A rapid method for simultaneous determination of 52 marker compounds in Xiao‐Qing‐Long‐Tang by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry†

Xiao‐Qing‐Long‐Tang (XQLT) is a classical Chinese medicine formula. It is generally used for the treatment of common cold, bronchial asthma, and allergic rhinitis in Asia. In this study, a multicomponent quantification fingerprinting approach based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry has been developed for the analysis of compounds in XQLT in 14.5 min. A total of 52 compounds were identified by co‐chromatography of sample extract with authentic standards and comparing the retention time, UV spectra, molecular ions and characteristic fragment ions with those of authentic standards, or tentatively identified by MS^E determination along with Mass Fragment software. Moreover, the method was validated for the simultaneous quantification of 16 components in XQLT commercial products. The method is practical for comprehensive standardization of XQLT and holistic comparison of its commercial products from different manufacturers.     

Analytical method for dimethenamid-P in selected raw agricultural commodities by gas chromatography with electron capture detection

A sensitive and simple method for the extraction and quantification of the herbicide dimethenamid-P from several raw agricultural commodities (RAC) is presented. The method provides sensitive and well-defined chromatography with baseline resolution in all matrixes tested. Homogenized samples were extracted with methanol-water, filtered, and then extracted with hexanes. A Florisil solid-phase extraction was then applied for the final cleanup. Sample separation and quantification were performed by gas chromatography with an electron capture detector. The instrument detection limit was 0.007 microg/mL, and the practical quantification limit was 0.003 microg/g (w/w) based on a 25 g sample. Recoveries for a series of fortified plant tissues ranged from 69 to 103%. The study demonstrated selective and sensitive recovery of dimethenamid-P from the RAC tested.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Separation and quantification of terpenoids of Boswellia serrata Roxb. extract by planar chromatography techniques (TLC and AMD)

An high-performance TLC (HPTLC) method for the separation of boswellic acids, the active constituents in Boswellia serrata extract, has been developed and TLC of these compounds on silica by automated multiple development (AMD) using solvent gradients was performed. Enhancement of the separation of boswellic acids on HPTLC plates was carried out by AMD chromatography. Densitometric analysis of the developed plate was carried out to quantify the four boswellic acids. 11-Keto-beta-boswellic acid (KBA) and acetyl-11-keto-beta-boswellic acid (AKBA) were quantified by densitometric scanning of the developed plate at 254 nm. beta-Boswellic acid (BA) and acetyl-beta-boswellic acid (ABA) were quantified after derivatization with anisaldehyde sulfuric acid reagent at 560 nm. The AMD system provided a clean separation according to polarity for each of the four groups studied and good results were obtained. The proposed HPTLC method for the simultaneous quantification of the major boswellic acids BA, ABA, KBA, and AKBA was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control and for the quantification of these compounds in plant materials. The study of market products revealed significant variations in the content of these pharmacologically active compounds in commercial samples.     

Detection and validated quantification of nine herbal phenalkylamines and methcathinone in human blood plasma by LC-MS/MS with electrospray ionization

The herbal stimulants Ephedra species, Catha edulis (khat), and Lophophora williamsii (peyote) have been abused for a long time. In recent years, the herbal drug market has grown owing to publicity on the Internet. Some ingredients of these plants are also ingredients of cold remedies. The aim of the presented study is to develop a multianalyte procedure for detection and validated quantification of the phenalkylamines ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, methylpseudoephedrine, cathinone, mescaline, synephrine (oxedrine), and methcathinone in plasma. After mixed-mode solid-phase extraction of 1 ml of plasma, the analytes were separated using a strong cation exchange separation column and gradient elution. They were detected using a Q-Trap LC-ESI-MS/MS system (MRM mode). Calibration curves were used for quantification using norephedrine-d3, ephedrine-d3, and mescaline-d9 as internal standards. The method was validated according to international guidelines. The assay was selective for the tested compounds. It was linear from 10 to 1000 ng/ml for all analytes. The recoveries were generally higher than 70%. Accuracy ranged from - 0.8 to 20.0%, repeatability from 2.5 to 12.3%, and intermediate precision from 4.6 to 20.0%. The lower limit of quantification was 10 ng/ml for all analytes. No instability was observed after repeated freezing and thawing or in processed samples. The applicability of the assay was tested by analysis of authentic plasma samples after ingestion of different cold medications containing ephedrine or pseudoephedrine, and after ingestion of an aqueous extract of Herba Ephedra. After ingestion of the cold medications, only the corresponding single alkaloids were detected in human plasma, whereas after ingestion of the herb extract, all six ephedrines contained in the plant were detected. The presented LC-MS/MS assay was found applicable for sensitive detection and accurate and precise quantification of all studied analytes in plasma.     

