Benzidine detection

That the aromatic amine benzidine is mutagenic only at the TK locus in L5178Y TK+,/ cells. The most disturbing finding was that benzidine (detectable without metabolism by S9 mix) did not produce detectable DNA adducts (as shown by 32P-post-labeling) in L5178Y cells. Thus, the mechanism... 

M. p. 168-169 C. This reagent has superseded benzidine for blood detection, showing same sensitivity and specificity but it is non-carcinogenic in doses at which benzidine produces a high incidence of neoplasms. 

It is known that not all reactions proceed in the same manner on all adsorbent layers because the material in the layer may promote or retard the reaction. Thus, Ganshirt [209] was able to show that caffeine and codeine phosphate could be detected on aluminium oxide by chlorination and treatment with benzidine, but that there was no reaction with the same reagent on silica gel. Again the detection of amino acids and peptides by ninhydrin is more sensitive on pure cellulose than it is on layers containing fluorescence indicators [210]. The NBP reagent (. v.) cannot be employed on Nano-Sil-Ci8-100-UV2S4 plates because the whole of the plate background becomes colored. 

In addition the role played by the sorbent on which the chromatography is carried out must not be neglected. For instance, it is only on aliuninium oxide layers and not on silica gel that it is possible to detect caffeine and codeine by exposure to chlorine gas and treatment with potassium iodide — benzidine [37]. The detection limits can also depend on the sorbent used. The detection limit is also a fimction of the h/ f value. The concentration of substance per chromatogram zone is greater when the migration distance is short than it is for components with high h/ f values. Hence, compounds with low h/ f values are more sensitively detected. 

Horseradish peroxidase (HRP) an enzyme routinely used in immunohisto-chemistry for labeling antibodies. Histochemichal detection of peroxidase is based on the conversion of aromatic phenols or amines, such as diamino-benzidine (DAB), into water-insoluble pigments in the presence of hydrogen peroxide (H202). 

Fig. 3.72. (a) Standard HPLC chromatogram. (1) Benzidine, (2) o-toluidine, (3) 4-chloroaniline, (4) 3,3-dimethoxybenzidine, (5) 3,3-dimethylbenzidine, (6) 4-aminobiphenyl (7) 3,3-dichloroben-zidine. Detection wavelength 280 nm amino concentration approximately 50 pg/ml. HPLC conditions are described in the text, (b) Chromatogram of a genuine leather sample treated with the MAE procedure. Reprinted with permission from C. S. Eskilsson et al. [140]. 

Fig. 3.105. HPLC chromatograms for enzymatic reduction, (a) Xylidine ponceau-2R (1 = 2,4-xyli-dine, 2 = 2,6-xylidine, 3 = 2,4,5-trimethylaniline). (b) Direct black-38 (1 = benzidine, 2 = 4-aminophenyl). (c) Direct brown-1 (1 = benzidine, 2 = 4-aminobiphenyl). Conditions mobile phase, acetonitrile (A) and water (B) flow rate, 0.7 ml/min 25°C injection volume, 10 p gradient elution 0 min, A 23 per cent, B 77 per cent 0-21 min, A 34 per cent and B 66 per cent 21-30 min, A 60 per cent and B 40 per cent 30-34 min, A 70 per cent and B 30 per cent 34-37 min, A 90 per cent and B 10 per cent and 37—40 min, A 23 per cent and B 77 per cent. Detection at 280 nm. Reprinted with permission from M. Bhaskar et al. [159].

Fig. 3.154. Electropherogram for the working solution of aromatic amines. Peaks 1 = 4,4 -diamin-odiphenylmethane 2 = 4,4 -oxidianiline 3 = benzidine 4 = aniline 5 = 2,4-diaminoanisole 6 = 2,4 -toluilendiamine 7 = o-toluidine 8 = 3,3 -dimethylbenzidine 9 = 3,3 -dimethoxyben-zidine 10 = p-cresidine 11 = 2-naphtylamine 12 = p-chloroaniline 13 = 4-aminodiphenyl 14 = 1-naphtylamine 15 = 4-chlorotoluidine all at 10 ng/jul. Conditions buffer = 50 mM phosphate 10 per cent methanol pH = 3.1 fused-silica capillary recovered with polyamide, 52 cm X 75 pm i.d. applied potential = +22 kV UV detection at 214 nm. Reprinted with permission from S. Borros et al. [195].

