Validated HPLC Method for Simultaneous Quantitation of Bergenin,Arbutin, and Gallic Acid in Leaves of Different Bergenia Species

Bergenia species (Saxifragaceae) are important sources of herbal medicines in Asia, mainly in Russia. Various plant parts are valued for their antibacterial, anti-inflammatory, antioxidant sand adaptogenic effect, and used for the dissolution of kidney and bladder stones. In this study a rapid reversed phase liquid chromatography (RP-HPLC) method has been developed for rapid screening and identifying of the main active components in leaf samples of Bergenia accessions. The main goal of this study was to develop an efficient method for the simultaneous identification and detection of arbutin, bergenin and gallic acid from Bergenia leaf samples, which were extracted with a methanolic solvent mixture [methanol:water = 1:1 (v/v)]. Chromatographic separations were performed on a reversed phase Luna C18(2)-HST HPLC column. This chromatographic system provided increased speed and efficiency for separations, without the need for ultra-high pressures. Reversed phase HPLC coupled with diode array detector method was used for the analysis. The method was validated using ICH guidelines. The level of gallic acid was significantly higher in Bergenia crassifolia samples compared to Bergenia cordifolia. However, the samples of the two Bergenia species did not differ substantially regarding the concentrations of arbutin and bergenin. The novel method proved to be fast and allowed sufficient separation and quantification of arbutin, bergenin and gallic acid, the most important bioactive compounds of Bergenia leaves; thus facilitating rapid screening and quality assessment of Bergenia samples of various botanical and geographical origins.     

Separation and evaluation of free radical-scavenging activity of phenol components of green, brown, and black leaves of Bergenia crassifolia by using HPTLC-DPPH* method

A new procedure has been developed to separate and quantify the free radical-scavenging activity of individual compounds from green, brown, and black leaves of Bergenia crassifolia based on the combination of high performance TLC (HPTLC) with a diode array detector (DAD) and postchromatographic 1,1-diphenyl-2-picrylhydrazyl radical (DPPH(*)) derivatization. Free gallic and ellagic acids, arbutin, hydroquinone, and bergenin in the B. crassifolia leaves' extracts were separated by HPTLC and identified. All compounds of the extract excluding bergenin were capable of scavenging DPPH * radicals. From the estimated ID(50) values, it can be seen that the increasing order of activity was gallic acid > arbutin > ellagic acid > hydroquinone > ascorbic acid. The antiradical activity of leaves of B. crassifolia is probably associated to the presence of phenol.     

Simultaneous determination of bergenin and gallic acid in Bergenia ligulata wall by high-performance thin-layer chromatography

A simple and reproducible high-performance thin-layer chromatographic method was developed for the simultaneous determination of bergenin and gallic acid in Bergenia ligulata. Water and methanol were used as the extracting solvents. The concentrations of bergenin and gallic acid in both of these solvents were found to be almost the same. The method involves separation of the components by thin-layer chromatography on a precoated Silica Gel 60 F254 plate with a solvent system of ethyl acetate-formic acid-acetic acid-water (100 + 11 + 11 + 27). The sensitivity of the method for bergenin was 0.30 microg, whereas for gallic acid it was 0.25 microg. The proposed method is precise and sensitive and can be used for the detection, monitoring, and simultaneous quantification of bergenin and gallic acid in B. ligulata.     

Rapid characterization the chemical constituents of Bergenia purpurascens and explore potential mechanism in treating osteoarthritis by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry combined with network pharmacology

