Liquid Chromatography-diode Array Detection-mass Spectrometry Research Articles

Aruncus dioicus var. kamtschaticu: A Newly Identified Source of Lactucaxanthin (ε,ε-Carotene-3,3′-diol)

Green leafy vegetables are rich in lipophilic metabolites such as carotenoids, phytosterols, tocopherols, and fatty acids, known for their significant health benefits. Goat’s beard (Aruncus dioicus var. kamtschaticus), a wild leafy vegetable native to Ulleungdo Island, South Korea, is reported to possess various biological activities and bioactive compounds. However, the content and profiles of lipophilic metabolites, especially carotenoids, have not been reported. This study investigated the composition of lipophilic metabolites in the foliage of goat’s beard using liquid chromatography–diode-array detection–mass spectrometry, gas chromatography–mass spectrometry, and gas chromatography–flame ionization detection. Five carotenoids (violaxanthin, neoxanthin, lactucaxanthin, lutein, β-carotene) and α-tocopherol were identified and quantified using liquid chromatography–diode-array detection–mass spectrometry. Lactucaxanthin, previously discovered in lettuce, was identified for the first time in goat’s beard foliage, with a higher concentration (45.42 ± 0.80 µg/g FW) compared to red lettuce (19.05 ± 0.67 µg/g FW). Furthermore, total carotenoids and α-tocopherol contents were higher in goat’s beard than in red lettuce. Gas chromatography–mass spectrometry analysis showed the presence of three phytosterols, namely, campesterol (3.53 ± 0.20 µg/g FW), stigmasterol (65.30 ± 4.87 µg/g FW), and β-sitosterol (89.54 ± 2.46 µg/g FW). Gas chromatography–flame ionization detection analysis revealed the presence of five essential fatty acids, with α-linolenic acid (57.03 ± 0.47%) being the most abundant, contributing to a favorable polyunsaturated FA/saturated FA ratio. These findings underscore the nutritional potential of a goat’s beard, suggesting its promising use in dietary supplements and the commercial extraction of valuable lipophilic antioxidants, particularly lactucaxanthin, for nutraceuticals and functional foods.

Gastrointestinal digestion of olive leaf waste from olive mill production chain in a simulator of the gastrointestinal tract.

Currently, olive leaves are considered waste, although their high phenolic content makes them a source of antioxidants that could be used directly. The aim of this work was to study the behavior of phenolic compounds contained in olive leaf from the olive mill production chain during their gastrointestinal digestion. Phenolic compounds in the gastric digestion analyzed by high-performance liquid chromatography-diode array detection-mass spectrometry increased by 58% to 314.5% compared to the meal, while in the intestinal stage they ranged from 1.87 to 9.04 times higher. An increase of between 187% and 903% in bioaccessibility of phenolic compounds in the intestinal phase was observed, except for verbascoside. Furthermore, such compounds were fully bioavailable, except for apigenin-7-O-glucoside, which showed a bioavailability of 56%. The current study showed that the concentration of oleuropein, apigegin-7-O-glucoside, hydroxytyrosol hexoside and hydroxytyrosol contained in olive leaf continued to be extracted during the gastrointestinal digestion process. Furthermore, the results obtained with respect to their bioaccessibility and bioavailability suggest a good disposition to pass into the bloodstream where they could exert beneficial effects. Therefore, these results are promising for olive leaf becoming a consumable by-product that could be directly ingested through a simple infusion. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Towards an index of damage of olive paste during virgin olive oil extraction: The role of β-OH-acteoside diastereomers for risk assessment of the fusty/muddy sediment sensory defect

The aim of this study was to propose an index/marker of thermal/oxidative damage of olive paste during virgin olive oil extraction to assess the risk of sensory defects in the obtained oil. Several olive oil extraction trials were carried out over two production years applying different time, temperature, and oxygen exposure conditions during malaxation. The phenolic composition of the olive pastes was analyzed by High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS). The chemical (legal quality indexes, phenolic compounds by HPLC-DAD-MS, volatile compounds by Head Space-Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS)) and sensory characteristics of the obtained oils were analyzed. The content of the β-OH-acteoside diastereomers in olive paste increased linearly with malaxation time as a function of malaxation temperature and oxidative conditions, following a pseudo-zero order kinetic. At the same time, two volatile compounds linked to sensory defects (2- and 3-methylbutanal) increased in the obtained oils when increasing malaxation time and temperature, and a relationship between them and the fusty/muddy sediment defect in olive oil samples was observed. A further direct relationship between the β-OH-acteoside content in the olive pastes and the 2+3-methylbutanal content in the oils was observed. Therefore, the β-OH-acteoside diastereomers content in olive pastes has been suggested as an index/marker to indicate the risk of sensory defects in virgin olive oil as a function of the thermal and oxidative conditions during malaxation. A threshold value of 100 mg/kg of the content of β-OH-acteoside diastereomers was proposed, above which the fusty/muddy sediment defect is perceptible.

