Ultra-high Performance Liquid Chromatography Method Research Articles

Development and validation of a rapid ultra-high performance liquid chromatography method for the assay of benzalkonium chloride using a quality-by-design approach

A rapid robust reversed-phase UHPLC method has been developed for the analysis of total benzalkonium chloride in preserved drug formulation. A systematic Quality-by-Design (QbD) method development approach using commercial, off the shelf software (Fusion AE®) has been used to optimize the column, mobile phases, gradient time, and other HPLC conditions. Total benzalkonium chloride analysis involves simple sample preparation. The method uses gradient elution from an ACE Excel 2 C18-AR column (50mm×2.1mm, 2.0μm particle size), ammonium phosphate buffer (pH 3.3; 10mM) as aqueous mobile phase and methanol/acetonitrile (85/15, v/v) as the organic mobile phase with UV detection at 214nm. Using these conditions, major homologs of the benzalkonium chloride (C12 and C14) have been separated in less than 2.0min. The validation results confirmed that the method is precise, accurate and linear at concentrations ranging from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The recoveries ranged from 99% to 103% at concentrations from 0.025mg/mL to 0.075mg/mL for total benzalkonium chloride. The validation results also confirmed the robustness of the method as predicted by Fusion AE®.

An ultra-high performance liquid chromatographic-tandem mass spectrometric method for the determination of sinomenine in human plasma after transdermal delivery of the zhengqing fengtongning injection.

A sensitive, precise and selective ultra-high performance liquid chromatography method coupled with triple-quadrupole mass spectrometry was developed and validated for the determination of trace amounts of sinomenine (ng/mL) in minute volumes of human plasma. Fifty microliter plasma samples were precipitated using methanol to extract sinomenine. Separation was carried out on a C18 column with a water and acetonitrile mobile phase gradient with formic acid as an additive. The mass spectrometry data were obtained in the positive ion mode, and the transition of multiple reactions was monitored at m/z 330.2→181.0 for sinomenine quantification. The working assay range for sinomenine was linear from 0.1173 to 15.02 ng/mL with the lower limit of quantification of 0.1173 ng/mL. The precision and accuracy of the method was less than 15% in intra-day and inter-day experiments with a matrix effect of less than 6.5%. After validation, the quantitative method was applied to analyze sinomenine levels in human plasma after transdermal delivery of the Zhengqing Fengtongning Injection. The results showed that some samples contained sinomenine within the concentration range 0.4131–4.407 ng/mL.

Determination of Thiophanate-Methyl and Carbendazim in Rapeseed by Solid–Phase Extraction and Ultra–High Performance Chromatography with Photodiode Array Detection

A rapid and reliable ultra-high performance liquid chromatography method using a photodiode array detector with QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation was developed to determine thiophanate-methyl and carbendazim in rapeseed. Samples were extracted with acetonitrile and partitioned with a mixture of NaCl and anhydrous MgSO4. The cleanup step was performed using dispersive solid-phase extraction with anhydrous MgSO4 and primary secondary amine. This step was repeated to improve the results. The limits of detection and quantification of thiophanate-methyl and carbendazim in rapeseed were 15 and 50 μg kg−1, respectively. The recoveries of thiophanate-methyl and carbendazim were from 77.44–100.9%, with relative standard deviations between 2.20% and 6.62%. The method was employed to determine thiophanate-methyl and carbendazim in rapeseed. This study has established a novel method and a safety evaluation system for the analytes in rapeseed.

Development and validation of ultra-high performance supercritical fluid chromatography method for determination of illegal dyes and comparison to ultra-high performance liquid chromatography method

