Fast profiling of anthocyanins in wine by desorption nano-electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) mass spectrometry appears to be a useful technique applicable in different areas (e.g. analysis of pharmaceuticals, identification of biologically active compounds in tissues, imaging mass spectrometry). Its modification termed desorption nano-electrospray (nano-DESI) was tested for analysis of anthocyanins. Acidifying of samples and acidic spray liquid (methanol:water = 75:25 with 0.2% HCOOH) were essential for obtaining good quality spectra. Profiles of main anthocyanins in wine samples, two vintages (2005 and 2007) of three cultivars (Alibernet, Neronet and Rubinet), were successfully acquired. They were in agreement with results of LC/MS experiments (anthocyanins isolated by solid phase extraction were separated by μ-HPLC with gradient elution and detected by ESI-MS). Nano-DESI-MS data also allowed to determine ratio of two cultivars (Neronet and Rubinet) in their mixture and to detect coloring of wine by tenturier or elderberry extract. Detection of main anthocyanins in slices of wine grape, chokeberries and elderberries or in a wine stain on cotton fabric is also presented.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

On-line trapping/capillary hydrophilic-interaction liquid chromatography/mass spectrometry for sensitive determination of RNA modifications from human blood

With the developed on-line trapping/cHILIC/MS analytical platform, the detection limits of RNA modifications of m⁶A and 5-mC can reach to 0.06 fmol and 0.10 fmol. We then investigated the contents of m⁶A and 5-mC in human blood RNA from healthy persons at the age of 6-14 and 60-68 years. Our results showed that both m⁶A and 5-mC contents were significantly decreased in elder persons, suggesting the RNA modifications of m⁶A and 5-mC are correlated to aging.     

High-efficiency high performance liquid chromatographic analysis of red wine anthocyanins

The analysis of anthocyanins in natural products is of significant relevance in recent times due to the recognised health benefits associated with their consumption. In red grapes and wines in particular, anthocyanins are known to contribute important properties to the sensory (colour and taste), anti-oxidant- and ageing characteristics. However, the detailed investigation of the alteration of these compounds during wine ageing is hampered by the challenges associated with the separation of grape-derived anthocyanins and their derived products. High performance liquid chromatography (HPLC) is primarily used for this purpose, often in combination with mass spectrometric (MS) detection, although conventional HPLC methods provide incomplete resolution. We have previously demonstrated how on-column inter-conversion reactions are responsible for poor chromatographic efficiency in the HPLC analysis of anthocyanins, and how an increase in temperature and decrease in particle size may improve the chromatographic performance. In the current contribution an experimental configuration for the high efficiency analysis of anthocyanins is derived using the kinetic plot method (KPM). Further, it is shown how analysis under optimal conditions, in combination with MS detection, delivers much improved separation and identification of red wine anthocyanins and their derived products. This improved analytical performance holds promise for the in-depth investigation of these influential compounds in wine during ageing.     

Validation of a confirmatory method for the determination of melamine in egg by gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry

A sensitive and reliable method was developed and validated for detection and confirmation of melamine in egg based on gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Trichloroacetic acid solution was used for sample extraction and precipitation of proteins. The aqueous extracts were subjected to solid-phase extraction by mixed-mode reversed-phase/strong cation-exchange cartridges. Using ultra-performance liquid chromatography and electrospray ionization in the positive ion mode, melamine was determined by LC-MS/MS, which was completed in 5 min for each injection. For the GC-MS analysis, extracted melamine was derivatized with N,O-bis(trimethylsilyl)trifluoracetamide prior to selected ion monitoring detection in electron impact mode. The average recovery of melamine from fortified samples ranged from 85.2% to 103.2%, with coefficients of variation lower than 12%. The limit of detection obtained by GC-MS and UPLC-MS/MS was 10 and 5 μg kg⁻¹, respectively. This validated method was successfully applied to the determination of melamine in real samples from market.     