Chemical characterization, multivariate analysis and comparison of biological activities of different parts of Fraxinus mandshurica.

Fraxinus mandshurica (Oleaceae) is used as a traditional medicinal plant for the treatment of red eyes, menstrual disorders, excessive leucorrhea, chronic bronchitis and psoriasis. To perform chemical characterization of the secondary metabolites of F. mandshurica roots, bark, stems and leaves, 32 samples were collected from eight provinces in this study. A total of 64 chemical components were detected from four different parts of F. mandshurica by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Meanwhile, a total of nine secoiridoids were obtained by natural product chemical extraction, isolation and identification methods. Quantitative analysis by high-performance liquid chromatography-diode array detection-mass spectrometry showed the highest total content of secoiridoids in the bark, which is also consistent with the traditional medicinal parts. The results of methodological validation showed that the correlation coefficient (R2) values were all >0.9993, indicating a good linear range of the standard curve, while the relative standard deviations of precision, reproducibility and stability were <3%, and the spiked recoveries ranged from 98.22 to 102.27%, indicating that the experimental method was reliable and stable. In addition, fingerprinting and a heatmap were established to demonstrate the content trends of F. mandshurica more visually from different origins. Multivariate analysis, including principal component analysis and partial least squares discriminant analysis, was performed to determine the chemical characteristics of different parts of F. mandshurica, and six characteristic secoiridoids that could be used to distinguish different origins were screened. Finally, the inhibition of tyrosinase, α-glucosidase, acetylcholinesterase and pancreatic lipase activities by the nine characteristic compounds and extracts from different parts were investigated, and the results showed that they all exhibited different degrees of enzyme activity inhibition and thus have potential applications in whitening and blemish removal, hypoglycemia, anti-Alzheimer's disease and anti-obesity as a new source of natural enzyme activity inhibitors. This study establishes an identification and evaluation method applicable to phytochemistry of different origins, which is a guideline for quality control, origin evaluation and clinical application of traditional medicinal plants. This is also an unprecedented study on the identification of the chemical composition of different parts of F. mandshurica, characteristic compounds and the inhibition of enzyme activity of extracts from different parts.

Sterilisation of biopharmaceuticals: Effect of gamma irradiation, e-beam irradiation and nitrogen dioxide on human insulin

Biopharmaceuticals are administered parenterally and therefore sterility is required. Sterility can be obtained via different processes including exposure to steam or dry heat. Sterilisation studies on biopharmaceuticals, which are highly sensitive medicinal products, are scarce. This study investigates the effect of different sterilisation processes on recombinant human insulin in solid state (gamma and e-beam irradiation (w/wo dry ice), nitrogen dioxide (NO2)) and in aqueous solution (gamma irradiation (w/wo dry ice, w/wo glycerin)) using ultra-high performance liquid chromatography-diode array detection-mass spectrometry. It is observed that NO2 substantially degrades the solid samples, while gamma and e-beam irradiation result in lower levels of degradation (mean normalized peak areas of 95.2–96.2 % with respect to the non-sterilised samples). Gamma irradiation of insulin solutions with and without dry ice at 2.5 kGy results in mean normalised peak areas of 85 % and <40 % with respect to the non-sterilised samples, respectively. It is concluded that sterilisation using ionising radiation of liquid biopharmaceuticals with insulin and sterilisation of insulin dry powder using NO2 is less suitable with the set-ups used here because of substantial degradation. In contrast, evidence is presented in favour of sterilisation of insulin dry powder using ionising radiation.

GC-MS and LC-DAD-MS Phytochemical Profiling for Characterization of Three Native Salvia Taxa from Eastern Mediterranean with Antiglycation Properties.

