Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Characterization of the aromatic composition of some liquid foods by nuclear magnetic resonance spectrometry and liquid chromatography with nuclear magnetic resonance and mass spectrometric detection

This paper describes the first application of NMR spectroscopy and LC-NMR/MS to the direct analysis of the aromatic composition of beer, grape juice and a wine phenolic extract. NMR spectroscopy provides non-invasive information on the overall aromatic profile and enables the identification of some compounds. However, a more comprehensive assignment is hindered by the low peak intensity and strong signal overlap in the low-field spectral region, as well as by the inherent lack of scalar coupling information for many aromatic compounds present. LC-NMR/MS can overcome these problems and is shown to aid significantly in the identification of aromatic compounds composing all samples analyzed. Some examples are the identification of several cinnamic acids (e.g. p-coumaric, trans-coutaric and trans-caftaric) in grape juice, the identification of 2-phenylethanol, tyrosol and tryptophol in beer and the detection of phenolics such as catechin, epicatechin, trans-resveratrol, tyrosol and caffeic acid in the wine extract.     

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.     

Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides

Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip‐based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C₁₈ LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C₁₈ chips. On the other hand, hydrophobic glycopeptides were better retained on C₁₈ chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1 pmol). The performance of both media also appeared comparable when analyzing a four‐protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural characterization of constituents with molecular diversity in fractions from Lysidice brevicalyx by liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance

A combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography (HPLC/ESI-MSn), and hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR), have been extensively utilized for on-line analysis of natural products, analyzing metabolite and drug impurity. In our last paper, we reported an on-line analytical method for structural identification of trace alkaloids in the same class. However, the structural types of the constituents in plants were various, such as flavanoids, terpenoids and steroids. It is important to establish an effective analytical method for on-line structural identification of constituents with molecular diversity in extracts of plants. So, in the present study, the fragmentation patterns of some isolated stilbenes, phloroglucinols and flavanoids from Lysidice rhodostegia were investigated by ESI-MSn. Their fragmentation rules and UV characteristics are summarized, and the relationship between the spectral characteristics, rules and the structures is described. According to the fragmentation rules, NMR and UV spectral characteristics, 24 constituents of different types in the fractions from L. brevicalyx of the same genus were structurally characterized on the basis of HPLC/HRMS, HPLC-UV/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Of these, six (10, 13, 14, 16, 17 and 23) are new compounds and all of them are reported from L. brevicalyx for the first time. The aim is to develop an effective analytical method for on-line structural identification of natural products with molecular diversity in plants, and to guide the rapid and direct isolation of novel compounds by chemical screening.     

Multi-residue liquid chromatography/tandem mass spectrometric analysis of beta-agonists in urine using molecular imprinted polymers

Ion suppression, a matrix effect that affects quantitative mass spectrometry, is one of the main problems encountered in liquid chromatography/tandem mass spectrometry. Two different clean-up steps for the multi-residue analysis of beta-agonists in urine were evaluated with respect to minimisation of ion suppression, namely, a mixed-phase solid phase extraction (SPE) column, i.e., clean screen Dau (CSD), and a molecular imprinted polymer (MIP) SPE column. Ion suppression experiments revealed that CSD sample clean-up can lead to false negative results for some beta-agonists, and that clean-up using MIP columns is more selective for beta-agonists than the use of CSD columns.     

Use of liquid chromatography/tandem mass spectrometric molecular fingerprinting for the rapid structural identification of pharmaceutical impurities

Use of liquid chromatography/tandem mass spectrometric (LC/MSⁿ) molecular fingerprinting is systematically demonstrated as a very effective tool for rapid structural elucidation of pharmaceutical impurities through a case study in which three isomers of betamethasone sodium phosphate (BSP) were rapidly identified as degradants formed due to the D‐homoannular ring expansion of the steroid core structure of BSP in the solid state. The rapid structural elucidation of these degradants was achieved by matching or closely matching the UV profiles, molecular weights, and more importantly the fragmentation patterns obtained from the LC/MSⁿ (n = 1 to 3) analysis of their enzyme‐catalyzed hydrolytic products, respectively, with those of a D‐homoannular isomer of betamethasone available in our LC/MSⁿ molecular fingerprint database. This strategy of using LC/MSⁿ molecular fingerprinting to obtain high‐confidence structures of unknown species is then validated by structure verification through one‐ (1D) and two‐dimensional (2D) nuclear magnetic resonance (NMR) experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a capillary high-performance liquid chromatography/electrospray tandem mass spectrometric method for the quantification of bisphenol A in air samples

