Exclusive Papers Collection of Scholars Invited by Editorial Board Members of Separations

Diabetes and its complications, such as delayed wound healing, are increasing at an alarming rate in India, putting an enormous strain on the country’s limited healthcare resources. Hence, the present study proposes to screen/identify the possible mechanisms and to study the effect of the flavonoid-enriched fraction of Selaginella bryopteris extract against human keratinocyte cell lines (HaCaT) and streptozocin (STZ)-induced diabetic wounds in a male Wistar rat model. Chemical profiling was performed by an MTT assay. The obtained GC–MS analysis results showed the presence of amentoflavone, gallic acid, imidazole, palmitic acid, catechine, L-fucitol, lupeol, and myo-inositol as the major bioactive phytoconstituents. S. bryopteris induces the generation of ROS, the condensation of chromatin in the nucleus, and changes in the membrane potential of mitochondria in HaCaT cell lines. An S. bryopteris-dependent induction of apoptosis-mediated cell death in HaCaT cell lines was confirmed by an AO/PI analysis. Mitochondrial depolarization was reflected in JC-1 staining of cells. The wound size was reduced and epithelialization was enhanced. Keratinocyte migration decreased interleukins, TNF-α, IL-2, and IL-6 and the expression of pro-apoptotic (p53, caspase-3, caspase-9, and Bax) and anti-apoptotic (Bcl-2) genes in a dose-dependent manner. Keratinocyte migration increased antioxidant enzyme levels (CAT, SOD, MDA, and GSH). Wound healing is facilitated through the mitochondria-mediated apoptosis pathway, revealing a new area of diabetic wound therapy. Full article

Since inflammatory bowel disease is a progressive disease, optimal treatment early in the disease course is paramount. Still, the use of conventional drugs is accompanied by side effects. The herbal therapies for gastrointestinal disorders are traditionally widespread in Asia, while in Western countries have been adopted in recent years. Andrographis paniculata is a herbaceous plant used in anti-inflammatory treatment. To improve its clinical application, an analytical method with the potential to define a relationship between dose and plasma concentration of active ingredients is prerequisite. Considering the increasing demand for eco-friendly bioanalytical methods, our research aimed to implement new sample preparation technique microextraction by packed sorbent for extraction of marker compounds of A. paniculata from plasma. This green procedure using polymeric sorbent based on the poly (styrene-divinylbenzene) allowed miniaturization of sample volume (100 µL), decrease in consumption of solvents, and high recovery (92–98%). A highly selective and sensitive reversed-phase ultrahigh-performance liquid chromatography method has been developed and validated. The newest greenness assessment software AGREE calculator was used for evaluation of the method’s environmental performance. It suggests that our method conforms to the eco-friendly conditions (score 0.8). The sample preparation was identified as weak point of the bioanalytical methods which highlights the importance of microextraction. Full article

The inhibition effects of the date palm seed extract corrosion of mild steel in 0.5 M HCl at different concentrations are investigated by potentiodynamic polarisation (PDP), electrochemical impedance spectroscopy (EIS) and weight loss tests. Additionally, this study provides a fundamental understanding of aromatic adsorption on iron (Fe) surfaces. Furthermore, the surface morphology and the extracts are performed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The maximum inhibition efficiency of 95, 96, and 91% were realised at 1400 mg/L for PDP, EIS, and weight loss, respectively. The inhibitive action of the DPS extract against mild steel corrosion in an acid solution has been supported by SEM analysis. The FTIR showed that the extract contained hydroxyl (−OH) and methoxyl (−OCH₃) functional groups. The DFT depicted the adsorption sites at the oxygen (O) and carbon (C) atoms as deduced from the Fukui functions, Mulliken atomic charge, and the highest occupied molecular orbital-lowest occupied molecular orbital (HOMO-LUMO) analysis. The DPS preferred to form chemical bonds by donating electrons to the Fe surface. The coordinate bonds between the O and C atoms and the metal surface resulted in a high inhibition efficiency value. In conclusion, date palm seed extract is an effective inhibitor to protect mild steel from corrosion in an acidic medium. Full article

