Recombinant cytochrome p450 immobilization for biosensor applications

As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.     

高效液相色谱-电喷雾串联四级杆质谱法检测小鼠组织中花生四烯酸及其代谢产物

采用反相高效液相色谱梯度洗脱,电喷雾串联质谱多反应检测模式,同时分离测定小鼠组织中花生四烯酸类代谢产物.组织样品经低温超声破碎细胞,采用乙酸乙酯进行两次萃取,LC-MS/MS检测.实验用3个内标,标准溶液稀释法建立了19种花生四烯酸类物质的标准曲线.方法学确证表明: 各被测物质的线性相关系数均大于0.99;定量限(LOQ)为3～250 μg/L;相对回收率在70.7%～123.0%之间;方法重复性(RSD)均小于20%.运用本法在小鼠肺、肝、脾组织中检测到了几种花生四烯酸类代谢产物,并对肺组织里的8种代谢产物进行了含量测定,结果为11-羟基二十碳四烯酸(58.3±11.6)ng/g,15-羟基二十碳四烯酸(52.6±15.5)ng/g,前列腺素E2(210±102) ng/g,前列腺素D2(20.3±13.9)ng/g,前列腺素J2(39.2±15.9)ng/g,血栓素B2(58.3±28.4)ng/g,前列腺素F2α(3.8±6.0) ng/g,花生四烯酸(1980±544)ng/g.本方法灵敏度高、重复性良好,为研究生物组织样品中花生四烯酸类物质的代谢状况提供了分析基础.     

Kinetic isotope effects implicate the iron-oxene as the sole oxidant in P450-catalyzed N-dealkylation

Multiple oxidants have been implicated as playing a role in cytochrome P450-mediated oxidations. Herein, we report results on N-dealkylation, one of the most facile reactions mediated by P450 enzymes. We have employed the N-oxides of a series of para-substituted 13C2H2-labeled N,N-dimethylanilines to function as both substrates and surrogate oxygen atom donors for P450cam and P4502E1. Kinetic isotope effect profiles obtained using the N-oxide system were found to closely match the profiles produced using the complete NAD(P)H/NAD(P)-P450 reductase/O2 system. The results are consistent with oxidation occurring solely through an iron-oxene species.     

CYP450 Epoxygenase Metabolites,Epoxyeicosatrienoic Acids,as Novel Anti-Inflammatory Mediators

Inflammation plays a crucial role in the initiation and development of a wide range of systemic illnesses. Epoxyeicosatrienoic acids (EETs) are derived from arachidonic acid (AA) metabolized by CYP450 epoxygenase (CYP450) and are subsequently hydrolyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs), which are merely biologically active. EETs possess a wide range of established protective effects on many systems of which anti-inflammatory actions have gained great interest. EETs attenuate vascular inflammation and remodeling by inhibiting activation of endothelial cells and reducing cross-talk between inflammatory cells and blood vessels. EETs also process direct and indirect anti-inflammatory properties in the myocardium and therefore alleviate inflammatory cardiomyopathy and cardiac remodeling. Moreover, emerging studies show the substantial roles of EETs in relieving inflammation under other pathophysiological environments, such as diabetes, sepsis, lung injuries, neurodegenerative disease, hepatic diseases, kidney injury, and arthritis. Furthermore, pharmacological manipulations of the AA-CYP450-EETs-sEH pathway have demonstrated a contribution to the alleviation of numerous inflammatory diseases, which highlight a therapeutic potential of drugs targeting this pathway. This review summarizes the progress of AA-CYP450-EETs-sEH pathway in regulation of inflammation under different pathological conditions and discusses the existing challenges and future direction of this research field.     

