Vortex-assisted dispersive liquid-liquid microextraction based on the hydrophobic deep eutectic solvent-based ferrofluid for extraction and detection of myclobutanil.

A vortex-assisted dispersive liquid-liquid microextraction (VA-DLLME) procedure using hydrophobic deep eutectic solvent-based ferrofluid (HDES-FF) as an extractant was established. The developed sample preparation method coupled with high-performance liquid chromatography-diode array detector (HPLC-DAD) was applied to the pretreatment and determination of myclobutanil (MYC) in fruit juice. Hydrophobic deep eutectic solvent, synthesized by n-decanoic acid and DL-menthol, was as a carrier and combined with magnetic nanoparticles (Fe3O4@OA) to form HDES-FF as an extractant with high extraction capacity. The synthesized materials were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). Parameters affecting extraction efficiency were optimized using single-factor experiments and Box-Behnken design via response surface methodology (BBD-RSM). Parallel tests were performed three times under the optimal conditions predicted by the model, yielding an actual mean recovery of 94.77% with RSD of 2.7% (n = 3) and an enrichment factor of 41.8 ± 0.98 (mean value ± SD, n = 3). Under the optimal conditions, the linear range was 1.0-100.0 µg·mL-1; the limit of detection (LOD) and limit of quantification (LOQ) were 0.25 and 0.80 µg·mL-1, respectively. The average spiked recoveries in the samples ranged from 98.2 to 100.5% with intra-day relative standard deviations (RSDs) of 1.2-3.5% (n = 3) and inter-day RSDs of 1.1-3.8% (n = 3). Finally, the method was successfully applied to the determination of MYC antimicrobial agent in different fruit juice samples. The proposed HDES-FF-VA-DLLME/HPLC-DAD method was verified to widely apply to the extraction of triazole fungicides.

Polystichum braunii ameliorates airway inflammation by attenuation of inflammatory and oxidative stress biomarkers, and pulmonary edema by elevation of aquaporins in ovalbumin-induced allergic asthmatic mice.

Asthma is a chronic inflammation of pulmonary airways associated with bronchial hyper-responsiveness. The study was aimed to validate the folkloric use of Polystichum braunii (PB) against ovalbumin (OVA)-induced asthmatic and chemical characterization OF both extracts. Allergic asthma was developed by intraperitoneal sensitization with an OVA on days 1 and 14 followed by intranasal challenge. Mice were treated with PB methanolic (PBME) and aqueous extract (PBAE) orally at 600, 300, and 150 mg/kg and using dexamethasone (2 mg/kg) as standard from day 15 to 26. High performance liquid chromatography-diode array detector analysis revealed the presence of various bioactive compounds such as catechin, vanillic acid, and quercetin. The PBME and PBAE profoundly (p < 0.0001-0.05) declined immunoglobulin E level, lungs wet/dry weight ratio, and total and differential leukocyte count in blood and bronchial alveolar lavage fluid of treated mice in contrast to disease control. Histopathological examination showed profoundly decreased inflammatory cell infiltration and goblet cell hyperplasia in treated groups. Both extracts caused significant (p < 0.0001-0.05) diminution of IL-4, IL-5, IL-13, IL-6, IL-1ß, TNF-α, and NF-κB and upregulation of aquaporins (1 and 5), which have led to the amelioration of pulmonary inflammation and attenuation of lung edema in treated mice. Both extracts profoundly (p < 0.0001-0.05) restored the activities of SOD, CAT, GSH and reduced the level of MDA dose dependently. Both extracts possessed significant anti-asthmatic action mainly PBME 600 mg/kg might be due to phenols and flavonoids and could be used as a potential therapeutic option in the management of allergic asthma.

Analysis of benzo[c]phenanthridine alkaloids in Eschscholtzia californica cell culture using HPLC-DAD and HPLC-ESI-MS/MS.

