Atmospheric pressure chemical ionisation normal phase liquid chromatography mass spectrometry and tandem mass spectrometry of chlorophyll a allomers

Four oxidation products formed during the allomerisation reaction of chlorophyll a in methanol have been separated and identified using normal phase high-performance liquid chromatography (NP-HPLC) coupled with atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry (MS/MS) and photodiode array detection. Fragmentation pathways of the main stereoisomers of the four major products, Mg-purpurin-7 dimethyl phytyl ester, 15(1)-MeO-lact-chl a, 13(2)-MeO-chl a and 13(2)-HO-chl a, have been assigned using APCI-MS(n) aided by examination of deuteriated counterparts of the purpurin and the lactone. Diagnostic fragmentations have been determined, including those for Mg-purpurin-7 dimethyl phytyl ester and 15(1)-MeO-lact-chl a, which display very similar full MS and MS/MS spectra. The use of NP-HPLC offers a rapid and efficient method which, combined with MS/MS, reduces reliance on precursor ion mass for assignment of structure in the allomerisation reaction where major and minor components are often isobaric.     

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time‐consuming processes. In this study, a novel multiple‐stage tandem mass spectrometric method (MSⁿ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β‐lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data‐dependent LC/MSⁿ screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks?). The proposed data‐dependent LC/MSⁿ method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling. Copyright © 2010 John Wiley & Sons, Ltd.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry

Articular cartilage contains both chondrocyte cells and extracellular matrix (ECM) components. Currently, comprehensive information concerning the protein composition of human articular cartilage tissue is somewhat lacking. In this report we detail the use of tandem mass spectrometry (MS/MS) for a preliminary global identification of proteins from human articular knee cartilage tissue from patients diagnosed with osteoarthritis. Knee cartilage supernatant was fractionated using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), in-gel digested and peptide sequences were then determined by performing on-line nano-liquid chromatography (LC)/MS/MS experiments using an ion trap mass spectrometer. Altogether, over 100 different proteins from nearly 700 unique peptide sequences were detected by MS/MS. The majority of the proteins identified are involved in ECM organization (35%), signal transduction and cell communication (14%), immune response (11%) and metabolism and energy pathways (11%). Proteins observed included several well-known cartilage components as well as lower abundant lesser known ECM proteins. Possible degradation products in the cartilage sample, such as from cartilage link protein, could also be detected by our mass spectrometry methods. We show here that mass spectrometry can be utilized as a tool for a fast, accurate and sensitive analysis of a complex mixture of cartilage proteins. It is believed that this type of proteomic analysis will aid future work centered on investigating the pathology of this and other related joint diseases.     

Proteome analysis in the bovine adrenal medulla using liquid chromatography with tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS)-based proteomics has been used to identify soluble proteins in the bovine adrenal medulla. This gland is a major source of hormones, opioids, neurotransmitters, and several vital proteins. The adrenal medulla proteins were first purified using ammonium sulfate precipitation. The resulting proteins were then pre-fractionated with a C-4 high-performance liquid chromatography (HPLC) column. Each 2-min HPLC fraction was digested with trypsin, and separated further and analyzed using capillary liquid chromatography/tandem mass spectrometry (capLC/nanospray-MS/MS) to map the proteome of the adrenal medulla. The parent mass and sequence ion information thus obtained for tryptic peptides was used to search the NCBInr database using the SEQUEST search engine. A total of 195 proteins were identified, of which 71 had good scores (delta correlation value greater than 0.1, preliminary score above 200, and cross-correlation value above 2.5). The prominent proteins thus identified are secretogranin I precursor, chromogranin A, proenkephalin A precursor, myosin X, hemoglobin beta chain, hemoglobin alpha chain, heat shock protein 10 kDa, and replicase.     

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.     

Structural identification of isomers present in technical perfluorooctane sulfonate by tandem mass spectrometry

The structures of isomeric by-products of technical perfluorooctane sulfonate (PFOS, C8F17SO3-) were characterized by high-performance liquid chromatography coupled to mass spectrometry. Ion trap (IT) as well as triple quadrupole (TQ) tandem mass spectrometry (MS/MS) identified ten PFOS isomers with best results being achieved by ion trap MS/MS. In addition to the linear isomer, the main by-products were isomers with geminal diperfluoromethyl groups and perfluoromethyl-substituted compounds. Purified fractions of the perfluoromethyl isomers allowed the elucidation of structures and the assignment of the retention times of perfluoroisopropyl-, 5-perfluoromethyl-, 4-perfluoromethyl-, 3-perfluoromethyl- and 1-perfluoromethyl-PFOS. The concentration of 2-perfluoromethyl-PFOS was too low in technical PFOS for a proper identification. Retention times of the geminal substituted diperfluoromethyl isomers could not be properly assigned due to co-elutions. The number and position of the CF3 groups can be derived from the MS/MS spectra, which allow an unequivocal identification of the isomers. TQ-MS/MS also allowed us to identify most of the isomers.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Investigation of unknown related substances in commercial erythromycin samples with liquid chromatography/mass spectrometry