Bisphenol A (BPA) is a toxic industrial chemical that affects the endocrine system even at low concentrations. A new method, based on capillary high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) analysis, has been developed to determine BPA in atmospheric samples. The method involves collection of air samples (typically 2 m(3)) on glass fiber filters, with ultrasonic extraction and sample concentration under vacuum before analysis. HPLC analysis was performed isocratically at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and MS/MS analysis in negative ion multiple reaction monitoring (MRM) mode, using BPA-d(16) as internal standard. The present method provides linear response in the range 0.007-3.5 microg/filter (R(2) > 0.999) and is characterized by high accuracy (mean bias 2%) and good reproducibility (mean RSD 5%). High sensitivity (LOD = 2 ng/m(3) based on 2 m(3) of air collected), specificity, and speed of the analysis make the present method suitable for routine determination of BPA in the atmosphere, both for ambient and personnel monitoring.     

On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tandem mass spectrometry,ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with liquid chromatographic micro-fractionation

In our continuing search for new antifungal agents of plant origin, the investigation of Erythrina vogelii Hook. f. (Leguminosae), a plant used in the traditional medicine of Ivory Coast to treat various infectious ailments, was undertaken. In order to rapidly identify the active principles, the crude extract was analysed by low-flow LC-1H nuclear magnetic resonance spectrometry (NMR) which gave a sensitive detection of all the main peaks. LC microfractionation was performed just after LC-NMR detection and all peaks collected were submitted to antifungal bioautography assays against Cladosporium cucumerinum. By this means, the antifungal activity could be efficiently linked to three of the LC peaks. In order to obtain complementary on-line structural information for all peaks of interest, high-resolution LC-MS-MS together with LC-UV with post-column addition of UV shifts reagents was undertaken on the crude extract. This chemical screening strategy with integrated antifungal bioassays has permitted the on-line identification of numerous constituents and has given useful information for an efficient peak-guided isolation procedure.     

Determination of alkylphenol and bisphenol A in beverages using liquid chromatography/electrospray ionization tandem mass spectrometry

A comprehensive analytical method based on liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) with negative ionization mode has been developed for measuring of alkylphenols and bisphenol A in beverage samples. Concentration and clean up of samples were performed on 200 mg OASIS HLB solid extraction cartridges. The effects of mobile phases and additives on ionization were assessed. The recoveries for each compound ranged from 76.7 to 96.9% and reproducibilities were represented as having relative standard deviation (R.S.D.) below 10%. The limits of quantification (LOQ) of the method under multiple-reaction monitoring (MRM) acquisition mode were 0.04, 0.03 and 0.2 ng L⁻¹ for 2 L of mineral drinking water and 2.0, 1.8 and 8.0 ng L⁻¹ for 50 mL of soda beverages.     

Multiple hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy, mass spectrometry and beyond

The advent of sensitive and reliable HPLC-NMR and HPLC-MS systems has revolutionised the identification of compounds eluting from chromatographic systems. More recently systems have been described wherein both NMR and MS are used together to provide an immensely powerful means of characterising compounds in chromatographic eluents. Here the construction and application of combined HPLC-NMR-MS systems to the analysis of mixtures of pharmaceuticals, drug metabolites in biological fluids and natural products in plant extracts is reviewed. In addition preliminary work with alternative systems such as HPLC-UV-NMR-FTIR-MS is highlighted and the prospects for such complex systems considered.     

Bioanalytical applications of 'fast chromatography' to high-throughput liquid chromatography/tandem mass spectrometric quantitation

A fast chromatographic separation approach that enables rapid method development for high-throughput sample quantification is discussed. This approach has been used to analyze samples from various biological matrices. Data are presented from in vivo pharmacokinetic studies (plasma) and in vitro drug metabolism and transport studies (hepatic microsomes, hepatocytes, and Caco-2 cells).     

