Alok Lehri

DEVELOPMENT AND OPTIMIZATION OF HPLC-PDA-MS-MS METHOD FOR SIMULTANEOUS QUANTIFICATION OF THREE CLASSES OF FLAVONOIDS IN LEGUME SEEDS, VEGETABLES, FRUITS, AND MEDICINAL PLANTS

Certain plants of the daily human diet are an important source of beneficially healthy flavonoids, possessing significant pharmacological activities against various diseases. Flavonoids display an array of chemical diversity based on relative positioning of benzopyrano moiety and aromatic ring. Due to structural diversity of these molecules, simultaneous quantification of these molecules in plant materials becomes difficult. In the present study, a rapid, simple, and sensitive, High Performance Liquid Chromatography-Photo Diode Array Mass Spectrometry (HPLC-PDA-MS-MS) method was developed for the simultaneous determination of three different classes of flavonoids,namely, isoflavonoids, flavanones, and flavonols in legume seeds, vegetables, fruits, and medicinal plants. The method was evaluated for validation parameters, such as linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), specificity, selectivity, and compound stability. The developed method was applied on extracts of different plant samples for quantification of the flavonoids daidzein, genistein, naringenin, quercetin, kaempferol, and rutin. Method developed in this study can be used in the quality control and standardization of plant extracts as well as herbal drugs and formulations. Supplemental materials are available for this article. Go to the publishers online edition of the Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

SIMULTANEOUS SEPARATION AND QUANTIFICATION OF TARGETED GROUP OF COMPOUNDS IN PSORALEA CORYLIFOLIA L. USING HPLC-PDA-MS-MS

A rapid, sensitive, and simple high performance liquid chromatography photo diode array mass spectrometry (HPLC-PDA-MS-MS) method was developed for the simultaneous determination of nine compounds, namely, psoralen (furocoumarin), daidzein, genistein (isoflavonoids), daidzin and genestin (isoflavone glycosides), biochanin A (methoxylated isoflavone), and quercetin, kaempferol, and rutin (flavonols). Chromatographic separation of these nine molecules was performed on RP C18 column within 20 min. Elution was accomplished by the application of trifluoroacetic acid (0.05%) in water and methanol in gradient system with flow rate of 1.0 mL/min. PDA and mass spectrometry was employed for qualitative and quantitative analysis. The method is economical in terms of the time expended and the amount of solvent used for each analysis. The calibration curves were linear and ranged between 0.994 to 0.999; limit of detection (LOD) and quantification (LOQ) ranged between 1.02 to 1.18 and 2.23 to 3.34 and recovery ranged between 91.76% to 97.41%. The validated method was successively used to separate and quantitative measurement of these compounds in plant parts of Psoralea corylifolia. The developed method may be used in the quality control and standardization of plant extracts as well as herbal drugs and formulations. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

High-performance thin-layer chromatographic analysis for the simultaneous quantification of four phenolic compounds in green, red, and black fruits of Trapa natans var. bispinosa Roxb. (singhara)

The fruits of Trapa natans var. bispinosa Roxb. (singhara) of nutraceutical importance are commercially consumed in food commodities. It is nutritious while having several biological activities. In the present study, a simple, rapid, cost-effective, and sensitive highperformance thin-layer chromatography (HPTLC) method was developed for the simultaneous determination of four phenolic compounds viz. gallic (phenolic acid), caffeic acid (hydroxycinnamic acid), quercetin, and kaempferol (flavonols) in seeds and pericarp of green-, red-, and black-colored singhara fruits. The method is economical in terms of the time taken and the amount of solvent used for analysis. Simultaneous separation and quantification of compounds were achieved on HPTLC precoated silica gel 60 F254 aluminum plates using mobile phase of toluene-ethyl acetate-formic acid (13:11:2). Densitometric determination was carried out at λmax 282 nm. The calibration curves were linear ranging between 0.996 and 0.999; the limits of detection and quantification ranged between 86.8–135.0 ng µL−1 and 263.2–409.2 ng µL−1 and recovery ranged between 96.4% and 98.5%. The validated method was successively used to analyze the above compounds in fruit parts of singhara. The amount of phenolic compounds ranged from 0.04% kaempferol (green pericarp) to 2.11% gallic acid (red pericarp). The developed method may be used in the quality control and standardization of plant extracts as well as herbal drugs and formulations having polyphenols. As this study reveals the presence of specific phenolic compounds in green, red, and black pericarp of singhara, the agrowaste shall be a good source for isolation of the above compounds for industrial use.

