Kinetex c18 100 column

NTX was quantified using HPLC analysis on a Shimadzu LC-2030 Prominence iSeries system (Shimadzu, Kyoto, Japan) equipped with a LC-2030 pump, autosampler, and UV detector; data analysis was performed using Shimadzu^® LC Solution software (Shimadzu, Kyoto, Japan, Ver. 5.92). Reverse phase chromatographic separation was carried out using a Kinetex^® C18 100 Å column (150 × 4.6 mm i.d., 5μm; Phenomenex, Torrance, CA, USA). The mobile phase (A: 0.1% TFA and 0.06% v/v TEA in water and B: acetonitrile) was used in a gradient elution as follows: phase B concentration increased from 10% to 30% over 4 min, maintained at 30% for 1 min, and then decreased to 10% until 6 min for equilibration. Flow rate was 1.0 mL/min, oven temperature maintained at 30 °C, and injection volume was 10 µL. NTX was detected at wavelength of 226 nm with a retention time of 3.62 min. The standard curve was linear across the range of 0.4 to 100 µg/mL (R² ≥ 0.99). Method accuracy (relative error (RE), %) was <1.5%, and within-day and between-day precision (relative standard deviation (RSD), %) was <2.5%.

The LC-MS/MS procedure was adapted by utilizing an Exion LC AD system and a TQ 6500 mass spectrometer equipped with an electrospray ionization source (AB SCIEX, Framingham, MA, USA). An aliquot of 2 μL of all prepared samples was injected into the LC-MS/MS system, and chromatographic separation was conducted using a Kinetex C18 100 Å column (100 × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA) at 40 °C. The pump flow rate was 0.25 mL/min, and the autosampler temperature was set at 4 °C. The mobile phase was composed of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile with gradient elution. The predetermined gradient elution system was as follows: 0–1 min (35% B), 1–1.1 min (60% B), 1.1–2 min (60% B), 2–2.1 min (35% B), and 2.1–4 min (60% B).

LC chromatographic analyses were performed by UV/LC-MS with an Agilent 1100 LC/MSD VL system (G1946C) (Agilent Technologies, Palo Alto, CA) equipped with a vacuum solvent degassing unit, a binary high-pressure gradient pump, an 1100 series UV detector, and a 1100 MSD model VL benchtop mass spectrometer. Chromatographic separations were obtained using a Phenomenex Kinetex C18-100 Å column (150 × 4.6 mm) with 5 µm particle size and gradient elution with a binary solution; (eluent A: H₂O acidified with formic acid (FA) 0.1% v/v, eluent B: ACN/MeOH 1:1 v/v) at room temperature. The analysis started with 5% of B (from t = 0 to t = 1 min), then B was increased to 95% (from t = 1 to t = 10 min), then kept at 95% (from t = 10 to t = 15 min) and finally returned to 5% of eluent A in one minute. The instrument worked in positive mode and the UV detector operated at 254 nm.

A portion of stereocalpin B (1, 1.0 mg) was hydrolyzed in 6 N HCl (1 mL) at 100 °C overnight. The reaction mixture was evaporated to dryness and dissolved in 150 μL H₂O. To the solution, 70 μL of 1 M NaHCO₃ and 200 μL of 1% FDAA in acetone were added, and the reaction mixture was stirred at 37 °C for 1 hr. The reaction was quenched with the addition of 100 μL of 1 N HCl. The DAA derivatives were analyzed by LC-MS using an analytical Kinetex C18 100 Å column (100 mm × 2.1 mm i.d., 5 μm; Phenomenex, Torrance, CA, USA) at a flow rate of 0.3 mL/ min. The mobile phases were 0.1% (v/v) formic acid aqueous solution (A) and 0.1% (v/v) formic acid acetonitrile (B) with the following gradient: 10–60% B (0–40 min), 60–100% B (40–41 min), 100% B (41–51 min), 100–10% B (51–52 min), 10% B (52–62 min). Retention times were compared to derivatized amino acid standards. Elution of the target peaks was detected by the extraction of relevant ions. Retention times for the analytical standards were as follows: L-Phe 21.8 min, D-Phe 23.9 min, N-Me-L-Phe 22.1 min, and N-Me-D-Phe 22.4 min. The L-DLA-derivatized hydrolysate of 1 gave peaks at 21.7 min and 22.0 min, which confirmed the presence of L-Phe and N-Me-L-Phe (Figure S11).

Shimadzu LC-2030c 3D (Shimadzu Corporation, Kyoto, Japan) was used for CUR analysis [25 (link)]. The HPLC column was a Kinetex^® C18 100 Å column (250 mm × 4.6 mm id, 5 μm) (Phenomenex, Torrance, CA) and the mobile phase consisted of 0.1% trifluoroacetic acid in distilled water (DW) adjusted to pH 3.0 with ammonia solution (A) and acetonitrile (B) (55:45% v/v). The temperature of the column was kept at 40 °C, and the flow rate was 0.8 mL/min. The UV detection wavelength was 425 nm and the injection volume was 20 μL.

A mixture of acenocoumarol (1 μM) and liver microsomes (2 mg protein/ml) from wild-type (WT), hepatic P450 reductase–null (HRN), Cyp2c KO, Cyp2c/Cyp2d/Cyp3a KO, or CYP2C9 humanized mice in phosphate buffer (100 mM KH₂PO₄ pH7.4, 3.3 mM MgCl₂) was incubated in a water bath for 5 minutes at 37°C prior to the start of the reaction by addition of NADPH regenerating system (final concentrations: 1.3 mM NADPH, 4 mM glucose-6-phosphate, and 2 IU/ml glucose-6-phosphate dehydrogenase). The reaction mixture aliquots were taken at specified time points and mixed with an equal volume of acetonitrile containing warfarin (50 ng/ml) as an internal standard. The mixture was placed on ice for at least 20 minutes, centrifuged at 16.1 krcf for 15 minutes at +4°C, and 100 μl of the supernatant was transferred to a 96-well plate for analysis. The concentrations of S-acenocoumarol were measured by LC-MS/MS. Chromatographic separation was performed on a Kinetex C18 100 Å column (2.6 μm; 5.0 × 2.1 mm) (Phenomenex) using an injection volume of 10 μl and a run time of 7 minutes. The detector used was a Micromass Quattro Micro mass spectrometer (Waters Corporation) run in electrospray positive ion mode. The multiple reaction monitoring parameters for S-acenocoumarol and warfarin were 354.06 and 309.1 (parent ions) and 163.08 and 251.15 (collision ions), respectively.