Acquity xevo tq system

UPLC-MS/MS analysis was carried out on an Acquity-Xevo TQ system (Waters, Barcelona, Spain). The conditions used were: ionization in negative mode (ESI-), capillary voltage 3.5 kV, source temperature 120 °C, desolvation temperature 300 °C, gas flow of the nitrogen cone of 150 L/h, and desolvation flow of 680 L/h.
Separation conditions were selected to achieve appropriate chromatographic retention and resolution by using a C18 column (2.1 × 50 mm, 1.7 μm) (Acquity UPLC BEH) and pre-column (2.1 × 5 mm) from Waters. A binary mobile phase CH₃OH (0.1% v/v HCOOH):H₂O (0.1% v/v HCOOH) with gradient elution was used. The flow rate was 0.4 mL/min, the temperatures of column and the autosampler were 37 °C and 4 °C, respectively. The injection volume was 10 µL. The gradient started with 30% v/v CH₃OH (0.1% v/v HCOOH) (i.e., channel B) and from 1 to 4.0 min %B increased up to 90%. Finally, the mobile phase composition returned to the initial conditions at 4.1, and it was maintained for 3.9 min for system conditioning.
The detection was performed by multiple reaction monitoring using the acquisition parameters obtained in a previous study [18 (link),19 (link)].
For data acquisition and processing, MassLynx 4.1 and QuanLynx 4.1 softwares from Waters (Waters, Barcelona, Spain) were used, respectively. Linear response curves were calculated employing PGF_2α-d₄ as internal standard.

A reaction mixture containing enzyme, DiBP or MiBP in 10 mM citric acid-Na₂HPO₄ solution (pH 7.5) was incubated at 45°C with the total reaction volume was 1 mL. After incubating the reaction mixtures for indicated time, the reaction was stopped by adding 10% (v/v) of 1 N HCl to the mixture, and the reaction products were extracted using the same volume of ethyl acetate. After dried over Na₂SO₄, the samples were re-dissolved in methanol. The residual and separated DiBP, MiBP and PTH were calculated based on the resulting peak areas by using an Agilent (Agilent 1100) spectrometry system. The operating conditions of the mobile phase 0.1% phosphorous acid solution/acetonitrile (10:90, v/v) were applied. The detector, wavelength, and flow rate were DAD, 230 nm, and 1 mL∙min⁻¹, respectively. The column, temperature, and injection volume were Zorbax Eclipasse XDB-C18 (4.6 mm × 150 mm, 5 μm Agilent), 30°C and 3 μL, respectively. The chromatographical analysis was carried out on a Waters Acquity-Xevo TQ system (Waters, Milford, MA, USA) using positive and negative electrospray ionization (ESI) under the following conditions: capillary voltage was 2.0 kV, cone −30.0 V and 30 V, source temperature 350°C, desolvation temperature 200°C, desolvation gas flow 800 L∙h⁻¹; and cone gas flow 150 L∙h⁻¹. Full scan and daughter ions scan were used to monitor the analytes.