Ultra-performance Liquid Chromatography-photodiode Array Research Articles

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.

Identification and Quantitation of Anthocyanins in Purple-Fleshed Sweet Potatoes Cultivated in China by UPLC-PDA and UPLC-QTOF-MS/MS.

The identification and quantitation of the anthocyanins in 12 purple-fleshed sweet potato (PFSP) cultivars ('Jihei 1', 'Xuzi 3', 'Xuzi 6', 'Zhezi 4', 'Ningzi 1', 'Ningzi 2', 'Ningzi 3', 'Ning 2-2', 'Ning 6-8', 'Guangzi 1', 'Ziluolan', and 'Qinzi 1') in China were carried out using a combination of ultraperformance liquid chromatography-photodiode array (UPLC-PDA), quadrupole-time-of-flight mass spectrometry (QTOF-MS), and tandem mass spectrometry (MS/MS) analyses. Thirteen acylated anthocyanins were tentatively characterized, including two new PFSP anthocyanins, cyanidin 3-caffeoyl-vanilloyl sophoroside-5-glucoside and peonidin 3-caffeoyl-vanilloyl sophoroside-5-glucoside. The quantitative analyses of these anthocyanins were conducted using cyanidin 3-O-glucoside as a standard. The total anthocyanin content of the PFSPs depended on the cultivar. The five PFSP cultivars with the highest content of anthocyanins were 'Jihei 1', 'Xuzi 3', 'Zhezi 4', 'Ziluolan', and 'Qinzi 1'. This is the first report of the 'Ningzi 2', 'Ningzi 3', and 'Ning 2-2' PFSP cultivars containing only diacylated anthocyanins and of the 'Xuzi 6' cultivar containing single anthocyanidin-based anthocyanins.

Three-way analysis of the UPLC–PDA dataset for the multicomponent quantitation of hydrochlorothiazide and olmesartan medoxomil in tablets by parallel factor analysis and three-way partial least squares

An application of parallel factor analysis (PARAFAC) and three-way partial least squares (3W-PLS1) regression models to ultra-performance liquid chromatography–photodiode array detection (UPLC–PDA) data with co-eluted peaks in the same wavelength and time regions was described for the multicomponent quantitation of hydrochlorothiazide (HCT) and olmesartan medoxomil (OLM) in tablets. Three-way dataset of HCT and OLM in their binary mixtures containing telmisartan (IS) as an internal standard was recorded with a UPLC–PDA instrument. Firstly, the PARAFAC algorithm was applied for the decomposition of three-way UPLC–PDA data into the chromatographic, spectral and concentration profiles to quantify the concerned compounds. Secondly, 3W-PLS1 approach was subjected to the decomposition of a tensor consisting of three-way UPLC–PDA data into a set of triads to build 3W-PLS1 regression for the analysis of the same compounds in samples. For the proposed three-way analysis methods in the regression and prediction steps, the applicability and validity of PARAFAC and 3W-PLS1 models were checked by analyzing the synthetic mixture samples, inter-day and intra-day samples, and standard addition samples containing HCT and OLM. Two different three-way analysis methods, PARAFAC and 3W-PLS1, were successfully applied to the quantitative estimation of the solid dosage form containing HCT and OLM. Regression and prediction results provided from three-way analysis were compared with those obtained by traditional UPLC method.

Mechanistic Evidence of Viscum schimperi (Viscaceae) Antihyperglycemic Activity: From a Bioactivity-guided Approach to Comprehensive Metabolite Profiling.

Diabetes mellitus is possibly the world's largest growing metabolic disorder. Effective treatment of diabetes is increasingly dependent on active constituents of medicinal plants capable of controlling hyperglycemia as well as its secondary complications. Viscum schimperi Engl. is a plant growing in Saudi Arabia and known for its antidiabetic activity. The potential antidiabetic activity of its methanol extract as well as its chloroform, n-butanol, and the remaining water fractions was evaluated in streptozotocin-induced diabetic rats at two dose levels. The antidiabetic activity was assessed through the determination of fasting blood glucose level, insulin levels, area under the curve (AUC) in oral glucose tolerance test, glucose absorption in isolated rat gut assay, and glucose uptake by psoas muscle. Moreover, large-scale untargeted metabolite profiling of methanol extract was performed via UPLC-PDA and qTOF-MS (ultra-performance liquid chromatography photodiode array detection and quadrupole time-of-flight mass spectrometry) respectively, to explore its chemical composition and standardization of its extract. Multivariate statistical analysis including principal component analysis and orthogonal projection to latent structures discriminant analysis was used to determine bioactives in its fractions. In conclusion, oleanane triterpenes and O-caffeoyl quinic acid conjugates were the major compounds that might account for antihyperglycemic effect of the plant.

