Chromatography Forum

I've developed a gradient method for analysis of Sulfaquinoxaline using 0.1% formic acid in ACN and 0.1% formic acid in UPW as mobile phases. The gradient takes 6 minutes where the previous isocratic method took 15 minutes.

The results of a finished product (with matrix) on the isocratic method are about 20% higher than the results I measure on the new gradient method. I've checked trueness by comparing our working standard and a USP standard. The results of this test are good.

The sample prep for both methods is exactly the same, so I'm wondering how the results can differ this much?

Did you check the mobile phase composition in the gradient at the time when your target compound is eluted is the same as with the isocratic method?

The gradient method uses different mobile phases than the isocratic method.
The isocratic method is with NH4H2PO4 buffer in water + ACN.

The gradient method was developed because it is faster and it has better peak shape.

Hi artsjeroen

You may utilize an excelent tool that is free from LCResouces the SCOUTING GRADIENTS.xls that I think will be very helpfull to your problem.

Best Regards

Fernando

There is no problem with the gradient.

A nice sharp peak elutes at Rt = 3 min. No peak in blank samples, excellent reproducibility, linearity, control samples RSD is <1%.
A sulfaquinoxaline USP standard was compared with a Sigma standard. Result was 99%.

The results for my product must be 25 mg/g Sulfaquinoxaline (in matrix)

With isocratic method I find about 27 mg/g
With the gradient method I find 22.2 mg/g

The exact same sample preparation (same vials) were used for both methods (even the same sample set was used, with a different method).

My question is, how it is possible that I find about 20% lower results when I use a gradient method?
The isocratic method has always been used before without too many problems.

Just a guess: is your compound peak with the isocratic conditions pure or is a compound coeluting at the same retention time. Maybe that compound is now eluting before or after your sulfaquinoxaline peak

How are you calculating your results? Via external standard? If yes, the first thing I'd look at is if the 20% lower result is due to lower peak area of the sample or higher peak area of the standards (taking into account, of course, possible changes in injection volume and flow-rate).

Almost forgotten: Is this reproducible? With new preparations of samples and standards?

I think Gerhard has the answer. The results are repoducible.
In the gradient I can see more (smaller) peaks than I can see in chromatograms of the isocratic method. I guess these are some related substances or maybe degradation products (due to excipients that are present in the matrix).