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Marine microalgae are emerging as promising sources of polyphenols, renowned for their health-promoting benefits. Recovering polyphenols from microalgae requires suitable treatment and extraction techniques to ensure their release from the biomass and analytical methodologies to assess their efficiency. This review provides a comprehensive comparison of traditional and cutting-edge extraction and analytical procedures applied for polyphenolic characterization in marine microalgae over the past 26 years, with a unique perspective on optimizing their recovery and identification. It addresses (I) cell disruption techniques, including bead milling, high-speed homogenization, pulsed electric field, ultrasonication, microwave, freeze-thawing, and enzymatic/chemical hydrolysis; (II) extraction techniques, such as solid–liquid extraction, ultrasound and microwave-assisted extraction, pressurized-liquid extraction, and supercritical CO2; (III) analytical methods, including total phenolic and flavonoid content assays and advanced chromatographic techniques like GC-MS, HPLC-DAD, and HPLC-MS. Key findings showed bead milling and chemical hydrolysis as effective cell disruption techniques, pressurized-liquid extraction and microwave-assisted extraction as promising efficient extraction methods, and HPLC-MS as the finest alternative for precise phenolic characterization. Unlike previous reviews, this study uniquely integrates both extractive and analytical approaches in one work, focusing exclusively on marine microalgae, a relatively underexplored area compared to freshwater species, offering actionable insights to guide future research and industrial applications.

This article presents the development of a reversed-phase (RP) high-performance liquid chromatographic (HPLC) method for determination of process-related impurities in a celecoxib drug substance following Analytical Quality by Design (AQbD) principles. The method from European Pharmacopeia (EP) for celecoxib drug substance does not sufficiently separate celecoxib from its EP impurity B because the system suitability criterion is not achieved (resolution NLT 1.8). The same issue was observed with the proposed method from United States Pharmacopeia (USP) for celecoxib capsules, where EP impurity A elutes under the main peak. A new HPLC method was developed that eliminates the disadvantages of the two pharmacopeial methods and is capable of efficiently separating and determining all seven impurities listed in EP and the proposed USP monographs. The development of a new HPLC method started with method scouting, in which various C18 and phenyl stationary phases were tested. Improved selectivity was obtained only with a chiral stationary phase. An immobilized Chiralpak IA-3 column used in RP mode turned out to be the most appropriate for method optimization. The ratio of acetonitrile in the mobile phase, flow rate, and column temperature were recognized as critical method parameters (CMPs) and were further investigated using a central composite face response-surface design. A multiple linear regression (MLR) method was applied to fit the mathematical models on the experimental data to determine factor–response relationships. The models created show adequate fit and good prediction abilities. The Monte Carlo simulation method was used to establish the design space. The method developed was verified in terms of precision, sensitivity, accuracy, and linearity, and the results showed that the new method is suitable for determination of seven process-related impurities of celecoxib.

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.

This study aimed to investigate the potential of four sea water microalgae, isolated and cultivated at M′diq Bay in Morocco, as a new source of natural antioxidants. These microalgae belong to different classes, including Phaedactylium tricornitum (Bacillariophyceae), Nannochloropsis gaditana (Eustigmatophyceae), Nannochloris sp (Trebouxiophyceae), and Tetraselmis suecica (Chlorodendrophycea). The antioxidant properties were screened by the use of in vitro assays, namely 2,2-difenil-1-picrylhydrazyl, Ferric reducing antioxidant power, and Ferrous ions chelating activity, and compoundidentification was carried out in methanol and acetone extracts of both dried and fresh microalgae biomass by HPLC–PDA–MS analysis. Among the investigated microalgae, Phaedactylium tricornutum was the richest one regarding its carotenoid (especially all-E-fucoxanthin) and phenolic (especially protocatechuic acid) contents, as well as antioxidant activity (65.5%), followed by Nannochloris sp, Tetraselmis suicica, and Nannochloropsis gaditana, with antioxidant activity of 56.8%, 54.9%, and 51.1%, respectively.

Context: Despite some studies related to Juniperus phoenicea L. (Cupressaceae), phytochemical and biological investigations of this plant remain unexplored.Objective: This work is the first report dealing with the identification and characterization of volatile components and flavonoids in hexane and methanol extracts from J. phoenicea leavesMaterials and methods: Antioxidant activity of hexane, and methanol extracts from J. phoenicea leaves were determined by DPPH-radical scavenging assay. α-Amylase inhibitory activity was evaluated by enzyme inhibition using in vitro assay (each extract was dissolved in DMSO to give concentrations of 50, 100 and 200 mg/mL). The chemical composition of fractions (Fr1-Fr3) from methanol extract was determined by high-performance liquid chromatography coupled with mass spectroscopy (HPLC-MS) analysis.Results and discussion: The hexane extract was analyzed by GC-MS technique which allowed the identification of 32 compounds. The main constituents were α-humulene (16.9%), pentadecane (10.2%) and α-cubebene (9.7%). Fraction Fr 2 exhibited a strong DPPH radical-scavenging activity (IC50 = 20.1 μg/mL) compared to that of BHT as well as the highest α-amylase inhibitory activity (IC50 = 28.4 μg/mL). Three flavonoids were identified in these fractions using HPLC-MS analysis: Quercetin 3-O-glucoside, isoscutellarein 7-O-pentoside and quercetin 3-O-pentoside. In addition, the more active fraction (Fr 2) was purified with semi-preparative HPLC affording one pure compound (amentoflavone) using 1H NMR analysis. This compound exhibited powerful DPPH radical-scavenging (IC50 = 14.1 μg/mL) and α-amylase inhibition (IC50 = 20.4 μg/mL) effects.Conclusion: This study provides scientific support to some medicinal uses of J. phoenicea found in North Africa.

