Establishment of mass spectrometric fingerprints of novel synthetic cholesteryl neoglycolipids: The presence of a unique <Emphasis Type="Italic">C</Emphasis>-glycoside species during electrospray ionization and during collision-induced dissociation tandem mass spectrometry

In this study we evaluated the fragmentation pattern of 16 novel amphiphilic neoglycolipid cholesteryl derivatives that can be efficiently used to increase cationic liposomal stability and to enhance gene transfer ability. These neoglycolipids bear different sugar moieties, such as D-glucosamine, N-acetyl-D-glucosamine, N-trideuterioacetyl-D-glucosamine, N-acetyllactosamine, L-fucose, N-allyloxycarbonyl-D-glucosamine, and some of their per-O-acetylated derivatives. Regardless of the structure of the tested neoglycolipid, QqToF-MS analysis using electrospray ionization (ESI) source showed abundant protonated [M+H]+ species. We also identified by both QqToF-MS and low-energy collision tandem mass spectrometry (CID-MS/MS) of the [M+H]+ ion, the presence of specific common fingerprint fragment ions: [Cholestene]+, sugar [oxonium]+, [(Sugar-spacer-OH)+H]+, [oxonium-H2O]+, and [(Cholesterol-spacer-OH)+H]+. In addition, we observed a unique ion that could not be rationally explained by the expected fragmentation of these amphiphilic molecules. The structure of this ion was tentatively proposed with that of a C-glycoside species formed by a chemical reaction between the sugar portion and the cholesterol. MS/MS analysis of this unique [C-glycoside]+ confirmed the validity of the proposed structure of this ion. The presence of an amino group at position C-2 and free hydroxyl groups of the sugar motif is crucial for the formation of a "reactive" sugar oxonium ion that can form the [C-glycoside]+ species. In summary, we precisely established the fragmentation patterns of the tested series of neoglycolipid cholesteryl derivatives and authenticated their structure as well; moreover, we speculated on the formation of a C-glycoside with the ESI source under atmospheric pressure and in the collision cell during MS/MS analysis.     

Determination of the fatty acyl profiles of phosphatidylethanolamines by tandem mass spectrometry of sodium adducts

Phosphatidylethanolamines (PEs) are one of the major constituents of cellular membranes, and, along with other phospholipid classes, have an essential role in the physiology of cells. Profiling of phospholipids in biological samples is currently done using mass spectrometry (MS). In this work we describe the MS fragmentation of sodium adducts of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and 2-linoleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (PLPE). This study was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) using three different instruments and also by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All MS/MS spectra show product ions related to the polar head fragmentation and product ions related to the loss of acyl chains. In ESI-MS/MS spectra, the product ions [M+Na-R1COOH-43]+ and [M+Na-R2COOH-43]+ show different relative abundance, as well as [M+Na-R1COOH]+ and [M+Na-R2COOH]+ product ions, allowing identification of both fatty acyl residues of PEs, and their specific location. MALDI-MS/MS shows the same product ions reported before and other ions generated by charge-remote fragmentation of the C3-C4 bond (gamma-cleavage) of fatty acyl residues combined with loss of 163 Da. These fragment ions, [M+Na-(R2-C2H3)-163]+ and [M+Na-(R1-C2H3)-163]+, show different relative abundances, and the product ion formed by the gamma-cleavage of sn-2 is the most abundant. Overall, differences noted that are important for identification and location of fatty acyl residues in the glycerol backbone are: relative abundance between the product ions [M+Na-R1COOH-43]+ > [M+Na-R2COOH-43]+ in ESI-MS/MS spectra; and relative abundance between the product ions [M+Na-(R2-C2H3)-163]+ > [M+Na-(R1-C2H3)-163]+ in MALDI-MS/MS spectra.     