Chromatographic determination of plant saponins

The methods used for saponin determination in plant materials are presented. It is emphasised that the biological and spectrophotomeric methods still being used for saponin determination provide, to some extent, valuable results on saponin concentrations in plant material. However, since they are sensitive to the structural variation of individual saponins they should be standardized with saponin mixtures isolated from the plant species in which the concentration is measured. However, one plant species may contain some saponins which can be determined with a biological test and others which cannot. That is why biological and colorimetric determinations do not provide accurate data and have to be recognized as approximate. Thin-layer chromatography on normal and reversed-phases (TLC, HPTLC, 2D-TLC) provides excellent qualitative information and in combination with on-line coupling of a computer with dual-wavelength flying-spot scanner and two-dimensional analytical software can be used for routine determination of saponins in plant material. The densitometry of saponins has been very sensitive, however, to plate quality, spraying technique and the heating time and therefore appropriate saponin standards have to be run in parallel with the sample. Gas-liquid chromatography has limited application for determination since saponins are quite big molecules and are not volatile compounds. Thus, there are only few applications of GC for determination of intact saponins. The method has been used for determination of TMS, acetyl or methyl derivatives of an aglycones released during saponin hydrolysis. However, structurally different saponins show different rates of hydrolysis and precise optimisation of hydrolysis conditions is essential. Besides, during hydrolysis a number of artefacts can be formed which can influence the final results. High performance liquid chromatography on reversed-phase columns remains the best technique for saponin determination and is the most-widely used method for this group of compounds. However, the lack of chromophores allowing detection in UV, limits the choice of gradient and detection method. The pre-column derivatisation with benzoyl chloride, coumarin or 4-bromophenacyl bromide has been used successfully in some cases allowing UV detection of separation. Standardisation and identification of the peaks in HPLC chromatograms has been based on comparison of the retention times with those observed for authentic standards. But new hyphenated techniques, combining HPLC with mass spectrometry and nuclear magnetic resonance are developing rapidly and allow on-line identification of separated saponins. Capillary electrophoresis has been applied for saponin determination only in a limited number of cases and this method is still being developed.     

Mutants of Arabidopsis as Tools to Understand the Regulation of Phenylpropanoid Pathway and UVB Protection Mechanisms

Abstract— Plants accumulate certain phenylpropanoid compounds in the vacuoles of their epidermal and subepidermal cell layers thereby protecting the underlying tissue against UVB-induced damage. However, a number of mutants of Arabidopsis thaliana are known that fail to synthesize these protective pigments, thereby allowing harmful UVB radiation to penetrate into their dermal layers. Study of several of these nonlethal mutants, defective in various aspects of flavonoid and lignin biosynthesis, has led to a better understanding of the coordinate regulation and expression of important genes as well as of mechanisms involved in plant defense against UVB radiation. The characteristics of the various phenylpropanoid mutants of Arabidopsis, viz. flavonoid mutants (banyuls [ baity , increased chalcone synthase expression 1 [ icx1 ]; transparent testa [ tt ] and ultraviolet sensitive [ uvs ]) and hydroxycinnamic acid ester mutants (ferulic acid hydroxylase 1 [ fahl ] and sinapoylglucose accumulator 1 [ sng1 ]) are discussed in detail. We have briefly touched upon, wherever relevant, the unique aspects in other plant species too.     

Determination of arsenic compounds in reference materials by HPLC-(UV)-HG-AFS

Arsenic compounds were determined in six reference materials of biological origin. None of them has yet been certified for arsenic compounds but some are in the process of certification; for most of these reference materials indicative literature values are available. Eight commonly used arsenic standards were used for quantification using a recently developed hyphenated speciation system comprising high performance liquid chromatography (HPLC) and atomic fluorescence spectrometry (AFS), interfaced via a UV-photoreactor and a hydride generation (HG) unit. Absolute detection limits were ca. 0.2 and 0.4 ng As for separation on anion and cation exchange columns, respectively. Our results agree well with indicative literature values which were generated by different authors using various separation and detection methods. The HPLC-(UV)-HG-AFS system validated in this way is suitable for quantification of eight arsenic compounds. Moreover, the system is capable of separation of at least six more compounds in the mentioned reference materials, of which two could be attributed to arsenosugars (OH and phosphodiester form) but due to the lack of standards, quantification was not possible. For accurate and extensive speciation analysis the availability of certified reference materials and standards for arsenic compounds should be promoted.     

