Mass Spectro Research Articles

Antibacterial activity of three ethanolic extracts of propolis from the municipalities of Sevilla, Cartago, and Armenia, Colombia, against MRSA (Methicillin-Resistant Staphylococcus aureus)

Introduction: Staphylococcus aureus is a pathogen commonly associated with hospital and community-acquired infections, particularly methicillin-resistant strains that impose limi- tations on treatment options. The resistance of these strains to beta-lactam antibiotics has spurred the search for new antimicrobial sources, and natural products such as propolis extracts have demonstrated efficacy against Methicillin-Resistant Staphylococcus aureus. The an- timicrobial activity of propolis depends to the flora used by the bees to make the propolis. Aim: This study aims to determine the antibacterial activity of three ethanolic extracts of propolis from the municipalities of Sevilla, Cartago, and Armenia, Colombia, against Methicillin-Resistant Staphylococcus aureus. Methodology: The methodology involved obtaining ethanolic extracts of propolis collected from different regions of Colombia, followed by fractionation and identification of the propolis with the highest bactericidal activity. Subsequently, an analysis using gas chromatography coupled with mass spectro- metry (GC-MS) was identified the predominant compound in the selected fractions. Re- sults: All three propolis samples evaluated in this study demonstrated bactericidal activity at concentration of 10 mg/mL, with the Cartago propolis showing concentrations below 5 mg/mL. Chromatographic analysis identified 3-Hexadecyloxycarbonyl-5-(2-hydroxye- thyl) as the predominant compound. Conclusion: In conclusion, the need for additional research to understand the pharmacological potential of Colombian propolis, specifically the bactericidal activity of Cartago propolis against Methicillin-Resistant Staphylococcus aureus.

P–283 Combination between amino acids profile of the spent culture media and morphokinetics parameters of human embryos to determine its viability

Abstract Study question How to determine human embryo viability noninvasively before embryo transfer? Summary answer We propose that the combination of the amino acid profile of an individual embryo with its morphokinetics will provide noninvasive tool to determine its viability. What is known already It was already known that human embryos at early cleavage require non-essential amino acids, while at the 8-cell to blastocyst stages, a mixture of non-essential and essential amino acids. Amino acids have important roles during embryo development. Acting as biosynthetic precursors,buffers of intracellular pH in the embryo, antioxidants, energy sources and regulators of metabolic function and signaling pathways. Many studies have used time-lapse to analyze human embryonic development including the process of fertilization and assessment of early events and introducednoninvasive prognostic markers which predict embryo development and correlate it to IVF treatment outcomes. Study design, size, duration This study was a prospective cohort study approved by the Clinical Trial Ethical Committee of Faculty of Medicine, Alexandria University according to ethical standards of scientific research (Serial number: 0303721).Thirty females aged 30.13 ± 4.83 years undergoing ICSI cycle in the Madina Fertility Center, during the period of March 2018 to November 2019.202 MII oocytes were incubated individually in embryoscope. Participants/materials, setting, methods Embryos (n = 161) were divided on Day 5 into two groups –developed embryos “Group D” (embryos that developed to blastocyst) and arrested embryos “Group A” (embryos remain at cleavage stage and fail to develop to blastocys).Developed embryos (Group D) included 99 embryos, and Arrested embryos (Group A) included 62 embryos. For each group, morphokinetic developmental points using embryoscope and the different amino acids concentrations in spent culture medium were analyzed using LC- mass spectro etry. Main results and the role of chance On one hand, the first appearance of pronuclei (TPNa), t2, t4 and CC2 in group D occurred significantly earlier than those of Group A.Analysis of 19 essential and non-essential amino acids in spent culture medium of each embryo in the two studied groups D and A showed a significantly higher concentration of two essential amino acids L-Valine (145.73 ± 150.96) and L-Phenylalanine (61.59 ± 55.78) in Group D than their concentration in Group A ( 104.58 ± 33.58, 44.24 ± 14.61, respectively , p ≤ 0.05).and significantly lower concentration of three non-essential amino acids L-Tyrosine (62.56 ± 41.03) , L-Cysteine (19.48 ± 11.90), and L-Alanine (136.0 ± 389.83) observed in Group D when compared to Group A (69.57 ± 20.78, 22.37 ± 8.59,145.33 ± 165.22, respectively, Limitations, reasons for caution It is important to note, that results were developed on a data set from one clinic with different stimulation protocols, a multicenter data and a correlation with the stimulation protocol used should be involved in future studies, in addition a larger sample size to avoid high standard deviation is recommended Wider implications of the findings: We can conclude that amino acid turnover is independent of the traditional morphological assessment of embryos and it may reflect its viability. The prospective combined use of amino acids profile of individual embryo and its morphokinetic parameters may contribute to introduce a new noninvasivs tool that may improve implantation rate Trial registration number 0303721

Inhibition properties of free and conjugated leupeptin analogues.

Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The specificity of leupeptin is most likely due to the Leu–Leu–Argininal sequence, and its C‐terminal aldehyde group has been suggested to enhance the binding efficiency and to be essential for function. The terminal aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether the inhibitory function of leupeptin toward serine proteases is retained after oxidation or reduction of the aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced inhibitory properties as compared with the oxidized form. Based on these results, new synthetic leupeptin analogues, 6‐aminohexanoic acid (Ahx)–Phe–Leu–Arg–COOH and Ahx–Leu–Leu–Arg–COOH, were prepared by solid‐phase peptide synthesis using the Fmoc strategy. In these analogues, the N‐terminal capping acetyl group was replaced with a 6‐aminohexanoyl group to allow conjugation. The structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry. Determination of the inhibitory properties against trypsin (IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1) revealed that these further modified tripeptides were tight binding inhibitors to their target enzyme, similar to the naturally occurring leupeptin, with Ki values generally in the micromolar range. The Ahx–Phe–Leu–Arg–COOH analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads. All conjugates exhibited inhibitory activity in the same range as for the free peptides.

Investigation of Exhaled Breath Biomarkers for LUNG Cancer By TD-GC-MS

Introduction Human exhaled breath contains more than 1,000 of the vast variety of volatile organic compounds (VOCs), providing valuable information about the metabolic process in human beings. The information of breath could include current state of disease, leading to great potential of noninvasive diagnosis in medical industry 1-6. The concentration of the exhaled breath VOCs varies in sub-ppm or even lower in ppb level in healthy people7. However, when disease occurs in the human body, the metabolic process becomes disbalance. Hence, the concentration profile of exhaled breath VOCs drastically increases. Recently, deaths caused by lung cancer have reached 1.6 million each year8. Early screening and diagnosing of lung cancer are big challenges in the healthcare industry. There are many technologies to detect and diagnose lung cancer, such as low-dose chest computed tomography (CT), which enhances the likelihood of early-stage tumor diagnosis9, resulting in an increase in the survival rate10. However, the aforementioned technique still suffers from a large rate of false positive due to cross reactive responses10. In addition, due to the existence of a low dose of ionizing radiation in the CT, employing this technique increases the risk of cancer. Therefore, a noninvasive technology for breath analysis is desired to diagnose lung cancer. In this paper, we report a fast screening method of the lung cancer biomarker in exhaled breath by using gas chromatography-mass spectroscopy coupled with thermal desorption (TD-GC-MS), which is one of the noninvasive technologies that is able to perform this task. In the experiments, Tenax TA material is used as absorbent to absorb the exhaled breath VOCs, thermal desorption system is used to desorb the VOCs, which are separated by the gas chromatography column and further detected by the mass spectrometer. Method A 1 Liter Tedlar bag was used to sample the exhaled breath for the lung cancer patients at the NTU hospital Hsinchu. Further, the breath sample was transferred from Tedlar bag to Tenax TA tube with a flow rate of 40 cc/min for 25 minutes. An Agilent type7890A GC system with 5975C inert MSD with a triple-axis detector along with Perkin Elmer thermal desorption system (Turbo matrix 100) was used. Breath VOCs were separated by Elite-5 MS column (30 m × 0.25 mm, film thickness 0.25μm, Perkin Elmer) while working in a constant pressure mode (10 psi). The mass spectrometer was set to scan mode. The program of column temperature was maintained at 80 for 4 min, and then increased at a rate of 15 per min, to 230 and held at 230 for 4 min11. After breath sampling, Tenax TA absorbent was used to absorb the volatile organic compound, then the Tenax TA was connected to the Perkin Elmer thermal desorption system. Results and Conclusions Gas chromatography mass Spectro meter coupled with the thermal desorption system was used in the detection of lower concentration of the volatile organic compound in the exhaled breath for lung cancer patients. The thermal desorption process was used to desorb the volatile organic compound from the Tenax TA absorbent. The desorbed samples from Tenax TA absorbent were transferred through the column. The VOCs were moved into the column by the inert gas mobile phase; then, they were separated by the stationary phase fixed into the column. The separation efficiency depended upon the gas chromatography column. The detection principle of the GC-MS was based on the mass to charge ratio (M/Z) of the ionized atom for the detection of biomarkers in the lung cancer patients. After separation in gas chromatography, VOCs were detected by the mass spectrometer. The peak area of various kind of VOCs for lung cancer have been obtained. We have found some biomarkers from lung cancer’s exhaled breath such as acetone, toluene, ethyl benzene, decane, etc. In future, we will sample the breath for the control group, after analysis by GC-MS compare the result with lung cancer patient to identify the unique biomarkers. The Figure 1 shows the profile of the peak area with respect to the various kind of VOCs for the lung cancer patient. Therefore, the TD-GC-MS is an effective technique for noninvasive diagnosis by employing exhaled breath VOCs for the health care industry. These exhaled breath biomarkers can be used to screen the early lung cancerous disease to save millions of lives worldwide. Figure 1