An Ultra-Performance Liquid Chromatography Photodiode Array Detection Tandem Mass Spectrometric Method for Simultaneous Determination of Seven Major Bioactive Constituents in Xiaochaihutang and Its Application to Fourteen Compatibilities Study.

A rapid and sensitive ultra-performance liquid chromatography photodiode array detection tandem mass spectrometric method (UPLC-PDA-MS-MS) was developed and validated to simultaneously determine seven major bioactive constituents in the formula of traditional Chinese medicines Xiaochaihutang (XCHT). To investigate the discipline of compatibility in XCHT, 14 kinds of compatibilities designed by orthogonal array were also analyzed. The separation was performed on an ACQUITY UPLC™ BEH C18 column (100 × 2.1 mm, 1.7 µm) using gradient elution with a mobile phase of 0.1% formic acid and acetonitrile at a flow rate of 0.2 mL/min. Two detection techniques of PDA detector and MS-MS detector were proposed, respectively. The concentrations of baicalin and wogonoside were high enough for PDA detection while low-concentration bioactive constituents including saikosaponin a, ginsenoside Rg1, liquiritin, baicalein and wogonin were quantified by MS-MS detection. The proposed method was fully validated in terms of sensitivity, linearity, specificity, precision, repeatability and recovery. This is the first report on the simultaneous determination of the major bioactive constituents of XCHT by UPLC-PDA-MS-MS, which could be used to evaluate the quality of XCHT and to investigate the discipline of compatibility in XCHT.

Selective removal of ATP degradation products from food matrices II: Rapid screening of hypoxanthine and inosine by molecularly imprinted matrix solid-phase dispersion for evaluation of fish freshness

A water compatible molecularly imprinted polymer (MIP), synthesized using theophylline (TPH) as dummy-template and acrylamide (AM) as functional monomer, has been employed as supporting material in matrix solid-phase dispersion combined with ultra performance liquid chromatography–photodiode array detection (MSPD–UPLC–PDA) for selective determination of adenosine triphosphate (ATP) derivatives in fish samples. ATP degradation products are used as freshness index for assessment of fish quality. The solid sample was directly blended with MIP in MSPD procedure resulting in sample disruption and subsequent adsorption of the compounds on the MIP. By using n-hexane and ammonium hydroxide aqueous solution at pH 9 as the washing and elution solvent, respectively, satisfactory recoveries and clean chromatograms have been obtained. Good linearity for hypoxanthine (HYP) and inosine (INO) has been observed with correlation coefficients (R2) of 0.9987 and 0.9986, respectively. The recoveries of the two ATP derivatives at three different spiked levels ranged from 106.5% to 113.4% for HYP and from 103.1% to 111.2% for INO, with average relative standard deviations lower than 4.2% in both cases. This new method, which is rapid, simple and sensitive, can be used as an alternative tool to conventional tedious methods.

Dissipation and residue of metalaxyl and cymoxanil in pepper and soil.

A simple and accurate method of determining metalaxyl and cymoxanil in pepper and soil was developed by ultra-performance liquid chromatography-photodiode array detection. The limits of detection were 0.015mg/kg for metalaxyl and 0.003mg/kg for cymoxanil. The limits of quantification were 0.05mg/kg for metalaxyl in pepper and soil as well as 0.01mg/kg for cymoxanil in pepper and soil. Recoveries of pepper and soil were investigated at three spiking levels and ranged within 77.52 to 102.05% for metalaxyl and 87.15 to 103.21% for cymoxanil, with relative standard deviations below 9.30%. For field experiments, the half-lives of metalaxyl were 3.2 to 3.9days in pepper and 4.4 to 9.5days in soil at the three experimental locations in China. At harvest, pepper samples were found to contain metalaxyl and cymoxanil well below the maximum residue limit MRLs of the European Union (EU) following the recommended dosage and the interval of 21days after last application.

Anti-Inflammatory Effects of the Bioactive Compound Ferulic Acid Contained in Oldenlandia diffusa on Collagen-Induced Arthritis in Rats.

