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Netilmicin analysis according to EP 9.2, Au electrode, application note 217_031, aminoglycoside antibiotics
Shift in retention times, high noise and bad peak shape in Netilmicin Sulphate analysis according to EP 9.2 method
Cause and remedy
- Retention time. The method for analysis of netilmicin sulphate according to the EP9.2 method is based on ion-pairing HPLC separation on a C18 column. A significant shift in retention time can be experienced when working under the specified conditions without regular regeneration of the column or when the column was not stored properly. Flush/regenerate the column on a regular basis according to the instructions in the column user manual when retention times and separation are deteriorating too much. Also be aware that ion-pairing separation takes more time to stabilize..
- Noise: a specific source of noise for this application is the combination of a saltbridge reference electrode in the flow cell and sodium hydroxide solution needed for detection in PAD-mode. The cotton frit in this reference electrode can get contaminated in a short period of time, resulting in increased baseline noise levels. A regular replacement of the cotton frit is advised. An alternative reference electrode (but not conform the EP method) is our maintenance-free HyREF. See out other kb paper about high noise levels for more general possible causes
- Bad peak shape: overloading the system with high concentrations of netilmicin can resulted in distorted peak shapes. The use of highest AST setting nr 3 in case of the SenCell or 130 um spacer in case of the FlexCell result in best peak shapes.
- Bad peak shape: a contamination on the auxiliary electrode or gold electrode can affect the peak shape. Polishing and cleaning these electrodes is advised.