Ultra-high Performance Liquid Chromatography Diode Array Detector Research Articles

Effects of different postharvest drying processes on flavonoid content and enzymatic activity of Styphnolobium japonicum (L.) Schott flowers for industrial and medicinal use

Traditionally, fresh S. japonicum flowers (SJF) and S. japonicum flowers buds (SJFB) are dried prior to further processing and use. Here, we investigated the ways in which drying techniques, including sun drying (SD), steam drying (STD), microwave drying (MD), hot air drying (HAD, 40 °C, 60 °C, 80 °C, 100 °C), and freeze drying (FD), alter the flavonoid composition of freshly-harvested SJF and SJFB. The flavonoid content of dried samples was determined by Ultra High Performance Liquid Chromatography-Diode Array Detector (UPLC-DAD). Overall, different drying techniques had significantly different effects on the RU content, ranging from 10.63 % (HAD-80 °C) to 34.13 % (HAD-100 °C) in SJF and from 18.91 % (HAD-100 °C) to 29.16 % (HAD-40 °C) and 30.53 % (SD) in SJFB. To clarify the mechanism by which drying affects the RU content of S. japonicum flowers, we studied the activity of a rutin-hydrolyzing enzyme (RHE) isolated from SJF and SJFB using multiple separation and assay methods. According to the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE) results, the apparent molecular weight of the purified RHE was approximately 38 kDa . According to UPLC-DAD, RHE catalyzes the production of quercetin (QU) from rutin (RU), but not from other flavonoid glycosides. Drying fresh SJF and SJFB at low and high temperatures can inhibit RHE activity and prevent RU hydrolysis. Therefore, subjecting freshly-harvest SJF to HAD-100 °C, and freshly-harvest SJFB to SD or HAD-40 °C, can greatly increase the RU content. In particular, HAD is viable for large-scale application due to its simplicity and industrial feasibility.

Incorporation of Partial Least Squares-Discriminant Analysis with Ultra-High-Performance Liquid Chromatography Diode-Array Detector for Authentication of Skin Gelatine Sources

This research seeks to (1) authenticate sources of skin gelatine by combining putative 17 aminoacids (AAs) analysis with chemometrics by Ultra-High-Performance Liquid ChromatographyDiode-Array Detector (UHPLC-DAD) and (2) create AA profiles in skin gelatines. Theclassification capability of partial least square-discriminant analysis (PLS-DA) models wasassessed to determine the most effective discriminant model. Principal component analysis(PCA) with quartimax rotation was utilised to accurately organise gelatine clusters and assign thesignificantly contributing AAs to each cluster. The PLS-DA model with 13 AAs (PLS-DAVIPAA)outperformed the PLS-DA model with 17 AAs (PLS-DAAA) because its R2Y (0.938), R2X (0.881),and Q2 (0.929) values were greater. With 13 significant AAs, the PLS-DAVIPAA model obtainedcluster classification accuracy of 100% on training and cross-validation datasets and 93.3% ontesting and verification datasets. The chemical structure of gelatines may shed light on theinteractions between AAs. Following six quartimax rotations, the gelatines were groupedcorrectly. The PCA showed the dominant presence of these AAs: L-Valine, L-Phenylalanine andL-Tyrosine in porcine gelatine; Glycine, L-Threonine, L-Arginine, L-Methionine, L-Histidine andL-Serine in fish gelatine; and L-Hydroxyproline, L-Leucine and L-Proline in bovine gelatine. Theauthority could use this technique to set a standard for authenticating skin gelatine samples.

