Acquity h class system

Plasma samples were quantified for pramipexole concentration using a validated ultra performance liquid chromatography with mass spectrometry (UPLC-MS/MS) detector (Waters, Acquity^® H-Class system with Xevo^® TQD Detector, Waters Corporation, Milford, MA, USA) in positive ion electrospray ionization mode, using a multiple reaction monitoring (MRM) method and atenolol as the internal standard (IS). The transitions used were 212.1 → 153.1 for pramipexole and 267.1 → 190.1 for the IS. The UPLC-MS/MS was equipped with an Acquity UPLC^® BEH Amide (1.7 µm, 2.1 × 50 mm) analytical column. Formic acid with a 0.1% concentration in a mixture of water and acetonitrile (15:85 v/v) as a mobile phase was pumped at a rate of 0.4 mL/min through the column. Before being injected into the chromatography system, the plasma sample was treated according the sample extraction procedure as described below. The validated condition of the UPLC-MS/MS system as indicated by its adequate sensitivity, specificity, linearity, accuracy, and precision (both within and between days) is presented in Table 1.

Sample analysis was performed using UPLC with Waters Acquity H-Class system equipped with a PDA detector and controlled by Empower software (EMP 2 Feature release 5, Built 2154). The analytical method followed the previous publication [28 (link)], and it is detailed in Table 1. The ambient temperature of the column and samples was used. Linear (R² = 0.999) calibration curves were obtained for each pH in the relevant drug-concentration ranges. Inter- and intraday coefficients of variation were lower than 1%. The stability of sildenafil over the experimental course was verified.

Chromatographic runs were performed in an ultra-performance liquid chromatography (UPLC, Acquity H–Class system, Waters Inc, Milford, MA, USA), with a C18 HSS 2.1 × 100 mm column and particle size of 1.8 μm (Waters Inc, Milford, MA, USA). The mobile phase was composed of 0.1% formic acid in acetonitrile (eluent A), 0.1% formic acid in ethyl acetate (eluent B) and 0.1% formic acid in methanol (eluent C), at an elution rate of 0.37 mL min^-1 and an isocratic run, with a proportion of 10% A/40% B/50% C for 5 min, and sample injection volume of 10 μL. Column temperature was kept at 30°C, while the auto injector was at 10°C. The UPLC detection system was composed of a coupled mass spectrometer single quadrupole SQ Detector 2. Capilar voltage was 5.5 kV and cone voltage 50 V, the desolvation temperature was 350°C, and gas flow was 650 L h^-1. Data acquisition was carried out in full scan mode, for masses between 100 and 1000 Da in positive ionization. Acquisition of chromatograms and mass spectra was performed with the software MassLynx™ (Waters Inc, Milford, MA, USA).

After growth of mycelia on solid supplemented YGT media, 220 ml of 2:1 acetone:water was added and shaken vigorously for 16 hours, then allowed to separate. The supernatant was extracted with 150 ml methylene chloride, which was then evaporated under filtered nitrogen gas. Each dried sample extract was resuspended in 1 ml acetonitrile, then transferred to a 2 ml, 0.45 μm nylon filter centrifuge tube (Spin-X; Corning Inc., Corning, NY) and centrifuged at 14000 rpm for 1 min. Injections (1 μl) of filtered extract were analyzed by UPLC. AFB₁ and AFB₂analysis was performed with modifications to a procedure previously described [33 ]. UPLC analyses were performed with a Waters Acquity H-Class System combined with an Acquity fluorescence (FLR) detector (Waters, Milford, MA). Sample extract (1 μl) was injected for separation through an Acquity BEH C18 column (1.7 um, 2.1 x 50 mm) at 30 °C. Run time was 3 min with an elution flow rate of 0.4 ml/min and isocratic mobile phase consisting of methanol:water (40:60). AFB₁ detection wavelength was 365 nm (excitation) and 440 nm (emission) and retention time was 2.1 min. A calibration curve with high linearity (R² = 0.9993) was constructed for AFB₁ from a series of diluted standards (Sigma-Aldrich). Statistical analysis conducted using Graphpad Prism 7 (Graphpad Software Inc).