Detection and characterization by mass spectrometry of radical adducts produced by linoleic acid oxidation

The formation of linoleic acid radical species under the oxidative conditions of the Fenton reaction (using hydrogen peroxide and Fe (II)) was monitored by FAB-MS and ES-MS using the spin trap 5,5-dimethyl-1-pyrrolidine-N-oxide, DMPO. Both the FAB and ES mass spectra were very similar and showed the presence of ions corresponding to carbon- and oxygen centered spin adducts (DMPO/L*, DMPO/LO*, and DMPO/LOO*). Cyclic structures, formed between the DMPO oxygen and the neighboring carbon of the fatty acid, were also observed. Electrospray tandem mass spectrometry of these ions was performed to confirm the proposed structure of these adducts. All MS/MS spectra showed an ion at m/z 114, correspondent to the [DMPO + H]+, and a fragment ion due to loss of DMPO (loss of 113 Da), confirming that they are DMPO adducts. ES-MS/MS spectra of alkoxyl radical adducts (DMPO/LO*) showed an additional ion at m/z 130 [DMPO - O + H]+, while ES MS/MS of peroxyl radical adducts (DMPO/LOO*) showed a fragment ion at m/z 146 [DMPO - OO + H]+, confirming both structures. Other fragment ions were observed, such as alkyl acylium radical ions, formed by cleavage of the alkyl chain after loss of water and the DMPO molecule. The identification of fragment ions observed in the MS/MS spectra of the different DMPO adducts suggests the occurrence of structural isomers containing the DMPO moiety both at C9 and C13. The use of ES tandem mass spectrometry, associated with spin trapping experiments, has been shown to be a valuable tool for the structural characterization of carbon and oxygen-centered spin adducts of lipid radicals.     

Identification by electrospray tandem mass spectrometry of spin-trapped free radicals from oxidized 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine

GPC radical species formed during oxidation of a glycerophosphocholine (16:0/18:1) under the Fenton reaction conditions were detected using a spin trap, 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO). The stable spin-trapped radical adducts were identified by mass spectrometry (MS) using electrospray (ES) as ionization method and characterized by tandem mass spectrometry (MS/MS). Radical adducts of oxidized free sn-2 fatty acid and of oxidized intact GPC, containing one, two and three additional oxygen atoms, were assigned. DMPO adducts of oxidized intact GPC were observed as singly and doubly charged ions in ES-MS, while adducts of oxidized free fatty acids were observed as singly charged ions. Oxidized free sn-2 fatty acids and intact GPC-DMPO adducts correspond to carbon- and oxygen-centered radicals that were identified by MS/MS as alkyl, hydroxy-alkyl, alkoxyl, hydroxy-alkoxyl, peroxyl and hydroperoxide-alkoxyl spin adducts. The DMPO molecule was attached predominantly at C(9) of the oleic chain. The fragmentation pathway of spin adducts with two DMPO molecules strongly suggests the presence of species that were simultaneously carbon- and oxygen-centered radicals. Several fragments identified are consistent with the presence of isomeric structures contributing to the same ions.     

Identification of isomeric spin adducts of Leu-Tyr and Tyr-Leu free radicals using liquid chromatography-tandem mass spectrometry

Free radical species are generally short-lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine-leucine (Tyr-Leu) and leucine-tyrosine (Leu-Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC-MS and MS(n) . We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MS(n) . Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr-Leu, while radicals were identified in Leu and Tyr during oxidation of Leu-Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N-terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N-terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical.     

Tandem mass spectrometry of intact oxidation products of diacylphosphatidylcholines: evidence for the occurrence of the oxidation of the phosphocholine head and differentiation of isomers

Three glycerophosphatidylcholine (GPC) phospholipids (oleoyl-, linoleoyl- and arachidonoylpalmitoylphosphatidylcholine) were oxidized under Fenton reaction conditions (H(2)O(2) and Fe(2+)), and the long-chain oxidation products were detected by electrospray mass spectrometry (ES-MS) and characterized by ES-MS/MS. The intact oxidation products resulted from the insertion of oxygen atoms into the phospholipid structure. The tandem mass spectra of the [MNa](+) molecular ion showed, apart from the characteristic fragments of GPC, fragment ions resulting from neutral losses from [MNa](+), and combined with loss of 59 and 183 Da from [MNa](+). These ions resulted from cleavage of the bond near the hydroxy group by a charge-remote fragmentation mechanism, allowing its location to be pinpointed. The fragments thus formed reflected the positions of the double bonds and of the derivatives along the unsaturated fatty acid chain, giving very useful information, as they allowed the presence of structural isomers and positional isomers to be established. The identification of the fragment ion at m/z 163, which is 16 Da higher than the five-membered cyclophosphane ion (m/z 147), in some tandem mass spectra, is consistent with the oxidation of the phosphocholine head. Some ions were found to occur with the same m/z value; in two of the phospholipids and based on the MS/MS data, structural and positional isomers were differentiated. Our findings indicate that MS/MS is a valuable tool for the identification of the wide complexity of structural features occurring in oxidized phosphatidylcholines during lipid peroxidation in cellular membranes.     

