Development and Validation of a UHPLC Method for Paroxetine Hydrochloride

An ultrahigh-pressure liquid chromatography (UHPLC) method was developed to separate paroxetine from several of its related compounds using a systematic screening protocol that monitors combinations of selectivity factors including column chemistry, organic modifier, and pH.
Oct 01, 2007
Volume 25, Issue 10, pg 1042–1049



Paroxetine is a potent and selective serotonin (5-hydroxytryptamine) reuptake inhibitor. The activity of the drug on brain neurons is thought to be responsible for its antidepressant effects. Paroxetine hydrochloride is the active ingredient of Paxil, a prescription drug used to treat panic disorder, social anxiety disorder, generalized anxiety disorder, obsessive–compulsive disorder, posttraumatic stress disorder, renal–hepatic impairment, and depression (1). Any related compounds, excipients, or impurities that exceed peak areas greater than 0.1% of the active ingredient must be fully identified, explained, and quantified for ICH guidelines to be met (2).

An ultrahigh-pressure liquid chromatography (UHPLC) method was developed to separate paroxetine from several of its related compounds by utilizing a systematic screening protocol that monitors combinations of selectivity factors including column chemistry, organic modifier, and pH. After the best combination of these factors was selected, the method was optimized by varying gradient slope and temperature.

As shown by MacNair and colleagues (3,4), the use of extremely high pressures in LC can improve the efficiency and reduce analytical time for columns packed with small particles. High performance LC (HPLC) methods typically use columns packed with 3-μm particles or greater on pumps operating at pressures as high as 6000 psi, while UHPLC methods use 1.7-μm particle columns operating at pressures as high as 15,000 psi. The smaller particle columns and larger pressure capacity offer significantly improved speed and peak capacity (5). Using 1.7-μm particles and a holistically designed system provide significantly more resolution (information) while reducing run times and improving sensitivity for the analyses of many types of compounds (6). UHPLC methods take advantage of smaller particle sizes and higher linear velocities, which produce higher efficiency at faster optimum flow rates (7). The time savings combined with increase in sensitivity while maintaining resolution provide a tremendous advantage to using UHPLC instead of HPLC methods and columns.

The 2006 USPC, Inc. Official 8/1/06 – 4/30/07 USP Monographs: Paroxetine hydrochloride, uses three different chromatographic runs on three different analytical columns to separate related compounds B, D, F and G from the active pharmaceutical ingredient (8). The total analysis time using HPLC columns and technology is over 180 min. Equivalent or greater resolution of related compounds B, D, F, and G from paroxetine was achieved in a single chromatographic run in less than 5 min using an UHPLC system and a 50 mm × 2.1 mm,1.7-μm column.

The method was validated using method validation software. The software enabled method validation, experimental design, and statistical analysis of the data to be preformed completely within the software. Paroxetine was treated as a drug substance and validated as a purity assay in phase II of development.

This article addresses the successful development and partial validation of an LC method for the analysis of paroxetine and its related compounds.

Experimental

Materials: USP reference standards: USP Paroxetine Hydrochloride RS, USP Paroxetine System Suitability Mixture A RS, USP Paroxetine Related Compound B RS, USP Paroxetine Related Compound C RS, USP Paroxetine Related Compound E Mixture RS, USP Paroxetine Related Compound F RS, USP Related Compound G RS were all purchased from the United States Pharmacopeia (Rockville, Maryland). Water was purified with a Milli-Q system (Millipore, Billerica, Massachusetts). Acetonitrile, formic acid, and ammonium hydroxide were purchased from Fisher Scientific (Fair Lawn, New Jersey). Ammonium bicarbonate and ammonium formate were purchased from Sigma-Aldrich (St. Louis, Missouri).


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