Metabolism of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane in the mouse

The metabolites of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) found in the urine of female Swiss mice are reported. The metabolites of DDT are DDD, 1-chloro-2,2-bis(p-chlorophenyl)ethene (DDMU), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene (DDE), 2,2-bis(p-chlorophenyl)acetic acid (DDA), 2-hydroxy-2,2-bis(p-chlorophenyl)acetic acid (αOH-DDA) and 2,2-bis(p-chlorophenyl)ethanol (DDOH), while DDD afforded DDMU, DDE, DDA, αOH-DDA and DDOH. The relative excreted levels of DDA and DDOH and the absence of 2,2-bis(p-chlorophenyl)acetaldehyde (DDCHO) are not consistent with the generally accepted path way for DDA formation, which involves sequential metabolism of DDT and DDD via DDOH to afford DDA. The quantitative results are interpreted to mean that DDA is formed by hydroxylation at the chlorinated sp³-side chain carbon of DDD to give 2,2-bis(p-chlorophenyl)acetyl chloride (DDA-Cl), which in turn is hydrolyzed to DDA. The excretion of αOH-DDA from both DDT- and DDD-treated mice has never been previously observed. It is suggested that this metabolite arises from the initial epoxidation of DDMU, a metabolite of DDT and DDD, to yield 1,2-epoxy-1-chloro-2,2-bis(p-chlorophenyl)ethane (DDMU-epoxide). This chloroepoxide is then hydrolyzed and oxidized to produce the αOH-DDA.     

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.     

Biased sex ratio and niche restriction in Baruscapillaria obsignata (Madsen 1945) (Nematoda, Capillariidae) from Columba livia (Aves, Columbidae)

In the present study populations of the avian nematode species Baruscapillaria obsignata are described from Columba livia. Male and female individuals were obtained from 27 birds, fixed in alcohol/formalin/acetic acid (AFA) and preserved in 70% ethanol. Nematodes were identified and then counted under a stereoscopic microscope. Baruscapillaria obsignata were much more frequent in the anterior third of the small intestine, and females were more abundant than males in all infra populations. The prevalence was 55.6%, mean intensity was 11.8 (median 11.0; range 1-31) and abundance 6.56. In the present study, we observed an aggregated distribution of parasite infrapopulations, as demonstrated by the value of the exponent of the negative binomial distribution, K?=?0.2773; by the discrepancy index, D?=?0.656 and by the variance/mean ratio, 12.44. The female/male sex ratios found in all infrapopulations were always greater than 1, showing a bias in favour of female abundance. This tendency was especially marked in infrapopulations containing fewer individuals. The sizes of infrapopulations ranged from 5 to 31 individuals. The mean sex ratio observed was 2.69?±?3.28 (median 1.83; range 0-11). In infrapopulations with 5-15 individuals, the sex ratios observed varied from 2.6 to 11, while in those with 17-31 individuals, the sex ratios were lower, ranging from 1.7 to 2.4. There was a negative correlation between the intensity of infection and the sex ratio of infrapopulations. Results are discussed in terms of possible factors influencing the processes that lead to niche restriction and biased sex ratios in parasite infrapopulations.     

A simple HPLC method for plasma level monitoring of mitotane and its two main metabolites in adrenocortical cancer patients

Mitotane (o,p'-DDD or (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane, DDD) is the drug of choice for non-resectable and metastatic adrenocortical carcinomas (ACC). Measurement of mitotane and metabolites, o,p'-DDE (1,1-dichloro-2-[p-chlorophenyl]-2-[o-chlorophenyl]ethene, DDE) and o,p'-DDA (1,1-[o,p'-dichlorodiphenyl] acetic acid, DDA) provides a better understanding of mitotane pharmacokinetics and pharmacodynamics. We have developed a simple, robust and efficient high performance liquid chromatography (HPLC) method to measure mitotane and its two main metabolites, DDE and DDA. The method involves a single ethanol extraction of mitotane, DDE, DDA, and an internal standard (int std) p,p'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) with an extraction efficiency of 77-88%. All compounds are measured simultaneously using a reversed-phase phenyl HPLC column with an isocratic elution of mobile phase at a flow rate of 0.6 ml/min followed by UV detection at λ 226 nm. Inter and intra-day validation demonstrates good reproducibility and accuracy. Limits of quantitation are 0.2 μg/ml for mitotane and DDE, and 0.5 μg/ml for DDA. The method has been evaluated in plasma from 23 patients on mitotane therapy, revealing DDA concentrations 1-18 times higher than the parent compound.     

