Typically for smaller sets of samples, a single column is sufficient, resulting in an analysis time of approximately 16 minutes per sample. However, if throughput needs to be improved, a dual column setup enables re-equilibration of one column while analyzing a sample on a second column, resulting in an analysis time of 13 minutes per sample. A diagram of the dual column setup is shown below.

Example of switching valve plumbing to allow automated alternating column regeneration

The liquid chromatography system is coupled to an Agilent 6490/6495C triple quadrupole LC/MS with an Agilent Jet Stream technology ionization source. Mass spectrometer and source parameters are shown below. Agilent Jet Stream source parameters were optimized using Agilent Source Optimizer software to balance sensitivity across all classes. A cycle time of 650 ms was chosen to maintain an adequate number of points across the peak for reliable quantitation.

MS instrument parameters

Dynamic multiple reaction monitoring

A critical aspect of this methodology is the use of dynamic multiple reaction monitoring (dMRM) within the mass spectrometer. For the instrument to measure the large number of lipid species in the extended lipidome, the specific retention time window within which each compound elutes must be defined, such that the instrument only scans for a given lipid species at specific points across the total sample run time. This retention time window varies for different MRMs and is dependent largely on differences in peak width. This approach improves the instrument duty cycle and extends the average dwell time per compound, as seen below.