P. nigrum L. extracts and the piperine alkaloid have important antimicrobial, anti-inflammatory, and antioxidant properties. Therefore, in this study, we evaluated the antimicrobial activity and cytotoxicity of P. nigrum L. extracts and piperine, a compound isolated from the extracts of P. nigrum L. Extracts obtained via maceration, soxhlet, and purification steps, in addition to isolated piperine, were used in this study. Spectroscopic methods, such as nuclear magnetic resonance, scanning electron microscopy, X-ray diffraction, thermogravimetry, and differential scanning calorimetry, were used to characterize piperine. In the microbiological analyses, the extract obtained via maceration-derived sample showed high efficiency in inhibiting Salmonella spp. (MIC < 100 μg/mL). The extract obtained via a soxhlet-derived sample showed promising inhibitory activity against almost all microorganisms, with negligible inhibition of Pseudomonas aeruginosa. Favorable inhibition coefficients were also observed against Staphylococcus aureus and Salmonella spp. (MIC < 100 μg/mL) for the extract obtained via purification of the steps-derived sample. Piperine showed an excellent inhibition coefficient against most microorganisms, with inactivity only observed against P. aeruginosa. Cytotoxicity evaluation assays in cancer cell lines revealed that piperine exhibited inhibitory potential on all tested tumor cell lines, causing a decrease in cell viability and achieving an IC₅₀ of less than 30 μg/mL. The analyzed extracts from P. nigrum L. seeds showed cytotoxic activity against tumor and non-tumor cell lines. Full article

Arthrospira platensis (A. platensis) is a microalga with a wide range of commercial uses. One of the main concerns that needs to be addressed in microalgae biorefineries is the costs associated with the harvesting and concentration steps. Filtration has been shown to be an effective technique for concentrating microalgae and recent studies have attempted to enhance membrane filtration by applying an external electric field to the filtration cell. This study consisted of assessing the use of electrically assisted filtration (electrofiltration) at 60 A/m² and 1 bar for the dewatering of A. platensis, as well as the effect of pretreating the microalgae with ultrasounds (US) on the filtration process. Untreated A. platensis exhibited better filtration kinetics than US-treated A. platensis, and electrofiltration was found to increase the cake dryness. More protein and pigments were present in the US-treated microalgae solution compared to the untreated microalgae, which led to the presence of higher concentrations of protein and pigments in the filtrate streams after pressure filtration at 1 bar without the application of an external electric field. Electrofiltration was found to consume less energy compared to traditional drying techniques used for A. platensis. However, electrofiltration degrades the biocompounds present in the filtrate and cake due to pH changes and other electrophoresis phenomena, which shows the need to optimize the process in future work. Full article

Over the past few years, research studies on the therapeutic benefits of medicinal plants with potent antioxidant activity and few side effects have grown significantly. This has sparked interest in determining whether naturally occurring antioxidants could take the place of synthetic antioxidants, which are currently being constricted because of their toxic and carcinogenic properties. The identification and quantification of phytochemicals in the methanolic extract of Kigelia pinnata fruits was measured using gas chromatography–mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography–mass spectrometry (UPLC-MS/MS) techniques. Additionally, the methanolic extract of fruits was used to determine antioxidant activity. Free radical-scavenging (DPPH) and ferric ion-reducing antioxidant power were measured using spectrophotometry, and total antioxidant capacity (TAC) was compared with two common antioxidants, vitamin C and α-tocopherol. Moreover, mature fruits have high DDPH, ferric ion-reducing antioxidant power and total antioxidant capacity. Furthermore, mature fruits have high levels of total phenolic, flavonoid, and tannin content; these compounds are thought to be the sources of the antioxidant activity. The major constituents of the methanolic extracts from the mature fruits of K. pinnata were found to be larixinic acid, 3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one (DMDP), and 5-Hydrxoymethylfurfural. We performed the elemental analysis of the whole fruit. Huh-7 (liver cancer), PANC-1 (pancreatic cancer), Colo-205 (colorectal cancer), HT-29 (colorectal cancer), SNU-16 (gastric carcinoma), SW620 (colorectal adenocarcinoma) and HCT116 (colon carcinoma) were tested in vitro for anticancer activity. Both methanolic and ethyl acetate extracts of mature fruits had a positive effect on all cancer cell lines as compared to the doxorubicin drug. In addition, the methanolic extracts of mature fruits showed more potent cytotoxic effects than the ethyl acetate extracts. Moreover, the most pronounced cytotoxic effects of the methanolic extract were detected in SW620 (colorectal adenocarcinoma), with an IC₅₀ value of 6.79 μg/mL, SNU-16 (gastric carcinoma), with and IC₅₀ value of 8.69 μg/ ml, and in PANC-1 (pancreatic cancer) with an IC₅₀ value of 10.34 μg/mL. Moreover, the results show that the water, ethyl acetate and methanolic extracts of mature fruits have antioxidant capacity, ferric ion-reducing antioxidant power, DPPH scavenging activity and also anticancer activity. Therefore, the present study suggests that the phytochemical profiles of mature fruits of K. pinnata may be used as potential natural antioxidants and anti-cancer cell lines. Full article