Site-specific incorporation of (aminooxy)acetic acid into proteins

By employing a general biosynthetic method for the elaboration of proteins containing unnatural amino acid analogues, we incorporated (aminooxy)acetic acid into positions 10 and 27 of Escherichia coli dihydrofolate reductase. Introduction of the modified amino acid into DHFR was accomplished in an in vitro protein biosynthesizing system by readthrough of a nonsense (UAG) codon with a suppressor tRNA that had been activated with (aminooxy)acetic acid. Incorporation of the amino acid proceeded with reasonable efficiency at codon position 10 but less well at position 27. (Aminooxy)acetic acid was also incorporated into position 72 of DNA polymerase beta. Peptides containing (aminooxy)acetic acid have been shown to adopt a preferred conformation involving an eight-membered ring that resembles a gamma-turn. Accordingly, the present study may facilitate the elaboration of proteins containing conformationally biased peptidomimetic motifs at predetermined sites. The present results further extend the examples of ribosomally mediated formation of peptide bond analogues of altered connectivity and provide a conformationally biased linkage at a predetermined site. It has also been shown that the elaborated protein can be cleaved chemically at the site containing the modified amino acid.     

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Envelope protein(s) derived from influenza virus

Eckert, Edward A. (University of Michigan, Ann Arbor). Envelope protein(s) derived from influenza virus. J. Bacteriol. 91:1907-1910. 1966.-Lipids were extracted from influenza virus, strain PR8, with methanol-chloroform, and the protein residue was dissolved in 67% glacial acetic acid. Hemagglutinating activity and complement-fixing reactivity were markedly reduced or lost during lipid extraction, and then increased after acetic acid treatment and subsequent dialysis. Evidence is presented that the envelope protein(s) responsible for these activities is dissociated in acetic acid and reassociated at neutral pH.     

LC-MS analysis of trimethoxyamphetamine designer drugs (TMA series) from urine samples

A sensitive liquid chromatography-mass spectrometric (LC-MS) method for quantification of an active psychedelic hallucinogenic drugs (trimethoxyamphetamines) in human urine after solid-phase extraction (SPE) with C(18) cartridge was developed and validated. Chromatographic separation was achieved on reversed-phase Phenomenex 3.0 microm Polar Plus column (150 mm x 2.1 mm) with acetonitrile -0.2% acetic acid as mobile-phase and the step gradient elution resulted in a total run time of about 20 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in selected ion monitoring (SIM) mode. In the evaluated concentration range (10-200 ng/mL) (R(2) > or = 0.998) a good linear relationship was obtained. The lower limits of detection (LLODs) and quantification (LLOQs) ranged from 4.26 to 9.12 ng/mL and from 13.18 to 29.22 ng/mL, respectively. Average recoveries ranged from 68.52 to 97.90% in urine at the concentrations of 25, 50 and 100 ng/mL. Intra- and inter-day relative standard deviations were 3.70-10.77% and 7.63-12.94%, respectively. This LC-MS method proved to be robust and reliable, and suitable for the use as a confirmation method in clinical urine drug testing.     