Effective HPLC-DAD and HPLC-ESI-MS/MS methods have been developed for the analysis of eight benzo[c]phenanthridine alkaloids (sanguinarine, chelirubine, macarpine, chelerythrine, dihydrosanguinarine, dihydrochelirubine, dihydromacarpine and dihydrochelerythrine), which are important metabolites in Eschscholtzia californica cell culture. By adopting a ternary gradient pump system, the dihydro-form alkaloids hardly separable from each other could be successfully separated, and all the target alkaloids could be simultaneously quantified with the LOD values of 0.01-0.79 µg/mL and the LOQ values of 0.03-3.59 µg/mL. This HPLC-DAD method was further confirmed by HPLC-ESI-MS/MS system in multiple reaction monitoring mode. Each separated HPLC peak was identified as the target alkaloid, showing its relevant ionized molecule and selected fragment ion. By applying the established method, alkaloid production during the E. californica cell culture could be successfully monitored and some valuable information on its metabolism could be deduced.

Development of a validated method for rapid quantification of up to sixteen cannabinoids using ultra-high-performance liquid chromatography diode-array detector with optional electrospray ionization time-of-flight mass spectrometry detection.

Due to the recent legalization of medical and recreational Cannabis in many countries in the world, there has been an increasing demand for accurate quantification of a growing number of cannabinoids. To meet this challenge, a method for rapid quantification of up to sixteen cannabinoids using ultra-high-performance liquid chromatography diode-array detector (UHPLC-DAD) has been developed, validated and used in the analysis of hemp concentrates. While published LC-UV methods were usually for twelve or less cannabinoids and might not achieve baseline separation of some critical pairs of cannabinoids, e.g., CBG/CBD (cannabigerol/cannabidiol) and Δ9-THC/Δ8-THC (tetrahydrocannabinol), in this study a systematic separation optimization led to a resolution of 1.7 for both pairs. The linear calibration range of all cannabinoids were between 0.02 to 25 µg/mL in methanol, leading to the quantification of 0.1 to 125% (w/w) individual cannabinoids in hemp concentrates after they were mixed with methanol at 20 µg/mL and analyzed after ultrasonication, centrifugation and filtration. The analytical results showed that none of the nine analyzed samples contained any of the seven acidic cannabinoids, but all the nine neutral cannabinoids were detected, with average content ranging from 0.10 to 93.23% (w/w) and RSD (relative standard deviation) values from 0.3 to 11.2% in triplicates. Particularly, two hemp concentrates, i.e., delta 8 hemp distillate and delta 8 hemp shatter contained 9.35 and 11.33% (w/w) Δ9-THC, respectively. While published recovery experiments were limited by the unavailability of cannabinoid-free matrix and the high cost of cannabinoid standards, this problem was solved by spiking abnormal CBD, a cannabinoid not naturally present in Cannabis plants and commercially available with a reasonable price, into the samples. The obtained average recovery ranged from 94.8 to 103.6% with RSD values from 1.5 to 9.0% in triplicates for the nine analyzed samples. Electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS) confirmed the good specificity of the UHPLC-DAD method, i.e., without any false positive identification of individual cannabinoids, and discovered six untargeted cannabinoids that were structural isomers of Δ9-THC in the nine hemp concentrate samples.

Characterization and Discrimination of Commercial Portuguese Beers Based on Phenolic Composition and Antioxidant Capacity.

Beer has been highly appreciated due to its phenolic composition and antioxidant capacity conjugated with its low alcohol content. Although some studies exist regarding the phenolic composition and antioxidant capacities of beers, there are no studies related to the determination of these parameters in the most commonly consumed commercial beers in Portugal. The phenolic composition and antioxidant capacity of 23 Portuguese commercial beers of different styles and types were studied for the first time. The total phenolic content, ortho-diphenols, and flavonoids ranged between 0.15 ± 0.01 and 0.82 ± 0.07 g Gallic Acid (GA) L-1; 0.07 ± 0.02 and 1.80 ± 0.09 g GA L-1, and 0.02 ± 0.00 and 0.15 ± 0.02 g Catechin (CAT) L-1, respectively. An accurate quantitative phenolic analysis was also performed, and the compound identified with a higher amount was gallic acid, followed by syringic acid. Concerning flavonoids, gallo-catechin was the most abundant compound identified (from 21.44 ± 2.87 and 144.00 ± 10.93 µg mL-1). A significant correlation between ortho-diphenols and the antiradical capacity (ABTS and DPPH) was found, the latter being negatively correlated. Flavonoids content was also positively correlated with total phenols and antiradical capacity determined by the ABTS assay. These results evidence that phenolic composition is affected by several factors inherent to beers, namely ingredients, fermentation type, and brewing process.

