HPLC-electrospray Mass Spectrometry Research Articles

Hepatic Metabolism of Sakuranetin and Its Modulating Effects on Cytochrome P450s and UDP-Glucuronosyltransferases.

Sakuranetin (SKN), found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug–herb interactions through the modulation of drug metabolizing enzymes (DMEs). HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT) 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP) 3A4 gene.

Non-targeted metabolomic analysis of orange (Citrus sinensis [L.] Osbeck) wild type and bud mutant fruits by direct analysis in real-time and HPLC-electrospray mass spectrometry

The direct analysis in real-time (DART) ion source and HPLC-electrospray mass spectrometry were applied in non-targeted metabolomic analyses of fruits of an orange bud mutant, ‘Hong Anliu’ along with its parental wild-type, ‘Anliu’. Fruits of the two isogenic cultivars were sampled at three different ripening stages, i.e. 120, 170 and 220 days after flowering. More than 130 metabolites were tentatively identified, including acids, sugars, flavonoids, alkaloids, limonoids, coumarins, amino acids, and plant hormones. Metabolomic analyses revealed that, compared to its wild type, the bud mutant fruit is characterized by higher levels of monosaccharides and disaccharides and lower levels of organic acids such as citric acid, malic acid and quinic acid, which agrees well with the anticipated fruit quality benefits of the mutation. In addition, many secondary metabolites, such as flavonoids, showed significant differences between the two genotypes, indicating that the whole fruit metabolome is significantly changed due to the bud mutation. This study provided a comprehensive assessment of metabolites in orange fruits, and revealed metabolomic differences in fruits between two isogenic orange genotypes. The results are helpful for understanding how the bud mutation in ‘Hong Anliu’ impacts the physiological and biochemical processes of orange fruits.

IDENTIFYING THE PLANT ORIGIN OF ARTISTS' YELLOW LAKE PIGMENTS BY ELECTROSPRAY MASS SPECTROMETRY

Dyes containing flavonoids are found in yellow lake pigments used as artists' materials. Pre-treating a sample of a flavonoid-based lake pigment with an acidic ion exchange resin followed by reversed-phase HPLC electrospray mass spectrometry enables the identification of key diagnostic yellow compounds, even when the pigment is bound in paint media. Reference raw plant materials of weld and buckthorn berries were also treated similarly for comparison with the lake pigments derived from these plants. This method is suitable for microsamples and thus suitable for analysis of samples that can be ethically removed from works of art in the course of their conservation. The flavonoid content is analysed and plant origins are suggested for yellow lake samples taken from Reclining Tiger by Jean-Baptiste Oudry and the 1893 version of The Scream by Edvard Munch.

Functional Ubiquitin Conjugates with Lysine-ε-Amino-Specific Linkage by Thioether Ligation of Cysteinyl-Ubiquitin Peptide Building Blocks

The modification of ubiquitin to defined oligo-ubiquitinated conjugates has received considerable interest due to the finding that isomeric oligo-ubiquitin conjugates exhibit distinct differences in their biochemical functions, depending on the specific lysine-epsilon-amino linkage used for conjugate formation. Here, we report the design and development of a thioether linkage-based approach for the synthesis of oligo-ubiquitin conjugates with lysine-specific branching by thioether ligation of a linear ubiquitin peptide containing a C-terminal cysteine residue as the "donor" component, with a corresponding lysine-epsilon-amino-branched haloacyl-activated ubiquitin "acceptor" peptide. This approach was successfully used for the synthesis of a lysine-63-linked diubiquitin conjugate by ligation of the modified ubiquitin(1-52)-Cys- donor peptide to the N-terminal Arg-54 residue of the branched Lys-63-linked acceptor peptide, ubiquitin(54-76)(2). Advantages of the present approach are as follows: (i) the conjugation reaction is performed in solution using suitable preformed donor ubiquitin peptides with a C-terminal Cys residue, and (ii) different corresponding N-chloroacetylated ubiquitin acceptor peptides containing the branched Lys residue are employed, providing broad applicability to the preparation of isomeric oligo-ubiquitin conjugates. The Lys-63-diubiquitin conjugate 7 described here was purified by semipreparative HPLC, and its structure and homogeneity ascertained by HPLC and high-resolution MALDI and electrospray-mass spectrometry. CD spectra and molecular modeling indicate a conformationally stable structure of the conjugate with spatial separation of the ubiquitin parts of the Lys-63 linkage. Moreover, the activity of the thioether-linked diubiquitin conjugate was ascertained by in vitro autoubiquitination assay. These results indicate the feasibility of this approach for the preparation of functional oligo-ubiquitin conjugates.

