On-line identification of phenanthroindolizidine alkaloids in a crude extract from Tylophora atrofolliculata by liquid chromatography combined with tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with sequential mass spectrometry (LC/MS(n)) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MS(n), LC/UV-MS and LC/MS(n) techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS).     

Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column

Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin. Lactose, which was immobilized on monolithic silica, was used as a capture ligand for ricin extraction from the sample solution, and the silica was supported in a disk-packed spin column. Recovery of ricin was more than 40%. After extraction, the extract was digested with trypsin and analyzed by LC/MS. The accurate masses of molecular ions and MS/MS spectra of the separated peptide peaks were measured by Fourier transform-MS and linear iontrap-MS, respectively. Six peptides, which were derived from the ricin A-(m/z 537.8, 448.8 and 586.8) and B-chains (m/z 701.3, 647.8 and 616.8), were chosen as marker peptides for the identification of ricin. Among these marker peptides, two peptides were ricin-specific. This method was applied to the determination of ricin from crude samples. The monolithic silica extraction removed most contaminant peaks from the total ion chromatogram of the sample, and the six marker peptides were clearly detected by LC/MS. It takes about 5 h for detection and identification of more than 8 ng/ml of ricin through the whole handling, and this procedure will be able to deal with the terrorism using chemical weapon.     

Liquid chromatography/tandem mass spectrometry of unusual phenols from Yucca schidigera bark: comparison with other analytical techniques

Qualitative and quantitative analyses of phenolic compounds are of interest for both medicinal and food plants. In the present work, the phenolic fraction from Yucca schidigera, a plant bearing the GRAS (Generally Recognized as Safe) label approved by the US Food and Drug Administration, was studied. Crude extracts of Y. schidigera bark were investigated by liquid chromatography/UV spectrophotometry with diode-array detection, liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), in order to develop and optimize simple and rapid techniques to determine both stilbenes and yuccaols for the purposes of quality control of collected material. With optimal LC and MS conditions, stilbenes and yuccaols were quantified with all the proposed methods and the results were compared. Sensitivity was evaluated and the results indicated that MS/MS detection in the multiple reaction monitoring mode is easily applicable to this plant and allows the rapid and direct identification and quantification of these peculiar compounds in crude plant extracts.     

Determination of senkirkine and senecionine in Tussilago farfara using microwave-assisted extraction and pressurized hot water extraction with liquid chromatography tandem mass spectrometry

Tussilago farfara (Kuan Donghua) is an important Chinese herbal medicine which has been shown to contain many bioactive compounds and widely used to relieve cough and resolve phlegm. However, besides therapeutic bioactive compounds, this herb has been found to contain toxic pyrrolizidine alkaloids (PAs), mainly senkirkine and traces of senecionine. In this report, conditions for microwave-assisted extraction (MAE) and pressurized hot water extraction (PHWE) were optimized for the extraction of the PAs. The results were compared against heating under reflux. It was found that the binary mixture of MeOH:H₂O (1:1) acidified using HCl to pH 2-3 was the optimal solvent for the extraction of the PAs in the plant materials. Liquid chromatography (LC) with ultra-violet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) in the positive mode was used for the determination and quantitation of senkirkine and senecionine in the botanical extract. The proposed extraction methods with LC/MS allow for the rapid detection of the major and the minor alkaloids in T. farfara in the presence of co-eluting peaks. With LC/MS, the quantitative analysis of PAs in the extract was done using internal standard calibration and the precision was found to vary from 0.6% to 5.4% on different days. The limits of detection (LODs) and limits of quantitation (LOQs) for MAE and PHWE were found to vary from 0.26 μg/g to 1.04 μg/g and 1.32 μg/g to 5.29 μg/g, respectively. The method precision of MAE and PHWE were found to vary from 3.7% to 10.4% on different days. The results showed that major and minor alkaloids extracted using MAE and PHWE were comparable to that by heating under reflux. Our data also showed that significant ion suppression was not observed in the analysis of senkirkine and senecionine in the botanical extracts with co-eluting peaks.     