Development of a deep eutectic solvent-based ultrasound-assisted homogenous liquid-liquid microextraction method for simultaneous extraction of daclatasvir and sofosbuvir from urine samples.

An ultrasound-assisted homogenous liquid-liquid microextraction method using a new deep eutectic solvent was proposed for the extraction of daclatasvir and sofosbuvir from urine. The analytes were determined by high performance liquid chromatography-diode array detector. The deep eutectic solvent was prepared by mixing p-aminophenol with tetrabutyl ammonium chloride. It was used in the extraction procedure as an extraction solvent. The amine group in structure of the prepared deep eutectic solvent led to its various solubility in different pHs. In this method, urine sample was placed in a glass test tube and then mixed with sodium chloride and its temperature adjusted at 50 °C. Then, the deep eutectic solvent was dissolved in the solution by manually shaking. In the following, an ammonia solution was added to the solution and the mixture was sonicated for 4 min. After centrifugation, an aliquat of the sedimented phase was injected into the determination system. Low limits of detection (daclatasvir 1.0 and sofosbuvir 1.3 µg/L) and quantification (daclatasvir 3.3 and sofosbuvir 4.0 µg/L), high enrichment factor (daclatasvir 96 and sofosbuvir 90) and extraction recovery (daclatasvir 96 and sofosbuvir 90 %), and good percision (relative standard deviation ≤9.3 %) were obtained. The introduced method was successfully applied in the determination of daclatasvir and sofosbuvir concentrations in urine samples.

Salting-out assisted liquid-liquid extraction for the determination of ciprofloxacin residues in water samples by high performance liquid chromatography-diode array detector.

BACKGROUND: The occurrence of emerging pollutants like pharmaceuticals and related compounds in the aquatic and terrestrial environments is of increasing concern. Ciprofloxacin is one of the pharmaceuticals which is active against a wide range of bacteria. The main objective of this research is to develop a simple method for the extraction and determination of ciprofloxacin residues in environmental water samples. RESULTS: A salting-out assisted liquid-liquid extraction (SALLE) method for the determination of ciprofloxacin in water samples by high-performance liquid chromatography with diode array detector (HPLC-DAD) was developed. The calibration curve was linear over the range of 0.1-100 µg/L with coefficient of determination (r2) of 0.9976. The limits of detection (LOD) and quantification (LOQ) of the method were 0.075 and 0.25 µg/L, respectively. The reproducibility in terms of relative standard deviation (% RSD) was less than 10%. The applicability of the developed method was investigated by analyzing tap water, bottled mineral water and waste water and demonstrated satisfactory recoveries in the ranges of 86.4-120%. CONCLUSION: The method offered a number of features including wide linear range, good recovery, short analysis time, simple operation process and environmental friendly. The developed method can be utilized as an attractive alternative for the determination of ciprofloxacin residues in environmental water matrices.

Nutraceutical Properties of Mulberries Grown in Southern Italy (Apulia).

