Patai S., Rappoport Z. 1996 The chemistry of functional groups. The chemistry of amino, nitroso, nitro and related groups. Part 2 / 1041 1168

An HPLC-UVD method was developed for the determination of the radiosensitizing agent N-(3-nitro-4-quinoline)morpholino-4-carboxamidine (EGIS-4136, 270) in plasma, using an internal standard and measuring at 330 nm. The assay was validated with respect to linearity, sensitivity, accuracy, precision, stability and recovery. The method was applied to pharmacokinetic studies in male rats, monitoring 270 concentrations in the range of 5 10 mg/L553.

NH

N O

HN

NO2

N

(270)

3-Nitro-1,2,4-triazole (271) was determined by solvent peak paper chromatography in the presence of impurities554. The crystallography, morphology, kinetics and mechanism of the thermal decomposition of 3-nitro-1,2,4-triazol-5-one (272) have been studied, applying DTA, DSC, TGA, IR spectroscopy, XRD and hot-stage microscopy. Cleavage of the C NO2 bond with rupture of the adjacent C N bond appears to be the primary step in the thermolysis of 272. The evolved gases were analyzed by IR spectroscopy555.

E. Aliphatic Compounds

The RP-HPLC retention times of nitroalkanes (e.g. MeNO2, EtNO2, n-PrNO2, i-PrNO2, c-HexNO2), their nitronates and their nitronic acid degradation products (including alkyl oximes, nitrooximes and pseudonitroles) were determined using a Nova-Pak C18 radial column556.

3-Nitropropanoyl esters of glucose from the roots of Lotus pendunculatus Cav. were determined by analysis of nitrate released on alkaline hydrolysis. This method was validated for quantitation of both total nitro compounds in ethanolic extracts and for individual components from TLC separations557.

Low levels of nitrogen dioxide react with the polyunsaturated fatty acids under anaerobic conditions to give allylic nitro and allylic nitrite derivatives of methyl linoleate and methyl linolenate. These were identified by NICI-MS558.

F. Nitrates

In Table 5 are listed some industrial organic nitrates and protocols containing analytical methods.

A method for the spectrophotometric analysis of nitroglycerin (273) in gaseous effluents was developed. The compound is absorbed in an alkaline solution and converted with hydrogen peroxide to nitrite ions. These can be analyzed spectrophotometrically by reacting with a mixture of sulfanylamide (103) and phosphoric acid (diazotization), coupling with N-(1-naphthyl)ethylenediamine (106) and measuring the absorption at 540 nm559. The determination of nitrite ions is a modification of the Bratton Marshall method (see Section IV.D.3.g).

CH2 ONO2

CHONO2

CH2 ONO2

(273)

A rapid and sensitive capillary GC-ECD method was used to evaluate the nitroglycerin (273) content in human blood serum; LOD was 50 ng/L. Significant amounts of the active metabolites 1,2- and 1,3-dinitroglycerine could be demonstrated560. A major problem in the analysis of 273 and its metabolites is due to adsorption of the nitro compounds on the glassware used during sample preparation or injection. The adsorption problem was overcome by the use of triethylamine, resulting in a simpler sample preparation and accurate results. Coupling this technique to a capillary GC-ECD method gave high precision in the determination of 273 and its metabolites in plasma at low nanomolar level; LOD was ca 0.2 nM in plasma561. A similar, specific capillary GC-ECD method for the simultaneous determination of 273 and its diand mononitrate metabolites included an extraction step; LOD was 0.4 mg/L plasma, with recoveries >76% for 273 and the mononitrates and >95% for the dinitrates. The assay was applied to pharmacological studies562.

A combination of FTIR and TGA is very effective for the quantitative and qualitative analysis of gunpowder563.

Four nitrosamines, seven nitramines, three nitroesters and the explosives Semtex 10 and Composition B have been investigated by TGA. Linear dependence was confirmed between the position of the TGA onsets, as defined in the sense of Perkin-Elmer’s TGA- 7 standard program, and the samples’ weights. The slope of this dependence is closely related to the thermal reactivity and molecular structure. The intercept values of the dependence correlate with the autoignition temperatures and with the critical temperatures of the studied compounds, without any clear influence from molecular structure. Results show that Semtex 10 exhibits approximately the same thermostability as its active component pentaerythrityl tetranitrate (PETN, 274). Results also show that TGA data for Composition B do not correlate with analogous data for pure nitramines564.

