Determination of the appetite suppressant P57 in Hoodia gordonii plant extracts and dietary supplements by liquid chromatography/electrospray ionization mass spectrometry (LC-MSD-TOF) and LC-UV methods

Hoodia gordonii is traditionally used in South Africa for its appetite suppressant properties. P57AS3 (P57), an oxypregnane steroidal glycoside, is the only reported active constituent from this plant as an appetite suppressant. Effective quality control of these extracts or products requires rapid methods to determine P57 content. New methods of liquid chromatography/mass spectrometry (LC/MS) and LC-UV for analysis of P57 from H. gordonii have been developed. The quantitative determination of P57 was achieved with a Phenomenex Gemini (Torrance, CA) reversed-phase column using gradient mobile phase of water and acetonitrile, both containing 0.1% acetic acid. The method was validated for linearity, repeatability, and limits of detection and quantification. Good results were obtained in terms of repeatability (relative standard deviation <5.0%) and recovery (98.5-103.5%). The developed methods were applied to the determination of P57 for H. gordonii plant samples, one related genus (Opuntia ficus-indica), and dietary supplements that claim to contain H. gordonii.     

Quantification of appetite suppressing steroid glycosides from Hoodia gordonii in dried plant material, purified extracts and food products using HPLC-UV and HPLC-MS methods

High-performance liquid chromatography (HPLC)-UV and HPLC-Mass Spectrometry (MS) methods were developed for the quantitative analysis of the family of Hoodia gordonii steroid glycosides with appetite suppressing properties in dried plant material, in purified and enriched extracts and in various prototype food-products fortified with H. gordonii extracts. For solid materials, e.g. dried plants or for non-fatty foods, extraction of the steroid glycosides is performed using methanol. For products where the steroid glycosides are present in an oil matrix, direct injection of the oil after dilution in tetrahydrofuran is applied. The HPLC separation is performed on an octyl-modified reversed-phase column in the gradient mode with UV detection at lambda = 220 nm. Quantification is performed against an external calibration line prepared using either one of the pure steroid glycosides or geranyl-tiglate. Short- and long-term repeatabilities of the methods are better than 3 and 6%, respectively. Recoveries are better than 85%, even in the analysis of the least abundant steroid glycosides in a complex yoghurt drink. Linearity is better than 3-4 orders of magnitude and the detection limits are below approximately 2 microg g(-1) for the individual steroid glycosides in dried plant material and food products. HPLC-MS is used to confirm that the steroid glycosides contain the characteristic steroid core, the carbohydrate chain and the tigloyl group.     

Identification and structural characterization of steroidal glycosides in Hoodia gordonii by ion-trap tandem mass spectrometry and liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

Electrospray ion-trap tandem mass spectrometry (ESI-MS/MS) and high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) were used to identify and characterize eight C-21 steroidal glycosides in Hoodia gordonii. A generalized fragmentation pathway was proposed by comparing the spectra acquired for eight C-21 steroidal glycosides. The steroidal glycosides in Hoodia gordonii have been classified into two major core groups: hoodigenin A and calogenin. Using the ESI-TOF method, the major core peak ions generated by hoodigenin A glycosides are m/z 313 and 295 and by calogenin glycosides are m/z 479, 461, 299 and 281, respectively. In the MS/MS spectra, fragmentation reactions of the [M+Na](+) ion were recorded to provide structural information about the glycosyl and aglycone moieties. The data illustrates the ability of positive mode ESI for the identification of hoodigenin A and calogenin glycosides, including the nature of the hoodigenin A and calogenin core, the number of sugar residues and the type of saccharide moiety.     

Chemical fingerprint of Hoodia species,dietary supplements,and related genera by using HPTLC

A HPTLC method was developed for simple and rapid chemical fingerprint analysis of four Hoodia species, dietary supplements that claim to contain Hoodia gordonii, and plants from genera related to Hoodia. HPTLC was performed on precoated silica 60F₂₅₄ plates with dichloromethane/methanol/water 75:17:2.2 by volume, as mobile phase. Evaluation of the HPTLC plates was done by using the CAMAG DigiStore2 digital system with winCATS software. The authentication of H. gordonii was achieved by comparing the band colors and R_f values for TLC fingerprints with those of 11 standard compounds including P57. The developed method was successfully applied for the identification of the 11 pregnane glycosides for four different species of Hoodia, 24 related genera and 13 dietary supplements that claim to contain H. gordonii. Different sample matrices were successfully analyzed, providing a wide range of applicability for this method, including gels, capsules, tablets, sprays, teas, snack bars, powders, and juices. The developed method was validated for specificity, stability, repeatability, and robustness. The results of HPTLC method were verified by LC‐UV‐MS method.     

