Ultra-high Performance Liquid Chromatography Method Research Articles

Evaluation of Crocins in Cheeses Made with Saffron by UHPLC

Saffron is a widespread consumed spice with potential health promoting compounds. In dairy technologies it is often used to enhance color and flavor of cheeses, so it would be recommended to know the content of saffron functional compounds in cheese made with this spice, as they could still have potential bioactivity. For this purpose, an UHPLC (ultra high performance liquid chromatography) method was developed, with the aim of reducing analysis time and solvent consumption. A methanol:water solution (80:20 v/v) was used to extract saffron compounds from cheese with stirring for 1 h in the dark at room temperature, then the samples were centrifuged at 3500 rpm, for 5 min at 4 °C and the residues were extracted twice. A linear gradient elution of acetonitrile in water allowed to simultaneously determine picrocrocin and crocins in saffron in a short time (16 min), and allowed a quantitative determination of crocins in commercial cheeses.

A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract using UHPLC core-shell column with F5 stationary phase

In this paper, a new ultra-high performance liquid chromatography (UHPLC) method using a core–shell column with a pentafluorophenyl stationary phase for separation of seven active compounds of a Silybum marianum extract was developed and validated. Silymarin, an extract of Silybum marianum, is known for its abilities to protect the liver from toxic substances, hepatitis therapy, and anti-tumour activity. Silymarin is currently being widely used in commercial preparations and herbal teas. Separation of seven compounds contained in the Silybum marianum extract (taxifolin, silychristin, silydianin, silybin A, silybin B, isosilybin A, isosilybin B) and other substances occurring in real samples was performed on the Kinetex 1.7μ F5 100A (150×2.1mm), 1.7μm particle size core–shell column, with a mobile phase methanol/100mM phosphate buffer pH 2.0 according to the gradient program. A mobile phase 0.35mLmin−1 flow rate and 50°C temperature was used for the separation. The detection wavelength was set at 288nm. Under optimal chromatographic conditions, good linearity with a correlation coefficient of R2 >0.999 for all compounds was achieved. The available commercial samples of herbal teas and food supplements were extracted with methanol using an ultrasonic bath. After dilution with water and centrifugation, a 2μL sample of the filtered supernatant was directly injected into the UHPLC system. The use of a pentafluorophenyl stationary phase with methanol as the organic component of the mobile phase showed new ways to effectively separate isomeric compounds in herbal extracts, which could not be done with the conventional C18 stationary phase.

On Track for a Truly Green Propolis—Fingerprinting Propolis Samples from Seven Countries by Means of a Fully Green Approach

The production of quality bee products, as well as bee survival itself, depends on the health conditions of the environment, but ironically, harmful solvents are often employed by scientists and traders to monitor the quality of these products. Many types of propolis have been recognized around the world, but specific biological activities can be expected for specific types of propolis. This work aimed to develop a new and green ultrahigh performance liquid chromatography method for the identification of green propolis type. The method was able to discern this type of propolis in a set of samples from seven countries as well as to cluster these samples by fingerprint similarity based on principal component analysis and partial least squares–discriminant analysis. This proved to be efficient, reproducible, and greener than methods previously reported in the literature for similar purposes and compatible with the cheap, largely available food grade ethanol produced from sugar cane

Determination of amino acids in colon cancer cells by using UHPLC-MS/MS and [U-13C5]-glutamine as the isotope tracer

Rapid and simple quantitative analysis of intracellular metabolites is a critical tool for monitoring the alteration of biologically significant metabolites in cell lines or in vivo. We established an ultra-high performance liquid chromatography (UHPLC) method, equipped with hydrophilic interaction liquid chromatography (HILIC) column coupled to tandem mass spectrometry (MS/MS) for the simultaneous determination of 19 amino acids and 2 related derivatives in human cell lines. Chromatographic separation was achieved within 20min using a BEH amide column, with aqueous mobile phase containing 20mM ammonium acetate and 20mM ammonium hydroxide, and acetonitrile as the organic mobile phase. Amino acids were analyzed in positive ion multiple reaction monitoring (MRM) mode without the need of derivatization. Intra- and inter-day precisions were less than 13.7%. The method was successfully applied to simultaneously detect the 21 compounds in a human colon cancer cell line DLD1. Moreover, metabolite fate of glutamine-derived carbons into amino acids in DLD1 cells was successfully traced by using [U-13C5] glutamine as the isotope tracer. Metabolic consequences of glutaminolysis inhibition on amino acid metabolism were evaluated. Analysis of 12C- and U-13C-labeled amino acids revealed the significantly decreased incorporation of [U-13C5]-glutamine derived carbons into aspartate, alanine and ornithine, indicating impaired metabolic flux via the tricarboxylic acid cycle and the urea cycle.

