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The final method uses a Kromasil column and the mobile phase comprises dioxane, water and ammonium formate solution pH 7.7.
The mobile phase comprises methanol, water, acetonitrile (ACN) and acetic acid (47:42:10:1, v/v/v/v) at a flow rate of 1.0 ml/min.
The mobile phase comprises 78% phosphate buffer (pH 5.5) and 20% acetonitrile, 1% triethylamine and 1% acetic acid degassed via sonication.
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The mobile phase comprised of A: methanol-10 mM KH2PO4-triethylamine in a ratio of 96:6:0.01 (v/v/v) and B: methanol.
The mobile phase comprised of acetonitrile (40%) and water containing 0.1% w/v triethylamine with pH adjusted to 3 with ortho-phosphoric acid (60%).
The sample was subjected to an elution gradient with a mobile phase comprising 35 85% (v/v) acetonitrile for 25 min followed by 85 100% (v/v) acetonitrile for 5 min.
Separation was achieved using a Phenomenex® Luna 5 μm (C18) column and a mobile phase comprised of phosphate buffer (adjusted to pH 3.0): acetonitrile in a ratio of 70 30 (v/v).
One work [18] describes simultaneous determination of FEX and its two impurities A and B using C8 column as stationary phase and a mobile phase comprising 1% triethylamine phosphate (pH 3.7), acetonitrile and methanol in the ratio 60 20 20 (v/v/v).
A gradient elution with a mobile phase comprising 0.1% formic acid in water (A) and methanol (B) was used as follows: 0 min, 60% B; 16 min, 60% B; 16.1 min, 100% B; 20 min, 100% B; 20.1 min, 60% B; and 25 min, 60% B. The detector wavelengths were set at 270 nm.
The best resolution of peaks were achieved with an isocratic elution by a mobile phase comprising acetonitrile:water:formic acid (65 35:0.1%, v/v/v) at a flow-rate of 0.2 mL/min, on Acquity UPLC BEH™ C18 column (50 × 2.1 mm, i.d. 1.7 μm).
The HPLC mobile phase comprised of acetonitrile buffer (20∶80, v/v, buffer 50 mM KH2PO4, pH of 6.8 adjusted with KOH).
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