Optimization and validation of a high-performance liquid chromatography method for the analysis of cardiac glycosides in Digitalis lanata

In this study, a simple and reliable HPLC method for the qualitative and quantitative analysis of cardiac glycosides in Digitalis lanata Ehrh. raw material was developed and applied to healthy and phytoplasma-infected plants. The target analytes cover a broad range of secondary metabolites, including primary, secondary and tertiary glycosides and the corresponding aglycones. The sample preparation was carried out by sonication of the plant material with 70% (v/v) aqueous methanol at room temperature, followed by reversed-phase solid-phase extraction purification from interfering pigments. The HPLC analyses were performed on a Symmetry C₁₈ column (75 mm × 4.6 mm I.D., 3.5 μm), with a gradient elution composed of water and acetonitrile, at a flow rate of 1.0 mL/min. The column temperature was set at 20 °C and the photodiode array detector monitored the eluent at 220 nm. The method was validated with respect to ICH guidelines and the validation parameters were found to be highly satisfactory. The application of the method to the analysis of D. lanata leaves indicated that air-drying was the optimum method for raw material processing when compared with freeze-drying. The analysis of healthy and phytoplasma-infected plants demonstrated that the secondary metabolite mainly affected by the pathogen presence was lanatoside C (153.2 μg/100 mg versus 76.1 μg/100 mg). Considering the importance of D. lanata plant material as source of cardiac glycosides, the developed method can be considered suitable for the phytochemical analysis and for the quality assurance of D. lanata used for pharmaceutical purpose.     

Cardenolide and oxypregnane glycosides from the root of Asclepias incarnata L

Twenty-nine new oxypregnane glycosides were obtained along with two known cardenolides, frugoside and gofruside, and three known 12-O-acylated pregnane glycosides from the roots of Asclepias incarnata L. (Asclepiadaceae). By detailed studies of the 1H- and 13C-NMR spectra, the structures were determined to be tri- to penta glycosides of isolineolon, 12-O-acetyllineolon, ikemagenin, 12-O-benzoylisolineolon, and two new 12-O-acylated pregnanes.     

Expeditious synthesis of saponin P57, an appetite suppressant from Hoodia plants

Pregnane glycoside P57, the appetite suppressant component from Hoodia, was synthesized expeditiously, featuring preparation of the aglycone Hoodigogenin A from digoxin and assembly of the deoxytrisaccharide with glycosyl o-alkynylbenzoates as donors.     

Determination of flavonoids in cultivated sugarcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography-UV detection

A high-performance liquid chromatography (HPLC) method with photo-diode array (DAD) detection was developed to separate and quantify flavonoids in sugarcane leaves and bagasse (= the crushed sugarcane refuse from juice extraction), and in sugarcane juice. Sugarcane flavonoids consist of a complex mixture of aglycones and glycosides (including flavonolignan glycosides), and the HPLC-UV method herein proposed is suitable for their quantification as total flavonoids. This method was applied to analyze samples of cultivated sugarcane, commercial juice and transgenic sugarcane leaves. Sugarcane leaves proved a promising source of flavonoids: an average of 1.10 mg of total flavonoids/g plant material was found in fresh leaves. Moreover, the flavonoid content of sugarcane juice (0.6 mg/mL) is comparable to other food sources of flavonoids previously reported. Transgenic sugarcane leaves ("Bowman-Birk" and "Kunitz") were compared with non-modified ("control") plant samples using the proposed HPLC-UV method, which indicated that the content of total flavonoids in transgenic plants is different from that in non-modified sugarcane.     

