Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography |
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Authors: | María Castro-Puyana Ivonne Lammers Joost Buijs Cees Gooijer Freek Ariese |
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Institution: | (1) Department of Biomolecular Analysis & Spectroscopy, LaserLaB, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, the Netherlands; |
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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. |
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