Performance Liquid Chromatography-photodiode Array Research Articles

Technical note: Simultaneous carotenoid and vitamin analysis of milk from total mixed ration-fed cows optimized for xanthophyll detection

Concentrations of retinol, α-tocopherol, and major carotenoids in dairy products are often determined simultaneously by liquid chromatography. These compounds have different polarity and solubility; thus, extracting them simultaneously can be difficult and inefficient. In milks with low carotenoid concentrations, the xanthophylls lutein and zeaxanthin may not be completely resolved using common extraction techniques. A simplified method was developed to optimize extraction efficiency and the limit of detection and limit of quantification (LoQ) of lutein and zeaxanthin in bovine milk without decreasing sensitivity to other vitamins or carotenoids. The developed method evaluates lutein, zeaxanthin, β-carotene, retinol, and α-tocopherol simultaneously by ultra-high performance liquid chromatography-photodiode array detection. Common saponification temperatures (40-60°C) and concentrations of KOH in water (10-50% KOH wt/vol) were evaluated. Multiple solvents were evaluated for optimal xanthophyll extraction (diethyl ether, dichloromethane, hexane, and tetrahydrofuran) following saponification. The limit of detection and LoQ were defined as 3:1 and 10:1 signal-to-noise ratio, respectively. All experiments were performed in triplicate. The optimal saponification procedure was a concentration of 25% KOH at either 40 or 50°C. Saponified extracts solubilized in solutions containing diethyl ether had greater concentrations of lutein- than hexane- or tetrahydrofuran-based solutions, with peak areas above LoQ values. The solution containing diethyl ether solubilized similar concentrations of retinol, α-tocopherol, and β-carotene when compared with other solutions. The proposed optimized method allows for the simultaneous determination of carotenoids from milk with increased lutein and zeaxanthin sensitivity without sacrificing recovery of retinol, α-tocopherol, and β-carotene.

Chromatographic analysis of lypophilic compounds in eggs from organically fed hens

Egg yolks represent a common foodstuff in the human diet and are an important source of nutrients including lipids and carotenoids. The aim of this study was to compare the carotenoid, fatty acid and vitamin E contents of the yolk of eggs from nine hen breeds (Barred Plymouth Rock, Speckled Italian, Black Italian, Red Italian, Rhode Island, Silver Laced Wyandotte, Gold Araucana, Partridge Brahma and Yellow Cochin) raised in barns in an enclosed house. Gas chromatography-mass spectrometry (GC-MS) was used for the fatty acid analyses, and reversed phase-high performance liquid chromatography-photodiode array detection (RP-HPLC-PDA) for carotenoids and vitamin E quantification. The major carotenoids identified were lutein and zeaxanthin, which together represented more than 93% of the total carotenoids, followed by β-cryptoxanthin and β-carotene.The results indicated that the total carotenoid content of the eggs ranged from 16.84–87.31 μg/g egg yolk (average value 49 μg/g egg yolk). The most representative fatty acids found were palmitic acid, stearic acid, oleic acid and linoleic acid. Significant differences in the proportions of α-linolenic acid were observed among the samples. The α-tocopherol content was directly correlated with the total carotenoid content. The results show that the chemical composition of egg yolk varies greatly among hen breeds.

A Snapshot of Viennese Textile History using Multi-Instrumental analysis: Benedict codecasa’s swatchbook

AbstractThe Habsburg Empire was a sovereign dynasty ruled by the Habsburgs between the 15th and 20th centuries. Although its borders were not defined before the 19th century, what is now Austria, Hungary, some areas of the Czech Republic, the Netherlands and Italy were at some point part of the Empire. Starting in the 17th century, the Empire had Vienna as the capital, which was a hub for culture and craft where silk was a valued commodity. Despite the political and cultural importance of the Empire, little is known of its trade practices and sources of raw material. Using a combination of X-Ray Fluorescence (XRF) and High Performance Liquid Chromatography-Photodiode Array Detector (HPLC-PDA) for the study of a Viennese swatch book, we conducted the first systematic approach to understanding the industry. Benedict Codecasa, a prominent merchant active in Vienna between the late 18th and early 19th century sold silk and other textile goods. Authorized by the Royal Court, Codecasa was assumed to sell luxurious and high-quality textiles. However, our results suggested colored goods were dyed with more focus on aesthetics (finding a similar color) rather than quality through unique recipes. This greatly contrasts with other contemporary textile industries praised for their quality and which, in turn, might be related to comparatively lesser quality textiles sold in Vienna.