High-performance liquid chromatographic determination of secondary cardiac glycosides in Digitalis purpurea leaves

An analytical method for the determination of secondary cardiac glycosides in Digitalis purpurea leaves by high-performance liquid chromatography (HPLC) is described. The procedure consisted of extraction of dry leaf powder with ethanol-chloroform (2:1) and clean-up by Sep-Pak cartridges prior to HPLC analysis. HPLC was performed on an octylsilyl bonded silica column, using acetonitrile-methanol-water (4:4:5) for trisdigitoxosides and acetonitrile-methanol-water (8:30:43) for strospeside; the effluent was monitored by ultraviolet detection (at 220 nm). Quantitation of these cardiac glycosides was carried out by the internal standard method. The amounts of digitoxin, gitoxin, gitaloxin and strospeside per 100 mg of dry leaf powder were estimated to be 22.6, 14.0, 54.7 and 1.9 micrograms, respectively. The method is sufficiently sensitive and reproducible to assay secondary glycosides in Digitalis purpurea leaves.     

Qualitative and quantitative analysis of phenolic acid glycosides in Ginkgo biloba L. leaf,G. biloba leaf extract and its injection

Ginkgo biloba L. leaf (GBL) is one of the most commonly used medicinal plants in the world. Phenolic acids with biological activities have a relatively high content in G. biloba leaf extracts (GBE); therefore they are of great significance for the quality control of GBL, GBE and its preparations. However, there have been few studies focused on their analysis. In this work, 12 phenolic acids, including 11 phenolic acid glycosides, were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC–Q-TOF/MS). Then, a method combining enzymolysis with HPLC was established for quantification of phenolic acid glycosides. It was found that the aglycones of phenolic acid glycosides mainly comprised five phenolic acids: 2,4,6-trihydroxybenzoic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid and p-coumaric acid. The quantitative method was validated, and the correlation coefficient (0.9993–0.9999), recovery (≥88.4%), repeatability (≤0.8%), and inter-day precision (≤5.5%) were satisfactory. Finally, the contents of glycosides of five phenolic acids in GBL, GBE and GBE injection from different sources were determined by the developed method. The method was accurate, repeatable and practicable, which could be helpful for the quantification of phenolic acid glycosides in other products containing GBL or GBE.     

Analysis and retention behavior of isoflavone glycosides and aglycones in Radix Astragali by HPLC with hydroxypropyl‐β‐cyclodextrin as a mobile phase additive

In order to determine isoflavone glycosides (calycosin‐7‐O‐β‐d ‐glucoside and formononetin‐7‐O‐β‐d ‐glucoside) and aglycones (calycosin and formononetin), a simple HPLC method with isocratic elution employing hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as a mobile phase additive was developed. Various factors affecting the retention of isoflavone glycosides and aglycones in the C₁₈ reversed‐phase column, such as the nature of cyclodextrins, HP‐β‐CD concentration, and methanol concentration, were systematically studied. The results show that HP‐β‐CD, as a very effective mobile phase additive, can markedly reduce the retention of isoflavone glycosides and aglycones, and the decrease magnitudes of isoflavone aglycones are more than those of their glycosides. The role of HP‐β‐CD in the developed HPLC method is attributed to the formation of the inclusion complexes between isoflavone glycosides (or aglycones) and HP‐β‐CD. So, the apparent formation constants of the isoflavone glycosides (or aglycones)/HP‐β‐CD inclusion complexes also were investigated. Isoflavone glycosides (and aglycones) form the 1:1 inclusion complexes with HP‐β‐CD, and the isoflavone aglycones/HP‐β‐CD complexes are more stable than the isoflavone glycosides/HP‐β‐CD complexes. Finally, the optimized method was successfully applied for the determination of isoflavone glycosides and aglycones in Radix Astragali samples.     

Analysis of flavanone-7-O-glycosides in citrus juices by short-end capillary electrochromatography

The separation of the major flavanone-7-O-glycoside constituents of Citrus was carried out by isocratic reversed phase capillary electrochromatography using a 75 microm i.d. silica fused column packed with 5 microm ODS silica gel. In comparison to HPLC mode, capillary electrochromatography resolution of flavanone glycosides was obtained with a high selectivity factor. Optimum separation conditions were found using a mixture of ammonium formate (pH 2.5)--acetonitrile (8:2, v/v) as the mobile phase by the short-end injection mode. Under these conditions all the investigated flavanones were baseline-resolved within short analysis time (i.e. between 5 and 10 min). A study, evaluating the intra- and inter-day repeatability as well as limit of detection and method linearity, was developed in accordance with the analytical procedures for method validation. The developed method was applied for the quantitative analysis of flavanone glycosides in commercial fruit juices (sweet orange, lemon and grapefruit).     

