Precursor Ion Analysis Research Articles

Unsupervised Structural Classification of Dissolved Organic Matter Based on Fragmentation Pathways.

Dissolved organic matter (DOM) is considered an essential component of the Earth's ecological and biogeochemical processes. Structural information of DOM components at the molecular level remains one of the most extraordinary analytical challenges. Advances in determination of chemical formulas from the molecular studies of DOM have provided limited indications on structural signatures and potential reaction pathways. In this work, we extend the structural characterization of a wetland DOM sample using precursor and fragment molecular ions obtained by a sequential electrospray ionization-Fourier transform-ion cyclotron resonance tandem mass spectrometry (ESI-FT-ICR CASI-CID MS/MS) approach. The DOM chemical complexity resulted in near 900 precursors (P) and 24 000 fragment (F) molecular ions over a small m/z 261-477 range. The DOM structural content was dissected into families of structurally connected precursors based on neutral mass loss patterns (Pn-1 + F1:n + C) across the two-dimensional (2D) MS/MS space. This workflow identified over 1900 structural families of DOM compounds based on a precursor and neutral loss (H2O, CH4O, and CO2). The inspection of structural families showed a high degree of isomeric content (numerous identical fragmentation pathways), not discriminable with sole precursor ion analysis. The connectivity map of structural families allows for the visualization of potential biogeochemical processes that DOM undergoes throughout its lifetime. This study illustrates that integrating effective computational tools on a comprehensive high-resolution mass fragmentation strategy further enables the DOM structural characterization.

Center-of-Mass iso-Energetic Collision-Induced Decomposition in Tandem Triple Quadrupole Mass Spectrometry.

Two scan modes of the triple quadrupole tandem mass spectrometer, namely Collision Induced Dissociation Precursor Ion scan and Neutral Loss scan, allow selectively pinpointing, in a complex mixture, compounds that feature specific chemical groups, which yield characteristic fragment ions or are lost as distinctive neutral fragments. This feature of the triple quadrupole tandem mass spectrometer allows the non-target screening of mixtures for classes of components. The effective (center-of-mass) energy to achieve specific fragmentation depends on the inter-quadrupole voltage (laboratory-frame collision energy) and on the masses of the precursor molecular ion and of the collision gas, through a non-linear relationship. Thus, in a class of homologous compounds, precursor ions activated at the same laboratory-frame collision energy face different center-of-mass collision energy, and therefore the same fragmentation channel operates with different degrees of efficiency. This article reports a linear equation to calculate the laboratory-frame collision energy necessary to operate Collision-Induced Dissociation at the same center-of-mass energy on closely related compounds with different molecular mass. A routine triple quadrupole tandem mass spectrometer can operate this novel feature (iso-energetic collision-induced dissociation scan; i-CID) to analyze mixtures of endogenous metabolites by Precursor Ion and Neutral Loss scans. The latter experiment also entails the hitherto unprecedented synchronized scanning of all three quadrupoles of the triple quadrupole tandem mass spectrometer. To exemplify the application of this technique, this article shows two proof-of-principle approaches to the determination of biological mixtures, one by Precursor Ion analysis on alpha amino acid derivatized with a popular chromophore, and the other on modified nucleosides with a Neutral Fragment Loss scan.

Liquid chromatography – quadrupole Orbitrap mass spectrometry method for selected pharmaceuticals in water samples

This study developed an analytical approach for sub-ppb level detection and confirmation of 13 pharmaceuticals and personal care products (PPCPs) in water samples using ultra high pressure liquid chromatography hyphenated with a quadrupole Orbitrap mass spectrometer (UHPLC- Q-Orbitrap-MS). Sample preparation was performed by using solid phase extraction (SPE) employing hydrophilic-lipophilic balance cartridges, with elution of sorbed analytes using methanol. Acceptable automatic gain control (AGC) target and maximum injection time (IT) were 1×106 and 200ms, respectively, resulting in a mass accuracy <2ppm. High response signals with sufficient data points per peaks (20–30) were obtained whilst maintaining high resolution of approximately 70,000 full width at half maximum. Extracted ion chromatograms provided quantitative analysis with linearity (R2) ranging from 0.9875 to 0.9993 and method detection limits ranging from 0.01–0.61ngmL−1. Compounds were further analysed by MS/MS analysis, with the MS operated in parallel reaction monitoring (PRM) mode under precursor ion analysis intervals and collision energies chosen for the different PPCPs. The developed method was applied to analyse water samples obtained from sources in Victoria, Australia.

Rapid screening and determination of nerve agent metabolites in human urine by LC-MS/MS

Qualitative screening procedures have been developed for the rapid detection and identification of the metabolites of nerve agents in the urine samples and extracts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The combination of negative electrospray ionization (ESI) using a C18 column and water-methanol mobile phase modified with ammonium formate provides a rapid screening procedure for nerve agent degradation products with limit of detection of 1 ng/mL in the precursor-ion analysis. Also, determination of the alkyl methylphosphonic acids was carried out by the SRM scan mode with the limit of detection of 0.1 ng/mL. These procedures will be applicable to the trace analysis of metabolites of nerve agents in human urine matrices in the Organisation for the Prohibition of Chemical Weapons (OPCW) proficiency test.