Solid-phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species

In the present paper a new extraction technique based on the combination of solid-phase/supercritical-fluid extraction (SPE/SFE) with subsequent reversed-phase HPLC is described. The SPE/SFE extractor was originally constructed from SPE-cartridge incorporated into the SFE extraction cell. Selected groups of benzoic acid derivatives ( p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acid), hydroxybenzaldehydes (4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde) and cinnamic acid derivatives ( o-coumaric, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acid) were extracted. Cyclic addition of binary extraction solvent system based on methanol:water (1:1, v/v) and methanol/ammonia aqueous solution was used for extraction at 40 MPa and 80 °C. The p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic and chlorogenic acid; 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde were identified by HPLC-electrospray mass spectrometry in SPE/SFE extracts of acid hydrolyzates of microalga ( Spongiochloris spongiosa) and cyanobacterial strains ( Spirulina platensis, Anabaena doliolum, Nostoc sp., and Cylindrospermum sp.). For the identification and quantification of the compounds the quasi-molecular ions [M−H] − and specific fragments were analysed by quadrupole mass spectrometry analyzer. Our analysis showed that the microalgae and cyanobacteria usually contained phenolic acids or aldehydes at μg levels per gram of lyophilized sample. The proposed SPE/SFE extraction method would be useful for the analysis of different plant species containing trace amount of polar fraction of phenols.

Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts Scientific Opinion of the Panel on Contaminants in the Food chain.

Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts Scientific Opinion of the Panel on Contaminants in the Food chain.

Analysis of flavonoids from propolis by on-line HPLC–electrospray mass spectrometry

In this paper, the qualitative and quantitative separation and determination of the polyphenolic component of propolis preparations in the form of ethanolic extract, usually used for commercial pharmaceutical preparations, has been investigated by means of on-line HPLC–ESI/MS technique. Propolis of different origin have been evaluated for their components and a specific fingerprint has been determined potentially useful for the quality control of extracts in pharmaceutical preparations. The ethanolic extracts of propolis from Argentina, Italy and Spain shows approximately the same total ion chromatogram (TIC) profile due to the presence of the same molecular species, identified by the negative ESI–MS. On the contrary, the samples from Azerbaijan, China, Ethiopia and Kenya show a very peculiar TIC profiles. By using many purified flavonoids and calibration curves over a wide concentration range, from 0.05 (5 μg/ml) to 5 μg (500 μg/ml), an accurate assessment of the contents of several bioactive compounds in extract samples was performed. The propolis from Argentina, Italy and Spain show a great amount of pinocembrin (approximately 49%, 48% and 39% of the total identified flavonoids, respectively) and variable but similar percentages of the other species. On the contrary, the propolis from China, Azerbaijan and Ethiopia have a great amount of pinocembrin (approximately 63%, 46% and 62%, respectively) but no presence of genistein, kaempferol, apigenin and chrysin for the sample from China, genistein, kaempferol, acacetin and chrysin for the propolis from Azerbaijan, and no kaempferol and acacetin for the sample from Ethiopia. The ethanolic extract from propolis of Kenya has no identified flavonoid species but just a peak possessing a m/ z of 253.0. Finally, an evaluation of the presence of total flavonoids for the various propolis samples was performed, with extracts from Argentina, Italy and Spain more rich in polyphenols than those from Azerbaijan, China, Ethiopia and Kenya. The HPLC–ESI/MS under the experimental conditions illustrated represents a valuable method for the qualitative and quantitative assay of the most relevant components of propolis. On-line HPLC–ESI/MS analysis constitutes an alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis polyphenolic components.