A novel simple, fast and efficient ultra-high performance supercritical fluid chromatography (UHPSFC) method was developed and validated for the separation and quantitative determination of eleven illegal dyes in chili-containing spices. The method involved a simple ultrasound-assisted liquid extraction of illegal compounds with tetrahydrofuran. The separation was performed using a supercritical fluid chromatography system and CSH Fluoro-Phenyl stationary phase at 70°C. The mobile phase was carbon dioxide and the mixture of methanol:acetonitrile (1:1, v/v) with 2.5% formic acid as an additive at the flow rate 2.0mLmin−1. The UV–vis detection was accomplished at 500nm for seven compounds and at 420nm for Sudan Orange G, Butter Yellow, Fast Garnet GBC and Methyl Red due to their maximum of absorbance. All eleven compounds were separated in less than 5min. The method was successfully validated and applied using three commercial samples of chili-containing spices – Chili sauce (Indonesia), Feferony sauce (Slovakia) and Mojo sauce (Spain). The linearity range of proposed method was 0.50–9.09mgkg−1 (r≥0.995). The detection limits were determined as signal to noise ratio of 3 and were ranged from 0.15mgkg−1 to 0.60mgkg−1 (1.80mgkg−1 for Fast Garnet) for standard solution and from 0.25mgkg−1 to 1.00mgkg−1 (2.50mgkg−1 for Fast Garnet, 1.50mgkg−1 for Sudan Red 7B) for chili-containing samples. The recovery values were in the range of 73.5–107.2% and relative standard deviation ranging from 0.1% to 8.2% for within-day precision and from 0.5% to 8.8% for between-day precision. The method showed potential for being used to monitor forbidden dyes in food constituents. The developed UHPSFC method was compared to the UHPLC-UV method. The orthogonality of Sudan dyes separation by these two methods was demonstrated. Benefits and drawbacks were discussed showing the reliability of both methods for monitoring of studied illegal dyes in real food constituents.

An ultrahigh-performance liquid chromatography method with electrospray ionization tandem mass spectrometry for simultaneous quantification of five phytohormones in medicinal plant Glycyrrhiza uralensis under abscisic acid stress.

An efficient simplified method was developed to determine multiple classes of phytohormones simultaneously in the medicinal plant Glycyrrhiza uralensis. Ultrahigh-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) with multiple reaction monitoring (MRM) in negative mode was used for quantification. The five studied phytohormones are gibberellic acid (GA3), abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid, and salicylic acid (SA). Only 100mg of fresh leaves was needed, with one purification step based on C18 solid-phase extraction. Cinnamic acid was chosen as the internal standard instead of isotope-labeled internal standards. Under the optimized conditions, the five phytohormones with internal standard were separated within 4min, with good linearities and high sensitivity. The validated method was applied to monitor the spatial and temporal changes of the five phytohormones in G. uralensis under ABA stress. The levels of GA3, ABA, JA, and SA in leaves of G. uralensis were increased at different times and with different tendencies in the reported stress mode. These changes in phytohormone levels are discussed in the context of a possible feedback regulation mechanism. Understanding this mechanism will provide a good chance of revealing the mutual interplay between different biosynthetic routes, which could further help elucidate the mechanisms of effective composition accumulation in medicinal plants.

A novel ultra-high performance liquid chromatography method for the rapid determination of β-lactoglobulin as heat load indicator in commercial milk samples

The level of undenatured acid-soluble β-lactoglobulin can be used as an indicator to assess the heat load applied to liquid milk, thus further allowing the discrimination between milk originating from different thermal production processes. In this work, a new UHPLC method for the rapid determination of bovine β-lactoglobulin in 1.8min only (total runtime 3min) is presented using simple UV detection at 205nm. Separation selectivity for possibly co-eluting other major whey proteins (bovine serum albumin, lactoferrin, α-lactalbumin, immunoglobulin G) was verified, and the method validated for the analysis of liquid milk samples regarding linearity (20–560μg/mL, R2>0.99), instrumentation precision (RSDs<2.8%), limits of detection and quantification (7 and 23mg/L milk), repeatability of sample work-up (RSDs≤2.6%) and method recovery (103%). In total, 71 commercial liquid milk samples produced using different preservation techniques (e.g., thermal or mechanical treatment), hence featuring different applied heat loads, were profiled for their intrinsic undenatured acid-soluble β-lactoglobulin levels. As expected, pasteurized milk showed the highest concentrations clearly above 3000mg/L due to pasteurization being the mildest thermal treatment, while in contrast, ultra-high temperature heated milk featured the lowest amounts (<200mg/L). For extended shelf life (ESL) milk, quite diverse levels were determined ranging from ∼100 up to 4000mg/L, thus clearly illustrating variable applied heat loads and impacts on the “nativeness” of milk essentially due to the fact that the production technologies used for ESL milk may differ significantly, and are currently not regulated in the EU.

