Determination of angiotensin II in blood–brain barrier permeability studies using microbore LC with p-nitrophenyl-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether as a pre-separation electrochemical labeling reagent Original Research Article

Angiotensin II has been determined using microbore LC after pre-column derivatization with the novel derivatization reagent, p-nitrophenyl 2,5-dihydroxyphenylacetate, bis-tetrahydropyranyl ether (NDTE). Derivatization of trace concentrations of angiotensin II, angiotensin III and angiotensin II3–8 was complete after 4 h at 25°C and resulted in the formation of an electrochemically active derivative possessing a hydroquinone. After suitable sample preparation, samples were analyzed using gradient elution microbore LC-EC with a radial-flow electrochemical thin-layer detection cell set at a potential of +250 mV vs. Ag/AgCl, [Cl−]=15 mM. The concentration limit of detection for angiotensin II was found to be 62.5 nM (39 fmol injected). Samples obtained during in vitro blood–brain barrier transport studies were subjected to an appropriate preparation protocol subsequently and analyzed for angiotensin II using the described method. Semi-linear permeation of angiotensin II across a cultured, bovine brain microvessel endothelial cell monolayer was monitored for 2 h at concentrations ranging from 80 to 495 nM (84–518 ng ml−1).