Liquid chromatography instrumentation

Plasma samples are separated using an Agilent 1290 Infinity/Infinity II liquid chromatography system.


There are several considerations in running larger sets of samples without sensitivity loss. Solutions to these problems that we implement in our laboratory include:

Divert valve
To ensure cleaner instrumentation and avoiding sensitivity loss, consider diverting the first 0.8 minutes of the run - containing residual salts and polar compounds - into waste rather than the mass spectrometer. This time is dependent on the column volume of the LC setup and needs to be adjusted as necessary.
In-line frit
To maintain column longevity and avoid blockages, an inline filter with 0.3 μm frit (part number 5067-6189) can prevent any precipitates from building up in the system.


Reducing extra-column volume ensures run times are reduced. The plumbing requirements for a single column setup is listed below. Dual column setups will require appropriate customization.

Liquid chromatography tubing details for single column setup

Location Part description Agilent part number
binary pump mixer V35 JetWeaver mixer G7120-68135
tubing from pump to autosampler 0.17 mm ID, 600mm stainless steel tubing G1312-67305
needle needle G4226-87201
needle seat needle seat G4226-87012
tubing to analytical head Capillary SST 0.12x50mm long Socket 5500-1243
sample loop assembly 20ul loop assembly G4226-60310
tubing to column compartment Capillary SST 0.12x50mm long Socket 5500-1243
in-line filter (including frit) 1290 Infinity II inline filter (0.3um) 5067-6189
heat exchanger Standard flow heat exchanger G7116-60015
connection to column Quick Connect Assy ST 0.12x105mm 5067-5957
connection from column Quick Turn LC Fitting 5067-5966
peek tubing to MS 1/16" red stripe 14-6935-002

Dual column setup

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

dual column setup

Mass spectrometer instrumentation

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

Parameter Agilent 6495C Triple Quadrupole Mass Spectrometer
Ion source Agilent Jet Stream
Polarity Positive and Negative Switching
Gas Temperature 150 °C
Drying Gas (Nitrogen) 17 L/min
Nebulizer Gas 20 psi
Sheath Gas 200 °C
Sheath Gas Flow 10 L/min
Capillary Voltage 3,500 V (+), -3,000 V (-)
Nozzle Voltage 1,000 V (+), -1,500 V (-)
iFunnel High/low pressure RF 200/110 V (+), 150/60 V (-)
Scan Type Dynamic MRM (DMRM)
Q1/Q2 Resolution Unit (0.7 amu)
Delta EMV 100 V (+), 0 V (-)
Cell Acceleration Voltage 4-5 V
Cycle Time 650 ms
Time Filter Width 0.07 min
Total number of MRMs 665 (positive: 646, negative: 19)

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.

concurrent dMRMs