In situ formation of C-glycosides during electrospray ionization tandem mass spectrometry of a series of synthetic amphiphilic cholesteryl polyethoxy neoglycolipids containing <Emphasis Type="Italic">N</Emphasis>-acetyl-D-glucosamine

In this communication, the structural analysis of six synthetic O-Linked amphiphilic cholesteryl polyethoxy neoglycolipids containing N-acetyl-D-glucosamine was performed by electrospray ionization mass spectrometry in the positive ion mode, with a QqTOF-MS/MS hybrid instrument. The MS/MS analyses provided evidence for the "in situ" formation, in the collision cell of the tandem mass spectrometer, of an unexpected and unique [C-glycoside]+ product ion, resulting from an ion-molecule reaction between the N-acetyl-D-glucosamine oxonium ion and the neutral cholesta-3,5-diene molecule. Quasi MS3 analysis of this ion resulted in the dissociation of the precursor [C-glycoside]+ ion, which produced the expected third generation N-acetyl-D-glucosamine oxonium and the protonated cholesta-3,5-diene product ions.     

Characterization of acylphosphatidylglycerols from <Emphasis Type="Italic">salmonella typhimurium</Emphasis> by tandem mass spectrometry with electrospray ionization

Acylphosphatidylglycerol (Acyl-PG), a polar lipid class containing three fatty acyl groups, was isolated from Salmonella bacteria and characterized by tandem quadrupole and quadrupole ion-trap mass spectrometric methods with electrospray ionization. The structural characterization of the acyl-PG with various acyl groups (A-B/C-PG, where A not equal B not equal C) is based on the findings that the carboxylate anions (R(x)CO(2)(-)) arising from sn-2 (R(2)CO(2)(-)) is more abundant than that arising from sn-3' (R(3')CO(2)(-)), which is much more abundant than that arising from sn-1 (R(1)CO(2)(-)). This information provides a simple method for determination of the fatty acyl moieties and their positions in the molecule. The structural identification of the molecule can also be achieved by the findings that the fragment ion reflecting the ketene loss at sn-2 is more prominent than that reflecting the acid loss (i.e., [M - H - R'(2)CH=CO](-) > [M - H - R(2)CO(2)H](-)), while the ion arising from acid loss at sn-1 or sn-3' is, respectively, more abundant than the corresponding ketene loss (i.e., [M - H - R(1)CO(2)H](-) > [M - H - R'(1)CH=CO](-); [M - H - R(3')CO(2)H](-) > [M - H -R'(3')CH=CO](-)). The identity of the acyl moiety at sn-3' can be confirmed by an acyl-glycerophosphate anion observed in the product-ion spectrum obtained with a triple-stage quadrupole (TSQ) instrument, but not in that obtained with an ion-trap mass spectrometer (ITMS). However, the MS(2)-spectrum obtained with an ITMS is featured by the ion series that abundances of [M - H - R'(2)CH=CO - R(3)CO(2)H - 74](-) > [M - H - R'(2)CH=CO - R(1)CO(2)H - 74](-) z.Gt; [M - H - R'(1(or 3'))CH=CO - R(3'(or 1))CO(2)H - 74](-). This information also facilitates structural elucidation of the acyl-PG subclass that contains various acyl substituents. Structural identifications of molecular species having two identical fatty acyl substituents at sn-1, sn-2, or sn-3' or consisting of more than one isomeric structures are also demonstrated. The identities of the minor isomeric species in the molecules can be revealed by the aforementioned structural information arising from the various ion series combined.     

Characterization of inositol phosphorylceramides from <Emphasis Type="Italic">Leishmania major</Emphasis> by tandem mass spectrometry with electrospray ionization

We describe tandem mass spectrometric approaches, including multiple stage ion-trap and source collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) to characterize inositol phosphorylceramide (IPC) species seen as [M - H](-) and [M - 2H + Li](-) ions in the negative-ion mode as well as [M + H](+), [M + Li](+), and [M - H + 2Li](+) ions in the positive-ion mode. Following CAD in an ion-trap or a triple-stage quadrupole instrument, the [M - H](-) ions of IPC yielded fragment ions reflecting only the inositol and the fatty acyl substituent of the molecule. In contrast, the mass spectra from MS(3) of [M - H - Inositol](-) ions contained abundant ions that are readily applicable for assignment of the fatty acid and long-chain base (LCB) moieties. Both the product-ion spectra from MS(2) and MS(3) of the [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) ions also contained rich fragment ions informative for unambiguous assignment of the fatty acyl substituent and the LCB. However, the sensitivity of the ions observed in the forms of [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) (Alk = Li, Na) is nearly 10 times less than that observed in the [M - H](-) form. In addition to the major fragmentation pathways leading to elimination of the inositol or inositol monophosphate moiety, several structurally informative ions resulting from rearrangement processes were observed. The fragmentation processes are similar to those previously reported for ceramides. While the tandem mass spectrometric approach using MS(n) (n = 2, 3) permits the structures of the Leishmania major IPCs consisting of two isomeric structures to be unveiled in detail, tandem mass spectra from constant neutral loss scans may provide a simple method for detecting IPC in mixtures.     