Derivatization of protonated peptides via gas phase ion—molecule reactions with acetone

The protonated [M + H]+ ions of glycine, simple glycine containing peptides, and other simple di- and tripeptides react with acetone in the gas phase to yield [M + H + (CH3)2CO]+ adduct ion, some of which fragment via water loss to give [M + H + (CH3)2CO - H2O]+ Schiff's base adducts. Formation of the [M + H + (CH3)2CO]+ adduct ions is dependent on the difference in proton affinities between the peptide M and acetone, while formation of the [M + H + (CH3)2CO - H2O]+ Schiff's base adducts is dependent on the ability of the peptide to act as an intramolecular proton "shuttle." The structure and mechanisms for the formation of these Schiff's base adducts have been examined via the use of collision-induced dissociation tandem mass spectrometry (CID MS/MS), isotopic labeling [using (CD3)2CO] and by comparison with the reactions of Schiff's base adducts formed in solution. CID MS/MS of these adducts yield primarily N-terminally directed a- and b-type "sequence" ions. Potential structures of the b1 ion, not usually observed in the product ion spectra of protonated peptide ions, were examined using ab initio calculations. A cyclic 5 membered pyrrolinone, formed by a neighboring group participation reaction from an enamine precursor, was predicted to be the primary product.     

Direct qualitative analysis of triacylglycerols by electrospray mass spectrometry using a linear ion trap

Triacylglycerols (TAGs) isolated from a biological sample provide a challenge for mass spectrometric analysis because of the complexity of naturally occurring TAGs, which may contain different fatty acyl substituents resulting in a large number of molecular species having the identical elemental composition. We have investigated the use of mass spectrometry to obtain unambiguous information as to the individual TAG molecular species present in a complex mixture of triacylglycerols using a linear ion trap mass spectrometer. Ammonium adducts of TAGs, [M+NH4]+, were generated by electrospray ionization, which permitted the molecular weight of each TAG molecular species to be determined. The mechanisms involved in the decomposition of the [M+NH4]+ and subsequent fragment ions were investigated using deuterium labeling, MS/MS, and MS3 experiments. Collision induced decomposition of [M+NH4]+ ions resulted in the neutral loss of NH3 and an acyl side-chain (as a carboxylic acid) to generate a diacyl product ion. MS/MS data were used to identify each acyl group present for a given [M+NH4]+ ion, and this information could be combined with molecular weight data to identify possible TAG molecular species present in a biological extract. Subsequent MS3 experiments on the resultant diacyl product ions, which gave rise to acylium (RCO+) and related ions, enabled unambiguous TAG molecular assignments. These strategies of MS, MS/MS, and MS3 experiments were applied to identify components within a complex mixture of neutral lipids extracted from RAW 264.7 cells.     

Data-directed scan sequence for the general assignment of C-glycosylflavone O-glycosides in plant extracts by liquid chromatography-ion trap mass spectrometry

An ion trap LC-MS/MS method is described for the analysis of C-glycosylflavone O-glycosides in crude methanolic extracts of plants. The method employs survey scans with and without the application of up-front collision induced dissociation (CID) to generate diagnostic ions for data-directed MS/MS. The spectra acquired allow assignment of the C-linked sugar to either the C-6 or C-8 position of the aglycone and provide data on the molecular mass of the compound, the number and type of O-linked sugars and the molecular mass of the flavone aglycone. These data for the majority of C-glycosylflavone O-glycosides in an extract are obtained automatically in one LC-MS/MS analysis without manual pre-programming. Key to the assignment of the C-6 or C-8 site of C-glycosylation is the generation, by up-front CID, of the (0,1)X+ product ion formed by internal cleavage of the C-linked sugar. MS/MS of this ion is found to have diagnostic value in addition to the (0,2)X+ product ion described by other authors. Ion trap MS/MS spectra of [M+H]+ of the 6,8-di-C-glycosylflavones schaftoside and isoschaftoside show an additional and previously unreported diagnostic product ion that is useful in determining the type of sugar at the C-6 position. The product ion spectra of protonated kaempferol 3-O-glucosylrhamnosides show similarities to the spectra of C-glycosylflavone O-glycosides; this is a potential source of confusion if the analysis of such glycosides is limited solely to MS/MS of [M+H]+.     