In recent years, direct and indirect evidence has been found of the efficacy of the traditional Chinese medicine Bergenia purpurascens in treating arthritis and osteoarthritis. Several major components, such as bergenin and 11‐O‐galloylbergenin, have good anti‐inflammatory activity. Since research on the chemical components of Bergenia purpurascens and related mechanisms for the treatment of osteoarthritis has never been performed, this study aimed to analyze the chemical components of Bergenia purpurascens through ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry technology and the UNIFI screening platform to predict the underlying mechanisms in treating osteoarthritis by analyzing the network pharmacology. In total, 43 chemical constituents were identified, mainly flavonoids (18), phenolic glycosides (13), and organic acids (7). Among them, 16 components were found in Bergenia purpurascens for the first time. Through the analysis of network pharmacology, several potential candidate targets and pathways were initially predicted, including AKT1, MAPK1, and MAPK3, as well as the apoptosis, estrogen, and MAPK signaling pathways. Bergenin, 11‐O‐galloylbergenin, arbutin, catechin‐3‐O‐gallate, and other components play a synergistic role in treating osteoarthritis. This study analyzed the chemical components of Bergenia purpurascens and preliminarily revealed potential mechanisms of treating osteoarthritis, providing a basis for further evaluating the drug's efficacy.     

An LC‐MS/MS method for the simultaneous determination and pharmacokinetic studies of bergenin,chlorogenic acid and four flavonoids in rat plasma after oral administration of a QingGanSanJie decotion extract

A rapid, simple and sensitive, liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for simultaneous determination of bergenin, chlorogenic acid and four flavonoids in a QingGanSanJie preparation in rat plasma. Puerarin was selected as the internal standard (IS). Plasma samples were precipitated with methanol and separated with a reverse phase Agilent Poroshell 120 EC‐C₁₈ column using a gradient mobile phase of methanol–water containing 0.1% formic acid (v/v). A triple quadruple mass spectrometer was used for quantification (limit of detection 0.36–5.55 ng/mL). Intra‐day and inter‐day precisions were within 15% and the average extraction recoveries ranged from 85 to 115% for each analyte. The method allowed simultaneous quantification for the first time of the pharmacokinetics of bergenin, chlorogenic acid and four flavonoids after intragastric administration of a QingGanSanJie extract in Sprague–Dawley rats. It was found that bergenin and chlorogenic acid had typical extravascular administration concentration–time curves; flavonoids had a bimodal distribution improving bioavailability and extending the pharmacodynamics period. Copyright © 2014 John Wiley & Sons, Ltd.     

Validated high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in fruits of Terminalia chebula,Phyllanthus emblica,and Quercus infectoria

A simple and rapid high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in dried fruits of Terminalia chebula, Phyllanthus emblica, and Quercus infectoria has been developed. The chromatographic development was carried out on precoated silica gel 60 F₂₅₄ plates in a mixture of toluene:ethyl acetate:chloroform:formic acid (4:8:1:3 v/v/v/v). The plate was scanned densitometrically at a wavelength of 280 nm. The retention factor value of gallic acid and ellagic acid was found to be 0.63 ± 0.2 and 0.53 ± 0.1, respectively. The developed method was validated in terms of linearity, precision, accuracy, sensitivity, robustness, specificity and stability as per the international conference of harmonization guidelines. The method showed good linear relationship over a range of 100–600 ng/band (gallic acid) and 100–500 ng/band (ellagic acid) with a regression coefficient (r²) of 0.997 (gallic acid) and 0.996 (ellagic acid). The method showed high accuracy (99.65%–100.85%). The percentage relative standard deviation of intra-day and inter-day precision studies was not more than 2%. The method is highly robust and has displayed high specificity. The developed method is new, simple, and accurate and can be successfully employed in routine analysis of raw materials and formulations containing gallic acid and ellagic acid.     

Simultaneous Determination of Phenolic Compounds in Red Wines by HPLC‐UV

Abstract

Ten samples of commercially Italian red wines were analyzed in order to determine the phenolic content. Variations in wine types are largely due to differences in concentration and composition of these compounds. Polyphenolic compounds are a large and complex group of substances which constitute one of the most important quality parameters of wine. These constituents of red wine contribute to organoleptic characteristics and to antioxidant and anti‐inflammatory properties. Moderate wine consumption is associated with several beneficial physiological effects, which include anticancer activities, inhibition of platelet aggregation, and inhibition of LDL oxidation which constitutes the initial stage of the pathogenesis of arteriosclerosis.

