Tsq quantum discovery triple quadrupole mass spectrometer

Plasma concentrations of midazolam and 1′-hydroxymidazolam were quantified using HPLC-MS/MS on a system consisting of a Surveyor HPLC autosampler, Surveyor MS quaternary pump, and TSQ Quantum Discovery triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA). After liquid-liquid extraction, midazolam and metabolite concentrations were measured simultaneously with electrospray ionization in the positive mode as described previously.^{13 (link)} midazolam, midazolam d4, 1′-hydroxymidazolam, and 1′-hydroxymidazolam-d4 were purchased from Cerilliant (Round Rock, TX). The lower limit of quantification for both midazolam and 1′-hydroxymidazolam was0.2 ng/ml, and the accuracy and precision were within 7%.

Acetaminophen (APAP) was extracted from plasma by liquid-liquid extraction with acidified HPLC-grade methanol (Fisher Scientific). Briefly, 10 μL plasma was enriched with 10 ng deuterium-labelled acetaminophen (APAP-d4; Santa Cruz Biotechnology Inc) as internal standard, 0.8 mL methanol (w/ 0.2% acetic acid, Sigma Aldrich) was added, and the samples were then vortexed and incubated for 20 min on ice. After centrifugation (3000 g, 10 min, 10°C), the supernatant was reduced to dryness under nitrogen at 40°C, reconstituted in mobile phase (200 μL water/methanol (65:35, v/v), and centrifuged for a second time.
Chromatographic separation was achieved using an Aria CTC autosampler and Allegros pump on an ACE Excel 2 SuperC18 column (150 x 3 mm; 2 μm) protected by a Kinetex KrudKatcher (Phenomenex) at 20°C, and acetaminophen was detected on a TSQ Quantum Discovery triple quadrupole mass spectrometer (Thermo Fisher Scientific). The mobile phase consisted of 0.1% formic acid (Sigma Aldrich) in water and 0.1% formic acid in methanol (B) at a flow rate of 0.3 mL/min. Gradient elution was achieved with a total run time of 9 min from 35% to 5% B. APAP eluted at 3.95 mins. The mass spectrometer was operated in positive ion electrospray mode (300°C, 3 kV). Transitions monitored were m/z 152 ➔ 110 and m/z 156.1 ➔ 114.1 for APAP and APAP-d4, respectively.