title: Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography.
abstract: Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of L-MTX and its enantiomeric impurity D-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10(-7) M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of L-MTX. Furthermore, L-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples.
authors.affiliation: Department of Biomolecular Analysis & Spectroscopy, LaserLaB, VU University Amsterdam, the Netherlands.
authors.forename: María
authors.initials: M
authors.lastname: Castro-Puyana
authors.forename: Ivonne
authors.initials: I
authors.lastname: Lammers
authors.forename: Joost
authors.initials: J
authors.lastname: Buijs
authors.forename: Cees
authors.initials: C
authors.lastname: Gooijer
authors.forename: Freek
authors.initials: F
authors.lastname: Ariese
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000302:isolation & purification*; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D005493:Folic Acid Antagonists / Q000302:isolation & purification*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008727:Methotrexate / Q000302:isolation & purification*; D004364:Pharmaceutical Preparations / Q000737:chemistry; D013237:Stereoisomerism
chemical_list: D000964:Antimetabolites, Antineoplastic; D005493:Folic Acid Antagonists; D004364:Pharmaceutical Preparations; D008727:Methotrexate