Influence of amino acid relative position on the oxidative modification of histidine and glycine peptides

The radical oxidation of isomeric peptides containing one reactive amino acid [histidine (H)] and another less reactive amino acid [glycine (G)] in the form of dipeptides (HG and GH) and tripeptides (HGG, GHG, and GGH) was studied by mass spectrometry coupled to liquid chromatography (LC-MS) for detection and LC-MSⁿ for structural characterization. The oxidation products identified were keto, hydroxy, keto-hydroxy, and hydroperoxide derivatives for both di- and tripeptides. Among these, it was found that insertion of oxygen atoms occurred at histidine for HG and HGG, and both histidine and glycine for GH, GHG, and GGH. In addition, oxidation products formed by alkoxyl rearrangement reactions with cleavage of the peptide chain were also identified for GH, GHG, and GGH, corroborating hydrogen abstraction step in G residues. These findings were supported through the identification of radical intermediate species formed and trapped with 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) spin trap. The observation of DMPO adducts bearing two spin trap molecules reinforced the abstraction of two hydrogen atoms from the same molecule.     

Identification of ozone-oxidation products of oxycodone by electrospray ion trap mass spectrometry

The products of oxycodone oxidized by ozone were characterized by electrospray ionization-tandem mass spectrometry (ESI--MS/MS). Liquid Chromatography(LC)--MS analyses revealed that the main constituents in the oxidation reaction mixture included the protonated molecules m/z 316, corresponding to oxycodone, and m/z 332, m/z 348, m/z 366, corresponding to the oxidation products. ESI--MS/MS and MS(n) spectra were used to study oxycodone fragmentation in detail and to characterize the structures of oxidation products. The results show that the oxidation products were formed by addition of one or two oxygen atoms or by addition of three oxygen and two hydrogen atoms to oxycodone. The fragmentation of the oxidation products also shows that the aromatic ring oxidizes due to rupture of the C-3--C-4 bond during product formation.     

Identification of oxidation products and free radicals of tryptophan by mass spectrometry

New oxidation products and free radicals derived from tryptophan (Trp) oxidation under Fenton reaction conditions were identified using mass spectrometry. After the oxidation of tryptophan using hydrogen peroxide and iron (II) system (Fenton reaction), mono- and dihydoxy tryptophans and N-formylkynurenine were identified using electrospray mass spectrometry (ES-MS) and ES-MS/MS. Besides these products, new products resulting from the reaction of tryptophan and oxidized tryptophan and 3-methyl indole derivatives were also identified. The 3-methyl indole derivatives resulted, most probably, from the oxidation process and not from in-source processes. A dimer formed by cross-linking between two Trp radicals (Trp-Trp), similar to the previously described tyrosine dimer was observed, as well as the corresponding monohydroxy-dimer (Trp-Trp-OH). Tandem mass spectrometry was used to identify the structures of these new oxidation products. Free radicals derived from tryptophan oxidation under Fenton reaction were detected using as spin trap the DMPO. The free radical species originated during the oxidation reaction formed stable adducts with the spin trap, and these adducts were identified by ES-MS. New adducts of oxidized tryptophan radicals, namely monohydroxy-tryptophan and dihydroxy-Trp dimer radicals, with one and two DMPO spin trap molecules where identified. Tandem mass spectrometry was used to confirm the proposed structure of the observed adducts.     

