Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag⁺ after their separation by non‐aqueous reversed‐phase liquid chromatography (NARP‐LC) before MS to improve MS sensitivity. Electrospray ionization–MS (ESI‐MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved tuning and calibration in liquid chromatography/thermospray mass spectrometry using acetic acid cluster ions

Tuning and calibration of a thermospray ion source can cause rapid contamination of the source, and can have the disadvantage of introducing substances or solvents that are not used in the subsequent analytical work on the mass spectrometer. The use of aqueous 0.05 M ammonium acetate alone to produce acetic acid clusters for calibration in the mass range up to m/z 800 is reported. A low temperature of the vaporizer and the source block is used in order to maintain the clusters formed. The low temperature makes possible a very good signal-to-noise ratio, since the only ions formed are clusters of the general formula [(CH₃COOH)_m(NH₃)_n(H₂O)_pNH₄]⁺. Owing to the use of the ammonium acetate mobile phase, no interference from other sources was observed, e.g. clusters of solvents with ammonium acetate or impurities.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Automated screening of reversed‐phase stationary phases for small‐molecule separations using liquid chromatography with mass spectrometry

There are various reversed‐phase stationary phases that offer significant differences in selectivity and retention. To investigate different reversed‐phase stationary phases (aqueous stable C₁₈, biphenyl, pentafluorophenyl propyl, and polar‐embedded alkyl) in an automated fashion, commercial software and associated hardware for mobile phase and column selection were used in conjunction with liquid chromatography and a triple quadrupole mass spectrometer detector. A model analyte mixture was prepared using a combination of standards from varying classes of analytes (including drugs, drugs of abuse, amino acids, nicotine, and nicotine‐like compounds). Chromatographic results revealed diverse variations in selectivity and peak shape. Differences in the elution order of analytes on the polar‐embedded alkyl phase for several analytes showed distinct selectivity differences compared to the aqueous C₁₈ phase. The electron‐rich pentafluorophenyl propyl phase showed unique selectivity toward protonated amines. The biphenyl phase provided further changes in selectivity relative to C₁₈ with a methanolic phase, but it behaved very similarly to a C₁₈ when an acetonitrile‐based mobile phase was evaluated. This study shows the value of rapid column screening as an alternative to excessive mobile phase variation to obtain suitable chromatographic settings for analyte separation.     

Identification of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone by reversed‐phase high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A reversed‐phase high‐performance liquid chromatography/electrospray tandem mass spectrometry method was developed for the characterization of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone (C6‐HSL), a member of the N‐acylhomoserine lactone (AHL) class of microbial quorum‐sensing signaling molecules identified in many Gram‐negative strains of bacteria. Six products were identified: four with molecular weight (MW) of 213 and two with MW of 260. The characteristic product ions formed through collision‐induced dissociation (CID) provided diagnostic structural information. One of the photolysis products was determined to be N‐(3‐oxohexanoyl)homoserine lactone (3OC6‐HSL), a highly active quorum‐sensing signal, by comparison with a reference standard. Three structural isomers with the same mass as 3OC6‐HSL were identified as acyl side chain oxidized C6‐HSL (keto/enol functionalized) by accurate mass measurement and the structures of these products were proposed from CID spectral interpretation. Two structural isomers formed from concurrent oxidation and nitration of C6‐HSL were also observed and their structures were postulated based on CID spectra. In addition to the six hydroxyl radical oxidation products formed from the C6‐HSL precursor, five additional compounds generated from combined oxidation and lactonolysis of C6‐HSL were identified and structures were postulated. Copyright © 2009 John Wiley & Sons, Ltd.     

Proteomic profiling of human urinary proteome using nano-high performance liquid chromatography/electrospray ionization tandem mass spectrometry

Urine, a blood filtrate produced by the urinary system, is an ideal bio-sample and a rich source of biomarkers for diagnostic information. Many components in urine are useful in clinical diagnosis, and urinary proteins can be strong indication for many diseases such as proteinuria, kidney, bladder and urinary tract diseases. To enhance our understanding of urinary proteome, the urine proteins were prepared by different sample cleanup preparation methods and identified by nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry followed by peptide fragmentation pattern. The experimental results demonstrated that a total of 2283 peptides, corresponding to 311 unique proteins, were identified from human urine samples, in which 104 proteins with higher confidence levels. The present study was designed to establish optimal techniques to create a proteomic map of normal urinary proteins. Also, a discussion of novel approaches to urine protein cleanup and constituents is given.     

Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry

Fragmentation pathways of five iridoid glycosides have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). The first-stage MS data of the five iridoid glycosides were compared. The MS spectra showed that the adduct ions of iridoid glycosides and the formate anion were diagnostic ions to distinguish iridoid glycosides with a carboxyl group at the C-4 position or an ester group at the C-4 position. The MS fragmentation pathways of the five iridoid glycosides were also studied. Analyzing the product ion spectra of iridoid glycosides, some neutral losses were observed, such as H(2)O, CO(2) and glucose residues, which were very useful for the identification of the functional groups in the structures of iridoid glycosides. Furthermore, specific loss of one molecule of methyl 3-oxopropanoate or 3-oxopropanic acid was firstly discussed, which corresponded to the isomerization of the hemiacetal group in the structure of iridoid aglycone. According to the fragmentation mechanisms and HPLC/MS(n) data, the structures of five iridoid glycosides in a crude extract of Gardenia jasminoisdes fruit have been identified. Three compounds were compared with standards and the other two were identified as shanzhiside and genipin gentibioside by their MS(n) data without standard compounds. In order to further validate the veracity of the deduction, genipin gentiobioside was isolated from the extract of Gardenia jasminoisdes fruit using Purification Factory and was further identified by C- and H-NMR.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry

A phospholipid mixture extracted from cultured cells was directly analyzed by capillary (Cap) liquid chromatography (LC)/electrospray ionization (ESI) mass spectrometry (MS). Using a quadrupole mass spectrometer, we analyzed positive molecular ions, negative molecular ions, positive fragment ions and negative fragment ions under four different functions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map, in which the first dimension is elution time and the second dimension is mass, proved useful. Consequently, four different maps can be obtained by each of four different functions. Among them, from negative fragment ions at high cone voltage in the negative ion mode, ions that originated from acyl fatty acid and phosphorylcholine, phosphorylethanolamine and cyclic inositol phosphate can be detected at specific elution times. The map from positive fragment ions at high cone voltage in the positive ion mode indicated ions such as diradylglycerol and derivatives of 1-alkyl or 1-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine (PE), and phosphorylcholine from choline-containing phospholipids. The map produced from positive molecular ions indicated choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and PE. The map of negative molecular ions effectively indicated acidic phospholipids such as phosphatidylinositol. We were able to obtain more than 500 molecular species of phospholipids by this method within a few hours immediately after extraction from culture cells using a mixture of chloroform and methanol (2:1). In this context, we concluded that the combination of Cap-LC and ESIMS seems to be very effective in the analysis of phospholipid classes and their molecular species.     

Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies

The glycosylation profile of intact antibody due to the galactose and fucose heterogeneity in the N-linked sugars was determined with instrument resolution of 5000 and 10,000. After deconvolution of electrospray ionization mass spectra to complete convergence, several extra peaks appeared in addition to the peaks observed in the original mass spectra. The artificial peaks were avoided if deconvolution was stopped after a smaller number of iterations. A standard antibody was used as an external calibrant to minimize mass measurement errors during long-period experiments. Precision of four consecutive LC/MS measurements of the same antibody was 10 ppm (+/-1.5 Da). By using this approach, the masses of 11 intact antibodies were measured. All antibodies containing N-terminal glutamines had a negative mass shift due to the formation of pyroglutamate (-17 Da). Although the pyroglutamate variant of intact antibody was not resolved from the unmodified variant, this modification led to a mass shift proportional to the percentage of N-terminal pyroglutamate. By accurately measuring the mass shift we were able to quantify the abundance of pyroglutamic acid on intact antibodies. Mass accuracy in measuring different antibodies was below 30 ppm (+/-4 Da). The accurate mass measurement can be an effective tool for monitoring chemical degradations in therapeutic antibodies.     

Analysis of underivatized oligosaccharides by liquid chromatography/electrospray ionization tandem mass spectrometry with post‐column addition of formic acid

Underivatized oligosaccharides were analyzed by electrospray ionization (ESI) using a linear ion trap mass spectrometer in the negative ion mode with post‐column addition of an aqueous solution of formic acid. Under these conditions all oligosaccharides showed the presence of the corresponding formate adduct [M + HCOO]^? with high intensity and easy subsequent low‐energy collision‐induced dissociation (CID) fragmentation using successive MSⁿ experiments. A careful examination of the mass spectra obtained from these MSⁿ experiments pointed out some significant differences useful to identify and quantify the single components in mixtures of coeluted disaccharides. This new sensitive and rapid method was successfully applied to the quantification of oligosaccharides in some juices minimizing sample handling. Copyright © 2009 John Wiley & Sons, Ltd.     

Sensitive and simple analysis of sorbic acid using liquid chromatography with electrospray ionization tandem mass spectrometry

Sorbic acid (SA: CH(3)-CH=CH-CH=CH-COOH) and its salts are widely used as preservatives in foodstuff because of their growth inhibitory effects on mold, yeast and a wide range of bacteria. However, it is still unclear whether SA and its salts are actually incorporated in these organisms and a higher organisms like mammalian cells. Acidic compounds such as SA are usually analyzed by HPLC with eluents containing acetic acid, formic acid and their ammonium acetates, but such acidic buffers may suppress the ionization efficiency of the acidic compounds in negative-mode electrospray ionization (ESI). In this study, we present a sensitive and simple method for analysis of SA by HPLC with non-acidic solvents such as CH(3)CN/CH(3)OH-H(2)O by negative ion mode ESI-LC/MS. As a result, SA at less as 30 fmol was selectively determined by the selected reaction monitoring (SRM) mode. It was defined as the peak area with a signal-to-noise ratio (S/N) of 3. Good linearity was obtained in the range from 55 fmol (S/N 3) to 500 fmol (r(2)=0.9968) for SA by using LC/MS with the SRM mode. We also show that the method is useful to analyze SA level in the cytosol of mastocytoma cells, which were pretreated with SA. These results suggest the applicability of this method for the highly sensitive determination of SA in the mammalian tissues and cells.