Ultra-performance liquid chromatography (UPLC) has an advantage over conventional High-performance liquid chromatography (HPLC) as UPLC offers substantial resolution, speed, and sensitivity during analysis. This advanced chromatographic technique uses sub-2μm particles for the stationary phase. As a result, it saves time and reduces solvent consumption, which allows it to take less run time and makes it highly efficient.

In this work, we present a novel method for determining the prescribed dosages of Dapagliflozin (DAPA), Vildagliptin (VIL), and Metformin (MET) all at once. The goal of this research is to create and test a new RP-HPLC technique that can simultaneously measure DAPA, VIL, and MET in formulation and bulk materials.The goal of this research is to create and test a new RP-HPLC technique that can simultaneously measure DAPA, VIL, and MET in formulation and bulk materials. An isocratic elution method was used with a flow rate of 1.0 ml min-1 and a diode array detector operating at 261nm to perform the chromatographic separation on a kromasil-C18 column(4.5 x 250mm; 5µm). Using orthophosphoric acid to get the pH down to 3.5, the mobile phase consisted of a combination of 0.05 mmol potassium dihydrogen phosphate buffer and acetonitrile in an 80:20 v/v ratio. With concentrations ranging from 0.1-1.0µg/ml and 2-25µg/ml, as well as DAPA, VIL, and MET values from 10 to 120µg/ml, the calibration curve displayed linearity. The research found that DAPA had a limit of detection and quantification of 0.0122µg/ml while VIL had a limit of 0.0323µg/ml. The upper limits for MET were 0.232µg/ml and 0.635µg/ml, while the lower limits were 1.124µg/ml and 3.124µg/ml, respectively. We have developed and validated a new reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of vildagliptin and metformin. This method is very sensitive, easy to use, and stable. The suggested technique could be used to routinely measure DAPA, VIL, and MET.

Vancomycin is being used for the treatment of a variety of infections caused by methicillin resistant Staphylococcus aureus and methicillin susceptible Staphylococcus aureus. Therapeutic drug monitoring (TDM) is highly recommended for ensuring the safe and effective therapy with vancomycin. A reliable and cost-effective bioanalytical method is required for TDM as well as pharmacokinetic studies of vancomycin.BACKGROUNDVancomycin is being used for the treatment of a variety of infections caused by methicillin resistant Staphylococcus aureus and methicillin susceptible Staphylococcus aureus. Therapeutic drug monitoring (TDM) is highly recommended for ensuring the safe and effective therapy with vancomycin. A reliable and cost-effective bioanalytical method is required for TDM as well as pharmacokinetic studies of vancomycin.A selective, sensitive, and cost effective HPLC method was developed and validated for quantification of vancomycin concentrations in human plasma. The mobile phase was a mixture of buffer (50 mM ammonium dihydrogen phosphate, pH 2.4) and acetonitrile 88 : 12 v/v. The separation was carried on C18 column (125 × 4.6 mm, particle size 5 µm) with isocratic flow rate of 0.370 mL/min at room temperature with UV detection at 215 nm. The method was validated for sensitivity, accuracy, and precision as well as stability of vancomycin in human plasma by following European Medicine Agency (EMA) guideline. Therapeutic drug monitoring of vancomycin was performed by quantifying the trough concentrations of vancomycin in 65 human plasma samples after administration of therapeutically relevant dose.MATERIALS AND METHODSA selective, sensitive, and cost effective HPLC method was developed and validated for quantification of vancomycin concentrations in human plasma. The mobile phase was a mixture of buffer (50 mM ammonium dihydrogen phosphate, pH 2.4) and acetonitrile 88 : 12 v/v. The separation was carried on C18 column (125 × 4.6 mm, particle size 5 µm) with isocratic flow rate of 0.370 mL/min at room temperature with UV detection at 215 nm. The method was validated for sensitivity, accuracy, and precision as well as stability of vancomycin in human plasma by following European Medicine Agency (EMA) guideline. Therapeutic drug monitoring of vancomycin was performed by quantifying the trough concentrations of vancomycin in 65 human plasma samples after administration of therapeutically relevant dose.The developed method was sensitive enough to quantify vancomycin concentrations as low as 0.25 mg/L in human plasma. Moreover, the method was proved accurate and precise in terms of quantifying the unknown concentration of vancomycin. The evaluation of short-term, long-term, and freeze-thaw stability proved the stability of vancomycin in human plasma. The TDM of vancomycin by using this method showed that 39 (60%) samples were within the target trough concentration range (TTCR), i.e. 10 - 20 mg/L, while 23 samples (35.4%) were below the TTCR, and 3 samples (4.6%) were above this range.RESULTSThe developed method was sensitive enough to quantify vancomycin concentrations as low as 0.25 mg/L in human plasma. Moreover, the method was proved accurate and precise in terms of quantifying the unknown concentration of vancomycin. The evaluation of short-term, long-term, and freeze-thaw stability proved the stability of vancomycin in human plasma. The TDM of vancomycin by using this method showed that 39 (60%) samples were within the target trough concentration range (TTCR), i.e. 10 - 20 mg/L, while 23 samples (35.4%) were below the TTCR, and 3 samples (4.6%) were above this range.The developed method is sensitive and cost effective for quantification of vancomycin in human plasma. The results of sample analysis shows that the developed method can be used reliably for TDM of vancomycin.CONCLUSIONThe developed method is sensitive and cost effective for quantification of vancomycin in human plasma. The results of sample analysis shows that the developed method can be used reliably for TDM of vancomycin.

