A simplified screening procedure for determination of total N-NO groups (TNG) and nitrite (NO2−) in commercial low-molecular-weight heparins (LMWH) by selective chemical denitrosation followed by high-sensitivity chemiluminescence detection (NO-analyzer

The proposed technique is based on the development of a pre-analytical device, coupled to a chemiluminometer, constituted by three sequentially connected and commercially available purge vessels, where selective reagents are employed for the conversion of NO2− and N-NO to nitric oxide (NO). In detail, NO2− was determined in the first chamber and non-volatile and volatile TNG in the second and third. This method was validated for selectivity, sensitivity, linearity, accuracy and precision. The method was shown to be selective, with a quantitative linear range of 1-1000 ppb). The bias, intra- and inter-day percent relative error was lower than 1%. The contamination of NO2− and TNG in nadreparin was below the limits; for dalteparin NO2− fell within the limit, but there was a huge amount of TNG (15.80 ± 0.05 ppm-6.69 ± 0.02 ppm). Preliminary investigation on the solvent-extractable material from dalteparin showed the majority of chemiluminescence retained in the aqueous residue to indicate that this N-NO groups may belong to solvent unextractable material or be tightly bound to the dalteparin backbone.