Sensitive quantification of omeprazole and its metabolites in human plasma by liquid chromatography–mass spectrometry

A sensitive method was developed for the simultaneous determination of omeprazole and its major metabolites 5-hydroxyomeprazole and omeprazole sulfone in human plasma by HPLC-electrospray mass spectrometry. Following liquid-liquid extraction HPLC separation was achieved on a ProntoSil AQ, C18 column using a gradient with 10 mM ammonium acetate in water (pH 7.25) and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH(+) ions, m/z 346 for omeprazole, m/z 362 for 5-hydroxy-omeprazole and omeprazol-sulfone and m/z 300 for the internal standard (2-{[(3,5-dimethylpyridine-2-yl)methyl]thio}-1H-benzimidazole-5-yl)methanol. The limit of quantification (LOQ) achieved with this method was 5 ng/ml for 5-hydroxyomeprazole and 10 ng/ml for omeprazole and omeprazole-sulfone using 0.25 ml of plasma. Intra- and inter-assay variability was below 11% over the whole concentration range from 5 to 250 ng/ml for 5-hydroxyomeprazol and from 10 to 750 ng/ml for omeprazole and omeprazole-sulfone. The method was successfully applied to the determination of pharmacokinetic parameters of esomeprazole and the two major metabolites after a single dose and under steady state conditions.

Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry

Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) is a species-common, immunogenic surface lipoprotein. In this study, the psaA gene was expressed as a nonfusion acylated protein in an Escherichia coli expression system. Yields of pure recombinant PsaA (rPsaA) were 8–10 mg/liter of fermentation culture. Analysis of rPsaA tryptic digests by HPLC–electrospray mass spectrometry (MS) confirmed 98% of the expected protein sequence. GC/MS data demonstrated very similar acylation of native and rPsaA by C12:0–C22:0 fatty acids, with C16 and C18 predominating. Negative ion electrospray MS/MS analysis of the rPsaA lipid anchor released by Pronase-E confirmed that the structure was based on an N-terminal palmitoylcysteine (Pam 3Cys). Electrospray MS heterogeneity analysis of intact rPsaA indicated that all of the observed heterogeneity could be accounted for by the fatty acid distributions. The availability of well-characterized rPsaA will facilitate the continued research and development of protein-based vaccines for the prevention of pneumococcal disease.

Liquid chromatographic separation of phosphoramidate diastereomers on a polysaccharide-type chiral stationary phase

To improve the therapeutic potential of anti-HIV nucleoside analogues (d4T, AZT, 3TC and ddI), the delivery of the corresponding monophosphate from neutral, membrane-permeable prodrugs has been realised by the synthesis of lipophilic phosphoramidate triester prodrugs, such as the simple phenyl- l-alaninephosphate derivatives. However, the present non-stereoselective synthesis results in a mixture of 1:1 diastereomers, which differ from the configuration of the phosphorus atom asymmetric center. Since each diastereomer may have different biological activity and pharmacokinetic profile, analytical methods have to be developed for their separation. This work aims at showing the ability of a polysaccharide-type chiral stationary phase to resolve such diastereomers in reversed-phase high-performance liquid chromatography. The influence of operating parameters has been studied to optimise the separation; a thermodynamic approach has also been investigated to gain an insight in the retention mechanism of the prodrugs. Preliminary validation study (linearity, accuracy, repeatability) has yielded good results; in addition, the feasibility of HPLC–electrospray-mass spectrometry (HPLC–ESI-MS) coupling has been demonstrated and it is expected that this will lead to lower detection limits.