The aerosol constituents generated from JUUL Menthol pods with 3.0% and 5.0% nicotine by weight (Me3 and Me5) are characterized by a non-targeted approach, which was developed to detect aerosol constituents that are not known to be present beforehand or that may be measured with targeted methods. Three replicates from three production batches (n = 9) were aerosolized using two puffing regimens (intense and non-intense). Each of the 18 samples were analyzed by gas chromatography electron ionization mass spectrometry and by liquid chromatography electrospray ionization high-resolving power mass spectrometry. All chemical constituents determined to differ from control were identified and semi-quantified. To have a complete understanding of the aerosol constituents and chemistry, each chemical constituent was categorized into one of five groups: (1) flavorants, (2) harmful and potentially harmful constituents, (3) leachables, (4) reaction products, and (5) chemical constituents that were unable to be identified or rationalized (e.g., chemical constituents that could not be categorized in groups (1–4). Under intense puffing, 74 chemical constituents were identified in Me3 aerosols and 68 under non-intense puffing, with 53 chemical constituents common between both regimens. Eighty-three chemical constituents were identified in Me5 aerosol using an intense puffing regimen and seventy-five with a non-intense puffing regimen, with sixty-two chemical constituents in common. Excluding primary constituents, reaction products accounted for the greatest number of chemical constituents (approximately 60% in all cases, ranging from about 0.05% to 0.1% by mass), and flavorants—excluding menthol—comprised the second largest number of chemical constituents (approximately 25%, ranging consistently around 0.01% by mass). The chemical constituents detected in JUUL aerosols were then compared to known constituents from cigarette smoke to determine the relative chemical complexities and commonalities/differences between the two. This revealed (1) a substantial decrease in the chemical complexity of JUUL aerosols vs. cigarette smoke and (2) that there are between 55 (Me3) and 61 (Me5) unique chemical constituents in JUUL aerosols not reported in cigarette smoke. Understanding the chemical complexity of JUUL aerosols is important because the health effects of combustible cigarette smoke are related to the combined effect of these chemical constituents through multiple mechanisms, not just the effects of any single smoke constituent. Full article

Optimized and reliable DNA/RNA extraction protocols are a vital tool in clinical practice in the context of molecular testing. Here, we present our successful attempt to enhance the quantity of RNA isolated from clinical specimens, which we originally found challenging (breast and testis). We compared several purification methods with special focus on two AllPrep system-based protocols (QIAGEN). Our data suggest that addition of proteinase K may markedly increase RNA and, in some cases, also DNA yield. The extraction kit used, AllPrep DNA/RNA/miRNA universal kit, provides RNA amounts comparable with the phenol-chloroform extraction method; however, part of the final yield consisted of small RNAs, visible as a thick band in the bioanalyzer gel-like image (5S peak). The 5S peak, albeit in some cases dominating the bioanalyzer image, plays only a small role in RT-qPCR analysis, and Qubit or NanoDrop measurements can still be used as a reliable estimate of starting amounts of mRNA for downstream analyses. In conclusion, we showed that implementing a protocol containing a step of proteinase K digestion markedly increases RNA yield. The AllPrep DNA/RNA/miRNA Universal Kit can be successfully used for simultaneous extraction of DNA and total RNA, irrespective of the tissue of origin, and does not present inconveniences related to phenol-chloroform extraction. Full article