Liquid chromatography/tandem mass spectrometric determination of inhibition of human cytochrome P450 isozymes by resveratrol and resveratrol-3-sulfate

trans-Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to both have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities, and to be a weak inhibitor of cytochrome P450 (CYP)3A4, which might have significance for drug-drug interactions. Since trans-resveratrol is rapidly converted in vivo to primarily trans-resveratrol-3-sulfate, a rapid, selective, and sensitive method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed to investigate human cytochrome P450 inhibition by trans-resveratrol-3-sulfate. Effects of trans-resveratrol and trans-resveratrol-3-sulfate on the metabolism of selective cytochrome P450 substrates (CYP1A2/ethoxyresorufin, CYP2C9/diclofenac, CYP2C19/(S)-mephenytoin, CYP2D6/bufuralol, CYP3A4/testosterone) were monitored using cDNA-expressed human recombinant isozymes. For method validation, LC/MS/MS was used to measure the inhibition of various cytochrome P450 isozymes by different concentrations (0-50 microM) of known selective inhibitors. IC(50) values of 3.2, 1.4, 8.9, 0.2, and 0.3 microM were obtained for the standard isozyme inhibitors CYP1A2/furafylline, CYP2C9/sulfaphenazole, CYP2C19/tranylcypromine, CYP2D6/quinidine, and CYP3A4/ketoconazole, respectively, which were in good agreement with literature values. trans-Resveratrol showed IC(50) values of 11.6 microM for CYP2C19 and 1.1 microM for CYP3A4, but the IC(50) values exceeded 50 microM for all the other CYP isozymes, which indicated no inhibition. No enzyme inhibition was observed for trans-resveratrol-3-sulfate. Our results indicate that trans-resveratrol is a marginal inhibitor of CYP3A4 and a weak inhibitor of CYP2C19, but its major metabolite trans-resveratrol-3-sulfate is not an inhibitor of any of the cytochrome P450 isozymes investigated.     

Direct and simultaneous profiling of epoxyeicosatrienoic acid enantiomers by capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection

Despite first evidence for the cytochrome P450-mediated enantioselective biosynthesis and activity of cis-epoxyeicosatrienoic acids (EETs), as yet little is known about the stereospecifity of EET generation and physiology, because the existing chiral methods are time consuming, labor intensive, and not sensitive enough. We present a method for highly sensitive, direct, and simultaneous chiral analysis of all eight EET enantiomers consisting of (i) solid-phase extraction, (ii) reversed-phase high-performance liquid chromatographic purification followed by (iii) consecutive regio- and enantiomeric separation of the four underivatized EET regioisomers within one chromatographic run employing capillary tandem column chiral-phase liquid chromatography with (iv) reliable dual online photodiode array and gentle electrospray ionization tandem mass spectrometric identification and quantitation of the eluting optical antipodes. This one-step, simple, expeditious, and highly sensitive measurement allows profiling of all eight EET enantiomers at once, thus avoiding substance loss and enabling high sample throughput. Limits of quantification in the low picogram range were achieved by the use of capillary columns with typical high quantitative sensitivity instead of conventional columns with low chromatographic signal intensity employed by previous methods. Application to tissue homogenates demonstrated the suitability of this approach for routine and reliable “enantioprofiling” of free endogenous EETs, i.e., EETs not esterified into cellular membrane phospholipids, typically occurring at very low concentrations. The technique can readily be employed for preparative purification of enantiomers in the microgram range using large-inner-diameter columns. Figure Direct and simultaneous enantioprofiling of the four free endogenous epoxyeicosatrienoic acids (EETs) from a complex biological matrix, like the cardiopulmonary system, within one chromatographic run by highly sensitive, one-step capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection (CapTC-CP-LC-PDAD-ESI-MS²) enables accurate, systematic, and routine correlation between the absolute configuration of EETs and their physiological actions Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti-CYP antibodies

A sensitive and rugged LC/MSMS method was developed for a comprehensive in vitro metabolic interaction screening assay with N-in-1 approach reported earlier. A cocktail consisting of ten cytochrome P450 (CYP)-selective probe substrates with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8) tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were simultaneously analysed with a single LC/MSMS run. Altogether, 13 metabolites and internal standard phenacetin were analysed in multiple reaction mode. Polarity switching mode was utilized to acquire negative ion mode electrospray data for hydroxychlorzoxazone and positive ionization data for the rest of the analytes. Fast gradient elution was applied, giving total injection cycle of 8 min. The method was modified for two different LC/MSMS systems, and was validated for linear range, detection limit, accuracy and precision for each metabolite. In addition, cocktail inhibition system was further tested using monoclonal anti-CYP antibodies as inhibitors for each probe reaction.     