Oxidation of Selected Phenothiazine Drugs During Sample Preparation: Effects of Varying Extraction Conditions on the Extent of Oxidation.

Characterization of degradation products formed from selected phenothiazine drugs during standard solid-phase extraction (SPE) approaches is described. An analytical method for promethazine (PMZ), chlorpromazine (CPZ) and their respective N-desmethyl and sulfoxide metabolites in biological samples (bone tissue extract and blood) by ultra performance liquid chromatography-photodiode array detection, using mixed-mode SPE for basic drugs was developed. When ethyl acetate:isopropanol:ammonium hydroxide (80:17:3) was used as the elution solvent during method development, extraneous peaks were observed that were absent in the negative controls. Analysis of extracts of PMZ and CPZ individually showed extraneous peaks, including peaks with retention time and UV spectra suggesting the formation of the sulfoxide metabolites, amongst others. Analytes were then extracted individually and analyzed by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The results confirmed the oxidation of PMZ to its sulfoxide and N-oxide metabolites and oxidation of CPZ to its sulfoxide metabolite. Oxidation was also observed in analysis of whole blood, and thus was not specific to bone tissue extract. To determine if extraction with minimal oxidation was possible, extractions using SPE with a different elution solvent system (dichloromethane:isopropanol:ammonium hydroxide) and filtration/pass through extraction (FPTE) with and without evaporation were evaluated. The results demonstrated that the sample preparation method highly influenced the extent of oxidation. FPTE without an evaporation step was the only method that did not measurably induce analyte oxidation.

Chemical profiling of infusions and decoctions of Helichrysum italicum subsp. picardii by UHPLC-PDA-MS and in vitro biological activities comparatively with green tea (Camellia sinensis) and rooibos tisane (Aspalathus linearis)

Several medicinal plants are currently used by the food industry as functional additives, for example botanical extracts in herbal drinks. Moreover, the scientific community has recently begun focusing on halophytes as sources of functional beverages. Helichrysum italicum subsp. picardii (everlasting) is an aromatic halophyte common in southern Europe frequently used as spice and in traditional medicine. In this context, this work explored for the first time H. italicum subsp. picardii as a potential source of innovative herbal beverages with potential health promoting properties. For that purpose, infusions and decoctions were prepared from roots, vegetative aerial-organs (stems and leaves) and flowers and evaluated for in vitro antioxidant and anti-diabetic activities. Samples were also assessed for toxicity in different mammalian cell lines and chemically characterized by spectrophotometric methods and ultra-high performance liquid chromatography–photodiode array–mass-spectrometry (UHPLC-PDA-MS). Results were expressed relating to ‘a cup-of-tea’ and compared with those obtained with green tea (Camellia sinensis) and rooibos tisane (Aspalathus linearis). Tisanes from the everlasting’s above-ground organs, particularly flowers, have high polyphenolic content and several phenolics were identified; the main compounds were chlorogenic and quinic acids, dicaffeoylquinic-acid isomers and gnaphaliin-A. The antioxidant activity of beverages from the everlasting’s above-ground organs matched or surpassed that of green tea and rooibos. Its anti-diabetic activity was moderate and toxicity low. Overall, our results suggest that the everlasting is a potential source of innovative and functional herbal beverages.

Validation of a straightforward high performance liquid chromatographic method for morphine quantitation

BackgroundMorphine is illegal to use unless it is prescribed by a medical doctor. This compound is commonly abused in the form of illicit heroin.MethodsAn easy-to-use method for determining morphine in illicit heroin using high performance liquid chromatography-photodiode array detector (HPLC-PDA) was developed. With the aid of 50:50 acetonitrile:ammonium formate, this target compound traveling in a pentafluorophenyl (PFP) column was successfully separated from other compounds frequently present in the heroin matrix.ResultsThe method was precise with an intra-day relative standard deviation (RSD) ≤ 0.8% and inter-day RSD < 5%. It was able to detect as low as 0.001 mg/mL morphine. The detector’s response was linear (R2 > 0.999) and reliable for morphine quantitation from 0.005 up to 1 mg/mL. Through recovery studies, accuracy of the method was averagely estimated to be 89.54–101.91%.ConclusionsThis method saves time in terms of mobile phase preparation and cuts cost by excluding additional purchase of expensive chemicals. More importantly, it proves to be able to determine the target analyte with sufficient accuracy and precision.