Covalent Labeling with an α,β-Unsaturated Carbonyl Scaffold for Studying Protein Structure and Interactions by Mass Spectrometry.

A new covalent labeling (CL) reagent based on an α,β-unsaturated carbonyl scaffold has been developed for studying protein structure and protein-protein interactions when coupled with mass spectrometry. We show that this new reagent scaffold can react with up to 13 different types of residues on protein surfaces, thereby providing excellent structural resolution. To illustrate the value of this reagent scaffold, it is used to identify the residues involved in the protein-protein interface that is formed upon Zn(II) binding to the protein β-2-microglobulin. The modular design of the α,β-unsaturated carbonyl scaffold allows facile variation of the functional groups, enabling labeling kinetics and selectivity to be tuned. Moreover, by introducing isotopically enriched functional groups into the reagent structure, labeling sites can be more easily identified by MS and MS/MS. Overall, this reagent scaffold should be a valuable CL reagent for protein higher order structure characterization by MS.

Diagnostic of graphene on Ge(100)/Si(100) in a 200 mm wafer Si technology environment by spectroscopic ellipsometry/reflectometry

Comprehensive diagnostics is a prerequisite for the application of graphene in semiconductor technologies. Here, the authors present long-term investigations of graphene on 200-mm Ge(100)/Si(100) wafers under clean room environmental conditions. Diagnostic of graphene was performed by a fast and nondestructive metrology method based on the combination of spectroscopic ellipsometry and reflectometry (SE/R), realized within a wafer optical metrology tool. A robust procedure for unambiguous thickness monitoring of a multilayer film stack, including graphene, interface layer GeOx underneath graphene, and surface roughness is developed and applied for process control. The authors found a relationship between the quality of graphene and the growth of GeOx beneath graphene. Enhanced oxidation of Ge beneath graphene was registered as a long-term process. SE/R measurements were validated and complemented using atomic force microscopy, scanning electron microscopy, Raman spectroscopy, and secondary ion mass spectrometry. This comparative study shows a high potential for optical metrology of graphene deposited on Ge/Si structures, due to its great sensitivity, repeatability, and flexibility, realized in a nondestructive way.

The Collection and Identification of Gunshot Residues to Distinguishing 4 Types of Bullets by ICP-MS

The identification of gunshot residue (GSR) dispersed in a shooting is important for forensic evidence to solve the firearm shooting crimes. In this experimental study, the residue of specific elements, namely: Lead (Pb), Antimony (Sb) and Barium (Ba) were compared using Inductively Coupled Plasma Mass Spectro photometer (ICP-MS) from 4 types of 9 mm ammunition labeled as A is Lead Round Nose (LRN) 135 gr., B is Jacketed Hollow Point (JHP) 115 gr., C is Full Metal Jacketed (FMJ) 124 gr. all of Bullet Master and D is Full Metal Jacketed (FMJ) of Thai Arms. The Bullet holes from wooden and metal targets at 7 firing ranges; 0.00, 0.15, 1.00, 1.50, 2.00 and 2.50 meters were investigated by cotton swab dripped with 5% nitric acid. The analytical results showed that at the firing range of 0.00 meters, very high concentrations of Pb was found more than Sb and Ba, while the outer side of the hole found more amount of the elements than the inner side. Moreover, the results shown the amount of Pb, Sb and Ba of ammunition A (LRN), B (JHP) and C (FMJ) of Bullet Master are significant difference, and classify the ammunition C (FMJ) of Bullet Master from the ammunition D (FMJ) of Thai Arms using Pb and Sb concentration. From the results of this study can be useful scientific evidence for forensic science investigation in order to estimate the ammunition and firing distances.

