Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 µg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-ß-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

Assessing the mutagens ethylnitrolic acid and 2-methyl-1,4-dinitro-pyrrole in meat products: Sample preparation and simultaneous analysis by LC-MS/MS.

The simultaneous use of nitrite and sorbate as preservatives in meat products may produce mutagenic compounds such as the ethylnitrolic acid and 2-methyl-1,4-dinitro-pyrrole. We developed a sensitive analytical method with high metrological reliability. After assessing several extraction approaches and chromatographic separation modes, a modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) approach was chosen for sample preparation, which were analyzed by reversed-phase liquid chromatography (with C18 as stationary phase) coupled to tandem mass spectrometry. After validation, we confirmed that this method is fit-for-purpose, since it was applied to the analysis of several meat products. Limits of detection were set from 5 to 20 µg kg-1. Satisfactory results were obtained for both compounds, such as precision (CV > 20%) and recoveries (77-92%). This method determine these carcinogenic compounds in processed meats, contributing to the preservation of public health and the improvement of food regulation and control.

Simultaneous analysis by LC-MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue.

This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 µl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11ß-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11ß-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.

Analysis of Metabolites from the Tricarboxylic Acid Cycle for Yeast and Bacteria Samples Using Gas Chromatography Mass Spectrometry.

We here explain step by step the implementation of gas chromatography coupled with tandem mass spectrometry for the quantitative analysis of intracellular metabolites from the tricarboxylic acid (TCA) cycle such as citrate, isocitrate, alpha-ketoglutarate, succinate, malate, and fumarate. Isotope dilution is used to correct for potential metabolite losses during sample processing, matrix effects, incomplete derivatization, and liner contamination. All measurements are performed in selected reaction monitoring (SRM) mode. Standards and samples are first diluted with a fixed volume of a mixture of fully 13 C-labeled internal standards and then derivatized to give trimethylsilyl-methoxylamine derivatives prior GC-MS/MS analysis.

Sequential derivatization of polar organic compounds in cloud water using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, N,O-bis(trimethylsilyl)trifluoroacetamide, and gas-chromatography/mass spectrometry analysis.

Cloud water samples from Whiteface Mountain, NY were used to develop a combined sampling and gas chromatography-mass spectrometric (GCMS) protocol for evaluating the complex mixture of highly polar organic compounds (HPOC) present in this atmospheric medium. Specific HPOC of interest were mono- and di keto-acids which are thought to originate from photochemical reactions of volatile unsaturated hydrocarbons from biogenic and manmade emissions and be a major fraction of atmospheric carbon. To measure HPOC mixtures and the individual keto-acids in cloud water, samples first must be derivatized for clean elution and measurement, and second, have low overall background of the target species as validated by GCMS analysis of field and laboratory blanks. Here, we discuss a dual derivatization method with PFBHA and BSTFA which targets only organic compounds that contain functional groups reacting with both reagents. The method also reduced potential contamination by minimizing the amount of sample processing from the field through the GCMS analysis steps. Once derivatized only gas chromatographic separation and selected ion monitoring (SIM) are needed to identify and quantify the polar organic compounds of interest. Concentrations of the detected total keto-acids in individual cloud water samples ranged from 27.8 to 329.3ngmL(-1) (ppb). Method detection limits for the individual HPOC ranged from 0.17 to 4.99ngmL(-1) and the quantification limits for the compounds ranged from 0.57 to 16.64ngmL(-1). The keto-acids were compared to the total organic carbon (TOC) results for the cloud water samples with concentrations of 0.607-3.350mgL(-1) (ppm). GCMS analysis of all samples and blanks indicated good control of the entire collection and analysis steps. Selected ion monitoring by GCMS of target keto-acids was essential for screening the complex organic carbon mixtures present at low ppb levels in cloud water. It was critical for ensuring high levels of quality assurance and quality control and for the correct identification and quantification of key marker compounds.

Chemical discrimination between dC and 5MedC via their hydroxylamine adducts.

