Synergi fusion rp column

For the determination of lovastatin residues in muscle and organ (kidney, liver, brain) tissues, 1 g of the samples were each ground to homogeneity with 5 mL of methanol. The extracts were then dried at 70 °C for 10 hours before re-suspension with 60% acetonitrile and centrifuged at 20,000 g for 20 min. The upper layer of the extract was analyzed for lovastatin residues using HPLC performed according to the method as described previously [15 ]. Since the concentration of lovastatin residues in all samples were below the detectable level (1 μg) by HPLC, one sample of each tissue from the highest lovastatin treatment group (6 mg/kg BW) were sent for further analysis using Trap Liquid Chromatography Tandem Mass Spectrometer (LCMS/MS) (AB Sciex 3200Q) (Toronto, Canada) coupled with Perkin Elmer Flexar FX15 Ultra High Performance Liquid Chromatography (UHPLC) system (Massachusetts, USA). The LCMS was equipped with Synergi Fusion RP column (Phenomenex, 100 mm x 2.0 mm x 4 μM). The mobile phases were water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B), with total run time of 8 minutes. Chromatography was achieved with Multiple Reaction Monitor (MRM) method. The gradient run program was set at 10% to 90% mobile phase B from 0.01 min to 4 min, and hold for 1 min, before going back to 10% mobile phase B and re-equilibrated for 3 min.

Qualitative HPLC-PDA/UV-ESI-MS/MS analyses were performed using a Surveyor LC pump, a Surveyor autosampler, coupled with a Surveyor PDA detector, and a LCQ Advantage ion trap mass spectrometer (ThermoFinnigan) equipped with Xcalibur 3.1 software. Analyses were performed using a 4.6 × 250 mm, 4 µm, Synergi Fusion-RP column (Phenomenex). The eluent was a mixture of methanol (solvent A) and a 0.1% v/v aqueous solution of formic acid (solvent B). A linear gradient of increasing 55% to 85% A was developed within 45 min. The column was successively washed for 15 min with methanol and equilibrated with 55% A for 10 min. Elution was performed at a flow rate of 0.8 ml/min with a splitting system of 2:8 to MS detector (160 ml/min) and PDA detector (640 ml/min), respectively. The volume of the injected methanol solutions was 20 μl. Analyses were performed with an ESI interface in the positive mode. The ionization conditions were optimized and the parameters used were as follows: capillary temperature, 270 °C; capillary voltage, 29.0 V; tube lens offset, 50.0 V; sheath gas flow rate, 60.00 arbitrary units; auxiliary gas flow rate, 3.00 arbitrary units; spray voltage, 4.50 kV; scan range of m/z 150–1200. N₂ was used as the sheath and auxiliary gas. PDA data were recorded with 200–600 nm range with preferential channel as the detection wavelength 260 nm.

The standards and extracts were injected into the Agilent 1290 Infinity UHPLC system via a Phenomenex Synergi Fusion RP column (2.1 × 100 mm, 3 μm) (Phenomenex, Torrance, CA, USA). The AB SCIEX QTRAP® 5500, which was operated in the MRM mode, was used to detect acrylamide. The Turbo VTM source was used with an ESI probe in positive polarity. The calibration standards comprised of acrylamide at five different levels: 1, 50, 250, 500, and 1000 ng g⁻¹. ¹³C₃-labeled acrylamide at a concentration of 50 ng g⁻¹ was added to the calibration standards. The injection volume was set to 20 μL.
Acrylamide was separated under gradient conditions using water with 0.1% formic acid and 5 mM ammonium formate (A) and acetonitrile with 0.1% formic acid and 5 mM ammonium formate (B) as the mobile phases at a flow rate 250 μL/min. The gradient programme for acrylamide quantification using LC-MS/MS was 10% B to 90% B from 0.01 min to 4.0 min, hold for 1 min and back to 10% B in 0.1 min and reequilibration for 3 min. The run time for the analysis was 8 min.

Identification of the quinidine (QND) concentration in the dialysate samples was performed on a Sciex 6500 QTrap hybrid tandem mass spectrometer coupled to an Agilent 1100 HPLC system. Electrospray ionization was used in positive ion detection mode with MRM transitions of 325.2/307.2 (quantifier) and 325.2/172 (qualifier) with a collision energy of 31 and 45 V, respectively. The dwell time of the transitions was 300 ms. Source conditions were: curtain gas: 45 arbitrary unit (au), spray voltage: 5,000 V, source temperature: 450°C, nebulizer gas: 40 au, drying gas: 40 au, and declustering potential: 171 V. The samples were introduced to the system via an HPLC system consisting of a binary pump, an autosampler, and a column compartment unit. A Phenomenex Synergi Fusion RP column (50 mm × 2 mm, 4 μm, 80 Å) column was applied for the separation using 0.1% formic acid in water as eluent A and 0.1% formic acid containing acetonitrile as eluent B in gradient elution mode. The gradient started at 90% of eluent A, and the eluent B was increased to 95% by 3 min and kept at that concentration for 0.5 min then decreased to the initial composition by 0.3 min and kept there for 2.2 min. The overall run time was 6 min. Then, 10 μl of samples was injected. The column was kept at ambient temperature. A 5-point calibration curve was used in the range of 0.1–100 ng/ml.