Salvia fruticosa and S. pomifera subsp. calycina are native to Eastern Mediterranean and S. pomifera subsp. pomifera is endemic to Greece. The primary aim of this study was to develop an analytical methodology for metabolomic profiling and to study their efficacy in combating glycation, the major biochemical complication of diabetes. After sequential ultrasound-assisted extraction of 2 g of leaves with petroleum ether and 70% methanol, the volatile metabolites in the petroleum ether extracts were studied with GC-MS (Gas Chromatography-Mass Spectrometry), whereas the polar metabolites in the hydroalcoholic extracts were determined and quantified by UHPLC-DAD-ESI-MS (Ultra-High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry). This methodology was applied to five populations belonging to the three native taxa. 1,8-Cineole was the predominant volatile (34.8-39.0%) in S. fruticosa, while S. pomifera had a greater content of α-thujone (19.7-41.0%) and β-thujone (6.0-39.1%). Principal Component Analysis (PCA) analysis of the volatiles could discriminate the different taxa. UHPLC-DAD-ESI-MS demonstrated the presence of 50 compounds, twenty of which were quantified. PCA revealed that not only the taxa but also the populations of S. pomifera subsp. pomifera could be differentiated. All Salvia samples inhibited advanced glycation end-product formation in a bovine serum albumin/2-deoxyribose assay; rosmarinic and carnosic acid shared this activity. This study demonstrates the antiglycation activity of S. fruticosa and S. pomifera extracts for the first time and presents a miniaturized methodology for their metabolomic profiling, which could aid chemotaxonomic studies and serve as a tool for their authentication and quality control.

Combined, sequential dye analysis and radiocarbon dating of single ancient textile yarns from a Nazca tunic

The accessioning of ancient textiles into museum collections often requires objective information regarding the object’s appropriateness and authenticity before purchase or gift acceptance. In the case of colored fabrics, the identification of dyestuffs consistent with the attributed time period and culture builds confidence and reduces the chances of the object being a simple forgery or fake produced using modern materials. Moreover, this information adds to the technical, cultural, and conservation knowledge regarding the object. Increasingly, chronometric age estimates in the form of radiocarbon dating are also needed to establish the object’s age or to further prove the materials match the purported date range of the textile. Each of these analyses consumes a small sample of the object, and typically they are conducted separately by different laboratories on individual sample yarns. This report demonstrates for the first time the sequential, combined analysis of dyes by liquid chromatography-diode array detection-mass spectrometry and radiocarbon dating of the same residual dye-extracted sample. The chemicals and solvents used in various dye extraction protocols are shown not to contaminate the extracted yarns for radiocarbon dating purposes. The approach was used in the authentication study of an ancient Nazca tunic made from natural fibers (wool) and dyes (indigoids, anthraquinones, and flavonoids) shown to have most likely been produced between 595 and 665 CE.

Restoring trilinearity with the purpose of advanced modeling: towards a more effective analysis of Pericarpium Citri Reticulatae during storage periods

To accurately quantify Pericarpium Citri Reticulatae samples, trilinear structure was restored in the stacked fingerprints for more robust modeling. Initially, liquid chromatography - diode array detector - mass spectrometry (LC-DAD-MS) and head space-solid phase micro extraction coupled to gas chromatography - mass spectrometry (HS-SPME/GC-MS) were utilized to analyze Pericarpium Citri Reticulatae. Faced with the time-shifts in two-dimensional (2D) matrices across different samples, three algorithms were developed to synchronize them. Furthermore, bilinear and trilinear models were used to realize the quantifications with different principles. Through real cases based on LC-DAD, the advantages and disadvantages of trilinear decomposition over multivariate curve resolution-alternating least-squares can be clarified in the quantification of raw or synchronized fingerprints. Also in the data processing, a modification version of multi-scale peak alignment (mMSPA) was proved to be more suitable for trilinearity restoring than the other two algorithms. Recognizing these facts, restoring trilinearity were developed for more robust modeling in the application of the Pericarpium Citri Reticulatae fingerprints from different storage periods. After effective analysis, the upward/downward trend of 13 flavonoids were drawn accurately; and several flavour components having the highest contribution rate during storage were outlined reasonably. In conclusion, more robust modeling can be realized in trilinear data synchronized by appropriate algorithms, leading to an accurate quantification in herbal quality researches.