Comparative In Vitro Dissolution of Two Commercially Available Er-Zhi-Wan Herbal Medicinal Products.

In vitro dissolution test is an essential tool to assess the quality of herbal medicinal products in the solid dosage forms for oral use. Our work aimed to evaluate the dissolution behavior of Er-Zhi-Wan, in the formulations of water-honeyed pill and formula granule. Different media (water, 30% EtOH, 0.1 M HCl, acetate buffer, pH 4.5 and phosphate buffer, pH 6.8) were used following United States Pharmacopoeia and Chinese Pharmacopeia. An ultra-high performance liquid chromatography method was developed and validated to detect simultaneously six active ingredients for quantification and dissolution study (salidroside, specnuezhenide, nuezhenoside, luteolin, apigenin, oleanolic acid). As we observed, contents of main active ingredients were close in the two formulations for daily dose. In each medium, more ingredients dissolved from formula granule with higher Ymax and Ka. The mean dissolution time of the most ingredients in granule was significantly shorter than that in pill in acetate buffer, pH 4.5 and phosphate buffer, pH 6.8. Furthermore, salidroside, specnuezhenide and luteolin dissolved more than 80% in 30 min from formula granule, which indicated higher solubility along the intestinal tract according to biopharmaceutics classification system. The dissolution test developed and validated was adequate for its purposes and could be applied for quality control of herbal medicine. This work also can be used to provide necessary information on absorption for its biopharmaceutical properties.

Ultra-High Performance Liquid Chromatography (UHPLC) Method for the Determination of Limonene in Sweet Orange (Citrus sinensis) Oil: Implications for Limonene Stability.

The citrus-derived bioactive monoterpene limonene is an important industrial commodity and fragrance constituent. An RP isocratic elution C18 ultra-HPLC (UHPLC) method using a superficially porous stationary phase and photodiode array (PDA) detector has been developed for determining the limonene content of sweet orange (Citrus sinensis) oil. The method is fast with a cycle time of 1.2 min, linear, precise, accurate, specific, and stability indicating, and it satisfies U.S. Pharmacopeia suitability parameters. The method may be useful in its present form for limonene processing, or modified for research on more polar compounds of the terpenome. A forced-degradation experiment showed that limonene is degraded by heat in hydro-ethanolic solution. PDA detection facilitates classification of minor components of the essential oil, including β-myrcene.

Determination and occurrence of phenoxyacetic acid herbicides and their transformation products in groundwater using ultra high performance liquid chromatography coupled to tandem mass spectrometry.

A sensitive method was developed and validated for ten phenoxyacetic acid herbicides, six of their main transformation products (TPs) and two benzonitrile TPs in groundwater. The parent compounds mecoprop, mecoprop-p, 2,4-D, dicamba, MCPA, triclopyr, fluroxypr, bromoxynil, bentazone, and 2,3,6-trichlorobenzoic acid (TBA) are included and a selection of their main TPs: phenoxyacetic acid (PAC), 2,4,5-trichloro-phenol (TCP), 4-chloro-2-methylphenol (4C2MP), 2,4-dichlorophenol (DCP), 3,5,6-trichloro-2-pyridinol (T2P), and 3,5-dibromo-4-hydroxybenzoic acid (BrAC), as well as the dichlobenil TPs 2,6-dichlorobenzamide (BAM) and 3,5-dichlorobenzoic acid (DBA) which have never before been determined in Irish groundwater. Water samples were analysed using an efficient ultra-high performance liquid chromatography (UHPLC) method in an 11.9 min separation time prior to detection by tandem mass spectrometry (MS/MS). The limit of detection (LOD) of the method ranged between 0.00008 and 0.0047 µg·L−1 for the 18 analytes. All compounds could be detected below the permitted limits of 0.1 µg·L−1 allowed in the European Union (EU) drinking water legislation [1]. The method was validated according to EU protocols laid out in SANCO/10232/2006 with recoveries ranging between 71% and 118% at the spiked concentration level of 0.06 µg·L−1. The method was successfully applied to 42 groundwater samples collected across several locations in Ireland in March 2012 to reveal that the TPs PAC and 4C2MP were detected just as often as their parent active ingredients (a.i.) in groundwater.