Dissociation pathways of alkali-cationized peptides: Opportunities for C-terminal peptide sequencing

Dissociation pathways of alkali-cationized peptides have been studied using multiple stages of mass spectrometry (MSx) with a quadrupole ion trap mass spectrometer. Over 100 peptide ions ranging from 2 to 10 residues in length and containing each of the 20 common amino acids have been examined. The formation of the [b(n-1) + Na + OH]+ product ion is the predominant dissociation pathway for the majority of the common amino acids. This product corresponds to a sodium-cationized peptide one residue shorter in length than the original peptide. In a few cases, product ions such as [b(n-1) + Na - H]+ and those formed by loss, or partial loss, of a sidechain are observed. Both [b(n-1) + Na + OH]+ and [b(n-1) + Na - H]+ product ions can be selected as parent ions for a subsequent stage of tandem mass spectrometry (MS/MS). It is shown that these dissociation patterns provide opportunities for peptide sequencing by successive dissociation from the C-terminus of alkali-cationized peptides. Up to seven stages of MS/MS have been performed on a series of [b + Na + OH]+ ions to provide sequence information from the C-terminus. This method is analogous to Edman degradation except that the cleavage occurs from the C-terminus instead of the N-terminus, making it more attractive for N-terminal blocked peptides. The isomers leucine and isoleucine cannot be differentiated by this method but the isobars lysine and glutamine can.     

Analysis of neutral oligosaccharides for structural characterization by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

We have acquired multi-stage mass spectra (MSn) of four branched N-glycans derived from human serum IgG by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF-MS) in order to demonstrate high sensitivity structural analysis. [M+H]+ and [M+Na]+ ions were detected in the positive mode. The detection limit of [M+Na]+ in MS/MS and MS3 measurements for structural analysis was found to be 100 fmol, better than that for [M+H]+. The [M+H]+ ions subsequently fragmented to produce predominantly a Y series of fragments, whereas [M+Na]+ ions fragmented to give a complex mixture of B and Y ions together with some cross-ring fragments. Three features of MALDI-QIT-CID fragmentation of [M+Na]+ were cleared by the analysis of MS/MS, MS3 and MS4 spectra: (1) the fragment ions resulting from the breaking of a bond are more easily generated than that from multi-bond dissociation; (2) the trimannosyl-chitobiose core is either hardly dissociated, easily ionized or it is easy to break a bond between N-acetylglucosamine and mannose; (3) the fragmentation by loss of only galactose from the non-reducing terminus is not observed. We could determine the existence ratios of candidates for each fragment ion in the MS/MS spectrum of [M+Na]+ by considering these features. These results indicate that MSn analysis of [M+Na]+ ions is more useful for the analysis of complicated oligosaccharide structures than MS/MS analysis of [M+H]+, owing to the higher sensitivity and enhanced structural information. Furthermore, two kinds of glycans, with differing branch structures, could be distinguished by comparing the relative fragment ion abundances in the MS3 spectrum of [M+Na]+. These analyses demonstrate that the MSn technology incorporated in MALDI-QIT-TOF-MS can facilitate the elucidation of structure of complex branched oligosaccharides.     

Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method using an atmospheric pressure chemical ionisation source was used to measure the metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivative (1) in human liver microsomes. After 15 min incubation with human liver microsomes, compound 1 exhibited metabolic turnover of 44%. Data-dependent tandem mass spectrometry (MS/MS) scanning was used to generate product ion spectra from the protonated ions of the compound and its metabolites. An unusual metabolite at m/z 407 corresponding to the [M-24+H]+ ion was identified for compound 1. Interestingly, the formation of the [M-24+H]+ ion was not observed in the analogues wherein the fused thieno double bond was substituted (2) and the thieno group replaced by a fused benzo derivative (3). Compounds 2 and 3 exhibited metabolic turnovers of 24 and 30%, yielding oxidative metabolites corresponding to [M+16] and [M+32]+, respectively. Based on these facts the mechanism for [M-24]+ formation in compound 1 through an initial epoxide formation on the double bond of the fused thieno ring followed by hydrolytic ring opening and deacylation is envisaged.     