The stereochemistry of fatty acid hydroxylation by cytochrome P450BM3

The stereochemical preference for the cytochrome P450_BM3-catalysed hydroxylation of tetradecanoic and pentadecanoic acids has been determined via comparison with authentic non-racemic standards utilising enantioselective HPLC. The sub-terminal hydroxylation of these fatty acids by P450_BM3 is highly selective for the formation of the R-alcohols. This is the same enantioselectivity as is seen for hexadecanoic acid oxidation but contrasts with a previous report of S-hydroxylation of pentadecanoic acid by P450_BM3.     

Comparison of liquid chromatography-isotope ratio mass spectrometry (LC/IRMS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) for the determination of collagen amino acid δ13C values for palaeodietary and palaeoecological reconstruction

Results are presented of a comparison of the amino acid (AA) δ(13)C values obtained by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography-isotope ratio mass spectrometry (LC/IRMS). Although the primary focus was the compound-specific stable carbon isotope analysis of bone collagen AAs, because of its growing application for palaeodietary and palaeoecological reconstruction, the results are relevant to any field where AA δ(13)C values are required. We compare LC/IRMS with the most up-to-date GC/C/IRMS method using N-acetyl methyl ester (NACME) AA derivatives. This comparison involves the analysis of standard AAs and hydrolysates of archaeological human bone collagen, which have been previously investigated as N-trifluoroacetyl isopropyl esters (TFA/IP). It was observed that, although GC/C/IRMS analyses required less sample, LC/IRMS permitted the analysis of a wider range of AAs, particularly those not amenable to GC analysis (e.g. arginine). Accordingly, reconstructed bulk δ(13)C values based on LC/IRMS-derived δ(13)C values were closer to the EA/IRMS-derived δ(13)C values than those based on GC/C/IRMS values. The analytical errors for LC/IRMS AA δ(13)C values were lower than GC/C/IRMS determinations. Inconsistencies in the δ(13)C values of the TFA/IP derivatives compared with the NACME- and LC/IRMS-derived δ(13)C values suggest inherent problems with the use of TFA/IP derivatives, resulting from: (i) inefficient sample combustion, and/or (ii) differences in the intra-molecular distribution of δ(13)C values between AAs, which are manifested by incomplete combustion. Close similarities between the NACME AA δ(13)C values and the LC/IRMS-derived δ(13)C values suggest that the TFA/IP derivatives should be abandoned for the natural abundance determinations of AA δ(13)C values.     

Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry

We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.     

Evaluation of ethoxynonafluorobutane as a safe and environmentally friendly solvent for chiral normal-phase LC-atmospheric pressure chemical ionization/electrospray ionization-mass spectrometry

Coupling normal-phase LC separation methods to atmospheric pressure ionization (API)-mass spectrometry (MS) for detection can be problematic because of the possible detonation hazard and because nonpolar solvents do not support ionization of the analyte. Unlike achiral separations, enantiomeric separations can be very sensitive to small changes in the separation environment. Thus, completely substituting the main mobile phase component of a normal-phase LC solvent for an environmentally friendly, nonflammable fluorocarbon-ether as a safe and effective solvent must be thoroughly evaluated before it can be recommended for enantioselective separations with API-MS detection. Ethoxynonafluorobutane (ENFB) was used as a normal-phase solvent for the enantioselective separation of 15 compounds on two macrocyclic glycopeptide chiral stationary phases (CSPs) and a new polymeric chiral stationary phase. The chromatographic figures of merit were compared between results obtained with the ENFB mobile phases and traditional heptane-based mobile phases. In addition, the limits of detection (LOD) using the API-MS compatible ENFB were examined, as well as flow rate sensitivities and compatibilities with common polar organic modifier. ENFB is a safe and effective solvent for enantioselective normal-phase/API-MS analyses.     