The identification of the critical quality attributes (CQAs) of monoclonal antibodies (mAbs) is a key component of quality by design. Traditional detection methods for the identification of mAbs, such as peptide mapping and weak cation‐exchange chromatography (WCX‐HPLC), require sophisticated equipment, experienced staff, and a considerable amount of time. In this study, the novel identification of mAbs was performed using Raman spectroscopy, combined with the evaluation of other CQAs, such as the appearance and purity of size‐exclusion high‐performance liquid chromatography(SEC‐HPLC), charge heterogeneity of WCX‐HPLC, and bioassay. Raman spectroscopy achieved comparable results to the conventional, but complex, approach to identifying anti‐vascular endothelial growth factor antibodies. Raman spectroscopy was used to identify and distinguish between different antibody types. Additionally, the Raman technique with principal component analysis of multivariate algorithms is rapid, efficient, and accurate for mAb identification; this technique has great potential to support biopharmaceutical development and counterfeiters. Raman spectroscopy was used to identify and distinguish between different antibody types. Additionally, the Raman technique with principal component analysis of multivariate algorithms is rapid, efficient, and accurate for mAb identification, this technique has great potential to support biopharmaceutical development and counterfeiters.

Wild ginseng (W-GS), ginseng under forest (F-GS, planted in mountain forest and growing in natural environment), and cultivated ginseng (C-GS) were compared via HPLC-DAD and HPLC-IT-TOF-MSn. A total of 199 saponins, including 16 potential new compounds, were tentatively identified from 100 mg W-GS (177 saponins in W-GS with 11 new compounds), F-GS (56 saponins with 1 new compound), and C-GS (60 saponins with 6 new compounds). There were 21 saponins detected from all the W-GS, F-GS, and C-GS. Fifty saponins were only detected from W-GS, including 23 saponins found in ginseng for the first time. Contents of ginsenosides Re (12.36–13.91 mg/g), Rh1 (7.46–7.65 mg/g), Rd (12.94–12.98 mg/g), and the total contents (50.52–55.51 mg/g) of Rg1, Re, Rf, Rb1, Rg2, Rh1, and Rd in W-GS were remarkably higher than those in F-GS (Re 1.22–3.50 mg/g, Rh1 0.15–1.49 mg/g, Rd 0.19–1.49 mg/g, total 5.69–18.74 mg/g), and C-GS (Re 0.30–3.45 mg/g, Rh1 0.05–3.42 mg/g, Rd 0.17–1.68 mg/g, total 2.99–19.55 mg/g). Contents of Re and Rf were significantly higher in F-GS than those in C-GS (p < 0.05). Using the contents of Re, Rf, or Rb1, approximately a half number of cultivated ginseng samples could be identified from ginseng under forest. Contents of Rg1, Re, Rg2, Rh1, as well as the total contents of the seven ginsenosides were highest in ginseng older than 15 years, middle–high in ginseng between 10 to 15 years old, and lowest in ginseng younger than 10 years. Contents of Rg1, Re, Rf, Rb1, Rg2, and the total of seven ginsenosides were significantly related to the growing ages of ginseng (p < 0.10). Similarities of chromatographic fingerprints to W-GS were significantly higher (p < 0.05) for F-GS (median: 0.824) than C-GS (median: 0.745). A characteristic peak pattern in fingerprint was also discovered for distinguishing three types of ginseng. Conclusively, wild ginseng was remarkably superior to ginseng under forest and cultivated ginseng, with ginseng under forest slightly closer to wild ginseng than cultivated ginseng. The differences among wild ginseng, ginseng under forest, and cultivated ginseng in saponin compositions and contents of ginsenosides were mainly attributed to their growing ages.