Tandem mass spectra of transition-metal ion adducts of glycosyl dithioacetals; distinction among stereoisomers

Electrospray ionization mass spectra of some glycosyl dithioacetals recorded in the presence of transition-metal chlorides, XCl2 (where X = Co, Mn and Zn), give abundant adduct ions such as [M+XCl]+ and [2M-H+X]+ and minor ions such as [M-H+X]+ and [2M+XCl]+. The tandem mass spectra of these adducts show characteristic elimination of neutral molecules such as H2O, HCl, EtSH, CH2O, C2H4O2/C2H4O. [M+XCl]+ ions fragment readily and the fragmentation appears to be stereochemically controlled as the relative abundances of the fragments are different for three stereoisomers. The added metal is lost as neutral molecules in the form of XCl(OH) and XCl(SEt). This is a predominant pathway in the ZnCl+ adducts. [2M+XCl]+ ions fragment preferentially by elimination of HCl, indicating strong metal interactions in the resulting dimeric [2M-H+X]+ ion. As there are several electron-rich centers in the molecule, the dimeric complex [2M-H+X]+ can have several structures and the observed fragmentations may reflect the sum of those of all these structures. The dimeric complexes fragment by elimination of neutral molecules leaving the dimeric interactions intact. The extent of fragmentation varies for the stereoisomers, leading to stereochemical differentiation.     

Tandem mass spectra of divalent metal ion adducts of glycosyl sulfides, sulfoxides and sulfones; distinction among stereoisomers

The tandem mass spectra of the divalent metal ion (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Ni2+, Co2+ and Zn2+) adducts of acetylated 1,2-trans-glycosyl sulfides, sulfoxides and sulfones were examined using low energy collision-induced dissociation on a Quattro II quadrupole tandem mass spectrometer. Abundant doubly charged ions, such as [3M + Met]2+ and [2M + Met]2+, were observed with alkaline earth metal chlorides. The other ions observed were [M + MetCl]+, [M + MetOAc]+, [M + MetO2SPh]+ and [2M + MetCl]+. The deprotonated metal adducts [M + Met-H]+ were seen only in the sulfones. The divalent metal ion adducts showed characteristic fragmentation pathways for the glycosyl sulfides, sulfoxides and sulfones, depending on the site of metal attachment. The doubly charged metal ion adducts dissociate to two singly charged ions, [M + MetOAc]+ and [M - OAc]+, in the sulfides and sulfoxides. In the sulfones, the adducts dissociate to [M + MetO2SPh]+ and [M - O2SPh]+. In contrast to the alkaline earth metals, which attach to the acetoxy functions, the transition metals attach to the sulfide and sulfoxide functions. The metal chloride adducts display characteristic fragmentation for the sulfides, sulfoxides and sulfones. The glucosyl, mannosyl and galactosyl sulfides, sulfoxides and sulfones could be differentiated on the basis of the stereochemically controlled MS/MS fragmentations of the metal chloride adducts.     

Fast-atom bombardment mass spectrometry of conjugated benzo(a)pyrene metabolites

Fast-atom bombardment mass spectrometry has been used to obtain spectra of conjugated benzo(a)pyrene (bap) metabolites using a 1:1, glycerol + thioglycerol matrix, bap Glucuronides give positive- and negative-ion spectra with peaks due to [M + H]+ and [M - H]- ions and a major fragment peak (base peak) at [bap-OH]+ and [bap-O]-. bap Sulfates (sodium salts) give similar negative-ion spectra with [M - Na]- and [bap-O]- peaks, but the positive-ion spectra are dominated by sodium and glycerol adducts of the bap sulfates.     

Structural determination of cyclitol derivatives by fast-atom bombardment tandem mass spectrometry

Three cyclitol derivatives were isolated from the marine sponge Sarcotragus sp. by reversed-phase high-performance liquid chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Their structural elucidation was carried out with FAB tandem mass spectrometry (FAB-MS/MS). FAB-MS spectra produced a significant abundance of the sodium adducts [M+Na]+ and [M+2Na-H]+ from a mixture of m-NBA and NaI. In addition, trifluoroacetylation of the cyclitol derivatives was used for confirmation of the presence of the cyclitol ring. High abundance [M-5H+5CF3CO+Na]+ ions were observed in the FAB-MS spectra of the trifluoroacetyl-cyclitol derivatives. Collision-induced dissociation (CID) of the [M+Na]+ ions produced diverse product ions via a series of dissociative processes. Charge-remote fragmentation (CRF) patterns of [M+Na]+ ions were very useful for the identification of product ions which are characteristic for the cyclitol ring and long hydrocarbon chains substituted at the glycerol backbone. Moreover, the CID-MS/MS spectra of the [M+Na]+ ions yielded characteristic product ions at m/z 53, 83, 113, 155 and 171 for the cyclitol moiety, and at m/z 213, 229 and 245 for the glycerol backbone attached to the cyclitol ring.     