Rapid Determination of Trace Multiresidues of 18 Sulfonamides in Chicken Eggs Using a Modified QuEChERS Method Coupled with Ultrahigh Performance Liquid Chromatography

A modified QuEChERS method was used and an ultrahigh performance liquid chromatography (UHPLC) method was developed for the rapid determination of 18 kinds of sulfonamide residues in chicken eggs. Sample preparation and cleanup conditions were carefully evaluated, and factors such as the adsorbent type and adsorption condition were key parameters in improving the cleanup. The modified QuEChERS method removed matrix interferences, and the sensitivity of the method increased about 5% for recovery and efficiency of the method. Under the optimized UHPLC method with UV detection, all 18 sulfonamide residues were simultaneously separated and rapidly identified within 15 min. The qualitative and quantitative method limits of the 18 sulfonamide residues were 2.06 to 4.12 and 6.86 to 13.7 μg·kg-1, respectively. A close linear relationship (R2 = 0.990 to 0.999) was observed within the concentration range of 0.10 to 2.25 μg·ml-1. Recovery was satisfactory (71 to 102%) for all the sulfonamides in three standard spiked levels, with relative standard deviations of <9.7%. After the modified sample pretreatment, the speed of sample pretreatment, purification, and analysis efficiency were all significantly increased. This method is suitable for the rapid detection of multisulfonamide residues in chicken eggs and other animal-derived foods.

Development and validation of an ultrafast chromatographic method for quantification of the immunosuppressant mycophenolic acid in canine, feline and human plasma

Mycophenolic acid (MPA) is the active metabolite of the prodrug mycophenolate mofetil. In this study, we developed and validated a novel ultra-high performance liquid chromatography (UHPLC) method for the rapid quantification of MPA in plasma from dogs, cats and humans. Following the protein precipitation, calibration standards and quality controls were separated by UHPLC reversed-phase on a 1.5μm 2.1×100mmC18 column and quantified using UV detection at 215nm. The procedure produced a linear curve (r2>0.997) over the concentration range 0.4–50μg/mL and exhibited a high degree of repeatability (CV% <11%). The limit of detection (LOD) and lower limit of quantitation (LLOQ) were 0.1 and ≤0.4μg/mL, respectively and the overall recovery was ≥87%. By combining isocratic conditions with a UHPLC column containing solid core particles, we were able to elute MPA and the internal standard (mycophenolic acid carboxybutoxy ether) within 3.0min. The short total run time makes this method ideal to study the disposition of MPA in large batches of plasma samples and/or monitor plasma drug concentrations, as recommended for patients that require optimized immunosuppression.

Analysis of Amikacin in Human Serum By UHPLC With Fluorescence Detector Using Chloro-Formate Reagent With Glycine

Background: Amikacin belongs to aminoglycosides family, commonly administered in the treatment of systemic infections due to gram negative bacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicity and ototoxicity. Objective: Optimize an ultra-high performance liquid chromatography (UHPLC) based analytical method for the determination of amikacin sulfate in human serum using derivatizaon with FMOCCl and glycine. Methods: Pre-column derivatization reaction of amikacin performed using fluorescence reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) at ambient temperature in the presence of borate buffer (0.2 M). Stabilizing reagent glycine (0.1 M) added into the reaction mixture solution after completion of the derivatization reaction for stabilization of fluorescent complex product. Fluorimetric detection of amikacin was performed at excitation and emission wavelength of 265 nm and 315 nm respectively, using C18 UHPLC column. The reported method was validated by performing linearity, precision, recovery and ruggedness. Results: The optimum mobile phase composition was found to be Acetonitrile:water in the ratio of 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin in serum samples ranging from 0.5-10 μg/ml was obtained, with correlation co-efficient of 1.00. The limit of detection (LOD) was found to be 50 ng/ml. Both inter- and intra-day analysis co-efficient values were found to be less than 10%. Conclusion: The developed UHPLC method will be useful for pre-clinical and pharmacokinetic study of amikacin in human serum. Key words: Amikacin Sulphate, Ultra High Performance Liquid Chromatography (UHPLC), 9-Fluorenylmethyl chloroformate (FMOC-Cl) reagent, Borate buffer, Glycine, Fluorescence detector.