Simultaneous determination of ten compounds in two main medicinal plant parts of Tibetan herb, &lt;i&gt;Pterocephalus hookeri&lt;/i&gt; (CB Clarke) Höeck, by ultra-high performance liquid chromatography-photodiode array

Purpose: To develop an ultra-high performance liquid chromatography (UPLC) - photodiode array (PDA) method to compare the chemical composition of two different medicinal components of Pterocephalus hookeri.Methods: Samples were chromatographically separated in succession using Waters Acquity UPLCR BEH C18 column (2.1 × 100 mm, 1.7 μm) and gradient elution (0.2 % phosphoric acid aqueous -acetonitrile). Using partial least squares discriminant analysis and one-way analysis of variance, attempts were made to distinguish different medicinal parts of P. hookeri.Results: Regression equation for 10 compounds showed good linear regression (R2 &gt; 0.9994). The relative standard deviations of precision, stability, repeatability and recovery were under 5 %. Compared with the aerial plant part, the root had significantly higher levels of sylvestroside I (p &lt; 0.01), cantleyoside (p &lt; 0.001), dipsanosides B (p &lt; 0.01) and dipsanosides A (p &lt; 0.01), but significantly lower levels of loganic acid (p &lt; 0.001), chlorogenic acid (p &lt; 0.01), and isochlorogenic acid (p &lt; 0.01). There were no significant differences between loganin, sweroside and isochlorogenic acid C.Conclusion: The described method is simple, accurate and reproducible, and can be used for the simultaneous determination of 10 major compounds of P. hookeri. The results demonstrate that there is variation in the chemical composition of the aerialpart and root of P. hookeri and that loganic acid and cantleyoside are the primary chemical biomarkers.Keywords: Tibetan medicine, Pterocephalus hookeri, Medicinal parts, Loganic acid and Cantleyoside, UPLC-PDA

Dissipation kinetics of beta-cyfluthrin and imidacloprid in tea and their transfer from processed tea to infusion

Dissipation kinetics of mixed formulation consisting beta-cyfluthrin and imidacloprid in tea crop under an open field ecosystem was investigated. The mixed formulation was applied on tea plant at recommended (27 + 63) and double the recommended (54 + 126g a.i./ha) dose and residues were determined using gas chromatography-electron capture detector and high performance liquid chromatography-photodiode array detector for beta-cyfluthrin and imidacloprid, respectively. The limit of quantification of analytical method was 0.05µg/g and the average recoveries were ranged from 88.36% to 103.49% with relative standard deviations of less than 6% at three spiked levels. The experimental results showed that in the green tea leaves imidacloprid dissipated faster than beta-cyfluthrin with the half-life ranging between 1.20–1.39 and 2.89–3.15days, respectively. The beta-cyfluthrin residues present in the processed tea not transferred into the tea infusion during the infusion process and imidacloprid transferred in the range 43.12–49.7%. On the basis of the transfer of residues from processed tea to infusion, a waiting period of 17 days for tea plucking after pesticide application at recommended dose may be suggested.

Simultaneous estimation of lisofylline and pentoxifylline in rat plasma by high performance liquid chromatography-photodiode array detector and its application to pharmacokinetics in rat.

Lisofylline (LSF) is an anti-inflammatory and immunomodulatory agent with proven activity in serious infections associated with cancer chemotherapy, hyperoxia-induced acute lung injury, autoimmune disorders including type-1 diabetes (T1DM) and islet rejection after islet transplantation. It is also an active metabolite of another anti-inflammatory agent, Pentoxifylline (PTX). LSF bears immense therapeutic potential in multiple pharmacological activities and hence appropriate and accurate quantification of LSF is very important. Although a number of analytical methods for quantification of LSF and PTX have been reported for pharmacokinetics and metabolic studies, each of these have certain limitations in terms of large sample volume required, complex extraction procedure and/or use of highly sophisticated instruments like LC-MS/MS. The aim of current study is to develop a simple reversed-phase HPLC method in rat plasma for simultaneous determination of LSF and PTX with the major objective of ensuring minimum sample volume, ease of extraction, economy of analysis, selectivity and avoiding use of instruments like LC-MS/MS to ensure a widespread application of the method. A simple liquid-liquid extraction method using methylene chloride as extracting solvent was used for extracting LSF and PTX from rat plasma (200μL). Samples were then evaporated, reconstituted with mobile phase and injected into HPLC coupled with photo-diode detector (PDA). LSF, PTX and 3-isobutyl 1-methyl xanthine (IBMX, internal standard) were separated on Inertsil® ODS (C18) column (250×4.6mm, 5μm) with mobile phase consisting of A-methanol B-water (50:50v/v) run in isocratic mode at flow rate of 1mL/min for 15min and detection at 273nm. The method showed linearity in the concentration range of 50-5000ng/mL with LOD of 10ng/mL and LLOQ of 50ng/mL for both LSF and PTX. Weighted linear regression analysis was also performed on the calibration data. The mean absolute recoveries were found to be 80.47±3.44 and 80.89±3.73% for LSF and PTX respectively. The method was successfully applied for studying the pharmacokinetics of LSF and PTX after IV bolus administration at dose of 25mg/kg in Wistar rat. In conclusion, a simple, sensitive, accurate and precise reversed-phase HPLC-UV method was established for simultaneous determination of LSF and PTX in rat plasma.

