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.

Highly sensitive analysis of low-molecular-mass aldehydes in beverages using a hydroxylamine reagent by high-performance liquid chromatography with fluorescence detection.

In this study, a fluorescent reagent, 4-((aminooxy)methyl)-7-hydroxycoumarin (AOHC), was for the first time applied to label the low-molecular-mass aldehydes (LMMAs) through reductive oxyamination reaction to afford single N,O-substituted oxyamine derivatives at room temperatures with derivatization efficiencies as high as 96.8%. In the following high-performance liquid chromatography with fluorescence detection analysis, 12 LMMAs, including furfurals, aromatic aldehydes, and aliphatic aldehydes, were baseline-separated on an ODS column and detected with low limits of detection (LODs) (0.2-50 nM), and good precisions (intraday relative standard deviations [RSDs] were 2.40-4.68%, and interday RSDs were 4.65-8.91%). This approach was then adopted to analyze six alcoholic beverages and five dairy products, and nine LMMAs with concentrations in the 0.28-798.16 µM range were successfully detected with excellent accuracies (recoveries were 92.2-106.2%). Finally, the results were statistically analyzed and discussed. The proposed method has several advantages, including high sensitivity, room-temperature labeling, and the avoidance of further extraction and/or enrichment procedures, demonstrating its great utility for monitoring LMMAs in various complex matrices.

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.

Insights into N2O turnovers under polyethylene terephthalate microplastics stress in mainstream biological nitrogen removal process.

The ubiquitous microplastics in wastewater have raised growing concerns due to their unintended effects on microbial activities. However, whether and how microplastics affect nitrous oxide (N2O) (a potent greenhouse gas) turnovers in mainstream biological nitrogen removal (BNR) process remain unclear. This work therefore aimed to fill such knowledge gap by conducting both long-term and batch tests. After over 100 days of feeding with wastewater containing polyethylene terephthalate (PET) microplastics (0-500 µg/L), the long-term results showed that both production and reduction of N2O during denitrification were reduced, as well as the N2O production during nitrification. Accordingly, 60% reduction in N2O accumulation and 70% reduction in N2O production were observed in the denitrification and nitrification batch tests, respectively. Nevertheless, the long-term N2O emission factors under PET microplastics stress were comparable to that in the control reactor, mainly because PET microplastics led to more nitrite accumulation in anoxic period. With the aid of online N2O sensors and site-preference analysis, it was demonstrated that the heterotrophic bacteria pathway and ammonia oxidizing bacteria denitrification pathway for N2O production were negatively affected by PET microplastics, whereas a clear increase in the contribution of hydroxylamine pathway (+ 22.9%) was observed. Further investigation revealed that PET microplastics even at environmental level (i.e. 10 µg/L) significantly reshaped the BNR sludge characteristics (e.g. much larger particle size) and microbial communities (e.g. Thauera, Rhodobacte and Nitrospira) as well as the nitrogen metabolism pathways, which were chiefly responsible for the changes of N2O turnovers and N2O production pathways under the PET microplastics stress.

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.

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.

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.

Hemolysis induced by 6-N-hydroxylaminopurine riboside, an adenosine analogue.

6-N-Hydroxylaminopurine riboside (HAPR) was studied in man because of its therapeutic activity in several transplanted animal neoplasms. It was not cross-resistant to other antimetabolites useful in the treatment of human neoplasia. HAPR produced marked hemolytic anemia at doses far below those that might have produced any cytotoxic or therapeutic effect. There was evidence of hemolysis at total doses as low as 0.5 mg/kg given intravenously. For man, HAPR is one of the most active hemolytic drugs.

Detection and removal of contaminating hydroxylamines from the spin trap DEPMPO, and re-evaluation of its use to indicate nitrone radical cation formation and S(N)1 reactions.

A previous report that the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) allows DEPMPO radical cation formation to be detected via the production of a carbon-centred radical adduct (assigned as the cis-hydroxyethyl species, formed by an intramolecular process) is shown to be incorrect. Rather, this and other paramagnetic species arise from the facile oxidation of trace hydroxylamine impurities present in commercial DEPMPO samples. As a result, techniques for the detection and elimination of such hydroxylamine impurities from DEPMPO solutions were developed and are described; these should prove to be of general use in EPR spin trapping experiments.

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.

Automated spectrophotometric assay of cefazolin.

An automated, stability-indicating, UV spectrophotometric assay for cefazolin is presented. The method employs a reaction with hydroxylamine and derives its stability-indicating power through comparison of reacted and unreacted aliquots of the sample. A double-probe sampling procedure is used. Good agreement with microbiological assays is obtained, and the coefficient of variation is about 1%.

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.