Electrophoresis-assisted open-tubular liquid chromatography/mass spectrometry for the analysis of lipooligosaccharide expressed by Campylobacter jejuni

Lipooligosaccharide (LOS) is the major component of the external membrane of Campylobacter jejuni. LOS contains a hydrophobic moiety, lipid A, and a hydrophilic moiety, oligosaccharide. Due to the unique mimicry of human ganglioside structures and potential involvement in the induction of the autoimmune polyneuropathies, Guillain-Barré and Miller Fisher syndromes, the structural characterization of C. jejuni LOS has received much attention. We have been using capillary zone electrophoresis-mass spectrometry to analyze O-deacylated LOS from C. jejuni. In an attempt to optimize the separation conditions, the effect of methanol on the separation of LOS was investigated. It was found that methanol resulted in stronger adsorption of LOS onto the wall of fused-silica capillary. In this paper, we applied this adsorption to perform electrophoresis-assisted open-tubular liquid chromatography electrospray mass spectrometry for the analysis of O-deacylated LOS mixtures from C. jejuni. The analytical potential of the proposed strategy for the analysis of O-deacylated LOS glycoforms from five bacterial colonies is demonstrated. Online tandem mass spectrometry is shown to provide a powerful tool for characterization of variations in the hexosamine backbone, phosphorylation of the lipid A, and sialic acid sequence information.     

Metabolomic profiling of rat serum associated with isoproterenol-induced myocardial infarction using ultra-performance liquid chromatography/time-of-flight mass spectrometry and multivariate analysis

An ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS)-based metabolomic approach was developed to characterize the metabolic profile associated with isoproterenol (ISO)-induced myocardial infarction (MI). Analysis of the serum samples revealed distinct changes in the biochemical patterns of ISO-induced rats. A multivariate statistical method, supervised partial least squares-discriminant analysis (PLS-DA), was then used for screening of potential biomarkers. As a result, 13 lipid biomarkers, including lysophosphatidylcholines (Lyso-PCs) and fatty acids were identified by the accurate mass measurement of TOF-MS. The relationship between abnormal lipid metabolism and the formation of MI were also studied. This work demonstrates the utility of UPLC/TOF-MS-based metabolic profiling combined with multivariate analysis as a powerful tool to further investigate the pathogenesis of cardiovascular diseases.     

Development of a broad toxicological screening technique for urine using ultra-performance liquid chromatography and time-of-flight mass spectrometry

Withdrawal of the support for the REMEDi HS drug profiling system has necessitated its replacement within our laboratories with an alternative broad toxicological screening technique. To this end, a novel method, based on ultra-performance liquid chromatography (UPLC) and time-of-flight (TOF) mass spectrometry, was developed for the routine analysis of urine samples. Identification was achieved by comparison of acquired data to libraries containing more than 300 common drugs and metabolites, and was based on a combination of retention time, exact mass and fragmentation patterns. Validation data for the method is presented and comprised an evaluation of the following parameters: precision; transferability of the methodology between the six collaborating laboratories; specificity; extraction recovery and stability of processed samples; matrix effects and sensitivity.This paper presents the benefits of supplementary fragmentation data with particular regard to increasing specificity and confidence of identification and its usefulness with overdosed samples. The utility of the method was assessed by the parallel analysis of 30 authentic urine samples using the REMEDi HS and UPLC-TOF. The latter provided enhanced detection, leading to the identification of twice as many drugs. Furthermore it did not miss any compounds that were identified by REMEDi HS. The UPLC-TOF findings were further verified by a combination of data from three other conventional screening techniques, i.e., GC-MS, HPLC-DAD and UPLC-MS/MS.     

Characterization of low-molecular weight peptides in champagne wine by liquid chromatography/tandem mass spectrometry

This paper reports the use of an on-line LC–ESI–MS/MS method for the identification and quantification of di- and tripeptides in champagne wine without laborious sample pretreatment. The identification of these compounds, in their underivatised form, is based on identical retention times and ESI–MS spectra to those of reference standards. The presence of nine dipeptides (Arg–Ile, Ile–Arg, Ile–Val, Lys–Phe, Lys–Tyr, Phe–Lys, Tyr–Gln, Tyr–Lys, Val–Ile) and the absence of two tripeptides (Phe–Arg–Arg and Lys–Met–Asn) have been evidenced in the matrix. Calibration curves for each analyte were established using Phe–Arg as internal standard. The calibration curves were linear in the concentration range 0.1–10 mg L⁻¹ with a determination coefficient, r², better than 0.992. The accuracy for the calibration standard was estimated at between 92 and 102%. This method allows high recovery and satisfies the necessary requirements with respect to accuracy, repeatability and sensitivity. The first application of this analytical method to the measurement of di- and tri-peptides in different vintages of champagne wine is reported. Compositional changes in the peptides occurred depending on the vintage.     