New Insight into Enzyme Inhibitory and Anti-proliferative Activity of Echinops ritro L. (Asteraceae)

Echinops ritro L. is a perennial herbaceous plant in the tribe Cardueae, Asteraceae family, widely distributed, up to 1000 m a.s.l. In the present study, the enzyme inhibitory and anti-proliferative activity of E. ritro leaves and flowering heads extracts were assayed. Quantitative determination of 22 analytes, including eleven acylquinic and one hydroxycinnamic acids, and ten flavonoids was performed by Ultra high-performance liquid chromatography – diode array detector (UHPLC-DAD) analyses. The major compound was 1,5-dicaffeoylquinic acid, being presented at $$20.48\pm 0.22$$ µg/mg in the flowering heads, while the leaves demonstrated very high levels of chlorogenic acid ($$117.29\pm 14.20$$ µg/mg) and 3,5-dicaffeoylquinic acid ($$12.79\pm 0.282$$ µg/mg). The flavonoids were dominated by isoquercitrin and rutin ($$3.13\pm 0.033$$ and $$3.25\pm 0.057$$ µg/mg, respectively) together with metoxylated aglycones nepetin, hispidulin and jaceosidin (up to 0.51 µg/mg). The leaves extract revealed prominent acetylcholinesterase ($$2.33\pm 0.09$$ mg GALAE/g) and α-glucosidase inhibitory potential ($$0.37\pm 0.01$$ mmol ACAE/g). Nevertheless, the flowering heads extract showed distinct butyrylcholinesterase ($$1.41\pm0.35$$ mg GALAE/g), tyrosinase ($$45.66\pm0.84$$ mg KAE/g), amylase ($$0.37\pm0.02$$ mmol ACAE/g), and lipase ($$9.06\pm1.43$$ mg OE/g) inhibitory activity. The anti-proliferative assay was completed towards THP-1 monocyte leukemic cells, where at a concentration $$>1000$$ µg/mL, a significant dose dependent reduction of cell viability was observed for the leaves extract. In conclusion, the data revealed a new insight into the taxon for its health benefit application.

Optimization of Polyphenol Extraction with Potential Application as Natural Food Preservatives from Brazilian Amazonian Species Dalbergia monetaria and Croton cajucara

Scientific interest has currently focused on natural products as a feasible approach for new food additives to replace synthetic preservatives. Thereby, the objective of this work was to optimize the polyphenol extraction from native Amazonian plant species Dalbergia monetaria L.f. and Croton cajucara Benth., and they were determined by the total content of these compounds. Accordingly, the hydroalcoholic extract’s phytocomposition was analyzed by ultra-high-performance liquid chromatography–diode array detector (UPLC-DAD) and various assays to determine the antioxidant capacity (e.g., 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, ferric reduction power, peroxidation inhibition). In addition, response surface methodology applying a central composite design was used to optimize the antioxidant compound extraction conditions. Extract phytochemical profiles identified polyphenols such as (-)-epigallocatechin gallate, rutin, and hyperoside in both species. Furthermore, D. monetaria and C. cajucara extracts displayed significant antioxidant capacity, exhibiting similar values compared to the standard synthetic antioxidant butylated hydroxytoluene. Nevertheless, C. cajucara showed more antioxidant efficiency compared to D. monetaria. These results were consistent with the distribution matrix obtained by a Central Composite Design since the C. cajucara extracts exhibited the best response to the adopted optimization model. Therefore, data are promising for obtaining potential options for natural additives for the food industry.

Development of a Novel Flavored Goat Cheese with Gentiana lutea Rhizomes.

Gentiana lutea rhizomes, generally used as a bittering agent in food, were harvested from two geographical sites (Massif Central: MC and Jura: J) to evaluate their potential use in the flavoring step during goat cheesemaking. Gentian flavored goat cheeses (MCGC and JGC) were elaborated by a one-night immersion of unflavored goat cheeses (CGC) into gentian-infused whey. The impregnation of gentian in goat cheeses was evaluated by chemical and sensory analysis. The chemical composition of cheeses was analyzed by HS-SPME-GC-MS (Head-Space-Solid Phase MicroExtraction-Gas Chromatography-Mass Spectrometry) for volatile compounds (alcohols, ketones, aldehydes, esters, alkenes, alkanes, acids, terpenes) and UHPLC-DAD (Ultra High-Performance Liquid Chromatography-Diode Array Detector) for gentian bitter compounds (seco-iridoids). The sensory analysis consisted of a bitterness rating and a free description of cheeses by 17 trained panelists. Results of the study highlighted that unflavored goat cheeses presented higher unpleasant notes (goaty and lactic whey) and higher amounts of hexanoic acid and toluene compared to gentian flavored goat cheeses. The bitterness of gentian flavored goat cheeses was higher compared to unflavored cheeses and could be explained by loganic acid transfer from yellow gentian to flavored cheeses. Other free descriptors of gentian flavored goat cheeses revealed more complex notes (herbal, vegetal, floral, sweet, spicy and creamy) and higher relative amounts of volatile compounds such as 3-methyl butanoic acid, 2-methyl propanoic acid, 4-methyl decane, 2,3-butanediol, ethanol, diacetyl, methyl acetate and 2-phenylethyl acetate, compared to unflavored cheeses. Phenylethyl acetate was the only volatile compound that enabled differentiation of gentian origin on gentian flavored goat cheeses. Gentian rhizomes could be considered a promising flavoring agent contributing to the olfactive and gustative complexity of flavored goat cheeses and the reduction of their goaty perceptions.