Factors Affecting Organic Acid Production by Sourdough (San Francisco) Bacteria

Previous workers from this laboratory observed considerable variation in the proportions of acetic and lactic acids produced in pure broth culture as compared to consistently high proportions of acetic acid produced in the sourdough and flour suspension systems. In the latter the proportion of acetic acid was always in the range of 20 to 35% of the total, whereas in pure broth culture frequently less than 5% acetic acid was produced. In the natural environment, the sourdough bacteria, tentatively identified as lactobacilli, coexist with a yeast, Saccharomyces exiguus, and this study was undertaken to determine whether this yeast or flour ingredients including glucose or other factors were involved in this variable production of acetic acid. The proportion of acetic acid produced in broth culture on maltose, the preferred carbohydrate source, was found to depend almost entirely on the degree of aeration. Essentially anaerobic conditions, as obtained by thorough evacuation and flushing with CO(2) or N(2), resulted in very low (5% or less) proportions of acetic acid. Aerobic conditions, achieved by continuous shaking in cotton-plugged flasks, yielded high levels (23 to 39% of the total) of acetic acid. Similar effects of aeration were observed with glucose as the substrate, although growth was considerably slower, or in nonsterile flour suspension systems. It is theorized that, under aerobic conditions, the reduced pyridine nucleotides generated in the dissimilation of carbohydrate are oxidized directly by molecular oxygen, thereby becoming unavailable for the reduction of the acetyl phosphate intermediate to ethyl alcohol, the usual product of anaerobic dissimilation of glucose by heterofermentative lactic acid bacteria. Comparative studies with known strains of homo- and heterofermentative lactobacilli showed similar effects of aeration only on the heterofermentative strains, lending additional support to the tentative grouping by previous workers from this laboratory of the sourdough bacteria with the heterofermentative lactobacilli.     

Bovine colostric transforming growth factor-beta-like peptide that induces growth inhibition and changes in morphology of human osteogenic sarcoma cells (MG-63)

TGF-beta like peptide, termed TGF(BC-1), was partially purified from defatted and decaseinated bovine colostrum by a sequence of DEAE-Sephacel chromatography and Sephadex G-50 gel filtration in 1M acetic acid. TGF(BC-1) was distinct from well-known 25K TGF-beta in chemical properties: TGF(BC-1) was sensitive to acid ethanol extraction (Roberts et al., 1980). Its apparent molecular weight ranged from 21k to 11k by gel filtration and it was composed of low MW peptides (15k, 13k, 10k and 7.3k but not 25k) as examined by SDS-PAGE under non-reducing conditions. However, TGF(BC-1) shares some biological properties with the prototype TGF-b. TGF(BC-1) remarkably suppressed growth of osteogenic sarcoma cells (MG-63), and this was intriguingly accompanied by a striking change in morphology.     

Inhibition of C4 photosynthesis by (benzamidooxy)acetic acid

(Benzamidooxy)acetic acid (common name benzadox) which has herbicidal properties was evaluated as a potential inhibitor of photosynthesis in C₄ plants. Among enzymes of the C₄ pathway, it was a relatively strong inhibitor of alanine aminotransferase in in vitro experiments at concentrations of 5mM. In benzadox treated leaves of Panicum miliaceum, a NAD-malic enzyme type C₄ species, there was strong inhibition of both alanine and aspartate aminotransferase and of photosynthetic O₂ evolution within one hour. Consistent with the inhibition of these enzymes of the C₄ cycle, the pool sizes of metabolites of the cycle was altered: the aspartate level was increased two fold, while the levels of other metabolites such as pyruvate, alanine, oxalacetate and malate were decreased. Kinetic studies with partially purified alanine aminotransferase showed that benzadox is a competitive inhibitor with respect to alanine and a noncompetitive inhibitor with respect to 2-oxoglutarate. Comparisons between the structures and inhibitory actions of benzadox and (aminooxy)acetic acid, the latter a potent inhibitor of alanine and aspartate aminotransferases, suggest that in vivo, benzadox may exert its effect through metabolism to (aminooxy)acetic acid.Abbreviations benzadox (benzamidooxy)acetic acid - DTE dithioerythritol This research was supported in part by gift funds from Monsanto Agricultural Products Company. St. Louis, Missouri, and by NSF Grant PCM-8107953.     