The presence of the methylated nucleobase (5Me)dC in CpG islands is a key factor that determines gene silencing. False methylation patterns are responsible for deteriorated cellular development and are a hallmark of many cancers. Today genes can be sequenced for the content of (5Me)dC only with the help of the bisulfite reagent, which is based exclusively on chemical reactivity differences established by the additional methyl group. Despite intensive optimization of the bisulfite protocol, the method still has specificity problems. Most importantly ∼95% of the DNA analyte is degraded during the analysis procedure. We discovered that the reagent O-allylhydroxylamine is able to discriminate between dC and (5Me)dC. The reagent, in contrast to bisulfite, does not exploit reactivity differences but gives directly different reaction products. The reagent forms a stable mutagenic adduct with dC, which can exist in two states (E versus Z). In case of dC the allylhydroxylamine adduct switches into the E-isomeric form, which generates dC to dT transition mutations that can easily be detected by established methods. Significantly, the (5Me)dC-adduct adopts exclusively the Z-isomeric form, which causes the polymerase to stop. O-allylhydroxylamine does allow differentiation between dC and (5Me)dC with high accuracy, leading towards a novel and mild chemistry for methylation analysis.

Gas-phase chemistry of (alpha-terpineol with ozone and OH radical: rate constants and products.

A bimolecular rate constant, kOH+alpha-terpineol, of (1.9 +/- 0.5) x 10(-10) cm3 molecule(-1) s(-1) was measured using gas chromatography/mass spectrometry and the relative rate technique for the reaction of the hydroxyl radical (OH) with alpha-terpineol (1-methyl-4-isopropyl-1-cyclohexen-8-ol) at (297 +/- 3) K and 1 atm total pressure. Additionally, a bimolecular rate constant, kO3+alpha-terpineol, of (3.0 +/- 0.2) x 10(-16) cm3 molecule(-1) s(-1) was measured by monitoring the first order decrease in ozone concentration as a function of excess alpha-terpineol. To better understand alpha-terpineol's gas-phase transformation in the indoor environment, the products of the alpha-terpineol + OH and alpha-terpineol + 03 reactions were also investigated. The positively identified alpha-terpineol/OH reaction products were acetone, ethanedial (glyoxal, HC(=O)C(=O)H), and 2-oxopropanal (methyl glyoxal, CH3C(=O)C(=O)H). The positively identified alpha-terpineol/O3 reaction product was 2-oxopropanal (methyl glyoxal, CH3C(=O)C(=O)H). The use of derivatizing agents O-(2,3,4,5,6-pentalfluorobenzyl)hydroxylamine (PFBHA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) clearly indicated that several other reaction products were formed. The elucidation of these other reaction products was facilitated by mass spectrometry of the derivatized reaction products coupled with plausible alpha-terpineol/OH and alpha-terpineol/O3 reaction mechanisms based on previously published volatile organic compound/ OH and volatile organic compound/O3 gas-phase reaction mechanisms.

Development and validation of an HPLC-UV method for the analysis of methoxyamine using 4-(diethylamino)benzaldehyde as a derivatizing agent.

Methoxyamine (MX) is a potential new anti-cancer drug. In this paper, a quantitative HPLC-UV method for MX using 4-(diethylamino)benzaldehyde (DEAB) as a derivatizing agent has been developed and validated. The studies showed that MX reacts with DEAB under acidic conditions to form protonated 4-(diethylamino)benzaldehyde o-methyloxime (DBMOH+). The equilibrium between DBMOH+ and its conjugate base 4-(diethylamino)benzaldehyde o-methyloxime (DBMO) is affected by both buffer concentration and organic solvent content in the solution. The method developed uses a reversed phase C18 column for the separation of MX derivatives, an internal standard benzil for method calibration, and a UV detector at a wavelength of 310 nm for analyte detection. The MX derivatives can be resolved in ca. 20 min. The method has a linear calibration range from 0.100 to 10.0 microM with a correlation coefficient of 0.999 for MX and a detection limit of 5 pmol with a 50 microl sample size. The intra-assay and inter-assay precision expressed in terms of percent relative standard deviation were < or =5 and 8%; and the intra-assay and inter-assay accuracy defined as the measured value divided by the accepted value multiplied by 100% were 94.2-100 and 92.6-111%, respectively. This method may be used for the analysis of MX in pharmaceutical preparations.

Rapid determination of C6-aldehydes in tomato plant emission by gas chromatography-mass spectrometry and solid-phase microextraction with on-fiber derivatization.