Development and optimization of natural deep eutectic solvent-based dispersive liquid-liquid microextraction coupled with UPLC-UV for simultaneous determination of parabens in personal care products: evaluation of the eco-friendliness level of the developed method.

Dispersive liquid-liquid microextraction (DLLME) combined with ultra-high performance liquid chromatography-diode array detector (UHPLC-DAD) method has been developed and validated for the determination of parabens in personal care products. In this study, a natural deep eutectic solvent (NADES) composed of menthol and formic acid at a molar ratio of 1 : 2 was prepared and used as an extraction solvent. The influencing variables on the extraction efficiency such as extraction solvent type and volume, composition of NADES, salt addition, vortex and centrifugation time were investigated. The proposed method exhibited good linearity with determination coefficients of ≥0.9992. The relative recoveries for the studied analytes ranged from 82.19 to 102.45%. Limits of detection and limits of quantification were in the range of 0.17-0.33 ng mL-1 and 0.51-0.99 ng mL-1, respectively. To evaluate the applicability of the developed method, it was successfully applied to determine four parabens in personal care products. Additionally, the eco-friendliness level of the presented method was evaluated using Eco-Scale Assessment, Green Analytical Procedure Index and Analytical GREEnness metric. The developed method is simple, environmentally friendly and cost effective and it could be employed for determination of parabens in personal care products without harming the environment.

PhUGT78A22, a novel glycosyltransferase in Paeonia ‘He Xie’, can catalyze the transfer of glucose to glucosylated anthocyanins during petal blotch formation

BackgroundFlower color patterns play an important role in the evolution and subsequent diversification of flowers by attracting animal pollinators. This interaction can drive the diversity observed in angiosperms today in many plant families such as Liliaceae, Paeoniaceae, and Orchidaceae, and increased their ornamental values. However, the molecular mechanism underlying the differential distribution of anthocyanins within petals remains unclear in Paeonia.ResultsIn this study, we used an intersectional hybrid between the section Moutan and Paeonia, hereafter named Paeonia ‘He Xie’, which has purple flowers with dark purple blotches. After Ultra-high performance liquid chromatography-diode array detector (UPLC-DAD) analysis of blotched and non-blotched parts of petals, we found the anthocyanin content in the blotched part was always higher than that in the non-blotched part. Four kinds of anthocyanins, namely cyanidin-3-O-glucoside (Cy3G), cyanidin-3,5-O-glucoside (Cy3G5G), peonidin-3-O-glucoside (Pn3G), and peonidin-3,5-O-glucoside (Pn3G5G) were detected in the blotched parts, while only Cy3G5G and Pn3G5G were detected in the non-blotched parts. This suggests that glucosyltransferases may play a vital role in the four kinds of glucosylated anthocyanins in the blotched parts. Moreover, 2433 differentially expressed genes (DEGs) were obtained from transcriptome analysis of blotched and non-blotched parts, and a key UDP-glycosyltransferase named PhUGT78A22 was identified, which could use Cy3G and Pn3G as substrates to produce Cy3G5G and Pn3G5G, respectively, in vitro. Furthermore, silencing of PhUGT78A22 reduced the content of anthocyanidin 3,5-O-diglucoside in P. ‘He Xie’.ConclusionsA UDP-glycosyltransferase, PhUGT78A22, was identified in P. ‘He Xie’, and the molecular mechanism underlying differential distribution of anthocyanins within petals was elucidated. This study provides new insights on the biosynthesis of different kinds of anthocyanins within colorful petals, and helps to explain petal blotch formation, which will facilitate the cultivar breeding with respect to increasing ornamental value. Additionally, it provides a reference for understanding the molecular mechanisms responsible for precise regulation of anthocyanin biosynthesis and distribution patterns.

