Characterization, purification, and analysis of solvent yellow 33 and solvent green 3 dyes.

Thin-layer chromatography and high-performance liquid chromatography (HPLC) with linear photodiode array detection (LPDA) were used to separate impurities from two commercial dyes. Gravity flow liquid chromatography was used to purify reference standards of the dyes. Normal phase HPLC with LPDA detection was used to determine purities of submitted samples by comparing responses to those of the reference standards. Electron impact mass spectrometry and nuclear magnetic resonance were utilized to confirm structures of the dyes and their impurities.

Metabolism of a dimethylbenzidine-based dye in rats and hamsters as determined by analysis of the urine for potentially carcinogenic aromatic amines.

Direct Red 2 was given as an aqueous solution to rats and hamsters to determine whether the dye is cleaved to potentially carcinogenic aromatic amines. Sensitive and specific assays of the urine from treated animals by EC/GC revealed appreciable levels of 3,3'-dimethylbenzidine, mono- and di-acetyldimethylbenzidine, and alkaline hydrolyzable conjugates. Peak concentrations of the metabolites in urine occurred 12 to 24 hours after administration to rats, and within 12 hours in hamsters. The levels of all metabolites and conjugates diminished rapidly in both species after peak concentrations were reached, with no residues detected after 96 hours. The results conclusively demonstrated in vivo cleavage of the dye in both species, and it is proposed that the analytical methods employed can be used for chemical monitoring of human urine.

Chromatographic assays for traces of potentially carcinogenic metabolites of two azo dyes, Direct Red 2 and Direct Blue 15, in rat, hamster and human urine.

Specific, precise and sensitive methods are described for determining traces of potentially carcinogenic metabolites of Direct Red 2 and Direct Blue 15 in rat and hamster urine and to monitor the urine from workers who may be occupationally exposed to these dyes. This methodology is generally applicable to metabolites of azo dyes based on benzidine or three of its congeners. The benzidine-congener free amines, their mono and diacetylated analogs and alkaline hydrolyzable conjugates were determined after appropriate extraction and hydrolysis by HPLC or EC/GC. Residues of metabolites in rat, hamster, and human urine were determined at levels as low as 1 ppb. Supplementary information is also presented concerning hydrolysis of diacetylated metabolites and the stability of Direct Red 2 and Direct Blue 15 in rat, hamster and human urine.

Metabolism of nine benzidine-congener-based azo dyes in rats based on gas chromatographic assays of the urine for potentially carcinogenic metabolites.

Metabolism experiments were conducted with rats dosed with nine azo dyes based on dimethyl-, dimethoxy-, or dichlorobenzidine to determine whether the free amine congeners, their monoacetyl or diacetyl metabolites, or alkaline hydrolyzable conjugates were excreted in the urine. After preliminary tests of the dyes, 2-mg doses were administered to each animal and urine samples were collected at intervals up to 96 hours. EC/GC procedures were based on the analysis of heptafluorobutyryl derivatives of the free amine congener moieties or their monoacetyl metabolites. Peak levels of metabolites were excreted either 0-12 or 12-24 hours after administration and, in seven of nine instances, no metabolites persisted in the urine after 48 hours. Minimum detectable levels of all metabolites were 12 ppb or less. All nine dyes were shown to be converted to measurable levels of their benzidine-congener-based metabolites in rats.

Benzidine-congener-based azo dyes: assays for purity and residues in feces from dosed rats.

Analytical methods were required to determine purities of benzidine-congener-based azo dyes and residues of the intact dyes in feces from rats before valid metabolism studies of such compounds could be conducted. A procedure is described for purity assays based on reduction of the dyes with stannous chloride followed by gas chromatography of the released free amine. Sixteen different samples of commercial dyes based on three benzidine congeners were assayed; purities ranged from 26.4 to 83.4%. Several dyes were also shown to be partially purified by cold water washes. A method to determine two intact dyes in feces from dosed rats, which consisted of extraction with dimethylformamide: water, clean-up by a rapid procedure using an octadecylsilane column, and quantification by ion-pair high-pressure liquid chromatography (HPLC) is reported. minimum detectable levels of both dyes in feces are 0.2 ppm. Excretion profiles based on parallel HPLC and radioassays of feces from rats dosed with 14C-labeled Direct Blue 15 and Direct Red 2 are presented. Based on radioassays, about 74% of each dose was excreted via the feces; however, HPLC assays showed that only about 11% of each dose was present as intact dye in the excrement.

Metabolism and distribution of two 14C-benzidine-congener-based dyes in rats as determined by GC, HPLC, and radioassays.