Multicomponent dye detection with californium-252 particle-desorption mass spectrometry

Particle-desorption mass spectrometry (PDMS) by using ²⁵²Cf was applied to multicomponent dye samples. A mixture of cesium iodide with three cationic dyes (rhodamine B, methyl violet and methylene blue), and a crude carotenoid sample extracted from a carrot were examined. In the positive-ion detection mode, the individual components of the dye mixture were identified by the detection of intactly desorbed preformed cations. In the carotenoid extract, two neutral components, carotene and lutein (xanthophyll), provided adduct ions, (M+H)⁺ In both examples, further fragmentation occurred, providing peaks useful for structural interpretation. These examples illustrate the ability of PDMS to acquire qualitative information from mixtures without a prior separation step.     

Effects of first dimension eluent composition in two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC×LC) has received a great deal of attention during the past few years because of its extraordinary resolving power. The biggest advantage of this technique is that very high peak capacities can be generated in a relatively short time. Numerous approaches to maximize the peak capacity in LC×LC have been employed. In this work we investigate the impact of the first dimension mobile phase on selectivity. LC×LC has several potential advantages over one-dimensional LC (1DLC) in that unconventional solvents, at least in reversed-phase LC, can be used. For example, solvents which strongly adsorb in the UV in the first dimension are not problematic in LC×LC. This so because the UV detector is placed after the second dimensional column, as pulses of the first dimension eluent arrive at the second dimensional column, they elute well before the solutes of interest and therefore do not interfere at all with detection of solute peaks. So far, the most widely used solvents in reversed-phase 1DLC are methanol and acetonitrile. However, the "UV advantage" of 2DLC allows us to employ UV active solvents, such as acetone. We compare their differential selectivities to that of acetonitrile for the separation of 23 indole acetic acids of interest in plant biology. We also apply them to the separation of a maize seed extract, a very complex sample. In both sample sets, mobile phase composition can be an important parameter to increase the orthogonality of the two dimensions and thus, to increase the effective peak capacity of LC×LC.     

Liquid chromatography with ultraviolet absorbance–mass spectrometric detection and with nuclear magnetic resonance spectrometry: a powerful combination for the on-line structural investigation of plant metabolites

In order to discover new bioactive compounds from plant sources which could become new leads or new drugs, extracts should be submitted at the same time to chemical screening and to various biological or pharmacological targets. Metabolite profiling using hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR, quickly provides plenty of structural information, leading to a partial or a complete on-line de novo structure determination of the natural products of interest. As a complement to this approach, bioassays performed after LC/microfractionation of the extracts allow efficient localisation of the bioactive LC-peaks in the chromatograms. The combination of metabolite profiling and LC/bioassays provides the possibility of distinguishing between already known bioactive compounds (dereplication) and new molecules directly in crude plant extracts. Thus, the tedious isolation of compounds of low interest can be avoided and targeted isolation of new bioactive products or constituents presenting novel or unusual spectroscopic features can be undertaken. Several examples of rapid localisation of bioactive compounds, based on post-chromatographic bioautographic testing of LC/NMR microfractions and subsequent on-line identification will be illustrated. Application of hyphenated techniques for the efficient characterisation of labile constituents or constituents difficult to separate at the preparative scale will also be mentioned. The possibilities and limitations of LC/UV/NMR/MS and LC/bioassay as well as future development expected in this field will be discussed.     

