Rapid and efficient chiral method development for lamivudine and tenofovir disoproxil fumarate fixed dose combination using ultra-high performance supercritical fluid chromatography: A design of experiment approach

• Published in: Journal of Chromatography A 2020-08, Vol.1625, p.461257, Article 461257
• Main Authors: Kurmi, Moolchand, Jayaraman, Karthik, Natarajan, Saravanan, Kumar, Gandhi Santosh, Bhutani, Hemant, Bajpai, Lakshmikant
• Format: Article
• Language: English
• Subjects: Chiral separation; Design of experiment (DoE); Fixed dose combination (FDC); Lamivudine (3TC); Tenofovir disoproxil fumarate (TDF); Ultra-high performance supercritical fluid chromatography (UHPSFC)
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2020.461257

•First chiral method for separation of 6 stereoisomers of TDF and 3TC in FDC.•Use of UHPSFC with sub-2 micron particle size column for time efficient separation.•3 Step DoE strategy (selection, screening and optimization) for method development.•Use of I-optimal, Taguchi OA and CCD experimental designs to evaluate CMPs.•Final optimized method well aligned with regulatory requirements for quantification. Fixed dose combination (FDC) of tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) is one of the most preferred FDC for the treatment of acquired immunodeficiency syndrome (AIDS)/human immunodeficiency virus (HIV) infection. To the best of authors’ knowledge there are no reported methods for chiral purity estimation of both drugs simultaneously from a FDC. The current study was focused on the development of a single chiral method uisng supercritical fluid chromatography (SFC) for separation of stereoisomers of TDF and 3TC combination employing design of experiment (DoE) approach. Method development was planned in three steps by using different experimental designs for each step. I-optimal, Taguchi orthogonal array and face-centred central composite designs (CCD) were employed for primary parameter selection, secondary parameter screening and final method optimization, respectively. All six stereoisomers were separated in a 10 minute run on Chiralpak IA column with carbon di-oxide /methanol (containing 0.5 % v/v n-butylamine) as mobile phase at 1.5 mL/min in gradient mode. The optimized method was verified for performance through establishing specificity, precision, linearity, accuracy, limit of quantification, and solution stability. Resolution between each isomeric pair was more than 1.5. The method was found to be linear from 1.5 µg/mL to 7.5 µg/mL for 3TC and 7.5 µg/mL to 37.5 µg/mL for TDF stereoisomers. The R2 values for all the linearity curves for undesired isomers were greater than 0.995. The method proved to be rapid, reproducible and efficient to quantify stereoisomers of both drugs in a single run.