Absorption, metabolism and tissue distribution studies were conducted in the rat with 14C-biphenyl ring-labeled Direct Blue 15, a 3,3'-dimethoxybenzidine (DiMxBzd) based azo dye; Direct Red 2, based on 3,3'-dimethylbenzidine (DiMeBzd) and corresponding benzidine congener amines. Single oral doses of the 14C-labeled dyes (12 mg/kg, 62 microCi/kg) and molar equivalent doses of the respective amines were administered and urine and fecal samples collected at intervals up to 192 hours. Urine specimens were analyzed for 14C content and further characterized by EC/GC for free amines, acetylated metabolites, and conjugates. Feces were assayed for 14C content and for unchanged dosed dyes or amines by HPLC. A comparison of the metabolism of Direct Blue 15 with its base DiMxBzd, indicated that the base was more extensively metabolized and that most of the 14C in various extracts was identified as known metabolites. The metabolism of Direct Red 2 compared with its base, DiMeBzd, indicated that the base was more extensively metabolized, yet only a small percentage of the 14C in extracts was identified as known metabolites. Most of the 14C present in the urine could not be extracted with benzene nor chloroform, indicating high polarity. Distribution studies conducted with both dyes showed that liver, kidney, and lung accumulated and retained higher levels of 14C than other tissues (at 72 hrs). Peak levels of 14C, which occurred 8-12 hours after dosing, were significantly higher with Direct Red 2 than Direct Blue 15. Tissue distribution data (72 hr) for rats dosed with the free amines compared with the dyes showed a generally lower but similar distribution pattern.

Chemical monitoring of urine from workers potentially exposed to benzidine-derived azo dyes.

Benzidine (Bzd) and monoacetylbenzidine (MoAcBzd) were found in the urine of workers exposed to benzidine-based azo dyes. A colorimetric screening method, based on the reaction of extracted free aromatic amines with 2,4,6-trinitrobenzene-sulfonic acid (TNBS), was used with a specific electron-capture gas chromatographic (EC-GC) method. Alkaline hydrolyzable conjugates of Bzd and 2,4-diaminoazobenzene (DiAmAzBz) were found together with free DiAmAzBz and traces of 3,3'-dimethylbenzidine (DiMeBzd) and 3,3'-dimethoxybenzidine (DiMxBzd). The presence of a known human bladder carcinogen (Bzd) and its metabolites in the urine of workers exposed to benzidine-based azo dyes is a cause for concern.

Analysis, purification and stability: requirements for a metabolism study of an azo dye and pigment.

Impurities of aromatic amines in the azo dye and pigment, Direct Black 38 and Pigment Yellow 12, and in vitro stability of the dye were determined. These factors can affect the results of studies designed to ascertain whether the two compounds are metabolized to potential carcinogens in hamsters. Procedures for removing impurities from the two compounds are also presented. Electron-capture gas chromatography of the heptafluorobutyryl derivatives of the impurities and degradation products was used to satisfy all the analytical requirements of the experiments. Major impurities found in Direct Black 28 were benzidine, 4-aminobiphenyl and 2,4-diaminoazobenzene; whereas, only 3,3'-dichlorobenzidine was found in Pigment Yellow 12. Stability studies of the purified dye conducted in water and in urine from hamsters and humans indicated that the dye would not degrade under the conditions used for collecting and assaying samples from a metabolism experiment. However, within 48 hours at 25 and 37.5 degrees C, the dye did degrade to known carcinogens in both hamster and human urine. Such degradation not only points out the need for proper storage of samples from metabolism studies but suggests that industrial effluents containing the dye should be properly treated before release into the environment.

Trace analysis of potentially carcinogenic metabolites of an azo dye and pigment in hamster and human urine as determined by two chromatographic procedures.

Analytical chemical procedures are described for determining traces of possible metabolites of two azo compounds. Direct Black 38 and Pigment Yellow 12, in hamster urine and to monitor the urine from workers who may be occupationally exposed during the manufacture or use of the dye and pigment. These methods were required for metabolism studies designed to assess the hazards that may occur if the two compounds are converted by in vivo mechanisms to potential carcinogens. Salient elements of the procedure are: extraction of the free aromatic amines and neutral compounds; alkaline hydrolysis of the aqueous phase and extraction of any hydrolyzed conjugates as free amines, and the analysis of the free amines and acetylated metabolites directly by high pressure liquid chromatography or by electron capture gas chromatography after conversion of the amines to heptafluorobutyryl derivatives. Residues of metabolites in hamster and human urine were determined at levels as low as 1 ppb. Ancillary data concerning hydrolysis of diacetylated metabolites and partition values for possible metabolites in various solvent systems are also presented.