Qualitative and quantitative analyses of Epimedium wushanense by high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem mass spectrometry

A new HPLC-DAD-ESI-MS(n) method was developed for rapid separation, characterization and quantitation of flavonoids in Epimedium wushanense, a popular Chinese herbal medicine. For qualitative identification, a total of 37 compounds were characterized from the underground and aerial parts of E. wushanense. Among them, 28 compounds were prenylated flavonoids, and 23 were confirmed by comparing with reference standards. For quantitative analysis, 12 major flavonoids including kaempferol glycosides, desmethylicaritin glycosides, and icaritin glycosides were simultaneously determined by HPLC/UV. Samples were separated on a Waters Symmetry C(18) column at 35 °C eluted with a gradient three-component mobile phase of acetonitrile, methanol, and water containing 0.03% v/v formic acid. All the flavonoids showed good linearity (r(2) ≥0.9997). The recoveries varied from 92.6 to 106.1% at three concentration levels. This method was applied to the determination of 20 samples of different geographical sources, harvesting time, and plant parts. Contents of the predominant flavonoid, epimedin C, ranged from 1.4 to 5.1% in aerial parts and 1.0 to 2.8% in underground parts. The methods established in this paper were simple and reliable and could be used for the quality control of E. wushanense.     

Structural Elucidation and HPLC Analysis of Six Flavone Glycosides from Artemisia frigida Willd.

An investigation of the EtOAc-soluble fraction from the aerial parts of Artemisia frigida Willd.(A. frigida) led to the isolation of three new flavonoid glycosides together with three known compounds. Their structures were elucidated by spectral experiments. At the same time, high-performance liquid chromatographic(HPLC) method was used for the simultaneous determination of the six flavonoid glycosides from the aerial parts of A. frigida. The separation by gradient elution was performed on a Hypersil ODS-2 column(250 mm×4.6 mm, 5 μm) at 30 ℃ with acetonitrile and water as the mobile phase, and monitored by absorbance at 276 nm. The parameters of linearity, precision, accuracy and specificity of the method were evaluated. The recovery of the method is 96.50%―98.01%, and linearity(r>0.9992) was obtained for all the flavonoid glycosides. A high degree of specificity as well as repeatability and reproducibility(relative standard deviation values less than 2.0%) were also achieved. This assay was applied to the determination of six flavonoid glycosides in ten samples. The results indicate that the developed assay method was rapid, accurate, reliable and could be readily utilized as a quantitative analysis method for A. frigida.     

Simultaneous determination of saponins and a flavonoid from aerial parts of Zygophyllum coccineum L

Triterpenoid saponins are a class of glycosides with a wide range of bioactivities, which make them interesting research candidates. Zygophyllum coccineum is an Egyptian desert plant rich in triterpenoid saponins. Reviewing the relevant literature, no data concerning the HPLC or ultra-performance LC (UPLC) analysis of Zygophyllum content were found. This paper presents two methods, HPLC-UV and UPLC-UV-evaporative light scattering detector (ELSD)/MS, for the simultaneous determination of 10 compounds in the alcohol extract of Z. coccineum. The HPLC method uses a C18 column and water-acetonitrile (both containing 0.1% trifluoroacetic acid) gradient system. The separation was achieved within 32 min. The developed UPLC method simultaneously detects and quantifies the 10 compounds using an Acquity UPLC BEH Shield RP18 column and reagent alcohol-acetonitrile (80/20, v/v) and water (both containing 0.5% formic acid) gradient system within 14 min with UV, ELS, and MS detectors. The methods were used to analyze another species, Z. simplex, and results revealed a great variation between the secondary metabolite pattern of both species.     

Rapid quantification of iridoid glycosides analogues in the formulated Chinese medicine Longdan Xiegan Decoction using high-performance liquid chromatography coupled with mass spectrometry [corrected

Longdan Xiegan Decoction (LXD) is a formulated preparation composed of 10 ingredient herbs, with iridoids as the main bioactive components. In this study, a rapid, simple and reliable method of simultaneous determination of four iridoid glycosides in LXD using high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (MS) was first developed and validated. The four iridoid glycosides references were isolated from LXD extract and purified using a preparative HPLC chromatography. The sample preparation for quantification comprised of a simple ultrasonic extraction and the satisfactory chromatographic separation of the four structurally similar iridoid glycosides was effected in less than three minutes on a CAPCELL PAK C(18) MGII column (3 microm, 100 mm x 2.0 mm), using an elution system of 10% methanol and their concentrations in different batches of LXD and ingredient herbs were simultaneously determined by HPLC-MS/MS using a multiple reaction monitoring (MRM) mode. The method was validated with respect to the overall intra- and inter-day variation (RSD less than 8%) and the limits of quantification for the four iridoid glycosides were 35, 20, 37 and 33 ng/mL, respectively.     