PENGARUH TEMPERATUR TERHADAP NILAI SUN PROTECTING FACTOR (SPF) PADA EKSTRAK KUNYIT PUTIH SEBAGAI BAHAN PEMBUAT TABIR SURYA MENGGUNAKAN PELARUT ETIL ASETAT DAN METANOL

White turmeric (Curcuma Mangga) is rhizome that plantyful in Pandeglang Banten, It has not optimally applied. White turmeric is a potential antioxidant that can be used as skin sun screen. The aim of the research is to observe the potential of curcuma manga to be a sunscreen. This study was initially prepared by slicing the rhizome into the small size, after that blanching by adding 0.05% of citric acid into the ryzome, and then followed by exctracting process in different temperature (30;50;70 o C). The next one was vaporization solvent (ethyl acetate and methanol) using water bath at 40-45 o C and dried at 37 o C for 8 hours with oven. The extract was then analyzed by spectrophotometry UV-Vis analysis. The result was then measured show that white turmeric the product potentially used for sun screen. It gave the the highest SPF value about 4.107 of SPF using solvent of ethyl acetate and 3.295 by using methanol solvent. From the GC-MS (Gas Chromathography – Mass Spectro) analysis show that white turmeric contain Oroselone, Xanthone, Curcumin, Calamen, And Palmitic Acid. Key words : antioxidant, White turmeric, Sun Protection Factor (SPF)

UPAYA ISOLASI β-ASARONE PADA EKSTRAK n-HEKSAN RIMPANG DRINGO (Acorus calamus L.) ASAL KABUPATEN PINRANG

The isolation of n-hexane extract from Sweet Flag rhizome (Acorus calamus L. ) was done. The aim of this research is to obtain β-asarone isolate as marker compound of Sweet Flag (Acorus calamus L .). Sample from 1 kg of dried rizhome, mascerate using n-hexane. This process produce 31 ml thick extract. Isolation of n-hexane extract using column chromatography with isocratic methode using n-hexane etil acetat 8 : 2. The isolate identified by UV-Vis spectroscopy, IR spectroscopy, ( 1 H) NMR and Gas Chromatography Mass Spectro. From the data result show that isolate is β-asarone.

Structure, function and evolution of the mitochondrial replicative DNA helicase (570.1)

Mitochondrial DNA (mtDNA) helicase is an essential part of the mtDNA replisome. The mtDNA helicase shows high amino acid sequence homology with bacteriophage T7 gene 4 protein (T7gp4), which has both DNA primase and DNA helicase activities in its N‐terminal domain (NTD) and C‐terminal domain (CTD), respectively. Although the NTD of mtDNA helicase in metazoa does not have detectable primase activity, retention of extensive amino acid sequence homology throughout most eukaryotic lineages suggests other important roles, such as in protein stability and/ or protein‐protein interactions. Here, we investigate the structural and biochemical properties of the NTD of Drosophila mtDNA helicase by constructing two N‐terminal truncated variants: a full‐length NTD (including both the zinc binding domain (ZBD) and the RNA polymerase domain (RPD)), and a ZBD only construct. We show that NTD and ZBD constructs of Drosophila mtDNA helicase contain an iron‐sulfur cluster, most likely a 2Fe‐2S, as determined by mass spectrometry, and UV‐visible and atomic absorption spectroscopy. Mutagenesis studies show that the four conserved cysteine residues in the ZBD responsible for binding zinc in T7gp4, bind the iron‐sulfur cluster. We extend our biochemical studies in vivo through several approaches to identify partner proteins of the Drosophila mtDNA helicase that may elucidate the functional role of its iron‐sulfur cluster.Grant Funding Source: NIH GM45295

