Separations

In this study, a method for the determination of trace elements in snacks using inductively coupled plasma–atomic emission spectrometry (ICP-AES) is presented. The examined elements were Pb, Ni, Cr, Cu, Mg, Zn, Fe, Al, Ba, Ca, Co, Mn and Cd. Under the optimized conditions, digestion of 300 mg of the snack samples was performed by the addition of 5 mL of nitric acid in a Teflon autoclave and by heating the obtained mixture at 120 °C for 75 min. In order to evaluate the efficiency of the proposed protocol, method linearity, accuracy precision, limits of detection (LODs) and limits of quantification (LOQs) were evaluated. The relative standard deviations (RSD%) for all elements were lower than 13.5%, demonstrating that the method offered good precision. The relative recoveries values (R%) ranged between 80–120%, demonstrating that the method offered good accuracy. The LODs for all the trace elements ranged between 0.18 and 3.75 μg g⁻¹, while the LOQs ranged between 0.60 and 12.50 μg g⁻¹. Finally, the proposed protocol was implemented for the analysis of a wide variety of savory snack samples including commercial snacks from corn, potato chips, popcorns, puffed rice cake and crackers. Full article

Symbiotic arbuscular mycorrhizal fungi (AMF) play a major role in plant development, growth, and relationships with the environment through a change in the accumulation of secondary metabolites; hence, we planned to investigate AMF’s influence on sennoside A and B accumulation in Senna alexandrina (SA) and Senna italica (SI). Seeds of SA (S. alexandrina free of mycorrhizae) and SI (S. italica free of mycorrhizae) were planted in two types of soils: +mycorrhiza and—mycorrhiza. The plant leaves of SA, SI, S. alexandrina with mycorrhizae (SAM) and S. italica with mycorrhizae (SIM) were collected and extracted (with 85% methanol), and sennoside A and B content was evaluated by the HPLC–UV method. The antioxidant activity of SA, SI, SAM and SIM was evaluated by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods, while antimicrobial properties were evaluated by the minimum inhibitory concentration method (MIC). The AMF colonization was 85.66% and 85%, respectively, in the roots of SA and SI. The HPLC analysis showed a significant increase in (%) the content of sennoside A/sennoside B by 71.11/88.21, respectively, in SAM and 6.76/36.37 in SIM, which clearly indicated positive AMF effects. The DPPH/ABTS [The half maximal inhibitory concentration (IC₅₀): 235.9/321.5 µg/mL] scavenging activity of SAM was comparatively higher and it also exhibited strong antibacterial action (MIC: 156.25 µg/mL), which supported the increase in sennoside content. This finding may be useful for further investigations of the symbiotic relation of mycorrhizal fungi with other plant species. Full article

Development of a rapid approach for universal microbial detection is required in the healthcare, food and environmental sectors to aid with medical intervention, food safety and environmental protection. This research investigates the use of enzymatic hydrolysis of a substrate by a microorganism to generate a volatile organic compound (VOC). One such enzyme activity that can be used in this context is nitroreductase as such activity is prevalent across a range of microorganisms. A study was developed to evaluate a panel of 51 microorganisms of clinical interest for their nitroreductase activity. Two enzyme substrates, nitrobenzene and 1-fluoro-2-nitrobenzene, were evaluated for this purpose with evolution, after incubation, of the VOCs aniline and 2-fluoroaniline, respectively. Detection of the VOCs was done using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) with obtained limits of quantitation (LOQ) of 0.17 and 0.03 µg/mL for aniline and 2-fluoroaniline, respectively. The results indicated that both enzyme substrates were reduced by the same 84.3% of microorganisms producing the corresponding volatile anilines which were detected using HS-SPME-GC-MS. It was found that nitroreductase activity could be detected after 6–8 h of incubation for the selected pathogenic bacteria investigated. This approach shows promise as a rapid universal microbial detection system. Full article

The sample analysis and data interpretation is the most challenging step of fire debris analysis, due to the presence of combustion and pyrolysis products in the substrate material. In this study, a headspace solid phase microextraction (HS-SPME) procedure was applied to the extraction of combustion and pyrolysis products from three commonly used carpet substrate materials, made of nylon 6,6 and polyesters. Each carpet sample was burned with and without two different ignitable liquids (ILs), i.e., gasoline and kerosene, and the Total Ion Chromatograms (TICs) and Extracted Ion Profiles of characteristic class compounds of ILs were obtained and compared to those of unburned neat ILs, using gas-chromatography mass spectrometry (GC-MS), to study the possible interferences of these substrate materials in fire debris analysis. Full article