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.     

Ionization of anabolic steroids by adduct formation in liquid chromatography electrospray mass spectrometry

The ionization of 46 anabolic steroids has been studied. The absence of basic or acidic moieties in most of these analytes makes their direct ionization as [M + H]+ by atmospheric pressure interfaces difficult. The formation of adducts with different components of the mobile phase has been found to be an efficient way to ionize anabolic steroids by electrospray. Different mobile phases using methanol (MeOH) or acetonitrile as organic solvent and HCOOH, Na+ or NH4+ as additives have been tested to favor the adduct formation. A direct correlation between the chemical structure of the anabolic steroid and the possibility to ionize it in a particular chromatographic condition has been found. According to their ionization, anabolic steroids can be divided into seven different groups depending on both the nature and the relative position of their functional groups. The formation of different adducts such as [M + Na + MeOH]+ or [M + H + CH3 CN - H2O]+ is required in order to ionize some of these groups and the optimal mobile phase composition for each group of anabolic steroids is proposed. Despite the ionization limitations due to their chemical structure, most of tested anabolic steroids could be ionized using the adduct formation approach.     

Comparison of mass spectra of peptides in different matrices using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG.

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.     

5-Ethyl-2-mercaptothiazole as matrix for matrix-assisted laser desorption/ionization of a broad spectrum of analytes in positive and negative ion mode

A preliminary investigation of the use of 5-ethyl-2-mercaptothiazole as matrix in matrix-assisted laser desorption/ionization (MALDI) of a broad spectrum of analytes is reported. The analytes studied are substance P, insulin, beta-cyclodextrin, triacylglycerols of coconut oil and polypropylene glycol 2000 (PPG 2000). In the positive ion mass spectra of the matrix/analyte combinations, the formation of [M + H]+ and [M + cation]+ species were observed and compared with those obtained by using well-established matrices such as alpha-cyano-4-hydroxycinnamic acid, genticic acid, sinapinic acid and dithranol. In addition, the usefulness of this new matrix for MALDI in negative ion mode is also described using substance P and beta-cyclodextrin as examples.     

Improved protonation, collision-induced decomposition efficiency and structural assessment for 'red tide' brevetoxins employing nanoelectrospray mass spectrometry

Brevetoxins are a group of natural neurotoxins found in blooms of red tide algae. Previous electrospray mass spectrometry (ES-MS) studies show that all brevetoxins have high affinities for sodium ions, and they form abundant sodium adduct ions, [M + Na]+, in ES-MS, even when trace contamination is the only source of sodium ions. Attempts to obtain informative product ions from the collision-induced decomposition (CID) of [M + Na]+ brevetoxin precursor ions resulted only in uninformative sodium ion signals, even under elevated collision energies. In this study, a nano-ES-MS approach was developed wherein ammonium fluoride was used to form cationic [M + NH4]+ adducts of brevetoxin-2 and brevetoxin-3; a significant increase in the abundance of protonated brevetoxin molecules [M + H]+ also resulted, whereas the abundance of sodium adducts of brevetoxins [M + Na]+ was observed to decrease. Under CID, both [M + NH4]+ and [M + H]+ gave similar, abundant product ions and thus underwent the same types of fragmentation. This indicated that ammonium ions initially attached to brevetoxins forming [M + NH4]+ easily lose neutral ammonia in a first step in the gas phase, leaving protonated brevetoxin [M + H]+ to readily undergo further fragmentation under CID.     

Tandem electrospray mass spectrometric studies of proton and sodium ion adducts of neutral peptides with modified N- and C-termini: synthetic model peptides and microheterogeneous peptaibol antibiotics

The fragmentations of [M+H]+ and [M+Na]+ adducts of neutral peptides with blocked N- and C-termini have been investigated using electrospray ion trap mass spectrometry. The N-termini of these synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C-termini are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N-terminal blocking group occurs quite readily in the case of MS/MS of [M+Na]+, the cleavage of the C-terminal methoxy group seems to be a facile process in the case of MS/MS of [M+H]+ * Fragmentation of the protonated adducts yields only bn ions, while yn and a(n) ions are predominantly formed from the fragmentation of sodium ion adducts. The a(n) ions arising from the fragmentation of [M+Na](+) lack the N-terminal Boc group (and are here termed a(n)* ions). MS/MS of [M+Na]+ species also yields b(n) ions of substantially lower intensities that lack the N-terminal Boc group (b(n)*). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N-terminal acetyl group. An example of the fragmentation of the H+ and Na+ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of b(n) ions derived from [M+H]+ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C-termini, for example, peptaibols.     