G. Nitramines

In Table 5 are listed some industrial nitramines and protocols containing analytical methods. Nitramines are used as explosives and propellants, they are toxic and might cause

1144 Jacob Zabicky and Shmuel Bittner

TABLE 6. Examples of environmental and occupational protocols for nitroso compounds of industrial significance

a Nomenclature may vary from source to source. See also Reference 69. bEntry number in Reference 70.

c Codes beginning with I and N denote FTIR spectra in Reference 71 and NMR spectra in Reference 72, respectively. d A code of two letters followed by seven digits is a reference to RTECS of NIOSH/OSHA. Standard samples are commercially available for most compounds with reference to EPA protocols.

The mechanism of electrochemical reduction of nitrosobenzene to phenylhydroxylamine in aqueous medium has been examined in the pH range from 0.4 to 13, by polarographic and cyclic voltametry. The two-electron process has been explained in terms of a nine-membered square scheme involving protonations and electron transfer steps565. This process is part of the overall reduction of nitrobenzene to phenylhydroxylamine, shown in reaction 37 (Section VI.B.2). Nitrosobenzene undergoes spontaneous reaction at pH > 13, yielding azoxybenzene471.

C. Nitrosamines

A review appeared, discussing the determination of nitrosamines in cosmetics and cosmetic raw materials, including analytical procedures and LOD568. The nitrosamines connected with tobacco are discussed in Section VI.D below.

1. Gas chromatography

Various gas chromatographic techniques combined with plentiful detection methods were used to separate and quantify volatile N-nitrosamines. Preconcentration methods were usually applied for separating these compounds. Thus, a method was developed for determination of N-nitrosodimethylamine (278a) in minced fish or frankfurters, based on SPE followed by GC-CLD-TEA; RSD was 0.56 to 2.25%569. This method has been adopted by AOAC. A similar GC method using NPD was described for the determination of 278a in fish products570. Steam distillation can also be used to isolate volatile

components and was applied to separate the 278a found as impurity in thiram (279) formulations, followed by SPE and determination by GC-MS-SIM571.

The nitrosamines on the EPA list (see Table 6) were determined in samples of groundwater and drinking water at the sub-ppb level (0.1 ppb). The method consisted of either LLE with methylene chloride or SPE on a series of two adsorbents (C8 and an activated C cartridge), followed by capillary GC-NPD572. Of all foods, nitrite-cured meats have been investigated most thoroughly for the presence of nitrosamines, many of which are carcinogens. Thus, varying levels (1 48 ppb) of N-nitrosodimethylamine (278a), N- nitrosopyrrolidine (280) and N-nitroso-N-methylaniline (281) were detected in Icelandic smoked mutton using GC-CI-MS. Low levels of N-nitrosothiazolidine (282, 0.6 2.4 ppb) and N-nitrosothiazolidine-4-carboxylic acid (283, 56 475 ppb) were also present. It was suggested that the formation of all the above nitrosoamines can be minimized by changing or modifying the method of smoking573.

A GC-tandem CI-MS method, using a quadrupole ion storage mass spectrometer, has been developed for the determination of N-nitrosodimethylamine (278a) in complex environmental matrices. No interference from chlorobenzene, ethylbenzene and the xylenes was detected; LOD was in the subpicogram range574. An alternative method for 278a involves preconcentration by SPE, extraction with CH2Cl2 and GC-MS, using isotope dilution with hexadeuterated 278a. LOD 1.0 ppt in water, accuracy of 6% at 10 ppt575. 278a was also determined in drinking water and fruit drinks by GC using both TEA and MS-SIM detection; LOD was 15 pg/g in drinking water and 1 pg/g in fruit drinks576.

Volatile nitroso compounds were determined in hams processed in elastic rubber nettings by SPE and GC-CLD577. By a similar method N-nitrosodibenzylamine (278b), a semivolatile nitrosamine, was determined in these products by SPE followed by GC interfaced to a nitrosamine-specific TEA-CLD detector; the coefficient of variation was 10.6% at the 2.1 ppb level578. The nitrosamines detected in ham most likely originate from the amine precursors in rubber and from the nitrite commonly used in the meat curing process.

A method involving SPE was developed for the determination of ten N-nitroso amino acids in cured meat products. These compounds were derivatized with diazomethane followed by O-acylation of hydroxyl groups with acetic anhydride-pyridine reagent. The methyl esters and their acylated derivatives were separated by GC on a DB-5 fused silica capillary column and quantified with a TEA-CLD specific for the nitric oxide derived from the thermal denitrosation of nitrosamines; recovery exceeded 75% at the 10 ppb level579.

