内标法及外标法方法、原理、优缺点.docVIP

An internal standard should be used when performing MS quantitation. An appropriate internal standard will control for extraction, HPLC injection and ionization variability. In a complex matrix it is not uncommon for two different standard levels in SRM integrated plots, at the lower end of the standard curve, to give nearly an identical response. It is only when an internal standard is used that the two points can be differentiated. Some researchers attempt to prepare standard curves and run samples without an internal standard and find moderate success. Often without an internal standard % RSDs of replicates can be as high as 20%. Using an internal standard the % RSDs can be brought down to approximately 2%. We run triplicates at each level of our standard curve. How do I choose an internal standard? The best internal standard is an isotopically labeled version of the molecule you want to quantify. An isotopically labeled internal standard will have a similar extraction recovery, ionization response in ESI mass spectrometry, and a similar chromatographic retention time. If you are performing non-clinical PK quantitation it may be difficult to justify such a standard since a special synthesis of an isotopically labeled standard can be expensive and time consuming. Often if you are working with medicinal chemists they will have a library of compound analogs that can be used as internal standards. These analogs were made in the evolution of the compound to be tested and will be similar to the compound to be quantified and more importantly will be slightly different by parent mass. Try to avoid using de-methylated (-14) or hydroxylated (+16) analogs as internal standards since these are the most common mass shifts observed in naturally occuring metabolites of the parent compound. A common internal standard is a chlorinated version of the parent molecule. A chlorinated version of the parent molecule will commonly have a similar chromatographic retention time which is a