In this work, for the first time, were analyzed mulberry genotypes grown in Apulia (Southern Italy, Salento region) were analyzed. Two local varieties of Morus alba (cv. Legittimo nero and cv. Nello) and one of Morus nigra were characterized for content in simple sugars, organic acids, phenols, anthocyanins; fruit antioxidant activity (AA) was also evaluated by three different methods (2,2-Diphenyl-1-picrylhydrazyl, DPPH; 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS; and Ferric reducing antioxidant potential, FRAP test). The results showed that the sugars amount ranged between 6.29 and 7.66 g/100 g fresh weight (FW) while the malic and citric acids content was low, at about 0.1-1 g/100 g FW. Mulberries are a good source of phenols which are present in higher values in M. nigra and M. alba cv. Legittimo nero (485 and 424 mg Gallic Acid Equivalent (GAE)/ 100 g FW, respectively). The high performance liquid chromatography/diode array detector/mass spectrometry (HPLC/DAD/MS) analysis identified 5 main anthocyanin compounds present in different concentrations in each variety of mulberry: cyanidin 3-sophoroside, cyanidin 3-glucoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, pelargonidin 3-rutinoside. The highest concentration of anthocyanins was determined in Morus alba Legittimo (about 300 mg/100 g FW) while the lowest content (about 25 mg/100 g FW) was measured in M. alba cv. Nello. Morus nigra showed a good AA in comparison with the different M. alba genotypes with all the used methods; its AA was equal to 33, 26 and 21 µmols Trolox/g FW when using DPPH, ABTS and FRAP tests, respectively. All genotypes showed an anti-inflammatory activity (measured by cyclooxygenase (COX) inhibitory assay) which was also compared with two commercial anti-inflammatory drugs. The data obtained support the high biological qualities of mulberry fruits and their diffusion in human nutrition.

Effect of Byrsonima sericea DC. leaf extracts on mice gastrointestinal tract.

Byrsonima sericea DC. (Malpighiaceae) leaves are popularly folk medicine in Brazil used to treat gastro-intestinal disorders including diarrhea and gastric diseases. Ethanol extract (BSEE), ethyl acetate extract (BSEAE) and hexane extract (BSHE) of the leaf part of Byrsonima sericea DC were characterized for their total phenolics, proanthocyanidins and flavonoids content. The total antioxidant capacity of extracts was determined. The ethnopharmacological use of B. sericea leaves was evaluated by assaying BSEE for gastroprotective activity in stomach ulcer induced by indomethacin, intestinal motility and toxicity. Abundance of phenols mainly tannins was found in BSEE. Total phenolics, flavonoids and proanthocyanidins content in BSEE were found to be 0.371, 0.172 and 1.3 × 10-4 (mg/g) respectively. BSEE showed concentration dependent significant scavenging of DPPH values 90.0 (%) respectively. Moreover, oral doses of 500 and 1000 mg/kg did not cause mortality, and there was no difference in animals weight, organs relative weight and alanine transaminase (ALT) and aspartate transaminase (AST), as compared to the control group. Doses of 250, 500 and 1000 mg/kg inhibited the gastric lesions induced by indomethacin in 52, 60 and 62 % respectively. The dose of 1000 mg/kg decreased intestinal motility in animals. The presence of phenolic compounds, including tannins could be associated with the anti-diarrheal action and the antioxidant properties could collaborate to the gastroprotective and anti- diarrheal activities, confirming its popular use of the plant.

Validated reverse phase HPLC diode array method for the quantification of intact bevacizumab, infliximab and trastuzumab for long-term stability study.

The aim of the present study was to develop suitable and reliable method for quantification three of the most worldwide used therapeutic monoclonal antibodies (mAbs) -bevazizumab (BVZ), infliximab (INF) and trastuzumab (TTZ)- to be used in long-term stability studies. Reverse phase (RP) was selected by its greater sensibility and reproducibility comparing with other chromatographic modes. Then a high performance liquid chromatography with diode array detection (RP)HPLC/DAD method was checked. Since the three mAbs represent the active ingredient in the medicines in which they are formulated, the selected method was validated for each one in accordance with the International Conference on Harmonization (ICH) guidelines for pharmaceuticals for human use. Then method was validated in terms of linearity, accuracy, precision, (repeatability, intermediate precision) specificity (by forced degradation studies), robustness and system suitability. Spectral peak purity analysis strategy was used to test mAb degradations. Comparative study of the results indicated similar behavior for the three mAbs. Forced degradation studies also provided deep knowledge of these important bio-macromolecules. At last the method was successfully used to quantify BVZ, INF and TTZ in long-term stability studies performed under hospital conditions of use and they showed great stability regarding quantification during the time of the study.

