Methods for measuring mutagenicity in urine of rats dosed with [14C]di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP) is extensively used as a plasticizer for vinyl plastic articles. It has been found to be positive in an NCI rodent bioassay but has generally given negative results in in vitro genotoxicity tests. We therefore decided to test the urine of rats fed [14C]DEHP for mutagenic activity in the Ames Salmonella test. The recovery of radioactivity from the urine of rats dosed with [14C]DEHP was examined by solvent extraction and XAD-2 resin absorption procedures. Both of these procedures were inadequate for quantitative recovery of urinary metabolites required for subsequent mutagenicity testing using the Ames Salmonella/microsome procedure. Recoveries of less than 5% were observed using standard solvent extraction techniques whereas the XAD-2 adsorption technique gave about 67% at high resin/urine ratios. Treatment of the urine with beta-glucuronidase/aryl sulfatase did not affect these recoveries. The direct urine plating procedure represents a viable alternative to the above concentration procedures for this phthalate ester. The effects of L-histidine and the beta-glucuronidase/aryl sulfatase preparation on the background reversion frequencies of the Ames tester strains is discussed.     

Impact of urine matrix and isolation procedure on retention behaviour of basic drugs in thin-layer chromatography

The influence of three different isolation procedures, namely liquid-liquid extraction, Extrelut column extraction and XAD-2 column extraction, and of the urine matrix on the standardised Rf values and variability of Rf values of some selected basic drugs was investigated. It appears that the liquid-liquid extraction may give a significant deviation of standardised Rf values in respect of pure drugs, which is dependent on the TLC system. For all three isolation procedures, the search window for substance identification by means of data collection based on standardised Rf values of pure drugs should be slightly wider after extraction than when using pure drugs. The TLC system cyclohexane-toluene-diethylamine (75:15:10, v/v/v) showed the best accuracy and precision of Rf values.     

Encapsulated XAD-2 extraction technique for a rapid screening of drugs of abuse in urine

This paper describes a novel technique for the rapid screening of drugs of abuse in urine involving extraction with encapsulated Amberlite XAD-2 resin followed by gas chromatographic analysis. Each capsule is fabricated as a rigid, porous, polypropylene sphere, 2 cm in diameter, and contains 300 mg of the XAD-2 resin. Two mL of urine is shaken with one capsule under slight positive pressure for eight minutes. A sequential extraction is employed to obtain separate elution of the acidic-neutral and basic drugs. Identification is by dual column gas chromatography. The results, as given for spiked urine samples containing 16 commonly encountered drugs, were found to be linear and reproducible over both the therapeutic and toxic ranges. Extraction efficiencies using the encapsulated XAD-2 technique are comparable to those reported in literature for XAD-2 column chromatography. The purity of the extracts, as seen from the gas chromatographic analysis, is demonstrably better than that seen with the conventional procedures. There was minimal interference from polymer bleed. The total volume of solvents used in the sequential scheme is 5 mL. Total extraction time to achieve dried residues is 15 minutes.     

Impact of biological matrix and isolation methods on detectability and interlaboratory variations of TLC Rf-values in systematic toxicological analysis

The retention behavior of eight basic and neutral drugs, extracted from plasma, blood, and liver by five different methods (XAD-2, Extrelut, Elut-X, Elut-C18, chloroform) and developed in three chromatographic systems [MeOH, Me OH:BuOH:NaBr, CHCl3:MeOH (KOH)] was observed in parallel in two laboratories. The corrected Rf-values were compared with reference data from a data base with data for pure drugs. The biological matrix and/or the extraction severely lowered the precision and, to a lesser extent, the accuracy of the Rf-values as compared to pure drug data and to reference Rf-values. The intra- and interlaboratory variation was smallest in the MeOH system and largest in the CHCl3:MeOH (KOH) system. The observed irreproducibility is caused by the biological matrix (extraction has a large negative impact on the potentials of TLC in identification procedures). Precision and accuracy of extracted drugs were independent of the biological matrix and on the extraction method used.     

