750 mM), GCDA (0.022-12.750 mM), TDCA (0.008-4.255 mM)] as well as in kinetic binding study (GCA + GCDA + TDCA) 0.050-3.000 mM (used for 3.0mM) and 0.050-0.300 (used for 0.3mM).

A rapid, reproducible, simple, specific, sensitive and highly separation efficient liquid chromatography tandem mass spectroscopy method is now available which was effectively applied in conducting in-vitro bioequivalence study as per FDA guidance.

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