Isolation and determination of paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode-array detection

A liquid chromatographic (LC) method is described for the isolation and determination of the tremorgenic mycotoxins paxilline (Penicillium paxilli NRRL 6110), paspaline, paspalinine and paspalicine (Claviceps paspali). Following a Soxhlet extraction of a mould-contaminated matrix using chloroform, the crude extract was partitioned between hexane and 80% aqueous methanol. The latter fraction, containing the desired toxin(s), was evaporated to dryness, the residue dissolved in methylene chloride and the solution analysed by liquid chromatography using a Supelcosil LC-Si column eluted with methylene chloride-diethyl ether (9 + 1, v/v). A mixture containing standards of these compounds was similarly analysed. All toxins were detected using a UV diode-array detector. The generated UV spectra and chromatographic data of the standard toxins were stored in a computer as a library and used to identify these toxins in a crude mixture. The purity of the separated peaks and the amount of toxin in the crude mixture were also determined. The toxins were isolated by selectively collecting the eluted peaks using a programmable fraction collector equipped with a peak level sensor. Further confirmation of compound identity was achieved by mass spectrometry using the direct inlet probe method. In comparison with methods used previously to isolate these toxins, the present technique is fast and allows the acquisition of complete UV spectral information and chromatographic data and the isolation of multiple toxins in a single chromatographic operation.     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Analysis of meat samples for anabolic steroids residues by liquid chromatography/tandem mass spectrometry

A rapid, specific and highly sensitive multi-residue method for the determination of anabolic steroid residues in bovine, pork and poultry muscle tissues was developed. The sample preparation involves enzymatic digestion followed by extraction with methanol. The crude extract was cleaned up by solid-phase extraction (SPE) combining C18 and NH2 columns. The detection was carried out by a highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using both positive and negative ionization modes. Natural and synthetic steroids covering different polarities could be extracted, concentrated and purified using one single method. Mobile phase composition and additives were optimized to achieve the highest sensitivity. The linearity was not good enough for quantitative analysis but the method was well-suited for qualitative confirmation. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.5 ng g(-1) for all the compounds in the three types of meat studied. The developed method is suitable for routine analysis in our laboratories.     

Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue determination of antifungal drugs in chicken tissues

A method involving LC coupled with MS/MS (LC/MS/MS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole, griseofulvin, clotrimazole, bifonazole, econazole, ketoconazole, itraconazole, miconazole, terconazole, and fluconazole) in the liver and muscles of chickens. Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation. A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes, coupled with MS/MS using an electrospray ionization source. The accuracy of the method was confirmed with a mean recovery of 71-121%, and acceptable coefficients of variation (4-23%, n = 6). The detection capability of these compounds in two different matrixes was 0.50-2.82 microg/kg. This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues.     

Comprehensive normal-phase x reversed-phase liquid chromatography coupled to photodiode array and mass spectrometry detection for the analysis of free carotenoids and carotenoid esters from mandarin

In the present work, a novel strategy including the use of two different comprehensive HPLC methods has been employed to study the whole carotenoid composition of mandarin essential oil. Thus, two different fully orthogonal two-dimensional HPLC methods have been used. A silica microbore column was coupled to a C(18) monolithic column to study the mandarin saponified extract, while the coupling of a cyano microbore column to a C(18) monolithic column was employed to study the intact mandarin essential oil sample in order to characterize the native carotenoid esters composition. Detection was performed by connecting a photodiode array detection (DAD) system in parallel with a MS detection system operated with an atmospheric pressure chemical ionization (APCI) interface. Thus, the carotenoid identification was carried out by combining the information provided by the DAD and MS systems and the peaks relative position in the two-dimensional chromatograms.     

Development and validation of a liquid chromatography/electrospray ionization time-of-flight mass spectrometry method for relative and absolute quantification of steroidal alkaloids in Fritillaria species