Determination of fexofenadine in human plasma and urine by liquid chromatography–mass spectrometry

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC–electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C 18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 m M ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm×2.0 mm I.D., particle size 3 μm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH + ions, m/ z 502.3 for fexofenadine and m/ z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 μl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.

Growth hormone abuse in the horse: preliminary assessment of a mass spectrometric procedure for IGF-1 identification and quantitation.

Previous studies have shown that insulin-like growth factor 1 (IGF-1) is a promising marker for the detection of growth hormone (GH) abuse in the horse. The significant increases observed with GH administration in comparison to natural levels imply the possibility of setting a threshold level for IGF-1 that would be indicative of GH abuse. Although an immunoradiometric assay (IRMA) has been identified as a reliable screening method, a more specific IGF-1 quantification method needs to be developed for the prosecution of GH abuse by horseracing authorities. This study describes such an HPLC electrospray mass spectrometry (LC/ESI-MS) method that was developed and then assessed for the specific analysis of IGF-1 at the low levels encountered in serum. The structural identity of IGF-1 was confirmed by endoproteinase Asp-N digestion followed by LC/MS and LC/MS/MS characterisation. This was followed by quantification of IGF-1 as the intact molecule against an internal standard.

Kyanomycin, a complex of unusual anthracycline-phospholipid hybrids from Nonomuria species.

A blue secondary metabolite complex, named kyanomycin (2), in addition to epsilon-rhodomycinone (1), was detected in the mycelium extract of Nonomuria sp. NN 22303 by HPLC-diode array and HPLC-electrospray mass spectrometry screening. The chemical structures of the novel compounds were determined to be unusual anthracycline-phosphatidylethanolamine hybrids by spectroscopic and chemical degradation experiments.

Nano-electrospray and microbore liquid chromatography-ion trap mass spectrometry studies of copper complexation with MHC restricted peptides.

The formation of copper/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for major histocompatibility complex (MHC) class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Tandem mass spectrometry of copper/peptide complex ions provides peptide sequence information and insight into the peptide chelation sites. Copper associated y fragment ions dominate the product ion spectrum for non-histidine containing peptides, but both b and y copper complex ions were observed for the histidine containing MHC class I associated peptide gp70.

Carbohydrate moieties in human secretory component

Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide- N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc 2Hex 5HexNAc 4, Fuc 3Hex 5HexNAc 4, NeuAcFucHex 5HexNAc 4, NeuAcFuc 2Hex 5HexNAc 4, NeuAc 2Hex 5HexNAc4 and NeuAc 2FucHex 5HexNAc 4. Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed.

Study of temoporfin metabolism by HPLC and electrospray mass spectrometry.

The in vivo and in vitro metabolism of temoporfin (m-THPC), one of the most potent photosensitizers for the treatment of cancer by photodynamic therapy, has been studied in detail by HPLC with fluorescence and spectrophotometric detection and on-line HPLC-electrospray mass spectrometry. The results showed that temoporfin was not metabolized in vivo and was excreted unchanged via the biliary system into the faeces. No temoporfin or metabolites were detected in the urine. In vitro incubation of temoporfin with human and rat liver microsomal preparations in the presence of NADPH resulted in no metabolite production, even after enzyme induction with cytochrome P-450 isoenzyme inducers such as phenobarbitone, dexamethasone and 3-methylcholanthrene. No conjugation of temoporfin by phase II cytosolic enzymes was observed. It is concluded that the possible 'metabolites' previously observed were artifacts generated by photochemical oxidation of temoporfin to hydroxylated derivatives during the sample administration, collection, preparation and extraction procedures or were impurities already present in the original drug before administration for metabolic studies. These have been confirmed experimentally.

