Separation and identification of peptide mixtures in a synthesis crude of carbetocin by liquid chromatography/electrospray ionization mass spectrometry

In order to separate and characterize the target peptide and the side-product peptide compounds of a synthesis crude of the peptide hormone carbetocin, liquid chromatography coupled to high-flow electrospray ionization mass spectrometry (LC/eS-MS) has been used. Carbetocin is an important drug with recognized therapeutical application for stimulation of uterine contractions to facilitate parturition. Stepwise solid phase peptide synthesis (SPPS) commonly results in unwanted side products associated with incomplete peptide chains. Consequently, this procedure requires extensive purification and characterization of the final synthesis crude. The linear solvation energy relationship (LSER) method has been applied to optimize the proportion of organic modifier of the mobile phase used in the established LC method. On the other hand, ES-MS has allowed rapid and reliable identification of the target peptide and the other impurities present in the carbetocin synthesis products.     

Separation of potentially therapeutic peptide hormones by liquid chromatography. Optimisation of the composition and pH of the mobile phase

The aim of this work is to optimise the proportion of the organic modifier and the pH of the mobile phase, in order to separate a series of peptide hormones with therapeutic interest in the molecular mass range from 500 to 6000. The composition of the mobile phase was optimised by establishing relationships between retention parameters and either the scale of solvent polarity, or the Kamlet–Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships. Likewise, linear correlations between the chromatographic retention and Reichardt’s E^N_T parameter were obtained. These relationships allowed an important reduction of the experimental retention data needed for developing a given separation. In addition, a model describing the effect of the correctly measured pH of the mobile phase on retention in LC was established and tested for the series of selected peptides using an octadecylsilica column. The proposed equations permit the prediction of the optimum pH and also permit the determination of the acidity constants of the peptides in the hydro-organic mixtures using a minimum number of measurements.     

Separation and characterization of underivatized oligosaccharides using liquid chromatography and liquid chromatography-electrospray ionization mass spectrometry

Native cyclodextrin-based columns are particularly useful for the analysis of oligosaccharides because the retention of these carbohydrates is based mainly on the hydrogen bonding interactions of oligosaccharide hydroxyl groups with the stationary phase. Thus, the retention time predictably increases with the number of analyte hydroxyl groups, which corresponds to the elongation of the oligosaccharide chain. High-performance liquid chromatography (HPLC) coupled to electrospray ionization (ESI) mass spectrometry (MS) was used for the separation and characterization of underivatized oligosaccharide mixtures. With the limits of detection as low as 50 pg, all individual components of oligosaccharide mixtures (up to 11 glucose units long) were baseline resolved on a Cyclobond I 2000 column and detected using ESI-MS. Low flow rates and narrow I.D. columns increase the ESI-MS sensitivity significantly. The method showed potential usefulness for the sensitive and quick analysis of hydrolysis products of polysaccharides, and for trace levels of individual oligosaccharide or oligosaccharide isomers from biological systems.     

Capillary high-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic columns and carbon fiber electrospray ionization emitters

Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based monolithic column, which was prepared by photo-polymerization of octylstyrene (OS) and divinylbenzene (DVB) were compared with that of the methacrylate-based monolithic column composed of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA). The sample loading ability of tryptic digested protein in poly-OS (POS)-DVB column was higher than that of poly-LMA (PLMA)-EDMA column, possibly due to the irregular and rugluous surface offering a greater surface area of POS-DVB stationary phase. The POS-DVB column also provided better separation efficiency in the separation of high concentration (10 microg) of tryptic digested albumin bovine serum (BSA). Due to the successful interface of a highly efficient monolithic column and a stable, durable carbon fiber emitter, low femtomole levels of peptides were successfully separated and identified in the presence of large amounts of tryptic digested protein.     

Separation and characterization of complex crude mixtures produced in the synthesis of therapeutic peptide hormones by liquid chromatography coupled to electrospray mass spectrometry (LC-ES-MS)

Native peptides and peptidomimetics can be synthesized in a routine way by rapid and efficient procedures. However, the final products always result in complex mixtures, in which the target peptide is contaminated with undesired side products and other impurities. Thus, it is imperative to develop analytical methods for the evaluation of the target peptide’s purity in order to obtain an effective, safe and legal pharmaceutical product. LC-ES-MS is used here in order to separate and characterize the side-products associated with several synthetic hormones with therapeutic interest: carbetocin, eledoisin, leuprolide, goserelin and triptorelin. General directions for LC-ES-MS analysis of the synthetic peptide mixtures are established. Mass information obtained offers a significant advantage for the purity assessment of therapeutic hormones and gives a key tool to enhance their process of synthesis.     

