Criticality of pH for accurate fluorometric measurements of dipyridamole levels in biological fluids

Abstract

Extended release dipyridamole (DIP) is widely used in clinical practice as an Aggrenox formulation, which is proven to improve outcomes for secondary stroke prevention in patients after acute vascular events. However, presently established fluorometry techniques are not suitable for trace amount determinations, because of the variable background fluorescence. The authors sought to determine whether biological fluid pH is important for the serial measures of DIP levels in the animal experiments and in patients treated with Aggrenox after ischemic stroke. Post-stroke patient (n = 34) and mice (n = 25) samples were tested to determine DIP levels by established techniques with FluoroMax 3 spectrofluorometer. Both the absorption and emission spectra of DIP were affected by modifications in pH. Fluorescence of DIP was found to be maximal at a wavelength of 490 nm (excitation 420 nm) and the spectral pattern was independent of pH. The intensity of fluorescence, however, was drastically lower at low pH (at pH 2.6, fluorescence was 4% of intensity at pH 9.8). Background plasma fluorescence, however, was completely unaffected by changes in pH. Using these fluorometric characteristics, a regression model that facilitates the efficient and sensitive determination of DIP concentration in biological fluids was formulated. Exploiting pH-dependent characteristics of DIP versus serum fluorescence patterns permits a convenient mathematical model to determine DIP concentration. This relatively inexpensive and time-efficient procedure can quantify drug levels in human/animal plasma/serum, thereby directly determining the level of patient adherence to the prescribed drug regimen, be it in the context of clinical trials or compliance with the animal protocol.