The UPLC method was developed for the analysis of the process related impurities of avanafil. The chromatographic analysis was performed on an Agilent 1260 UHPLC system (Agilent Technology Co., Ltd, California, USA). The chromatographic analysis was carried out on a Waters ACQUITY HSS C18 with 50 mm × 2.1 mm and 1.8 μm particle size column due to well resolution. The mobile phase composing of 20 mM ammonium formate aqueous solution; pH was adjusted to 5.00 ± 0.05 with dilute formic acid (mobile phase-A) and acetonitrile (mobile phase-B). It was degassed and filtered, before delivering into the system, through 0.22 μm filters under vacuum. The linear gradient programme was optimized by the percentage change of mobile phases and set as follows: T time/mobile phase-A:B (%): T0/90:10, T1/90:10; T8/20:80; T10/90:10; T11/90:10; with flow rate of 0.3 mL min−1. Injection volume used was 1.0 μL, and kept the column temperature at 35 °C. The analytes were detected in photo diode array detector at 239 nm.
Agilent 1260 uhplc system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1260 UHPLC system is a high-performance liquid chromatography instrument designed for analytical and preparative applications. It features a compact design, high-pressure capabilities, and advanced software for method development and data analysis. The system is capable of performing a wide range of chromatographic separations with high resolution and sensitivity.
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3 protocols using agilent 1260 uhplc system
1
UPLC Method for Avanafil Impurities
2
UHPLC Analysis of Compounds
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An Agilent 1260 UHPLC system (Agilent, Santa Clara, CA, USA) was used for the analysis. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm, Waters, Wexford, Ireland) with an ACQUITY UPLC HSS T3 VanGuard Pre-Column (2.1 mm × 5 mm, 1.8 µm, Waters, Wexford, Ireland) kept at 35 °C. With a flow rate of 0.30 mL/min, the mobile phase was a gradient system consisting of water infused with 0.1% formic acid (A) and acetonitrile (B), with the gradient as follows: 0–2 min, 8–20% B; 2–20 min, 20–50% B; 20–26 min, 50–98% B; 26–33 min, an isocratic elution of B. The injection volume was set at 10 µL.
3
UHPLC-FT-ICR-MS Analysis of Compounds
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The apparatus consisted of an Agilent 1260 UHPLC system (Agilent, USA), with quadruple pump, auto-sampler, online degasser, column compartment diode-array detector and a Bruker Solarix 7.0T FT-ICR-MS system (Bruker, Germany). Analytical column was a ACQUITY UPLC™ BEH C18 column (100 mm × 2.1 mm, 1.7 μm; Waters, USA). The mobile phase was 0.1% formic acid water–acetonitrile (78 : 22, v/v) with a flow rate of 0.3 mL min−1. The auto-sampler and the column temperature was set at 4 °C and 35 °C, respectively. The injection volume was 5 μL.
The mass spectrometric detection was operated on a Solarix 7.0T FT-ICR mass spectrometer equipped with electrospray ionization interface and performed in positive ion mode. The ion source parameters were set as following: capillary voltage, 4500 V; dry gas temperature, 250 °C; dry gas flow, 8 L min−1; nebulizing gas, high-purity nitrogen (N2); nebulizer gas pressure, 4 bar; collision gas, high-purity argon (Ar). Mass spectra recorded was in the range of m/z 50–1000. The collision energy was 10 eV for the MS/MS experiments.
The mass spectrometric detection was operated on a Solarix 7.0T FT-ICR mass spectrometer equipped with electrospray ionization interface and performed in positive ion mode. The ion source parameters were set as following: capillary voltage, 4500 V; dry gas temperature, 250 °C; dry gas flow, 8 L min−1; nebulizing gas, high-purity nitrogen (N2); nebulizer gas pressure, 4 bar; collision gas, high-purity argon (Ar). Mass spectra recorded was in the range of m/z 50–1000. The collision energy was 10 eV for the MS/MS experiments.
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