High-performance liquid chromatography-electrospray ionization mass spectrometry and multiple mass spectrometry studies of hyperforin degradation products

The alcoholic extract of the aerial parts of Hypericum perforatum L. finds wide application because of its antidepressant activity. The extract contains a number of constituents with documented biological activity including chlorogenic acid, a broad range of flavonoids, naphthodianthrones and phloroglucinols. Hyperforin and adhyperforin are the major phloroglucinol constituents found in the lipophilic fraction of the extracts. Since the stability of hyperforin has been shown to be limited, an investigation of the hyperforin degradation products using HPLC-electrospray ionization mass spectrometry and multiple mass spectrometry was undertaken.     

Liquid chromatography-electrospray mass spectrometry of multicomponent peptide mixtures. Characterization of a mixture from the synthesis of the hormone goserelin

In order to separate and characterize the target peptide and the side-product peptide compounds of a synthesis mixture of the peptide hormone goserelin, liquid chromatography coupled to high-flow electrospray ionization mass spectrometry (LC-ES-MS) has been used. Goserelin is an important drug with recognized therapeutical application for palliative treatment of prostatic and breast carcinomas. Stepwise solid-phase peptide synthesis commonly results in unwanted side-products associated with incomplete peptide chains. Consequently, this procedure requires extensive purification and characterization of the final synthesis mixture. The method of linear solvation energy relationships has been applied to optimize the proportion of organic modifier of the mobile phase used in the established LC method. On the other hand, ES-MS has allowed rapid and reliable identification of the target peptide and the other impurities present in the goserelin synthesis products.     

Quantitative analysis of caffeic acid phenethyl ester in crude propolis by liquid chromatography-electrospray ionization mass spectrometry

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antinflammatory, and immunomodulatory properties. The paper describes a rapid and simple liquid chromatography-electrospray ionisation mass spectrometry method for qualitative and quantitative determination of CAPE. The chromatographic separation was performed with a Luna RP-C18 column using a water-acetonitrile linear gradient. The method was linear over a 0.125-80 ng/mL range (LOD = 62.5 pg/mL). The method was applied for the quantitation of caffeic acid phenethyl ester in crude propolis samples, which were analysed directly after extraction with ethyl acetate solution.     

Determination of the antibiotic chloramphenicol in meat and seafood products by liquid chromatography-electrospray ionization tandem mass spectrometry

A confirmatory method based on isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry is described for the determination of the antibiotic chloramphenicol (CAP) in foods. The method is quantitative and entails liquid-liquid extraction followed by a clean-up step on a silica gel solid-phase extraction cartridge. Mass spectral acquisition is done in the negative ion mode applying multiple reaction monitoring of two diagnostic transition reactions for CAP (m/z 321 --> 257 and m/z 321--> 152). In addition, the presence of two chlorine atoms in the CAP molecule provides further analyte certainty by assessing the 37Cl/35Cl ratio using the transition reactions m/z 323 --> 257 and m/z 323 --> 152. Validation of the method in chicken meat is conducted according to the latest European Union criteria for the analysis of veterinary drug residues at levels of 0.05, 0.10, and 0.20 microg/kg, employing [2H5]-chloramphenicol as internal standard. The decision limit and the detection capability were calculated at 0.01 microg/kg and 0.02 microg/kg, respectively. At the lowest fortification level (i.e. 0.05 microg/kg), precision values below 14 and 17% were achieved under repeatability and within-laboratory reproducibility conditions, respectively. The accuracy of the method was within 20, 15, and 5% of the target values at the 0.05, 0.10, and 0.20 microg/kg fortification levels, respectively. The applicability of this procedure was demonstrated by the analysis of other meat (turkey, pork, beef) and seafood (fish, shrimps) products. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.     

Determination of tetrodotoxin in puffer-fish by liquid chromatography-electrospray ionization mass spectrometry

A simple and reliable method using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for the determination of tetrodotoxin in the puffer-fish has been developed. The LC separation was performed on a Shodex RSpak NN-414 column (15 cm x 4.6 mm id) using 20 mM ammonium acetate-methanol (75 + 25) as the mobile phase at a flow rate of 0.5 ml min(-1). The positive ionization produced the typical [M + H]+ molecular ion of tetrodotoxin (m/z 320). The calibration graph for tetrodotoxin was rectilinear from 0.01 to 1 microg ml(-1) with selected ion monitoring (SIM). Tetrodotoxin was extracted with 0.1% acetic acid by heating in a boiling water-bath and the extracts were cleaned up on a Bond Elut C18 (500 mg) cartridge. The recoveries of the tetrodotoxin from the puffer-fish fortified at 1 microg g(-1) were 77.7-80.7% and the detection limit was 0.1 microg g(-1) (equivalent to ca. 0.5 mouse units per gram).     