Tuberculosis (TB) is a massive problem for public health and is the leading cause of illness and death worldwide. Rosemary (Rosmarinus officinalis) is used traditionally to treat many diseases, such as infections of the lungs including pulmonary TB. R. officinalis was collected from Al Anbar Governorate, Iraq, and was extracted with deep eutectic solvents (DESs) of many different kinds and with conventional water solvent. The antimycobacterial activities of the R. officinalis extracts were tested against multidrug-resistant (MDR) Mycobacterium tuberculosis by agar disc diffusion assay. Minimum inhibitory concentrations were measured spectrophotometrically at 570 nm. Then, a time-kill assay and cell membrane integrity analysis were conducted to investigate the effects of the most active extracts on cell growth. The in vitro cytotoxicity of the most active extracts was evaluated against Rat Embryonic Fibroblasts (REF) cell line by MTT assay. Liquid chromatography-mass spectrometry (LC-MS) was conducted to analyze the chemical components of the most active extracts. At 200 mg/mL concentration, a significant inhibition activity was seen in DES2: Tailor (DIZ = 17.33 ± 1.15 mm), followed by DES3: ChGl, DES1: LGH and DES4: ChXl. The best result was DES2: Tailor, which had a MIC of 3.12 mg/mL and an MBC of 12.5 mg/mL. The DES2 extract exhibited a high drop in the number of colonies over time, killing more than 80 colonies. The main phytochemical compounds of the R. officinalis extract were camphene, camphenilol, α-pinene, limonene, apigenin, camphor, carnosol, linalool and myrcene. R. officinalis extracts obtained by DESs have shown evident power in treating tuberculosis, and extraction by DES is a greener procedure than the methods involving conventional extraction solvents. As a result, additional research into the application of DES should be considered. Full article

Sparfloxacin, a synthetic antibiotic belonging to the third-generation fluoroquinolones, has numerous pharmacokinetic and microbiological advantages which can make it an excellent candidate for the treatment of infections in sheep. The objective of this study was to develop and validate an analytical HPLC method to quantify sparfloxacin in sheep plasma, following the recommendations set out in the Guideline on Bioanalytical Method Validation of the European Union (EMEA/CHMP/EWP/192217/2009). The HPLC mobile phase consisted of acetonitrile and monopotassium phosphate buffer (1.36 g/L) 49:51 (v/v). Genabilic acid was used as internal standard. Mean retention times for sparfloxacin and genabilic acid were 2.6 and 5.8 min, respectively. The method met all specifications of the EMA guideline, being selective and linear in the range of 0.2–10 µg/mL (R² ≥ 0.99). Within-run precision ranged between 0.00 and 0.88%, with an accuracy of 90.3–118.0% for the lower limit of quantitation (LLOQ). The LLOQ was 0.2 µg/mL, and no interference from the biological matrix was found. The stability of sparfloxacin in the biological matrix was demonstrated under different storage conditions. Therefore, the method can be used to determine sparfloxacin concentrations in sheep plasma in different types of studies. Full article

Artemisinin (ART) is a sesquiterpene lactone and a popular malaria drug used in many parts of the world. Artesunate (ARTS) is a semi-synthetic derivative of ART with improved pharmacokinetic properties. However, the half-life of ARTS is less than an hour in vivo. The analysis of this drug in vitro in different solvent systems using LC-MS/TOF showed a solvent-driven breakdown. ARTS breakdown formed several derivatives, including dihydroartemisinin (DHA), artemether (ARTM) and DHA-dimer among others, at different rates in different solvent composition systems. The change in temperature from room temperature to physiological temperature (37 °C) was found to enhance the rate of the ARTS breakdown. In methanol, ARTS mainly formed ARTM with a chromatographic peak decrease of about 3.13%, while methanol and water (90:10) v/v mainly gave rise to DHA and ARTM with about an 80% chromatographic peak decrease. On the other hand, ARTS in methanol and ammonium acetate (85:15) v/v formed DHA, ARTM, DHA-dimer and other reaction peaks with about a 97% peak decrease and the formation of an orange solution pointing to a molecular re-arrangement reaction. These results have an important bearing on research on the analysis of artemisinin drugs conducted on these common solvents. Full article