A selective reversed phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of erythromycin impurities and related substances in commercial erythromycin samples. Mass spectral data are acquired on a LCQ ion trap mass spectrometer equipped with an electrospray interface operated in positive ion mode. The LCQ is ideally suited for identification of impurities and related substances because it provides on-line LC/MS(n) capability. Compared with UV detection, this hyphenated LC/MS(n) technique provides as a main advantage efficient identification of novel substances without time-consuming isolation and purification procedures. Using this method four novel related substances were identified in commercial samples.     

High-resolution liquid chromatography/electrospray ionization time-of-flight mass spectrometry combined with liquid chromatography/electrospray ionization tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber

Grape antioxidant dietary fiber (GADF) is a dietary supplement that combines the benefits of both fiber and antioxidants that help prevent cancer and cardiovascular diseases. The antioxidant polyphenolic components in GADF probably help prevent cancer in the digestive tract, where they are bioavailable. Mass spectrometry coupled to liquid chromatography is a powerful tool for the analysis of complex plant derivatives such as GADF. We use a combination of MS techniques, namely liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) on a triple quadrupole, for the identification of the polyphenolic constituents of the soluble fraction of GADF. First, we separated the mixture into four fractions which were tested for phenolic constituents using the TOF system in the full scan mode. The high sensitivity and resolution of the TOF detector over the triple quadrupole facilitate the preliminary characterization of the fractions. Then we used LC/ESI-MS/MS to identify the individual phenols through MS/MS experiments (product ion scan, neutral loss scan, precursor ion scan). Finally, most of the identities were unequivocally confirmed by accurate mass measurements on the TOF spectrometer. LC/ESI-TOF-MS combined with MS/MS correctly identifies the bioactive polyphenolic components from the soluble fraction of GADF. High-resolution TOF-MS is particularly useful for identifying the structure of compounds with the same LC/ESI-MS/MS fragmentation patterns.     

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.     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Versatility of tandem mass spectrometry for focused analysis of oxylipids

Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) in the multiple reaction monitoring (MRM) scan mode has been the primary MS method applied for the target identification of specific and minor oxylipids in complex matrices, such as eicosanoids and docosanoids, which are potent lipid mediators derived from polyunsaturated fatty acid oxygenation. However, the high specificity of MRM can limit the detection of species with m/z MRM transitions not covered by the method. In addition to MRM, tandem‐quadrupole mass analyzers enable other experiments to be conducted, by fragmenting ions via collision‐induced dissociation process (CID). This paper presents the potential of tandem mass spectrometry for the focused analysis of oxylipids. We have successfully developed an LC‐MS/MS method for the identification of precursor ions of m/z 115, a diagnostic product ion of 5‐hydroxy‐ and 5‐epoxy‐fatty acids. As a proof of concept, the developed method was used to discover several oxylipids oxidized at C5 derived from arachidonic acid (C20 : 4) oxygenation in a hypothalamus rat extract that were not identified using the target MRM methodology. The proposed focused MS/MS‐based approach in a tandem mass analyzer has proven to be a powerful strategy to accelerate the identification of oxylipids with structural similarities and assist the field of lipidomic research. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural analysis of monoterpene glycosides extracted from Paeonia lactiflora Pall. using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Paeoniflorin standard was first investigated by electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) using a sustained off-resonance irradiation (SORI) collision-induced dissociation (CID) method at high mass resolution. The experimental results demonstrated that the unambiguous elemental composition of product ions can be obtained at high mass resolution. Comparing MS/MS spectra and the experimental methods of hydrogen and deuterium exchange, the logical fragmentation pathways of paeoniflorin have been proposed. Then, the extracts of the traditional Chinese medicine Paeonia lactiflora Pall. were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). By comparison with the ESI-FTICR-MS/MS data of paeoniflorin, the isomers paeoniflorin and albiflorin in Paeonia lactiflora Pall. have been identified using HPLC/MS with CID in an ion trap and in-source CID. Furthermore, using the characteristic fragmentation pathways, the retention times (t(R)) in HPLC and MS/MS spectra, the structures of three other kinds of monoterpene glycoside compounds have been identified on-line without time-consuming isolation. Thus an HPLC/ESI-MS method for the analysis of constituents in Paeonia lactiflora Pall. has been established.     