The Antimicrobial Properties of Poplar and Aspen-Poplar Propolises and Their Active Components against Selected Microorganisms, including Helicobacter pylori.

There is a noticeable interest in alternative therapies where the outcome is the eradication of the Gram-negative bacterium, Helicobacter pylori (H. pylori), for the purpose of treating many stomach diseases (chronic gastritis and peptic ulcers) and preventing stomach cancer. It is especially urgent because the mentioned pathogen infects over 50% of the world’s population. Recent studies have shown the potential of natural products, such as medicinal plant and bee products, on the inhibition of H. pylori growth. Propolis is such a bee product, with known antimicrobial activities. The main scope of the study is the determination of the antimicrobial activity of ethanolic extracts from 11 propolis samples (mostly from Poland, Ukraine, Kazakhstan, and Greece) against H. pylori, as well as selected bacterial and yeast species. The most effective against H. pylori was the propolis from Ukraine, with an MIC = 0.02 mg/mL while the rest of samples (except one) had an MIC = 0.03 mg/mL. Moreover, significant antimicrobial activity against Gram+ bacteria (with an MIC of 0.02–2.50 mg/mL) and three yeasts (with an MIC of 0.04–0.63 mg/mL) was also observed. A phytochemical analysis (polyphenolic profile) of the propolis samples, by ultra-high-performance liquid chromatography-diode array detector-mass spectrometry (UPLC-DAD-MS), was performed. An evaluation of the impact of the propolis components on antimicrobial activity, consisting of statistical analyses (principal component analysis (PCA) and hierarchical fuzzy clustering), was then performed. It was observed that the chemical composition characteristics of the poplar propolis correlated with higher antibacterial activity, while that of the poplar and aspen propolis correlated with weaker antibacterial activity. To summarize the activity in vitro, all tested propolis samples indicate that they can be regarded as useful and potent factors in antimicrobial therapies, especially against H. pylori.

Determination of Illegally Used Chemical Dyes in the Chinese Herbal Medicine "Red Flower Oil".

Red flower oil is a group of herbal medicinal liniments widely used in China and Southeast Asia. The color of red flower oil is adjusted to red or brownish-red by adding natural dyes. The objective of this study is to identify and quantify the synthetic dyes illegally used in red flower oil. Thirty-two batches of red flower oil (from nine manufacturers) were collected from different cities in China.High-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) analysis was performed to identify the chemical dyes in the samples, and high-performance liquid chromatography-diode array detection (HPLC-DAD) was used to quantify the chemical dyes. Sudan I, Sudan IV, and Solvent Red 207 were detected in nine batches of preparations (from three manufacturers) with concentration ranges of 101.7-214.9 μg/mL for Sudan I, 24.0-41.0 μg/mL for Sudan IV, and 147.5-221.7 μg/mL for Solvent Red 207. In present study, sudan I, sudan IV, and solvent red 207 were detected in red flower oil. The control of chemical dyes in food and drug should be further studied and not limited to sudan dyes. It is the first report about the detection of solvent red 207 in food and drug. The illegal use of those chemical dyes should be regarded as serious violation of good manufacturing practice (GMP) and might be dangerous for the patients.

Characterization of an Endophytic Strain Talaromyces assiutensis, CPEF04 With Evaluation of Production Medium for Extracellular Red Pigments Having Antimicrobial and Anticancer Properties.