Magnetic deep eutectic solvent-based dispersive liquid–liquid microextraction for enantioselectively determining chiral mefentrifluconazole in cereal samples via ultra-high-performance liquid chromatography

A simple, rapid, and efficient pretreatment method of mefentrifluconazole enantiomers in cereal samples was established by dispersive liquid–liquid microextraction coupled with ultra-high-performance liquid chromatography-diode array detector (UHPLC-DAD). In this study, a novel ternary magnetic deep eutectic solvent (MDES) [octyltrimethylammonium bromide][cobalt chloride][acetic acid] was synthesized as the extractant. Acetic acid was used as the dispersant to promote the in situ dispersion of binary MDES [octyltrimethylammonium bromide][cobalt chloride]. The microdroplets of binary MDES containing mefentrifluconazole were separated by an external magnet. Device-assisted dispersion and time-consuming centrifugation steps were eliminated to ensure simplicity and rapidity of the pretreatment. Good linearity ranging from 0.01 μg g−1 to 2 μg g−1 was obtained. The extraction recovery varied from 82.9 to 95.0%. The limit of detection was 0.003 μg g−1. Finally, this established approach has been applied for the enantioselective detection of chiral mefentrifluconazole in corn, rice, wheat, millet, and sorghum samples.

Lamotrigine Extraction and Quantification by UPLC-DAD in Plasma from Patients with Bipolar Disorder.

A sensitive and efficient analytical process for detecting lamotrigine in acidic solution based in ultra-high-performance liquid chromatography-diode array detector (UPLC-DAD) was developed; the stationary phase used was a C8, 150 × 4.6 mm, 2.6 µm. The mobile phase consisted of acetonitrile/acidified water (0.01% H3PO4 and 0.005% triethylamine, pH 2.4) (25 : 75 v/v). Limits of detection and quantification were 0.02 µg/mL and 0.05 µg/mL, respectively. The working interval for the evaluation of the method ranged from 0.05 to 12 µg/mL, and the linear fit of the experimental data has a value of r2≥0.98. Before quantifying lamotrigine in plasma of patients with bipolar disorder, lamotrigine was released from plasma proteins with a 0.2 M sodium hydroxide solution, and then proteins were removed by precipitation with acetonitrile. Afterward, the lamotrigine base was dissolved in ethyl acetate. This extract was reconstituted in potassium phosphate solution (pH 2.4) to obtain more than 98% of lamotrigine protonated in N2, which was detected and quantified as indicated above. The absolute percentage of lamotrigine recovery is ≥80% for all tested concentration levels. The accuracy and precision of the method have %CV values <4% for the lamotrigine levels of 3, 6, and 9 µg/mL. The correlation coefficient for the used concentration range is 0.99. The analytical method is precise and sensitive to measure lamotrigine levels expected in plasma of BD patients and these levels were in the therapeutic dose range.

Fractionation of Extracts from Black Chokeberry, Cranberry, and Pomegranate to Identify Compounds That Influence Lipid Metabolism.