A novel stability-indicating UPLC method development and validation for the determination of seven impurities in various diclofenac pharmaceutical dosage forms.

An innovative simple, fast, precise and accurate ultra-high performance liquid chromatography (UPLC) method was developed for the determination of diclofenac (Dic) along with its impurities including the new dimer impurity in various pharmaceutical dosage forms. An Acquity HSS T3 (C18, 100×2.1mm, 1.8μm) column in gradient mode was used with mobile phase comprising of phosphoric acid, which has a pH value of 2.3 and methanol. The flow rate and the injection volume were set at 0.35ml·min(-1) and 1μl, respectively, and the UV detection was carried out at 254nm by using photodiode array detector. Dic was subjected to stress conditions from acid, base, hydrolytic, thermal, oxidative and photolytic degradation. The new developed method was successfully validated in accordance to the International Conference on Harmonization (ICH) guidelines with respect to specificity, limit of detection, limit of quantitation, precision, linearity, accuracy and robustness. The degradation products were well resolved from main peak and its seven impurities, proving the specificity power of the method. The method showed good linearity with consistent recoveries for Dic content and its impurities. The relative percentage of standard deviation obtained for the repeatability and intermediate precision experiments was less than 3% and LOQ was less than 0.5μg·ml(-1) for all compounds. The new proposed method was found to be accurate, precise, specific, linear and robust. In addition, the method was successfully applied for the assay determination of Dic and its impurities in the several pharmaceutical dosage forms.

Variation of the Phytochemical Constituents and Antioxidant Activities of Zingiber officinale var. rubrum Theilade Associated with Different Drying Methods and Polyphenol Oxidase Activity.

The effects of different drying methods (freeze drying, vacuum oven drying, and shade drying) on the phytochemical constituents associated with the antioxidant activities of Z. officinale var. rubrum Theilade were evaluated to determine the optimal drying process for these rhizomes. Total flavonoid content (TFC), total phenolic content (TPC), and polyphenol oxidase (PPO) activity were measured using the spectrophotometric method. Individual phenolic acids and flavonoids, 6- and 8-gingerol and shogaol were identified by ultra-high performance liquid chromatography method. Ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used for the evaluation of antioxidant activities. The highest reduction in moisture content was observed after freeze drying (82.97%), followed by vacuum oven drying (80.43%) and shade drying (72.65%). The highest TPC, TFC, and 6- and 8-shogaol contents were observed in samples dried by the vacuum oven drying method compared to other drying methods. The highest content of 6- and 8-gingerol was observed after freeze drying, followed by vacuum oven drying and shade drying methods. Fresh samples had the highest PPO activity and lowest content of flavonoid and phenolic acid compounds compared to dried samples. Rhizomes dried by the vacuum oven drying method represent the highest DPPH (52.9%) and FRAP activities (566.5 μM of Fe (II)/g DM), followed by freeze drying (48.3% and 527.1 μM of Fe (II)/g DM, respectively) and shade drying methods (37.64% and 471.8 μM of Fe (II)/g DM, respectively) with IC50 values of 27.2, 29.1, and 34.8 μg/mL, respectively. Negative and significant correlations were observed between PPO and antioxidant activity of rhizomes. Vacuum oven dried rhizomes can be utilized as an ingredient for the development of value-added food products as they contain high contents of phytochemicals with valuable antioxidant potential.