Direct eicosanoid profiling of the hypoxic lung by comprehensive analysis via capillary liquid chromatography with dual online photodiode-array and tandem mass-spectrometric detection

Eicosanoids are arachidonic acid-derived mediators, with partly contradictory, incompletely elucidated actions. Thus, epoxyeicosatrienoic acids (EETs) are controversially discussed as putative vasodilatative endothelium-derived hyperpolarizing factors in the cardiovascular compartment but reported as vasoconstrictors in the lung. Inconsistent findings concerning eicosanoid physiology may be because previous methods were lacking sensitivity, identification reliability, and/or have focused on special eicosanoid groups only, ignoring the overall mediator context, and thus limiting the correlation accuracy between autacoid formation and bioactivity profile. Therefore, we developed an approach which enables the simultaneous assessment of 44 eicosanoids, including all representatives of the arachidonic acid cascade, i.e., cytochrome P450, lipoxygenase, cyclooxygenase products, and free isoprostanes as in vivo markers of oxidative stress, in one 50-minute chromatographic run. The approach combines (i) source-specific sample extraction, (ii) rugged isocratic and high-sensitivity capillary liquid-chromatographic separation, and (iii) reliable dual online photodiode-array and electrospray ionization tandem mass-spectrometric identification and quantitation. High sensitivity with limits of quantification in the femtogram range was achieved by use of capillary columns with typical high peak efficiency, due to small inner diameters, and virtually complete substance transfer to the mass spectrometer, due to flow rates in the low microliter range, instead of large inner diameter columns with low chromatographic signal and only partial analyte transfer employed by previous methods. This expeditious, global and sensitive technique provides the prerequisite for new, accurate insights regarding the physiology of specific mediators, for example EETs, in the context of all relevant vasoactive autacoids under varying conditions of oxidative stress by direct comparison of all eicosanoid generation profiles. Indeed, application of comprehensive “eicoprofiling” to hypoxically ventilated rabbit lungs revealed at a glance the enhanced biosynthesis of free EETs in the overall mediator generation context, thus suggesting their hypothetical contribution to hypoxic pulmonary vasoconstriction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Direct electrochemistry of enzymes from the cytochrome P450 2C family

The human cytochromes P450 are responsible for the clearance of ∼90% of xenobiotics yet comparatively little is known about their electrochemistry. Here we report the first direct electrochemistry of P450s from the 2C subfamily; one of the major groups of enzymes from this family. Specifically, the proteins that we have examined are recombinant human P450s 2C9, 2C18 and 2C19 and reversible Fe^III/II couples are seen in the absence of dioxygen. Even in the presence of trace amounts of dioxygen, a pronounced cathodic response is seen which is assigned to catalytic reduction of the bound dioxygen ligand by the ferrous P450.     

Analysis of frankincense from various Boswellia species with inhibitory activity on human drug metabolising cytochrome P450 enzymes using liquid chromatography mass spectrometry after automated on-line extraction

In our search for herbal remedies with inhibitory activity on cytochrome P450 (CYP) enzymes, we identified extracts of the gum-resin of Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata as equally potent, non-selective inhibitors of the major drug metabolising CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4. LC/LC/ESI-MS fingerprint analyses of the boswellic acids 11-keto-beta-boswellic acid, alpha-boswellic acid, beta-boswellic acid and their 3-O-acylated derivatives were used for the authentication of the commercially obtained frankincense samples. Although the boswellic acids could be identified as moderate to potent inhibitors of the applied CYP enzymes, they are not the major CYP inhibitory principle of frankincense.     