Isolation of drugs from autopsy material by XAD-2 adsorption-elution technique a routine procedure

The method of adsorption of drugs on Amberlite XAD-2 resin, followed by differential elution was under study during 1 1/2 year of routine use. The method allows a separation of acidic and basic drugs and — due to acid hydrolysis step before adsorption — assures better recovery of conjugated and proteinbound drugs. The amounts of various drugs, found in autopsy cases by the XAD-2 method were usually higher than those found by solvent extraction. The method applied requires 20 g of sample (biofluid or tissue) for general toxicological analysis.     

Determination of beta2-agonists in bovine urine: comparison of two extraction/clean-up procedures for high-resolution gas chromatography-mass spectrometry analysis

Two extraction/clean-up analytical procedures were investigated and compared regarding their recovery and matrix-purification efficiency for screening beta2-agonist residues in fortified bovine urine by high-resolution gas chromatography-mass spectrometry (GC-MS). The first procedure, based on an analytical method originally developed for detecting anabolic steroids, consists of the employment of the nonionic resin, Amberlite XAD-2, a styrene-divinylbenzene copolymer for solid-phase extraction (SPE), followed by liquid-liquid extraction with diethyl ether. The second focuses on the use of a mixed SPE cartridge (reversed-phase and ion-exchange sorbent, Bond Elut Certify). In both cases, the trimethylsilylated derivatives were analyzed by GC-MS with an ion-trap detector. Clenbuterol, salbutamol, and terbutaline were used to spike urine samples during the comparison experimental phase. Afterwards, tulobuterol, mabuterol, mapenterol, cimbuterol, and brombuterol were included in the evaluation of the second procedure (the Bond Elut Certify procedure). At this stage, the detection was accomplished by GC-MS (quadrupole mass analyzer) with selective ion monitoring acquisition. The isotopic dilution method with the hexadeuterated analogues of clenbuterol and salbutamol was applied to prepare calibration curves and calculate recovery percentages. With XAD-2 resin, terbutaline and salbutamol (resorcinol and phenol-type beta2-agonists, respectively) could not be detected at 20 ng/mL or at 40 ng/mL. In spite of clenbuterol having been detected at 20 ng/mL, the results obtained were not reproducible. The use of the reversed-phase and ion-exchange sorbent Bond Elut Certify allowed multiresidue detection and showed several advantages for the screening of clenbuterol such as higher recoveries, cleaner final extracts, reduced sample preparation time, less labor intensive, and easier solvent consumption and disposal. Recoveries over 88% (concentrations ranging from 0.5 to 10 ppb) and limits of detection equal to 0.5 ppb were met for all the beta2-agonists studied with the last method.     

Analysis of naltrexone urinary metabolites

A reversed-phase HPLC method using ion-pair formation has been developed for the simultaneous determination of naltrexone and three urinary metabolites. The extraction of the free and conjugated metabolites was studied by liquid—solid procedures using styrene—divinylbenzene copolymers (Amberlite XAD-2) and bonded octadecyl silica supports (ODS-silica). Optimum recovery was obtained with ODS-silica extraction using 25% acetonitrile in a 5 mM diammonium phosphate buffer pH 2.1 as elution solvent. The chromatographic behaviour of naltrexone metabolites and naloxone (internal standard) was examined by varying the mobile phase composition. Increments of both the diammonium phosphate buffer concentration and the percentage of organic solvent in the eluent decreases the retention of compounds in a non-linear manner. Increments of the dodecyl sulphate (counter-ion) concentration, increases the retention time. The method was applied to determine the urinary levels of major naltrexone metabolites in a volunteer receiving a 50 mg oral dose. This is the first method reported which permits the simultaneous quantitative determination of naltrexone and its metabolites, 6β-naltrexol, naltrexone glucuronide and 6β-naltrexol glucuronide, in urine.     