Relationship of Mycotoxins Accumulation and Bioactive Components Variation in Ginger after Fungal Inoculation.

Ginger has got increasing worldwide interests due to its extensive biological activities, along with high medical and edible values. But fungal contamination and mycotoxin residues have brought challenges to its quality and safety. In the present study, the relationship of content of mycotoxins accumulation and bioactive components variation in ginger after infection by toxigenic fungi were investigated for the first time to elucidate the influence of fungal contamination on the inherent quality of ginger. After being infected by Aspergillus flavus and Aspergillus carbonarius for different periods, the produced mycotoxins was determined by an immunoaffinity column clean-up based ultra-fast liquid chromatography coupled with tandem mass spectrometry, and the main bioactive components in ginger were analyzed by ultra performance liquid chromatography-photodiode array detection. The results showed that consecutive incubation of ginger with A. flavus and A. carbonarius within 20 days resulted in the production and accumulation of aflatoxins (especially AFB1) and ochratoxin A, as well as the constant content reduction of four bioactive components, which were confirmed through the scanning electron microscope images. Significantly negative correlation was expressed between the mycotoxins accumulation and bioactive components variation in ginger, which might influence the quality and safety of it. Furthermore, a new compound was detected after inoculation for 6 days, which was found in our study for the first time.

Simultaneous Analytical Method Development of 6-Mercaptopurine and 6-Methylmercaptopurine in Plasma by High Performance Liquid Chromatography-Photodiode Array

Objective: 6-Mercaptopurin is antineoplastic drug that is included in antimetabolite group and is used in acute lymphoblastic leukemia medication. 6-Mercaptopurin is inactive pro-drug that will be metabolized into metabolites. One of its metabolites is 6-methylmercaptopurine. This study is aimed to optimize the analytical conditions and perform validation for the analysis of 6-mercaptopurine and 6-methylmercaptopurine in plasma. Method: Separation was performed using Waters 2996 HPLC, C18 SunfireTM column (5μm, 250 x 4.6 mm) with the mobile phase containing water-methanol-acetonitrile with gradient elution, and detected at 303 nm. 5-Fluorouracil was used as internal standard. Plasma extraction was done by liquid-liquid extraction using dichloromethane. Result: The method was linear at concentration range of 2.0 – 200.0 ng/mL with r > 0.9991 for 6-mercaptopurine and 20 – 2000 ng/mL with r > 0.9993 for 6-methylmercaptopurine. Accuracy and precision within-run and between-run fulfill the acceptance criteria with % RE and relative standard deviation (% RSD) ≤ 20% (LLOQ) and ≤ 15% (QC samples). 6-Mercaptopurine and 6-methylmercaptopurine was stable in plasma at least for 21 days when stored at -20oC. Conclusion: The bio-analytical method was sensitive, selective and all the parameters fulfilled the acceptance criteria of the EMA Bio-analytical Method Validation Guideline, 2011. Keywords: 5-fluorouracil, 6-mercaptopurine, 6-methylmercaptopurine, HPLC, validation

Identification and evaluation of antioxidant components in the flowers of five Chimonanthus species