Considering the worldwide demand for colorants of natural origin, the utilization of ascomycete fungi as a prolific pigment producer unfolds a novel way to obtain these pigments for various applications, including food, cosmetic, and medical use. The presence of very few natural red pigment alternatives in the market also attracts research and industry priorities to unearth novel and sustainable red pigment producers. The present work is an attempt to identify a novel source of red color obtained from endophytic fungi isolated from terrestrial and marine habitats. Based upon the fungal capacity for pigment production, seven isolates of endophytic fungi were recognized as prospective pigment producers. Out of all, fungal isolate CPE04 was selected based upon its capacity to produce profuse extracellular red pigment. The isolate was identified as Talaromyces assiutensis, employing morphological features and phylogenetic characterization by internal transcribed spacer (ITS) sequences. To understand the chemical behavior of pigment molecules, an investigation of the chemical profile of fungal culture filtrate dried powder (CFDP) was performed using ultra-high-performance liquid chromatography-diode array detector-mass spectrometry (UPLC–DAD–MS). In total, eight compounds having pigment and pharmaceutical application were tentatively identified using UPLC–DAD–MS. Considering the commercial aspect of the stated work, an effort was also made for standardizing the upscaling of the pigment molecule. Investigations were performed for optimum medium and culturing conditions for maximum pigment production. CFDP was found to have a significant antibacterial activity against the bacterial pathogens Staphylococcus aureus (MTCC737), Vibrio cholerae (N16961), and methicillin-resistant S. aureus (MRSA) (ATCC BAA811). The CFDP showed a minimum inhibitory concentration at 64, 128, and 256 μg/ml against S. aureus, MRSA, and V. cholerae. A concentration-dependent (50–400 μg/ml) anticancer effect on HeLa cancer line was also observed, having a half-maximal inhibitory concentration (IC50) at 300 μg/ml. The antioxidant potential of CFDP has also been proven with the help of an antioxidant assay against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (IC50, 32.01 μg/ml); DNA nicking assay and reactive oxygen species were generated in HeLa cancer line cells. The CFDP was also found to have no cytotoxicity toward HEK 293 T cell line using alamar blue (resazurin), a cell metabolic activity reagent.

Identification of aloe and other dyes by means of SERS and HPLC-DAD-MS in the embroidery of a 15th century English folded almanac

The folded almanac Western MS.8932 (Wellcome Collection, London), produced in England between 1387 and 1405, is a calendar with astrological tables and diagrams used by medical practitioners to harness astrological information relating to health. Apart from the great interest for its use in medicine, this object has a unique feature in its exquisite, unique embroidered binding that indicates a prestigious artefact. Considering the scarceness of information on such items, a diagnostic study on the materials used for the outstanding embroidery of MS.8932 is a unique occasion of having information on the methods of production of medieval embroideries. A diagnostic campaign has been carried out to identify the dyes used for this task and the colourants used for the illuminations, and to evaluate them with reference to their commercial value. Preliminary information on the dyes was yielded by means of UV–visible diffuse reflectance spectrophotometry with fibre optics (FORS) and spectrofluorimetry with fibre optics (FOMF). Then a final identification was yielded by micro-invasive analysis involving Surface Enhanced Raman Spectroscopy (SERS) and High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS) [1]. Analysis on the micro samples (5–10 mm of very thin threads) taken from the embroidery revealed the presence of orchil for pink hues, indigo/madder double dyeing for purple hues, red safflower for salmon pink hues and indigo/weld double dyeing for green hues. Particularly remarkable was the additional identification of aloe in the purple threads, possibly used as mordant and/or as antibacterial agent, which evidence is the first ever recorded in such materials. This information is very important to understand the significance of the artefact and its historical-artistic location, and will allow to make comparison with similar artworks.

Impact of Plant Origin on Eurasian Propolis on Phenolic Profile and Classical Antioxidant Activity.

Propolis is a bee product with known medical properties, including antioxidant activity. The scope of the study is profiling 19 different Eurasian propolis samples (mostly from Russia and Kazakhstan, Kyrgyzstan, Poland, Ukraine, and Slovakia). Profiles of propolises were investigated by ultra-high-performance liquid chromatography–diode array detector–mass spectrometry (UPLC-DAD-MS). Classical antioxidant properties, which are based on electron donation mechanism, were assessed by DPPH, ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) assays. Total phenolic and flavonoid contents were also evaluated by colorimetric tests. Most of the samples exhibited significant content of polyphenols (from 30.28 to 145.24 mg GAE/g of propolis) and flavonoids (from 10.45 to 82.71 mg GAE/g of propolis). Most of the propolis samples exhibited potent antiradical (DPPH test—from 8.83 to 64.47 mg GAE/g of propolis) and reducing activity (FRAP test—from 0.08 to 1.17 mmol Fe2+/g of propolis). Based on the occurrence of marker compounds, propolis samples were classified as poplar, aspen–birch, aspen–poplar, and aspen–birch–poplar type. Main markers present in propolis of poplar (e.g., chrysin, pinocembrin, galangin, and 3-O-acetyl-pinobanksin), birch (ermanin and acacetin) and aspen (2-acetyl-1,3-di-p-coumaroylglycerol) origin were used. DPPH, FRAP, and ORAC tests results were correlated with flavonoids, total polyphenols, or the polyphenols other than flavonoids content. In term of activity, poplar propolis type was variable, while aspen–birch–poplar type usually exhibited high DPPH and FRAP activity.