N-Glucuronidation of N-hydroxy aromatic amines: A mechanism for their transport and bladder-specific carcinogenicity

Glucuronide conjugates of carcinogenic N-hydroxy metabolites of the primary aromatic amines, 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 1-naphthylamine (1-NA) were isolated from the urine of dogs administered the respective primary amine and from the in vitro incubation of N-hydroxy metabolites with uridine-5′-disphosphoglucuronic acid-fortified dog liver microsomes. The urinary and microsomal conjugates were purified by several sequential chromatographic procedures, including Sephadex G-15, Amberlite XAD-2, and cellulose CF-11 chromatography for microsomal conjugates and Sephadex G-10, DEAE, and Amberlite XAD-2 chromatography for urinary conjugates. The infrared spectra of purified urinary and microsomal conjugates of these three N-hydroxy aromatic amines were identical to spectra of authentic N---C glucuronides prepared by two different synthetic procedures. The urinary and microsomal conjugates comigrated with synthetic N---C glucuronides in two solvent systems. These observations in conjunction with previous studies provide evidence that N---C glucuronidation represents a general metabolic reaction of carcinogenic N-hydroxy aromatic amines which provides the means of transport of these compounds to their site of action in the bladder.     

Genotoxicity of wastewaters used for irrigation of food crops

In most towns of India, wastewater coming from both industrial and domestic sources and without any treatment is used to irrigate the agricultural crops. This practice has been polluting the soil, and pollutants could possibly reach the food chain. For the above reasons, the wastewaters of Ghaziabad City (India), which is used for irrigation, were sampled (at two different sites) and monitored for the presence of genotoxic agents from January 2005 to June 2007. Gas chromatographic analysis showed the presence of certain OC (DDE, DDT, Dieldrin, Aldrin, and Endosulfan) and OP (Dimethoate, Malathion, Methlyparathion, and Chlorpyrifos) pesticides in both the sampling sites. Wastewater samples were concentrated using XAD resins (XAD-4 and XAD-8) and liquid-liquid extraction procedures, and the extracts were assayed for genotoxic potential by Ames Salmonella/microsome test, DNA repair defective mutants, and bacteriophage lambda systems. The test samples exhibited significant mutagenicity with TA98, TA97a, and TA100 strains with the probable role of contaminating pesticides in the wastewater. However, XAD-concentrated samples were more mutagenic in both sites as compared to liquid-liquid-extracted samples. The damage in the DNA repair defective mutants in the presence of XAD-concentrated water samples were also found to be higher to that of liquid-liquid-extracted water samples at the dose level of 20 muL/mL culture. All the mutants invariably exhibited significant decline in their colony-forming units as compared to their isogenic wild-type counterparts. The survival was decreased by 81.7 and 75.5% in polA(-) strain in site I, and 76.0 and 73.5% in site II in polA(-) under the same experimental conditions after 6 h of treatment with XAD-concentrated and liquid-liquid-extracted samples, respectively. A significant decrease in the survival of bacteriophage lambda was also observed when treated with the test samples.     

Genotoxicity of water extracts from the River Yamuna at Mathura,India

Water samples were collected from the River Yamuna at Mathura, India, and concentrated by using XAD resins (Amberlite XAD-4 and XAD-8) and liquid-liquid extraction procedures. The genotoxicities of the extracted water samples were evaluated by the Ames Salmonella/mammalian microsome test, DNA repair of defective mutants, and bacteriophage lambda systems. The results of the Salmonella test demonstrated that the XAD-concentrated water samples had maximum mutagenicity with the TA98 strain, both with and without metabolic activation. The XAD-concentrated water samples collected in the summer showed maximum mutagenic responses compared with those collected in other seasons, whereas the liquid-liquid extracted samples exhibited maximum mutagenic activity during the postmonsoon season. The damage brought about during DNA repair of defective mutants in the presence of XAD-concentrated water samples was found to be remarkably high compared with the liquid-liquid extracted water samples at a dose level of 20 microL/mL of culture. All the mutants invariably exhibited significant decline in their colony-forming units compared with their isogenic wild-type counterparts. Survival was decreased by 86.7% and 65.1% in the polA(-) strain after 6 h of treatment with XAD-concentrated and liquid-liquid extracted water samples, respectively. A significant decrease in the survival of bacteriophage lambda was also observed when treated with test samples. The damage was more pronounced in lexA mutants when the phage was treated with XAD-concentrated samples. The recA, lexA, and polA mutants of E. coli K-12 were found to be sensitive to the test samples, suggesting damage to the DNA of exposed cells as well as to the role of recA(+), lexA(+), and polA(+) genes in coping with the hazardous effect of the pollutants. The results demonstrated substantial genotoxicity and mutagenicity in the water samples tested.     