Quantitative liquid chromatography/time-of-flight mass spectrometry

Over the last decade, time-of-flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals.     

Characterization of mixtures of biocidal oligoguanidines by capillary electrophoresis and high-performance liquid chromatography coupled to mass spectrometry

The polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) leads to various types of oligomeric guanidines exhibiting a broad spectrum of biocidal activities. In the present work a reversed-phase high-performance liquid chromatographic method with a gradient consisting of aqueous 0.1% trifluoroacetic acid and acetonitrile as mobile phase has been developed to separate these oligoguanidines according to type and chain length. The combination with electrospray mass spectrometry allowed the identification of the various compounds. By this technique, some structures already suggested in the literature could be confirmed, and several additional oligoguanidines not yet reported could be identified. As a complementary technique, capillary zone electrophoresis was investigated. Best results were obtained with carrier electrolytes consisting of phosphoric acid in water/acetonitrile mixtures. Although the number of peaks that could be separated by the electrophoretic method was considerably lower than in case of the chromatographic method, capillary electrophoresis in combination with UV detection at 195 nm may still be a fast method suitable for quantitation of some of the major compounds and for monitoring the reaction rate during the polycondensation reaction.     

Open tubular column supercritical fluid chromatography/mass spectrometry on a benchtop mass spectrometer

A benchtop SFC/MS system is described which utilizes supercritical carbon dioxide in a 50 micron diameter open tubular column interfaced directly to an unchanged commercially available benchtop capillary GC/MS system equipped with a chemical ionization (CI) source. A small amount of methane reagent gas was admitted co-axially to a capillary restrictor at the exit of the capillary chromatographic column. This make-up gas served as the CI reagent gas and appeared to optimize the sensitivity of the system while providing abundant (M+1) ions for the analytes investigated in this study. Good chromatographic intergrity was obtained for the GC/MS test compound, decafluorotriphenylphosphine (DFTPP), but the capillary restrictor appeared to cause some tailing of the ion current profiles resulting from low nanogram levels of caffeine and some fatty acid esters. Improvements in the SFC/MS capillary restrictor interface and the pumping system of the benchtop GC/MS system should increase the capability of this system for future applications.     

Analysis of proteoglycans derived sulphated disaccharides by liquid chromatography/mass spectrometry

A method has been developed for the identification and quantitative determination of sulphated disaccharides derived from chondroitin sulphate (CS) and dermatan sulphate (DS) chains attached to proteoglycans (PGs). After digestion with Chondroitinase ABC, the pool of disaccharides can be directly separated by liquid chromatography on a porous graphitized carbon (PGC) column and identified by on-line electrospray mass spectrometry under negative ionization conditions. The relative intensities of the fragment ions obtained by MS/MS allow to distinguish the sulphate position. Calibration with standard disaccharides allows the quantification of the different isomers. The method showed good repeatability in terms of relative standard deviation (RSD < 2%) and linearity between 0.5 and 50 ng (total injected amount) for both 4- and 6-sulphated disaccharides. The limit of detection achieved in full scan mode was 0.1 ng. The methodology was applied to different types of biological samples obtained from patients suffering from chronic lung inflammation such as: lung tissue, bronchoalveolar lavage fluid (BALF), induced sputum and urine.     