Ultra-high-throughput SPE-MALDI workflow: Blueprint for efficient purification and screening of peptide libraries

At the forefront of synthetic endeavors in the pharmaceutical industry, including drug discovery and high-throughput screening, timelines are tight and large quantities of pure chemical targets are rarely available. In this regard, the development of novel and increasingly challenging chemistries requires a commensurate level of innovation to develop reliable analytical assays and purification workflows with rapid turnaround that enables accelerated pharmacological evaluation. A small-scale automation platform enabling high-throughput analysis and purification to streamline the selection of candidate leads would be a transformative advance. Herein, we introduce an automation-friendly solid-phase extraction-matrix-assisted laser desorption/ionization (SPE-MALDI) platform applied to the high-throughput purification and analysis of peptide libraries. This advance enabled us to purify peptides from microgram levels in less than a day with results comparable to traditional high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS).

Comprehensive Chemical and Sensory Assessment of Wines Made from White Grapes of Vitis vinifera Cultivars Albillo Dorado and Montonera del Casar: A Comparative Study with Airén.

The ability to obtain different wines with a singular organoleptic profile is one of the main factors for the wine industry’s growth, in order to appeal to a broad cross section of consumers. Due to this, white wines made from the novel grape genotypes Albillo Dorado and Montonera del Casar (Vitis vinifera L.) were studied and compared to the well-known Airén at two consecutive years. Wines were evaluated by physicochemical, spectrophotometric, high-performance liquid chromatography–diode array detection–mass spectrometry, gas chromatography–mass spectrometry and sensory analyses. The chromatic characteristics of the new wines were defined by more color purity than Airén, with greenish highlights. In general, the phenolic profile of the Albillo Dorado wines showed a higher flavonol and hydroxycinnamic acid derivative content. Several volatile compounds were determined, and their odor activity values were calculated to determine their impact on wine aroma. A fruity series dominated the wine aromatic composition, but spicier and greener notes characterized the aroma profile of Airén wines. Albillo Dorado and Montonera del Casar were sensory evaluated as wines with a less fresh taste compared to Airén. Unique chemical and sensory profiles were determined for wines made from these novel grape genotypes, providing alternative monovarietal wines to encourage the wine market growth and extend the offer to consumers.

Study of the effect of marination treatment on garlic bioactive compounds through an innovative HPLC-DAD-MS method for alliin and curcuminoids analysis

The pungent aroma of garlic is associated to the formation of organosulfur compounds (OSCs) through alliinase activation. Therefore, to alleviate garlic pungency, food treatment preventing the formation of thiosulfinates (allicin) are needed. This study aims thus, to study the effect of marination on garlic OSCs to assess the efficiency of this process on garlic pungency. This was done through the development of a high-performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS) method. Moreover, different extraction solvents have been tested and the highest recoveries were obtained using methanol with formic acid 0.05%. The developed method was applied on marinated and non-marinated garlic samples. Alliin concentrations were significantly reduced in non-marinated garlic samples (29.9 ± 3.0 μg g−1) respect to marinated samples (296.4 ± 50.2 μg g−1). Furthermore, the chromatogram profiles of fresh garlic samples showed the presence of very high peaks corresponding to allicin and allicin degradation products. This was not observed in marinated samples proving that the marination process can inactivate alliinase. Moreover, the presence of curcuminoids such as curcumin (13.4 ± 2.7 μg g−1) in a garlic preparation made of marinated garlic and turmeric (Curcumaglio®) confirmed that marination could be considered as a process to transfer bioactive compounds in food.