Chimonanthus Lindl. has seven family members with Ch. praecox blooming in winter and others in autumn. The plants are widely used in landscaping, fragrance industry, as well as production of medicinal material because of their flower fragrance. Both Chimonanthus flowers and leaves contain abundant secondary metabolites. But it is unclear whether the flowers from different species have distinct ingredients and antioxidant activity. To address the question, we evaluated the main compositions of the flowers from five Chimonanthus species and analyzed the antioxidant activity of the compositions. The results indicated that flavonoids and coumarins are the two major types of substances with antioxidant capacity in Chimonanthus flowers. Totally nine antioxidative compounds, including scopolin and scopoletin, were identified based on the high performance liquid chromatography-photo diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD/ESI–MS). All the antioxidant compounds were confirmed by pre-column 2, 2-diphenyl-1- picryhydrazyl (DPPH) assay. Rutin (highest in content), isoquercitrin and quercetin appeared stronger scavenging DPPH radical ability than the other five compounds, mainly due to their ortho-dihydroxyl groups. Among the five Chimonanthus species, Ch. praecox flower is most abundant in secondary metabolites and shows most potent antioxidant activity. The other four flowers contain the same types of ingredients with Ch. praecox, but exhibit lower antioxidant activity. The results obtained from this comprehensive analysis provide a theoretical basis for the utilization of the plant resources of Chimonanthus genus.

Deciphering the influence of column chemistry and mass spectrometry settings for the analyses of geometrical isomers of L-chicoric acid.

Resolving the chemo-diversity of plant extract samples is an essential step for in-depth analyses of natural products which often exhibit promising biological activities. One of the challenges in this endeavor has been the confident differentiation of geometrical isomers. In this study, we investigated these aspects in chromatography (column chemistry and mobile phase composition) and mass spectrometry settings with regards to better differentiation of geometrical isomers. A standard of a hydroxycinnamic acid (HCA) derivative, L-chicoric acid (L-CA) - a di-acylated caffeoyltartaric acid ester found in a number of plant families - was used. Geometrical isomers of L-CA were formed by exposing the compound to ultraviolet (UV) radiation, to mimic the natural environment. The high performance liquid chromatography photo-diode array (HPLC-PDA) and ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) platforms were used to analyze the trans and cis geometrical isomers of L-CA. The HPLC-PDA results confirmed the generation of two cis geometrical isomers following UV exposure of the authentic trans-L-CA standard. Furthermore, the HPLC-PDA analyses demonstrated that the changes in both column chemistry (reverse-phase: C18, biphenyl, phenyl-hexyl and pentafluorophenyl propyl) and mobile phase composition (aqueous acetonitrile and aqueous methanol) affect the chromatographic elution profiles of the L-CA isomers. The MS results, on the other hand, revealed undisputed fragmentation differences between the geometrical isomers of L-CA. Thus, this study demonstrates that the identification of the L-CA isomers can be achieved more efficiently and confidently with good chromatography coupled to well-optimized mass spectrometry conditions, a requirement which has been proven impossible with other types of HCA derivatives. Moreover, differences in the binding modes of L-CA geometrical isomers to the HIV type 1 integrase enzyme were observed, suggesting a synergistic anti-HIV-1 activity of these isomers.

An initial assessment of naproxen, ibuprofen and diclofenac in Ladysmith water resources in South Africa using molecularly imprinted solid-phase extraction followed by high performance liquid chromatography-photodiode array detection

An initial assessment of naproxen, ibuprofen and diclofenac in Ladysmith water resources in South Africa using molecularly imprinted solid-phase extraction followed by high performance liquid chromatography-photodiode array detection

A fabric phase sorptive extraction-High performance liquid chromatography-Photo diode array detection method for the determination of twelve azole antimicrobial drug residues in human plasma and urine.