Formation of [b(n−1)+OH+H]+ ion structural analogs by solution-phase chemistry

Derivatization of a variety of peptides by a method known to enhance anhydride formation is demonstrated by mass spectrometry to yield ions that have elemental composition and fragmentation properties identical to [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation. The [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation and the solution-phase [b(n-1) + OH + H]+ ion structural analogs formed by derivatization chemistry show two different forms of dissociation using multiple-collision CAD in a quadrupole ion trap and unimolecular decomposition in a TOF-TOF; one group yields identical product ions as a truncated form of the peptide with a free C-terminal carboxylic acid and fragments at the same activation energy; the other group fragments differently from the truncated peptide, being more resistant to fragmentation than the truncated peptide and yielding primarily the [b(n-2) + OH + H]+ product ion. Nonergodic electron capture dissociation MS/MS suggests that any structural differences between the specific-fragmenting [b(n-1) + OH + H]+ ions and the truncated peptide is at the C-terminus of the peptide. The specific-fragmentation can be readily observed by MS(n) experiments to occur in an iterative fashion, suggesting that the C-terminal structure of the original [b(n-1) + OH + H]+ ion is maintained after subsequent rearrangement and fragmentation events in peptides which fragment specifically. A mechanism for the formation of specific-fragmenting and nonspecific-fragmenting [b(n-1) + OH + H]+ ions is proposed.     

A non-covalent dimer formed in electrospray ionisation mass spectrometry behaving as a precursor for fragmentations

A non-covalent-bonded dimer was detected in the positive ion electrospray ionisation (ESI) mass spectra of a synthetic impurity. In tandem mass spectrometry (MS/MS) experiments using collision-induced dissociation (CID), the ion was found to behave as a [M+H]+-type precursor ion for fragmentation until MS5. The dimer was probably formed through multi-hydrogen bonds over a proton bridge. When the fragmentation occurred at the center of the bridge, the dimer was broken apart to give monomer fragments at MS6. However, no corresponding deprotonated dimer [2M-H]- was found in the negative ion ESI spectra. The dimer was extremely stable, and it could still be observed when a fragmentation voltage of up to 50 V was applied in the ionisation source. The formation of the non-covalent dimer was also found to be instrument-dependent, but independent of sample concentration. Accurate mass measurements of the [2M+H]+ and [M+H]+ ions, and their MSn product ions, provided the basis for assessing the fragmentation mechanism proposed for [2M+H]+. The fragmentation pathway was also illustrated for the deprotonated molecule [M-H]-.     

Characterization of electrospray ionization mass spectrometry for N-diisopropyloxyphosphoryl dipeptide methyl esters

A systematic study of the fragmentation pattern of N-diisopropyloxyphosphoryl (DIPP) dipeptide methyl esters in an electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was presented. A combination of accurate mass measurement and tandem mass spectrometry had been used to characterize the major fragment ions observed in the ESI mass spectrum. It was found that the alkali metal ions acted as a fixed charge site and expelled the DIPP group after transferring a proton to the amide nitrogen. For all the N-phosphoryl dipeptide methyl esters, under the activation of a metal ion, the rearrangement product ion at m/z 163 was observed and confirmed to be the sodium adduct of phosphoric acid mono-isopropyl esters (PAIE), via a specific five-membered penta-co-ordinated phosphorus intermediate. However, no rearrangement ion was observed when a beta-amino acid was at the N-terminal. This could be used to develop a novel method for differentiating isomeric compounds when either alpha- or beta-amino acid are at the N-terminus of peptides. From the [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters (DIPP Xaa1 Xaa2 OMe), the peaks corresponding to the [M+Na Xaa1 C3H6]+ were observed and explained. The [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters with Phe located in the C-terminal, such as DIPPValPheOMe, DIPPLeuPheOMe, DIPPIlePheOMe, DIPPAlaPheOMe and DIPPPhePheOMe, had characteristic fragmentation. Two unusual gas-phase intramolecular rearrangement mechanisms were first proposed for this fragmentation. These rearrangements were not observed in dipeptide methyl ester analogs which did not contain the DIPP at the N-terminal, suggesting that this moiety was critical for the rearrangement.     