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6'-dibehenate)-a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6'-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4 degrees C and 25 degrees C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-gamma cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-gamma was identified as CD4 T cells.     

The synthesis of tris(hydroxymethyl)acetic acid by fermentation of pentaerythritol

A process was devised for the synthesis of tris(hydroxymethyl)acetic acid by means of bio-oxidation of pentaerythritol. A flavobacterium able to grow on pentaerythritol was isolated from the soil. Mutants were obtained that, were deficient, in their pentaerythritol oxidation system. These mutants could not grow on pentaerythritol, but when grown on an assimilable carbon source; they oxidized pentaerythritol to tris(hydroxymethyl)acetic acid. This conversion readily took place in a medium consisting of 20 g pentaerythritol, 10 g yeast extract, and 2 g acetic acid (neutralized) per liter. Since the mutants were unable to metabolize tris(hydroxymethyl)acetic acid, theoretical conversion yields were attainable. When pentaerythritol was added stepwise and the acid formed was neutralized continuously, 95–100% yields were obtained in concentrations of the order of 60 g/l.     

Effects of partial acid hydrolysis on physical and chemical properties of agar from a newly reported Japanese agarophyte (Gracilariopsis lemaneiformis)

Physical and chemical properties of alkali-treated agar polymers extracted from Gracilariopsis lemaneiformis, newly reported Japanese agarophyte, were investigated after partial acid hydrolysis. The alkali-treated agar was hydrolyzed in boiling 0.1 N, 0.01 N, and 0.001 N sulfuric acid, oxalic acid, acetic acid, and citric acid solutions, for 1, 2, and 3 h at 100 °C. Partial acid hydrolysis of the agar polymers indicated strong effects on the physical properties. Different kinds of acid used for hydrolysis gave different agar properties. Gelling polymers were obtained from the agar hydrolysed in boiling 0.001 N acetic acid, oxalic acid, and citric acid solutions, and in 0.01 N acetic acid solution. High gel strength (715 ± 74.6 g cm-2) with low viscosity (2.47 cP) was obtained from 1 h treatment by 0.001 N acetic acid on hydrolysed agar. The results indicated that partial hydrolysis of agar under appropriate conditions probably improve agar quality and produce good grade agar from the Japanese agarophyte. This revised version was published online in August 2006 with corrections to the Cover Date.     

The identification of gonadotropin-releasing hormone (GnRH) in hypothalamic and extrahypothalamic loci of the human nervous system.

Immunoreactive-like GnRH activity has been identified in 24 of 26 separate loci of the human central nervous system. Tissues, secured from 5 brains at autopsy, were dissected, extracted sequentially with 2N and glacial acetic acid, lyophilized, and eluted in buffered saline for GnRH determinations by specific radioimmunoassay. GnRH concentrations (ng/mg protein) ranged from 8.96 (infundibulum) to 0.001 (cerebellum. middle lobe). Highest extrahypothalamic concentrations of GnRH were found in mamillary body (0.076) and thalamus (0.002). Extrahypothalamic GnRH was identical to synthetic and hypothalamic GnRH by criteria of immunoidentity. No post-mortem GnRH peptidolysis, evaluated experimentally in rats, was evident between 0 and 16 hrs in intact tissues maintained at 4 degrees C. These data suggest that GnRH is distributed throughout regions of the human brain outside the hypothalamus and suggest new, non-endocrine functions for GnRH in the human CNS, analogous the those reported recently for GnRH in experimental animals.     