There is no information on the metabolism of 3,3 -dichlorobenzidine in children. Limited data in humans suggest that N-acetylation is an important metabolic pathway (Belman et al. 1968), and a detoxification mechanism. N-Acetylation in humans is likely done by one of two families of N-acetyltransferases. One of these families, NAT2, is developmentally regulated (Leeder and Kearns 1997). Some enzyme activity can be detected in the fetus by the end of the first trimester. Almost all infants exhibit the slow acetylator phenotype between birth and 2 months of age. The adult phenotype distribution is reached by the age of 4-6 months, whereas adult activity is found by approximately 1-3 years of age. Also, UDP-glucurono-syltransferase, responsible for the formation of glucuronide conjugates, seems to achieve adult activity by 618 months of age (Leeder and Kearns 1997). These data suggest that metabolism of 3,3 -dichloro-benzidine by infants will differ from that in adults in extent, rate, or both. 

No studies were located that monitored human tissues for content of 3,3 -dichlorobenzidine or its metabolites. 3,3 -Dichlorobenzidine is excreted in urine. If 3,3 -dichlorobenzidine and metabolites can be detected and correlated with exposure, it may be possible to correlate urinary levels of 3,3 -dichloro-benzidine or its metabolites, with systemic effects. 

Because the chemical has no agricultural or food chemical application, it is very imlikely that 3,3 -dichloro-benzidine occurs in food in general. [ C]-3,3 -Dichlorobenzidine was foimd to rapidly accumulate in bluegill sunfish as a result of their exposiue to water in which either 5 or 100 g/L of the chemical was intentionally added. Residues were distributed in both the edible and nonedible portions (Appleton and Sikka 1980). However, 3,3 -dichlorobenzidine was not detected in fish samples obtained from rivers near nine textile dyestuff manufacturers known to use 3,3 -dichlorobenzidine-based pigments (Diachenko 1979). 

Armentrout DN, Cutie SS. 1980. Determination of benzidine and 3,3 -dichlorobenzidine in wastewater by liquid chromatography with UV and electrochemical detection. J Chromatogr Sci 18 370-374. 

Kawahara FH, Dunn JR, Fiutem RA, et al. 1982. Determination of benzidines by gas chromatographic separation of derivatives with electron capture detection. Anal Chim Acta 138 207-220. 

Trippel-Schulte P, Zeiske J, Kettrup A. 1986. Traee analysis of seleeted benzidine and diaminodiphenylmethane derivatives in mine by means of liquid ehromatography using preeolumn sample preconcentration, UV and electrochemical detection. Chromatographia 22(1-6) 138-148. 

The currently accepted name for (1) in Chemical Abstracts is dibenzothiophene, although biphenylene, diphenylene, or biphenylylene sulfide are still used, especially in the Russian literature. Occasionally 9-thia-fluorene is used and recently, in one instance, dibenzo[6,d]thiophene. An alternative numbering system for (1) is met with on occasions in which C-4 is taken as C-1, as in carbazole. In the absenee of moleeular diagrams this system must be detected by reference to the chemistry described. For example, in a few dyestuff patents 2,7-diaminodibenzothiophene 5,5-dioxide is referred to and this is obviously the 3,7-diamino compound, also known as benzidine sulfone. 