For the analysis, reversed‐phase high performance liquid chromatography (HPLC) method coupled with UV‐Vis detection was used. The method uses a gradient elution to identify nine biologically active phenolic constituents: catechin; epicatechin; trans‐ and cis‐resveratrol; gallic, chlorogenic and caffeic acid; rutin and quercetin in red wine samples. The samples are injected directly without any pretreatment. The method is simple, fast, not expensive and shows good linearity for all constituents, and the detection limits ranged from 0.3–1.6 µg/ml for trans‐resveratrol and gallic acid, respectively. Moreover, the samples were analyzed in different times for estimation of stability of these compounds.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

Speciation of phenyltin(IV) compounds using high‐performance liquid chromatography: Part 1. The direct analysis of mixed standard solutions of tetraphenyltin,triphenyltin chloride,triphenyltin hydroxide and triphenyltin acetate

A rapid speciation high‐performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of phenyltin compounds. The commercially important products of triphenyltin‐chloride, ‐acetate, ‐hydroxide and tetraphenyltin were separated by reversed‐phase HPLC on a Waters Spherisorb S5W ODS‐2 (octadecylsilica) column using an isocratic mixture of 90:10 (v/v) acetonitrile:water as the mobile phase at a flow rate of 1 ml min^?1. The phenyltin compounds were detected by UV detection at 254 nm and the total elution time is 8 min. The elution order is triphenyltin‐chloride, ‐acetate, ‐hydroxide and tetraphenyltin. Detection limits were 0.01 ppm for each of the triphenyltin compounds and 0.02 ppm for tetraphenyltin. Spiked water samples containing the three biocidal triphenyltin compounds could also be analysed simultaneously by the above method without the need for any prior derivatization, following extraction with toluene. The versatility of the method in sensing substituent group variations on the phenyl ring was also demonstrated by the successful resolution of the hydroxides, tris(p‐chlorophenyl)tin hydroxide, diphenyl(p‐chlorophenyl)tin hydroxide and triphenyltin hydroxide. Copyright © 2002 John Wiley & Sons, Ltd.     

Determination of Gallic Acid in Rat Plasma by LC-MS-MS

Liquid chromatography–electrospray ionization tandem mass spectrometry has been used for rapid, selective, and sensitive quantitative analysis of gallic acid in rat plasma. Sample pretreatment involved a one-step extraction using ethyl acetate with protocatechic acid as internal standard. Separation was on a C₁₈ column using an isocratic mobile phase, consisting of methanol-0.1% aqueous formic acid (40:60, v/v) at 0.2 mL min⁻¹. The stability of gallic acid was evaluated in acidified and non-acidified plasma. The method was validated then successfully applied to a pharmacokinetic study in rats after oral administration of rhubarb extract.

     

Development and Validation of a LC Method for the Analysis of Phenolic Acids in Turkish Salvia Species

An accurate, simple, reproducible, and sensitive method for determination of rosmarinic, caffeic, chlorogenic, and gallic acids in 12 Salvia species growing naturally in Anatolia, has been developed and validated. The phenolic acids were separated using a μBondapack C₁₈ column by gradient elution with a flow rate of 1.0 mL min⁻¹, which was adjusted to deliver firstly o-phosphoric acid 0.085% in water, 0.085% in methanol, and 0.085% in 2-propanol (80:10:10, v/v/v), then decreased gradually (60:20:20, v/v/v) during 20 min with a flow rate of 1.0 mL min⁻¹. The samples were monitored at 220 nm for gallic acid and 330 nm for rosmarinic, caffeic, and chlorogenic acids using photo-diode array detection. The linear range of detection for gallic, chlorogenic, caffeic, and rosmarinic acids were between 0.051–101.4, 0.207–103.6, 0.100–100, and 0.201–100.5 μg mL⁻¹, respectively. The linearity, range, peak purity, selectivity, system performance parameters, precision, accuracy, and robustness had also acceptable values. The developed method was applied to the flower, leaf, stem, and root parts of the Salvia species.

