Acquity uplc hss t3 1.8 μm column

Analysis was performed using a UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a quadrupole time-of-flight (AB Sciex TripleTOF 6600) at Shanghai Applied Protein Technology Co., Ltd. To achieve HILIC separation, we used a 2.1 × 100 mm ACQUITY UPLC BEH 1.7 μm column (Waters, Ireland). The mobile phases for both ESI⁺ and ESI⁻ modes consisted of A, which was 25 mmol/L ammonium acetate and 25 mmol/L ammonium hydroxide in water, and B, which was acetonitrile. To obtain RPLC separation, we used a 2.1 × 100 mm ACQUITY UPLC HSS T3 1.8-μm column (Waters, Ireland). For ESI⁺ mode, the mobile phases included A, which was water with 0.1% formic acid, and B, which was acetonitrile with 0.1% formic acid. On the other hand, for ESI⁻ mode, the mobile phases comprised A, which was 0.5 mmol/L ammonium fluoride in water, and B, which was acetonitrile. During the MS-only acquisition, the instrument was set to capture data within the m/z range of 60–1,000 Da, with an accumulation time of 0.20 s/spectra for the TOF MS scan. Meanwhile, in auto MS-MS acquisition, the instrument captured data within the m/z range of 25–1,000 Da, with an accumulation time of 0.05 s/spectra for the product ion scan. To conduct the product ion scan, we used information-dependent acquisition, specifically selecting the high-sensitivity mode (11 (link)).

Chromatographic separation was performed by a gradient elution on the ACQUITY UPLC HSS T3 1.8 μm column (2.1 × 100 mm) with VanGuard pre‐column (Waters Corporation). The mobile phase solvents were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate 0.4 ml/min for 5 min. The gradient program mobile phase conditions were 85% of A and 15% of B for the first 2.5 min, then changed linearly to 10% of A and 90% of B from 2.5–4.0 min, then immediately back to 85% of A and 15% of B from 4.0–5.0 min to re‐equilibrate at the initial conditions. The column temperature was 40°C, the autosampler temperature was maintained at 10°C and the injection volume was 8 μl. The positive electrospray ionization mode (ESI [+]) was used with the multiple reactions monitoring (MRM). The tune page source voltages were 0.7 kV and 84 V for the capillary and cone, respectively. The source desolvation temperature was 500°C. The source desolvation gas flow was 800 L/h and the cone gas was 50 L/h. The MS file cone voltage setting was 74 V with collision energy setting of 48 V. Argon was used as the collision gas and nitrogen as the desolvation and cone gases. Quantification was performed using the transition Parent (m/z): 468.34 and Daughter (m/z): 83.77 with a retention time of 2.43 min.

To improve hydrophobicity of INH, we derivatizated INH with cinnamaldehyde. To improve the retention of highly polar compounds in the chromatography column, we added ion-pairing reagent (HFBA) to the sample diluted solution. After optimizing the method for sample preparation, we compared the elution solution (B) of acetonitrile (ACN) and methanol, and solution A of 0.1% FA water, 5 mM NH₄FA, 5 mM NH₄FA (pH 4.0), 10 mM NH₄FA, and 10 mM NH₄FA (pH 4.0). Two kinds of spiked plasma standards with concentration of upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ) were used. Chromatographic separation was performed on an Acquity UPLC HSS T3 1.8 μm column (2.1 × 100 mm, Waters) at a flow rate of 0.35 mL/min with HPLC gradient of 0–0.5 min 2% B, 1.5–2.5 min 80% B, and 3.0–4.0 min 2% B. The column temperature was 35 °C. For each sample, 5.0 μL was injected with a total running time of 4.0 min per sample.