Estrone

Estradiol and estrone were quantified in human serum or plasma from pre and post-menopausal females and males using the validated approach.

Custom-made stably overexpressing hHSD17B13-Myc/DDK HEK293 cells
(LakePharma, Inc.) and estradiol were prepared in serum free Dulbecco’s
modified Eagle’s medium (DMEM) medium containing 10% heat inactivated
FBS, 1× Glutamax, and 1× sodium pyruvate; 24 h prior to
compound testing, 25 μL of a 0.4 × 10⁶ cells/mL
dilution were seeded on 384-well Microplate (culture plate, PerkinElmer,
Cat# 6007680). Compounds were serially diluted in 100% DMSO and 50
nL of the compound dilution were spotted on the preseeded cell plate
by a Labcyte Echo 55× (1% DMSO in the Assay) and incubated for
30 min at 37 °C in a humidified incubator (rH = 95%, CO₂ = 5%). After that incubation step, 25 μL of a 60 μM
estradiol dilution were added to each well of the microtiter plate
and incubated for 3 h at 37 °C in the humidified incubator. Finally,
20 μL of supernatant were taken and 2.5 μL of d₄-estrone as an internal standard (final conc.
50 nM) were added. To derivatize the analytes, 5 μL of Girard’s
Reagent P (6.5 mM final) dissolved in 90% methanol and 10% formic
acid were added to the samples followed by overnight incubation at
RT before adding 60 μL of dH₂O to increase the sample
volume for the RapidFire MS/MS measurements of estrone levels as described
previously. To exclude impact on cell viability as cause for reduced
estrone levels, we also performed a CellTiter-Glo Luminescent Cell
Viability Assay (CellTiter-Glo, Promega, Cat# G9242) with the remaining
cell samples of the initial assay plate, according to manufacturer
protocol. Luminescence was measured using a PHERAstar FSX (BMG Labtech,
Ortenberg, Germany).

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).