Polyphenols show a spectrum of bioactive effects, including an influence on lipid metabolism. In this study, we performed activity-guided fractionations of black chokeberry (aronia), cranberry, and pomegranate extracts to identify the biologically active compounds. The extracts were prepared from fruit juice concentrates with the adsorbent resin Amberlite XAD-7 and were separated into a copigment and an anthocyanin fraction, followed by fractionation into a polymer and monomeric fraction by means of hexane precipitation. For further fractionation of the cranberry and pomegranate copigment fractions, high-performance countercurrent chromatography (HPCCC) was used. The compounds in each fraction were identified by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS), and the quantification was performed by ultra high-performance liquid chromatography-diode array detector (UHPLC-DAD) analyses. Each of the (sub-)fractions was tested in three in vitro assays: phosphodiesterase 3B (PDE) activity, lipid accumulation, and lipolysis in 3T3-L1 cells. The results showed that various fractions and subfractions can inhibit lipid accumulation and PDE activity as well as increase lipolysis, particularly copigments. Overall, our results indicate an influence of polyphenol-rich (sub-)fractions on the lipid metabolism.

Establishing forensic food models for authentication and quantification of porcine adulterant in gelatine and marshmallow

This study aims at authenticating gelatine sources using the incorporation of amino acid (AA) analysis via Ultra-High-Performance Liquid Chromatography Diode-Array Detector (UHPLC-DAD) with forensic food models such as discriminant analysis (DA) and principal component analysis (PCA). The multiple linear regression (MLR), principal component regression (PCR) and partial least square regression (PLSR) were compared to select the best model to (1) quantify the porcine adulterant in non-porcine gelatines and (2) quantify porcine adulterant in fish and bovine gelatine. The method linearity was 37.5–1000 pmol/μL with a determination coefficient (R2) of 0.96–1.00 and a total recovery of 85%–111%. The training, testing and validation datasets were established by the AA of fish, bovine and porcine skin gelatines. The DA had successfully classified 100% fish, bovine and porcine gelatines while the PCA identified dominant AA in each gelatine sources. The discriminating model of non-porcine and porcine gelatines (NPPDM) and discriminating model of fish, bovine and porcine gelatines (FBPDM) had 100% correctly classified (1) non-porcine and porcine gelatines and (2) fish, bovine and porcine gelatines. Using the fish and bovine gelatin (PFBG) training dataset, the PCR model was ineffective in quantifying the porcine adulterant in non-porcine gelatines. The MLR was a better model than PCR to quantify porcine adulterant in non-porcine gelatines using porcine adulterant in fish gelatine (PFG) and porcine adulterant in bovine gelatine (PBG) training datasets due to its lower relative error range and average relative error than of the PCR. Likewise, the PCR model of the PFBG training dataset was ineffective to quantify the porcine adulterant, specifically in fish and bovine gelatine. In contrast, the MLR of PFG and PBG training datasets was the best model for quantifying porcine adulterant in fish and bovine gelatines, respectively. The MLR was ineffective to classify porcine gelatine in marshmallow using the PFG training dataset. However, the MLR had successfully quantified porcine gelatine in marshmallow using PFG and PBG training datasets. Since the training, testing and validation datasets were established by the fish, bovine and porcine skin gelatines, the NPPDM, FBPDM and MLR were best applied for these gelatines. This study anticipated that the regulatory bodies might adopt this approach to establish a standard of authentication of gelatine products.

Black rice cultivar from Java Island of Indonesia revealed genomic, proteomic, and anthocyanin nutritional value.

Black rice is considered to be functional food containing anthocyanins as bioactive compounds. This study examined the genomic and proteomic patterns in local black rice from Java Island, Indonesia, with attention to the mechanism of anthocyanin synthesis. Three kinds of black rice from Java Island, including black rice from East Java (BREJ), black rice from Central Java (BRCJ), and black rice from West Java (BRWJ), were studied in comparison to white rice (WREJ) and red rice (RREJ). Genomic profiling was done by simple sequence repeat (SSR) analysis, and sequencing of red coleoptile (Rc) and glycosyltransferase (GT) genes, followed by in silico analysis. Total anthocyanin was investigated by ultra-high performance liquid chromatography- diode array detector (UHPLC-DAD). The proteomic profiles were determined by liquid-chromatography and mass spectrometry of tryptic peptides. The SSR profiles showed a specific band in each black rice variant. The Rc gene exon-2 sequences were similar in the three black rice cultivars. The GT gene sequence was identified as a new variant that correlates with the purple stem, leaf, bran, and whole grain morphology seen exclusively in the BRWJ cultivar. The anthocyanin composition in Java black rice is diverse. The highest cyanidin level was seen in BRWJ and the highest level of peonidin-3-O-glucoside in BREJ. Proteomic profiling of the black rice cultivars demonstrated that the expression of proteins that might be related to the levels of anthocyanin synthesis varied. These studies conclude that the genomic, proteomic and anthocyanins composition of Java black rice cultivars may be used the improvement of their functional nutrition values.

