Two-Step Quantification Approach Used in MDMS-SL

After separation of different lipid classes in the ESI source (i.e., intrasource separation) and MDMS identification of individual species (see Section 3.2.3.3), quantification of the identified individual species of a lipid class of interest is performed in a two-step procedure in MDMS-SL [11, 35]. This procedure can be conducted automatically [29]. 

It is worthy to note that when only two species, including the preselected internal standard, meet the criteria for selection as the standards for the second step of quantification in MDMS-SL, this second step of quantification is virtually identical to that used in tandem MS-based shotgun lipidomics with a linear standard curve [24]. In this case, the presence of different numbers of double bonds might affect the accurate quantification of those overlapping and/or low-abundant species performed in the second step of quantification. This effect is relatively small in MDMS-SL, especially for the total content of the class, with the following reasons (1) the selected, endogenously obtained standard usually contains a certain number of double bonds and (2) the species determined in the full MS mode in the first step of quantification largely contributes to the total content of the lipid class of interest. 

There is another big difference between the second step of quantification in MDMS-SL and that used in tandem MS-based shotgun lipidomics. All quantified 

Finally, MDMS-SL possesses other advantages in comparison to the tandem MS-based shotgun lipidomics. For example, the second step of quantification in MDMS-SL can apply any or all head group-related PIS and/or NLS of the lipid class, if present and sensitive enough, for quantification of individual species in the second step. This redundant process is very useful to refine the data and serves as an internal validation of the determined results. Moreover, with the second step of quantification, an over 5000-fold linear dynamic range for many lipid classes can be readily achieved [36] due to the second step of quantification serving as a relay for the dynamic range. 

Two caveats of this two-step quantification approach should be recognized. 

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