New flavonoid-<Emphasis Type="Italic">C</Emphasis>-glycosides from <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Two new flavonoid-C-glycosides named triticuside A (1a) and triticuside B (1b) were isolated from bran of Triticum aestivum L. The structures of the two new compounds were elucidated by spectral techniques including ¹H NMR, ¹³C NMR as well as HSQC, HMBC, and COSY. Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 135–137, March–April, 2008.     

Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry

Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation.     

Reduction of cationic free-base <Emphasis Type="Italic">meso</Emphasis>-tris-<Emphasis Type="Italic">N</Emphasis>-methylpyridinium-4-yl porphyrins in positive mode electrospray ionization mass spectrometry

Reductions involving more than one electron with formation of the M⁺ and [M+2H]⁺ ions were observed for electrosprayed meso-tris(N-methylpyridinium-4-yl)porphyrin iodides, MI₃. These reductions were studied by using different solvents and flow rates. Formation of the [M+2H]⁺ ions occurred only for protic solvents and to a larger extent at lower flow rates. The type of the fourth substituent does not seem to affect the reduction processes. Formation of the two reduced species, M⁺ and [M+2H]⁺ ions, may occur through the participation of counter ion/solvent clusters. Reduction of multiply charged, non-metallated species with formation of [M+nH]⁺ ions (n>1) was not observed before in positive mode electrospray mass spectrometry.     

Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.     

Using amino acids for probing structural information of cytochrome <Emphasis Type="Italic">c</Emphasis> by electrospray ionization mass spectrometry

By using electrospray ionization mass spectrometry (ESI-MS), protein complexes of cytochrome c with amino acids were studied. Different amino acids were investigated to explore these complexes. Using these amino acids, a strategy for probing the structure of cytochrome c was established. It was found that L-Arg and L-Glu could bind with cytochrome c to form noncovalent complexes. At low pH solution, complexes between the cytochrome c molecule with several L-Arg molecules (multiple L-Arg adducts) were formed, and the number of binding ligands depended on the charge state of cytochrome c. While in neutral solution, the cytochrome c molecule complexed with only one L-Arg molecule (single L-Arg adducts). As for L-Glu, only single L-Glu adducts were formed in both acidic and neutral solutions.     

Analysis of triterpenoids in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> using liquid chromatography coupled with electrospray ionization mass spectrometry

Triterpenoids extracted from Ganoderma lucidum (Leyss. ex Fr.) Karst were separated and characterized using optimized reversed-phase liquid chromatography with diode array detection and electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MS(n)). They could be classified into five types depending on the fragmentation behavior. All triterpenoids gave [M - H](-) and [2M - H](-) ions by electrospray ionization monitored in the negative ion mode; in addition, compounds of types III and IV gave prominent [M - H - H(2)O](-) ions and the unsaturated bond at C-20, 22 would reduce the abundance of [M - H - H(2)O](-) ion. The key fragmentation information was cleavage at C- and D-rings despite the predominant losses of H(2)O and CO(2). Compounds with hydroxyls at C-7 and C-15 would produce a list of b, b - 1, b - 2, and b - 16 ions attributed to cleavage of D-ring; if the second alcohol at C-15 were oxidized to ketone, the prominent cleavage would occur at C-ring and produce a group of ions of a; if C-7 were oxidized to ketone, transference of two hydrogen atoms would occur during the cleavage of rings and a list of ions about a + 2 and/or b + 2 would appear instead. The above fragmentations and regularities in fragmentation pathways were reported for the first time, and were implemented for the analysis of triterpenoids in G. lucidum. The chloroform extract was separated on a Zorbax SB-C(18) column, eluting with an acetonitrile-0.2% acetic acid gradient. A total of 32 triterpenoids, including six new ones, were identified or tentatively characterized based on the tandem mass spectra of the HPLC peaks.     