Quantification of lidocaine and several metabolites utilizing chemical-ionization mass spectrometry and stable isotope labeling.

Quantification of the suspected metabolites of lidocaine in humans was carried out using the direct insertion probe and chemicalionization mass spectrometry. Deuterated analogs of the metabolites of lidocaine were added to serial human plasma and urine samples and were used as internal standards following oral administration of 250 mg of lidocaine hydrochloride monohydrate to two male subjects and 202 mg of lidocaine free base to one male subject. The average results after analysis of the 0-24 hr urine samples, before beta-glucuronidase-sulfatase treatment, indicated the presence of seven of the possible metabolites in the following amounts (percent of administered dose based on the free base): lidocaine, 1.95; omega-ethylamino-2,6-dimethylacetanilide, 4.90; omega-amino-2,6-dimethylacetanilide, 0.88; m- and/or p-hydroxylidocaine, 0.73; m- and/or p-hydroxy-omega-ethylamino-2,6-dimethylacetanilide, 0.56; 2,6-dimethylaniline, 0.97; and 4-hydroxy-2,6-dimethylaniline, 63.5. Both N-ethyl- and N,N-diethylglycine were detected in human and Rhesus monkey urine, although quantification was not achieved.

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.

Oxidation of aliphatic hydroxylamines in aqueous solutions.

The effects of pH, buffer constituents, duration of storage, presence of air, heavy metal ions, extracting solvents and various additives and cofactors on the aerial oxidation of some aliphatic primary and secondary hydroxylamines were investigated. Copper ions were particularly effective catalysts of the oxidation reaction. Conditions to minimize this transformation are described.

Derivatization to stabilize some aliphatic primary hydroxylamines for g.l.c. analysis.

By appropriate choice of trimethylsilylating and trifluoroacetylating reagents and organic solvents for extraction, stable derivative of aliphatic primary hydroxylamines metabolites, N-hydroxyphentermine, N-hydroxychlorphentermine, N-hydroxymexiletene, N-hydroxyphenethylamine, N-hydroxyamphetamine, and N-hydroxy-3,4-dimethoxyamphetamine, were obtained and examined by g.l.c. analysis without decomposition and without interference from the parent drug or other metabolic products.

Synthesis of the O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine oximes of selected carbonyl compounds and their determination by liquid chromatography with ultraviolet detection.

The aims were to develop a liquid chromatographic (LC) method with ultraviolet detection (UVD) for O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) O-oximes of common aldehydes and ketones, and to define the steric limits of the synthetic reaction used to make the PFBHA O-oxime standards for gas chromatographic (GC) and LC methods. Ten new O-oximes were synthesized with the new optimized method, and their purities were demonstrated by GC/electron-capture detection (ECD), GC/mass spectrometry (MS), ultraviolet spectroscopy, infrared spectroscopy, and proton and 13C-nuclear magnetic resonance spectroscopy. Ketones substituted at both beta-carbons relative to the carbonyl carbon, like diisobutyl ketone and 2,4-hexanedione, showed lower synthetic yields by wet chemistry methods. A new C18 reverse-phase LC method with UVD at 200 nm and acetonitrile-water in both the isocratic and gradient-elution modes was then developed to sensitively resolve a mixture of 13 pure PFBHA O-oximes. The detection limit was near 100 ng O-oxime/mL or about 14-50 ng aldehyde/mL and the least quantifiable limits were near 500 ng/mL or about 70-250 ng aldehyde/mL, with lower limits for glyoxal, methylglyoxal, benzaldehyde, and acetophenone. Carbonyl compounds in 500 mL water samples were then determined in distilled water and tap water by gradient elution. Vapors of n-valeraldehyde and acrolein generated in gas bags at concentrations near occupational guidelines were also sampled, desorbed, and then determined by either isocratic or gradient elution at 200 or 254 nm within 30-45 min.

Spectrophotometric method for the determination of nifedipine with 4-(methylamino)phenol and potassium dichromate.

A new simple, sensitive and reproducible spectrophotometric method for the determination of nifedipine in pure and dosage forms has been proposed. It is based on the reduction of nifedipine with Zn/NNH4Cl, followed by coupling with N-methyl-1,4-benzoquinoneimine--the oxidation product of 4-(methylamino)phenol, to give a chromophore which absorbed maximally at 525 nm. The experimental conditions were optimised and Beer's law was obeyed over the concentration range of 5-175 microg ml(-1). The molar absorptivity, detection limit, recovery and RSD were found to be 1.9 x 10(3) l mol(-1) cm(-1), 1.1 microg ml(-1), 99.7-100.5% and 0.3-0.8%, respectively. The proposed method was compared favourably with the official B.P. method.