Of 25 workers involved in benzidine manufacture, 13 developed urinary bladder tumors and 4 renal tumors also occurred. The average duration of exposure was 13.6 years, and the average induction time from first exposure to detection of the first tumor was 16.6 years. Initial tumors made their appearance as late as 9 years after cessation of exposure. Airborne benzidine concentrations were estimated to have ranged from 0.005 to 17.6mg/m It is not known whether the cancers were influenced by concurrent exposure to other chemicals in the occupational environment. ... 

Figure 5.3 Chromatograms of amines with mobile phases containing (a) water adjusted to pH 3 with HCl and (b) 30 mmol/L l-ethyl-3-methylimidazolium tetra-fluoroborate. Chromatographic conditions C18 column (5 pm, 150 X 4.6 mm ID) rate flow 1.0 mL/min detection at 254 nm. Peaks (1) benzylamine (2) benzidine (3) N,N-dimethylaniline and (4) N-ethylaniline. (Adapted from Xiaohua, X., Liang, Z., Xia, L., and Shengxiang, Anal. Chim. Acta, 519, 207-211, 2004.)...

This system resolved the aniline peak (retention time (rt) = 2.67 min) from the benzidine peak (rt = 2.27 min) as can be seen in Figure 2. Other potential interferences were selected for study by looking at the expected fragments from the reduction of various dyes. Reduced dye samples were spiked with aniline (rt = 2.67 min), -aminophenol (rt = 1.97 min), -phenylenediamine (rt = 1.93 min) and -nitroaniline (rt = 3 16 min). None of these materials interfered with the detection of the benzidine peak. To determine if other types of dyes might interfere with the analysis, two sets of filters were spiked at low and high levels separately with C.I. Direct Red 28 (13 7 yg and 137 yg), C.I. Direct Blue 53 formulation (o-tolidine-based) (21.2 yg and 212 yg) and C.I. Direct Blue 8 formulation (o-dianisidine-based)(23.3 yg and 233 yg). 

Determination of Benzidine in Hair Dyes. Experiments were conducted to determine whether or not benzidine could be detected in benzidine based dyes and/or hair dye products containing benzidine based dyes. The benzidine based dye Direct Blue 6 was obtained commercially as were two shades of a particular hair dye product. One shade, "Lucky Copper , contained Direct Blue 6 whereas another shade, Silver Lining", did not contain any benzidine based dyes. 

Assay extract for benzidine by HPLC with electrochemical detection. 

The work illustrates the ability of HPLC/EC to detect trace quantities of benzidine in hair dye products, although positive results should be verified by GC-MS or other spectroscopic means to be certain of the component identity. Unfortunately, the sensitivities of such techniques are generally much poorer than HPLC/EC, thus making absolute confirmation impractical. 

Benzidines (Method 605). The method chosen for the determination of benzidine and 3,3 -dichlorobenzidine uses HPLC. Lichrosorb RP-2 (5 jLtm) is used as the analytical column, and acetonitrile and an acetate buffer are used as the mobile phase. A relatively selective electrochemical detector is used to detect and measure the benzidines. The instability of 1,2-diphenylhydrazine, which decomposes to azobenzene, caused it to be eliminated from consideration. 

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

The most recent paper on this topic has been published by Lu and Huang (213). The method consists of an online enrichment of the aromatic amines on a carboxymethyl-bonded silica precolumn and an HPLC-UV (at 254 nm) analysis. The mobile phase, ACN-acetate buffer (pH 4.66) (40 60, v/v), was used to desorb the analytes and for the subsequent separation. The method was applied to the determination of several compounds (4-aminoazobenzene (4-AAB), benzidine (Bz), 3,3 -methylbenzidine (DMBz), 4-aminobiphenyl (4-ABP), 3,3 -dichlorobenzi-dine (DCBz), and 2-naphthylamine (2-NA) together with some substituted naphthalens and phenols) in aqueous solution of four food dyes Direct Blue 6, Amaranth, Sunset Yellow FCF, and D C Orange No. 4. Detection limits ranged between 0.6 and 1.6 fig/g. Most part of the methods developed for this kind of determination are reported in Table 3. 

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