Liquid chromatography instrumentation

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

The system includes the following components:

Agilent 1290 Infinity II Multisampler (G7167B)
Agilent 1290 Infinity II High-Speed (binary) Pump (G7120A)
Agilent 1290 Infinity II Multicolumn Thermostat (G7116B)

Considerations

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 1.0 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 0.2 μm frit (part number 5067-1603) can prevent any precipitates from building up in the system.

Plumbing

Reducing extra-column volume ensures run times are reduced. The plumbing requirements for a single column setup is listed below.

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	High pressure seat assembly 0.075 mm	G4267-87020
tubing to analytical head	Capillary SST 0.12x50mm long Socket	5500-1243
sample loop assembly	20ul loop assembly	G4226-60310
tubing to column compartment	Quick Turn capillary stainless steel 0.075 x 105 mm	5500-1198
in-line filter (including frit)	InfinityLab quick change inline filter (0.2 µm)	5067-1603
heat exchanger	InfinityLab Quick-Connect heat exchanger, ultralow dispersion. 0.075 mm id, 1 µL	G7116-60021
connection to column	InfinityLab Capillary ST 0.075 mm x 105 mm with Quick Connect fitting	5500-1174
connection from column	Quick Turn LC Fitting	5067-5966
peek tubing to MS	Capillary ST 0.075 mm x 500 mm, long socket	5500-1205

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 900 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	200 °C
Drying Gas (Nitrogen)	17 L/min
Nebulizer Gas	20 psi
Sheath Gas	280 °C
Sheath Gas Flow	10 L/min
Capillary Voltage	3,500 V (+), -2,500 V (-)
Nozzle Voltage	1,000 V (+), -1,000 V (-)
iFunnel High/low pressure RF	210/160 V (+), 200/160 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	900 ms
Time Filter Width	0.07 min
Total number of MRMs	938 (positive: 925, negative: 13)

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.