Chromatography Forum

Hello all,
I am developing a method for a catecholamine and an associated impurity present at 5ppm and 0.025 ppm respectively in the HPLC sample prep. The main chromophore is primarily the same for both compounds. I am using fluorescence detection. I do not have a reference std for the impurity. I am planning to use the catecholamine ref std as both a working std. (at 5ppm conc) for the main peak and as a surrogate std for the impurity peak. I have a response factor generated from the slopes obtained from 5 concentrations (0.025 ppm to 5 ppm range) of the main peak and the impurity. Is this acceptable? Traditionally, the sample and working std. should be at the same concentration.

The entire logic depends on the assumption that your catecholamine and the impurity have the same fluorophore. If I were auditing that method, I would want to see some evidence that the assumption is justified.

Depending on the fluorophore and the mobile phase, the fluorescence yield can differ across a gradient elution. For instance, OPA/thiol derivatives are weakly quenched by methanol, so later eluting peaks have a lower molar response factor. Acetonitrile has almost the same molar response across the gradient for the same set. (I learned this while doing EPA 531.1 for carbamate insecticides in water.)

Thank you all for the replies. You have a good point . Luckily I am using isocratic conditions. It does bring up a point about fluorescence methods being challenging from the view point of demonstrating specificity. Unlike UV detection where there is a fallback to diode array, a coeluting (or closely eluting degradant that has the same fluorophore may be hard to distinguish.

TV

Well, some people use fluorescence derivatization to increase the info in relation to that obtainable with UV spectrometry. In HPLC derivatization the development was and is in the direction of strong fluorescing substances which are not much influenced by the derivatized molecule nor the dissolution medium (another advantage of FMOC-CL over OPA).