A simple, rapid, sensitive, and solvent-free method was developed for determination of plant-signalling compounds, the three C6-aldehydes hexanal, (Z)-3-hexenal, and (E)-2-hexenal, in tomato plant emission by gas chromatography-mass spectrometry (GC-MS) and solid-phase microextraction (SPME) with on-fiber derivatization. In this method, O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine (PFBHA) in aqueous solution was first headspace adsorbed onto a 65 microm poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber at 25 degrees C for 5 min, and then the fiber with adsorbed PFBHA was used for headspace extraction of tomato plant emission at 25 degrees C for 6 min. Finally, the resulting oximes adsorbed on the fiber were desorbed and analyzed by GC-MS. Extraction conditions and method validation were studied. The proposed method had low detection limit values for the three aldehydes from 0.1 to 0.5 ng/L and good precision (RSD less than 10%). In this work, the method was applied to investigation of tomato plant defense response to Helicoverpa armigera.

Determination of carbonyl compounds in pool water with O-(2,3,4,5,6-pentafluorobenzyl)hydroxyamine hydrochloride and gas chromatographic-tandem mass spectrometric analysis.

To avoid microbiological decay pool water is disinfected, a procedure which results into a lot of disinfection by-products, like carbonyl compounds, as well as a large number of others. The carbonyl compounds dissolved in pool water were derivatisized with O-(2,3,4,5,6-pentafluorobenzyl)hydroxyamine hydrochloride (PFBHA) and extracted using n-hexane. Measuring with the help of GC-electron-capture detection is hardly possible because of interferents like halogenated organics. Another method to detect the PFBHA derivates is the use of tandem mass spectrometry. Calibration ranges and precision are applicable and sufficient to determine carbonyl compounds in pool water.

A new sensitive HPLC assay for methoxyamine and its analogs.

Methoxyamine (MOA) and its analogs are polymerization regulators, building blocks and intermediates for agrichemicals and pharmaceuticals. MOA induces mutagenesis of nucleic acids and has been considered for anti-cancer and anti-virus therapy. It has been studied as a DNA repair modifier in anti-cancer therapy. HPLC procedures available in the literature for MOA are all based on electrochemical detection, which is not commonly available. This paper describes the development and validation of a HPLC assay with UV detection for MOA and its analogs. The analytes are first reacted with o-phthalaldehyde to form an oxime derivative before chromatography with an ODS column. Detection is achieved by UV at 254 nm. The chromatography resolves MOA from its decomposition products and analogs. The assay is reproducible (R.S.D. < 0.8%), linear (r(2) = 0.9997), and accurate (error < 1%). The method is sensitive and has a lower detection limit of 5 pmol (0.4 ng of MOA.HCl), which is comparable to that of electrochemical detection.

[Biochemical diagnosis of pheochromocytoma and neuroblastomas]. / Diagnostic biochinique du phéochromocytome et du neuroblastome.

Pheochromocytoma and neuroblastoma are distinct tumours, but their biological diagnosis is based on secretion increase of one or several catecholamines. Assays have to be very sensible and specific for an early diagnosis. 24 hours urinary catecholamines and metabolites are currently measured, but technical improvements permit plasma metanephrine assay, an excellent indicator of pheochromocytoma. HPLC coupled to electrochemical detection represents the most efficient methodology. After a review of urinary and plasma assay methods, the authors show usual values of catecholamines, metanephrines, HVA and VMA, according to ages, and give examples of results encountered in classical or not tumours and in falsely positive cases. Urinary metanephrine assay is the most sensible and specific in biological diagnosis of pheochromocytoma, while catecholamines and VMA assays lack of sensibility. Results have to be given by 24 hours and by creatinine ratio. Metanephrine assay can be performed also in plasma and exhibits the same interest. However, in urine as in plasma, in case of renal failure, results cannot be interpreted. Neuroblastoma biological diagnosis is based classically on HVA, VMA, and dopamine assays, nowadays only in 24 hours urine (or in urinary micturition for screening), and results are also expressed as creatinine ratio. But even if several assays are advisable, 5% of the neuroblastoma cases do not produce increased catecholamine values. In some cases, metanephrine assay could be of interest. After the age of 12 months, clinical expression of neuroblastoma is dramatic in 70% of cases. So, a biological screening has been experimented in several countries including France. A French translation of the consensus conference report (1998) is appended, which shows the complexity of neuroblastoma screening. Now, there is no evidence that early tumour detection by screening lessens the mortality rate, but a weak benefit is not excluded.

