Validation of a shielded-hydrophobic-phase high-performance liquid chromatography method for the determination of residual methotrexate in recombinant protein biopharmaceuticals

A shielded-hydrophobic-phase (SHP) HPLC method for the determination of res idual methotrexate in recombinant protein biopharmaceuticals was validated. The method requires no removal of protein or other prior sample "clean-up" and detects quantities of methotrexate as low as 2.5 ng in the presence of up to 25 mg/ml of protein. Methotrexate was fully resolved from a recombin ant IgG1 monoclonal antibody and associated matrix components. Accuracy was demonstrated by measuring "spiked" recoveries at the limit of quantitation (found 90-120% recovery with R.S.D.s less than or equal to 10%). Other val idation parameters studied included range, precision, ruggedness, robustnes s and stability of "spiked" samples. (C) 1998 Published by Elsevier Science B.V. All rights reserved.