Separation of four isomeric tropane alkaloids from Schizanthus grahamii by non-aqueous capillary electrophoresis

The potential of non-aqueous capillary electrophoresis was investigated for the separation of four isomeric tropane alkaloids, namely 3alpha-senecioyloxy-7beta-hydroxytropane, 3alpha-hydroxy-7beta-senecioyloxytropane, 3alpha-hydroxy-7beta-angeloyloxytropane and 3alpha-hydroxy-7beta-tigloyloxytropane extracted from Schizanthus grahamii. The composition of the organic solvent and the nature of the electrolyte were of considerable importance with respect to selectivity. Different organic solvents (i.e. methanol, ethanol, acetonitrile, tetrahydrofuran) and mixtures thereof were investigated. Moreover, different electrolytes such as formate, acetate and trifluoroacetate were tested. After optimisation, an electrolyte consisting of 1 M trifluoroacetic acid and 25 mM ammonium trifluoroacetate in methanol:ethanol (40:60, v:v) was selected. It provided an efficient separation of the four positional isomers as well as a good repeatability of migration time (RSD < 0.2%). The method was successfully used with electrospray MS to confirm the molecular mass of the tropane alkaloids.     

Rapid screening and characterisation of antioxidants of Cosmos caudatus using liquid chromatography coupled with mass spectrometry

Ulam raja (Cosmos caudatus) is used traditionally for improving blood circulation. In this study, it was found that ulam raja had extremely high antioxidant capacity of about 2,400 mg l-ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh sample. Antioxidant peaks in extract of ulam raja were firstly characterized using free radical spiking test through high performance liquid chromatography coupled with mass spectrometry (MS). Upon reaction with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, intensities of antioxidant peaks will be significantly reduced. HPLC/MS(n) was further applied to elucidate the chemical structures of antioxidant peaks characterized in the spiking test. More than twenty antioxidants were identified in ulam raja, and their chemical structures were proposed. The major antioxidants in ulam raja were attributed to a number of proanthocyanidins that existed as dimers through hexamers, quercetin glycosides, chlorogenic, neo-chlorogenic, crypto-chlorogenic acid and (+)-catching. High content of antioxidants antioxidants contained in ulam raja could be partly responsible for its ability to reduce oxidative stress.     

Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry

The need for urgent diagnoses has propelled the development of automated analyses that can be performed in a short time at reasonable cost. One such method is immunoaffinity capillary electrophoresis. This emerging hybrid technology employs two powerful techniques coupled on-line for the direct and rapid determination of analytes present in biological fluids. The first technique, immunoaffinity, is used for the selective extraction of a molecule present in a complex matrix, utilizing a microscale-format chamber affinity device. An analyte (affinity target) present in serum or urine is captured by an immobilized molecular recognition antibody molecule (affinity ligand) bound to a solid support constituent (glass beads or an appropriate porous structure) of a microchamber affinity device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated affinity target material after elution from the microchamber affinity device. In this work, immunoaffinity capillary electrophoresis was developed for the identification and characterization of a single constituent of a complex matrix. Immunoreactive gonadotropin-releasing hormone was determined in serum and urine specimens derived from a normal individual and from a patient suffering from benign prostatic hyperplasia. Furthermore, the on-line immuno-separation system was coupled in tandem to mass spectrometry to obtain molecular mass information of the affinity isolated and CE separated neuropeptide. This hybrid immuno-analytical technology is simple, rapid, selective and sensitive. In addition, an attempt was also made to characterize other urinary constituents by CE–MS that may lead to marker activity in the urine of the diseased subject. The hyphenation of analytical techniques has proved valuable in enhancing their individual features. The future of bioanalysis using miniaturized affinity systems is discussed in this paper.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.     

Analysis of ellagic acid in pomegranate rinds by capillary electrophoresis and high-performance liquid chromatography

Two novel methods for the analysis of ellagic acid in pomegranate (Punica granatum) rinds are proposed. Capillary electrophoresis (CE) was performed in a bare fused-silica capillary using a buffer solution of tri(hydroxymethyl)aminomethane:potassium dihydrogen phosphate (pH 8.4) with an applied voltage of 20 kV and UV detection at 254 nm. HPLC analysis was performed with a Zobax SB C(18) column and a mobile phase consisting of methanol:ethyl acetate:potassium dihydrogen phosphate: phosphoric acid at a flow rate of 1.0 mL/min. Under optimised conditions, the HPLC retention and the CE migration times for ellagic acid were 10.32 and 12.23 min, respectively. Calibration curves of peak area vs. concentration gave correlation coefficients of 0.9999 for HPLC and 0.9990 for CE. The detection limits for HPLC and CE were 2.8 and 2.2 microg/mL, respectively. Average recoveries were 98.32 +/- 1.2% for HPLC and 96.52 +/- 2.8% for CE. Both methods were shown to be suitable for the determination of ellagic acid in pomegranate rinds extraction; however, the CE method required less solvent and gave better column efficiency, whilst the HPLC provided superior precision.     