The detection of red pigment-concentrating hormone (RPCH) in crustacean eyestalk tissues using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry: [M + Na]+ ion formation in dried droplet tissue preparations

Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.     

Electrospray tandem mass spectrometry of alkali-cationized BocN-carbo-alpha,beta- and -beta,alpha-peptides: Differentiation of positional isomers

Dissociation pathways of a series of alkali-cationized hybrid peptides, viz., Boc-alpha,beta- and -beta,alpha-carbopeptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and alpha-alanine (L-Ala), have been investigated by electrospray ionization tandem mass spectrometry. The positional isomers (six pairs) of the cationized alpha,beta- and beta,alpha-peptides can be differentiated by the collision-induced dissociation (CID) spectra of their [M + Cat-Boc + H]+ ions which give characteristic series of alkali-cationized C- (x(n)+, y(n)+, z(n)+) and N-terminal (a(n)+, b(n)+, c(n)+) ions. Another noteworthy difference is cationized beta,alpha-peptides eliminate a molecule of ammonia whereas this pathway is absent for alpha,beta-peptides. This is useful for identifying the presence of a beta-amino acid at the N-terminus. The CID spectra of [M + Cat-Boc + H]+ ions of these peptide acids show abundant rearrangement [b(n) + 17 + Cat]+ (n = 1 to n-1) ions which is diagnostic for distinguishing between alpha- and beta-amino acid at the C-terminus. MS(n) experiments of [b(n) + Li-H]+ ions from these hybrid peptides showed the loss of CO and 72 u giving rise to [a(n) + Li-H]+ and cationized nitrile product ions which render support to earlier proposals that b(n)+ or [b(n) + Cat-H]+ ions have protonated or cationized oxazolinone structures, respectively.     

The influence of electrostatic interactions on the detection of heme-globin complexes in ESI-MS

The heme-globin complexes of hemoglobin and myoglobin are investigated in positive-ion mode and negative-ion mode using a nano-ESI source coupled to a quadrupole ion trap MS and an orthogonal time-of-flight MS. The extent of dissociation of these noncovalent complexes upon collisional activation and thus their gas-phase stability is strongly dependent on the polarity of the ESI-MS experiment as well as on the charge of the prosthetic group (ferri-heme [Fe3+-heme]+ vs. ferro-heme [Fe2+-heme]+/-0). The results clearly point to the important role of electrostatic interactions on the gas phase stability of noncovalent complexes and therefore the ion signals observed in ESI-MS experiments.     

Exceptional gas-phase enantioselectivity of chiral tetramide macrocycles

Diastereomeric proton-bound complexes between some phenylalanine derivatives (A) and chiral tetramide macrocycles (M) exhibit an uncommon enantioselectivity when reacting with the enantiomers of 2-aminobutanes in the gas phase (B). The measured enantioselectivity depends mainly on two distinct factors: (i) the configuration of the A guest; and (ii) the structure and the relative stability of isomeric [MHA]+ complexes. No significant effects of the B configuration are observed. The diastereomeric [MHA]+ complexes with A = 1-naphthylalanine ethyl ester exhibit the largest enantioselectivity factor ever measured in the gas phase (khomo/khetero = 0.046). The origin of such an exceptional enantioselectivity is mainly attributed to the relative stability of the diastereomeric [MHA]+ complexes, as demonstrated by the comparison of the kinetic results with those from collision-induced dissociation of the trimeric [M2HA]+ adducts and with computational evidence.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an inorganic particle matrix for small molecule analysis

Fine metal or metal oxide powder as an alternative to conventional organic matrices in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been utilized successfully for lower molecular mass analytes, poly(ethylene glycol) 200 (PEG 200) and methyl stearate. Eleven kinds of particle, Al, Mn, Mo, Si, Sn, SnO2, TiO2, W, WO3, Zn and ZnO, were evaluated. The analyte was mixed with a metal or metal oxide powder (inorganic matrix) with particle diameter of tens of micrometers and liquid dispersant, followed by application to the sample target. Using a commercial MALDI-TOFMS instrument equipped with an internal 337 nm pulsed nitrogen laser, the analytes, PEG 200 and methyl stearate, were ionized as the alkali metal ion adducted molecules [M+Na]+ or [M+K]+ when the inorganic matrices Mn, Mo, Si, Sn, TiO2, W, WO3, Zn or ZnO were used. In the case of an Al matrix, PEG 200 was ionized as [M+K]+, whereas methyl stearate was ionized as [M+H]+ and [M+Al]+. These particles have potential as the matrix for MALDI. During our examination, however, only SnO2 particles did not ionize either PEG 200 or methyl stearate. Based on our protocol, when TiO2 powder was suspended with liquid paraffin, PEG 200 and methyl stearate gave their MALDI-TOF mass spectra with the lowest background noise and highest intensity. TiO2 powder seemed to be a broad potential matrix for low molecular mass polar or non-polar analytes. The results suggested that bulk particles caused rapid heating/vaporization processes and ionized analyte molecules under irradiation with a pulsed UV laser. The present method can be readily applied to obtain the low background noise MALDI-TOF mass spectra of small-sized compounds.     