A multicomponent pharmaceutical that contains loratadine, paracetamol, and pseudoephedrine was quantified using HPLC-PDA. The three analytes were well-separated and quantified in the dosage form on a C-18 column using a gradient mobile phase. A quality by design strategy was followed to achieve the challenging separation. Screening and optimization steps were carried out to investigate the effect of many factors on the studied responses with a minimum number of runs. The ANOVA of the factorial model showed that % acetonitrile (factor A), flow rate (factor B), and pH (factor C) were significant. The detection of the analytes’ peaks was carried out using a PDA detector at 248nm for loratadine and paracetamol, and 214 nm for pseudoephedrine. The second method was SPE-HPLC-MS, where the three analytes and desloratadine, the active metabolite of loratadine, were quantified in spiked plasma and urine, using betamethasone valerate as an internal standard. The recovery of the analytes from body fluids was above 96%, and the LOQ was below 0.5 ng/mL. The validation of the developed HPLC-PDA method was achieved as per ICH guidelines, whereas the HPLC-MS method was validated according to FDA guidelines for bioanalytical method validation. The results were compared with the reported method, and no significant differences were found. Full article

Application of Sustainable analytical chemistry concepts has become crucial in order to remove the environmentally harmful impacts originating from the routine use of analytical techniques. Here, a new LC method is developed and its parameters are analyzed, depending on a mixed micellar mobile phase. This was primarily aimed at getting rid of the use of organic solvents in conventional routine analyses. Combinations of tazobactam (TZB) with piperacillin (PPC) or cefepime (CFM) are commonly used as effective antimicrobial therapies, especially for resistant strains. Therefore, the three drugs were separated and quantified using an organic solvent-free mobile phase. The mixed micellar mobile phase was comprised of 15 mM Brij-35 with 38 mM SDS, adjusted to pH 3.5. Separation was performed by HPLC on monolithic RP-C18 column Chromolith® Performance RP-18e (100 mm × 4.6 mm) at a rate of 1 mL per minute of flow in conjunction with a measurement wavelength 210 nm. The method was found valid and applicable in accordance of precision, and accuracy within ranges of 5–100 µg mL⁻¹ for PPC and CFM and of 0.625–12.5 µg mL⁻¹ for TZB. The quality-by-design technique was used to analyze the effect of modifying the mixed micellar ratios on separation efficiency and conclude their behavior. Finally, the suggested approach was assessed applying the green analytical procedure index against the greenest published methodology to show superiority. Full article

Growing medical, engineering, biochemical, and biological interest has led to a steady pace of research and development into polymeric monolithic structures with densely interconnected pores for purifying bio compounds. Cryogels, which are generated by freezing a reactive polymerization mixture, are highlighted due to their versatility and low relative cost as macroporous, polymeric, monolithic adsorbents. The conversion of cryogels into affinity adsorbents is one possible alternative to their optimal application. Some of the most often utilized supports for immobilizing particular ligands are monolithic columns manufactured with epoxy radicals on their surfaces. The purification of biomolecules with a high degree of specificity, such as lectins and glycoproteins with an affinity for glycosylated groups, has garnered interest in the use of fixed non-traditional beds functionalized with ligands of particular interest. The interaction is both robust enough to permit the adsorption of glycoproteins and reversible enough to permit the dissociation of molecules in response to changes in the solution’s pH. When compared to other protein A-based approaches, this one has been shown to be more advantageous than its counterparts in terms of specificity, ease of use, and cost-effectiveness. Information on polymeric, macroporous, monolithic adsorbents used in the affinity chromatographic purification of lectins has been published and explored. Full article

Food, drugs, dyes, extracts, and minerals are all made up of complex elements, and utilizing unidimensional chromatography to separate them is inefficient and insensitive. This has sparked the invention of several linked chromatography methods, each of them with distinct separation principles and affinity for the analyte of interest. Multidimensional chromatography consists of the combination of multiple chromatography techniques, with great benefits at the level of efficiency, peak capacity, precision, and accuracy of the analysis, while reducing the time required for the analysis. Various coupled chromatography techniques have recently emerged, including liquid chromatography–gas chromatography (LC–GC), gas chromatography–gas chromatography (GC–GC), liquid chromatography–liquid chromatography (LC–LC), GCMS–MS, LCMS–MS, supercritical fluid techniques with chromatography techniques, and electro-driven multidimensional separation techniques. In this paper, the different coupled chromatography techniques will be discussed, along with their wide spectrum of applications for food, flavor, and environmental analysis, as well as their usefulness for the pharmaceutical, color, and dyes industries. Full article