Screening the anti-gout traditional herbs from TCM using an in vitro method

The aim of this work was to evaluate the ability of 33 herbal extracts in inhibiting the acute inflammation and xanthine oxidase (XOD) activity. The anti-inflammation effects of the herbal extracts were detected by an in vitro cell model, which was established by stimulating human umbilical vein endothelial cells (HUVEC) using sodium urate (MSU). In this model, the intercellular adhesion molecule-1 (ICAM-1) and interleukin-1 beta (IL-1β) were expressed, and the anti-inflammation effects of herbal extracts were evaluated by detecting the content changes of ICAM-1 and IL-1β in cell lysates and cell culture supernates using an enzyme-linked immunosorbent assay (ELISA). Moreover, an ultrahigh performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) method was used for the detection of XOD activity and the screening of XOD inhibitors in this research. The amount of uric acid from each analyte was directly detected using the multiple reaction monitoring mode and the uric acid level could be reduced via the addition of an inhibitor. Results indicated that Salviae Miltiorrhizae Radix et Rhizome, Rhei Radix et Rhizoma, Polygoni Cuspidati Rhizoma et Radix, Selaginellae Herba, Paeoniae Radix Rubra, especially Ginkgo Folium seemed to be more effective in anti-inflammation and inhibiting XOD activity. The anti-inflammation and enzyme inhibitory activities of the herbal extracts may be correlated with their bioactive components. And the differences between the herbal extracts were correlated with the amount of flavonoid and anthraquinone components. In our study, we have investigated the potential anti-inflammation bioactivity of 33 herbal extracts in vitro, which could provide a reference for further in vivo research in the prevention and treatment of gout.

Development and validation of bioanalytical UHPLC-UV method for simultaneous analysis of unchanged fenofibrate and its metabolite fenofibric acid in rat plasma: Application to pharmacokinetics

A simple, precise, selective and fast ultra-high performance liquid chromatography (UHPLC-UV) method has been developed and validated for the simultaneous determination of a lipid regulating agent fenofibrate and its metabolite fenofibric acid in rat plasma. The chromatographic separation was carried out on a reversed-phase Acquity® BEH C18 column using methanol–water (65:35, v/v) as the mobile phase. The isocratic flow was 0.3ml/min with rapid run time of 2.5min and UV detection was at 284nm. The method was validated over a concentration range of 100–10000ng/ml (r2⩾0.9993). The selectivity, specificity, recovery, accuracy and precision were validated for determination of fenofibrate/fenofibric acid in rat plasma. The lower limits of detection and quantitation of the method were 30 and 90ng/ml for fenofibrate and 40 and 100ng/ml for fenofibric acid, respectively. The within and between-day coefficients of variation were less than 5%. The validated method has been successfully applied to measure the plasma concentrations in pharmacokinetics study of fenofibrate in an animal model to illustrate the scope and application of the method.

Retention behavior of lipids in reversed-phase ultrahigh-performance liquid chromatography–electrospray ionization mass spectrometry

Reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) method using two 15cm sub–2μm particles octadecylsilica gel columns is developed with the goal to separate and unambiguously identify a large number of lipid species in biological samples. The identification is performed by the coupling with high-resolution tandem mass spectrometry (MS/MS) using quadrupole – time-of-flight (QTOF) instrument. Electrospray ionization (ESI) full scan and tandem mass spectra are measured in both polarity modes with the mass accuracy better than 5ppm, which provides a high confidence of lipid identification. Over 400 lipid species covering 14 polar and nonpolar lipid classes from 5 lipid categories are identified in total lipid extracts of human plasma, human urine and porcine brain. The general dependences of relative retention times on relative carbon number or relative double bond number are constructed and fit with the second degree polynomial regression. The regular retention patterns in homologous lipid series provide additional identification point for UHPLC/MS lipidomic analysis, which increases the confidence of lipid identification. The reprocessing of previously published data by our and other groups measured in the RP mode and ultrahigh-performance supercritical fluid chromatography on the silica column shows more generic applicability of the polynomial regression for the description of retention behavior and the prediction of retention times. The novelty of this work is the characterization of general trends in the retention behavior of lipids within logical series with constant fatty acyl length or double bond number, which may be used as an additional criterion to increase the confidence of lipid identification.

Automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry for the determination of ten antipsychotics in human plasma.

An automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry method was developed for the simultaneous determination of ten antipsychotics in human plasma. The plasma sample after filtration was injected directly into the system without any pretreatment. A Shim-pack MAYI-C8 (G) column was used as a solid-phase extraction column, and all the analytes were separated on a Shim-pack XR-ODS III column with a mobile phase consisting of 0.1% v/v formic acid in water with 5mM ammonium acetate and acetonitrile. The method features were systematically investigated, including extraction conditions, desorption conditions, the equilibration solution, the valve switching time, and the dilution for column-head stacking. Under the optimized conditions, the whole analysis procedure took only 10 min. The limits of quantitation were in the range of 0.00321-2.75μg/L and the recoveries ranged from 75.9 to 122%. Compared with the off-line ultra-high performance liquid chromatography and the reported methods, this validated on-line method showed significant advantages such as minimal pretreatment, shortest analysis time, and highest sensitivity. The results indicated that this automatic on-line method was rapid, sensitive, and reliable for the determination of antipsychotics in plasma and could be extended to other target analytes in biological samples.

Simultaneous Estimation of Loteprednol Etabonate and Gatifloxacin in Pharmaceutical Dosage Form by UHPLC Method

A reverse phase ultra-high performance liquid chromatography (RP-UHPLC) method was developed and validated for the simultaneous estimation of Loteprednoletabonate (LOTE) and Gatifloxacin (GAT) in pharmaceuticaldosage form. The separation was carried on zorbax eclipse plus column C18 (4.6 × 50 mm, 1.8 μ particle size) column with mobile phase (pH 6.0 withtriethylamine) containing Acetonitrile, Methanol and 0.02M potassiumdihydrogen phosphate in the ratio of 40:30:30 v/v/v with a flow rate of 0.7 mL/min and UV detection at 272 nm. The linearity was found to be in range of 15.34-46.03 μg/mL (R 2 = 0.999) and 24.94-74.81 μg/mL (R 2 = 0.999) for GAT and LOTE respectively. The method has shown good, consistent recoveries for GAT 98.88-101.24% and LOTE 98.42 - 100.94% respectively. The method was found to be accurate, precise, specific,robust and linear for the determination of GAT and LOTE in pharmaceutical dosage form.

Variation in secondary metabolite production as well as antioxidant and antibacterial activities of Zingiber zerumbet (L.) at different stages of growth.

BackgroundZingiber zerumbet (L.) is a traditional Malaysian folk remedy that contains several interesting bioactive compounds of pharmaceutical quality.Methods Total flavonoids and total phenolics content from the leaf, stem, and rhizome of Z. zerumbet at 3 different growth stages (3, 6, and 9 months) were determined using spectrophotometric methods and individual flavonoid and phenolic compounds were identified using ultra-high performance liquid chromatography method. Chalcone Synthase (CHS) activity was measured using a CHS assay. Antioxidant activities were evaluated by ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. The antibacterial activity was determined against Gram-positive and Gram-negative bacteria using the disc diffusion method.ResultsHighest content of total flavonoid [29.7 mg quercetin equivalents (QE)/g dry material (DM)] and total phenolic (44.8 mg gallic acid equivalents (GAE)/g DM) were detected in the rhizome extracts of 9-month-old plants. As the plant matured from 3 to 9 months, the total flavonoid content (TFC) and total phenolic content (TPC) decreased in the leaf, but increased significantly in the rhizomes. Among the secondary metabolites identified, the most abundant, based on the concentrations, were as follows: flavonoids, catechin > quercetin > rutin > luteolin > myricetin > kaempferol; phenolic acids, gallic acid > ferulic acid > caffeic acid > cinnamic acid. Rhizome extracts from 9-month-old plants demonstrated the highest CHS activity (7.48 nkat/mg protein), followed by the 6-month-old rhizomes (5.79 nkat/mg protein) and 3-month-old leaf (4.76 nkat/mg protein). Nine-month-old rhizomes exhibited the highest DPPH activity (76.42 %), followed by the 6-month-old rhizomes (59.41 %) and 3-month-old leaves (57.82 %), with half maximal inhibitory concentration (IC50) of 55.8, 86.4, and 98.5 μg/mL, respectively, compared to that of α- tocopherol (84.19 %; 44.8 μg/mL) and butylated hydroxytoluene (BHT) (70.25 %; 58.6 μg/mL). The highest FRAP activity was observed in 9-month-old rhizomes, with IC50 of 62.4 μg/mL. Minimal Inhibitory Concentration (MIC) of Z. zerumbet extracts against Gram-positive and Gram-negative bacteria ranged from 30 to >100 µg/mL. Among the bacterial strains examined, Staphylococcus aureus was sensitive to the leaf extract of Z. zerumbet, with MIC of 30.0 μg/mL and other strains were sensitive to the rhizome extracts.ConclusionsThree- and 9-month-old plants are recommended when harvesting the leaf and rhizome of Z. zerumbet, respectively, in order to obtain effective pharmaceutical quality of the desired compounds.