Identification and quantification of eight flavones in root and shoot tissues of the medicinal plant huang-qin (Scutellaria baicalensis Georgi) using high-performance liquid chromatography with diode array and mass spectrometric detection

A method of analysis of eight flavones using high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS) in root and aerial tissues of the medicinal plant Scutellaria baicalensis was developed. The identity of the analytes was confirmed using retention time, UV-vis and mass spectral comparisons to commercial standards. Both UV-vis and mass spectral patterns were characterized for glycosylated flavones. Two additional flavone glycosides were tentatively identified as chrysin-7-glucuronide and wogonoside, but not quantified. Greenhouse and in vitro-grown tissues were analyzed with flavone concentrations ranges of 0.14-150 and 0.030-1.7 microg/mg for greenhouse root and shoot tissue, respectively, and 0.0068-6.4 and 0.082-1.5 microg/mg for in vitro-grown roots and shoots, respectively.     

Direct identification of phenolic constituents in Boldo Folium (Peumus boldus Mol.) infusions by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry

A very simple and direct method was developed for the qualitative analysis of polyphenols in boldo (Peumus boldus Mol., Monimiaceae) leaves infusions by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSⁿ). The phenolic constituents identified in infusions of the crude drug Boldo Folium were mainly proanthocyanidins and flavonol glycosides. In the infusions, 41 compounds were detected in male and 43 compounds in female leaf samples, respectively. Nine quercetin glycosides, eight kaempferol derivatives, nine isorhamnetin glycosides, three phenolic acids, one caffeoylquinic acid glycoside and twenty one proanthocyanidins were identified by HPLC-DAD and ESI-MS for the first time in the crude drug. Isorhamnetin glucosyl-di-rhamnoside was the most abundant flavonol glycoside in the male boldo sample, whereas isorhamnetin di-glucosyl-di-rhamnoside was the main phenolic compound in female boldo leaves infusion. The results suggest that the medicinal properties reported for this popular infusion should be attributed not only to the presence of catechin and boldine but also to several phenolic compounds with known antioxidant activity. The HPLC fingerprint obtained can be useful in the authentication of the crude drug Boldo Folium as well as for qualitative analysis and differentiation of plant populations in the tree distribution range.     

Application of liquid chromatography/electrospray ionization tandem mass spectrometry to the analysis of polyphenolic compounds from an infusion of Byrsonima crassa Niedenzu

A fast and reliable method, based on high-performance liquid chromatography coupled to electrospray ionization ion trap tandem mass spectrometry (HPLC/ESI-ITMS), was developed to investigate the infusion prepared from the leaves of Byrsonima crassa Niedenzu (Malpighiaceae), a native plant used in Brazil against gastric disorders. The use of on-line reverse-phase HPLC/ESI-ITMS allowed separation of three major classes of compounds and identification of over 20 very polar compounds characterized as galloylquinic acids, proanthocyanidins, and flavonoid glycosides, as well as the dimeric flavonoid amentoflavone and minor amounts of galloyl hexose and galloyl saccharose. This approach provided data that will allow establishment of a method for a future standardization of the infusion.     

On-line high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry-chemiluminescence assay of radical scavengers in Epimedium

An on-line analysis method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining HPLC with hydrogen dioxide radical scavenging and HPLC-DAD-MS-CL system. The structural identification and activity characteristics of various constituents could be rapidly achieved by the on-line assay of UV, MS and CL in one run. In 4 species of Epimedium studied 32 compounds, including phenolic acids, 8-isopentenyl-flavonoid glycosides and flavonoid glycosides containing a ortho-hydroxyl group, were identified by comparison with authentic standards and published mass data. Among these compounds, phenolic acids and flavonoid glycosides containing an ortho-hydroxyl group could obviously inhibit CL, which suggested their strong radical scavenging activity. These four species each exhibited different active properties, which might correlate to their respective quality. The results indicated that the on-line HPLC-DAD-MS-CL system would be a potential method to rapidly and sensitively screen radical scavengers in herbal medicines, and could display an integrated fingerprint based on different detectors.