This paper reports a novel fabric phase sorptive extraction-high performance liquid chromatography-photodiode array detection (FPSE-HPLC-PDA) method for the simultaneous extraction and analysis of twelve azole antimicrobial drug residues that include ketoconazole, terconazole, voriconazole, bifonazole, clotrimazole, tioconazole, econazole, butoconazole, miconazole, posaconazole, ravuconazole, and itraconazole in human plasma and urine samples. The selected azole antimicrobial drugs were well resolved by using a Luna C18 column (250mm×4.6mm; 5μm particle size) in gradient elution mode within 36min. The analytical method was calibrated and validated in the range from 0.1 to 8μg/mL for all the drug compounds. Blank human plasma and urine were used as the sample matrix for the analysis; while benzyl-4-hydroxybenzoate was used as the internal standard (IS). The limit of quantification of the FPSE-HPLC-PDA method was found as 0.1μg/mL and the weighted-matrix matched standard calibration curves of the drugs showed a good linearity upto a concentration of 8μg/mL. The parallelism tests were also performed to evaluate whether overrange sample can be analyzed after dilution, without compromising the analytical performances of the validated method. The intra- and inter-day precision (RSD%) values were found ≤13.1% and ≤13.9%, respectively. The intra- and inter-day trueness (bias%) values were found in the range from -12.1% to 10.5%. The performances of the validated FPSE-HPLC-PDA were further tested on real samples collected from healthy volunteers after a single dose administration of itraconazole and miconazole. To the best of our knowledge, this is the first FPSE extraction procedure applied on plasma and urine samples for the simultaneous determination of twelve azole drugs possessing a wide range of logKow values (extending from 0.4 for fluconazole to 6.70 of butoconazole) and could be adopted as a rapid and robust green analytical tool for clinical and pharmaceutical applications.

Application of Response Surface Methodology for Optimisation of Simultaneous UHPLC-PDA Determination of Oleanolic and Ursolic Acids and Standardisation of Ericaceae Medicinal Plants

A fast and sensitive ultra-high performance liquid chromatography-photodiode array (UHPLC-PDA) method for simultaneous quantification of oleanolic acid (OA) and ursolic acid (UA) in plant materials was developed. A central composite design combined with a response surface methodology was utilized to establish optimal separation conditions. The final separation was accomplished on a Zorbax Eclipse XDB-C18 column (1.8 μm, 100 mm × 3 mm I.D., Agilent, Santa Clara, CA, USA) using a mixture 90:10 (v/v) of methanol and 1% (w/v) aqueous orthophosporic acid as a mobile phase at a flow rate of 0.44 mL/min and temperature of 18 °C. The analysis was completed in 6.2 min with satisfactory resolution of 1.5 between the target analytes. The developed method proved to be precise (relative standard deviations below 3.2%), accurate (recoveries in the range of 95.27%–98.60%), and sensitive (limits of detection (LODs) in the range of 0.047–0.051 mg/mL). The method was then successfully applied to evaluate OA and UA content in real samples of selected Ericaceae plant materials (leaves of Arctostaphylos uva ursi, Vaccinium myrtillus, Vaccinium vitis idaea, Gaultheria procumbens). The content of OA and UA in investigated samples varied in the range of 0.74–4.47 mg/g dry weight (dw) and 1.30–18.61 mg/g dw, respectively.

Simultaneous Determination of Eight Constituents in Fruits of Rubus chingii by UPLC

ObjectiveTo develop a simple, efficient, and reliable method for routine quantitative analysis of main constituents presented in the fruits of Rubus chingii, which is widely used in Chinese materia medica (CMM), known as Fupenzi (FPZ) in Chinese. MethodsAn ultra performance liquid chromatography-photo diode array (UPLC-PDA) system was employed for simultaneous quantification of eight compounds, i. e. adenosine, gallic acid, brevifolin carboxylic acid, ethyl gallate, ellagic acid, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucopyranoside, and tiliroside. The chromatographic analysis was performed on a C18 column using a gradient elution of acetonitrile −0.1% formic acid aqueous solution within a runtime of 25 min. ResultsAll calibration curves were linear (R2 > 0.9997) over the tested ranges. The intra- and inter-day precisions as determined from sample solutions were both less than 2.45% and 2.78%, respectively. The average recoveries for the eight constituents ranged from 94.77% to 101.35% with RSD ≤ 4.41%. The newly-developed method was applied to the quality assessment of various R. chingii samples, including both ripe and unripe fruits of R. chingii from different habitats. ConclusionThe relative levels of the investigated compounds vary remarkably in the fruits of R. chingii collected from different habitats. As only two of the eight compounds, adenosine and ellagic acid, are determined in the ripe fruits of R. chingii, the results may explain the reason why only the unripe fruits can be used in CMM.

