A Rapid, Rugged and Sensitive Method to Measure Therapeutic Concentrations of Dextromethorphan, Dextrorphan, Chlorpheniramine and Pseudoephedrine in Human Plasma by LC-MS-MS
Authors
C. Buggé, K. Adam, A. Ljungqvist, M. Sullivan, J. Niemeyer
Purpose
Develop a method to measure dextromethorphan (DM), dextrorphan (DR), chlorpheniramine (CP) and pseudoephedrine (PE) in human plasma following oral dosing with new formulation cough/cold medicine. Challenges included four analytes, three internal standards (IS); and disparate quantitation ranges (40 pg/ml LLOQ for DM, 1000 ng/ml ULOQ for DR).
Methods
Human K3-EDTA plasma (0.2mL) containing DM, DR, CP and PE is fortified with levallorphan, brompheniramine and pseudoephedrine-D3 internal standards and extracted with ethyl acetate/hexane at pH 9. The extract is injected directly onto a Sciex API 4000 LC-MS-MS (positive-ion MRM) equipped with a 50x4mm CN column. The peak areas of analyte product ions are measured against those of the internal standards: m/z 272>147 DM and 258>199 DR against m/z 284>199 levallorphan; m/z 275>230 CP against m/z 319>274 brompheniramine; and m/z 166>115 PE against 169>115 PE-D3. Quantitation used 1/x2-weighted linear least squares regression lines generated from freshly spiked calibration samples.
Results
Blank plasma extract chromatograms were free of interferences at analyte and IS retention times. Linearity was established for DM: 40 pg/ml - 20 ng/ml; DR: 2 - 1000 ng/ml; CP: 80 pg/ml - 40 ng/ml; and PE: 0.8 - 400 ng/ml. Inter-/intra-day precision and accuracy of LLOQ and QC samples were better than 15% for all analytes. Different plasma lots did not affect assay precision, accuracy or specificity. Extraction recovery was >80% for all analytes and IS. Room temperature stability was acceptable to 40h. Benchtop (6h at RT) and freeze/thaw (4 cycles) stability indicated acceptable stability for all analytes. Stability at –20°C was established for 5.5 weeks (<15% change).
Conclusions
A rapid, rugged and sensitive method was developed and validated for simultaneous determination of DM, DR, CP and PE in human plasma, resulting in over 1000 clinical study samples being analyzed.