Phosphorylcholine-containing lipid molecular species profiling in biological tissue using a fast HPLC/QqQ-MS method

A fast reversed-phase liquid chromatography-electrospray ionization triple quadrupole mass spectrometry method was developed for the molecular species profiling of glycerophosphocholine (GPC) and sphingomyelin (SM) in total lipid extracts. A two-stage mass spectrometry strategy was adopted to analyze in detail the composition of lipid molecular species. Precursor ion analysis was first conducted to obtain the preliminary composition profile of the phosphorylcholine-containing lipid. The product ion spectra were sequentially acquired for each recorded signal to determine the molecular structure of the lipid. A total of 150 GPCs and 12 SMs were identified in the fetal mouse lung with relative amounts ranging from 13.7% to less than 0.002% (normalizing by the total signal response). A column packed with core-shell particles was used to obtain excellent chromatographic separation with a shorter time demand in a conventional high-performance liquid chromatography system. Considering the compromise between the chromatographic efficiency and the electrospray signal response, the optimization of the mobile phase improves the chromatographic plate number to approximately 40,000 and the detection limits to less than 0.001 mg/L. The applicability of the method was validated through a study of chemically induced early lung maturation. The metabolic alteration in the fetal mouse lung was clearly reflected in the GPC and SM composition with several characteristics of the molecular structure that related to the character of the phospholipid layer upon the epithelial lining of alveoli and the relevant cell function. The results indicated that this analytical strategy is reliable for comprehensive molecular species profiling of GPC and SM and might be extended to the analysis of other phospholipids.

Study of anthocyanic profiles of twenty-one hybrid grape varieties by liquid chromatography and precursor-ion mass spectrometry

The anthocyanins of 21 hybrid red varieties produced by crossing V. vinifera, V. riparia, V. labrusca, V. lincecumii and V. rupestris species, the profiles for which have not yet been reported, were studied. Profiles were determined by LC/DAD, and identification of single anthocyanins was confirmed by LC/MS precursor-ion analysis. Anthocyanidin precursors (pelargonidin at m/z 271, dephinidin at m/z 303, cyanidin at m/z 287, petunidin at m/z 317, peonidin at m/z 301, and malvidin at m/z 331) and precursors of monoglucoside compounds allowed 24 different compounds to be identified. Analysis of precursor ions of monoglucoside anthocyanins at low capillary voltage revealed the signals of diglucosides only, providing a very selective method for analysis of diglucoside anthocyanins in grape. According to anthocyanin profile, the samples were subdivided into two groups: one characterized by the substantial presence of diglucoside compounds (particularly Seyve Villard 23-399 and Seyve Villard 23-369) and one by the scarce presence or practical absence of diglucosides (Seibel 10878, Burdin 4077, and Galibert 238-35). Particularly interesting for producing anthocyanin for the natural colorant industry were the varieties Siebel 8357, Bacò 30-12 and Terzi 100-31.

Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.

Selective analysis of phenolic compounds in propolis by HPLC‐MS/MS

Phenolic compounds (flavonoids and phenolic acid derivatives) are major active constituents of the resinous fraction of propolis, and also represent its allergenic principles. We have developed a chromatography electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) method to characterise the polyphenolic fraction of propolis rapidly and quali-quantitatively. With precursor ion scanning, selective detection of caffeic esters was easily achieved, confirming the identification of prenyl caffeate, benzyl caffeate and phenylethyl caffeate by comparison with synthetic standards. The ionisation and fragmentation behaviour of the major propolis flavonoids was rationalised and applied to selected real samples. Taken together, the results of this study show that the introduction of precursor ion analysis leads to a significant improvement in the characterisation of the phenolic fraction of propolis, paving the way to the establishment of a better quality control for this important natural remedy.

Direct Analysis of Pharmaceutical Drug Formulations Using Ion Mobility Spectrometry/Quadrupole-Time-of-Flight Mass Spectrometry Combined with Desorption Electrospray Ionization

A novel approach to the rapid analysis of pharmaceutical drug formulations using hyphenated ion mobility spectrometry (IMS) and time-of-flight mass spectrometry (ToF-MS) that requires no sample pretreatment or chromatographic separation is described. A modified quadrupole time-of-flight mass spectrometer containing an ion mobility drift cell was used for gas-phase electrophoretic separation of ions prior to ToF-MS detection. The generation of sample ions directly from tablets and cream formulations was effected by desorption electrospray ionization (DESI) using a modified electrospray ion source. The analysis of a range of over-the-counter and prescription tablet formulations is described, including histamine H2 receptor antagonist (ranitidine), analgesic (paracetamol), opiate (codeine), and aromatase inhibitor anticancer (anastrozole) drugs. The successful determination of active drugs from soft formulations, such as an antiseptic cream (chlorhexidine) and a nicotine-containing skin patch, is also presented. Limits of detection for the active drugs using the DESI/IMS/ToF-MS method fell within the high-picomole to nanomole range. In all cases, the use of ion mobility drift tube separation showed increased selectivity for active drug responses (present as low as 0.14% w/w) over excipient responses such as poly(ethylene glycol). Tandem mass spectrometric analysis of precursor ions separated by IMS allowed positive confirmation of active drugs with little loss of ion mobility efficiency. The ability to analyze hard or soft pharmaceutical formulations directly by DESI combined with ion mobility spectrometry/mass spectrometry in approximately 2 min demonstrates the potential applicability of this novel method to pharmaceutical screening of low-molecular-weight drug formulations with high selectivity over the formulation vehicle.

Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring

A systematic method for anthocyanin identification using tandems mass spectrometry (MS/MS) coupled to high-performance liquid chromatography (HPLC) with photo-diode array detection (PDA) was developed. Scan for the precursor ions of commonly found anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin, and peonidin) using LC/MS/MS on a triple quadrupole instrument allows for the specific determination of each category of anthocyanins. Further characterization of each anthocyanin was performed using MS/MS product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring (SRM). The method was demonstrated for analysis of anthocyanins in black raspberries, red raspberries, highbush blueberries, and grapes ( Vitis vinifera). Previous reported anthocyanins in black raspberries and red raspberries are confirmed and characterized. Common-neutral-loss analysis allows for the distinction of anthocyanin glucosides or galactoside and arabinosides in highbush blueberries. Separation and identification of anthocyanin glucosides and galactosides were achieved by LC/MS/MS using SRM. Anthocyanin isomers such as cyanidin sophoroside and 3,5-diglucoside were differentiated by their fragmentation pattern during product-ion analysis. Fifteen anthocyanins (all possible combinations of five anthocyanidins and three sugars) were characterized in highbush blueberries. Pelargonidin 3-glucoside and pelargonidin 3,5-diglucoside were detected and characterized for the first time in grapes. The present approach allows mass spectrometry to be used as a highly selective detector for rapid identification and characterization of anthocyanins and can be used as a sensitive procedure for screening anthocyanins in fruits and vegetables.

Probing Anthocyanin Profiles in Purple Sweet Potato Cell Line (Ipomoea batatas L. Cv. Ayamurasaki) by High-Performance Liquid Chromatography and Electrospray Ionization Tandem Mass Spectrometry

A purple line cell line (PL) generated from the storage root of purple-fleshed sweet potato (Ipomoea batatas L.) cv. Ayamurasaki produces a complex mixture of anthocyanins, and seven major anthocyanins have been isolated and identified to date. All these anthocyanins are exclusively cyanidin or peonidin 3-sophoroside-5-glucosides and their acylated derivatives. High-performance liquid chromatography (HPLC) coupled to photodiode array (PDA) detection and electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole instrument was employed to further investigate the anthocyanin composition of the PL extract. Precursor-ion analysis, product-ion analysis, and selected reaction monitoring (SRM) MS/MS experiments were conducted sequentially to screen and characterize anthocyanins in the aqueous extract of the PL cell line. Precursor-ion analysis specifically detected the molecular cations of each category of anthocyanins by scanning the precursors of anthocyanidins (cyanidin, peonidin, and pelargonidin). The detected molecular cation of each anthocyanin was fragmented using product-ion analysis by collisionally activated dissociation (CAD). MS/MS using SRM detection was conducted to further confirm the fragmentation observed during product-ion analysis. In comparison to the commonly used product-ion analysis technique, the combined use of precursor-ion analysis, product-ion analysis, and SRM is particularly useful for positive identification of anthocyanins in complex matrixes and provides important information to confirm the proposed structures. Twenty-six anthocyanins were detected and characterized in the aqueous extract of the PL cell line. Several anthocyanins, including two pelargonidin derivatives, were tentatively identified for the first time in these cells.

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.

Characterization of a complex mixture of ceramides by fast atom bombardment and precursor and fragment analysis tandem mass spectrometry

Fast atom bombardment, precursor and fragment ion analysis mass spectrometry were employed to characterize the fatty acid and long-chain base composition of a complex ceramide mixture prepared from beef brain lipids. Precursor ion analysis of a sphingenine-specific fragment allowed selective recognition of ceramides with different fatty acids, some of which escaped undetected in the source spectra owing to background contamination of the FAB matrix. The use of a triple quadrupole yielded far better results than linked scanning on a sector mass spectrometer, owing to unit mass resolution of the precursor ions, which allows discrimination of saturated from unsaturated acids with the same number of carbon atoms. The composition of the mixture of linked fatty acid was derived from the intensity of the molecular species both in the source and in the precursor spectra, and was compared with that obtained by capillary gas chromatography of the released fatty acid methyl esters. Some discrepancy between the values obtained with the two techniques can be attributed to very different surface activity of the short- and long-chain fatty acids. Analysis of the fragment ions following collision-induced decomposition of cationized species allowed localization of the position of the double bond in a ceramide containing a C-24:1 fatty acyl chain.