Comparison of UPLC and HPLC methods for determination of vitamin C

Ultra performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) methods for determination of ascorbic acid (AA) and total AA (TAA) contents (as the sum of AA and dehydroascorbic acid (DHAA) after its reduction to AA) in fruit beverages and in pharmaceutical preparations were compared. Both methods are rapid: total time of analysis was 15 and 6min for HPLC and UPLC methods, respectively. The methods were validated in terms of linearity, instrument precision, limits of detection (LOD) and quantification (LOQ), accuracy and recovery. Intra- and inter-day instrument precisions for fruit juices, expressed as RSD, were 2.2% and 2.4% for HPLC, respectively, and 1.7% and 1.9% for UPLC, respectively. For vitamin C tablets, inter- and intra-day precisions were 0.4% and 0.5%, respectively (HPLC), and 0.5% and 0.3%, respectively (UPLC). Both methods were sensitive: LOD was 0.049μg/mL for HPLC and 0.024μg/mL for UPLC while LOQs were 0.149 and 0.073μg/mL for HPLC and UPLC, respectively. These methods could be useful in the routine qualitative and quantitative analysis of AA or TAA in pharmaceutical preparations or fruit beverages. However, UPLC method is more sensitive, faster and consumes less eluent.

Determination of Four Types of Hazardous Chemicals in Food Contact Materials by UHPLC-MS/MS

A simple ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometric method for the identification and quantification of two photoinitiators 4-methylbenzophenone and 2-ethylhexyl-4-dimethylaminobenzoate; nine plasticizers including di(2-ethylhexyl) adipate and diisobutyl adipate; three primary aromatic amines 4-aminobiphenyl, 4-amino-2′,3-dimethylazobenzene and bis-(4-aminophenyl) methane and six bisphenols bisphenol F diglycidyl ether, bisphenol A diglycidyl ether, bisphenol A (BPA), bisphenol B (BPB), bisphenol E (BPE) and bisphenol F (BPF) in food contact materials has been developed. The chromatographic conditions, pre-treatment methods and matrix effects were studied and optimized. For the determination of the four bisphenols, BPA, BPB, BPE and BPF, the UHPLC method employed a mobile phase of aqueous ammonia and methanol in binary gradient mode, and measurement was based on a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ion mode. The remaining chemicals were determined using the ESI source in positive ion mode and using the [M + NH4]+ or [M + H]+ adducts as precursor ions for tandem mass spectrometry. The calibration graphs were linear with correlation coefficients of above 0.995. Detection limits for the method were in the range of 1–16 µg/kg. Analyte recovery values were in the range of 70–114%, and relative standard deviations were 1–14%. Under optimized conditions, the chromatographic separation was performed in 12 min. The validation data indicated that the method was effective for the determination of the four classes of hazardous chemicals in plastic packaging materials or in can lacquers. The optimized method was successfully applied to trace analysis of commercially available food contact materials. Copyright © 2014 John Wiley & Sons, Ltd.

Antibiotic residues in broiler and layer meat in Chittagong district of Bangladesh

Aim: The present study was described thin layer chromatography (TLC) and ultra-high performance liquid chromatography (UHPLC) method for the detection of antibacterial substances in poultry muscle (breast and thigh), kidney, and liver. Materials and Methods: TLC method was used for screening detection of tetracycline, amoxicillin, ciprofloxacin, and enrofloxacin residues in poultry tissues. The samples were extracted with trichloroacetic acid (30%), diethyl ether, followed by detection in pre-coated TLC paper on ultraviolet detector. The UHPLC method was used for the quantification of antimicrobial residues in poultry tissues. Results: The residues of tetracycline were 48% in livers, 24% in kidneys, 20% in thigh muscles, and 24% in breast muscles. Ciprofloxacin residues were found 44% in liver, 42% in kidneys, 34% in thigh muscles and 30% in breast muscles. Enrofloxacin residues were found 40% in liver, 34% in kidneys, 22% in thigh muscles, and 18% in breast muscles. Amoxicillin residues were found 42% in liver, 30% in kidneys, 26% in thigh muscles and 22% in breast muscles. Most of the cases highest residues were found in liver such as tetracycline (48%), ciprofloxacin (44%), enrofloxacin (40%) and amoxicillin (42%) and almost lowest in breast muscles. In addition, nine positive samples from broiler were selected for amoxicillin residue quantification by UHPLC. It was observed that the concentration of amoxicillin residue in liver was ranging from 16.92 μg/kg to 152.62 μg/kg and in breast muscle was 45.38 μg/kg to 60.55 μg/kg, respectively. The maximum and minimum peak time was 4.7-5.2 min. Among the poultry tissues, liver had the highest level of antibiotic residues in comparison to other samples but the variation was not significant (p>0.05). Conclusions: Evidence suggests that more judicious use of antimicrobials in food animals will reduce the selection of resistant bacteria and help to preserve these valuable drugs for both human and veterinary medicine. The method described in this study is a simple, easy inexpensive which can be readily adopted by any laboratory for the detection of antibiotic residues in tissues of food-producing animals.