Three approaches to the analysis of trace formaldehyde in bulk and dosage from pharmaceuticals.

Trace-level determinations for the presence of formaldehyde in both bulk and dosage form pharmaceuticals were developed using three innovative strategies. One system adapted the chromotropic acid spot test for formaldehyde. This was accomplished spectrophotometrically over a linear detection range against authentic control samples. The other two chromatographic approaches necessitated rapid derivatization. One derivative was its corresponding oxime, formaldoxime, which was resolved on a gas chromatographic porous polymer column and sensed by a nitrogen-specific detector. The other derivative, sodium formate, was detected and quantified on an ion chromatograph using an anion-exchange column and a conductivity detector. The chromotropic acid technique was sensitive but not specific for formaldehyde. The chromatographic techniques required a high degree of water solubility. All were subject to interferences that could preclude their use for a particular application. None of the tested samples, which included a penicillin analogue, a pharmaceutical dosage from additive, a vitamin, and biological proteins, showed the presence of formaldehyde at trace levels.

Gas chromatographic/mass spectrometric determination of alicyclic primary hydroxylamines in metabolic studies in vitro.

The combination of gas chromatography and mass spectrometry (GC/MS) is effective for separation and identification of the hydroxylamine metabolites of alicyclic primary amines after acetylation. These products give mass spectra containing diagnostic fragment ions which are of great value for identification of metabolites. The mass spectra of diacetyl alicyclic primary hydroxylamines gave prominent characteristic peaks at m/z (M - 42), (M - 42 - 42), (M - 101), 118 (AcNOAc) and 76 (AcNOH). GC/MS analysis of the incubation extracts has shown that the N-hydroxylamines are the major metabolites of alicyclic primary amines in rabbit liver microsomes.

The products of the reduction of doxyl stearates in cells are hydroxylamines as shown by oxidation by 15N-perdeuterated Tempone.

The use of nitroxides in functional biological systems has increased greatly as it has become evident that such studies can provide valuable biophysical and metabolic data. This has led to a need to understand the nature of the metabolism of nitroxides and their products. This paper presents data indicating the value of 15N-perdeuterated Tempone specifically to indicate the amount of hydroxylamines that are present in a cellular system. Using this technique, we found that in the mammalian cells that we studied the principal or only products of reduction of doxyl stearates were the corresponding hydroxylamines.

Determination of hydroxylamine traces in propionohydroxamic acid bulk drug and pharmaceutical preparations by capillary gas chromatography.

A specific, sensitive, accurate, and precise capillary gas chromatographic assay for determining trace levels of hydroxylamine, a well-known mutagen, in propionohydroxamic acid bulk drug and oral preparations is described. The analytical procedure involves derivatization in a nonaqueous medium with cyclohexanone and use of an internal standard. The derivative is then determined by capillary GC with a cool on-column injector and a nitrogen-selective detector. Effects of different matrices on the measurement were also determined. The lower limit of quantitation was 7 ppm, and the response was linear from 10 to 260 ppm. The procedure is simple and rapid enough for routine purposes.

Gas chromatography of sample monocarbonyls in cigarette whole smoke as the benzyloxyamine derivatives.

A qualitative and semi-quantitative method was established for the investigation of low-molecular-weight volatile carbonyl compounds in cigarette whole smoke. The carbonyls were trapped on a silica gel "column" and eluted with water. The aqueous solution was then treated with benzyloxyamine to form the corresponding oximes, which were then separated on a short (12 m) FFAP glass-capillary gas chromatographic column with temperature programming, and detected by a nitrogen selective detector. An internal standard was added both as a reference for retention time determinations, and as an aid in estimating the amounts of the individual carbonyls in the smoke samples.

Reaction of arylnitroso compounds with mercaptans.

1. The non-enzymic reactions of trans-4-nitrosostilbene, 4-nitrosobibenzyl, 2-nitrosofluorene and p-nitrosotoluene with glutathione were studied. 2. Three types of reaction products have been identified, namely, the corresponding hydroxylamine, amine, and a water-soluble adduct which hydrolyses under acidic or alkaline conditions to an amine and glutathione sulphinic acid. 3. Reduction to the amine is explained by formation of adducts which are reduced by glutathione with the production of oxidized glutathione. 4. The relevance of this reaction for metabolic activation and inactivation of aromatic amines in vivo is discussed.