S Sagi

Characterization and screening of pyrrolizidine alkaloids and N-oxides from botanicals and dietary supplements using UHPLC-high resolution mass spectrometry.

The UHPLC-QToF-MS analysis of pyrrolizidine alkaloids (PAs) from various parts of 37 botanicals and 7 products was performed. A separation by LC was achieved using a reversed-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. MS-MS detection was used because of its high selectivity, and ability to provide structural information. Free base and N-oxides were observed by this method. PAs were analyzed and detected in plants from three different families, viz., Asteraceae, Boraginaceae and Fabaceae. The Asteraceae family was found to contain senecionine and lycopsamine type PAs. The Boraginaceae family contained lycopsamine and heliotrine type PAs and the Fabaceae family contained senecionine and monocrotaline type PAs. These PAs may serve as important markers for the detection of these plant materials in food and dietary supplements. PAs were identified in 44 samples by comparing their retention times, accurate mass and mass fragmentation patterns with those of 25 reference standards.

Chemical profiling and quantification of monacolins and citrinin in red yeast rice commercial raw materials and dietary supplements using liquid chromatography-accurate QToF mass spectrometry: Chemometrics application.

Red yeast rice (RYR) is prepared by fermenting rice with various strains of the yeast Monascus spp of the Aspergillaceae family. Depending on the Monascus strains and the fermentation conditions, the products may contain monacolins, pigments and citrinin as secondary metabolites. Authentic and commercial RYR samples were analyzed using UHPLC-DAD-QToF-MS for monacolins, pigments and citrinin. A separation by UHPLC was achieved using a reversed-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. Accurate mass QToF spectrometry was used to distinguish isobaric monacolins. Principle component analysis (PCA), a chemometric technique was used to discriminate between authentic RYR, commercial RYR raw materials and dietary supplements. Three authentic RYR samples, 31 commercial RYR raw materials and 14 RYR dietary supplements were analyzed. Monacolin K content in 600mg of authentic RYR samples ranged from 1.2mg to 1.38mg. Amounts of monacolin K in dietary supplements labeled as containing 600mg of RYR varied more than 40-fold from 0.03mg to 2.18mg. Monacolin K content of dietary supplements labeled as containing 1200mg RYR varied more than 20-fold from 0.22mg to 5.23mg. In addition to large variations in quantity of monacolin K found in dietary supplements, RYR dietary supplements contained ratios of monacolins that differed significantly from authentic samples. The results indicated that RYR commercial products are of variable quality and the analytical method is suitable for quality control testing of a variety of RYR products.

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high-performance thin-layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin-7-O-β-glucoside, chamaemeloside, apigenin-7-O-β-glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid-β-ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra- and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6-18 and 16-55 ng/band for six flavonoids and one coumarin, respectively. The intra- and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high-performance thin-layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.

Identification and quantification of vinpocetine and picamilon in dietary supplements sold in the United States

Vinpocetine and picamilon are drugs prescribed in many countries to treat a variety of cerebrovascular disorders. In the United States, vinpocetine and picamilon have never been approved by the US Food and Drug Administration, but they are both available for sale directly to consumers as dietary supplements. We designed our study to determine the accuracy of supplement labels with regard to the presence and quantity of vinpocetine and picamilon. A validated ultra-high performance liquid chromatography-photodiode-array method was developed for the quantification of vinpocetine and picamilon. The separation was achieved using a reversed phase (C-18) column, photodiode array detection, and water/acetonitrile as the mobile phase. Vinpocetine and picamilon were detected at concentrations as low as 10 and 50 ng/mL, respectively. The presence of vinpocetine and picamilon was confirmed using reference standards. Twenty-three supplements labelled as containing vinpocetine were available for sale at two large supplement retail chains; 17 contained vinpocetine with quantities ranging from 0.3 to 32 mg per recommended daily serving. No vinpocetine was detected in six of the sampled supplements. The supplement label implied that vinpocetine was a constituent of lesser periwinkle in three of the supplements. Of the 31 picamilon supplements available for sale from a variety of retailers: 30 contained picamilon in quantities ranging from 2.7 to 721.5 mg per recommended daily serving. We found that consumers cannot obtain accurate information from supplement labels regarding the presence or quantity of vinpocetine and picamilon. Copyright

Identification and Characterization of Indole and Oxindole Alkaloids from Leaves of Mitragyna speciosa Korth Using Liquid Chromatography-Accurate QToF Mass Spectrometry.

Alkaloids have been reported to be the major physiologically active constituents in Mitragyna. An analytical method was developed to provide an alternative, fast method for characterization of alkaloids from various M. speciosa samples. The separation was achieved using an RP octylsilyl (C8) column, MS detection, and a water-acetonitrile with formic acid gradient as the mobile phase. Ultra-HPLC/quadrupole time-of-flight MS analysis and characterization were performed on 12 corynanthe-type indole/oxindole alkaloids obtained from the leaves of M. speciosa Korth. The indoles and oxindoles had an open E ring with or without substitution occurring at the C9 position. The full single mass spectrum of alkaloids showed a strong signal for the protonated molecule [M+H]+. The product ion spectrum of mitragynine type of alkaloids showed strong response at m/z=174.0901 suggestive of an ion containing an odd number of nitrogen atoms corresponding to formula C11H12NO, which is characteristic of indole alkaloids. A multivariate statistical analysis technique, principal component analysis, was used to show discrimination between the M. speciosa samples. The results indicated that the analytical method is suitable for QC testing of various Mitragyna commercial samples and can be used to evaluate market products purported to contain M. speciosa.

