Acquity uplc sample manager

The LC/MS/MS method was conducted as described in Reference [12 ]. The method was carried out using a Waters Acquity UPLC Sample Manager and a Waters Acquity UPLC Binary Solvent Manager connected to a Waters Quattro Premier XE triple-quadruple mass spectrometer and Mass Lynx 1.5.2 software (Waters, Milford, MA, USA). An Agilent Zorbax XDB-C₁₈ (3.5 mm, 2.1 mm × 50 mm) was also used. The gradient mode was used to achieve triptolide and internal standard using mixtures of 40 mM ammonium formate (mobile phase A) and methanol with 0.1% (v/v) formic acid (mobile phase B) at a flow rate of 0.5 mL/min. Mobile phase A was 98% at 0.1–0.6 min, then a linearly programmed gradient from 98% to 5% at 3–4.5 min, at last, mobile phase A ramp to 98% at 4.5–6 min toward the end of the analysis. The system was auto-injected with 10 μL of each analyte. Multiple reaction monitoring (MRM) mode was applied to achieve quantification with monitoring precursor-product ion transitions of m/z 361.0→105.0 for triptolide using electrospray ionization mass spectrometry (ESI-MS) on an API-4000, with Turbo Ionspray. The system was in positive ESI-mode during the run. The desolvation temperature was set at 550 °C and the nebulizer gas pressure was 60 psi.

Fentanyl in plasma was quantitated using a validated UPLC-MS/MS method consisting of a Waters Acquity UPLC sample manager coupled to a triple quadruple mass spectrometer operating in the multiple reaction monitoring mode (MRM) with positive ion electrospray ionization (Waters, Etten-Leur, The Netherlands). The multiple reaction monitoring transition was set at 337→188.
Chromatographic separation of fentanyl was achieved on an Acquity UPLC^® BEH C18 1.7 μm 2.1 x 100 mm column eluted at a flow-rate of 0.350 mL/min on a gradient of methanol. The overall cycle time of the method was 6 minutes. The calibration curves were linear over the range of 0.1 to 10 ng/mL with the lower limit of quantitation validated at 0.1 ng/mL for fentanyl. The within and between-run precisions at five tested concentrations, including the LLQ, were ≤ 5.5% and ≤ 6.1%, respectively, while the average accuracy ranged from 86.2% to 97.5%. The extraction of 200 μL of plasma involved a deproteinization step with acetone followed by a simple liquid extraction with ethyl acetate. The organic phase was evaporated and subsequently dissolved in 100 μL methanolic solutions, from which aliquots of 10 μL were injected into the UPLC-MS/MS system.

Plasma concentrations of oxycodone and its metabolites, oxymorphone, nor-oxycodone and nor-oxymorphone were quantified using a validated UPLC-MS/MS method consisting of a Waters Acquity UPLC sample manager coupled to a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode (MRM) with positive ion electrospray ionization (Waters, Etten-Leur, The Netherlands). Chromatographic separations were achieved on an Acquity UPLC^® (Waters, Etten-Leur, The Netherlands) BEH C18 1.7 µm 2.1 × 100 mm column eluted at a flow rate of 0.350 mL/min on a gradient of methanol combined with ammonium formate. The overall cycle time was 10 min. The calibration curves were linear with the lower limit of quantitation validated at 0.200 ng/mL for oxycodone and oxymorphone and 1.00 ng/mL for nor-oxycodone and nor-oxymorphone.