Radical identification by liquid chromatography/thermospray mass spectrometry

Radical adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) with hydroxyl, methanol-derived, and ethanol-derived radicals were detected by a combination of liquid chromatography with either electron paramagnetic resonance or thermospray mass spectrometry (LC/EPR or LC/TSP-MS) in the Fenton system (with methanol or ethanol). One radical adduct was observed in the reaction of DMPO with the hydroxyl radical or the methanol-derived radical, while two adducts were detected in the reaction of DMPO with ethanol-derived radicals. The LC/TSP-MS spectra showed quasi-molecular ions [M + H]+ at m/z 146 and m/z 160 for the methanol-derived and ethanol-derived radical adducts, respectively, and an apparent molecular ion M+ at m/z 130 for the hydroxyl radical adduct. Use of methyl-D3 alcohol (CD3OH) and ethyl-D5 alcohol (CD3CD2OH) indicated that carbon-centered radicals are formed. Experiments with partially deuterated ethanol (CD3CH2OH and CH3CD2OH) indicated that the two adducts observed in the reaction of DMPO with ethanol-derived radicals correspond to the two diastereomeric adducts of DMPO with the alpha-hydroxyethyl free radical.     

Identification of leucine-enkephalin radical oxidation products by liquid chromatography tandem mass spectrometry

The oxidation of the peptide leucine-enkephalin (YGGFL) induced by the hydroxyl radical (HO*), formed under Fenton-like conditions [Cu (II)/H(2)O(2)], was studied and monitored by LC-MS. The oxidation products identified included products resultant from (a) the insertion of oxygen atoms (1-5), (b) peptide backbone cleavage (short-chain products formed by diamide pathway) and (c) radical-radical crosslinking reactions. In order to identify the modified residues, LC-MS/MS spectra were obtained. The insertion of oxygen atoms into the peptide originated hydroxide, di-hydroxide and/or hydroperoxide derivatives. In addition it was found that the aromatic amino acids are most susceptible to being hydroxylated, while the aliphatic amino acids are more prone to forming hydroperoxides. Oxidation products with double bonds were also identified. The short chain products resulted from the alpha-carbon radical of terminal amino acids (Tyr and Leu). Products resulting from cross-linking reactions between intact carbon-centered peptide radical (with and without one HO group) and a side chain radical (*C(7)H(7)O) were identified. It was found that, although all amino acids residues of the peptide undergo modifications, the N-terminal seems to be prone to oxidative modifications under these conditions.     

Investigation of the electrochemical oxidation products of zotepine and their fragmentation using on-line electrochemistry/electrospray ionization mass spectrometry

When zotepine, an antipsychotic drug, was electrochemically oxidized using electrospray ionization mass spectrometry (ESI-MS) coupled with a microflow electrolytic cell, [M + 16 + H]+ (m/z 348), [M-H]+ (m/z 330) and [M-14 + H]+ (m/z 318) were observed as electrochemical oxidation product ions (M represents the zotepine molecule). Although a major fragment ion that was derived from the dimethyl aminoethyl moiety was observed only at m/z 72 in the collision-induced dissociation (CID) spectrum of zotepine, new fragments such as m/z 315 and 286 ions could be generated in the CID spectrum by combining electrochemical oxidation and CID. Since these fragments were relatively specific with high ion strength, it was thought that they would be useful for developing a sensitive LC-MS/MS assay. The S-oxide and N-demethylated products were detected by electrolysis assuring that a portion of P450 metabolites of zotepine could be mimicked by the electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) system.     

Detection and characterization of hydroxyl radical adducts by mass spectrometry

The study of the influence of free radicals in the biological process depends primarily on the capacity to detect these reactive species. In this work we have studied the application of mass spectrometry to the identification of hydroxyl radical species. The detection and identification by collisional activation mass-analyzed ion kinetic energy spectrometry (CA-MIKES) of a spin adduct of DMPO with the hydroxyl radical [(DMPO + O) + H]+ (m/z 130) has demonstrated that mass spectrometry can be a powerful tool in the detection and identification of spin adducts of DMPO with hydroxyl radical species. We were also able to detect the capture of secondary free radicals using ethanol by detecting and identifying the corresponding adduct [(DMPO + ethanol) + H]+. Other spin adducts have also been detected and identified. We consider that the use of mass spectrometry is a relevant technique for the detection of free hydroxyl radicals, especially in complex mixtures, since mass spectrometry is able to discriminate these adducts in such situations. Moreover, using this approach, it was possible to identify new spin adducts.     

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 μm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.     

