LAUSR

Tandem mass spectrometry (MS/MS)2, often combined with liquid chromatography (LC-MS/MS), has emerged as an extremely versatile platform for quantification of analytes in complex biological samples. The method is rendered highly quantitative by use of a chemically identical but isotopically differentiated internal standard. Such an internal standard corrects for losses of analyte signal in the assay owing to sample handing or MS/MS signal loss because of suppression of analyte ionization in the electrospray source. At a minimum, the true concentration of the analyte in a stock solution should be known as accurately as possible. One can then inject an absolute number of moles of analyte into the instrument (the true moles), together with an aliquot of a solution of internal standard, and determine the ratio of analyte to internal standard MS/MS response (response ratio). It is not required that the true moles of internal standard in the stock solution be accurately known. Even if a truly equimole mixture of analyte and internal standard is analyzed, the response ratio may not be 1 because of biases in the tuning of the quadrupole mass filters and/or an isotope effect on the fragmentation of the parent ion to give the product ions (no isotope effect is expected if a bond to the heavy isotope does not break in the fragmentation reaction). Thus, the measured response ratio will reflect the true difference in absolute moles of analyte and internal standard analyzed, tuning bias, and any isotope effects on fragmentation. For example, if one injected truly 1 nmol of analyte and nominally 1 nmol of internal standard in the MS/MS instrument and observed a response ratio of 1.2, one can obtain the true moles of analyte in a new sample via the following equation: ![Formula][1] The above equation is valid even if the absolute moles … [1]: /embed/mml-math-1.gif