Simultaneous Determination of Isothiazolinones and Parabens in Cosmetic Products Using Solid-Phase Extraction and Ultra-High Performance Liquid Chromatography/Diode Array Detector.

Isothiazolinones methylisothiazolinone (MI) and methylchloroisothiazolinone (MCI), and parabens methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) are the most common synthetic preservatives. They are all known to be potential skin allergens that lead to contact dermatitis. Thus, the identification of these unsafe chemicals in cosmetic products is of high importance. In the present study, solid-phase extraction (SPE) based on HyperSep reversed-phase C8/benzene sulfonic acid ion exchanger (HyperSep C8/BSAIE) and Sep-Pak C18 sorbents, and ultra-high performance liquid chromatography/diode array detector (UHPLC/DAD) were optimized for the simultaneous determination of MI, MCI, MP, EP, PP and BP in cosmetic products. HyperSep C8/BSAIE and UHPLC/DAD with the eluting solvent mixture (acetonitrile/methanol, 2:1, v/v) and detection wavelength (255 nm) were found to be the optimal conditions, respectively. The method illustrates the excellent linearity range (0.008–20 μg/mL) with coefficient of determination (R2, 0.997–0.999), limits of detection (LOD, 0.001–0.002 μg/mL), precision in terms of relative standard deviation (RSD < 3%, intra-day and <6%, inter-day) when examining a standard mixture at low (0.07 µg/mL), medium (3 µg/mL) and high (15 µg/mL) concentrations. A total of 31 cosmetic samples were studied, achieving concentrations (MI, not detected (nd)-0.89 µg/g), (MCI, nd-0.62 µg/g), (MP, nd-6.53 µg/g), (EP, nd-0.90 µg/g), (PP, nd-9.69 µg/g) and (BP, nd-17.80 µg/g). Recovery values ranged from 92.33 to 101.43% depending on the types of sample. To our knowledge, this is the first specific method which covers the theme and describes background amounts of such preservatives in cosmetics.

Identification of Abies sibirica L. Polyprenols and Characterisation of Polyprenol-Containing Liposomes.

The needles of conifer trees are one of the richest sources of natural polyprenols. Polyprenol homologs from Abies sibirica L. lipophilic 80% purified extract were analyzed and quantified. In total, 10 peaks (Prenol-11 to Prenol-20) were observed in the ultra-high-performance liquid chromatography–diode array detector (UHPLC-DAD) chromatogram of Siberian fir with the most abundant compound being Prenol-15 (relative amount 37.23 + 0.56% of the total polyprenol yield). Abies sibirica L. polyprenol solubility and incorporation efficiency into liposomes were studied in various commercially available lecithin mixtures (Phosal IP40, Phosal 75SA, and Lipoid P45). The resulting multilamellar polyprenol liposomes were morphologically characterized by Light and Transmission Electron Microscopy, and the liposome size was discovered to be polymodal with the main peak at 1360 nm (90% of the volume). As polyprenols are fully soluble only in lipids, a liposomal formulation based upon co-solubilization and a modified ethanol injection method of polyprenols into the ethanol-phospholipid system was developed for the entrapment and delivery of polyprenols for potential commercial applications in food supplement and cosmetic industries.

Cytotoxic Properties of Damiana (Turnera diffusa) Extracts and Constituents and A Validated Quantitative UHPLC-DAD Assay.