Olive Leaves Extract from Algerian Oleaster (Olea europaea var. sylvestris) on Microbiological Safety and Shelf-life Stability of Raw Halal Minced Beef during Display.

Oleaster (wild olive tree) by-products represent a renewable and low-cost source of biopolyphenols. Leaf extracts (sylv.OLE) of Algerian oleaster, locally called a'hachad (Olea europaea subsp. europaea var. sylvestris), were applied at 1 and 5% (v/w) to raw Halal minced beef (HMB) in order to test its safety and shelf-life prolongation during retail/display. The total phenolic compound content in the extract was 198.7 ± 3.6 mg gallic acid equivalent. Ten compounds were identified in the sylv.OLE by High Performance Liquid Chromatography/Diode Array Detector (HPLC/DAD), of which oleuropein was the most abundant (43.25%). Samples treated with 5% sylv.OLE had significantly higher antimicrobial and antioxidant effects than those treated with 1% extract (p < 0.05). The addition of sylv.OLE reduced psychrotrophic counts as well as the level of pathogens (Salmonella enterica ser. Enteritidis and Shiga toxin-producing Escherichia coli O157:H7). A thiobarbituric acid reactive substance (TBARS) value of 2.42 ± 0.11 was reached throughout six days of retail/display in control samples, while the addition of 5% sylv.OLE reduced TBARS value by 58% (p < 0.05). The presence of sylv.OLE at the tested concentrations did not negatively influence the overall acceptability and bitterness of HMB.

Extraction time and temperature affect the extraction efficiencies of coumarin and phenylpropanoids from Cinnamomum cassia bark using a microwave-assisted extraction method.

Microwave-assisted extraction (MAE), an efficient extraction tool, was employed to extract a coumarin and five phenylpropanoids (cinnamic acid, cinnamyl alcohol, cinnamaldehyde, 2-hydroxycinnamadehyde, and 2-methoxycinnamaldehyde) from Cinnamomum cassia bark using water as the extraction solvent. Six marker compounds were quantified by high-performance liquid chromatography-diode array detector using a validated analytical method. To investigate the influences of temperature and time on the extraction yields of the six marker compounds, the water extracts of C. cassia bark were prepared using a MAE method at six different extraction temperatures (70, 75, 80, 85, 90, and 95°C) and times (2, 4, 6, 8, 10, and 12min). Their influences were assessed by multiple regression analysis. The results obtained demonstrated that higher extraction temperature and longer extraction time positively affected coumarin and cinnamyl alcohol contents, but negatively affected extract contents of cinnamic acid, cinnamaldehyde and 2-hydroxycinnamaldehyde (all p-<0.05). The extraction of 2-methoxycinnamaldehyde was affected by both positively and negatively by increasing temperature and time. These changes during MAE were assumed by the chemical natures of the marker compounds with various functional groups. In conclusion, temperature and times significantly affected the extraction efficiencies of a coumarin and five phenylpropanoids from C. cassia bark when a water-based MAE method was used. This study provides a novel approach to the preparation of the water extract of C. cassia bark using MAE.

Theanine and Caffeine Content of Infusions Prepared from Commercial Tea Samples.