High-performance thin-layer chromatography hyphenated to high-performance liquid chromatography-diode array detection-mass spectrometry for characterization of coeluting isomers

The effect-directed analysis on a planar chromatogram allows for fast non-target screening, multi-imaging detection of effects (bioprofiling) and highly targeted characterization and isolation of bioactive compounds. For direct characterization by high-resolution mass spectrometry (HRMS), however, the orthogonal hyphenation of two different liquid chromatographic techniques (planar and column chromatography) is still underexplored. In particular, it can be helpful in case of coeluting compounds. Exemplarily, lemon balm (Melissa officinalis L.) leaf extract was analysed by high-performance thin-layer chromatography in combination with bioactivity assays for antibacterial (against the Gram-positive Bacillus subtilis and the Gram-negative Aliivibrio fischeri) and α-glucosidase-inhibitory compounds (HPTLC-UV/Vis/FLD-EDA). High-resolution mass spectra of two bioactive compound zones were directly recorded via an elution head-based interface. By HPTLC-HESI-HRMS, the compound in zone a inhibited A. fischeri and was identified as linolenic acid, whereas the two closely related constitutional isomers oleanolic acid and ursolic acid were present in zone b. This was proven by two-dimensional liquid chromatography. Heart-cutting HPTLC-UV/Vis/FLD-HPLC-DAD-MS allowed the separation of the two isomers and proved both to be present in the bioactive zone with ursolic acid at a much higher abundance.

Characterization of Arils Juice and Peel Decoction of Fifteen Varieties of Punica granatum L.: A Focus on Anthocyanins, Ellagitannins and Polysaccharides.

Pomegranate is receiving renewed commercial and scientific interest, therefore a deeper knowledge of the chemical composition of the fruits of less studied varieties is required. In this work, juices from arils and decoctions from mesocarp plus exocarp were prepared from fifteen varieties. Samples were submitted to High Performance Liquid Chromatography—Diode Array Detector–Mass Spectrometry, spectrophotometric and colorimetric CIEL*a*b* analyses. Antioxidant, antiradical and metal chelating properties, inhibitory activity against tyrosinase and α-amylase enzymes were also evaluated. All varieties presented the same main phenols; anthocyanins and ellagitannins were widely variable among varieties, with the richest anthocyanin content in the juices from the Wonderful and Soft Seed Maule varieties (approx. 660 mg/L) and the highest ellagitannin content in the peel of the Black variety (approx. 133 mg/g dry matter). A good correlation was shown between the colour hue and the delphinidin/cyanidin ratio in juices (R2 = 0.885). Total polysaccharide yield ranged from 3% to 12% of the peels’ dry weight, with the highest content in the Black variety. Decoctions (24.44–118.50 mg KAE/g) showed better in vitro antioxidant properties and higher inhibitory capacity against tyrosinase than juices (not active-16.56 mg KAE/g); the inhibitory capacity against α-amylase was similar and quite potent for juices and decoctions. Knowledge about the chemical composition of different pomegranate varieties will allow for a more aware use of the different parts of the fruit.

Does Fermentation Really Increase the Phenolic Content in Cereals? A Study on Millet.

Millet is underutilized in Europe, despite its advantages compared to other common cereals. In Asia and Africa, millet is mainly eaten in fermented form; its consumption has beneficial properties on human health. Three millet batches were compared in terms of free and bound phenols by High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS). The richest one in terms of bound phenols was selected for testing via a basic (0.1 M NaOH) and an acidic (1.2 M H2SO4) hydrolysis, in which 149.3 and 193.6 mg/100 g of phenols were recovered, respectively. The ability of fermentation, with yeast and Lactobacilli, to increase the content of phenolic compounds was verified. Five withdrawalswere performed to verify the influence of fermentation time on the total phenolic content. The greatest phenolic content was observed after 72 h. Fermentation increased the cinnamic acids and flavonoids contents by approximately 30%. Vitexin and vitexin 2″-O-rhamnoside contents were significantly higher in the fermented millet; these compounds partially inhibit the protein tyrosine phosphatase enzyme, which is overexpressed in type-2 diabetes. A molecular dynamic simulation showed the two flavonoids to be allosteric inhibitors. The phenolic extract from fermented millet demonstrated a higher level of antioxidant protection on human erythrocytes by ex vivo cellular antioxidant activity in red blood cells. In this context, functional foods based on fermented millet could represent a new trend in European markets.

Comprehensive online multicolumn two-dimensional liquid chromatography-diode array detection-mass spectrometry workflow as a framework for chromatographic screening and analysis of new drug substances.

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-μm columns in the first dimension in conjunction with several 3.0mm × 50mm RP columns packed with sub-3-μm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC×LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.