LC-HRMS analysis was
performed on a Waters Synapt XS HDMS coupled
with an ACQUITY UPLC I-Class system. Separation was carried out on
the Acquity Premier CSH C18 column (150 mm × 2.1 mm, 1.7 μm).
Mouse urine (25 μL) was diluted (10×) with solvent A. The
separation was performed using a binary gradient with 0.1% formic
acid in UHPLC-grade water (Honeywell) as solvent A and 0.1% formic
acid in acetonitrile as solvent B (UHPLC–MS, Thermo Scientific).
Gradient conditions: 0.0–11.0 min, 100–77% A; 11.0–14.6
min, 77–5% A; 14.6–17.0 min, 5% A; and 17.05–20.0
min, 100% A. The following settings were used: flow rate, 0.5 mL/min;
sample injection volume, 1 μL; column temperature, 50 °C;
sample temperature, 5 °C. Synapt XS HDMS data were acquired in
the negative ion MSe mode. Authentic standards of 3HPMA and 23HPMA
in water (100 ng/mL) were also prepared and analyzed using identical
conditions to confirm and validate the assignments in urine samples
(retention time, MS/MS, and external database match). The Waters UNIFI
software package was used for data analysis and metabolite identification.
The El-Maven and PollyPhi packages (Elucidata, MA) were used to assign 13C-labeled isotopologues, as well as to correct for the natural
abundance of 13C, as described previously.37 (link)
performed on a Waters Synapt XS HDMS coupled
with an ACQUITY UPLC I-Class system. Separation was carried out on
the Acquity Premier CSH C18 column (150 mm × 2.1 mm, 1.7 μm).
Mouse urine (25 μL) was diluted (10×) with solvent A. The
separation was performed using a binary gradient with 0.1% formic
acid in UHPLC-grade water (Honeywell) as solvent A and 0.1% formic
acid in acetonitrile as solvent B (UHPLC–MS, Thermo Scientific).
Gradient conditions: 0.0–11.0 min, 100–77% A; 11.0–14.6
min, 77–5% A; 14.6–17.0 min, 5% A; and 17.05–20.0
min, 100% A. The following settings were used: flow rate, 0.5 mL/min;
sample injection volume, 1 μL; column temperature, 50 °C;
sample temperature, 5 °C. Synapt XS HDMS data were acquired in
the negative ion MSe mode. Authentic standards of 3HPMA and 23HPMA
in water (100 ng/mL) were also prepared and analyzed using identical
conditions to confirm and validate the assignments in urine samples
(retention time, MS/MS, and external database match). The Waters UNIFI
software package was used for data analysis and metabolite identification.
The El-Maven and PollyPhi packages (Elucidata, MA) were used to assign 13C-labeled isotopologues, as well as to correct for the natural
abundance of 13C, as described previously.37 (link)