Separation and identification of DMPO adducts of oxygen-centered radicals formed from organic hydroperoxides by HPLC-ESR,ESI-MS and MS/MS

Many electron spin resonance (ESR) spectra of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) radical adducts from the reaction of organic hydroperoxides with heme proteins or Fe(2+) were assigned to the adducts of DMPO with peroxyl, alkoxyl, and alkyl radicals. In particular, the controversial assignment of DMPO/peroxyl radical adducts was based on the close similarity of their ESR spectra to that of the DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the peroxyl adducts from the DMPO/superoxide adduct. Although recent reports assigned the spectra suggested to be DMPO/peroxyl radical adducts to the DMPO/methoxyl adduct based on independent synthesis of the adduct and/or (17)O-labeling, (17)O-labeling is extremely expensive, and both of these assignments were still based on hyperfine coupling constants, which have not been confirmed by independent techniques. In this study, we have used online high performance liquid chromatography (HPLC or LC)/ESR, electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) to separate and directly characterize DMPO oxygen-centered radical adducts formed from the reaction of Fe(2+) with t-butyl or cumene hydroperoxide. In each reaction system, two DMPO oxygen-centered radical adducts were separated and detected by online LC/ESR. The first DMPO radical adduct from both systems showed identical chromatographic retention times (t(R) = 9.6 min) and hyperfine coupling constants (a(N) = 14.51 G, a(H)(beta) = 10.71 G, and a(H)(gamma) = 1.32 G). The ESI-MS and MS/MS spectra demonstrated that this radical was the DMPO/methoxyl radical adduct, not the peroxyl radical adduct as was thought at one time, although its ESR spectrum is nearly identical to that of the DMPO/superoxide radical adduct. Similarly, based on their MS/MS spectra, we verified that the second adducts (a(N) = 14.86 G and a(H)(beta) = 16.06 G in the reaction system containing t-butyl hydroperoxide and a(N) = 14.60 G and a(H)(beta) = 15.61 G in the reaction mixture containing cumene hydroperoxide), previously assigned as DMPO adducts of t-butyloxyl and cumyloxyl radical, were indeed from trapping t-butyloxyl and cumyloxyl radicals, respectively.     

Detection of Radical Adducts with Small Molecular Weights by Matrix-Assisted Laser Desorption/Ionization with Fourier Transform Mass Spectrometry

As an alternative method, matrix-assisted laser desorption/ionization with Fourier transform mass spectrometry （MALDI-FTMS） has been successfully used to detect and identify free radical adducts with small molecular weights of hydroxyl and 2-cyano-2-propyl radicals trapped with 5,5-dimethylpyrroline N-oxide （DMPO）. The detection and identification by MS/MS experiments using sustained offresonance irradiation collision-induced dissociation （SORI-CID） of [（DMPO＋·OH-·H）＋H^＋] （m/z 130.0868） and [DMPO＋2 ·CH（CH3）2CN＋H^＋] （m/z 250.1917） have demonstrated that MALDI-FTMS could be an effective method for detection and identification of free radical adducts. Other radical adducts have been also detected and identified. The approach of MALDI-FTMS is simple, fast, and sensitive which has potential for high-throughput analysis.     

人体尿液中双酚A与壬基酚的同位素稀释的LC-MS/MS分析

采用固相萃取、高效液相色谱-质谱/质谱仪同位素稀释技术,建立了尿中双酚A(BPA)和壬基酚(NP)的检测方法.液相色谱分离用C18柱,流动相为甲醇和水,负离子电喷雾模式电离,多反应离子监测方式检测,BPA和NP的定量离子对分别为m/z 226.9/132.9、219.3/118.8, 13C和氘代同位素化合物分别做回收率控制和内标定量.BPA和NP的基质加标回收率分别为86% ～106%、90% ～114%,定量下限分别为0.97、2.3 μg/L.实际尿样的测定验证了该方法的可行性.     

Peptide-phospholipid cross-linking reactions: Identification of leucine enkephalin-alka(e)nal-glycerophosphatidylcholine adducts by tandem mass spectrometry

The covalent interactions between peptides and lipid oxidation products, with formation of Schiff and Michael adducts, are known to occur during free radical oxidative damage. In this study, leucine-enkephalin-glycerophosphatidylcholine alka(e)nal adducts were analyzed by electrospray tandem mass spectrometry (MS/MS). Upon collision-induced dissociation of the Leucine enkephalin-2-(9-oxo-nonanoyl)-1-palmitoyl-3-glycerophosphatidylcholine, an alkanal Schiff adduct observed at m/z 1187.7, the main product ions were attributed to the phosphocholine polar head and loss of the peptide. Also, product ions resulting from characteristic losses of phosphatidylcholines and cleavages of the peptide chain (mainly b-type) were observed. Additional product ions formed by combined peptide and phosphatidylcholine fragmentations were identified. The fragmentation pattern of the leucine enkephalin-alkanal Schiff adduct and the leucine enkephalin-alkenal phosphatidylcholine Schiff and Michael adducts were similar, although the loss of the peptide for the Michael adduct should occur through a distinct mechanism. These fragmentation pathways differ greatly from those described for peptide-lipid Schiff and Michael adducts, in which only peptide chain cleavages are reported, probably due to charge retention in the glycerophosphatidylcholine polar head in peptide-glycerophosphatidylcholine adducts.     