UHPLC Determination of Enantiomeric Purity of Sertraline in the Presence of its Production Impurities

A fast and low reagents consuming ultra-high performance liquid chromatography method was developed for the determination of the enantiomeric purity of sertraline in presence of its stereoisomers and four other process-related chiral impurities. The optimum chromatographic conditions were achieved using a commercially available C8 column with sub-2-μm particle size, and a mobile phase of 10 mM tetrabutylammonium hydrogen sulfate buffer pH = 3-acetonitrile (87:13, v/v) and 12 mM 2-hydroxypropyl-β-cyclodextrin as chiral additive, with ultraviolet detection at 220 nm. The method was validated in terms of ruggedness, specificity, linearity, accuracy, precision, and limits of detection and quantification. Different variables affecting the enantioresolution and retention time were optimized, such as pH, buffer concentration and type, chiral additive concentration and type, flow rate, stationary phase, mobile phase, and column temperature. Total analysis time was reduced by 50 % compared with conventional HPLC; also 94 % of acetonitrile and 90 % of 2-hydroxypropyl-β-cyclodextrin were saved.

Simultaneous Determination of Cefepime and Tazobactam in Injectables by Ultra-High Performance Liquid Chromatography Method

Aim: Stability indicating ultra-high performance liquid chromatography (UHPLC) method was developed and validated for the determination of cefepime (CFPM) and tazobactam (TZB) in injectable dosage form. Materials and Methods: Separation was performed in a Dionex Ultimate 3000 UPHLC system equipped with chromeleon software using Acclaim 120 C 18 (250 × 4.6 mm, 5 μm particle size) column with mobile phase (pH 6.0) containing methanol and sodium acetate buffer in the ratio of 11:89 v/v with a fl ow rate of 1.8 mL/min and detection wavelength of 220 nm. Stress studies were performed using HCL, NaOH, H 2 O 2 , and ultraviolet radiation. Results: The method was found to be linear in the concentration range of 50-350 μg/ml (R 2 = 0.998) and 6.25-43.75 μg/ml (R 2 = 0.998) with the regression equation y = 11068 x + 115231 and y = 8317.1x – 9869.7 for CFPM and TZB, respectively. The percentage of relative standard deviation (%RSD) of 0.63 and 1.39 for intra-day and 0.64 and 0.54 for inter-day precision, respectively for CFPM and TZB suggest the precision of the method as all these values are Conclusion: The method was found to be accurate, precise, specifi c, robust, linear, and stability indicating for the determination of CFPM and TZB in injectable dosage form. Key words: Cefepime, injectables, simultaneous determination, tazobactam, ultra high performance liquid chromatography.

UHPLC Study on the Degradation Profiles of Olopatadine Hydrochloride in Eye Drops Subjected to Heat and Filtration Methods of Sterilisation