Parallel factor analysis and trilinear partial least squares applied to the UPLC-PDA data array for the quantification of brimonidine tartrate and timolol maleate in an eye drop formulation

ABSTRACTSimultaneous quantification of brimonidine tartrate (BRI) and timolol maleate (TIM) in an eye drop formulation was performed by applying parallel factor analysis (PARAFAC) and trilinear (three way) partial least squares to the ultra performance liquid chromatography-photodiode array (UPLC-PDA) data array. In PARAFAC and 3 W-PLS1 applications, the co-elution of the related compounds in their chromatograms obtained in the presence of ornidazole as an internal standard (IS) was resolved, and then analyses were performed. On the other hand, a new conventional ultra performance liquid chromatography (UPLC) method was developed after long and tedious studies to get desirable elution of BRI and TIM in a chromatogram using different column and mobile phase system than that of chromatographic conditions of PARAFAC and 3 W-PLS1 applications. The performance and validity of all the proposed methods were confirmed by analyzing independent validation samples consisting of synthetic mixture, intraday and interday samples, and standard addition samples. Analysis results of BRI and TIM in eye drop samples by chemometric PARAFAC and 3 W-PLS1, and conventional UPLC were statistically compared to each other. It was concluded that PARAFAC and 3 W-PLS1 have shortest analysis time and lower cost than the developed conventional UPLC method for the analysis of the related compounds in commercial eye drop preparation with adequate selectivity and sensitivity.

Quantification and evaluation of thymoquinone loaded mucoadhesive nanoemulsion for treatment of cerebral ischemia

Stroke is an important cause of deaths worldwide, resulting in an irreversible deterioration of the central nervous system. Finally, production of more free radicals. Therefore, Thymoquinone is having antioxidant property and reported to have a potential role in the amelioration of cerebral ischemia but due to low solubility and poor absorption; they exhibit low serum and tissue levels. Present work aims to prepare nanoemulsions in order enhance the bioavailability of drug and hence evaluate the drug targeting in brain via non-invasive nasal route administration. Thymoquinone Mucoadhesive Nanoemulsion (TMNE) was prepared by ionic gelation method; characterized for particles size, entrapment efficiency, zeta potential, and ex vivo permeation study. Optimized TMNE ended up with a mean globule size 94.8±6.61nm; zeta potential −13.5±1.01mV; drug content 99.86±0.35% and viscosity 110±12cp. Ultra Performance Liquid Chromatography-Photodiode Array (UPLC-PDA) based bioanalytical method was developed and validated for pharmacokinetics, biodistribution, brain-targeting efficiency (628.5786±44.79%) and brain drug-targeting potential (89.97±2.94%) studies via post intranasal administration which revealed enhanced bioavailability of TQ in brain as compared to intravenous administration. Improved neurobehavioural activity (locomotor and grip strength) was observed in middle cerebral artery occlusion induced cerebral ischemic rats after i.n. administration of TMNE.

Elsholtzia ciliata (Thunb.) Hylander attenuates renal inflammation and interstitial fibrosis via regulation of TGF-ß and Smad3 expression on unilateral ureteral obstruction rat model

BackgroundRenal interstitial fibrosis is characterized by excessive accumulation of extracellular matrix, which leads to end-stage renal failure. PurposeThe aim of this study was to explore the effect of Elsholtzia ciliata (Thunb.) Hylander ethanol extract (ECE) on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Study designAfter quantitative analysis of ECE using the high performance liquid chromatography-photodiode array (HPLC-PDA) method, an in vitro study was performed to assess the anti-inflammatory and anti-fibrotic effects of ECE, using lipopolysaccharide (LPS) and transforming growth factor-ß (TGF-ß), respectively. MethodsFor in vivo study, all male Sprague Dawley (SD) rats (n=10/group), except for those in the control group, underwent UUO. The rats were orally treated with water (control), captopril (positive control, 200mg/kg), and ECE (300 and 500mg/kg) for 14 days. ResultsIn ECE, luteolin and rosmarinic acid were relatively abundant among the other flavonoids and phenolic acids. ECE treatment ameliorated LPS-induced overexpression of nuclear factor-κB, tumor necrosis factor (TNF-α), and interleukin-6 and improved oxidative stress in RAW 264.7 cells. Furthermore, ECE treatment suppressed TGF-ß-induced α-smooth muscle actin and matrix metalloproteinase 9 expression in human renal mesangial cells. In the UUO model, 14 consecutive days of ECE treatment improved UUO-induced renal damage and attenuated histopathological alterations and interstitial fibrosis. Moreover, the renal expression of TNF-α, TGF-ß, and Smad 3 were inhibited by ECE treatment. ConclusionTaken together, the effects of ECE may be mediated by blocking the activation of TGF-ß and inflammatory cytokines, leading subsequently to degradation of the ECM accumulation pathway. Based on these findings, ECE might serve as an improved treatment strategy for renal fibrotic disease.