Separation of peroxidation products of diacyl-phosphatidylcholines by reversed-phase liquid chromatography-mass spectrometry

Lipid peroxidation process has attracted much attention due to the growing evidence of its involvement in the pathogenesis of age-related diseases. The monitoring of the lipid peroxidation products in phospholipids, formed under oxidative stress conditions, may provide new markers for oxidative stress signaling and for disease states, giving new insights in the pathogenesis process. Reversed-phase liquid chromatographic method coupled to mass spectrometry was developed for the separation of oxidized glycero-phosphatidylcholine (GPC) peroxidation products formed by the Fenton reaction that mimic in vivo oxidative stress conditions. The LC-MS conditions were applied for the separation of peroxidation products of oleoyl- (POPC), lineloyl- (PLPC) and arachidonoyl-palmitoyl phosphatidylcholine (PAPC). The peroxidation products separated included products resulting from the insertion of oxygen atoms in the sn-2 chain (long-chain), and products with the sn-2 chain shortened resulting from cleavage of oxygen-centered radicals (short-chain). Among long-chain products were the keto, hydroxy, hydroperoxide and poly-hydroxy derivatives, while short-chain products included dicarboxylic acids, aldehydes and hydroxy-aldehydes. Separation of long-chain products formed in each phosphatidylcholine was observed, and the reconstructed ion chromatogram of each ion showed an increase in the number of peaks with the increase in the number of oxygen atoms inserted into the phospholipid. Separation of short-chain products took place according to the functional group present at the sn-2 moiety that allowed the elution of dicarboxylic acids distinct from aldehydes. Separation between isomeric structures that were present in short- and long-chain products was also achieved.     

Characterization of polyethylenimine by electrospray ionization and matrix-assisted laser desorption/ionization

Branched polyethylenimines (PEIs) with lower average molecular weights (600, 1200 and 1800 Da) have been studied by Electrospray Ionization (ESI) and Matrix‐Assisted Laser Desorption/Ionization (MALDI) mass spectrometry. In both, ESI and MALDI mass spectra, the main distribution arises from protonated PEI oligomers with NH₂ end groups, [PEI + H]⁺, which are observed at m/z 43n + 18. A trace of sodium contamination in the PEI samples results in the presence of a series that appears at m/z 43n + 40 [PEI + Na]⁺. However, only the MALDI mass spectra show a [PEI + K]⁺ series at m/z 43n + 56, because of matrix contamination with potassium, and a series generated by condensation of the matrix with PEI at m/z 43n + 30. Collisionally activated dissociation tandem mass spectrometry (CAD (MS/MS)) of protonated PEI oligomers is shown to yield three fragment ion series b_n, and K_n. The experiments have demonstrated the capabilities of these mass spectrometry techniques, along with CAD MS/MS to detect and characterize such polar synthetic polymers. Copyright © 2011 John Wiley & Sons, Ltd.     

Tautomerization in gas-phase ion chemistry of isomeric C-8 deoxyguanosine adducts from phenol-induced DNA damage

Collision-induced dissociation (CID) of 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.     

Rapid quantification of lisinopril in human plasma by liquid chromatography/tandem mass spectrometry

An assay based on protein precipitation and liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative analysis of lisinopril in human plasma. After the addition of enalaprilat as internal standard (IS), plasma samples were prepared by one-step protein precipitation using perchloric acid followed by an isocratic elution with 10 mm ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)-methanol (70:30, v/v) on a Phenomenex Luna 5 mu C(18) (2) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing an electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 406 --> 246 for lisinopril and m/z 349 --> 206 for enalaprilat. Calibration curves of lisinopril in human plasma were linear (r = 0.9973-0.9998) over the concentration range 2-200 ng/mL with acceptable accuracy and precision. The limit of detection and lower limit of quantification in human plasma were 1 and 2 ng/mL, respectively. The validated LC-MS/MS method has been successfully applied to a preliminary pharmacokinetic study of lisinopril in Chinese healthy male volunteers.