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.
Department of Pharmaceutical Sciences Pietro Pratesi, Faculty of Pharmacy, University of Milan, via Mangiagalli 25, 20133 Milan, Italy. giangiacomo.beretta@unimi.it
Giangiacomo
G
Beretta
Fabrizio
F
Gelmini
Mario
M
Merlino
Sandra
S
Furlanetto
Roberto Maffei
RM
Facino
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