Separation of isomers <Emphasis Type="Italic">L</Emphasis>-alanine and sarcosine in urine by electrospray ionization and tandem differential mobility analysis-mass spectrometry

Sarcosine, an isomer of L-alanine, has been proposed as a prostate cancer progression biomarker [1]. Both compounds are detected in urine, where the measured sarcosine/alanine ratio has been found to be higher in prostate biopsy-positive group versus controls. We present here preliminary evidence showing that urine samples spiked with sarcosine/alanine can be partially resolved in 3 min via tandem differential mobility analysis-mass spectrometry (DMA-MS). Based on the calibration curves obtained for two mobility peaks, we finally estimate their concentration ratio in urine.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-and <Emphasis Type="Italic">C</Emphasis>-trimethylsilyl-substituded anililes

N-Metallation of bromoanilines with ethylmagnesium bromide followed by a reaction with trimethylchlorosilane provided N-mono and N-bis(trimethylsilyl)bromoanilines depending on the structure of substrate. The metallation of bissilylated bromoanilines with butyllithium permitted the introduction of a trimethylsilyl substituent in the aromatic ring. Previously unknown 2-bromo-N,N-bis(trimethylsilyl)aniline, 2,6-dibromo-N-trimethylsilylaniline, 2,6-dibromo-N,N-bis(trimethylsilyl)aniline, 2-bromo-6-trimethylsilylaniline, 2-bromo-6-trimethylsilyl-N,N-bis(trimethylsilyl)aniline, 2-bromo-6-trimethylsilyl-N-trimethylsilylaniline, 2,4,6-tribromo-N-trimethylsilylaniline, and 2,4,6-tribromo-N,N-bis(trimethylsilyl)aniline were prepared. The structures of the compounds obtained were established by the chromato-mass spectrometry and ¹H, ¹³C, and ²⁹Si NMR spectroscopy.     

Synthesis of propylene-ethylene copolymers in liquid propylene using <Emphasis Type="Italic">ansa</Emphasis>-metallocenes of the <Emphasis Type="Italic">C</Emphasis><Subscript>1</Subscript> symmetry

The copolymerization of ethylene with propylene in the liquid propylene initiated by ansa-metallocenes of the C ₁ symmetry, rac-[1-(9-η⁵-fluorenyl)-2-(5,6-cyclopenta-2-methyl-1-η⁵-indenyl)ethane]zirconium dichloride and rac-[1-(9-η⁵-fluorenyl)-2-(5,6-cyclopenta-2-methyl-1-η⁵-indenyl)ethane]hafnium dichloride, activated by methylaluminoxane has been studied. Triisobutylaluminum has been used as a cocatalyst. The propylene-ethylene copolymers thus prepared contain 5–60 mol % ethylene units. The reactivity ratios have been measured. In the case of the zirconocene-based catalyst, the molecular mass of the copolymers decreases with an increase in the content of ethylene units. The reverse situation is observed in the case of the hafnocene-based catalytic system. The copolymers are characterized by the low T _g values (down to ?45°C). Incorporation of a small amount of ethylene units (5 mol %) results in a rise in the elastomeric behavior of the polymers.     

Ortho effect in electron ionization mass spectrometry of <Emphasis Type="Italic">N</Emphasis>-acylanilines bearing a proximal halo substituent

Electron ionization (EI) mass spectra are not very helpful for characterizing ortho, meta, and para isomers of underivatized haloanilines since their spectra are virtually identical. In contrast, when the amino group of chloro-, bromo-, or iodoanilines is transformed to an N-formyl, N-acetyl, or N-benzoyl derivative, the spectra of the derivatives reveal a highly dramatic loss of a halogen radical, instead of an HX elimination usually expected from an "ortho effect." For example, the spectra of N-formyl, N-acetyl, and N-benzoyl derivatives of ortho isomers of chloro-, bromo-, and iodoanilines show a very prominent peak at m/z 120, 134, and 196, respectively, for the loss of the corresponding halogen atom.     

An electrospray ionization mass spectrometric study of the subunit structure of the giant hemoglobin from the leech <Emphasis Type="Italic">Nephelopsis oscura</Emphasis>

The subunit structure of the giant, extracellular hexagonal bilayer (HBL) hemoglobin (Hb) from the leech Nephelopsis oscura was investigated by electrospray ionization mass spectrometry (ESI-MS) employing a maximum entropy deconvolution of its complex, multiply charged ESI spectra. The denatured unreduced Hb consisted of three monomer globin chains (M), a1 = 16535 Da, a2 = 17171 Da and a3 = 17315 Da, five nonglobin linker chains, L1 = 24512 Da, L2 = 24586 Da, L3 = 24979 Da, L4 = 25006 Da, and L5 = 25566 Da and two subunits of 32950 Da and 33125 Da. ESI-MS of the denatured, reduced Hb showed that the latter were disulfide-bonded heterodimers (D) of globin chains b1 = 16322 Da and b2 = 16499 Da with chain c = 16632 Da. Time-of-flight ESI-MS of the Hb at pH 3.8, 4.5, 5.0, 5.8 and 7.0 revealed a distribution of charge states from 32(+) to 37(+) with masses decreasing from 211 to 208.5 kDa with increase in cone voltage from 60 to 160 V, indicating the presence of a subassembly comprising 12 globin chains. The subunit composition 6M + 3D + 12h, where M = 16993 Da and D = 33004 Da are the weighted masses and h = 616.5 Da, provides a calculated mass, 208.37 kDa that is closest to 208.5 kDa. Our experimental findings are consistent with the bracelet model of HBL Hbs, verified by the recent low-resolution crystal structure of Lumbricus Hb, wherein an HBL arrangement of 12 globin dodecamer subassemblies is tethered to a central complex of 36 linker chains for a total mass of 208.37 x 12 + 24.94 x 36 = 3398 kDa.     

