Abstract: The cited method depends on electroreduction of dissolved oxygen to form H2O2 to oxidize the acridinium ester (used as chemiluminescence labels in immunoassays), instead of addition of H2O2 from a separate reagent stream. Sample solution (10 µL) were injected into 0.15 M borate solution, pH 12 (3 mL min-1). The connecting tube to the flow cell ensured a delay time of 0.22 s and a residence time of 0.2 s. Hydrogen peroxide was generated at a vitreous carbon cathode at -1.0 V vs. Ag - AgCl, with a stainless steel auxilary electrode and chemiluminescence was measured with a photo-multiplier. Calibration graphs were rectilinear for 0.01 to 100 pM phenyl acridinium-9-carboxylate. For application to acridinium ester-labelled substrates in immunoassay, the method was used to determine ester-labelled lysine. Best results were obtained with an acidified (with HNO3) sample before measurement to break the coupling bond; the detection limit was 10 fmol injected. Effect of electrochemical generation of H2O2 paralleled the effect of its direct addition in all respects, including the doubling of light intensity after 2 mM hexadecyltrimethylammonium bromide was added. Acridinium esters are used as chemiluminescence (CL) labels in immunoassay. Acridinium ester CL is traditionally triggered by addition of a solution of H2O2. This paper is concerned with the generation of the reaction-initiating species in situ (electrochemically) to eliminate problems associated with solution addition 9-Ph acridinium-9-carboxylate shows no electrochemistry over the range -1.0 to +1.0 V. In the presence of dissolved oxygen, as the applied potential is stepped negative, electrogenerated chemiluminescence (ECL) emission intensity increases to a plateau region corresponding to the peroxide plateau of electrochemical oxygen reduction. ECL emission intensity increases as pH increases from 9 to 12, but decreases at higher pH. The rate of formation of nonchemiluminescent pseudobase between sample injection and CL observation was studied; at pH 12, that delay time should be limited to under 0.5 s. The working curve dynamic range covers 4 decades in concentration. The detection limit for acridinium ester-labeled lysine is 10 fmol.

Abstract: Flow injection electrospray mass spectrometry was used to detect the intermediates and products formed during the reaction of chemiluminescent acridinium salts under the conditions necessary for light emission. A stream of aqueous alkaline hydrogen peroxide was mixed with an aqueous solution of N-sulfonylacridinium-9-carboxamide salt immediately prior to entering the ESI-MS interface. The resulting negative-ion mass spectra corresponded to the expected 9-hydroperoxide adduct, the acridone end product normally seen in the chemiluminescent reaction, and unreacted acridinium salt, with no indication of the postulated spirodioxetanone intermediate or competing pseudobase.