A validated ultra-high performance liquid chromatography (UHPLC) method has been proposed, validated and used for the determination of olopatadine hydrochloride degradation products in olopatadine 1 mg mL−1 eye drops solution under the influence of two different sterilisation methods, heating and filtration, with good precision and accuracy. We found that the heat sterilization method yields a higher content of olopatadine hydrochloride degradation products in eye drops compared to unsterilized drug product or drug product sterilized by filtration, except for α-hydroxy olopatadine impurity, which remains stable with time and applied sterilization method. Contents of olopatadine related compound B shows a higher increase (from <0.005 to 0.044 %) when sterilised by heating than when subjected to aging and sterilization by filtration (increase up to 0.011 %). Similarly, total amount of all impurities is also increased from 0.13 to 0.49 % when the drug product is sterilised by heating instead of filtration (up to 0.39 %). Content of olopatadine related compound B and of all impurities is increased by aging, probably through thermal and oxidative degradation. Forced degradation studies were correlated with the sterilisation study and possible degradation pathways were identified. Olopatadine shows strong degradation under oxidative and moderate degradation under photolytic environment, with the olopatadine related compound B as the main degradation product. Sterilization of eye drops solution by filtration is recommended.

Inhibitory effect of eleven herbal extracts on advanced glycation end-products formation and aldose reductase activity

The formation of advanced glycation end-products (ages) and aldose reductase (ar) activity have been implicated in the development of diabetic complications. our study sought to characterize the capacities of eleven herbal extracts against the formation of ages and the ar activity. an ultrahigh performance liquid chromatography and tandem mass spectrometry (uplc-ms/ms) method was used for the detection of ar activity and the screening of ar inhibitors in this research. the amount of sorbitol from each analyte was directly detected using the multiple reaction monitoring mode and the sorbitol level could be reduced via the addition of an inhibitor. moreover, the bsa/glucose (fructose) system was applied to investigate their inhibitory activities of ages formation in glycation model reactions. compared with other screened herbs used in our study, flos sophorae immaturus and radix scutellariae seemed to be more effective on inhibiting the formation of ages and ar activity. the inhibiting capacities of herbal extracts against ar activity and ages formation may be correlated with the bioactive components of the herbal extracts. the differences were correlated with the amount of polyphenol and flavonoid components. in the study, we have investigated the potential anti-hyperglycemic bioactivity of eleven herbal extracts in vitro, which could provide a reference for further in vivo research in the prevention and treatment of diabetic complications. (c) 2014 feng-rui song. published by elsevier b.v.

An ultra-high performance chromatographic method for the determination of artemisinin

Objective: The goal of this study is to develop an ultra-high performance liquid chromatographic method for the quantitative determination of artemisinin at very low concentrations using selective ion mass spectroscopic detection.Materials and methods: Separation was conducted using a C4 100 mm× 2.1 mm column, and the mobile phase consisted of an isocratic two-component system consisting of 60% of a 0.1% aqueous solution of formic acid and 40% acetonitrile at a flow rate of 0.4 ml/min. The drug was detected by means of an electrospray mass spectrometer with selective ion monitoring of the [M-H2O+H]+ with m/z of 265.3 in positive ion mode.Results: The calibration curves of artemisinin obtained from the UPLC/MS system were linear in the three ranges analyzed, with a correlation coefficient of no less than 0.9996 for all sets of standards. The peak tailing factor for all measurements were ≤1.7. The method proved to have good repeatability and linearity.Discussion: The described analytical method reached a LOQ of 0.010 µg/ml with an isocratic system and enables an analysis rate of 20 samples per hour. The linearity of the standards was excellent for all sets of standards analyzed.Conclusion: The method presented in this study provides a rapid and suitable means for the determination of artemisinin at very low concentrations. This is especially significant when performing dissolution studies where, due to the low solubility of artemisinin, a method that can measure the drug at nanogram levels is necessary.

Phytochemical profile and nutraceutical potential of chia seeds (Salvia hispanica L.) by ultra high performance liquid chromatography

Chia seeds (Salvia hispanica) were analyzed for total phenolic compounds, antioxidant activity, and quantification of phenolic acids and isoflavones by ultra high performance liquid chromatography (UHPLC), in order to obtain a phenolic phytochemical profile. The total phenolic concentration was 1.8-fold higher than previous reports and the antioxidant activity using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assay showed 68.83% inhibition, which was higher than the values reported previously for chia and different plant foods. Additionally, a simple, reproducible and rapid UHPLC method was proposed for the analysis of phenolic acids and isoflavones in chia. The method demonstrated to perform well with regard to linearity, limits of detection and quantification, precision, accuracy, and sensitivity. The detection limits ranged from 0.05 to 0.4ng/mL and the recovery percentage from 23.62 to 162.48%. With this method the major compounds identified and quantified were: rosmarinic acid 0.92, protocatechuic ethyl ester 0.74, caffeic acid 0.02, gallic acid 0.01, and daidzin 0.006mg/g seed. In brief, this study demonstrates that chia could be considered a seed with high antioxidant capacity and novel isoflavone source that can be incorporated in human diet.

