An on-line liquid chromatography/tandem mass spectrometry (LC-MS/MS) procedure, using the Prospekt-2 system, was developed and used for the determination of the levels of the active ingredients of cough/cold medications in human plasma matrix. The experimental configuration allows direct plasma injection by performing on-line solid phase extraction (SPE) on small cartridge columns prior to elution of the analyte(s) onto the analytical column and subsequent MS/MS detection. The quantitative analysis of three analytes with differing polarities, dextromethorphan (DEX), dextrorphan (DET) and guaifenesin (GG) in human plasma presented a significant challenge. Using stable-isotope-labeled internal standards for each analyte, the Prospekt-2 on-line methodology was evaluated for sensitivity, suppression, accuracy, precision, linearity, analyst time, analysis time, cost, carryover and ease of use. The lower limit of quantitation for the on-line SPE procedure for DEX, DET and GG was 0.05, 0.05 and 5.0 ng mL−1, respectively, using a 0.1 mL sample volume. The linear range for DEX and DET was 0.05–50 ng mL−1 and was 5–5,000 ng mL−1 for GG. Accuracy and precision data for five different levels of QC samples were collected over three separate days. Accuracy ranged from 90% to 112% for all three analytes, while the precision, as measured by the %RSD, ranged from 1.5% to 16.0%.