BACKGROUND: Caffeine and L-theanine are pharmacologically important constituents of tea, especially due to their effects on the central nervous system. The effects of these two compounds are opposite: While caffeine is a well-known stimulant, theanine has a relaxing effect. Tea processing may influence the caffeine and theanine content of tea leaves. OBJECTIVE: The aim of our work was to quantify these constituents from a set of commercial products to reveal the possible correlations of caffeine and theanine content and processing methods. MATERIALS AND METHODS: Theanine and caffeine contents of 37 commercial white, green, oolong, black, and pu-erh tea samples were quantified by high-performance liquid chromatography-diode array detector. RESULTS: The mean L-theanine content of white, green, oolong, and black teas were 6.26, 6.56, 6.09, and 5.13 mg/g, respectively. The same values for caffeine content were 16.79, 16.28, 19.31, and 17.73 mg/g. CONCLUSION: Though the effect of processing on theanine content was evident, quantification for these analytes does not seem to be a good criterion to discriminate the different types of tea. Caffeine content provided no information on the effect of processing, and the theanine content of the samples was rather variable, independently from the type of the tea. The quantitative analysis of caffeine and theanine is essential to assess the stimulating effect of the tea, however, for chemical profiling further secondary metabolites have to be determined. SUMMARY: Thirty-seven commercial white, green, oolong, black, and pu-erh tea samples were analyzed for caffeine and theanine contentWhile the caffeine content was similar, the theanine contents of black teas were slightly lower and practically zero in pu-erhThe great variability of these two compound within the tea categories allows no discrimination of tea types based solely on theanine and caffeine quantificationContrary to the previous data, the way of processing has no determining effect on theanine content. FigureAbbreviations used: CZE: Capillary zone electrophoresis, DAD: Diode array detector, EEG: Electroencephalography, GC: Gas chromatography, HPLC: High-performance liquid chromatography, IR: Infrared spectroscopy, MEKC: Micellar electrokinetic capillary chromatography, MS: Mass spectrometry, RP: Reversed phase, RSD: Relative standard deviation, SD: Standard deviation, TLC: Tile liquid chromatography, UV: Ultraviolet.

Simultaneous quantification of crocetin esters and picrocrocin changes in Chinese saffron by high-performance liquid chromatography-diode array detector during 15 years of storage.

BACKGROUND: Saffron, which is made up of the dried stigmas of Crocus sativus L., has been successfully cultivated in China since 1970s and Zhejiang province is now the largest producing area in China, but the contents of crocetin esters and picrocrocin in saffron from Zhejiang province has not been determined simultaneously by high-performance liquid chromatography (HPLC) and changes of these constituents in Chinese saffron during storage for years has not been studied. OBJECT: To establish a simple method quantification of the five main compounds including picrocrocin and four crocetin esters in saffron from main producing areas of China and study the influence of storage time on the changes of saffron constituents. MATERIALS AND METHODS: A simple, sensitive, and accurate HPLC method was developed for simultaneous determination of five major active components in saffron and eight samples which collected from the same farm of Zhejiang province in different years were analyzed. RESULTS: The correlation coefficient values (R (2) > 0.9997) indicated good correlations between the investigated compounds' concentrations and their peak areas within the test ranges. The limits of quantification and detection of the five compounds were 0.53-2.76 µg/mL and 0.11-0.77 µg/mL, respectively. The recoveries ranged from 94.67% to 101.31%, and the overall relative standard deviations for intra-day and inter-day were lower than 3.49%. The method was applied to study the changes of crocetin esters and picrocrocin contents in saffron samples during 15 years of storage. The losses of crocetin esters and picrocrocin in saffron with 1 -year storage were 52.2% and 54.3%, respectively. The trend then declined during subsequent storage. CONCLUSION: The developed method can be applied to the intrinsic quality control of saffron.

Quantification of maltol in Korean ginseng (Panax ginseng) products by high-performance liquid chromatography-diode array detector.

BACKGROUND: Maltol, as a type of phenolic compounds, is produced by the browning reaction during the high-temperature treatment of ginseng. Thus, maltol can be used as a marker for the quality control of various ginseng products manufactured by high-temperature treatment including red ginseng. For the quantification of maltol in Korean ginseng products, an effective high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed. MATERIALS AND METHODS: The HPLC-DAD method for maltol quantification coupled with a liquid-liquid extraction (LLE) method was developed and validated in terms of linearity, precision, and accuracy. An HPLC separation was performed on a C18 column. RESULTS: The LLE methods and HPLC running conditions for maltol quantification were optimized. The calibration curve of the maltol exhibited good linearity (R (2) = 1.00). The limit of detection value of maltol was 0.26 µg/mL, and the limit of quantification value was 0.79 µg/mL. The relative standard deviations (RSDs) of the data of the intra- and inter-day experiments were <1.27% and 0.61%, respectively. The results of the recovery test were 101.35-101.75% with an RSD value of 0.21-1.65%. The developed method was applied successfully to quantify the maltol in three ginseng products manufactured by different methods. CONCLUSION: The results of validation demonstrated that the proposed HPLC-DAD method was useful for the quantification of maltol in various ginseng products.

