Use Guide to Internal Standards for Lipid Quantification by ESI-MS
Introduction
Cellular lipidomes are complex. The changes in the levels and/or composition of lipid species and/or classes occur after perturbation or during cell growth. Lipidomics was developed to identify and quantify these changes in order to study the function and interactions of lipids and to delineate the biochemical mechanisms of lipid changes under pathologic (physiological) conditions. Numerous modern technologies have been developed and applied to perform the lipidomics study, of which, recent advances on electrospray ionization mass spectrometry (ESI-MS) have greatly revolutionized the progress on lipidomics. Currently, ESI-MS based approaches are widely used in the field for absolute quantification of lipid species. When it comes to quantitative analysis, the use of internal standards is absolutely indispensable. Therefore, Alfa Chemistry has written a guide to use it for absolute quantification, and the main topics include what types of internal standards can be chosen, how many internal standards are needed, what amount is added, etc. It is our hope that the description presented herein can provide a foundation for absolute and accurate quantification and can serve as a reference for further expansion of one's knowledge in this important area.
Why Internal Standards Are Required for Accurate Quantification
In ESI-MS analysis, there is no definitive rule between the absolute intensity of an analyte ion and the solution concentration of analyte that yields the ion, and the ion intensity of an analyte measured with ESI-MS could be affected by many factors. Minor changes of these factors could lead to significant alterations in ion intensity from one condition to another. Thus, it is essentially impossible to replicate a measurement of the ion intensity of an analyte with ESI-MS, even from one operator to another in the same laboratory. Not to mention from an instrument to another instrument, from one laboratory to others. Therefore, to minimize the variations introduced at any step of analysis, addition of some kind of controls (e.g., analogs to the analytes of interest, i.e. internal standards) between the samples or between analyses becomes essential. In this manner, any unexpected changes of ion intensity during analysis can be either controlled internally or normalized.
What Kind of Lipid Species Can Be Used as Internal Standards
Internal standards should have identical chemical and physical properties to the analytes and to experience the identical experimental conditions from sample preparation to MS analysis. Thus, the ideal internal standard to quantify an analyte of interest is its stable isotope-labeled analog, which is nearly identical to the analyte and exhibits exactly the same response factor. In addition, Another primary requirement for used as an internal standard is the absence of any overlap of internal standard(s) with the endogenous species, or the overlapped endogenous species in very low abundance (e.g., <<1% of the most abundant species of the class) in lipid extracts.
How Many Internal Standards Are Necessary
The minimal number of internal standards that must be used for accurate quantification of species of a class varies from method to method, and largely depends on the existence of the variables in each method. Table 1 lists the minimal number of internal standards necessary for different methods [1].
Note: all the discussions are for quantification of individual species of a polar lipid class.
Table 1. Summary of variables present in lipidomics approaches and their required minimal number of internal standards for accurate quantification of a polar lipid class.
aMS, MDMS, and SL stand for "mass spectrometry", "multi-dimensional mass spectrometry" and "shotgun lipidomics", respectively.
How Much Individual Internal Standard Should Be Used
After knowing the minimum number of internal standards that must be added, the next question is how much of these internal standards are required. Generally, too much or too small of a number of internal standards both could lead to large experimental errors, and the added amount of internal standards must be optimized to make the relative intensity of the internal standard peak in the range of 20–500% in comparison to the ion peak that corresponds to the most-abundant species in the class.
Alfa Chemistry is a global leading supplier of analytical chemistry reagents. We provide a wide range of high quality internal standards and many other analytical chemistry products. If you cannot find the suitable products, Alfa Chemistry also offers you with custom synthesis service. If necessary, please don't hesitate to contact us.
Reference
- Wang M., et al. Selection of internal standards for accurate quantification of complex lipid species in biological extracts by electrospray ionization mass spectrometry—What, how and why?[J]. Mass spectrometry reviews, 2017, 36(6): 693-714.
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