Steroidal alkaloids are naturally occurring nitrogen-containing compounds in many edible or medicinal plants, such as potato, tomato, Fritillaria and American hellebore, which possess a variety of toxicological and pharmacological effects on humans. The aim of this study is to explore the potential of liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) method in the determination of these important alkaloids in plant matrices. The application of this method has been proven through 26 naturally occurring steroidal alkaloids in Fritillaria species. Accurate mass measurements within 4 ppm error were obtained for all the alkaloids detected out of various plant matrices, which allowed an unequivocal identification of the target steroidal alkaloids. The bunching factor for mass spectrometer, an important parameter significantly affecting the precision and accuracy of quantitative method, was firstly optimized in this work and satisfactory precision and linearity were achieved by the optimization of that parameter. The ranges of RSD values of intra-day and inter-day variability for all alkaloids were decreased remarkably from 41.8-159% and 13.2-140% to 0.32-7.98% and 2.37-16.1%, respectively, when the value of bunching factor was optimized from 1 to 3. Linearity of response more than two orders of magnitude was also demonstrated (regression coefficient >0.99). The LC/TOF-MS detection method offered improvements to the sensitivity, compared with previously applied LC (or GC) methods, with limits of detection down to 0.0014-0.0335 microg/ml. The results in this paper illustrate the robustness and applicability of LC/TOF-MS for steroidal alkaloids analysis in plant samples. In addition, relative quantitative determination of steroidal alkaloid with one popular analyte verticinone which is commercially available was also investigated in order to break through the choke point of lack of standards in phytochemical analysis. The accuracies of relative quantitative method for steroidal alkaloids determinations with verticinone were 90.6-110.0% (average 98.5%) suggesting that it is feasible to quantify steroidal alkaloids by the proposed relative quantitative determination method within acceptable errors.     

Antibacterial, antifungal, antispasmodic and Ca++ antagonist effects of Caesalpinia bonducella

Caesalpinia bonducella F. (Leguminosae) has been used as a folk medicine for a variety of ailments. The crude extract of C. bonducella and its fractions were studied for antibacterial, antifungal, antispasmodic and Ca++ antagonistic properties. The strongest antibacterial effect was displayed by the n-butanol (72%) and ethyl acetate (80%) fractions, followed by the crude extract (46% and 42%), against Escherichia coli and Bacillus subtilis, respectively. The plant extract and its fractions showed mild to excellent activity in antifungal bioassays, with maximum antifungal activity against Candida glaberata (80%) and Aspergillus flavus (70%) by the n-butanol and chloroform fractions, followed by the crude extract (70% and 65%). Caesalpinia bonducella extract caused concentration-dependent inhibition of spontaneous and high K+ (80 mM)-induced contractions of isolated rabbit jejunum preparations, similar to that caused by Verapamil. These results indicate that C. bonducella exhibits antibacterial, antifungal, spasmolytic and Ca++ channel blocking actions.     

Determination of pesticides in vegetables using large-volume injection column liquid chromatography-electrospray tandem mass spectrometry

Direct injection of a large volume (900 microl) of a sample extract onto a liquid chromatographic (LC) column, LC separation and electrospray tandem mass spectrometric detection were used for the quantitative analysis of a wide polarity range of pesticides in carrots and potatoes. Rapid sample preparation involved extraction of a small amount of sample (2 g) with a small volume of organic solvent (3 ml), clean-up over a filter and dilution of the organic extract with the aqueous LC eluent. The extraction efficiency for the selected pesticides was studied using methanol, acetone and acetonitrile as solvents. Evaluation of the performance of the overall method, using extraction with acetonitrile and detection in the selected-reaction-monitoring mode, showed excellent linearity in the range of 2-100 microg/kg with limits of detection of 0.5-2 microg/kg for both types of vegetable. With relative standard deviations of the MS peak area measurements of less than 6.5% (n=8) the repeatability of the method was fully satisfactory.     

Characterization of peptides from Aplysia using microbore liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry guided purification

Liquid chromatography (LC) has been used extensively for the separation and isolation of peptides due to its high selectivity and peak capacity. An approach combining microbore LC with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) detection is described to identify peptides in cells and guide the purification of peptides from the marine mollusc Aplysia californica. Direct MALDI-MS of neurons and processes provides molecular mass information for unknown peptides with almost no sample preparation, and LC-MALDI-MS allows the isolation and purification of these peptides from pooled samples, thus enabling new putative neuropeptides to be isolated from complex cellular samples. Both direct MALDI-MS and LC-MALDI-MS are compared in terms of detecting peptides from neuronal samples. Using both approaches, two peaks from Aplysia californica connectives having molecular masses of 5013 and 5021 have been isolated, partially sequenced and identified as novel collagen-like peptides.     

