High‐throughput polymer tip‐electrospray ionization mass spectrometry for enhanced detection of neopterin and biopterin in clinical urine samples

Urinary biopterin (Bio) and neopterin (Neo) are important markers for clinical diagnosis of hyperphenylalaninemia. Herein, we developed a high‐throughput analysis method based on electrospray ionization mass spectrometry (ESI‐MS) with polymer tips for the rapid quantitative detection of Bio and Neo in clinical urine samples. Different polymer tips were investigated. It is found that the best detection sensitivity was achieved with hydrophobic polymer tip, ie, polyethylene tips. The high‐throughput polymer tip‐ESI‐MS method allowed a rapid analysis speed at ~40 seconds per sample. The limits of quantification (LOQ) (S/N ≥ 10) for the detection of Bio and Neo were improved to be 5.0 ng/mL. Acceptable relative standard deviation (RSD) values for Neo and Bio were measured to be 12.2% and 13.4% for direct measurement of Bio and Neo in raw urine samples, respectively. Furthermore, Bio and Neo were directly quantified from 18 clinical urine samples by presented method. The ratios of urinary Bio‐to‐Neo were analyzed for diagnosis of hyperphenylalaninemia. The results demonstrated that the present polymer tip‐ESI‐MS method is a promising strategy for the rapid analysis of clinical samples.     

Selective linkage detection of O‐sialoglycan isomers by negative electrospray ionization ion trap tandem mass spectrometry

Sialylated O‐linked oligosaccharides are involved in many biological processes, such as cell‐cell interactions, cell‐substance adhesion, and virus‐host interactions. These activities depend on their structure, which is frequently determined by tandem mass spectrometry. However, these spectra are frequently analyzer‐dependent, which makes it difficult to develop widely applicable analytical methods. In order to deepen the origin of this behavior, two couples of isomers of sialylated O‐linked oligosaccharides, NeuAcα2‐3Galβ1‐3GalNAc‐ol/Galβ1‐3(NeuAcα2‐6)GalNAc‐ol and NeuGcα2‐3Galβ1‐3GalNAc‐ol/Galβ1‐3(NeuGcα2‐6)GalNAc‐ol, were analyzed by liquid chromatography/negative electrospray ionization ion trap tandem mass spectrometry (LC/ESI(?)‐MSⁿ) using both an ion trap and a triple quadrupole mass spectrometer. Results clearly showed that while ions obtained in the triple quadrupole instrument fitted very well with the standard fragmentation routes, in the ion trap several intense ions could not be explained by these rules, specially a fragment at m/z 597. Furthermore, this ion was observed in the mass spectrum of those isomers that sialic acid binds to GalNAc by an α2‐6 linkage. From the MS³ spectrum of this ion an unexpected structure was deduced, and it led to propose alternative fragmentation pathways. Molecular mechanics calculations suggested that the found atypical route could be promoted by a hydrogen bond located only in α2‐6‐linked oligosaccharides. It has also been demonstrated that this process follows a slow kinetic, explaining why it cannot be observed using an ion beam‐type mass analyzer. In conclusion, ion traps seem to be more appropriate than triple quadrupoles to develop a reliable analytical method to distinguish between isomeric O‐linked glycans. Copyright © 2010 John Wiley & Sons, Ltd.     

Oligonucleotide sequencing using guanine-specific methylation and electrospray ionization ion trap mass spectrometry

This paper describes a novel method to map guanine bases in short oligonucleotides using a simple chemical modification reaction and subsequent analysis by electrospray ionization ion trap mass spectrometry (ITMS). In situ guanine-specific methylation followed by gas-phase fragmentation permits the determination of the positions of all guanine residues. Collision-induced dissociation (CID) of the monomethylated oligonucleotide strand promotes rapid depurination and further collision (MS3) of the apurinic oligonucleotide leads to preferential cleavage of the backbone at the site of depurination. The mass of the resulting complementary product ions verifies the position of each guanine base in the sequence. The utility of this methodology is demonstrated for oligonucleotide sequences up to 10 bases in length. In addition, this technique successfully illustrates the use of selective fragmentation for sequencing oligonucleotides by ITMS.     

Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry

Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI‐MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,‐trimethoxyphenyl)phosphonium acetic acid (TMPP‐AA) to improve liquid chromatography (LC)/ESI‐MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl‐sn‐glycerol, dihydrotachysterol, octadecanol, and alpha‐tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI‐MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in 2009 by John Wiley & Sons, Ltd.     