Quantitative measurement of 3'-oxolutein from human retina by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry

3-Hydroxy-beta,epsilon-carotene-3'-one (3'-oxolutein) is the major oxidative metabolite of dietary carotenoids in the retina of the human eye. Elucidating the biochemical mechanism of its formation may provide helpful insight into the pathogenesis of age-related macular degeneration; however, it is found in relatively low quantities that require highly sensitive methods for quantitation from individual retinas. Normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry allowed us to do quantitative analysis of 3'-oxolutein from central and peripheral retinas obtained from individual human donors. The limit of quantification for 3'-oxolutein in human retina at a signal-to-noise ratio of 10 was 6 pg. The precision of the assay yielded a coefficient of variation ranging from 4.7 to 7.4% and accuracies of 106-108%. A statistically significant (R = 0.99, p < or = 0.001) linear working range was achieved between 5 and 7200 pg. The 3'-oxolutein contents from 8-mm punches of the central macula and peripheral retina were found to be 375+/-192 and 191+/-95 pg/tissue, respectively.     

Application of both high-performance liquid chromatography combined with tandem mass spectrometry shotgun and 2-D polyacrylamide gel electrophoresis for streptococcal exoproteins gave reliable proteomic data

Streptococci secrete a large number of exoproteins including virulence-associated toxins and enzymes. To construct a reliable database of streptococcal exoproteins, we integrated the results that were derived from two approaches: LC-based shotgun proteomic analysis and 2-D PAGE-based proteomic analysis. We identified 74 and 82 proteins by LC-based and gel-based analysis, respectively. Forty-five proteins were identified by both methods. In addition, two proteins, one identified by both methods and the other only by LC-based shotgun analysis, were newly annotated. We therefore found the importance of combinational analysis by the two methods for the construction of a more reliable database.     

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.     

Studies on the stability of diester-diterpenoid alkaloids from the genus Aconitum L. by high performance liquid chromatography combined with electrospray ionisation tandem mass spectrometry (HPLC/ESI/MSn)

The stability of diester-diterpenoid alkaloids (DDA) from plants of the genus Aconitum L. has been studied in different solvents and pH buffers. The HPLC/ESIMS method for analysing the concentration of DDA was established and DDA's decomposition products were elucidated by HPLC/ESI-MS/MS(n). In different solvents, e.g. dichloromethane, ether, methanol and distilled water, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the difference of their structures, in which substituents at the N atom and substituents at C-3 are different. The pyrolytic products of DDA, such as deacetoxy aconitine-type alkaloids, have been observed in the above solvents, whereas 8-methoxy-14-benzoyl aconitine-type alkaloids have been obtained only in methanol. Furthermore, the experimental results demonstrate that the stability of DDA depends on pH values of the buffer. Aconine as hydrolysate has been only found in pH 10.0 buffer, and the other hydrolysates and the pyrolyzates of DDA, such as benzoylaconine and deacetoxy aconitine, have been observed in all pH aqueous solutions. The decomposition pathways of DDA in buffers are related to the substituent on the C-3 position. The decomposition pathway of aconitine is similar to that of mesaconitine, but different from that of hypaconitine.     

Rapid isolation and characterization of bacterial lipopeptides using liquid chromatography and mass spectrometry analysis

The structural analysis of post‐translational modifications (PTMs) of lipoproteins is difficult due to the hydrophobic properties of their fatty acid moieties. At the present time, the relative positions of fatty acid components on the N‐acyl‐S‐diacylglycerylcysteine core structure has not been specifically identified in any natural or bacterial expressed recombinant lipoproteins. In this study, we describe a rapid solid‐phase extraction using acetonitrile and isopropanol method that can be performed manually to isolate large amounts of relatively pure lipopeptides generated by the limited tryptic‐digestion of recombinant lipoproteins. Using these lipopeptides and LC/MS mass spectra analysis, two groups of N‐terminal lipidated (diacyl or triacyl) molecules that differ by one fatty acid unit were successfully identified. This LC/MS method also provided the separation of lipopeptides differing by 14 Da for the on‐line MS identification. Multiple‐stage fragmentation analyses of the di‐ and triacyl lipopeptides using both the positive and negative ion modes enabled to identify the putative structure of the N‐acyl‐S‐diacylglycerylcysteine containing an amide bond to palmitic acid at the N‐terminal cysteine, a palmitic acid at sn1 position, and an unsaturated fatty acid of either hexadecenoic acid, cyclopropaneoctanoic acid, oleic acid and nonadecenoic acid at sn2 position of diacylglycerol residue through ester bonding. For diacyl lipoprotein, the saturated palmitoyl fatty acid group is absent at sn1 position of glycerol‐derived lipid residue of lipopeptide.     

Structure characterization and identification steroidal saponins from Ophiopogon japonicus Ker-Gawler (Liliaceae) by high-performance liquid chromatography with ion trap mass spectrometry

Introduction – Steroidal saponins are the main active constituents in Ophiopogon japonicus Ker‐Gawler (Liliaceae). However, because of their high polarity, non‐chromophores and low content in plants, steroidal saponins are difficult to be isolated from O. japonicus by conventional phytochemical methods. Objective – To develop a sensitive and rapid approach towards the structural analysis of steroidal saponins using HPLC/ESI‐MSⁿ. Methodology – The fragmentation behaviors of six known steroidal saponins in negative ESI‐MSⁿ were used to deduce their mass spectral fragmentation mechanisms. By using HPLC/ESI‐MSⁿ, the important structural information on aglycone types, sugar types and saccharide sequences can be obtained. Results – According to the HPLC retention behaviour, the molecular structural characteristics provided by multistage mass spectrometry spectra and the literature, a total of 8 steroidal saponins were tentatively identified or characterized in O. japonicus rapidly. Conclusion – This work has shown that HPLC‐ESI‐MSⁿ may be used as an effective and rapid method for the characterization and identification of steroidal saponins from O. japonicus. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.