Electrospray ionization tandem mass spectral analysis of oxidation products of precursors of sulfur mustards

Electrospray ionization tandem mass spectral (ESI-MSn) analysis of thiodiglycol, bis(2-hydroxyethylthio)alkanes (BHETAs) and their mono-, di-, tri-, and tetraoxygenated compounds was carried out to obtain their characteristic spectra for ESI-MS analysis. These compounds are important markers of chemical warfare agents, namely sulfur mustards. ESI-MSn (n > or = 3) analysis of a compound by collisionally induced dissociation in an ion trap gives rise to mass spectra that are somewhat similar to electron ionization mass spectra. These ESI-MSn spectra can be used for compound identification. Under ESI-MS and ESI-MS/MS the compounds mostly produced [M+NH4]+, [M+H]+ and [M+H--H2O]+ ions. Fragmentations of these even-electron precursors in the ion trap gave rise to characteristic product ions via neutral loss of O2, H2O, C2H4, HCHO, C2H4O, C2H4S, HSC2H4OH and C2H4SO. Fragmentation routes of these compounds are proposed that rationalize the formation of product ions in ESI-MSn analysis.     

Structural determination of saponins extracted from starfish by fast atom bombardment collision-induced dissociation mass spectrometry.

Three saponins were extracted and isolated from starfish by reversed-phase high performance liquid chromatography (HPLC), and analyzed by fast atom bombardment mass spectrometry (FAB-MS). Their molecular weight information could be obtained by the presence of abundant [M+Na]+ ions and weak [M+H]+ ions in FAB-MS spectra. Moreover, high resolution mass measurements of their [M+Na]+ ions were performed at the resolution of 10000 to elucidate the element composition of extracted saponins. The collision-induced dissociation (CID) of sodium-adducted molecules [M+Na]+ yielded diverse product ions via dissociated processes. In the collision-induced dissociation (CID)-MS/MS analysis of [M+Na]+ ion, the sulfate-containing saponins produced characteristic ions such as SO4Na+, [NaHSO4+Na]+, [M+Na-sugar]+ and [M+Na-2sugar]+ ions, whereas the sulfate-free compound showed characteristic ions produced by cleavage of sugar moiety and side chain of aglycone. The fragmentation patterns could provide information on the linkage position of sugar groups in aglycone and sulfate groups.     

Dehydrogenation of tertiary amines in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of various compounds synthesized in our laboratory, strong [M - H]+ ion peaks were often observed for the molecules with tertiary amino groups. In this work, the MALDI TOF MS behavior of two groups of compounds that incorporate tertiary amino moieties was investigated. One group is bisurea dimethylanilines (BUDMAs) prepared for the study of molecular recognition in thermoplastic elastomers, and the other group is the poly(propylene imine) diaminobutane dendrimers. The results clearly demonstrate the appearance of the [M - H]+ ions. In order to understand the possible mechanisms for the generation of these ions, a series of model compounds, ranging from primary to tertiary amines, were investigated. Unlike the tertiary amines, no [M - H]+ ion peaks were recorded for the primary amines, and only barely detectable ones, if any, for some secondary amines. It appears that the tertiary amino groups play an important role in the formation of these ions. In addition to MALDI TOF MS analysis, these samples were also applied to electrospray ionization (ESI) MS where no [M - H]+ ions were observed. The results indicate that the generation of [M - H]+ ion is due to the unique MALDI conditions and is likely to be formed via dehydrogenation of a protonated tertiary amine resulting in an N=C double bond. The absence of [M - H]+ ion peaks for the primary and secondary amines is probably because upon their formation these ions could easily transfer one proton to the corresponding amines in the MALDI gas-phase plume, yielding neutral imines that cannot be detected by MS.     