     

Development of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of fenofibric acid in rat plasma

A rapid and sensitive LC‐MS/MS method for the quantification of fenofibric acid in rat plasma was developed and validated. Plasma samples were prepared by liquid–liquid extraction with a mixture of N‐hexane–dichloromethane–isopropanol (100:50:5, v/v/v). Isocratic chromatographic separation was performed on a reversed‐phase Discovery C₁₈ column (2.1 × 50 mm, 5 µm). The mobile phase was methanol–water–formic (75:25:0.25, v/v/v). Detection of fenofibric acid and the internal standard (IS) diclofenac acid was achieved by ESI MS/MS in the negative ion mode using m/z 317 → m/z 213 and m/z 294 → m/z 250 transitions, respectively. The method was linear from 0.005 to 1.250 µg/mL when 100 μL plasma was analyzed. The lower limit of quantification was 0.005 µg/mL. The intra‐ and inter‐day precision values were below 8.2%, and accuracy ranged from ?0.9 to 2.1% in all quality control samples. The recovery was 90.3–94.7% and 83.3% for fenofibric acid and IS, respectively. Total run time for each sample analysis was 2.5 min. The validated method was successfully applied to a pharmacokinetic study in six rats after oral administration of fenofibrate, the ester prodrug of fenofibric acid (equivalent to fenofibric acid 5 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid fenofibric acid determination. Copyright © 2011 John Wiley & Sons, Ltd.     

Validated LC Method, with a Chiral Mobile Phase, for Separation of the Isomers of Fexofenadine Hydrochloride

A rapid, simple and sensitive high-performance liquid chromatographic method (HPLC) has been developed to assay atomoxetine HCl in capsules. The HPLC analysis used a reversed phase C18 (150 × 4.6 mm i.d. 5 μm particle size) analytical column and a mobile phase consisting of monobasic potassium dihydrogen orthophosphate and acetonitrile (95:5 v/v), with UV detection at 269 nm. The validation data showed that the assay is sensitive, specific and reproducible for determination of atomoxetine HCl in this dosage form. Calibration curves were linear from 1 to 10 μg mL⁻¹ (R ² > 0.997). The accuracy of the method ranged from 98.13 to 101.5%. Mean inter- and intra-assay relative standard deviations (RSD) were less than 1.0%. The proposed method provided an accurate and precise analysis of atomoxetine HCl in its pharmaceutical dosage form.     

Ionic liquids dispersive liquid–liquid microextraction and high‐performance liquid chromatographic determination of irbesartan and valsartan in human urine

An environmentally friendly ionic liquids dispersive liquid–liquid microextraction (IL‐DLLME) method coupled with high‐performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed‐phase C₁₈ column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL‐DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL‐DLLME‐HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration. Copyright © 2012 John Wiley & Sons, Ltd.     

A Rapid and Inexpensive Thin Layer Chromatographic Method for Quantitative Analysis of Bleomycin Complex

Abstract

A densitometric and a spectrophotometric method for rapid but accurate determination of different components of bleomycin injections has been described. The bleomycin components were separated by reversed phase thin layer chromatography on silanized silicagel plates using a mixture of aqueous ammonium nitrate (2.5%) : methanol :: 7 : 3 (v/v) as mobile phase. Assay was done at the absorption maxima of the components (291 nm) by in situ densitometry or by spectroscopy after extracting the drugs from the adsorbent with the mobile phase. Results obtained by both the methods agreed well with each other and with those obtained by an official HPLC method. The densitometric method described was highly suitable for routine quality control of bleomycines as a large number of samples could be analysed within a short time (68 samples/analyst/day).     

