a) Why does the mass spectral quantification of a compound need a calibration of signal versus concentration to be made if an internal standard is not available?
Ionization efficiencies vary from compound to compound. Although these variations are comparatively small as long as compared within a homologous series of compounds, they can be extremely large when different compound classes are compared. Therefore, realistic quantification results are only obtained, if the system is calibrated prior to analysis under fully identical conditions.
b) What are the advantages of internal standardization as compared to external calibration?
During elongated measurements the sensitivity of a chromatography-mass spectrometry system can shift resulting from contamination of injector or ion source, changing performance of the chromatographic column, etc. Furthermore, chemical noise may vary widely from sample to sample and clean-up procedures prior to the measurement can suppress the analyte of interest. An internal standard is added before clean-up, and thus suffers the same alterations as the analyte.
c) Which type of compounds represents the ideal internal standard? Why?
Isotopically labeled compounds are best suited for internal standardization. They can be assumed to have almost identical ionization efficiency, have almost identical retention times in chromatography, and are well separated from the analyte due to the mass shift from isotopic labeling.