Objectives. This study aimed to identify the active compounds in Oldenlandia diffusa (OD) decoction and the compounds absorbed into plasma, and to determine whether the absorbed compounds derived from OD exerted any anti-inflammatory effects in rats with collagen induced arthritis (CIA). Methods. The UPLC-PDA (Ultra Performance Liquid Chromatography Photo-Diode Array) method was applied to identify the active compounds both in the decoction and rat plasma. The absorbable compound was administered to the CIA rats, and the effects were dynamically observed. X-ray films of the joints and HE stain of synovial tissues were analyzed. The levels of IL-1β and TNF-α in the rats from each group were measured by means of ELISA. The absorbed compound in the plasma of CIA rats was identified as ferulic acid (FA), following OD decoction administration. Two weeks after the administration of FA solution or OD decoction, the general conditions improved compared to the model group. The anti-inflammatory effect of FA was inferior to that of the OD decoction (P < 0.05), based on a comparison of IL-1β TNF-α levels. FA from the OD decoction was absorbed into the body of CIA rats, where it elicited anti-inflammatory responses in rats with CIA. Conclusions. These results suggest that FA is the bioactive compound in OD decoction, and FA exerts its effects through anti-inflammatory pathways.

Protective Effect of Chlorophyllin and Lycopene from Water Spinach Extract on Cytotoxicity and Oxidative Stress Induced by Heavy Metals in Human Hepatoma Cells

The purpose of this study was to examine the inhibitory effects of ethanol extract of water spinach (EEWS) containing chlorophyll and lycopene on cytotoxicity and oxidative stress in liver induced by heavy metals. The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay and dichlorofluorescein (DCF) assay were conducted to measure cytotoxicity and inhibition of reactive oxygen species (ROS), respectively. Cytotoxicity was prevented at a concentration of 11.7 mg/L of EEWS. Both sodium copper chlorophyllin (SCC) and lycopene in EEWS were identified by ultraperformance liquid chromatography–photodiode array–electrospray ionization–mass spectroscopy (UPLC-PDA-ESI-MS/MSn) as major components at m/z 722.64 and 535.45, respectively. The concentrations of SCC and lycopene were 0.12 and 0.04 mg from 100 g of dried powder, respectively. Approximately 99% cytotoxicity induced by Cd was inhibited by EEWS. However, the inhibitory effect attributed to generation of ROS was similar with SCC, lycopene, and EEWS. Our results indicated that EEWS was effective in reducing cytotoxicity and oxidative stress produced by heavy metals in a HepG2 cell. Data suggest that the possible mechanism underlying the preventive action of SCC might be associated with diminished absorption of metal ions by chelating and blocking metal-mediated generation of ROS, while lycopene effects may be attributed to its high number of conjugated dienes that act as most potent singlet oxygen quenchers.

Advanced ultra-performance liquid chromatography–photodiode array–quadrupole time-of-flight mass spectrometric methods for simultaneous screening and quantification of triterpenoids in Poria cocos

A sensitive, precise and accurate method was developed to screen and quantify triterpenoids based on ultra-performance liquid chromatography–photodiode array–quadrupole time-of-flight mass spectrometry (UPLC–PDA–QTOF–MS). An exact neutral loss scan of 62.0004Da (CH2O3) was used to selectively detect triterpenoids in Poria cocos, followed by a survey scan for exact masses of precursor and fragment ions of these triterpenoids. The developed method was applied to quantify seven major triterpenoids in 40 P. cocos samples of different origins within 18min, and a total of 31 triterpenoids were unequivocally or tentatively identified. Principal component analysis of these samples showed a clear separation of three groups, and ten triterpenoids play key roles in differentiating these samples were obtained from the OPLS-DA variable influence on projection (VIP) plot and then unequivocally or tentatively identified. The developed method can be applied for rapid bitterness evaluation, quality control and authenticity establishment of P. cocos.

QUALITY CONTROL OF POLYGONUM CUSPIDATUM BY UPLC-PDA AND RELATED STATISTICAL ANALYSIS

A rapid and accurate ultra-performance liquid chromatography-photo-diode array (UPLC-PDA) detection method was established for simultaneous determination of 5 compounds including polydatin, resveratrol, emodin-8-glucoside, emodin, and physcion in Polygonum cuspidatum. Twenty-six batches of samples were collected from different areas and markets in China. The compounds were separated in less than 12 min using a C18 column with acetonitrile and water containing 0.5% acetic acid as the mobile phase at a flow rate of 0.3 mL/min. The precision (intra-day and inter-day assay), stability, limits of detection and quantitation, linearity, and recovery were evaluated for this method. RSD for intra-day precision, inter-day precision, and stability were less than 2.5%, 3.5%, and 2.19%, respectively. Recovery ranged from 96.78% to 104.58% (RSD < 2.71%). The content range of polydatin, resveratrol, emodin-8-glucoside, emodin, and physcion were 13.45–34.55, 0.97–13.12, 3.34–17.62, 6.53–13.69, and 1.71–4.23 mg/g, respectively. By applying principal component analysis and hierarchical cluster analysis, the quality of 26 specimens can be divided into 3 groups according to contents of compounds. This indicates the need to strengthen the quality control of P. cuspidatum. This is the first report to provide an effective UPLC method and related statistical methods to evaluate the quality of P. cuspidatum from different areas.