Optimization of Microwave-Assisted Extraction Conditions for Five Major Bioactive Compounds from Flos Sophorae Immaturus (Cultivars of Sophora japonica L.) Using Response Surface Methodology.

Microwave-assisted extraction was applied to extract rutin; quercetin; genistein; kaempferol; and isorhamnetin from Flos Sophorae Immaturus. Six independent variables; namely; solvent type; particle size; extraction frequency; liquid-to-solid ratio; microwave power; and extraction time were examined. Response surface methodology using a central composite design was employed to optimize experimental conditions (liquid-to-solid ratio; microwave power; and extraction time) based on the results of single factor tests to extract the five major components in Flos Sophorae Immaturus. Experimental data were fitted to a second-order polynomial equation using multiple regression analysis. Data were also analyzed using appropriate statistical methods. Optimal extraction conditions were as follows: extraction solvent; 100% methanol; particle size; 100 mesh; extraction frequency; 1; liquid-to-solid ratio; 50:1; microwave power; 287 W; and extraction time; 80 s. A rapid and sensitive ultra-high performance liquid chromatography method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (EIS-Q-TOF MS/MS) was developed and validated for the simultaneous determination of rutin; quercetin; genistein; kaempferol; and isorhamnetin in Flos Sophorae Immaturus. Chromatographic separation was accomplished on a Kinetex C18 column (100 mm × 2.1 mm; 2.6 μm) at 40 °C within 5 min. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile (71:29; v/v). Isocratic elution was carried out at a flow rate of 0.35 mL/min. The constituents of Flos Sophorae Immaturus were simultaneously identified by EIS-Q-TOF MS/MS in multiple reaction monitoring mode. During quantitative analysis; all of the calibration curves showed good linear relationships (R2 > 0.999) within the tested ranges; and mean recoveries ranged from 96.0216% to 101.0601%. The precision determined through intra- and inter-day studies showed an RSD% of <2.833%. These results demonstrate that the developed method is accurate and effective and could be readily utilized for the comprehensive quality control of Flos Sophorae Immaturus.

A UHPLC-UV Method to Quantify Skin Deposition and Transdermal Permeation of Tizanidine Hydrochloride.

Tizanidine hydrochloride is an α2-adrenergic agonist used for the symptomatic relief of spasticity associated with multiple sclerosis or with spinal cord injury or disease. The objective of this study was to develop an isocratic, robust and sensitive ultra-high performance liquid chromatography method using UV detection for use in a project to develop a transdermal therapeutic system to deliver tizanidine across the skin. Isocratic separation was achieved using a C18 column and a mobile phase comprising a 80:20 mixture of 0.004% trifluoroacetic acid in water and MeCN (pH* 3.2) at a flow rate of 0.2 mL min(-1) Tizanidine eluted at 1.499 min and the total run time was 2 min. The method was specific, robust and the response was accurate, precise and linear from 17.4 to 290 ng mL(-1) In contrast to existing methods, the method developed here was validated over a concentration range so as to include the low concentrations frequently observed in transdermal permeation studies and in samples extracted from the cutaneous matrix. Its suitability for use in transdermal permeation studies was subsequently tested and confirmed in preliminary experiments using porcine skin in vitro.

The anti-inflammation and pharmacokinetics of a novel alkaloid from Portulaca oleracea L.

