2487 dual λ absorbance detector

Acetaminophen (≥99%) was purchased from Sigma–Aldrich (Steinheim, Germany), while ibuprofen sodium (≥98%) was purchased from Fluka (Buchs, Switzerland). The main properties of these pharmaceuticals are depicted in Table 2.
The target pharmaceuticals were analysed by High Performance Liquid Chromatography (HPLC), using a Waters HPLC 600 equipped with a 2487 Dual λ Absorbance Detector, a phenomenex C18 column (5 μm, 110 Å, 250 × 4.6 mm), a Rheodyne injector and a 50 μL loop. The wavelengths of detection were 246 and 220 for Acetaminophen and ibuprofen, respectively. The mobile phase consisted of a mixture of acetonitrile:water (30:70, v/v) for the analysis of Acetaminophen and a mixture of methanol:water:orthophosphoric acid (75:25:0.3) for the analysis of ibuprofen. For the preparation of the mobile phase mixtures, HPLC quality acetonitrile (CH₃CN) and methanol (CH₃OH) were acquired from Sigma-Aldrich (Madrid, Spain), orthophosphoric acid (H₃PO₄) was purchased from Panreac (Barcelona, Spain), and ultrapure water obtained by a Milli-Q purification system from Merck Millipore (Darmstadt, Germany). Before use, each mixture was passed through a Millipore 0.45 µm pore size filter and degasified in an ultrasound bath for 30 min. For the chromatographic determination of concentration, four replicated injections were carried out under a mobile phase flow rate of 1 mL min⁻¹.

The Waters system used for chromatographic analysis of 5-HMF consisted of an ultraviolet (UV) detector (1525 binary HPLC pump and 2487 Dual λ Absorbance detector), and chromatographic separation was performed using a Zorbax EclipsePlus C18 (3.5 µm, 3.6 mm × 150 mm) column and a Luna^® 5 µm C18(2) 100 Å (250 × 4.6 mm) column purchased from Phenomenex (Torrance, CA, USA). The following conditions were used for chromatographic analysis of 5-HMF: a mobile phase of a solvent mixture of deionized water and methanol (90:10, v/v); a flow rate of 1 mL/min; and an injection volume of 20 µL. For calibration, several concentrations of the 5-HMF standard were prepared: 1 mg/L, 2 mg/L, 5 mg/L, and 10 mg/L. The sample preparation procedure involved dissolving 10 g of honey in 50 mL of ultrapure water and then filtering. In addition, each sample was prepared in triplicate.

The HPLC system comprised Waters 1525 binary HPLC pump, 2487 dual λ absorbance detector, 717 plus autosampler, and a Synergi™ 4‐μm fusion—RP80A (Phenomenex, Cheshire, UK) C18 analytical column (150 mm × 4.6 mm of internal diameter, 4‐μm particle size). Mobile phase: Isocratic elution of 100% 20‐mM potassium phosphate in ultrapure, filtered and degassed water, with 0.1% TFA and 0.1 ACN, pH 2.8 with 2.5% phosphoric acid. Chromatographic conditions: Flow rate: 1 ml·min⁻¹. Injection volume: 200 μl. Detection wavelengths: 255 nm (cGMP) and 256 nm (cAMP).