Agilent 1290 series hplc

Plasma levels of diacetyl were determined using LC–MS, as previously reported, with slight modifications^{57 (link)}. Briefly, 20 µL of plasma was added to a tube containing 80 µL ultrapure water and 200 µL CHCl₃/MeOH (2:1). The tube was vortexed, centrifuged, and the organic phase was harvested in new tubes. The residual aqueous phase was added to 200 µL of CHCl₃/MeOH (2:1), vortexed, and centrifuged, and the harvested organic phase was combined. The organic phase was mixed with 100 µL of acetonitrile containing 50 µg DH and 10 µg p-TsOH, vortexed, and incubated for 4 h in the dark. The sample was dried by evaporation, dissolved in 200 µL acetonitrile, and subjected to LC–MS analyses. LC–MS analyses were performed using an Agilent 1290 series HPLC coupled with a G6410B triple quadrupole tandem mass spectrometer. HPLC separation was performed using a ZORBAX Eclipse Plus C18 column (1.8 µm, 50 × 2.1 mm; Agilent Technologies) at 40 °C. The mobile phases A and B were ultrapure water (0.1% formic acid) and acetonitrile (0.1% formic acid), respectively. Diacetyl-DH was detected by monitoring its transition in the ESI-positive MRM mode 294.1 > 236. Under these conditions, the recovery rate was 81% (n = 3).