Energetic heterogeneity of the surface of a molecularly imprinted polymer studied by high-performance liquid chromatography

A sequential combination of reversed-phase liquid chromatography–mass spectrometry (LC–MS) and capillary electrophoresis (CE) has been explored in order to perform separation and characterization of a multicomponent peptide mixture from the synthesis of leuprolide. The mixture was first analyzed and fractionated by LC–MS, and the collected fractions were subsequently separated by CE. Unambiguous identification of the electrophoretic peaks was achieved by injecting the collected fractions separately and spiking the leuprolide crude mixture. Furthermore, structural information about the components of the mixture provided by several semi-empirical migration models has been used to check the accuracy of the structures previously proposed by LC–MS. Combination of the two orthogonal techniques results in an enhancement of their individual selectivity characteristics.     

Automated mass analysis of low-molecular-mass bacterial proteome by liquid chromatography-electrospray ionization mass spectrometry

Due to the presence of a large number of proteins in cell extracts, ion chromatograms of cell extracts obtained by electrospray ionization mass spectrometry (ESI-MS) can be quite complicated. It is found that the elevated baseline in an ion chromatogram contains many protein signals. One deficiency of current commercially available LC-ESI-MS data interpretation software is found to be the lack of functional operation that allows automated mass spectral integration and interpretation over signals hidden in the baseline. This current limitation can be overcome by a technique that involves the introduction of artificial pulses to an ion chromatogram by removing the solvent mixer in the HPLC pump. These artificial pulses are treated as chromatographic peaks by the software, thereby allowing automated spectral integration over the duration of a pulse. The reliability of mass analysis from the integrated spectra is shown to be dependent on spectral interpretation parameters such as mass spectral baseline threshold. The application of this method is demonstrated for rapid detection and mass analysis of low-molecular-mass proteins from cell extracts of Escherichia coli or Bacillus globigii.     

Separation and detection of oxidation products of fluorodeoxyglucose and glucose by high-performance liquid chromatography-electrospray ionisation mass spectrometry

2-Deoxy-2-[18F]fluoro-D-glucose ([18F] FDG), the most popular positron emitting radiopharmaceutical, may oxidise by autoradiolysis in aqueous solution. The aim of this work was to use LC-MS for determination of the oxidation products of fluorodeoxyglucose and glucose (Glc) obtained by oxidation with Fenton's reagent. Asahipak NH2P-50 polyamide silica column and acetonitrile-0.025% aqueous ammonium formate (80:20 (v/v)) eluent were utilised with an Agilent 1100 HPLC-MS instrument. Ten major oxidation products of FDG and Glc were separated and identified by mass spectrometry: 2-fluorogluconic acid, 2-fluoroglucuronic acid, 2-oxoerythronic acid, arabinose, arabonic acid, araburonic acid, erythrose, erythrulose, gluconic acid, and glucuronic acid. The most intensive electrospray ionisation signals were found in the negative ion spectra and were due to HCOO- adducts, the other acids being in their lactone forms.     

Determination of pyrrolizidine alkaloids in comfrey by liquid chromatography-electrospray ionization mass spectrometry

Symphytum officinale L. (comfrey) is a medicinal plant commonly used in decoctions and aliments. Besides therapeutic bioactive compounds present in the herb, it is found to contain hepatotoxic pyrrolizidine alkaloids (PAs), such as lycopsamine and others. In the present study, PAs such as lycopsamine, echimidine and lasiocarpine were determined using electrospray liquid chromatography-mass spectrometry (LC-MS) with the method precision (relative standard deviation, RSD) <10%. Detection of lycopsamine, symviridine and their N-oxides could be confirmed with a newly developed method based on HPLC ion-trap and orbitrap MS with electrospray ionization interface. With LC-MS, quantitative analysis of lycopsamine in the botanical extract was carried out. The effect of extraction solvent was optimized by sonication and methanol: H₂O (50:50) was selected. Then a rapid method based on pressurized hot water extraction (PHWE) was employed for the extraction of lycopsamine from comfrey followed by the comparison with heating under reflux with the RSD ranging from 2.49% to 19.32%. Our results showed a higher extraction efficiency for heating under reflux compared with PHWE. It was proposed that the lower extraction efficiency for PHWE was attributable to dissolved nitrogen from air which caused the reduction in the solubility of lycopsamine in the compressed hot solvent. In this study, quantitative analysis of PAs in comfrey was demonstrated. In addition, it was found that the use of subcritical water for extractions depended on the physical properties of the dissolved solutes and their tendency to degrade under the chosen extraction conditions.     

Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.     

Determination of abamectin in citrus fruits by liquid chromatography-electrospray ionization mass spectrometry

Liquid chromatography coupled to electrospray mass spectrometry (LC-ES-MS) with positive ion detection was used to determine abamectin in oranges. MS conditions were optimized to achieve maximum sensitivity. The main ion for abamectin was [M+Na]+ at a fragmentor voltage of 180 V. Abundant structural information can be obtained at different fragmentor voltages. The detection limit for the standard solution was 12 pg injected, and good linearity and reproducibility were observed. Abamectin residues were extracted using matrix solid-phase dispersion. Orange samples were homogenized with C18 bonded silica placed onto a glass column and eluted with dichloromethane. Recoveries of the abamectin from oranges fortified with approximately 0.01-10 mg/kg ranged from 94 to 99% with an overall average recovery of 96%. The quantification limit is 0.0025 mg/kg, which means detection limit for this analyte could be set at a few hundred picograms per gram of fruit. The presence in the electrosprayed solution of numerous citrus constituents did not interfere significantly with the ionization process of abamectin. The assay procedure provides a simple, rapid, and sensitive method for monitoring residues in oranges. The method was applied to field treatment orange samples.     

Application of liquid chromatography-electrospray ionization tandem mass spectrometry to the detection of 10 sulfonamides in honey

Liquid chromatography (LC) in combination with tandem mass spectrometry (MS-MS) has been applied to the separation and detection of 10 different sulfonamides in honey. The methodology encompasses a simple hydrolysis of the honey sample to liberate sugar-bound sulfonamides followed by liquid-liquid extraction of the 10 analytes, filtration, and analysis by LC-MS-MS. Conditions for reversed-phase LC and electrospray ionization (ESI) MS-MS in the positive ion mode were optimized for the 10 compounds under study, monitoring two characteristic mass transitions simultaneously for each analyte. The procedure is a qualitative confirmatory method for 10 sulfonamides at the low microg/kg level in honey. Typical recoveries of the analytes in honey ranged from 44 to 73% at a fortification level of 50 microg/kg. This study also addresses the issue of matrix-induced suppression of ionization, an effect often encountered in trace residue analysis of food matrices using LC-ESI-MS-MS based methods.     

Determination of betaine in Lycium chinense fruits by liquid chromatography-electrospray ionization mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-electrospray mass spectrometric method has been developed for the determination of betaine in Lycium chinense fruits. Betaine was analyzed on a system consisting of a NH2 stationary phase and a mobile phase of water-acetonitrile (25:75) by isocratic elution for 40 min. Betaine was identified and quantitated by electrospray ionization mass spectrometry with selected ion monitoring of the protonated ion [Betaine+H]+ and clustered ions [nBetaines+H]+. The limit of detection for betaine by this method was ca. 0.2 ng/ml and the relative standard deviations of the assay (intra- and inter-day) were less than 8.1%.     

Direct characterization of isoquinoline alkaloids in a crude plant extract by ion-pair liquid chromatography-electrospray ionization tandem mass spectrometry: example of Eschscholtzia californica

An ion-pair HPLC-ESI-MS-MS method has been developed for the direct and rapid characterization of isoquinoline alkaloids in a crudely purified extract of the aerial parts of Eschscholtzia californica (Papaveraceae). This plant was chosen because of its increasing use in pharmaceutical industries and because its well known alkaloid composition allows the optimization of the experimental procedure through an on-line analytical sequence. Thus, 14 isoquinoline alkaloids of different types were detected and characterized. The identities of these compounds were confirmed unambigously by their fragmentation and UV spectra obtained by LC-diode-array detection. Various experiments including tandem mass spectrometry and in-orifice collision induced dissociation were performed and prove that MS-MS is a very efficient technique to identify these compounds. An explanation for each isoquinoline alkaloid type MS-MS fragmentation pattern is proposed and indicates similar neutral and/or radical losses. The order of the fragmentation depended on the type of compound but the lost fragments were similar.