Enantiomeric analysis of methotrexate in pharmaceuticals by cyclodextrin-modified capillary electrophoresis

A capillary electrophoresis for the chiral separation of racemic methotrexate (rac-MTX) was developed and validated. The two enantiomers were separated by using fused-silica capillary and a running buffer containing phosphate and hydroxypropyl-β-cyclodextrin (HP-β-CD). Several parameters were studied, including concentration and pH of phosphate buffer, separation voltage, and type and concentration of CD. The quantitative ranges were 12.5–200.0 μM for each enantiomer. The intra- and inter-day relative standard deviations (R.S.D.) and relative errors (R.E.) (n=5) were all <5%. The detection limits were found to be about 4 μM (S/N=3, injection 5 s) at 280 nm. All recoveries were greater than 93%. This method was applied to the assay of l-MTX in pharmaceuticals.