Carcinogens and analogs: trace analysis of thirteen compounds in admixture in wastewater and human urine.

A gas chromatographic method is described for determining traces of 13 carcinogens and related compounds (aromatic amines and estrogens) in admixture in wastewater and human urine. This method was developed for use in toxicological research for monitoring the safe disposal of wastewater and to signal any accidental exposure of personnel to hazardous test substances. Salient elements of the procedure are: extraction of phenolic and neutral residues from the acidified sample, liquid--liquid partitioning cleanup and separation of neutral from phenolic residues at pH 14 and 10.2, acid hydrolysis of the neutral component, subsequent alkalinization of the sample and extraction of the basic residues as the free amines, conversion of all residues to the corresponding pentafluoropropionyl (PFP) derivatives and quantification by electron-capture gas chromatography. Residues were detectable in wastewater and urine at the 0.1 and 1 ppb levels, respectively. Additional information is provided concerning partition values for all PFP derivatives in five solvent systems, structure verification of the derivatives by mass spectrometry and the adaption of this method to the monitoring of surfaces and air in potentially contaminated work areas.

Trace analysis of estradiol in animal chow by electron-capture gas chromatography.

A gas chromatographic method is described for trace analysis of the natural steroidal hormone estradiol in animal chow at levels as low as 3 ppb. Salient elements of the method include extraction of the estradiol with methanol, an initial clean-up on a column of Sepadex LH-20, liquid-liquid partitioning at pH 14 and 10.2, additional cleanup on a slilica gel column, conversion of the estradiol to the pentafluoropropionly (PFP) derivative and analysis by electron-capture gas chromatography on a column of OV-25. Samples containing less than ppb of estradiol are subjected to further clean-up on silica gel after derivatization and prior to analysis.

Trace analysis of zearalenone and/or zearalanol in animal chow by high pressure liquid chromatography and gas-liquid chromatography.

An analytical method is described for determining residues of the estrogens zearalenone and/or zearalanol in animal chow at levels as low as 10 ppb. The chow is extracted with methanol and cleaned up by a 3-step procedure employing a Sephadex LH-20 column, liquid-liquid partitioning at pH 13 and 8.3, and a silica gel column. Residues of the 2 compounds, separated on silica gel, are assayed by using high pressure liquid chromatography with ultraviolet detection. Additional data are also included concerning p-values of the compounds in several solvent systems, Rf values from thin layer chromatography with 9 solvent systems, solubilities in 3 solvents, and a procedure for preparing their pentafluoropropionyl derivatives for analysis by electron capture gas-liquid chromatography.

Trace analysis of 2,4,5-trichlorophenoxyacetic acid, its glycineamide, and their alkaline hydrolyzable conjugates in mouse blood, urine, and feces.

Chemical methods were developed for the trace analysis of the herbicide 2,4,5-trichlorophenoxyacetic acid, its glycineamide, and their alkaline hydrolyzable conjugates in mouse blood, urine, and feces. The salient elements of the methods are extraction of the free acids with benzene, methylation, cleanup on a silica gel column, and quantification via electron-capture GLC. Any unextracted conjugates remaining in the substrates are then subjected to alklaline hydrolysis, and the liberated 2,4,5-trichlorophenoxyacetic acid is assayed. Data are presented concerning recoveries of the compounds from the three spiked substrates. The utility of the procedures is illustrated by a preliminary pharmacolinetic study employing parallel electron-capture GLC and radioassays of the three substrates from mice injected with a single intravenous dose of 14C-2,4,5-trichlorophenoxyacetic acid. GLC characteristics and partition values of the the compounds and hydrolysis of the glycineamide under various conditions also are discussed.

Removal of trace levels of 2-acetylaminofluorene (2-AAF) from wastewater.

An adsorption system is described for the removal of part per billion levels of the chemical carcinogen, 2-acetylaminofluorene (2-AAF), from industrial wastewater. The system consists primarily of filters and activated carbon and non-ionic polymeric adsorbents arranged in tandem. It is highly efficient, operates at low cost, and requires minimal attention. The chemical monitoring of the raw and/or cleaned-up wastewater is based on a highly sensitive and specific spectrophotofluorometric method that allows acceptance or rejection of samples at the 0.2 part per billion level. The system is presented as a model for evaluating the removal of traces of organic chemicals from wastewater prior to recycling or discharging it into the environment. Results of laboratory evaluations of several other approaches to the purification of 2-AAF-containing wastewater are also presented.