Simultaneous identification and quantification of bisphenol A and 12 bisphenol analogues in environmental samples using precolumn derivatization and ultra high performance liquid chromatography with tandem mass spectrometry

A method for the identification and quantification of bisphenol A and 12 bisphenol analogues in river water and sediment samples combining liquid–liquid extraction, precolumn derivatization, and ultra high‐performance liquid chromatography coupled with tandem mass spectrometry was developed and validated. Analytes were extracted from the river water sample using a liquid–liquid extraction method. Dansyl chloride was selected as a derivatization reagent. Derivatization reaction conditions affecting production of the dansyl derivatives were tested and optimized. All the derivatized target compounds were well separated and eluted in 10 min. Dansyl chloride labeled compounds were analyzed using a high‐resolution mass spectrometer with electrospray ionization in the positive mode, and the results were confirmed and quantified in the parallel reaction monitoring mode. The method validation results showed a satisfactory level of sensitivity. Linearity was assessed using matrix‐matched standard calibration, and good correlation coefficients were obtained. The limits of quantification for the analytes ranged from 0.005 to 0.02 ng/mL in river water and from 0.15 to 0.80 ng/g in sediment. Good reproducibility of the method in terms of intra‐ and interday precision was achieved, yielding relative standard deviations of less than 10.1 and 11.6%, respectively. Finally, this method was successfully applied to the analysis of real samples.     

Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection

Four cation-exchange materials, possessing propanesulfonic acid ligands, for use in capillary electrochromatography were prepared from different commercially available 5-microm bare-silica particles ranging from 80 to 800 A in pore size. The performance of the materials was investigated at different compositions of the mobile phase (pH, ionic strength, and acetonitrile content) using tricyclic antidepressants and related quaternary ammonium analogues as test analytes. The wide-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave, as could be expected, the best selectivity.     

Derivatization reaction of the mycotoxin moniliformin with 1,2-diamino-4,5-dichlorobenzene: structure elucidation of an unexpected reaction product by liquid chromatography/tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance spectroscopy

The derivatization reaction of the mycotoxin moniliformin with 1,2-diamino-4,5-dichlorobenzene was previously introduced to improve distinctly the sensitivity of an assay applying high-performance liquid chromatography prior to fluorescence detection. In the course of the implementation of this derivatization approach into a liquid chromatographic/mass spectrometric method, an unexpected derivatization product has now been discovered by mass spectrometry. In order to elucidate its structure, detailed investigations with liquid chromatography/tandem mass spectrometry and liquid chromatography coupled on-line with NMR spectroscopy were performed. These studies give evidence for a heterocyclic structure that has been formed by the loss of one water and one carbon monoxide molecule. A reasonable mechanism for this derivatization reaction is proposed.     

Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis

The implementation of nanoflow liquid chromatography offers unique opportunities for automation of proteomics research. We demonstrate that automated nanoflow LC/MS/MS allowed the unambiguous identification of proteins from the omnipotent bacterium Shewanella putrefaciens, based on similarity searches against the completely determined genome of related microorganisms and against non-redundant databases. Total protein extracts were separated by 2-dimensional polyacrylamide electrophoresis. Only 1/20th of a tryptic digest mixture obtained from a single Coomassie Blue stained spot was loaded on the nanoflow LC column using a preconcentration/desalting step, and analyzed on-line on a hybrid quadrupole time-of-flight mass spectrometer with an automated MS-to-MS/MS switching protocol. This method allowed the de novo peptide sequence determination of several tryptic fragments and the identification of different proteins. CopyrightCopyright 2000 John Wiley & Sons, Ltd.     

Combined use of liquid chromatography with mass spectrometry and nuclear magnetic resonance for the identification of degradation compounds in an erythromycin formulation

A commercial erythromycin formulation containing erythromycin A (EA) as the major compound showed the presence of an unknown degradation compound that was co-eluted with erythromycin E (EE) in the European Pharmacopoeia (Ph. Eur.) liquid chromatographic (LC) method. The amount of the degradation compound increased with respect to time. To separate this unknown (UNK1), investigation was performed with different LC methods coupled to ultraviolet detection (LC-UV). With the present Ph. Eur. method, the degradation compound could not be well separated. However, with the most selective LC-UV method (XTerra method), two more degradation products (UNK2 and UNK3) were found in the formulation which could not be observed using other methods because of their poor separation. By combining the results obtained with LC-UV, LC/MS and LC/NMR, the degradation products were identified as pseudoerythromycin A hemiketal (PsEAHK), erythromycin A enol ether carboxylic acid and erythromycin C enol ether carboxylic acid. PsEAHK is known to be a base-catalysed degradation product of EA, whereas the other two degradation products were newly identified.