A rapid method for the determination of nitrate and nitrite by chemiluminescence.

Nitrite and nitrate + nitrite can be determined by selective chemical reduction to nitric oxide which is measured using a chemiluminescence analyser. The reducing agents are sodium iodide in acetic acid for nitrite and ferrous ammonium sulphate-ammonium molybdate for nitrate + nitrite. The concentrations of the reducing agents have been optimized to obtain the maximum yield of nitric oxide and the minimum coefficient of variation. Under these conditions, it is possible to inject repeated samples into the refluxing reducing agents and to obtain rapid evolutions of nitric oxide from which the determinations can be made. Nitric oxide has also been produced using the nitrite reagents from organic nitrites, a S-nitrosothiol, a pseudonitrole and N-nitrosamines. Similarly, an organic nitrate and some C-nitroso compounds respond to the method for nitrate but only to the extent of a yield of nitric oxide of about 10% of the theoretical. Very low or zero responses were evident from aliphatic and aromatic C-nitro compounds but not omega-N-nitroarginine which gave a large yield of nitric oxide using the reagents for nitrate. In general, however, concentrations of nitrate will be in considerable excess of those of related compounds which would interfere with the determinations. Nitrate can be determined either by difference in its mixtures with nitrite or by prior removal of the nitrite using ascorbic acid provided oxygen and nitric oxide are removed by degassing with nitrogen.

Nitrosation of drugs under in-vivo conditions.

Application of the WHO Nitrosation Assay Procedure (NAP test) to a range of potentially nitrosatable drugs has given rise to considerable variations in the formation of volatile N-nitrosamines and N-nitroso compounds as a group. No nitrosation whatsoever was observed with 40 mM nitrite in some instances. In simulating more closely the conditions likely to be encountered in the human stomach, however, the order of susceptibility of the drugs to N-nitrosation has proved to be very different. At a constant nitrite concentration of 25 microM, which is considered to represent the upper limit of those likely to be encountered in the acidic human stomach, the drugs giving rise to the greatest yields of products reacting as N-nitroso compounds from a maximum adult dose were the penicillins, G, V, cloxacillin and ampicillin.

Susceptibilities of drugs to nitrosation under simulated gastric conditions.

Drugs of differing structures and pharmacological actions have been incubated at 37 degrees C and pH 2.0 under conditions simulating those within the normal fasting stomach. The nitrite concentration (25 microM) was kept as constant as possible for 3 hr in an attempt to mimic its in vivo replenishment from the saliva. The extents of N-nitrosation varied widely, but were less than those observed by Gillatt et al. (Fd Chem. Toxic. 1984, 22, 269) using the WHO Nitrosation Assay Procedure, in which the initial nitrite concentration is 40 mM, 1600 times greater, and the pH (3.0) is close to the optimum for the N-nitrosation of secondary amines. The highest yield of N-nitroso compound was obtained with the benzathine salt of penicillin G whereas some drugs, including hydrochlorothiazide and chlorthalidone, produced no detectable N-nitroso derivative. The degree of N-nitrosation was consistently reduced when the initial nitrite concentration of 25 microM was not replenished during the incubations, underlining the importance of simulating the continuous supply of nitrite from the saliva. In all instances, the reactions of the drugs with nitrous acid were inhibited and, in most cases, completely prevented by the presence of ascorbic acid (125 mg).

Susceptibilities of drugs to nitrosation under standardized chemical conditions.

Of 22 drugs with either a N,N-dimethylamino, N,N-diethylamino or N-morpholino group in the molecule, eight were converted to volatile N-nitrosamines by nitrosative cleavage in reactions of nitrite and drug in a molar ratio of 4:1 at pH 3. Under standardized conditions yields were greatest with aminopyrine and minocycline which contains two N,N-dimethylamino groups in the molecule. Oxytetracycline, chlortetracycline, tetracycline, promethazine, chlorpromazine, imipramine and disulfiram gave much lower yields and amitriptyline, clomiphene, clomipramine, dextropropoxyphene, diphenhydramine, disopyramide, erythromycin, mepyramine, methapyrilene, penicillin G procaine salt, procaine, tamoxifen, trimeprazine and tripelennamine yielded no detectable levels of volatile N-nitrosamines. Nitrosation products of 57 drugs were also examined by a group selective procedure estimating both volatile and non-volatile N-nitroso compounds. Virtually all of the yield obtained from aminopyrine or minocycline could be accounted for by N-nitrosodimethylamine (NDMA). However, compounds yielding excess N-nitrosamines compared to NDMA were obtained from the other three tetracyclines, presumably as a result of the cleavage of a methyl group from the N,N-dimethylamino substituent to form desmethyl-N-nitroso compounds. In general, the drugs giving the highest yields of N-nitroso compounds were those containing secondary rather than tertiary amino groups. A considerable range of susceptibilities towards nitrous acid was observed overall; ten drugs containing a secondary or tertiary amino- or amido- or hydrazido - group did not react with nitrous acid to form N-nitroso compounds.