Structural determination of monoacylglycerols extracted from marine sponge by fast atom bombardment tandem mass spectrometry

Five new monoacylglycerols (MAGs) were isolated from the marine sponge Stelletta sp. by reversed-phase high-performance liquid chromatography and analyzed by positive ion fast atom bombardment mass spectrometry (FAB-MS). FAB mass spectra of these compounds produced abundant sodium-adducted molecules [M+Na]+ from a mixture of 3-nitrobenzyl alcohol and sodium iodide. The structural elucidation of these sponge MAGs was carried out by FAB tandem mass spectrometry (MS/MS). To find diagnostic ions for the characterization of the MAGs, authentic MAGs were initially analyzed by collision-induced dissociation (CID) MS/MS. The CID MS/MS of [M+Na]+ precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge-remote fragmentation (CRF) provided important information for the characterization of acyl chains substituted at the glycerol backbone, and product ions at m/z 84, 97, 113 and 139 were diagnostic for the sodiated glycerol backbone. On the basis of these fragmentation patterns, the structures of five MAGs extracted from marine sponge were elucidated. In addition, high-resolution mass measurement was performed to obtain the elemental compositions of the MAGs.     

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.     

高效液相色谱-电喷雾银离子诱导电离飞行时间质谱定性分析高沸点多环芳烃

建立了高效液相色谱-电喷雾飞行时间质谱联用技术快速鉴别高沸点多环芳烃的方法。多环芳烃经色谱柱分离后,通过柱后添加AgNO3溶液诱导其在电喷雾离子源中电离,生成多环芳烃[M]+及其复合[M+Ag]+和[2M+Ag]+特征离子,根据所获得的各特征离子的精确分子量和分子式,可实现多环芳烃类化合物的快速鉴别。将本方法用于美国环保局(USEPA)规定的16种优先控制多环芳烃及原油中多环芳烃类化合物的分析鉴别,结果表明,四环以上的PAHs质谱信号良好,说明本方法适用于四环以上的高分子量、高沸点多环芳烃类化合物的分析鉴别。     

Identification of 17-hydroxyprogesterone and other steroid hormones in saliva from a normal child and patients with congenital adrenal hyperplasia by plasmaspray liquid chromatography/mass spectrometry

A very sensitive, selective and simultaneous method for the analysis of salivary steroid hormones was examined by discharge-assisted thermospray liquid chromatography mass spectrometry (plasmaspray LC/MS). Plasmaspray LC/MS gave [M + H]+ or [MH - H2O]+ as a predominant ion in most of the steroids in this work and in some cases fragment ions were also observed. Eight salivary steroid hormones, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, aldosterone, and testosterone were identified within 10 minutes using the selected ion monitoring technique in conjunction with plasmaspray LC/MS. Progesterone, 17-hydroxyprogesterone, cortisol, aldosterone and testosterone were detected in 1 mL of saliva from a normal child and two patients with congenital adrenal hyperplasia.     

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.     

Discovering metabolites of post-harvest fungicides in citrus with liquid chromatography/time-of-flight mass spectrometry and ion trap tandem mass spectrometry

In this study, we benefit from the combination of liquid chromatography (LC)/time-of-flight (TOF) MS accurate mass measurements to generate elemental compositions of ions and LC/ion trap multiple MS (MSn) providing complementary structural information, which is useful for the elucidation of unknown organic compounds at trace levels in complex food extracts. We have applied this approach to investigate different citrus fruits extracts, and we have identified two post-harvest fungicides (imazalil and prochloraz), the main degradation product of imazalil ([M + H]+, m/z 257) and a non-previously reported prochloraz degradation product ([M + H]+, m/z 282). The database-mediated identification of the parent compounds was based on the generated elemental composition obtained from accurate mass measurements and additional qualitative information from the high resolution chlorine isotopic clusters of both the protonated molecules (imazalil, [M + H]+ 297.0556, <0.1 ppm error, 2-Cl; prochloraz, [M + H]+ 376.0381, 1.9 ppm error, 3-Cl) and their characteristic fragments ions (imazalil: m/z 255 and 159; prochloraz: m/z 308 and 266). The correlation between the structural information provided by ion trap MS/MS fragmentation pathways of the parent species and the TOF accurate mass elemental composition data of the degradation products were the key to elucidate the structures of the degradation products of both post-harvest fungicides. Finally, where standards were not available (prochloraz), further confirmation was obtained by synthesizing the proposed degradation product by acid hydrolysis of the parent standard and confirmation by LC/TOF-MS.