Characterisation of Stevia rebaudiana by comprehensive two-dimensional liquid chromatography time-of-flight mass spectrometry

Comprehensive two-dimensional liquid chromatography (LC x LC) connected on-line to electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS) was employed for analysis of aqueous extract of Stevia rebaudiana. Different combinations of strong cation-exchange (SCX), amino (NH2), and octadecyl siloxane (C18) stationary phases were tested in the separation of all nine known sweet Stevia glycosides. A combination of C18 as the first-dimension column and NH2 as the second-dimension column fully separated all the glycosides from the matrix. The method proved to be quantitative and repeatable. The limit of detection (S/N=3) for stevioside, a widely used natural sweetener, was 43.4 ng/g in dry leaves. The RSD for retention times was <0.1% and that of peak areas 4.5%.     

Confirmation of moxidectin residues in cattle fat by liquid chromatography/mass spectrometry

Moxidectin, a potent new endo- and ectoparasitic agent, is determined in cattle tissues by liquid chromatography (LC) with fluorescence detection. The original confirmatory method for moxidectin in cattle fat, the target tissue for regulatory purposes, was LC with mass spectrometry (MS) using thermospray (TSP) ionization and selected ion monitoring. As newer ionization techniques for LC/MS made TSP obsolete and with the availability of a new generation of benchtop LC/MS instrumentation, the confirmation of moxidectin in cattle fat was re-evaluated. The ionization techniques of atmospheric pressure chemical ionization versus electrospray ionization, the detection techniques of single-stage MS versus tandem MS, and the instrumentation of ion trap versus quadrupole were investigated. The final confirmatory method was based on full-scan single-stage MS. Even with full-scan detection, the analysis required at least 10-fold less extract than the original TSP method. The applicability of this new confirmatory method was demonstrated on both ion trap and quadrupole instruments.     

Separation and characterization of silybin, isosilybin, silydianin and silychristin in milk thistle extract by liquid chromatography-electrospray tandem mass spectrometry

A selective and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the characterization of silymarin in commercially available milk thistle extract. In this study, six main active constituents, including silydianin, silychristin, diastereomers of silybin (silybin A and B) and diastereomers of isosilybin (isosilybin A and B) in silymarin, were completely separated on a YMC ODS-AQ HPLC column using a gradient mobile phase system comprised of ammonium acetate and methanol/water/formic acid. Identification and characterization of the major constituents were based not only on the product ion scan, which provided unique fragmentation information of a selected molecular ion, but also on the specific fragmentation of multiple reaction monitoring (MRM) data, which confirmed the retention times of LC chromatographic peaks. The method was applied in the analysis of human plasma samples in the presence of silymarin and appeared to be suitable for the pharmacokinetic studies in which the discrimination of silymarin constituents is essential.     

A novel approach for sensitivity enhancement in atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry of chlorophylls

Differences in the ionisation efficiency of chlorophylls and their phaeophytin counterparts result in lower sensitivity for atmospheric pressure chemical ionisation mass spectrometric detection of the former. Improvement in the sensitivity of detection of chlorophyll of around an order of magnitude at a concentration of 1 x 10(-6)mol L(-1) has been achieved using post-column addition of methanoic acid during analysis by liquid chromatography/mass spectrometry (LC/MS). The method gives linear response and is a simple strategy to improve sensitivity both for LC/MS and LC/MS/MS without loss of information relating to the precise nature of the tetrapyrrole distributions. Detection levels achieved exceed those obtained by absorbance detection.