Low results with new method

[artsjeroen]

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?

[Gerhard Kratz]

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?

[artsjeroen]

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.

[Fernando]

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

[artsjeroen]

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.

[Gerhard Kratz]

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

[HPLCaddict]

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?

[artsjeroen]

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).