Synthesis,characterization, and use of 2-[(2H(9))butoxy]acetic acid and 2-(3-methylbutoxy)acetic acid as an internal standard and an instrument performance surrogate,respectively, for the gas chromatographic-mass spectrometric determination of 2-butoxyacetic acid,a human metabolite of 2-butoxyethanol

2-[(2H(9))Butoxy]acetic acid and 2-(3-methylbutoxy)acetic acid were synthesized, mixed with 2-butoxyacetic acid, and separated by capillary gas chromatography on a fused-silica column with a length of 50 m, inside diameter of 0.200 mm, and a "free fatty acid phase" wall coating of 0.3 microm film. 2-[(2H(9))Butoxy]acetic acid, 2-butoxyacetic acid, and 2-(3-methylbutoxy)acetic acid were baseline resolved at retention times of 13.55, 13.78, and 15.20 min; 2-(3-methylbutoxy)acetic acid having a peak efficiency of 360,000. Mass spectrometric detection using selected ion monitoring at m/z 66, 57, and 71 showed linear analytical responses from 0.04 ng to at least 200 ng with a limit of detection of 0.04 ng for 2-butoxyacetic acid.     

Liquid chromatography method for quantifying N-(4-hydroxyphenyl)retinamide and N-(4-methoxyphenyl)retinamide in tissues

A simple and accurate high-performance liquid chromatography (HPLC) method was developed to measure levels of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its main metabolite N-(4-methoxyphenyl)retinamide (4-MPR) in tissue. Following ultrasonic extraction of fresh tissue in acetonitrile (ACN), 4-HPR and 4-MPR were measured by HPLC with UV absorbance detection at 340 nm, using isocratic elution with ACN, H(2)O, and acetic acid. N-(4-ethoxyphenyl)retinamide (4-EPR) was employed as an internal standard. The 4-HPR and 4-MPR recovery in bovine liver or bovine brain tissue samples spiked with known amounts of 4-HPR and 4-MPR ranged from 93 to 110%. The detection limit of the method was 50 ng/ml. The method was tested on actual samples from an athymic (nu/nu) mouse carrying a subcutaneous tumor xenograft originating from SMS-KCNR neuroblastoma cells. The tissues were harvested and analyzed following a 3 day long treatment with intraperitoneal injections of 4-HPR/Diluent-12. 4-HPR and the metabolite 4-MPR were detected and quantitated in the tested tissues including tumor, liver, and brain. This method can be used to quantify 4-HPR and 4-MPR in different tissues to determine the bioavailability of 4-HPR.     

Metabolic and mutagenicity studies on DDT and 15 derivatives. Detection of 1,1-bis(p-chlorophenyl)-2,2-dichloroethane and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (kelthane acetate) as mutagens in Salmonella typhimurium and of 1,1-bis(p-chlorophenyl) ethylene oxide, a likely metabolite, as an alkylating agent.

Using a novel in vitro technique, whereby microsomal enzymes were embedded in an agar layer to prolong their viability, 1,1-bis(p-chlorophenyl) ethylene(DDNU), a mammalian metabolite of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), was converted by microsomal mono-oxygenases of mouse liver into 1,1-bis(p-chlorophenyl)-1,2-ethanediol (DDNU-diol). The putative epoxide intermediate, 1,1-bis(p-chlorophenyl)ethylene oxide (DDNU-oxide), a new compound, was synthesized; it showed weak alkylating activity with 4-(4-nitrobenzyl)pyridine but was not mutagenic in Salmonella typhimurium strains TA100 and TA98. DDT and 13 of its metabolites or putative synthetic derivatives, including 1,1-bis(p-chlorophenyl)-2,2-dichloroethylene (DDE), 1 1,1-bis(p-chlorophenyl)-2-chloroethylene (DDMU), 1,1-bis(p-chlorophenyl)-2-chloroethane (DDMS)-DDNU, 2,2-bis(p-chlorophenyl)ethanol (DDOH), bis(p-chlorophenyl)acetic acid (DDA) and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethanol (Kethane), caused no mutagenic effects in S. typhimurium strains TA100 or TA98, either in the presence or absence of a mouse-liver microsomal fraction. 1,1-Bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (Kelthane acetate) was a direct-acting mutagen in strain TA100, whereas 1,1-bis(p-chlorophenyl)-2,2-dichloroethane (DDD) was mutagenic in TA98, only in the presence of a mouse-liver microsomal system. The results are discussed in relation to possible pathways whereby DDT is activated to mutagenic and/or carcinogenic metabolites.     

Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.