Isolation of drugs from blood by column chromatography on amberlite XAD-2

An investigation was carried out on the isolation of 28 drugs from blood by column chromatography on Amberlite XAD-2. The following substances were added to postmortem blood specimens at concentrations generally of 1–10 g/ml: barbital, heptabarbital, hexobarbital, pentobarbital, phenobarbital, phenylbutazone, monocrotophos, amidopyrine, carbromal, diazepam, meprobamate, methaqualone, nitrazepam, phenazetin, chlorpromazine, dibenzepin, diphenhydramine, haloperidol, imipramine, mescaline, methadone, morphine, pentazocine, pethidine, tilidine, triflupromazine, verapamil, N-propylajmalinium bitartrate. The samples were purified by column chromatography on Amberlite XAD-2 and simple solvent extraction and subsequently quantitated by gas chromatography. By systematic variation of the conditions for adsorption of the drugs and desorption from the resin, revoveries of more than 80% after XAD-2 column chromatography could be achieved for most drugs. Thereby it was demonstrated, that in cases of fatal poisoning or emergencies this procedure is a valuable tool for forensic and clinical toxicologists who have to find out the toxic agent by chemical analysis of the blood.

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Zusammenfassung Es wurden Untersuchungen über die säulenchromatographische Isolierung von 28 Arzneistoffen aus Blut an Amberlite XAD-2 durchgeführt. Die folgenden Substanzen wurden Leichenblutproben in Konzentrationen im allgemeinen von 1–10 g/ml zugesetzt: Barbital, Heptabarbital, Hexobarbital, Pentobarbital, Phenobarbital, Phenylbutazon, Monocrotophos, Amidopyrin, Carbromal, Diazepam, Meprobamat, Methaqualon, Nitrazepam, Phenacetin, Chlorpromazin, Dibenzepin, Diphenhydramin, Haloperidol, Imipramin, Meskalin, Methadon, Morphin, Pentazocin, Pethidin, Tilidin, Triflupromazin, Verapamil, N-Propylajmalinium Bitartrat. Die Blutproben wurden säulenchromatographisch an Amberlite XAD-2 und durch eine einfache Lösungsmittelverteilung gereinigt, und anschließend wurden die Arzneimittel durch Gaschromatographie quantitativ bestimmt. Durch systematische Optimierung der Bedingungen für die Adsorption und die Desorption vom Kunstharz konnten für die meisten Substanzen Wiederfindungen von mehr als 80% nach XAD-2 Säulenchromatographie erreicht werden. Dieses Verfahren ist also ein wertvolles Hilfsmittel für den forensischen und klinischen Toxikologen, der bei tödlichen oder akut lebensbedrohlichen Vergiftungsfällen das wirksame Agens durch die Untersuchung des Blutes nachweisen muß.