Simultaneous determination of the inhibitory potency of herbal extracts on the activity of six major cytochrome P450 enzymes using liquid chromatography/mass spectrometry and automated online extraction

Here we describe a liquid chromatography/mass spectrometry (LC/MS) method with automated online extraction (LC/LC/MS) to simultaneously determine the in vitro inhibitory potency of herbal extracts on six major human drug-metabolising cytochrome P450 enzymes. Substrates were incubated with a commercially available mixture of CYP1A2/2C8/2C9/2C19/2D6 and 3A4 from baculovirus-infected insect cells and the resulting metabolites were quantified with LC/LC/MS using electrospray ionisation in the selected ion monitoring mode. Consistent inhibitory activities were obtained for known inhibitors and plant extracts using the enzyme/substrate cocktail and the individual enzymes/substrates. Popular herbal remedies including devil's claw root (Harpagophytum procumbens), feverfew herb (Tanacetum parthenium), fo-ti root (Polygonum multiflorum), kava-kava root (Piper methysticum), peppermint oil (Mentha piperita), eucalyptus oil (Eucalyptus globulus), red clover blossom (Trifolium pratense) and grapefruit juice (GJ; Citrus paradisi) could be identified as inhibitors of the applied CYP enzymes with IC(50) values between 20 and 1000 microg/mL.     

Analysis of perfluorinated carboxylic acids in soils: detection and quantitation issues at low concentrations

Methods were developed for the extraction from soil, identification, confirmation and quantitation by LC/MS/MS of trace levels of perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA). Whereas PFOA, PFNA and PFDA all can be quantitated using the method of standard additions, PFOA also can be quantitated less laboriously using 13C4-PFOA as a matrix internal standard. The impact of extract matrices on signal varied between soils and temporally during analytical runs rendering 13C4-PFOA unsuitable as a matrix internal standard for quantitating perfluorinated carboxylic acids (PFCAs) other than PFOA, which co-elutes with 13C4-PFOA. In fact, for soil extracts, quantitation of PFCAs based on external calibrations proved about as accurate as use of matrix internal standards for target analytes that do not co-elute with the matrix internal standard. Also, 13C4-PFOA should be used carefully as a matrix internal standard for trace levels of PFOA because some 13C4-PFOA standards contain trace impurities of unlabelled PFOA. When the presence of PFCAs in soil extracts is being determined by LC/MS/MS, detection limits are best defined by statistical methods that quantify the significance of contrast between analytical signal and background noise using multiple analyses. Further, when developing a calibration of low concentrations using weighted regression, the central tendency of the calibration line is best fitted using graphical depictions of error. As the MDL for the transition-product quantitation ion is approached in LC/MS/MS, relatively weak signals of transition-product confirmation ions can be used as a rejection criterion by looking for anomalously high values of the ratio of the confirmation to the quantitation ion.     

Atmospheric pressure chemical ionization in gas chromatography-mass spectrometry for the analysis of persistent organic pollutants

Atmospheric pressure chemical ionization (APCI) was primarily applied as the ion source for liquid chromatography-mass spectrometry (LC–MS). While APCI started to be used in gas chromatography-mass spectrometry (GC–MS) in 1970s, GC-APCI-MS was not widely used until recently. As a soft ionization technique, APCI provides highly diagnostic molecular ions, which is favored for the wide-scope screening. With the capability of tandem mass spectrometry (MS/MS), GC-APCI-MS methods with high sensitivity and selectivity have been developed and applied in the analysis of persistent organic pollutants (POPs) in environment and biological samples at trace levels. The present review introduces the history of the APCI source, with emphasis on mechanisms of ionization processes under the positive and negative ionization modes. Comparison between GC-APCI-MS and GC–MS with traditional electron ionization (EI) and chemical ionization (CI) are provided and discussed for selectivity, sensitivity and stability for the analyses of POPs. Previous studies found that the GC-APCI-MS methods provided limits of detection (LODs) around 10–100 times lower than other methods. An overview of GC-APCI-MS applications is given with the discussions on the advantages and drawbacks of various analytical methods applied for the analyses of POPs.     

Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry

A liquid chromatography (LC)/mass spectrometry method was developed for the determination of selected biogenic amines in various fish and other food samples. It is based on a precolumn derivatization of the amines with succinimidylferrocenyl propionate under formation of the respective amides and their reversed-phase liquid-chromatographic separation with subsequent electrospray ionization mass-spectrometric detection. Deuterated putescine, cadaverine, and histamine are added prior to the derivatization as internal standards that are coeluted, thus allowing excellent reproducibility of the analysis to be achieved. Depending on the analyte, the limits of detection were between 1.2 and 19.0 mg/kg, covering between 2 and 3 decades of linearity. The limit of detection and the linear range for histamine are suitable for the surveillance of the only defined European threshold for biogenic amines in fish samples. Compared with the established ortho-phthalaldehyde (OPA)/LC/fluorescence method, the newly developed method allows an unambiguous identification of the biogenic amines by their mass spectra in addition to only retention times, a fivefold acceleration of the separation, and independency from the sample matrix owing to the isotope-labeled internal standards. Various fish, calamari, and salami samples were successfully analyzed with the new method and validated with an independent OPA/LC/fluorescence method.     

Development of an isotope dilution liquid chromatography/tandem mass spectrometry detection method for DNA adducts of selected aromatic amines

Polycyclic aromatic amines (arylamines) are a class of chemical carcinogens that are prevalent in environmental and industrial settings. They are metabolically activated to covalently bond to DNA, forming mutagenic adducts. In order to study the mechanisms of their toxicity, sensitive and selective quantitative LC/MS/MS detection methods were developed to measure the N-(adenin-8-yl)-benzidine adduct and N-(adenin-8-yl)-2-aminofluorene in total DNA extract samples. A novel synthetic method using a palladium catalyst was previously developed to prepare authentic and deuterated arylamine-adenine adducts to serve as standards. These standards were then used to develop an HPLC electrospray ionization tandem mass spectrometry, isotope dilution method. Sample detection limits in DNA samples were 22 pg on-column and 51 pg on-column for the N-(adenin-8-yl)-benzidine- and N-(adenin-8-yl)-2-aminofluorene-adenine adducts, respectively. This method has applications for the study of DNA adduct formation as a biological marker of exposure to carcinogens and for environmental and workplace monitoring of these aromatic amines.     

Recent progress in the analysis of sugar monomers from complex matrices using chromatography in conjunction with mass spectrometry or stand-alone tandem mass spectrometry

Analysis of trace levels of carbohydrate monomers in complex matrices requires excellent discrimination of the peaks of interest from background noise. Minimizing contaminating peaks introduced during sample preparation and chromatography is extremely important. However, the exquisite selectivity of the mass spectrometer is essential as a chromatographic detector in this regard. Traditionally gas chromatography-mass spectrometry (GC-MS) has been the method of choice for trace analysis of derivatized carbohydrates. Recent improvements in commercial tandem mass spectrometers (MS-MS) are encouraging the use of GC-MS-MS for improved specificity in trace analysis. There has also been an explosion in applications of electrospray ionization (ESI) for sensitive introduction of polar molecules (including sugars) into the mass spectrometer. This has encouraged ongoing developments in high-performance liquid chromatography-mass spectrometry (LC-MS) and MS-MS of underivatized carbohydrates. This has the potential to dramatically simplify sample preparation. However, as yet LC-MS and MS-MS do not match the sensitivity of GC-MS or GC-MS-MS. Developments in analysis of sugar monomers from complex matrices using chromatography (GC/LC) in conjunction with mass spectrometry (MS, MS-MS) or stand-alone MS-MS are discussed.