This study was to elucidate the pharmacokinetics of a novel alkaloid, 6-acetyl-2,2,5-trimethyl-2,3-dihydrocyclohepta[b]pyrrol-8(1H)-one, named oleracone isolated from Portulaca oleracea L., and to examine the anti-inflammatory ability with lipopolysaccharide (LPS) stimulated macrophages. The novel alkaloid, oleracone, was isolated from Portulaca oleracea L., and its structure was determined by spectroscopic analysis including HRESIMS, 2D NMR spectroscopic data and single-crystal X-ray diffraction. The activity of anti-inflammation was assayed via the test with RAW 264.7 activated by LPS, and the pharmacokinetics of oleracone in rat plasma after intravenous and oral administration at dose of 2.5 mg/kg was, respectively, investigated by a rapid and sensitive ultra high-performance liquid chromatography (UHPLC) method with bergapten as internal standard. Oleracone was a novel alkaloid first isolated from Portulaca oleracea L. and possessed unique structure in natural products, whose anti-inflammatory effecting on nitrite oxide production and several pivotal pro-inflammatory cytokines was found at the concentration of 50 μm, and the pharmacokinetic results indicated that oleracone was rapidly distributed with Tmax of 15.7 min after oral administration and presented a higher oral absolute bioavailability to be 74.91 ± 10.7%. Oleracone as novel alkaloid presented remarkably anti-inflammatory effect, which was rapid distributed in rat with high bioavailability of 74.91 ± 10.7%.

Forced degradation of nepafenac: Development and validation of stability indicating UHPLC method

This paper presents stability study of the nonsteroidal anti-inflammatory drug (NSAID) nepafenac. In order to investigate stability of nepafenac, it was subjected to forced degradation under different stress conditions: acid and base hydrolysis, oxidation, humidity, heat and light. A novel stability indicating reverse phase ultra high performance liquid chromatographic (UHPLC) method coupled to ultraviolet detector has been developed to separate nepafenac and all related compounds (2-aminobenzophenone, Cl-thionepafenac, thionepafenac, Cl-nepafenac, hydroxy-nepafenac, and cyclic-nepafenac). Efficient chromatographic separation was achieved on a Waters Acquity BEH C18 stationary phase with a gradient elution. Quantification was carried out at 235nm at a flow rate of 0.6mL/min−1. The resolution between nepafenac and six potential impurities is found to be greater than 2.0. The developed method was validated with respect to specificity, LOD, LOQ, linearity, precision, accuracy and robustness. The r2 values for nepafenac and six potential impurities were all greater than 0.999. The developed method is capable to detect impurities of nepafenac at a level of 0.005% with respect to test concentration of 1.0mg/mL. Significant degradation is observed in acid, base and oxidative degradation conditions and degradation products (DPs) were identified using mass spectrometry analysis; two of them were found to be a known process related impurities (hydroxy- and cyclic-nepafenac) whereas four degradation products were identified as new degradation impurities. The forced degradation samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.5%.

High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography

The development and manufacturing of safe and effective vaccines relies essentially on the availability of robust and precise analytical techniques. Virus-like particles (VLPs) have emerged as an important and valuable class of vaccines for the containment of infectious diseases. VLPs are produced by recombinant protein expression followed by purification procedures to minimize the levels of process- and product-related impurities. The control of these impurities is necessary during process development and manufacturing. Especially monitoring of the VLP size distribution is important for the characterization of the final vaccine product. Currently used methods require long analysis times and tailor-made assays. In this work, we present a size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method to characterize VLPs and quantify aggregates within 3.1min per sample applying interlaced injections. Four analytical SEC columns were evaluated for the analysis of human B19 parvo-VLPs and murine polyoma-VLPs. The optimized method was successfully used for the characterization of five recombinant protein-based VLPs including human papillomavirus (HPV) VLPs, human enterovirus 71 (EV71) VLPs, and chimeric hepatitis B core antigen (HBcAg) VLPs pointing out the generic applicability of the assay. Measurements were supported by transmission electron microscopy and dynamic light scattering. It was demonstrated that the iSE-UHPLC method provides a rapid, precise and robust tool for the characterization of VLPs. Two case studies on purification tools for VLP aggregates and storage conditions of HPV VLPs highlight the relevance of the analytical method for high-throughput process development and process monitoring of virus-like particles.