Analysis of carotenoids by field desorption mass spectrometry

The mass spectra of a variety of authentic carotenoids covering a range of structural types have been studied using the field desorption technique. All the compounds examined show the molecular ion as the base peak, even where this ion is of very low intensity in the electron-impact spectra. Little fragmentation is observed, but thermal decomposition can be induced in certain cases by using higher emitter currents. Application to a simple mixture of authentic compounds and a more complex mixture of sedimentary origin shows the potential of the method in the rapid qualitative screening of carotenoids in crude extracts.     

Separation of carotenoids by high-performance liquid chromatography with polymeric and monomeric octadecylsilica stationary phases

Summary In addition to their value in the nutritional context, the carotenoids have other important functions, including some epidemiological significance in disease prevention. With increasing interest in the carotenoids methods for their characterization and quantification in various matrices are essential particularly with regard to epidemiologic studies dealing with diet and health. Since high-performance liquid chromatography (LC) is the most promising analytical method for this purpose, having high reliability, high selectivity and quantification ability, this work presents the evaluation of reversed-phase LC methods using polymeric and monomeric octadecylsilica (ODS) stationary phases for the highly selective separation of carotenoids. It has been found that the polymeric ODS has a better selectivity for carotenoids, taking account of their molecular shape and size recognition, than the monomeric ODS phase and that the former is more suitable than the latter for separations of the carotenoids.     

Application of HPLC and Dual Wavelength Detection to the Analysis of Phenolic Compounds in Apples

Abstract

High-performance liquid chromatography, coupled with a programmable UV/visible detector, was applied to the identification and quantification of the phenolic compounds of apple tissue. The identity and purity of unknown components eluted from the HPLC column were established by comparing retention times and absorbance ratios with those of authentic compounds. In Golden Delicious apples, components with retention times agreeing with those of authentic catechin, epicatechin, chlorogenic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, and sinapic acid were found. However, comparison of absorbance ratios with those of authentic compounds suggested that many of the components that eluted in this system as single, symmetrical peaks were mixtures rather than individual compounds. This method helped to eliminate inaccurate identifications or faulty assumptions about homogeneity of HPLC “peaks”, errors that can occur when traditional HPLC methods that rely solely on detection at a single wavelength are employed. The method should have general applicability to plant extracts.     

High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach

Stable isotope dilution–selective reaction monitoring–mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Separation, identification, and quantification of selected withanolides in plant extracts of Withania somnifera by HPLC-UV(DAD)--positive ion electrospray ionisation-mass spectrometry

This paper describes a method for separation, identification, and quantification of selected withanolides in Withania somnifera plant extracts by HPLC-UV(DAD)-Mass Spectrometry (HPLC-MS). Withaferin-A (WS-3), 12-deoxywithastramonolide (WS-12DS), Withanolide A (WS-1), and Withanone (WS-2) were used as external standards. The compounds were isolated from Withania somnifera by repeated column chromatography of the root extract and their identity was established by 1H- and 13C-NMR and mass spectral data. The compounds were chromatographed on a Merck (250 x 4.6 mm ID, 5 microm) column and analyzed by Electrospray Ionization on a mass spectrometer in Selected Ion Mode (SIM). For quantification, [M + Na]+ ions were monitored. Linear calibration curves were obtained in the concentration range of 1.50 microg/mL to 6.5 microg/mL. The method was applied successfully to the detection and quantification of the said withanolides in a number of samples.     

Analytical determination of antioxidants in tomato: typical components of the Mediterranean diet

Diets in the countries of the Mediterranean basin are characterised by abundant plant foods (fruits, vegetables, breads, nuts, seeds, wine, and olive oil) and include fish and low-fat dairy products. Among the vegetables, tomatoes are a main component of the traditional Mediterranean diet, which has been associated with health protection and longevity. Eating tomatoes has been associated with reduced risks of some types of cancer and other diseases. These beneficial properties appear to be related to the antioxidant content of the fruit, particularly carotenoids (lycopene and beta-carotene), ascorbic acid, and phenols, which may play a role in inhibiting reactions mediated by reactive oxygen species. Due to the importance of antioxidant compounds in tomatoes and tomato products, we present here an overview of current analytical methods (from 2000 until the present date) for determining the different antioxidants. The analytical procedures used to determine individual compounds involve extraction from the sample, analytical separation, and quantification. The choice of analytical method depends on the particular focus of the analysis and the kind of product analysed. High-performance liquid chromatography is the technique of choice for the analysis of tomato antioxidants.     