Electrode‐assisted desorption electrospray ionization mass spectrometry

A new ion source has been developed for rapid, noncontact analysis of materials at ambient conditions. The method provides desorption of analytes under ambient conditions directly from different surfaces with little or no sample preparation. The new method, termed electrode‐assisted desorption electrospray ionization (EADESI), is on the basis of the ionization of molecules on different surfaces by highly charged droplets produced on a sharp‐edged high voltage tip, and ions produced are introduced into the mass spectrometer through a capillary. The EADESI technique can be applied to various samples including amino acids, peptides, proteins, drugs and human fluids such as urine and blood. EADESI is promising for routine analyses in different fields such as forensic, environmental and material sciences. EADESI interface can be fit to a conventional ion‐trap mass spectrometer. It can be used for various types of samples with a broad mass range. EADESI can also provide real‐time analysis which is very valuable for biomedical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid screening method for intact glucosinolates in Chinese medicinal herbs by using liquid chromatography coupled with electrospray ionization ion trap mass spectrometry in negative ion mode

An optimized method using liquid chromatography coupled with electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) in negative ion mode has been developed for screening different structural classes of intact glucosinolates in six Chinese medicinal herbs. The glucosinolates were extracted with hot methanol/water (70:30 v/v) and separation of the individual glucosinolates was achieved using a reversed-phase C18 column with an aqueous ammonium acetate/methanol gradient. Identification of the intact glucosinolates was based on the detection of compounds with a constant neutral loss of 242 Da corresponding to the combined loss of anhydroglucose (162 Da) and sulfur trioxide (80 Da) in collision-induced dissociation. The structures of the identified glucosinolates were confirmed with the use of group-specific product ions at m/z 195, 241, 259, 275 in their corresponding MS/MS product ion spectra. Differentiation of intact glucosinolates was achieved through their respective retention times and molecular masses as well as the characteristic product ions. The limits of detection were at the low nanogram level per injection, based on constant neutral loss scans. Significant variation in the compositions of intact glucosinolates was identified in the cruciferous herbs. This method was applied in the differentiation and quality control of two pairs of easily confused herbs. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Detection of microcystins using electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry/mass spectrometry

A combination of electrospray ionization, high-field asymmetric waveform ion mobility spectrometry, and mass spectrometry (ESI-FAIMS/MS) was used to analyze standard solutions of microcystins-LR, -RR, and -YR. The ability of FAIMS to separate ions in the gas phase reduced the amount of background in the mass spectrum without compromising the absolute signal for these microcystins. This reduction in background resulted in a ten-fold improvement in the signal-to-background ratio over conventional ESI-MS. Detection limits, using direct infusion, were determined to be 4, 2, and 1 nM for microcystins-LR, -RR, and -YR, respectively.     

The distinction of underivatized monosaccharides using electrospray ionization ion trap mass spectrometry

A convenient method for distinguishing underivatized isomeric monosaccharides has been established using electrospray ionization ion trap mass spectrometry (ESI-ITMS). Mass spectra of hexoses (glucose, galactose, and mannose), N-acetylhexosamines (N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine) and hexosamines (glucosamine, galactosamine, and mannosamine) dissolved in solvent containing 1 mM ammonium acetate were obtained in the positive ion mode. Glucose was distinguished from galactose and mannose in the MS(2) spectrum of the [M+NH(4)](+) ion at m/z 198. The MS(3) spectra generated from [M+NH(4)-H(2)O-NH(3)](+) at m/z 163 showed that galactose and mannose could be distinguished by the ratio of peak intensities at m/z 145 and 127, while the three N-acetylhexosamine and hexosamine stereochemical isomers could be identified by the relative abundance ratios of product ions observed in MS(3) spectra. The investigation of MS and MS(2) spectra from complexes of these monosaccharides with Na(+) and Pb(2+) failed to distinguish these monosaccharide isomers. Therefore, multiple stage mass analysis by ESI-ITMS using either [M+NH(4)](+) or [M+H](+) was useful to distinguish between the isomers of monosaccharides.     

Dual negative precursor ion scan approach for rapid detection of glutathione conjugates using liquid chromatography/tandem mass spectrometry

Screening for conjugates formed by the tripeptide glutathione with new chemical entities is an essential step during the drug discovery process, as the formation of these conjugates serves as an indicator for the presence of reactive electrophilic intermediates. To increase the selectivity and throughput of this analysis, various mass spectral scan types have evolved over time. Historically, samples were analyzed under positive ionization conditions for the neutral loss of m/z 129 (loss of the pyroglutamic acid moiety from glutathione); however, more recently, negative precursor ion scanning for the loss of m/z 272 (deprotonated gamma-glutamyl-dehydroalanyl-glycine from glutathione) has emerged as a more selective tool. Further increasing the selectivity, we report on an extension of this methodology by incorporating a simultaneous dual negative precursor ion scan for two commonly observed ion fragments from glutathione conjugates, m/z 272 and 254 (the dehydrated form of m/z 272). This negative dual precursor ion scan methodology was first validated using substrates known to undergo metabolic bioactivation (diclofenac, carbamazepine, and 3-methyl indole) and has then been applied to the routine analysis of proprietary compounds undergoing active lead optimization. In comparison to alternative scan methodologies, the increased selectivity offered by this simultaneous dual precursor method results in a reduction in the generation of false positive results as well as reduced data interpretation time.     