Detection of oxidative species for 4-phenoxyphenol derivatives during the electrospray ionization process

Analyses by flow injection as well as liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were performed with four 4-phenoxyphenol derivatives. When ambient temperature nitrogen gas was used to facilitate solvent evaporation, [M + H]+, [M + NH4]+, and [2M + NH4]+ ions were observed as the major ions. As the nitrogen gas temperature increased from ambient to 250 and 450 degrees C, [M]+*, [M - 1]+ and [M + 15]+ ions were the predominant ions. Heat-induced oxidation was found to be the primary source for the formation of oxidative species. Aqueous solvents were found to be essential for the formation of the [M + 15]+ ions. The [M]+* and [M + 15]+ ions were further characterized by tandem mass spectrometry. Based on the MS/MS data, it was proposed that the [M + 15]+ ions were the in-source generated 1,2-quinone ions.     

Mobile phase variations in thermospray liquid chromatography-mass spectrometry of pesticides

The effect of four different mobile phase compositions with reversed-phase methanol-water (50:50) + 0.05 M ammonium acetate, methanol-water (50:50) + 0.05 M ammonium formate, acetonitrile-water (50:50) + 0.05 M ammonium acetate and acetonitrile-water (50:50) + 0.05 M ammonium formate were compared in filament-on thermospray liquid chromatography-mass spectrometry for the determination of carbamate and chlorotriazine pesticides. In the positive-ion mode, [M + H]+ and [M + NH4]+ were generally the base peaks for the chlorotriazines and the carbamates, respectively. Depending on the mobile phase used, other adduct ions obtained corresponded to [M + CH3CN + H]+, [M + CH3OH + NH4]+, [M + CH3COONH4 + NH4 - 2H2O]+, [M + CH3CN + NH4]+, [M + CH3COONH4 + H - H2O]+ and the dimer [2M + H]+. In the negative-ion mode, [M - H]- and adducts with the ionizing additive [M + CH3COO]- or [M + HCOO]- were obtained. Other ions for the carbamates carbaryl and oxamyl corresponded to [M - CONHCH3 + CH3COOH]- and [M - CON(CH3)2 + HCOO]-, respectively. The variation of mobile phase composition provides additional structural information in thermospray liquid chromatography-mass spectrometry with no appreciable loss of sensitivity. Applications are reported for the determination of carbamate and chlorotriazine pesticides at the ng/g level in spiked and real soil samples, respectively.     

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.     

Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus

Derivatives were prepared from N-linked glycans by reductive amination from 2-aminobenzamide, 2-aminopyridine, 3-aminoquinoline, 2-aminoacridone, 4-amino-N-(2-diethylaminoethyl)benzamide, and the methyl, ethyl, and butyl esters of 4-aminobenzoic acid. Their electrospray and collision-induced dissociation (CID) fragmentation spectra were examined with a Q-TOF mass spectrometer. The strongest signals were obtained from the [M + Na]+ ions for all derivatives except sugars derivatized with 4-amino-N-(2-diethylaminoethyl)benzamide which gave very strong doubly charged [M + H + Na]2+ ions. The strongest [M + Na]+ ion signals were obtained from the butyl ester of 4-aminobenzoic acid and the weakest from 2-aminopyridine. The most informative spectra were recorded from the [M + Li]+ or [M + Na]+ ions. These spectra were dominated by ions produced by sequence-revealing glycosidic cleavages and "internal" fragments. Linkage-revealing cross-ring cleavage ions were reasonably abundant, particularly from high-mannose glycans. Although the nature of the derivative was found to have little effect upon the fragmentation pattern, 3-aminoquinoline derivatives gave marginally more abundant cross-ring fragments than the other derivatives. [M + H]+ ions formed only glycosidic fragments with few, if any, cross-ring cleavage ions. Doubly charged molecular ions gave less informative spectra; singly charged fragments were weak, and molecular ions containing hydrogen ([M + 2H]2+ and [M + H + Na]2+) fragmented as the [M + H]+ singly charged ions with no significant cross-ring cleavages.     

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.