     

人尿中卡鲁睾酮的代谢产物研究

使用GC/MS方法对人尿中卡鲁睾酮(calusterone)的代谢情况进行了研究。尿样经XAD-2树脂柱吸附、酶水解、乙醚萃取及三甲基硅烷衍生化处理后,使用毛细管柱进行GC/MS分析,鉴定了7种代谢物,从而推导出卡鲁睾酮在人体中的代谢模式。     

The Bacillus subtilis/microsome rec-assay for the detection of DNA-damaging substances in waters of a night soil treatment plant

A Bacillus subtilis/microsome Rec-assay was improved by the introduction of a period for the test sample to interact with the bacteria before cultivation in a Monod tube. This method is called the “liquid B. subtilis/microsome Rec-assay method.” Evaluation of the Rec-assay is presented in terms of R50, S-probit, and RS, which were analyzed with computer programs developed on Probit theory and Target theory. Results could be classified into four categories: strong DNA damaging, DNA damaging, not DNA damaging, and reverse. Application of the Rec-assay to water samples from a night soil treatment plant showed that raw night soils contained a considerable amount of DNA-damaging substances. The amount of DNA-damaging substances in test samples was measured in terms of a new indicator, “Rec-volume.” After the biological treatment of nitrification and denitrification process, and the tertiary treatment of chemical coagulation and floatation for phosphorus removal, DNA-damaging substances were effectively reduced in the treated water. Following the tertiary treatment, ozonation was practiced for disinfection and color removal in this plant. Ozonation also showed effective reduction of DNA-damaging substances in the treated waters. In order to concentrate DNA-damaging substances in test samples, the adsorption method of XAD-2 and XAD-8 resins followed by ether and methanol extraction was employed.     

Aridicins, novel glycopeptide antibiotics. II. Isolation and characterization

A new antibacterial antibiotic complex, aridicin, was produced by a new genus, Kibdelosporangium aridum (SK&F-AAD-216). The individual factors, aridicins A, B and C, were isolated from the fermentation broth by an Amberlite XAD-7 resin extraction and purified by preparative reversed phase HPLC. The aridicins were found to be novel members of the glycopeptide class of antibiotics as exemplified by ristocetin and vancomycin, based on chemical and spectroscopic data, their molecular weights as determined by FAB mass spectrometry (1,786, 1,800 and 1,814), the detection of actinoidinic acid in their acid hydrolysates, and detailed TLC and HPLC comparisons with representative members of this class.     

Solid-phase extraction of l-muscone from aqueous samples with amberlite XAD-4 for gas chromatographic assay

An efficient analytical method was devised for the accurate L-muscone assay in aqueous samples. It involves solid-phase extraction of L-muscone in adsorption mode using XAD-4 as the sorbent and dichloromethane modified with 10% (v/v) methanol as the eluting solvent. The gas chromatographic analysis of the eluate residue dissolved in toluene on a DB-5MS capillary column provided complete resolution of L-muscone from the co-extracted interferences. The overall method showed excellent linearity (r2 > or = 0.9994) in the range of 0.1 to 2.0 microg/mL with good intra- and inter-day precisions (% RSD = 2.5 to approximately 7.3) and with high extraction recovery rates (> or = 98.1%). When the present method was applied to a L-muscone herbal drink product, the within-batch RE (%) in the labeled concentration (1.5 microg/mL) for the three randomly chosen bottles were -2.4, -1.3 and -3.3 with high precision (% RSD < or = 3.1). The present method is considered to be suitable for quality control evaluation on liquid drinks and other complex formulations fortified with L-muscone.     

The use of gas chromatography/negative ion chemical ionization mass spectrometry for the determination of lorazepam in whole blood

The determination of lorazepam in blood by gas chromatography/mass spectrometry with selected ion monitoring (GC/MS/SIM) in the negative chemical ionization mode (NICI) was investigated. The method involves a single extraction with Amberlite XAD-2 resin and the use of [2H6]triazolam as an internal standard. The limit of detection is 0.5 ng/mL and standard curves are linear from 6.25 to 250 ng/mL. The applicability of the method to forensic toxicology is reported.     