Qualitative and quantitative high performance liquid chromatographic analysis of monoamine neurotransmitters and metabolites in cerebrospinal fluid and brain tissue using reductive electrochemical detection

The application of reductive coulometric electrochemical detection for analysis of the monoamine neurotransmitters norepinephrine, dopamine, and serotonin and their common metabolites in brain and cerebrospinal fluid following separation by isocratic high performance liquid chromatography is described. The high sensitivity and screening capabilities of coulometric electrodes permits the accurate quantitation of as little as 3-5 pg of these compounds in tissue following a simple single step purification procedure. Moreover, comparison of peak height ratios obtained from analysis of authentic reference standards and tissue samples at selected multiple electrode potentials provides a straightforward means for qualitative evaluation of peak identification and purity during analysis of biological samples. The method is comparatively inexpensive and precise within and between day coefficients of variation for most compounds range from 2-5%. Thirty samples can be run in duplicate in a 24 h period.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry monitoring of anthocyanins in extracts from Arabidopsis thaliana leaves

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.     

Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry

This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0-100microM, selectivity, short analysis time, and limits of detection of 0.1microM for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5microM for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase (pgm) mutant over a 12-h light/12-h dark growth cycle.     

A rapid densitometric method for simultaneous quantification of gallic acid and ellagic acid in herbal raw materials using HPTLC

Gallic acid and ellagic acid are two widely occurring phenolic compounds of plant origin, to which many biological activities including anticancer and antiviral activity have been attributed. A simple HPTLC method has been developed for the simultaneous quantification of gallic acid and ellagic acid. The method was validated for precision, repeatability, and accuracy. Instrumental precision was found to be 0.083 and 0.78, and the repeatability of the method was found to be 1.07 and 1.50 (% CV) for gallic acid and ellagic acid, respectively. The accuracy of the method was checked by a recovery study conducted at two different levels and the average percentage recovery was found to be 101.02% for gallic acid and 102.42% for ellagic acid. The above method was used for the quantification of gallic acid and ellagic acid content in seeds of Abrus precatorius Linn., whole plant of Phyllanthus maderaspatensis Linn., and flowers of Nymphaea alba Linn. The proposed HPTLC method for the simultaneous quantification of gallic acid and ellagic acid was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control of herbal raw materials and for the quantification of these compounds in plant materials.     

AUTHENTIC in vivo ABSORPTION SPECTRA FOR CHLOROPHYLL IN LEAVES AS DERIVED FROM in situ AND in vitro MEASUREMENTS*

On the basis of a comprehensive treatment of light propagation in plant tissue the first authentic in vivo absorption spectrum of chlorophyll in a leaf (of Catalpa bignonioides) has been elucidated. An experimental-theoretical procedure is described, which permits derivation of the authentic chlorophyll spectrum by purifying the in vivo absorption spectrum of a leaf from the distortions of optical origin: multiple scattering and distributional error. The procedure involves measurements of internal and external light fluxes, spatial characterization of tissue layers, cells, chloroplasts and grana stacks. Furthermore, one has to determine the local chlorophyll concentrations. The theories of radiation transfer and absorption statistics make up the theoretical basis of the procedure. The derived authentic chlorophyll spectrum differs both from the in vivo spectrum of the leaf and (although to a smaller degree) from the in vitro spectrum of chlorophyll-protein complexes extracted from the same leaf.     

Chromatographic determination of plant saponins--an update (2002-2005)

The developments during 2002-2005 in the methods used for saponin analyses in plant material are presented. There were number of papers published on isolation and identification of new saponins by chromatographic techniques. Some new developments can be found in separation techniques or solid and mobiles phases used. Separation of individual saponins is still complicated and time consuming. This is due to the fact that in most of the plant species saponins occur as a multi-component mixture of compounds of very similar polarities. Thus, to isolate single compound for structure elucidation or biological activity testing, a combination of different chromatographic techniques has to be used, e.g. first separation of the mixture to simpler sub-fractions on reversed phase C18 has to be followed by further purification on normal phase Silica gel column. Especially difficult is determination of saponins in plant material as these compounds do not possess chromophores and their profiles cannot be registered in UV. Most HPLC methods apply not only specific registration at 200-210 nm, but these methods are not applicable for determination of many saponins in plant material at levels lower than 200-300 mg/kg. Some new or improved techniques for quantification of saponins in plant material were published in reviewed period. These include further progress in the application of evaporative light scattering detection (ELSD) for saponin profiling and quantification, which is also not only specific but also more sensitive in comparison to 200-210 nm detection. Some progress in development of new applications for liquid chromatography-electrospray mass spectrometry (LC/ESI/MS) for saponin determination has also been done. This method gives highest sensitivity and on line identification of separated saponins and should be recommended for specialized analyses of extracts and pharmaceutical formulas like the validation of a new assay. From non-chromatographic techniques for saponin determination, a sensitive and compound specific ELISA tests for some saponins were developed.