In our continuing search for new cytotoxic agents, we assayed extracts, fractions, and pure compounds from damiana (Turnera diffusa) against multiple myeloma (NCI-H929, U266, and MM1S) cell lines. After a first liquid-liquid solvent extraction, the ethyl acetate layer of an acetone (70%) crude extract was identified as the most active fraction. Further separation of the active fraction led to the isolation of naringenin (1), three apigenin coumaroyl glucosides 2–4, and five flavone aglycones 5–9. Naringenin (1) and apigenin 7-O-(4″-O-p-E-coumaroyl)-glucoside (4) showed significant cytotoxic effects against the tested myeloma cell lines. Additionally, we established a validated ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method for the quantification of the isolated components in the herb and in traditional preparations of T. diffusa.

Determination of isoorientin levels in rat plasma by ultra-high performance liquid chromatography coupled with diode array detector and its application to a pharmacokinetic study

Background: Isoorientin is a C-glycosylflavone and a pharmacologically active ingredient found in various medicinal plants. Objective: The aim of this study is to develop a specific and reproducible ultra-high-performance liquid chromatography-diode array detector (UHPLC-DAD) based method to quantify isoorientin in rat plasma and to apply the devised method to a pharmacokinetic study in rats. Materials and Methods: Simple protein precipitation with methanol was utilized to extract isoorientin and rutin (the internal standard) from rat plasma. Analytes were separated on an UHPLC Phenomenex Luna Omega Polar C18 column (100 mm × 2.1 mm, 1.6 μm) by gradient elution using a mobile phase containing 1% aqueous acetic acid and 100% acetonitrile at the flow rate of 0.25 mL/min. Results: The developed UHPLC-DAD method showed good linearity (R2 = 0.9993) over the concentration range 20–5000 ng/mL with a lower limit of quantification of 20 ng/mL. Intra- and inter-day precisions were Abbreviations used: UHPLC: Ultra-high-performance liquid chromatography; DAD: Diode array detector; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LLOQ: Lower limit of quantification; IS: Internal standard; QC: Quality control; RSD: Relative standard deviation; Cmax: The maximum plasma concentration; Tmax: The time point to reach the maximum concentration; AUClast: The area under the curve from time zero to the last measurable point, AUCinf: The area under the curve from time zero to infinity; ke: Terminal elimination rate constant; t1/2: Terminal half-life; CL/F: Oral clearance; Vd/F: Apparent volume of distribution after oral administration; SD: Standard deviation; SPE: Solid-phase extraction.

Development and validation of a rapid ultra-high performance liquid chromatography diode array detector method for Verbena officinalis L.

This study presents a fast and validated ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method for the simultaneous determination of the major compounds in Verbena officinalis L. (Verbenaceae), a medicinal plant listed in the European, British, and, Chinese Pharmacopoeias. In order to get reference substances, nine compounds, belonging to iridoids, flavonoids, and phenylpropanoid glycosides, were isolated from the herb extract. Two of them, namely cistanoside D and leucosceptoside A, were found in this plant species for the first time. Chromatographic separation was achieved in less than 7 min on a Kinetex 1.7 u XB-C18 (50 × 2.10 mm) column by using a solvent gradient of water-acetonitrile modified with 0.1% formic acid. Method validation confirmed the assays sensitivity, linearity (R2 ≥ 0.997), precision (intraday precision ≤ 1.71%; interday precision ≤ 1.46%) and accuracy (recovery rates between 93.9% and 108.8%) for the quantitative analysis of the eight selected marker compounds. Identity and peak purity of the analytes was confirmed by coupling the UHPLC instrument to a quadrupole time-of-flight mass spectrometer via an electrospray ionization interface (ESI-QTOF-MS) operating in the negative ionization mode.Finally, the applicability of the developed UHPLC-DAD method was successfully proven for the sensitive quantitation of the major compounds in Verbena herb extracts, thereby providing a reliable tool for its rapid quality control.