Development and Validation of HPTLC Densitometric Method for Identification and Quantification of Geraniol in Palmarosa Oil

India is the main producer of palmarosa oil obtained from rosha grass (Cymbopogan martini var. motia) of family Graminae. The essential oil obtained from the grass is rich in geraniol content. The oil is commercially obtained by hydro-steam distillation of rosha grass. Oil of palmarosa and its separated fraction geraniol are widely used in perfumery industry. The palmarosa oil is valued due to geraniol and largely used as base for fine perfumery. In this article, a simple, rapid, cost-effective and accurate method using high-performance thin-layer chromatographic (HPTLC) method has been developed for separation, identification and quantification of geraniol in palmarosa oil. Separation and quantification of palmarosa are achieved by HPTLC using mobile phase of toluene-ethylacetate (92.5:7.5) followed by separation on precoated silica gel 60 F254 aluminium plates and densitometric determination carried out after derivatization with vanillin-sulfuric acid reagent at λmax, 400 nm, in absorption-reflectance mode. The calibration curves were linear in the range of (1–7 µg) and all the distinguished bands were observed at 400 nm. The method was also evaluated for different validation parameters such as linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), specificity, selectivity and sample stability. The amount of geraniol varied from 78.3% to 78.9% by HPTLC and 78.2% to 78.7% by gas chromatographyflame-ionization detector (GC-FID).

Simultaneous quantification of six phenolic compounds in various parts of Moringa oleifera Lam. using high-performance thin-layer chromatography

Fresh pods of Moringa oleifera with nutraceutical importance are widely consumed in food commodities as vegetables. It is nutritious and it also has several biological activities. In the present study, a simple, rapid, cost-efective, and sensitive high-performance thin-layer chromatography (HPTLC) method was applied for the simultaneous determination of six phenolic compounds, viz., gallic (phenolic acid), p-coumaric, cafeic acid (hydroxycinnamic acid), chlorogenic acid (cinnamic acid derivative), quercetin and kaempferol (flavonols) in flowers, pods, leaves, twigs, and seeds of M. oleifera. Simultaneous separation and quantification of compounds were achieved on HPTLC pre-coated silica gel 60 F254 aluminum plates using the mobile phase toluene—ethyl acetate—formic acid (14:10:1). Densitometric determination was carried out at λmax 282 nm. The calibration curves were linear, ranged between 0.984 and 0.998; the limit of detection and quantification ranged between 110.8 ng mL-1 and 142.3 ng mL-1, and 301.6 ng µL-1 and 410.8 ng µL-1; and recovery ranged between 96.2% and 97.9%. The validated method was successively used to analyze the above compounds in the plant parts of M. oleifera. The amount of the total phenolic content and specifc phenolic compounds ranged from 4.86 mg g-1 (gallic acid equivalent [GAE]) to 14.79 mg g-1 (GAE) and 0.007% quercetin (fower and fower with pods) to 0.099% gallic acid (pods of 15 days). This study reveals that the presence of specific phenolic compounds in M. oleifera shall be a good source for the isolation of the above-mentioned compounds for industrial use.

Biochemical composition of Betula utilis D. Don bark, collected from high altitudes of Indian Himalayas

Betula utilis (family Betulaceae) is an important medicinal plant that grows in high altitudes of Himalayan region of India. The present study is aimed to determine the biochemical composition in the bark of Betula utilis using HPLC, HPTLC and GCMS methods. The optimization of solvent system for maximum extraction yield has been carried out. Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and Antioxidant Activity (AA) were estimated in various solvent extracts. Maximum extractive yield (14.56%), TPC (14.47 mg/g), TFC (20.10 mg/g) in 75% ethanol and scavenging activity (92.89%) were found in 50% ethanol. Pentacyclic triterpenoids (betulin, tupeol, oleanolic acid, beta-sitosterol) were characterized and quantified using HPLC, HPTLC and GCMS found close within limits. Betulin was found maximum in ethanol.

Extraction of polyphenols from Trewia nudiflora L. and its antioxidant activity

Total phenoliccontent(TPC), specific polyphenols (Gallic, ellagic and protocatechuic acid),antioxidant activity (AOA), free radical scavenging activity (FRSA), lipid peroxidation (LPO), inhibition of nitroblue tetrazolium chloride (NBT) reduction caused by superoxide anions and reducing power (RP) through various extraction optimization parameters using solvents of different polarity, time, temperature and pH variation were evaluated in fruits, leaves, twigs and bark of Trewia nudiflora. The extraction parameters exhibited a broad range of TPC inplant parts varying from 7.9–668.6mg/g gallic acid equivalent(GAE), AOA 14.6–82.9% and RP 0.47–7.2ASE/ml. FRSA, measured as IC50 invarious antioxidant assays ranged from 0.014–4.1mg/ml. The extracts of all plant parts showed significant protective effect against Fentons reaction on supercoiled plasmid DNA pUC 18 assayed by agarose gel electrophoresis. Fruits were found to possess the highest TPC, AOA and FRSA in all the tested models, with reduction in the activity, in order of fruits > leaves > twigs > bark.