N-Nitrosodiethanolamine (284), N-nitroso-1,3-oxazolidine (285a) and N-nitroso-5- methyl-1,3-oxazolidine (285b) were detected in metalworking fluids in Canada, using GC-ECD. 284 was derivatized with trifluoroacetic acid anhydride, while 285a and 285b were converted to their corresponding nitramine analogs by oxidation with pertrifluoroacetic acid before analysis; LOD was 1.2 5 ng580.

A laboratory-assembled supercritical fluid extractor was designed for the efficient recovery of volatile nitrosamines from frankfurters. The nitrosamines were separated and detected using a GC-TEA-CLD. Recovery of 10 volatile aliphatic and alicylic nitrosamines from frankfurters spiked at the 20 ppb level was 84.3 104.8% with RSD 2.34 6.13%581.

2. Liquid chromatograpy

Computer simulation was applied for the development and optimization of a gradient chromatography method for the analysis of nitrosamines582. Interest in the analysis of nonvolatile N-nitrosamines has recently been renewed due to the development of novel interfaces to TEA or CLD after RP-HPLC. An interface was devised, incorporating a thermospray vaporizer, a counter flow gas diffusion cell to reduce the LC effluent to a dry aerosol and a single-stage momentum separator to form a particle beam of the nonvolatile analyte. This interface was used in the HPLC-TEA analysis of the nonvolatile N-nitrosodiethanolamine (284) and 2-ethylhexyl N-nitroso-N-methyl-p- aminobenzoate (286). These results are comparable to other LC-TEA interfacing methods; however, several advantages are ease of application, ruggedness and MS compatibility. Full scan EI-MS identification of the N-nitrosamine contaminants in cosmetics was used for confirming the TEA detection data583. Traces of 284 in triethanolamine, up to 10 mg/L, were determined by HPLC-UVD, using a strongly acidic cation exchanger PRPX200 column and aqueous HClO4 as eluent of high optical transparency, measuring at max 235 nm. The method takes advantage of difference in pKa values of the amine matrix and the nitroso impurity584.

Using TEA-CLD it was possible to determine rapidly total N-nitroso compounds and nitrite in fresh human gastric juice; LOD 1.0 pmol, RSD 1 6%585. Mixtures of volatile and nonvolatile N-nitroso compounds, including N-nitrosodipeptides, were determined by HPLC-TEA, using a water/acetonitrile gradient mobile phase; RSD was 3.0 and 5.1%, for 80 90 ng injections of N-nitrosoproline (287) and N-nitrosotrimethylurea (288a), respectively586.

A method for analysis of N-nitroso-N-alkylureas (288b) has been developed by forming fluorescent derivatives with sodium sulfide, taurine (77) and o-phthalaldehyde (73)

and separating by RP-HPLC. The method was applied to the determination of 288b in blood587.

2-(Hydroxymethyl)-N-nitrosothiazolidine (289) and 2-(hydroxymethyl)-N-nitrosothia- zolidine-4-carboxylic acid methyl ester (290) were determined in cured smoked meats by HPLC-TEA588.

Various nonvolatile nitrosamines were analyzed using HPLC-UV photolysis-CLD. This was applied for determination of N-nitrosamides in dried squid589 and N- nitrosodiphenylamine (278c) in treated apples590.

An improved HPLC photohydrolysis colorimetry method was validated for twentyeight reference nitrosamines. These were separated by HPLC and photolytically cleaved by UV radiation. The resulting nitric oxide was oxidized and hydrolyzed to nitrite ions, which were derivatized into an azo dye with Griess’ reagent and measured spectrophotometrically. The method was applied to separate and detect hitherto unknown nonvolatile nitrosamines in biological fluids and food extracts591.

Two conformers of N-nitrosoglyphosate (291) were separated by HPLC. NMR, spectrophotometric and electroanalytical measurements indicate that these conformers are always present in equilibrium, with slow interconversion592.

ON NCH2 CO2 H

CH2 PO3 H2

(291)

Microconcentrations of carcinogenic N-nitrosamines were determined in various rubber articles (tubing, stoppers, hoses, seals, etc.) for medical and food uses by extraction followed by HPLC-FLD593. N-Nitrosodiphenylamine (278c) present in diphenylamine formulations was determined by LC-TEA on a Zorbax CN column594.