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the quantification of clotrimazole (CTZ) plasma levels after intravaginal administration of the drug given at approved dosages. Plasma samples were extracted by liquid–liquid extraction and a single chromatographic run could be completed within about 2 min. The method was linear over the investigated range (0.488–250 ng/mL) with all the correlation coefficients, R², greater than 0.9903. All data were in the range of ±15.0% with respect to the nominal concentration for high QC and medium QC, and in the range ±20% with respect to the nominal concentration for low QC. This rapid and sensitive method was validated and could be applied to human plasma samples from a healthy volunteer, showing that the assay is able to detect plasma concentrations of CTZ in the range of those found after the administration of the drug at approved dosages in the clinical setting. Full article

The quantitative analysis of the chromatographic behavior of basic compounds measured with pentyl-, hexenyl-, and octyl-bonded silica gels were analyzed in silico employing model phases. The main retention force was the van der Waals (VW) interaction, and the main desorption force was an electrostatic (ES) interaction. The contribution of hydrogen bonding (HB) was weak compared to that for acidic compounds. The quantitative explanation was achieved utilizing the calculated VW, HB, and ES energy values obtained from a molecular mechanics program. The electron localization was observed at the molecular interaction-site calculated MOPAC program. This fundamental approach was like that of explaining chemical reactions. The difference was electron localization in chromatography or electron transfer in a chemical reaction. Full article

Currently, the medical use of food supplements containing Cannabis sativa has attracted the interest of consumers, as well as the medical and scientific community. With the increasing consumption of these products, there is also a risk of their abuse or discrepancy between the actual and declared contents of active substances by the manufacturer in these products. Thus, the development and elaboration of analytical procedures for determination of appropriate phytocannabinoids seems to be important. This work focuses on the development of a simple, fast and environmentally friendly liquid-liquid extraction method combined with fat freezing from an oil sample to isolate two phytocannabinoids: cannabidiol (CBD) and cannabidiolic acid (CBDA). The extraction method was optimized considering efficacy and repeatability of extraction, as well as minimalizing use of organic reagents and sample amount. Under the optimized conditions, extraction recovery for CBD was 97.3–109% and for CBDA was 69.1–69.5% with precision (RSD, %) 5.0–8.4 and 7.1–10.6, respectively. The evaluated main analytical parameters of the developed high pressure liquid chromatography with diode array detector (HPLC-DAD) method for both studied cannabinoids are satisfactory. The usability of the developed method was checked by analysis of real samples of a food supplement–hemp oil enriched with CBD. Full article

Recently, a new formulation containing metformin HCl (MFH) and remogliflozin etabonate (RGE) has been approved for the management of diabetes mellitus. However, only one analytical method has been reported for the simultaneous determination of both the analytes. Therefore, the current study was designed to develop simple UV derivative spectroscopic and rapid RP-HPLC methods for simultaneous determination of MFH and RGE. The chromatographic separation of MFH and RGE was performed using a monolithic C18 column with an optimized chromatographic conditions carried out by full factorial Box–Behnken design model. The spectroscopic technique was based on the determination of peak amplitude of second-order derivative UV spectra at zero crossings. Further, both the methods were validated and compared statistically using Student’s-t-test and F-test, and employed for the concurrent estimation of MFH and RGE in laboratory mixed solutions and formulations. Perturbation plots and response surface models showed the effect of chromatographic parameters and the final chromatographic condition was selected from 47 solutions suggested by the desirability function. Further, UV spectroscopic and HPLC procedures showed good linearity in the range of 1–24 µg/mL and 2–150 µg/mL for RGE and 2–30 µg/mL and 5–200 µg/mL for MFH, respectively. The average percent assay was found to be 99.51% and 99.80% for MFH and 99.60% and 100.07% for RGE by spectroscopic and HPLC methods, respectively. The proposed methods were simple, accurate, precise, and rapid. Therefore, they can be used for regular quality control of MFH and RGE formulations and dissolution studies as well. Full article