Liquid chromatography–tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co‐eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H]⁺ ions at m/z at 833 and/or m/z 835, co‐eluted with uroporphyrin I and uroporphyrinogen I by LC‐MS/MS and could be wrongly identified as uroporphomethenes. Copyright © 2013 John Wiley & Sons, Ltd.     

Fragmentation pathways of heroin-related alkaloids revealed by ion trap and quadrupole time-of-flight tandem mass spectrometry

The electrospray ionization (ESI) ion trap and quadrupole time-of-flight (QqToF) mass spectra of heroin and seven related alkaloids, i.e., morphine, codeine, O-6-monoacetylmorphine (6-MAM), thebaine, acetylcodeine, papaverine and narcotine, have been extensively investigated in this work. The ESI mass spectrometric fragmentation pathways of protonated 6-MAM, heroin, acetylcodeine, and thebaine were comprehensively elucidated for the first time with the aid of high-resolution mass spectrometry. It was found that cleavage of the piperidine ring was the featured fragmentation route of six of the compounds, although not of papaverine and narcotine. In addition, a simple high-performance liquid chromatography (HPLC)-based separation method gave baseline resolution of all eight components. This study could play an important role in the screening for these alkaloids in different matrices by HPLC coupled to tandem mass spectrometry (MS/MS).     

Analyses of Stemona alkaloids in Stemona tuberosa by liquid chromatography/tandem mass spectrometry

Alkaloid profiles in Stemona tuberosa were found to be highly variable. Six Stemona alkaloids isolated from the plant were subjected to on-line high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses. Their fragmentation patterns and products were useful for their characterization. The LC/MS fingerprints of these alkaloids, though variable among samples, could provide an overall characterization of the authenticity and quality of this species and help to differentiate it from S. japonica and S. sessilifolia, as all three species are recognized as genuine sources of the herb Radix Stemonae in the Pharmacopoeia of the People's Republic of China.     

Screening for anabolic steroids and related compounds in illegal cocktails by liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

Findings of illegal hormone preparations such as syringes, bottles, cocktails, and so on, are an important information source for the nature of the current abuse of anabolic steroids and related compounds as growth-promoting agents in cattle. A new screening method for steroids in cocktails is presented based on liquid chromatography (LC) with diode-array UV-absorbance detection and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS). Accurate mass measurements were performed at a mass resolution of 4000 using continuous introduction of a lock mass through a second (electro)sprayer. Similar experiments were carried out using dual-sprayer quadrupole time-of-flight mass spectrometry (ESI-QTOFMS/MS) at a mass resolution of 10 000 with data-dependent MS/MS acquisition; i.e. beyond an intensity threshold for the [M + H](+) ions, MS/MS spectra were automatically acquired at three different collision energies. Elemental compositions were calculated for precursor and product ions and it is shown that the combined information from LC retention behavior, UV spectra, elemental compositions, and accurate mass MS/MS spectra yield a fast impression of the steroids present in the complex mixture. Using a new software tool for structure elucidation of MS/MS spectra, an additional non-steroidal additive was identified as well.     

Analysis of curcuminoids by positive and negative electrospray ionization and tandem mass spectrometry

The curcuminoids are a group of diarylheptanoid molecules that possess important pharmacological activities, particularly acting as anti-inflammatory agents. The main purpose of this study was to investigate the fragmentation behavior of the three major curcuminoids in ion trap liquid chromatography/tandem mass spectrometry (LC/MS/MS). Both positive and negative mode electrospray ionization in tandem and multidimensional MS(n) experiments in quadrupole ion trap instruments and high-resolution and accurate mass MS and sustained off-resonance irradiation (SORI) MS/MS experiments in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer were used to elucidate the main fragmentation channels of these compounds. These experiments yielded essentially the same fragmentation results in both ion trap and ICR instruments for all three curcuminoids and for their phenolic monoacetates. Major and diagnostic fragment ions were identified and their origins are proposed.     

Rapid structural determination of amides in Piper longum by high-performance liquid chromatography combined with ion trap mass spectrometry

A reversed-phase liquid chromatography/tandem multistage mass spectrometry (MS/MS) method was developed for the characterization of amides from the extracts of Piper longum. The characteristic fragmentations of the amides found in P. longum showed diagnostic structural information. Extracted ion chromatography (EIC) and constant neutral losses were used to guide the search for the amides of interest. Amides of known structures that contain four subtypes of amides were rapidly determined, and novel amides were also identified for this plant. Forty-two amides were rapidly identified, of which 22 were found in this plant for the first time and 9 were new compounds. The method is convenient and sensitive, especially for minor components in the unpurified, complex mixture; the structures of unknown constituents could be determined, in the absence of authentic sample, by comparison of the fragmentation patterns with those of homologous compounds.