Development and Validation of A High-Performance Liquid Chromatography- Mass Spectroscopy Assay for the Bioequivalence Study of Guanfacine Extended Release (GXR) Tablet in Human Plasma

Guanfacine, an α2-adrenergic (α2A) agonist long indicated to treat hypertension, is now being used to treat attention deficit hyperactivity disorder (ADHD) in adolescents. A simple, rapid high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method to detect and quantify guanfacine provides a basis for studying its bioequivalence and pharmacokinetics in human plasma. This assay involves quantitation of guanfacine using its stable isotope-labeled internal standard (IS) guanfacine-¹³C-¹⁵N3 without the impact of ion suppression in the plasma matrix. Electrospray Ionization (ESI) in positive mode and Multiple Reaction Monitoring (MRM) was used for guanfacine and guanfacine-¹³C-¹⁵N3 at the transitions m/z 246.1→60.1 and m/z 250.0→159.1 respectively. This method's sample preparation is optimized with an accurate and simple protein precipitation method employing methanol. Linearity was demonstrated within the range of 0.0500-10.0 ng/mL for guanfacine in plasma with correlation coefficients greater than 0.99. The method showed excellent reproducibility. The Aaccuracy (Relative Error) ranged from -2. to 8.9%, even at the lower limit of quantification (LLOQ), and total precision, expressed as the coefficient of variation was between 1.6% and 10.5%. The average recoveries of guanfacine at three spiked levels of 0.150, 1.00 and 7.50 ng/mL were 103.93, 97.91 and 100.22%, respectively. The validated method was applied successfully to a bioequivalence study of a fixed-dose of extended-release guanfacine hydrochloride (GXR) tablet (Test formulation) and Intuniv® (Reference formulation) in healthy Chinese subjects, 42 subjects under fasting conditions and 30 subjects under feeding condition. Pharmacokinetic parameters were calculated using DAS 3.2.8, and 90% confidence intervals (CIs) of AUC0-t, AUC0-∞ and Cmax for guanfacine were all within 80.00- 125.00%, suggesting that the two formulations were bioequivalent in terms of rate and extent of absorption.