Lipids from <Emphasis Type="Italic">Halostachys caspica</Emphasis> and <Emphasis Type="Italic">Halocharis hispida</Emphasis>

The composition of lipids from the aerial parts of two species of halophytes from the family Chenopodiaceae, Halostachys caspica C. A. Mey. and Halocharis hispida Bge. was determined. Neutral lipids (NL, 62.1 and 54.2%, respectively) dominated the total lipids (TL) of these plants. More than a third of the NL were esters of aliphatic alcohols and phytosterols (FAE). Fatty acids 16:0, 18:1, and 18:2 dominated the acids of FAE; 16:0, 18:1, and 18:3, the phospholipids. The principal fatty acids of glycolipids were unsaturated acids (68.3 and 75.1%) with linolenic acid dominating (44.9 and 43.5%). Presented at the 7th International Symposium on the Chemistry of Natural Compounds, Tashkent, October 16–18, 2007. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 276–278, May–June, 2009.     

4<Emphasis Type="Italic">p</Emphasis><Superscript>2</Superscript> resonances in photoionization from 4<Emphasis Type="Italic">s</Emphasis>4<Emphasis Type="Italic">p</Emphasis> levels in neutral zinc

In neutral zinc the 4p ² configuration lies above the 3d ¹⁰4s ionization limit and its levels become perturbers in the continuum. Lines have been identified in the Zn I spectrum for the multiplet, whereas no lines have been found for transitions to 4p ^{2 1} D or ¹ S. In this paper, cross sections for photoionization from 4s4p levels are reported that reveal the positions and widths of the 4p ² resonances. Calculations were performed using the multiconfiguration Hartree-Fock (MCHF) and B-spline R-matrix (BSR) method. Results from Breit–Pauli calculations that ignore the background continua are also presented. Included in the latter are energies for the levels and transition data (transition energies, line strengths, f-values, and A-rates) for all E1 transitions between these levels. Transition energies and the agreement in the length and velocity values, particularly for allowed transitions, indicate the accuracy of the computational model. Line widths are compared with other estimates. Contribution to the Serafin Fraga Memorial Issue.     

Determination of the structural features of <Emphasis Type="Italic">O</Emphasis>-alkyl-<Emphasis Type="Italic">S</Emphasis>-2-(<Emphasis Type="Italic">N,N</Emphasis>-dialkylamino) ethyl alkylthiophosphonates from their electron ionization mass spectra

It is believed that information about the molecular structure of highly toxic O-alkyl-S-2(N,N-dialkylamino) ethyl alkylthiophosphonates (V-gases) obtained from their EI mass spectra is too insufficient. In particular, the determination of molecular weights and structures of radicals at phosphorus and oxygen atoms causes great difficulties. In this paper, solutions of these problems are proposed.     

Biodegradation studies of <Emphasis Type="Italic">N</Emphasis><Superscript>4</Superscript>-acetylsulfapyridine and <Emphasis Type="Italic">N</Emphasis><Superscript>4</Superscript>-acetylsulfamethazine in environmental water by applying mass spectrometry techniques

This work evaluates the biodegradation of N ⁴-acetylsulfapyridine (AcSPY) and N ⁴-acetylsulfamethazine (AcSMZ), metabolites of two of the most commonly used sulfonamides (SAs) in human and veterinary medicine, respectively. Aerobic transformation in effluent wastewater was simulated using aerated fixed-bed bioreactors. No visible changes in concentration were observed in the AcSMZ reactor after 90 days, whereas AcSPY was fully degraded after 32 days of experiment. It was also demonstrated that AcSPY transformed back to its parent compound sulfapyridine (SPY). The environmental presence of these two metabolites in wastewater effluent had been previously investigated and confirmed, together with three more SA acetylated metabolites and their corresponding parent compounds, in 18 different wastewater treatment plants in Hesse (Germany). Sulfamethoxazole (SMX) and SPY were the two SAs detected most frequently (90% and 89% of the samples, respectively) and in the highest concentrations (682 ng L⁻¹ for SMX and 532 ng L⁻¹ for SPY). To conclude, hazard quotients were calculated whenever toxicity data were available. None of the SAs studied posed an environmental risk.     

<Emphasis Type="Italic">N</Emphasis>-halohexamethyldisilazanes

The review is devoted to N-halohexamethyldisilazanes, perspective but scantily studied halogenating agents. Methods of preparation, physiochemical characteristics, and synthetic potential are considered. Mechanisms of a number of reactions are presented.