Method Development Placebo Interference

[sdo7575]

I am performing method development on chromium picolinate. However, I'm having issues with co-elution between the placebo (glycerin) and chromium picolinate at ~ 20% interference. At this point, I'm not sure if there is a way to separate the two analytes and I'm considering preparing a placebo for every sample analysis and subtracting out the peak area of the placebo from the sample preparation, which is not ideal. Standard and Sample are prepared in Water at 0.7mcg/mL. Diluent chosen was water because Placebo is ~ 60% Glycerin and glycerin is insoluble in organics like acetonitrile. pKa is 1.5 for chromium picolinate so that's why I chose Phosphate Buffer, pH 3.0. I tried to dilute and inject a smaller amount to eliminate the placebo peak, but the s/n ratio of the sample was too low to quantitate. Wavelength was selected at 264 because that is the only absorbance maxima for chromium picolinate. Glycerin peak has very similar UV spectrum to chromium picolinate. The retention time of chromium picolinate is ~ 9.5min. Email me directly at sean.olson@eaglelabsinc.com if you need any additional information. Any suggestions would be very helpful.

Here are my HPLC conditions:
Mobile Phase A: Phosphate Buffer, pH 3.0
Mobile Phase B: Acetonitrile
HPLC Column: Restek Raptor ARC-18, 2.7um, 4.6 x 150mm
Flow Rate: 0.7 mL/min
Inj Volume: 10uL
Column Temp: 30C
Autosampler Temp: 20C
Wavelength: 264nm
Gradient Conditions:
Time (min) % MPB
0.0 5
3.0 5
15.0 20
15.1 5
18.0 5

[H.Thomas]

Hi Sean,

Glycerin has no UV-absorption at 264 nm. You are probably observing an impurity in glycecin (or the placebo).

You can try to separate the analyte from the impurity by changing the pH of the eluent (e.g. pH 7 phosphate buffer) or changig the column. A phase with an aromatic group. like C18-AR, Phenylhexyl or PFP might do the job.