RapidFire-based Analytical Sample Handling

The analytical sample handling was performed
by a RapidFire autosampler system (Agilent, Waldbronn, Germany) coupled
to a triple quadrupole mass spectrometer (Triple Quad 6500, AB Sciex
Germany GmbH, Darmstadt, Germany). Liquid sample was aspirated by
a vacuum pump into a 10 μL sample loop for 250 ms and subsequently
flushed for 3000 ms onto a C18 cartridge for estrone and oxo-LTB₄ (Agilent, Waldbronn, Germany) and a C4 cartridge for retinal
with mobile phase A (for estrone: 99.9% water, 0.09% acetic acid,
0.01% TFA, flow rate 1.5 mL/min. For oxo-LTB₄: 1 L water
+ 50 μL of 25% NH₃, flow rate 1.5 mL/min. For retinal:
99.5% water, 0.49% acetic acid, 0.01% TFA, flow rate 1.5 mL/min).
The analyte was backflushed from the cartridge for 3000 ms with mobile
phase B (for estrone: 475 mL of methanol, 475 mL of ACN, 50 mL of
water, 90 μL of acetic acid, 10 μL of TFA, flow rate 1.25
mL/min. For oxo-LTB₄: 475 mL of methanol, 475 mL of acetonitrile,
50 mL of water, 50 μL of 25% NH₃, flow rate 1.5 mL/min.
For retinal: 49.75% methanol, 49.75% acetonitrile, 0.49% acetic acid,
0.01% TFA, flow rate 1.25 mL/min) and flushed into the mass spectrometer
for detection in MRM mode. The MRM transition for estrone-GP was Q1/Q3: 404.1/157.1 Da (declustering potential
27 V, collision energy 43 V) and for the internal standard D₄-estrone-GP Q1/Q3: 408.1/159.1
Da (declustering potential 27 V, collision energy 43 V). The mass
spectrometer was operated in positive ionization mode (curtain gas
35 Au, collision gas medium, ion spray voltage 4200 V, temperature
550 °C, ion source gas 1 65 Au, ion source gas 2 80 Au). The
MRM transition for the oxo-LTB₄ was Q1/Q3: 336/195.1 Da (declustering potential 27 V, collision
energy 43 V) and for the internal standard arachidonic acid Q1/Q3: 303.2/259.3 Da (declustering potential
27 V, collision energy 43 V). The MRM transition for retinal-GP3 was Q1/Q3: 418.3/94.9 (declustering potential
10 V, collision energy 23 V) and for the internal standard D₆-retinal-GP Q1/Q3: 424.3/136.9
(declustering potential 66 V, collision energy 39 V). Dwell time for
all analytes and each MRM transition was 25 ms and pause time between
MRMs was 5 ms. For oxo-LTB₄ and retinal: the mass spectrometer
was operated in negative ionization mode (curtain gas 35 Au, collision
gas medium, ion spray voltage 4200 V, temperature 550 °C, ion
source gas 1 65 Au, ion source gas 2 80 Au). The solvent delivery
setup of the RapidFire system consists of two continuously running
and isocratically operating HPLC pumps (G1310A, Agilent, Waldbronn,
Germany) and one binary HPLC pump channel B (G4220A, Agilent, Waldbronn,
Germany).