Identification and quantification of 1,3-dimethylbutylamine (DMBA) from Camellia sinensis tea leaves and dietary supplements

1,3-dimethylbutylamine (DMBA), is a CNS stimulant, which has recently been identified in multiple dietary supplements and sometimes labeled as a natural constituent of Pouchung tea. DMBA is an homologue of 1,3-dimethylamylamine (DMAA) which the US Food and Drug Administration has attempted to remove from all dietary supplements after DMAA consumption was linked to strokes, heart disease, and sudden death. To address questions concerning the natural origin of DMBA, three independent analytical methods were developed for analyzing authentic tea samples and dietary supplements. A high performance thin layer chromatography (HPTLC) method was developed for the fast screening and chemical fingerprint analysis. Chiral Gas Chromatography-Mass Spectrometry (GC-MS) was used to determine the enantiopurity and a validated Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry (UHPLC-QToF-MS) method was developed for the quantification of DMBA. Using these techniques the presence of DMBA was confirmed using a reference standard and was not detected in any of 25 authentic or commercial samples of Camellia sinensis tea leaves (green tea, black tea, Oolong tea, and Pouchung tea). Of 13 dietary supplements tested, 11 contained DMBA in racemic form and ranged from 0.1 to 214mg per daily dose.

Liquid Chromatography-Electrospray Ionization Mass Spectrometry Analysis of Limonoids and Flavonoids in Seeds of Grapefruits, Other Citrus Species, and Dietary Supplements

A selective UHPLC-DAD-QToF-MS method was developed to screen grapefruit seeds, and the seeds of other Citrus species for limonoid aglycones, acids, glucosides, and flavonoids. These classes of compounds were identified in positive and negative ion modes over a mass-to-charge range from 100-1500. Accurate mass values, elution times, and fragmentation patterns obtained by QToF-mass spectrometry were used to identify or tentatively characterize the compounds detected in the sample of this study. Limonin was the major limonoid in most of the seeds of Citrus species, followed by nomilin. This analytical method was successfully applied for the analysis of commercial extracts and dietary supplements claiming to contain grapefruit seed extract, or extracts made from the seed and other fruit parts such as the peel or pulp. Many commercial products contained large numbers of flavonoids, indicating the use of peel, pulp, or seed coat. This method also permitted detection of synthetic preservatives such as benzethonium chloride, methylparaben, and triclosan in commercial grapefruit seed extract products. Out of the 17 commercial products analyzed, two contained the synthetic antimicrobial agent benzethonium chloride.

Simultaneous Determination of Aegeline and Six Coumarins from Different Parts of the Plant Aegle marmelos Using UHPLC-PDA-MS and Chiral Separation of Aegeline Enantiomers Using HPLC-ToF-MS

A fast UHPLC-PDA method was developed for the simultaneous analysis of one alkaloid, aegeline, and six coumarins, viz., umbelliferone, scopoletin, marmesinin, 8-hydroxypsoralen, angelicin, and marmelosin, from the leaf, fruit, root, and bark of Aegle marmelos. The UHPLC method was validated for linearity, accuracy, repeatability, limits of detection and limits of quantification. The linearity range (r(2) > 0.99) of the seven compounds was found to be 0.5-250 µg/mL, and the limits of detection and limits of quantification for the seven compounds were found to be 0.1 and 0.5 µg/mL, respectively. The developed UHPLC method is simple, fast, and especially suitable for quality control analysis of coumarins and aegeline from A. marmelos and commercial dietary supplements. Single quadrupole mass spectrometry was used for the identification and confirmation of coumarins and aegeline from different plant parts and dietary supplements. In addition, a novel chiral HPLC-ToF-MS method was developed for the resolution of aegeline enantiomers. By applying this chiral method, the distribution of enantiomers of aegeline from different parts of A. marmelos and aegeline-containing dietary supplements is reported for the first time.

Application of anatomy and HPTLC in characterizing species of Dioscorea (Dioscoreaceae)

The edible tubers from different species of Dioscorea are a major source of food and nutrition for millions of people. Some of the species are medicinally important but others are toxic. The genus consists of about 630 species of almost wholly dioecious plants, many of them poorly characterized. The taxonomy of Dioscorea is confusing and identification of the species is generally problematic. There are no adequate anatomical studies available for most of the species. This study is aimed to fill this gap and provides a detailed investigation of the anatomy and micro-morphology of the rhizomes and tubers of five different species of Dioscorea, namely D. balcanica, D. bulbifera, D. polystachya, D. rotundata and D. villosa. The primary features that can help in distinguishing the species include the nature of periderm, presence or absence of pericyclic sclereids, lignification in the phloem, types of calcium oxalate crystals and features of starch grains. The descriptions are supported with images of bright-field and scanning electron microscopy for better understanding of these species. The diagnostic key of anatomical features included in this paper can help distinguish the investigated species unambiguously. Additionally, HPTLC analyses of authentic and commercial samples of the five species are described.