A Sensitive High Performance Liquid Chromatographic Method for U-80, 278A,A Substituted Aminotetralin,in Rat Plasma,Whole Blood and Brain Tissue

Abstract

A sensitive analytical method for U-80, 278A, a substituted aminotetralin analogue in rat plasma, whole blood and brain tissue has been developed. The method involves solid phase extraction, efficient reversed phase HPLC and fluorescence detection, and can measure 1 ng/ml from 50 μl samples. During method development, many analogues were investigated and a wide range of extraction and HPLC conditions were explored. These experiments enabled rapid modification and revalidation of the method to support animal experiments with novel analogues.     

A Combined HPLC-VIS Spectrophotometric Method for the Identification of Cosmetic Dyes

Abstract

Ion-pair reversed phase HPLC was observed to give very good separations of 20 representative cosmetic dyes whilst numerical analysis of VIS spectra provided an efficient additional means of identification when similar retention times for different dyes were encountered. The results strongly suggest that a combination of HPLC and rapid scanning VIS spectrophotometry should be very promising, especially when on-line computing facilities are available.     

In vivo metabolism study of bergenin in rats by HPLC‐QTOF mass spectrometry

Bergenin is the major component of Ardisia creanta sims and Rodgersia sambucifolia hemsl with many biological activities. Although bergenin has been used to treat human diseases in China for man years, there is no report regarding its metabolism. This is the first report to separate and identify the metabolites of bergenin in vivo. In the study, HPLC/Q‐TOF‐MS/MS was used to investigate the metabolites of bergenin in vivo by analyzing the rat body fluid and feces samples. Three metabolites of bergenin were finally identified by the TIC chromatograms, and the structures were also confirmed by their MS² spectra. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid determination of rifaximin in rat serum and urine by direct injection on to a shielded hydrophobic stationary phase by HPLC

A simple and rapid reversed‐phase HPLC method for determination of rifaximin in rat serum and urine was developed. Separation of rifaximin from biological matrix was achieved by direct injection of rat serum and urine onto a restricted‐access medium, Supelco LC‐Hisep, a shielded hydrophobic stationary phase, using acetonitrile:water:acetic acid (18:82:0.1 v/v/v) as a mobile phase. The linear range was 0.10–20 µg/mL (r² > 0.999, n = 6), intraday and interday variation was <6.10%. The limits of detection and quantification were 0.03 (signal‐to‐noise ratio >3) and 0.10 µg/mL (signal‐to‐noise ratio >10), respectively. The method was successfully applied to pharmacokinetic studies of rifaximin after an oral administration to rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of methotrexate, 7-hydroxymethotrexate,and 2,4-diamino-N10-methylpteroic acid by LC–MS/MS in plasma and cerebrospinal fluid and application in a pharmacokinetic analysis of high-dose methotrexate

A rapid and robust method for measuring methotrexate (MTX) and its two primary metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N¹⁰-methylpteroic acid (DAMPA), was developed for use in pharmacokinetic studies of plasma and cerebrospinal fluid samples collected from infants with malignant brain tumors. Sample aliquots (100?µL) were prepared for bioanalysis of MTX and metabolites using a Waters Oasis HLB microelution solid-phase extraction (SPE) plate. Chromatography was performed using a Phenomenex Synergi Polar-RP 4 µ 75?×?2.0?mm ID column heated to 40°C. A rapid gradient elution on a Shimadzu HPLC system was used, with mobile phase A consisting of water/formic acid (100/0.1?v/v) and mobile phase B consisting of acetonitrile/formic acid (100/0.1?v/v). Column eluent was analyzed using AB Sciex QTRAP 5500 instrumentation in electrospray ionization mode. The ion transitions (m/z) monitored were 455.2?→?308.1, 471.1?→?324.1, and 326.2?→?175.1 for MTX, 7-OHMTX, and DAMPA, respectively. The method was linear over 0.0022–5.5?µM for MTX, 0.0085–21?µM for 7-OHMTX, and 0.0031–7.7?µM for DAMPA. The method was applied to the analysis of serial plasma samples obtained from infants diagnosed with malignant brain tumors receiving high-dose methotrexate and results were compared to MTX concentrations from an immunoassay based on fluorescence polarization.