Chemical investigation of the seed extract of <i>Senna occidentalis l.</i>

The seeds Senna occidentalis were successively extracted with n-hexane and 95 % ethanol. The n-hexane extract highly rich in fatty acid was subjected to transmathylation to allow the sample ready for looms. Theextract (SH and SE) were analyzed using high resolution gas  chromatography and spectrometer. The comparison of their GC, retention indices and mass spectro characteristics with the stored laboratory massspectral library data, led to the identification of phyto-chemical aspect of the seed. The aim of the study was to qualitatively determine the organic compound of the seed of Senna occidentalis. Thirteen compounds were identified in n-hexane extract and in ethanolic extract twelve compounds were identified. The prevailing compound in n-hexane extract was palmetric acid (17.96 %). Other compounds such as aldehydre, lactone and chloride were present. Octadecanoic acid as fatty acid was identified as the main constituent (22.56%) of ethanolic extract. The study has gone a long way to validate scientifically the major phytochemical properties of the seed of Senna occendentalis.

Hair analysis interpretation of an unusual case of alleged scopolamine-facilitated sexual assault

The apparent victim in an alleged scopolamine- facilitated sexual assault case requested a toxicological hair analysis. Hair decontamination wash solvents were evapo- rated and stored until analysis. Hair was incubated with 2 ml of 0.01 M phosphate buffer (pH 8.4), and liquid-liquid extracted. Liquid chromatography-tandem mass spectro- metric analysis of hair extracts revealed the presence of scopolamine at concentrations lower than the limit of quantification. However, scopolamine was also present in the last of the wash solvents, with wash/hair ratios(4, sug- gesting external contamination of the hair rather than drug use. These findings emphasize the importance of analyzing wash solvents for the correct interpretation of results in toxicological hair analysis.

Evaluation and validation of two different chromatographic methods (HPLC and LC-MS/MS) for the determination and confirmation of ochratoxin A in pig tissues

This study was undertaken to compare two different analytical methods for the determination and confirmation of ochratoxin A (OTA) in blood serum, kidney and liver of pigs. Sample clean-up was based on liquid-liquid phase extraction. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with fluorescence detection (FL) or electro spray ionization (ESI+) tandem mass spectrometry (MS–MS). Comparative method evaluation was based on the investigation of 90 samples of blood serum, kidney and liver per animal originating from different regions of Serbia. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with (LC–MS–MS) liquid chromatography electro spray tandem mass spectro metry detection offered comparable good results at the sub-ppb concentration level. The results indicate that the liquid chromatography electro spray tandem mass spectrometric (LC-MS/MS) method was more specific and sensitive for the analysis and confirmation of ochratoxin A in pig tissues then high pressure liquid chromatography (HPLC) method after methylation of OTA.

Synthesis of Pheromones: Highlights from 2002-2004

This review summarizes the synthesis of pheromones published in the triennium 2002-2004. The syntheses of a total of 66 pheromone (65 from insects, 1 from a crustacean species), belonging to 9 different structural cate- gories, are presented in schemes. New methodologies, but also well-known reactions have been employed to achieve the (in many cases stereoselective) synthesis of the compounds. Since its very beginnings in the mid 19th century, or- ganic chemistry has made advances unimaginable to the early pioneers of the field. Among the experimental key techniques contributing to these advances are chromatogra- phy, which made possible the separation of complex mix- tures, and spectroscopic (UV, IR, NMR) and mass spectro- metric methods, giving organic chemists powerful tools for the structure determination of organic compounds. This, in turn, stimulated research in organic synthesis, as products and by-products of reactions could be easily isolated and characterized. As once stated by Meinwald, Organic chem- istry blossomed into a self-contained, inward-looking sci- ence, bringing order and understanding to the synthesis and reactions of millions of compounds (1). This order and un- derstanding allowed the development of countless reactions of carbon-carbon bond formation or modification of func- tional groups, applicable to all classes of organic molecules, which would later eventually be modified or improved, e.g. in terms of scope, yields, reaction conditions, or stereo- chemical selectivities. Research in chemical communication has been profiting from these decade-lasting and ongoing efforts, as the synthesis of pheromones is of great importance in the process of identifying these chemical messengers. Moreover, a sort of mutualism has been evolving, as many new synthetic methods have been developped with the ex- plicit aim to improve the synthetic route towards a phero- mone. Pheromones are which are produced and lib- erated by an individual, provocing a behavioural or physio- logical response in another individual of the same species. As pheromones are usually produced in minute amounts, thus preventing crystallographic or extensive NMR experi- ments, their independent synthesis is required to confirm the identification and to establish the absolute configuration in case of chiral compounds. Furthermore, chemical synthesis of pheromones provides the amounts necessary for the evaluation of their biological activity in the laboratory or in the field.