Ce(IV) in acidic medium is a suitable post-column reagent in the LC-amperometric determination of nonvolatile nitrosamines such as nitrosourea, nitrosoguanidine, nitrosourethane and nitrosoamino acids. The behavior of the Ce(IV) Ce(III) couple with a rotating disk electrode approaches the operational conditions of a ‘channel thin layer’ cell with solid electrodes, frequently used as detector for LC. Gold was found to be the most suitable electrode. The reaction between Ce(IV) and NO2 , the product of nitrosamine decomposition in warm acidic solution, was considered595. An improved LC-amperometric determination of nonvolatile nitrosamines was proposed, using an online detector system based on the Ce(IV) reagent in acidic medium. A two-line flow manifold coupled with a flow-through voltammetric detector equipped with twin gold electrodes, for both monoand biamperometric detection modes, was evaluated. Monoand biamperometric measurements allowed determination of linear dynamic ranges, sensitivities and LOD of nitrite under different experimental conditions of composition, liquid carrier and temperature of the reactor596. The use of iodide reagent in acidic medium was introduced for the LC-amperometric determination of nonvolatile nitrosamines. A two-line flow-injection manifold was used, coupled with a voltametric flow-through

detector. A peak current signal was obtained for the nitrite iodide reaction. The method

has high sensitivity and LOD of about 1 ð 10 8 M, which is better than with the Ce(IV) reagent597.

Optimized conditions were found for the separation of p-substituted N-nitroso-N- methylanilines (292), using RP-HPLC with a C18 chemically bonded stationary phase. Four detection techniques were studied: Direct UV photometry, polarography on a hanging Hg electrode, anodic voltammetry on a glassy carbon fiber array electrode and indirect anodic voltammetry after photolytic denitrosation of the analytes. UV photometry is the most universal with LOD around 10 6 M. Polarography exhibits the poorest sensitivity (LOD ca 10 5 M) but can be used for selective detection of the p-nitro derivative 292g. Direct voltammetric detection is selective for the oxidizable derivatives, and the LOD attained are lower than those obtained by UV photometry for 292f. When the analytes are photolytically denitrosated to yield oxidizable derivatives, the LOD of voltammetric detection of 292a, 292b, 292d and 292g are an order of magnitude lower than those of UV photometry598.

The diffusion-limited electrochemical oxidation of N-nitrosamines in an aqueous pH 1.5 buffer was demonstrated at a GCE coated with a film of mixed valence ruthenium oxides, stabilized by cyano crosslinks. This electrode was used in a potentiostatic amperometric detector for FIA and HPLC, to allow the determination of representative N- nitrosamines (278a, 278c and 278d); for 278c, LOD was 10 nM and RSD 2% at 0.80 mM (n D 5)599.

3. Miscellaneous methods

Hexetidine (293) and hexedine (294), common ‘formaldehyde releasing’ antimicrobial agents and drug constituents, can undergo nitrosation in the pH range 1 4.8. The major nitrosamine product, ‘HEXNO’ (295), can be characterized and analyzed using common spectroscopic methods. Rapid formation of 295 from 293 and 294 supports the hypothesis that tertiary geminal diamines produce nitrosamines rapidly, by a mechanism involving cleavage of a nitrosammonium ion with the assistance of the neighboring nitrogen atom600.

A linear correlation was found between the absorbance and the concentration (12.5 100 mg/L) of sixteen antineoplastic nitrosoureas, belonging to 4 distinct chemical classes, in the presence of ceftizoxime (296) in acidic media ( max 500 nm)601.

Sodium iodide in trifluroacetic anhydride reacts with nitrosamines and releases iodine. This was used for selective detection of nitrosamines after TLC separation602,603.

Denitrosation of N-nitrosamines to yield secondary amines affords an alternative way for detecting N-nitrosamines. Treatment with a hydrogen bromide acetic acid mixture and reacting the resulting amines with 4-(2-phthalimidyl)benzoyl chloride (297) gives fluorescent amides. N-Nitrosodialkylamines such as 278a, 278d, 278e, 280 and N-nitrosopiperi- dine (298) were used as model compounds604.

Quantification of total N-nitroso compounds in urine and gastric juice is achieved by combining photolytic denitrosation with TEA. Nitrite interference is effectively eliminated with sulfamic acid (H2NSO3H)605.

S-Nitroso derivatives of the biological thiols glutathione, cysteine (115) and homocysteine have been considered as bioactive intermediates in the metabolism of organic nitrates and the endothelium-derived relaxing factor with properties of nitric oxide. A simple, rapid and reproducible method for separating these thiols from their