Quantitative analysis of the flavonoid glycosides and terpene trilactones in the extract of Ginkgo biloba and evaluation of their inhibitory activity towards fibril formation of β-amyloid peptide.

The standard extract of Ginkgo biloba leaves (EGb761) is used clinically in Europe for the symptomatic treatment of impaired cerebral function in primary degenerative dementia syndromes, and the results of numerous in vivo and in vitro studies have supported such clinical use. The abnormal production and aggregation of amyloid β peptide (Aβ) and the deposition of fibrils in the brain are regarded as key steps in the onset of Alzheimer’s Disease (AD), and the inhibition of Aβ aggregation and destabilization of the preformed fibrils represent viable approaches for the prevention and treatment of AD. Flavonoid glycosides and terpene trilactones (TTLs) are the two main components of EGb761 which represent 24 and 6% of the overall content, respectively. In our research, seven abundant flavonoid glycosides 1–7 were isolated from the extract of Ginkgo biloba leaves and characterized by spectroscopic analysis. Furthermore, an ultra-high performance liquid chromatography method was established for the simultaneous quantification of these seven flavonoids. The inhibitory activities of these flavonoids, as well as four TTLs, i.e., ginkgolides A, B, and C and bilobalide (compounds 8–11), were evaluated towards Aβ42 fibril formation using a thioflavin T fluorescence assay. It was found that three flavonoids 1, 3 and 4 exhibited moderate inhibitory activities, whereas the other four flavonoids 2, 5, 6 and 7, as well as the four terpene trilactones, showed poor activity. This is the first report of the inhibition of Aβ fibril formation of two characteristic acylated flavonoid glycosides 6, 7 in Ginkgo leaves, on the basis of which the structure-activity relationship of these flavonoids 1–7 was discussed.

Validation and application of sub-2 μm core–shell UHPLC–UV–ESI–Orbitrap MS for identification and quantification of β-carotene and selected cleavage products with preceding solid-phase extraction

A validated ultrahigh-performance liquid chromatography method using 1.7 μm core–shell particles is presented for the identification and quantification of β-carotene (BC) and related cleavage products (CPs) in primary cell culture media. Besides BC, apo-4′-, apo-8′-, apo-10′-, and apo-12′-carotenals, as well as 5,6-epoxy-β-carotene, were selected as target analytes. Detection was performed via an 80-Hz diode array detector and an electrospray ionization–linear quadrupole ion trap–Orbitrap XL mass spectrometer, both hyphenated in series. Total analysis time was below 6 min with peak widths <12 s. Addition of trifluoroacetic acid and tetrahydrofuran to the mobile phase allowed for the mass spectrometric detection of BC and related CPs and reduced peak tailing due to improved solubility of hydrophobic analytes. Intra-day and inter-day precision for UV and mass spectrometric detection were ≤1.5 % for retention times and ≤5.1 % for peak areas. Instrumental linearity was confirmed by Mandel’s fitting test between 0.25 (or 1.00 μg/mL) and 5.00 μg/mL for UV detection. The higher sensitivity of mass spectrometric detection allowed for the coverage of three concentration domains between 0.025 and 5.00 μg/mL in linearity testing. Homoscedasticity was confirmed between 0.10 and 5.00 μg/mL for Orbitrap XL MS. The limits of quantification were between 52.6 and 889.4 ng/mL for UV detection and between 19.3 and 102.4 ng/L for mass spectrometric detection. Offline solid-phase extraction from culture media fortified with BC and CPs provided intra- and inter-day recoveries between 65.8 and 102.4 % with coefficients of variation ≤6.2 %. Primary rat hepatocyte cultures treated with BC and subjected to different oxidative stress conditions contained 5,6-epoxy-BC and apo-4′-carotenal besides residual BC. Apparently, 5,6-epoxy-BC was formed in the medium via autoxidation of BC by ambient oxygen.