Kinetex hilic column

The oseltamivir and oseltamivir carboxylate concentrations in the plasma and urine were determined using a highly specific and sensitive method of liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Agilent 6490 Triple Quadrupole, Agilent Technologies, Santa Clara, CA, USA). To prepare the samples for analysis, an aliquot of the plasma or urine specimen was mixed with acetonitrile in the presence or absence of the internal standard oseltamivir carboxylate-d3. The mixture was vortexed for 30 sec and then centrifuged for 10 min at 14,000 rpm. An aliquot of the supernatant was transferred to an autosampler vial, and 2 μL was injected onto the Kinetex HILIC column (50 mm × 2.1 mm, 5 μm; Phenomenex, Torrance, CA, USA) within a 3 min run at a flow rate of 0.3 mL/min using gradient elution. Mobile phase A consisted of 10 mM ammonium acetate in water, and mobile phase B consisted of 100% acetonitrile. Oseltamivir and oseltamivir carboxylate were quantitatively detected using positive ionization of triple-quadrupole mass spectrometry equipped with electrospray ionization. The method was validated within a ranges of 0.5–100 ng/mL and 20–20,000 ng/mL for oseltamivir, and 2–500 ng/mL and 500–100,000 ng/mL for oseltamivir carboxylate in plasma and urine, respectively.

Genomic DNA was extracted from approximately 25 mg of heart tissue using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. For each sample, 1 μg of DNA was degraded into individual nucleosides using DNA Degradase Plus™ (Zymo Research Corporation, Irvine, CA). Individual nucleosides were measured using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system consisting of a Dionex UltiMate® 3000 UHPLC system (Thermo Scientific, Sunnyvale, CA) containing a Kinetex HILIC column (2.1 × 100 mm, 1.7 μm; Phenomenex Inc., Torrance, CA) connected to a TSQ Vantage mass spectrometer (Thermo Scientific). Quantification was performed using area-based linear regression curves derived from calibration standards containing internal standard solutions. 5mC and 5hmC levels were calculated as a concentration percentage ratio of total deoxycytidine species; %5mC = 5-methyl-2′-deoxycytidine/(5-methyl-2′-deoxycytidine + 5-hydroxymethyl-2′-deoxycytidine + 2′-deoxycytidine) and %5hmC = 5-hydroxymethyl-2′-deoxycytidine/(5-methyl-2′-deoxycytidine + 5-hydroxymethyl-2′-deoxycytidine + 2′-deoxycytidine).

Bacterial lipids were separated by a Waters UPLC (Waters Corp., Milford, MA, United States) as described previously (Hines K. M. et al., 2017 (link); Hines KM. et al., 2017 (link)). Briefly, hydrophilic interaction liquid chromatography (HILIC) was performed with a Phenomenex Kinetex HILIC column (2.1 × 100 mm, 1.7 µm) maintained at 40°C at a flow rate of 0.5 ml/min. The solvent system consisted of: A) 50% acetonitrile/50% water with 5 mM ammonium acetate; and B) 95% acetonitrile/5% water with 5 mM ammonium acetate. The linear gradient was as follows: 0–1 min, 100% B; 4 min, 90% B; 7–8 min, 70% B; 9–12 min, 100% B. A sample injection volume of 5 µL was used for all analyses.

Brain uptake of PF8380 was studied using LC-MS/MS. Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight. The antagonist was administered by gavage to get an indication about oral bioavailability and uptake efficacy across the gastrointestinal epithelium. At the indicated times mice were transcardially perfused with ice-cold PBS under deep anesthesia, and the brains were dissected and snap frozen in liquid N₂. Brains were homogenized in a BioPulverizer (BioSpec Products, Bartlesville, OK) and tissue homogenates were weighed and extracted using a modified Bligh & Dyer HCl method [32 (link)]. External calibration was performed for PF8380 in a concentration range of 0.1–2 µM, LPA species were quantitated using LPA-C17 as internal standard. Quantitation of LPA and PF8380 was conducted by LC-MS/MS. Chromatographic separation was performed on a Phenomenex Kinetex HILIC column (2.1 × 100 mm, 2.6 µm). Detection was performed on a Thermo Orbitrap Velos Pro (Thermo Fisher Scientific Inc., Waltham, MA, USA) hybrid mass spectrometer, using a HESI II probe in negative ionization mode. Automated identification and quantitation of LPA and PF8380 was performed by lipid data analyzer, as previously reported [64 (link)].

The concentrations of metformin in the plasma and urine were determined using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS, Agilent 1260 HPLC system and Agilent 6490 Triple Quadrupole; Agilent Technologies, Santa Clara, CA, USA). Blood samples collected for PK analyses were centrifuged at 2095 g for 10 minutes at 4°C to separate plasma. Plasma (50 μL) and urine (20 μL) samples were mixed with 450 μL and 980 μL of internal standard working solution (phenformin, 50 μg/L in 100% acetonitrile), respectively. After the solution was vortexed for 10 minutes and centrifuged at 18341 g for 10 minutes at 4°C, the supernatant (100 μL) was transferred to an autosampling vial. The injection volume was 1 μL. The column used was a Kinetex HILIC column (50 × 2.1 mm, 5 μm; Phenomenex, Torrance, CA, USA) at 24°C. The mobile phase A consisted of 5 mM ammonium formate in distilled water (pH 6.2) and mobile phase B 100% acetonitrile (30:70, v/v). The calibration curves were linear over a range of 10–5,000 μg/L for plasma and 100–25,000 μg/L for urine. The intra- and inter-batch precisions of QC samples were < 9.003% and < 15.57% (at LLOQ level) for plasma and urine, respectively. The mean accuracy values were within ±4.0% and ±14.4% of nominal values for plasma and urine, respectively.

The quantification of MPI8 concentrations in extracted plasma samples was performed on a 6500+ Triple Quad LC-MS/MS System using a Kinetex HILIC Column (unbonded silica stationary phase, 2.6 µm, 50 × 2.1 mm, 100 Å, Phenomenex, CA, USA). The optimized method used binary gradient mobile phases with water (containing 1 mM ammonium acetate) as mobile phase A and acetonitrile as mobile phase B. A flow rate of 0.4 mL/min was used with an injection volume of 2 μL. The autosampler and oven temperature were set at 10 °C and 30 °C, respectively. The running time was 5 min. In this study, the HILIC column, widely considered as an NP column, was eluted in a typical RP time program of the gradient: Phase B was initially kept at 5% for 0.2 min, increased from 5% to 90% in the next 1.3 min, then held at 90% for 1 min, and then decreased to 5% in 1.5 min, and kept stably at 5% for 1 min. The unique behavior of MPI8 on a HILIC column, which resembles an RP characteristic, was most likely due to its hydrophobic nature.
Mass spectrometry data were recorded using ESI, positive ion detection, and MRM scanning. The ion spray voltage and temperature were set at 5000 v and 350 °C, respectively. The curtain gas and CAD were set at 45 and 8 psi; gases 1 and 2 were set at 40 and 50 psi, respectively. Data were acquired by Analyst software 1.6.3. The MS/MS parameters for MPI8 and IS are shown in Table 1.