Hi, our lab has been conducting HPLC to determine the monosaccharide composition of polysaccharide samples using the following method:Article Microwave-assisted extraction of polysaccharide from Cinnamo...

"The sample was treated with 3 M trifluoroacetic acid (8 h, 95 °C) followed by derivatization for 1 h at 70 °C using 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) in the presence of 0.3 M NaOH. The excessive PMP was removed using chloroform as the eluting solvent. The monosaccharide composition was assayed using the high-performance liquid chromatography (HPLC) system with the following test conditions: (1) column: Zorbax SB-C18 reversed-phase column (250 mm × 4.6 mm, 5 μm, Agilent, USA); (2) mobile phase: (A) acetonitrile (ACN), (B) 3.3 mM KH2PO4 containing 4.0 mM Tris-acetate–EDTA buffer and 10 % (v/v) ACN; (3) mobile phase gradient: 0–4 min: 94 % B; 4–9 min: from 94 to 88 % B; and 9–20 min: 88 % B; flow rate: 1.0 mL/min; and UV absorbance wavelength: 250 nm."

However, an unknown peak consistently shows up at ~11.6 min in every sugar standard and sample tested. We have freshly prepared each chemical and mobile phase, but the result still showed the same peak at ~11.6 min. The issue was resolved when we purchased a new bottle of PMP but after ~2 months the same problem occurred again. We have kept the PMP according to the instructions. We tried splitting it into smaller portions, wrapping it with aluminium foil and keeping it in the chiller for long-term storage but after some time the unknown peak occurred again.

Anyone had a problem with PMP before? How did you manage it? Or what else could have gone wrong?

Thank you for sharing.

Pei Gee