Analysis of Methotrexate from Formulations and Skin Specimens by HPLC

FRED L. MA, WEIPING WANG, JANE ZHENG, R.N.,GENEVA TSAO, STEVE JOHNSEN, ZHUHUI CAI AND DEAN S. HSIEH

Conrex Pharmaceutical Corporation 300 Kimherton Road, Suite 120, Phoenixville, PA 19460

ABSTRACT

   A method for quantitative analysis of methotrexate (MTX) from formulated gels/creams and from tissue/solution samples after percutaneous permeation studies is described, High performance liquid chromatography (HPLC) with a MicroBondapak C18, reverse-phase column was used. A calibration curve of doses versus peak areas for MTX showed linearity over the range of 0.010-14.056ug with a very high correlation coefficient (r) of 0.99998. Replicate analysis of ug of MTX showed that the peak area was 9627424±22111 counts ( mean ±SD; one count is 1/8 uV-sec , 1uV is 2x10-6a.u.) with a relative standard deviation of 0.23%. The resolution factor between MTX and folic acid was 33.35 when the applied doses of both were 1 ug each. Results demonstrated that the photolytic products of MTX, samples from gel and samples extracted from hairless guinea pig skin after percutaneous permeation and epidermis/dermis separation by microwave technique, could be well detected by this method, Preliminary results are satisfactory and meet the requirements of U.S. Pharmacopeia XXII(1). This report decribes novel approaches for determination of methotrexate content in the formulations and skin specimens. These approaches include the separation of epidermis and dermis by microwaving, the pretreatment procedures employed before analysis, and the use of guard column during HPLC analysis.

Key words: Methotrexate, Percutaneous Absorption, In Vitro, In Vivo.