경기도내 유통 수입과실류의 잔류농약 실태조사

Abstract We tested for pesticide residues in 124 samples of 22 different items of imported fruits circulatedin Gyeonggido. Total 218 pesticides were analyzed by multi-residue method using gas chromatography/nitrogen phosphorus detector-electron capture detector (GC/NPD-ECD), time of flight/mass spectrometer(TOF/MS), ultra performance liquid chromatography/photo diode array (UPLC/PDA), high performance liquidchromatography/fluorescence detector (HPLC/FLD) and mass spectrometer (LC/MS/MS). The pesticides weredetected in 18 fruits samples, ranging 0.003~0.3 mg/kg and no samples had violative residue. The separationtest to 14 sample pesticides detected was conducted to monitor the current status of pesticide residuesaccording to the partial characteristic. The pesticides were detected in 14 peels ranging 0.03~1.5 mg/kg and 2fleshes in less than detection limits. These results indicate that imported fruits are safe when the human takesnormally but even the small amount of pesticides is harmful when the human takes it in a prolonged period.Therefore, the pesticide residual amounts of imported friuts should be constantly monitored for food safety.Key words Pesticide residues, Imported fruits, Peel, Flesh, Test for disjoined parts

Comparison of SPE-TD-GC-FID with UPLC-PDA and GC-MS Methods for Analysis of Benzene, Toluene and Xylene Isomers in Solid-Liquid Mixing Paints

Three methods were developed for analysis of benzene, toluene, p-xylene, m-xylene and o-xylene (BTX) in a solid-liquid mixing paint. These methods were based on solid phase extraction-thermal desorption-gas chromatography with a flame ionization detector (SPE-TD-GC-FID), ultra performance liquid chromatography-photodiode array detector (UPLC-PDA) and gas chromatography-mass spectrometry (GC-MS). At their optimum conditions of operation, the developed methods were compared in terms of recovery, sensitivity, selectivity and universality. Although the time required for GC-MS analysis was shorter than for GC-FID and UPLC-PDA, it offered relatively poorer recoveries and suffered from matrix interferences. All of the mentioned methods were proven to be ideal for the analysis of targeted analytes; SPE-TD-GC-FID was particularly fit for the determination of trace level BTX residues present in the complex matrix. As one of the sample pretreatment techniques, the novel SPE also showed some selectivity towards BTX and was found to be superior to the conventional SPE.

UPLC and Spectrophotometric Analysis of Polyphenols in Wines Available in the Polish Market

Ultra performance liquid chromatography–photodiode array detection (UPLC–PDA) was used to analyze five biologically active polyphenolic compounds (catechin, epicatechin, rutin, trans-resveratrol, and quercetin) in different types of wine available in the Polish market. The results showed that the major polyphenols in chosen wines were catechin and epicatechin, the minor polyphenol was trans-resveratrol. The use of UPLC system allowed a shorter run time of up to six times in comparison with the conventional HPLC system. Total polyphenols were analyzed according to the Folin-Ciocalteu method using gallic acid as a standard and the results were presented as gallic acid equivalents. The wines were also analyzed for anthocyanins content using malvidin-3,5-diglucoside as a reference material in spectrophotometric analysis.

Identification of chemical constituents in extracts and rat plasma from Fructus Aurantii by UPLC‐PDA‐Q‐TOF/MS

Fructus Aurantii, as a component of several compound formulae, shows many bioactivities. As is well known, the constituents of traditional Chinese medicines are very complex and multiple constituents are responsible for the therapeutic effect. However, the concrete compounds are difficult to confirm. Therefore, studies on the constituents absorbed into serum and their metabolites are necessary. To search for the active constituents in Fructus Aurantii. An ultra-performance liquid chromatography-photodiode array detector-quadrupole time-of-flight mass spectrometry (UPLC-PDA-Q-TOF/MS) method was established to analyse the aqueous extract in Fructus Aurantii and the constituents absorbed into blood. Compound identification was made by matching the empirical molecular formula with those of the reported compounds and UV spectra, and further elucidated by lower energy CID mass spectra. Twelve flavonoid O-glycosides were detected, and nine compounds were tentatively identified as polymethoxylated flavones. Six parent compounds were identified and four metabolites were observed in rat plasma, two of which were identified as naringenin glucuronide and hesperetin glucuronide, respectively. The approach developed has proved useful in the study of the active constituents in traditional Chinese medicines.