Profiling of components of rhizoma et radix polygoni cuspidati by high-performance liquid chromatography with ultraviolet diode-array detector and ion trap/time-of-flight mass spectrometric detection.

INTRODUCTION: Rhizoma et Radix Polygoni Cuspidati (Huzhang in Chinese, HZ) is a traditional medicinal plant in China. Many of the components of HZ have been proved to be bioactive while it is difficult to conduct a comprehensive chemical profiling of HZ as a consequence of the absence of efficient separation system and sensitive detective means. We developed a simple and effective method for comprehensive characterization of constituents in HZ. OBJECTIVE: To develop a simple and effective method to characterize the components in HZ and provide useful information for subsequent metabolic studies of HZ. MATERIALS AND METHODS: The components in HZ aqueous extract were characterized by using high performance liquid chromatography with UV diode-array detector (HPLC-DAD) and ion trap/time-of-flight mass spectrometric detection (HPLC-IT/TOF). Stilbenes, anthraquinones, gallates and tannins, naphthalenes and some other compounds were identified and confirmed by diagnostic fragment ions with accurate mass measurements, characteristic fragmentation pathways and relevant published literatures. RESULTS: Among the 238 constituents detected in HZ, a total number of 74 constituents were identified unambiguously or tentatively, including 29 compounds reported for the first time in HZ. CONCLUSION: The identification and structure elucidation of these chemicals provided essential data for quality control and further in vivo metabolic studies of HZ. Key words: Polygonum cuspidatum, HPLC-DAD, HPLC-IT/TOF, qualitative analysis.

Fluorescence, electrophoretic and chromatographic fingerprints of herbal medicines and their comparative chemometric analysis.

The aim of the present study was to compare the polyphenolic compositions of 47 medicinal herbs (HM) and four herbal tea mixtures from Central Estonia by rapid, reliable and sensitive Spectral Fluorescence Signature (SFS) method in a front face mode. The SFS method was validated for the main identified HM representatives including detection limits (0.037mgL(-1) for catechin, 0.052mgL(-1) for protocatechuic acid, 0.136mgL(-1) for chlorogenic acid, 0.058mgL(-1) for syringic acid and 0.256mgL(-1) for ferulic acid), linearity (up to 5.0-15mgL(-1)), intra-day precision (RSDs=6.6-10.6%), inter-day precision (RSDs=6.4-13.8%), matrix effect (-15.8 to +5.5) and recovery (85-107%). The phytochemical fingerprints were differentiated by parallel factor analysis (PARAFAC) combined with hierarchical cluster analysis (CA) and principal component analysis (PCA). HM were clustered into four main clusters (catechin-like, hydroxycinnamic acid-like, dihydrobenzoic acid-like derivatives containing HM and HM with low/very low content of fluorescent constituents) and 14 subclusters (rich, medium, low/very low contents). The average accuracy and precision of CA for validation HM set were 97.4% (within 85.2-100%) and 89.6%, (within 66.7-100%), respectively. PARAFAC-PCA/CA has improved the analysis of HM by the SFS method. The results were verified by two separation methods CE-DAD and HPLC-DAD-MS also combined with PARAFAC-PCA/CA. The SFS-PARAFAC-PCA/CA method has potential as a rapid and reliable tool for investigating the fingerprints and predicting the composition of HM or evaluating the quality and authenticity of different standardised formulas. Moreover, SFS-PARAFAC-PCA/CA can be implemented as a laboratory and/or an onsite method.

Qualitative and quantitative analysis of an alkaloid fraction from Piper longum L. using ultra-high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry.