Novel glutathione conjugates of phenyl isocyanate identified by ultra‐performance liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance

Phenyl isocyanate is a highly reactive compound that is used as a reagent in organic synthesis and in the production of polyurethanes. The potential for extensive occupational exposure to this compound makes it important to elucidate its reactivity towards different nucleophiles and potential targets in the body. In vitro reactions between glutathione and phenyl isocyanate were studied. Three adducts of glutathione with phenyl isocyanate were identified using ultra‐performance liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR). Mass spectrometric data for these adducts have not previously been reported. Nucleophilic attack on phenyl isocyanate occurred via either the cysteinyl thiol group or the glutamic acid α‐amino group of glutathione. In addition, a double adduct was formed by the reaction of both these moieties. NMR analysis confirmed the proposed structure of the double adduct, which has not previously been described. These results suggest that phenyl isocyanate may react with free cysteines, the α‐amino group and also with lysine residues whose side chain contains a primary amine. Copyright © 2014 John Wiley & Sons, Ltd.     

A fragmentation study of isoflavones in negative electrospray ionization by MSn ion trap mass spectrometry and triple quadrupole mass spectrometry

This study has elucidated the fragmentation pathway for deprotonated isoflavones in electrospray ionization using MS(n) ion trap mass spectrometry and triple quadrupole mass spectrometry. Genistein-d(4) and daidzein-d(3) were used as references for the clarification of fragment structures. To confirm the relationship between precursor and product ions, some fragments were traced from MS(2) to MS(5). The previous literature for the structurally related flavones and flavanones located the loss of ketene (C(2)H(2)O) to ring C, whereas the present fragmentation study for isoflavones has shown that the loss of ketene occurs at ring A. In the further fragmentation of the [M-H-CH(3)](-*) radical anion of methoxylated isoflavones, loss of a hydrogen atom was commonly found. [M-H-CH(3)-CO-B-ring](-) is a characteristic fragment ion of glycitein and can be used to differentiate glycitein from its isomers. Neutral losses of CO and CO(2) were prominent in the fragmentation of deprotonated anions in ion trap mass spectrometry, whereas recyclization cleavage accounted for a very small proportion. In comparison with triple quadrupole mass spectrometry, ion trap MS(n) mass spectrometry has the advantage of better elucidation of the relationship between precursor and product ions.     

Deep‐ultraviolet laser ablation electrospray ionization mass spectrometry

A 193‐nm wavelength deep ultraviolet laser was used for ambient laser ablation electrospray ionization mass spectrometry of biological samples. A pulsed ArF excimer laser was used to ablate solid samples, and the resulting plume of the desorbed material merged with charged electrospray droplets to form ions that were detected with a quadrupole time‐of‐flight mass spectrometer. Solutions containing peptide and protein standards up to 66‐kDa molecular weight were deposited on a metal target, dried, and analyzed. No fragmentation was observed from peptides and proteins as well as from the more easily fragmented vitamin B₁₂ molecule. The mass spectra contained peaks from multiply charged ions that were identical to conventional electrospray. Deep UV laser ablation of tissue allowed detection of lipids from untreated tissue. The mechanism of ionization is postulated to involve absorption of laser energy by a fraction of the analyte molecules that act as a sacrificial matrix or by residual water in the sample.     

Studies on alkaloids binding to GC-rich human survivin promoter DNA using positive and negative ion electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding of 13 alkaloids to two GC-rich DNA duplexes which are critical sequences in human survivin promoter. Negative ion ESI-MS was first applied to screen the binding of the alkaloids to the duplexes. Six alkaloids (including berberine, jatrorrhizine, palmatine, reserpine, berbamine, and tetrandrine) show complexation with the target DNA sequences. Relative binding affinities were estimated from the negative ion ESI data, and the alkaloids show a binding preference to the duplex with higher GC content. Positive ion ESI mass spectra of the complexes were also recorded and compared with those obtained in negative ion mode. Only the 1 : 1 complex with berbamine was observed with lower abundance in the positive ion mass spectrum while complexes with the other alkaloids were absolutely absent. Collision-induced dissociation (CID) experiments indicate that the complexes with the protoberberine alkaloids (berberine, jatrorrhizine, and palmatine) dissociate via base loss and covalent cleavage. In contrast, product ion spectra of the complexes with the alkaloids reserpine, berbamine, and tetrandrine show the predominant loss of a neutral alkaloid molecule, accompanied by base loss and covalent cleavage to a lesser extent. A comparison of the gas-phase behaviors of complexes with the alkaloids to those with the traditional DNA binders has suggested an intercalative binding mode of these alkaloids to the target DNA duplexes.