Solid‐State Conversion of Processable 3,4‐Ethylenedioxythiophene (EDOT) Containing Poly(arylsilane) Precursors to π‐Conjugated Conducting Polymers

The thermal degradation products of poly(methyl meth acrylate) model compounds in an ambient atmosphere were mapped via high-resolution electrospray ionization quadrupole ion trap time of flight (Q-ToF) mass spectro metry.

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Fragmentation reactions of molecular ions and dications of indoleamines

The combination of techniques such as two-dimensional mass spectrometry and mass analysed ion kinetic energy spectrometry (with and without collision gas) in the study of gas-phase ion chemistry of indole, gramine, tryptamine and serotonine enabled us to estab lish the fragmentation pathways of their singly- and doubly-charged molecular ions generated by electron impact. The measurement of the kinetic energy released in the charge separation reactions of the molecular dications produced information on the intercharge distance on the transition state for the fragmentation, assuming that it arises entirely from the coulombic energy released. For the sin gly-charged molecular ions of indoleamines, loss of the neutral molecule CH 2=NR (R = H or CH3), by hydrogen transfer from the amino (or methyl) group to the indole nucleus, is the only fragmentation occurring in the third field-free region of the mass spectro meter. Upon collision of the molecular ion with He gas, a competing process of bond cleavage by loss of the radical ∞ CH2NH2 (or ∞ N(CH3)2 for gramine) is observed for tryptamine and serotonine. The doubly-charged ions dissociate by elimination of neutral molecules or radicals to generate doubly-charged fragment ions or by charge separation reactions. The intercharge distance, calculated from the kinetic energy released in the fragmentation, was, whenever possible, correlated with charge location in the ion.

Heteropolyatomic Gas-Phase Species Produced by Laser Ablation of Lead Titanate Oxide, Studied by Laser Microprobe Mass Spectrometry

Frequency tripled Nd:YAG (355 nm) laser ablations of lead titanate in a vacuum (10-6 mbar) have produced oxide cluster ions identified in positive and negative mode by laser microprobe mass spectro...

NADPH-dependent oxidation of benzidine by rat liver.

This study used liver microsomes from control an naphthoflavone-treated rats to evaluate NADPH-dependent oxidation of benzidine. With microsomes from beta-naphthoflavone-treated rats, the rates of formation of aqueous soluble metabolite (HPLC analysis) and protein and DNA binding were 835 +/- 81, 14.5 +/- 1.8 and 0.71 +/- 0.14 pmol/mg/min respectively. beta-Naphthoflavone treatment elicited 12.3-, 1.8- and 14.2-fold increases in benzidine metabolism compared with controls as judged by HPLC and protein and DNA binding respectively. For microsomes from treated animals, Km and Vmax values were 47 +/- 6 micromol and 1.13 +/- 0.16 nmol/mg protein/min respectively. All of the metabolic parameters were inhibited to varying degrees by glutathione (1 or 10 mM), N-acetylmethionine (10 mM) and ascorbic acid (10 mM). Following glutathione addition, at least two new metabolite peaks were observed, representing -6% of the total radioactivity recovered by HPLC. Neither metabolite was 3-(glutathion-S-yl)benzidine. Cytochrome P450 inhibitors (10 micro) specific for different members of cytochrome gene families 1-3 indicated that benzidine was metabolized by cytochrome P450 1A1/1A2. Ellipticine and alpha-naphthoflavone, specific 1A1/1A2 inhibitors, elicited 50% inhibition at -0.2 and 0.5 micro respectively. Electron impact and negative ion chemical ionization mass spectro- metry identified the aqueous soluble metabolite as 3-hydroxybenzidine. The lability of 3-hydroxybenzidine observed at pH > 7.0 was prevented by ascorbic acid. Thus, cytochrome P450 1A1/1A2 NADPH-dependent metabolism of benzidine to 3-hydroxybenzidine was demonstrated.