Novel voltammetric method for enantioseparation of racemic methotrexate - Determination of its enantiomeric purity in some pharmaceuticals

A carbon paste electrode is modified by hydrophobic molecules of hydroxypropyl P-cyclodextrin to form enantioselective membrane sensor. In neutral aqueous media, methotrexate (Mtx) enantiomers have been selectively accumulated at this enantioselective sensor depending on their hydrophilic/hydrophobic balance and then they can be quantified by voltammetric oxidation. The effect of solution and instrumental parameters on rac-Mtx enantioresolution is investigated by using window diagram approach. The optimal enantioresolution (R = 2.96) is achieved at pH 6.4 in phosphate buffer by applying osteryoung square-wave anodic stripping voltammetry. The obtained resolution is sufficient to evaluate distomer D-Mtx at 0.1% (m/m of L-Mtx, the main compound). Selectivity of the proposed procedure is estimated by testing recovery and adding the most interfering metal ions and/or organic compounds. The enantioselective sensor is satisfactorily used to investigate simultaneously ssDNA interactions with L-Mtx and D-Mtx. It is observed that the stability Of D-Mtx-ssDNA diasteriomeric complexation is higher than that Of L-Mtx-ssDNA. These results are crucial in medical chemistry for investigating many phenomena concerned with the carcinogenic effect of methotrexate. Using the commercially available certified reference materials Of L-Mtx and D-Mtx, the proposed enantioselective approach is validated to determine Mtx enantiopurity in its tablets and injections with recovery values falling within the labeled amount of 90-110% with R.S.D. less than 6.00% required by US Pharmacopeia. Furthermore, high accuracy for application of the method to formulations is achieved and statistically confirmed by calculating F-test and t-test values at 95% confidence level. (c) 2005 Elsevier B.V. All rights reserved.