Chromatography Forum

Hi all,

I am working on an isocratic method to analyse fluticasone and salmeterol.

HPLC System: Dionex 3000
Column: Hypersil ODS, 125 x 4, 5um
Column Temperature: 40C
Mobile Phase: 50 MeOH : 35 Acetonitrile : 15 Buffer (40mM)
Flow Rate: 1.0 ml/min
Injection Volume: 20ul
UV-Wavelength: 225 nm
Sample and Standard Solvent: 75% MeOH : 25% dH2O

The salmeterol peak retention time is 3.5 minutes and fluticasone 7 minutes. My problem is that I get a mysterious peak in my sample solutions right beside the salmeterol peak at 3.6 minutes. The strange thing is that the peak remains equally large throughout the run, despite the difference in sample concentrations. The peak is not present in the solvent (blank) and standard injections.

Could it be the column?

What is the nature of the sample?

What is the nature of the sample?

The sample consists of the active ingredient fluticasone and salmeterol mixed with the excipient lactose. The powder mixture is dissolved in solvent (75% MeOH) and analysed.

Concentration: approx. 7 mg/L Salmeterol
approx. 38 mg/L Fluticasone

The sample solutions are not filtered.

Three things to try

1. Use mobile phase as diluent

2. Try injecting a sample made by extracting excipients without actives.

3. Alter the conditions ie % organic, temp etc and see if the 'ghost peak' behaves like a normal peak.

If you could post a chromatogram that would be helpful.