Simultaneous Determination of Methotrexate and Sulfa- salazine in Plasma Using an Agilent 1290 Infinity LC System

This Application Note describes an HPLC/DAD method for the simultaneous determination of methotrexate (MTX) and sulfasalazine (SSZ) in plasma using an Agilent 1290 Infinity LC System. MTX and SSZ are used in combination for the treatment of rheumatoid arthritis. Using two different detector wavelengths, 304 nm for MTX and 358 nm for SSZ, we were able to selectively quantitate both analytes during the same chromatographic run. The method employed off-line SPE sample extraction. The chromatography was performed using an Agilent Poroshell Extend-C18 column 3.0 mm × 150 mm, 2.7 μm, and gradient elution using methanol (containing 10 mM ammonium acetate with 0.1 % formic acid) and water (containing 10 mM ammonium acetate with 0.1 % formic acid). The method has a Lower Limit of Quantification (LLOQ) of 0.02 ng/μL for MTX and 0.1 ng/μL for SSZ. The 15 minute method is linear for MTX and SSZ for a concentration range of the LLOQ to 100 ng/μL with a R2 coefficient > 0.998. The method was validated, and the results are presented, using quality control samples for critical analytical performance criteria of recovery, stability, reproducibility, selectivity, accuracy, and precision.