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.FigureA quenched phosphorescence method was developed and coupled to chiral EKC for the sensitive detection of the drug L-methotrexate and its impurity D-MTX; unspiked pharmaceutical formulation

Analytical methodologies for determination of methotrexate and its metabolites in pharmaceutical, biological and environmental samples

“…CE using low amount of sample, cheap capillaries, high speed, and high resolution and efficiency has been proposed as another technique to separate MTX in different matrices, by Castro-Puyana et al., Cheng et al., and Guichard et al. [[115], [116], [117], [118]]. Due to the importance of cerebrospinal fluid (CSF) during chemotherapy and relapse, the evaluation of MTX and its metabolites in CSF was performed by triple-stacking CE [116].…”

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis

“…Only few report the use of electromigration techniques for the determination of the stereochemical purity of drug substances. Recent examples are summarized in Table . In the majority of the cases, LODs of 0.1% or below could be realized.…”

New approaches in sensitive chiral CE

“…However, it is necessary to say that it has not been well established for chiral analysis to over the years. Only one method for the chiral separation of methotrexate has been developed in the last two years . In this EKC method with indirect phosphorescence detection (quenched phosphorescence), a strong phosphorophore compound (1‐bromo‐4‐naphthalenesulfonic acid) was directly excited and its phosphorescence quantum yield was reduced by biomolecular quenching process with an analyte molecule, which lead to negative peaks in the electropherogram.…”