Comparison of gas and liquid chromatography coupled to mass spectrometry for the residue analysis of pesticides in organges

Summary Liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure chemical ionization (APCI), and gas chromatography-mass spectrometry (GC-MS) with electron impact ionization (EI), are compared for the determination of eight pesticides in oranges. Seven of the selected pesticides, chlorpyriphos, chlorpyriphos-methyl, imazalil, α and β-endosulfan, endosulfan sulphate and dicofol, are commonly determined by GC whereas one, thiabendazole, can only be directly determined by LC. Primary ions [M-H]⁻ or [M-Cl+O]⁻ are obtained using LC-APCI-MS in negative ionization (NI) mode. In contrast, a high degree of fragmentation is reported with GC-MS. Both techniques were applied to oranges, which had been previously extracted with ethyl acetate and anhydrous sodium sulphate. The data indicate equivalent detection limits that range from 0.01 to 0.1 mgkg⁻¹ and similar degree of specificity. Mean recoveries ranged from 82% for α-endosulfant to 96% for imazalil, with relative standard deviation ranging from 7 to 17%.     

Rapid screening for anthocyanins and anthocyanin dimers in crude grape extracts by high performance liquid chromatography coupled with diode array detection and tandem mass spectrometry

A rapid and efficient method using high performance liquid chromatography coupled with diode array detection and tandem mass spectrometry (HPLC-DAD–MS/MS) for fast screening large numbers of anthocyanins and anthocyanin dimers in different grape skin extracts, without further sample clean-up procedures, was developed. A good separation of most detected anthocyanins was achieved in a run time of 15 min. Identification of anthocyanin pigments required a combination of several information: UV–vis spectra, MS and MS/MS spectra, and elution pattern. Many compounds have been here detected for the first time and their structures tentatively elucidated.     

Simultaneous analysis of anthocyanins and flavonols in petals of lotus (Nelumbo) cultivars by high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry

A fast and reliable HPLC method for the simultaneous separation of anthocyanins and flavonols in lotus petals was developed based on the study of four candidate solvent systems. Fifteen flavonoids were identified by high-performance liquid chromatography with photodiode array detection/mass spectrometry. Among them, two anthocyanins and nine flavonols were discovered in lotus petals for the first time. This work is valuable for both the hybrid breeding on lotus oriented to flower color and the utilization of lotus petals as functional food materials.     

Ultra-performance liquid chromatography-tandem mass spectrometry: a novel challenge in multiresidue pesticide analysis in food

Potential of ultra-performance liquid chromatography (UPLC) separation strategy coupled with tandem (in space) mass spectrometric detection (MS/MS) in multiresidue pesticide analysis was critically assessed. Performance parameters such as number of theoretical plates, height of theoretical plate, peak symmetry and peak capacity were measured/calculated on the basis of data generated by analysis of apple extracts containing 17 (semi)polar pesticides representing various classes of active ingredients of widely used crop protective preparations. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) procedure provided improved chromatographic parameters resulting in significantly increased sample throughput including lower solvent consumption and lower limits of quantitation (LOQs) for most of target analytes compared to common method employing conventional high-performance liquid chromatography (HPLC) separation.     

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.     

Open-access liquid chromatography/mass spectrometry in a drug discovery environment

The use of open-access mass spectrometry to monitor synthetic chemistry reactions, and also the integrity and purity of new chemical entities, has been a part of the medicinal chemist's tool-box for more than 5 years. Originally in our group at Wyeth Research there were two open-access methods available to the chemists, flow injection analysis (FIA) and liquid chromatography/mass spectrometry (LC/MS). The FIA method was approximately 3 min long, while the LC/MS method was approximately 20 min long (including an 8 min gradient). Within the first 2 years, the total number of open-access analyses increased by approximately 125%. It is interesting, however, that the number of LC/MS analyses increased by more than 285%. This is attributed to the fact that the chemists began using the LC/MS data to monitor reactions and also to check final product integrity and purity. In addition, the number of chemists performing parallel synthesis reactions has increased; thus, individual chemists can produce sample sets of up to 100 vials. This paper describes the implementation of new methodology, which accommodates the need for much faster run times and also the ability to acquire alternating positive and negative ion spectra within the same run. In addition, the instrument has been configured to e-mail the resulting processed data report to the submitting chemist. Several methods have been developed, including structure elucidation using in-source collision-induced dissociation (CID) and night-time analysis. The LC/MS methods for this system are described herein and are applicable to both industrial and academic synthetic chemistry optimization efforts.