Study of temoporfin metabolism by HPLC and electrospray mass spectrometry

The in vivo and in vitro metabolism of temoporfin (m-THPC), one of the most potent photosensitizers for the treatment of cancer by photodynamic therapy, has been studied in detail by HPLC with fluorescence and spectrophotometric detection and on-line HPLC-electrospray mass spectrometry. The results showed that temoporfin was not metabolized in vivo and was excreted unchanged via the biliary system into the faeces. No temoporfin or metabolites were detected in the urine. In vitro incubation of temoporfin with human and rat liver microsomal preparations in the presence of NADPH resulted in no metabolite production, even after enzyme induction with cytochrome P-450 isoenzyme inducers such as phenobarbitone, dexamethasone and 3-methylcholanthrene. No conjugation of temoporfin by phase II cytosolic enzymes was observed. It is concluded that the possible ‘metabolites’ previously observed were artifacts generated by photochemical oxidation of temoporfin to hydroxylated derivatives during the sample administration, collection, preparation and extraction procedures or were impurities already present in the original drug before administration for metabolic studies. These have been confirmed experimentally. Copyright © 1999 John Wiley & Sons, Ltd. Abbreviations used: ES-MS, electrospray mass spectrometry; PDT, photodynamic therapy; m-THPC, temoporfin, 5, 10, 15, 20-tetra (m-hydroxyphenyl) chlorin.

Intracellular localization and metabolism of the phenanthridinium trypanocide, ethidium bromide, by isolated rat hepatocytes

1. Confocallaser scanning microscopy (CLSM) has shown that ethidium (3,8-diamino5-ethyl-6-phenylphenanthridinium) bromide, an aromatic phenanthridinium trypanocide, is taken up rapidly into the nucleoli and nuclear membranes of isolated rat hepatocytes. 2. It is biotransformed by the hepatocytes and at least five metabolites have been detected by high-performance liquid chromatography (HPLC). 3. Two new metabolites, 3- and 8-N-glucuronosylethidium, have been identified by HPLC-electrospray mass spectrometry and they represent the major pathway of metabolism, accounting for 6.4±0.7 and 19.5±1.2% respectively of total recovered drug after incubation. A third metabolite, 3,8-diacetylethidium, is formed in trace quantities. 4. The other two metabolites, 3-acetylethidium and 8-acetylethidium, have been reported previously.

Investigations by HPLC-electrospray mass spectrometry and NMR spectroscopy into the isomerisation of salinomycin.

HPLC-MS studies have indicated that certain polyether ionophore veterinary drugs are prone to degradation when stored as water-methanol solutions at ambient temperature. Salinomycin and narasin were particularly susceptible, disappearing completely within weeks to produce more polar species, which were identified as isomers of the original compounds. Lasalocid appeared to be stable under such conditions. Structural elucidation of the principal ultimate salinomycin isomerisation product was achieved by 2D NMR spectroscopy. This indicated that the isomerisation process consists of the opening of the spiro rings in the salinomycin structure with the concomitant formation of a furan moiety. The MS data indicated that the isomers retain the ability to complex alkali metal ions and may therefore retain their pharmacological activity. These discoveries may have implications both for the development of legislation covering acceptable levels of polyether ionophore residues in foodstuffs and also for analytical protocols designed to detect them.

A Simplified Gradient Solvent Delivery System for Capillary Liquid Chromatography–Electrospray Ionization Mass Spectrometry

We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column. The shape of the gradients formed can be predicted from a simple theoretical model. Gradients of different slope can be formed by varying either the size of the chamber or the system flow rate. The system is robust, reproducible, and simple to operate. We provide a detailed protocol of how to construct a low-cost capillary HPLC system consisting of two syringe pumps, a capillary mixing chamber, a capillary column, and a zero dead-volume microelectrospray interface. We demonstrate that the coupling of this HPLC system to a mass spectrometer enabled us to identify proteins at the low femtomole level in solution-phase digests and at the picomole level in digests of samples separated on SDS–PAGE gels. We believe that the strategy presented will be useful as a general method for the characterization of proteins and peptides by capillary HPLC–electrospray mass spectrometry.