Enhancement of chemiluminescence for vitamin B12 analysis

In the current article, chemiluminescence (CL) from the vitamin B12 and luminol reaction was studied under alkaline conditions to develop a sensitive analytical method for vitamin B12 using the carbonate enhancement effect. The method was successfully applied to the determination of vitamin B12 in vitamin B12 tablets, multivitamin capsules, and vitamin B12 injections. Experimental parameters were optimized, including luminol concentration, urea–hydrogen peroxide (urea–H2O2) concentration, effect of pH, and sequence of addition of reactants for obtaining maximum CL, which was not explored previously. The limit of detection was 5 pg/ml, and the linear range was 10 pg/ml to 1 μg/ml with a regression coefficient of R2 = 0.9998. The importance of these experimental parameters and the carbonate enhancement effect is discussed based on the knowledge of the mechanism of oxidation of luminol and decomposition of urea–H2O2 in the presence of vitamin B12. Extraction of vitamin B12 was carried out, and the observed recovery was 97–99.2% with a relative standard deviation in the range of 0.30–1.09%. The results obtained were compared with those of the flame atomic absorption spectrometry method.