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, NOA).
Aim of this work was to set up a method for the sensitive and selective determination of nitrite (NO(2)(-)) and total N-nitroso groups (TNG) in dalteparin and nadroparin, commercial low- molecular-weight heparins (LMWH), prepared by deaminative depolymerization of heparin with nitrous acid. The European Pharmacopoeia VI ed. indicates respectively 5 ppm as the maximum content for contaminant NO(2)(-) in the former and 0.25 ppm for TNG in the latter and no clear indication is given for N-NO groups in dalteparin, i.e. TNG must be absent because of the specific manufacturing process. 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 NO(2)(-) and N-NO to nitric oxide (NO). In detail, NO(2)(-) 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 NO(2)(-) and TNG in nadreparin was below the limits; for dalteparin NO(2)(-) 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.
Beretta
Giangiacomo
G
Department of Pharmaceutical Sciences Pietro Pratesi, Faculty of Pharmacy, University of Milan, via Mangiagalli 25, 20133 Milan, Italy. giangiacomo.beretta@unimi.it
Gelmini
Fabrizio
F
Merlino
Mario
M
Furlanetto
Sandra
S
Facino
Roberto Maffei
RM
D000925:Anticoagulants / Q000032:analysis; D002138:Calibration; D017985:Dalteparin / Q000032:analysis; D006495:Heparin, Low-Molecular-Weight / Q000032:analysis*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D009569:Nitric Oxide / Q000032:analysis* / Q000737:chemistry; D009573:Nitrites / Q000032:analysis* / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry*; D010602:Pharmacopoeias as Topic; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions; D013997:Time Factors
D000925:Anticoagulants; D006495:Heparin, Low-Molecular-Weight; D009573:Nitrites; D012996:Solutions; D009569:Nitric Oxide; D009584:Nitrogen; D017985:Dalteparin