In a previous research, an alkaloid fraction and 18 alkaloid compounds were prepared from Piper longum L. by series of purification process. In this paper, a qualitative and quantitative analysis method using ultra-high performance liquid chromatography-diode array detector-mass spectrometry (UHPLC-DAD-MS) was developed to evaluate the alkaloid fraction. Qualitative analysis of the alkaloid fraction was firstly completed by UHPLC-DAD method and 18 amide alkaloid compounds were identified. A further qualitative analysis of the alkaloid fraction was accomplished by UHPLC-MS/MS method. Another 25 amide alkaloids were identified according to their characteristic ions and neutral losses. At last, a quantitative method for the alkaloid fraction was established using four marker compounds including piperine, pipernonatine, guineensine and N-isobutyl-2E,4E-octadecadienamide. After the validation of this method, the contents of above four marker compounds in the alkaloid fraction were 57.5mg/g, 65.6mg/g, 17.7mg/g and 23.9mg/g, respectively. Moreover, the relative response factors of other three compounds to piperine were calculated. A comparative study between external standard quantification and relative response factor quantification proved no remarkable difference. UHPLC-DAD-MS method was demonstrated to be a powerful tool for the characterization of the alkaloid fraction from P. longum L. and the result proved that the quality of alkaloid fraction was efficiently improved after appropriate purification.

Simultaneous determination of four neuroprotective compounds of Tilia amurensis by high performance liquid chromatography coupled with diode array detector.

BACKGROUND: Tilia amurensis consists of various compounds, such as flavonoids and terpenoids. OBJECTIVE: A simple and reliable high performance liquid chromatography (HPLC) coupled with the diode array detector (DAD) method has been established for simultaneous determination of epicatechin, nudiposide, lyoniside, and scopoletin isolated from Tilia amurensis. MATERIALS AND METHODS: Optimum separations were obtained with a SHISEIDO C18 column by gradient eluton, with 0.1% Trifluoroacetic acid (TFA) water-methanol as the mobile phase. The gradient elution system was completed within 40 minutes. The flow rate and detection wavelength were 1 mL/minute, 205 nm, 250 nm, and 280 nm, respectively. RESULTS: Validation of the analytical method was evaluated by linearity, precision, and the accuracy test. The calibration curve was linear over the established range with R(2) > 0.997. The limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.01-15.20 µg/mL and 0.03-46.06 µg/mL. The method exhibited an intraday and interday precision range of 96.25-105.66% and 93.52-109.92%, respectively (RSD <2.80%). The recoveries and relative standard deviation (RSD) of the four compounds in Tilia amurensis were in the range of 90.42-104.84% and 0.2-2.58%. CONCLUSION: This developed method was accurate and reliable for the quality evaluation of the four compounds isolated from Tilia amurensis.

Effects of Tapinanthus globiferus and Zanthoxylum zanthoxyloides extracts on human leukocytes in vitro.

OBJECTIVE: This study aimed at investigating the genotoxicity and cytotoxicity effect of Tapinanthus globiferus and Zanthoxylum zanthoxyloides to human leukocytes. In addition, the reductive potential and the chemical composition of the two plant extracts were also determined. MATERIALS AND METHODS: Human leukocytes were obtained from healthy volunteer donors. The genotoxicity and cytotoxicity of T. globiferus and Z. zanthoxyloides were assessed using the comet assay and trypan blue exclusion, respectively. The antioxidant activity of the plant extracts was evaluated by the reducing power assay. Furthermore, high-performance liquid chromatography-diode array detector was used to characterize and quantify the constituents of these plants. RESULTS: T. globiferus (10-150 µg/mL) was neither genotoxic nor cytotoxic at the concentrations tested, suggesting that it can be consumed safely at relatively high concentrations. However, Z. zanthoxyloides showed cytoxicity and genotoxicity to human leukocytes at the highest concentration tested (150 µg/mL). In addition, the total reducing power of T. globiferus was found higher than Z. zanthoxyloides in potassium ferricyanide reduction. Both plants extract contained flavonoids (rutin and quercetin) and phenolic acids (chlorogenic and caffeic). CONCLUSION: The results obtained support the fact that some caution should be paid regarding the dosage and the frequency of use of Z. zanthoxyloides extract.