Ultra Performance Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry for the Absolute Quantification of Proteins and Peptides

A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography−isotope dilution tandem mass spectrometry (UPLC−ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography−isotope dilution tandem mass spectrometry (HPLC−ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C₁₈ reversed-phase column (50 × 2.1 mm i.d., 1.7-μm particle size) with gradient elution at a flow rate of 300 μL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C₁₈ column with dimensions of 150 × 1.0 mm at a flow rate of 30 μL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 10−90 fmol/μL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC−ID/MS/MS method, thus demonstrating performance equivalent to conventional HPLC−ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and high-sample throughput, which expands laboratory emergency response capabilities over conventional HPLC.