title: Ion-pair reversed phase liquid chromatography with ultraviolet detection for analysis of ultraviolet transparent cations.
abstract: This paper describes the use of an anionic ion-pair reagent (IPR) to impove the ultraviolet (UV) detection and hydrophobic retention of polar and UV transparent cations. Anionic IPR added to the mobile phase forms an ion-pair with cations. Formation of the ion-pair causes a redshift in the absorption wavength, making it possible for direct UV detection of UV-inactive cations. The ion-pairs with increased hydrophobicity were separated by reversed phase liquid chromatography (RPLC). Different perfluorinated caboxylic acids (trifluoroacetic acid, heptafluorobutyric acid, nonafluoropentanoic acid) were evaluted as IPR in the separation and detection of the common cations sodium, ammonium and Tris(hydroxymethyl)aminomethane (Tris). The effects of the IPR type and concentration on separation and detection have been investigated to understand the separation and detection mechanisms. The optimal separation and detection condtions were attained with mobile phase containing 0.1% nonafluoropentanoic acid and with the UV detection at 210nm. UV detection and charged aerosol detection (CAD) were compared in the quantitation of the cations. The limit of quantitation (LOQ) of sodium and Tris with UV detection is comparable to that by CAD. The LOQ of ammonium with UV detection (1ppm or 3ng) is about 20-fold lower than that (20ppm or 60ng) by CAD. The RPLC-UV method was used to monitor ammonium clearance during ultrafiltration and diafiltration in the manfucaturing of biopharmceutical drug substance.
authors.affiliation: Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA. Electronic address: Yan.He2@pfizer.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Cook
authors.affiliation: Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Littlepage
authors.affiliation: BioProcess R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
authors.forename: John
authors.initials: J
authors.lastname: Cundy
authors.affiliation: BioProcess R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
authors.forename: Ratish
authors.initials: R
authors.lastname: Mangalathillam
authors.affiliation: Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Jones
mesh_terms: D000838:Anions; D002412:Cations; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D005466:Fluorocarbons / Q000032:analysis; D057927:Hydrophobic and Hydrophilic Interactions; D014269:Trifluoroacetic Acid / Q000032:analysis
chemical_list: D000838:Anions; D002412:Cations; D005466:Fluorocarbons; C033094:perfluorobutyric acid; D014269:Trifluoroacetic Acid
keywords: Cations; Improve UV detection; Ion-pair RPLC