Development, Validation, and Application of a New Method To Correct the Nonlinearity Problem in LC-MS/MS Quantification Using Stable Isotope-Labeled Internal Standards

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become an indispensable tool for bioanalysis. To quantify a small molecule with LC-MS/MS, a stable isotope-labeled analyte is routinely used as the internal standard. However, cross signal contributions between the analyte and its stable isotope labeled internal standard (SIL-IS) could cause problems when the signal response of the LC-MS/MS system is nonlinear. In the present work, we try to illustrate how the “cross talk” between the analyte and its SIL-IS may cause problems for a nonlinear system. We assume that the instrumental responses toward the analyte and its SIL-IS are the same. When the calibration curve is nonlinear, the addition of a SIL-IS would practically move the response of the analyte up along the parabolic line causing a change in the signal strength of the analyte (usually decrease). The more the SIL-IS is added, the larger change the analyte signal would become. Such a problem would only be corrected by making the calibration curve linear. To this end, we proposed a component equation (CE) as the calibration for nonlinearity correction. In this study, we contrasted the accuracy of CE with the common quantitative method using two drugs whose mass spectrometric responses are linear and nonlinear, respectively. The acceptable accuracy results demonstrated that the CE calibration was comparable with the regular quantitative SIL-IS method with a proper weighting factor and much better than that without weighting. Therefore, CE calibration may provide another reliable way for LC-MS/MS quantification.