Analysis of mutagenic activity in human urine after concentration on different resins and high-performance liquid chromatography

Smokers' urine was tested for mutagenic activity on Salmonella typhimurium strain TA1538 with metabolic activation after adsorption on different resins and desorption with organic solvents. The amounts of XAD-2 were 1.25 and 5 g/100 ml urine, the amounts of alumina, cyanopropyl and C18 were all 5 g/100 ml and extrelut 80 g/100 ml. Adsorbed organic chemicals were eluted with acetone from XAD-2, with dichloromethane from extrelut and with a series of solvents from the other resins (hexane, toluene, dichloromethane and methanol). All columns gave similar results, with the exception of extrelut, which had poor recovery of mutagenic activity. Higher resin/urine ratios and sequences of columns gave better results. The organic eluates from XAD-2 columns loaded with the urine of patients treated with cyclophosphamide and melphalan were mutagenic on strain TA1535 with S9, and some mutagenic activity was also detectable in the aqueous eluate. Cisplatin was adsorbed on XAD-2, C18 and extrelut, but was eluted only from extrelut using dimethylformamide as a solvent. Smokers' urine was separated into several fractions with high-performance liquid chromatography, using C-18 columns with a series of solutions of 2.5 mM phosphoric acid and acetone or with a gradient of methanol. Several fractions containing dissolved organic compounds and no histidine were mutagenic with metabolic activation, but the overall mutagenic activity was still lower than the one detected with one-step chromatography on XAD-2. Using XAD-2 resins with a high ratio of resin to urine still seems to be the method of choice for studying urinary mutagenicity.     

Identification of cocarcinogens and promoters in industrial discharges into and in the Illinois River

Organic compounds were isolated from grab or composite samples of industrial and municipal discharges and of the Illinois River by liquid-liquid extraction or adsorption on activated carbon or XAD-2 resin columns. Of the 213 different compounds identified and semiquantitated by gas chromatography-mass spectrometry in 16 samples, 74 were long-chain hydrocarbons or their derivatives. Although their toxicological significance in the environment at the levels found is unknown, the widespread presence of these cocarcinogens and promoters (often found in conjunction with known initiators) may make them significant environmental toxicants. Some evidence for this is the fact that serial dilutions of the extracts were highly toxic to Salmonella typhimurium in the Ames assay, while weak mutagenicity was occasionally detected.     

Studies related to the metabolism of anabolic steroids in the horse: The phase I and phase II biotransformation of 19-nortestosterone in the equine castrate

1. The metabolism of 19-nor[4-¹⁴C]testosterone has been studied in the equine castrate.

2. Following XAD-2 extraction of aliquots of the 0—24 h urine samples, the glucuronic acid and sulphate conjugates were separated by Sephadex LH-20 column chromatography. After hydrolysis of the conjugates, the neutral phase I metabolites of 19-nortestosterone were extracted, purified and identified by g.l.c.-mass spectrometry.

3. In phase I metabolism stereospecificity was observed in the reduction of the A-ring with the formation of the 5α,β-isomers of estranediol. Epimerization at C-17 and hydroxylation at C-16 were the other major pathways.

4. In phase II metabolism the C-17α steroid epimers were predominantly conjugated with glucuronic acid and the C-17β epimers with sulphuric acid.

5. One animal showed a slight variation in metabolism with a tendency towards the formation of polar metabolites.     

尿液中利尿剂、丙磺舒、咖啡因、匹莫林的固相提取和HPLC初筛分析

A solid-phase extraction and reversed phase high performance liquidchromatographic method(RP-HPLC) was developed for the rapid determination of 13 diuretics (bel-onging to five different pharmacological groups) ,probenecid, caffeine and pemoline in urine. Two mlurine sample was first adsorbed on a XAD-2 column, then eluted with ether--ethyl acetate(l: 1).The eluate was evaporated to dryness and reconstituted in methanol. The methanolic solution wasinjected into a HP LiChrosorb RP-18 column, using phosphate buffer (pH 3) and acetonitrile as the mo-bile phase and monitored at 216 nm, 230 nm, and 275 nm on a diode array ultraviolet detector. Theextraction recoveries of 16 drugs were above 75%. The limits of detection ranged from 0. 3～3.0μg/ml of urine. All drugs were separately administered to healthy volunteers, positive urine sampleswere collected, and urinary excretion--time curves of some drugs were reported.