Green Approach for Sample Preparation and Determination of Anthocyanins from Lycium ruthenicum Murr. Using a β-Cyclodextrin-Based Extraction Method Coupled with UPLC-DAD Analysis

This study aimed to develop and optimize a β-cyclodextrin (β-CD)-based technique for extracting anthocyanins from Lycium ruthenicum Murr. and to establish a ultra-high-performance liquid chromatography-diode array detector (UPLC-DAD) method for their analysis. β-CD solutions produced higher extraction yields of petunidin-3-O-(trans-p-coumaroyl)-rutinoside-5-O-glucoside and total anthocyanins from L. ruthenicum fruit than did pure water and aqueous hydroxypropyl-β-cyclodextrin (HPβ-CD) and ethanol and methanol solutions. Extraction at 50 °C for 30 min using 1.65% β-CD solution and a liquid/solid ratio of 15:1 produced the optimal extraction yield of L. ruthenicum anthocyanins. A UPLC-DAD method was developed for the determination of L. ruthenicum anthocyanins using an ethanol-based mobile phase, and the primary anthocyanins were identified using two-dimensional LC-MS/MS. Method validation showed that the developed method was accurate, stable, and reliable for the analysis of petunidin-3-O-(trans-p-coumaroyl)-rutinoside-5-O-glucoside and total anthocyanins from L. ruthenicum fruit. The present study showed that β-CD-based extraction coupled with UPLC-DAD analysis is an efficient and green method for the extraction and analysis of anthocyanins from L. ruthenicum fruit.

Determination of polycyclic aromatic hydrocarbons in edible oil by gel permeation chromatography and ultra-high performance liquid chromatography coupled with diode array detector and fluorescence detector

This paper develops an instrumental analytical approach for detection of fourteen polycyclic aromatic hydrocarbons (PAHs) in edible oil samples using gel permeation chromatography (GPC) and ultra-high performance liquid chromatography (UHPLC) coupled with diode array detector (DAD), and fluorescence detector (FLD). The GPC was used to remove triglycerides from edible oil samples. The extracted samples were then detected using UHPLC—DAD—FLD. In order to obtain good separation and high reproducibility, the UHPLC—DAD—FLD experimental condition was optimized. The PAHs including three groups of isomeric PAHs can be separated completely in 12 min using BEH Shield RP 18 column with a suitable gradient elution program. The mean recoveries were in the range of 73–110% with an acceptable reproducibility (RSD &lt; 10%, n = 3). During real sample analysis, the method can decrease the chance of false positives with both DAD and FLD being used simultaneously. The results indicate that the approach is simple, easy, and acceptably reproducible, thereby showing great potential as a method for detection of fourteen PAHs contained in edible oil samples.

Simultaneous separation of antihyperlipidemic drugs by green ultrahigh-performance liquid chromatography–diode array detector method: Improving the health of liquid chromatography

Statins in combination with fibrates show beneficial effects on the lipoprotein profile of patients because they have positive complimentary effects on lipid profile. A new green ultrahigh-performance liquid chromatography–diode array detector method for simultaneous analysis of simvastatin (SMV) and fenofibrate (FNF) in standard form, marketed formulations, and self-emulsifying drug delivery system formulations was developed and validated in the present investigation. The method utilized C18 as stationary phase and a combination of methanol:water (8:2) as an eluent. It was found that selected eluent provided short run time (2.5 minutes), better peak symmetry and satisfactory values of other chromatographic parameters such as resolution (Rs=2.325), capacity factor (k, 3.0 and 4.2 for SMV and FNF, respectively), selectivity (α =1.4), and number of theoretical plates (N, 4265 and 5285 for SMV and FNF, respectively). An excellent linear relationship (r2 0.998 and 0.997 for SMV and FNF, respectively) was observed for linear regression data for the calibration plots. The developed system was validated for accuracy, precision, robustness (˃ 2% for both drugs) and recovery (98–102% for both drugs). Results obtained from the statistical treatment of the values obtained for different parameters proved that the method is suitable, reproducible, and selective for the simultaneous analysis of SMV and FNF in bulk, marketed, and self-emulsifying drug delivery system formulations. The replacement of commonly applied toxic solvents with innocuous and environmentally benign solvents provides a better option than the more toxic processes in drug analysis.