Background: The metabolic changes associated with diabetes can lead to nephropathy eventually resulting in end-stage renal disease. Current antidiabetic therapies do not effectively prevent the onset of diabetic kidney diseases as well as progression. Aim: To evaluate the effect of Coccinia indica leaf extract alone and in combination with pioglitazone, an antihyperglycemic agent was used to modulate the progressive kidney damage induced by type 2 diabetes in rats. Hypotheses: Pioglitazone causes severe adverse effects when administered for long-term therapy. The hypotheses in this study is to examine the renoprotective effect of Coccinia indica leaf extract (200 mg/kg p.o.) when co-administered with low-dose pioglitazone (7 mg/kg) in type-2-diabetes-induced nephropathy in rats and simultaneously evaluate the hypoglycemic response as well. Methods: Rats (Males, Sprague Dawley) were kept on a high-fat diet and were given a single dose of streptozotocin (35 mg/kg, i.p.) to induce diabetic nephropathy. Treatment groups received either Coccinia indica leaf extract or pioglitazone or pioglitazone with Coccinia indica extract, fenofibrate, or lisinopril for 7 weeks. Blood glucose, antioxidant status, triglycerides, total cholesterol, creatinine, blood urea nitrogen, and proteinuria levels were estimated and compared with the normal control and disease control (untreated) groups. Results: The untreated diabetic rats showed increased blood glucose levels, lipid profiles, and renal oxidative stress, along with an increase in nephropathy markers such as blood urea nitrogen, creatinine, and proteinuria. Histopathological examination revealed glomerular damage. Combination treatment with Coccinia indica leaf extract and a low dose of pioglitazone normalized the nephropathic markers as well as histopathological changes. Conclusion: Coccinia indica leaf extract when co-administered with a low dose of pioglitazone as antidiabetic therapy showed good glycemic control and a beneficial renoprotective effect. Combination therapy would lower the dose of pioglitazone and also protect kidneys from drug-induced toxicity as observed from normalized nephropathic markers in a diabetic rat model. Full article

Globotriaosylsphingosine (LysoGb₃) is a biomarker for Fabry disease (OMIM 301500) that contains long-chain bases (LCBs) as a building block. There have been several studies proposing that LysoGb₃ forms with distinct LCBs could be putative disease subtype-related biomarkers for this congenital disorder; however, there have been no detailed multiple reaction monitoring-based studies examining the LCB distribution in this lysosphingolipid. To achieve this, we established an assay procedure that aimed at elucidating the LCB-targeted lipidome using liquid chromatography–tandem mass spectrometry. Consistent with previous studies, we found d18:1 to be the major LCB species of the LysoGb₃ in pooled human plasma, while some atypical LCBs, such as d18:2, d18:0, t18:1, d16:1, and d17:1, were detected as minor fractions. When the same methodology was applied to fetal bovine serum (FBS) as a positive control, we identified additional unique LCB species, such as t18:0, d20:1, t19:1, and t21:1, in herbivore LysoGb₃. Furthermore, we found an elevation of sphingosine and LysoGb₃, which are N-deacylated forms of ceramide and Gb₃, respectively, in FBS, suggesting that ceramidase activity may be involved in this process. Thus, our LCB-targeted lipidomics data revealed that mammalian LCBs in glycosphingolipids have a greater variety of molecular species than previously expected. Full article

Minor cannabinoid and non-cannabinoid molecules have been proposed to significantly contribute to the pharmacological profile of cannabis extracts. Phytoplant Research has developed highly productive cannabis cultivars with defined chemotypes, as well as proprietary methods for the extraction and purification of cannabinoids. Here, we investigate the effect of solvent selection and decarboxylation on the composition and pharmacological activity of cannabis extracts. A library of forty cannabis extracts was generated from ten different cannabis cultivars registered by Phytoplant Research at the EU Community Plant Variety Office. Plant material was extracted using two different solvents, ethanol and hexane, and crude extracts were subsequently decarboxylated or not. Cannabinoid content in the resulting extracts was quantified, and biological activity was screened in vitro at three molecular targets involved in hypoxia and inflammation (NF-κB, HIF-1α and STAT3). Changes in transcriptional activation were strongly associated to solvent selection and decarboxylation. Two decarboxylated extracts prepared with hexane were the most potent at inhibiting NF-κB transcription, while HIF-1α activation was preferentially inhibited by ethanolic extracts, and decarboxylated extracts were generally more potent at inhibiting STAT3 induction. Our results indicate that solvent selection and proper decarboxylation represent key aspects of the standardized production of cannabis extracts with reproducible pharmacological activity. Full article

A chiral analytical methodology was developed by nano-liquid chromatography (nano-LC) enabling the enantiomeric separation of two chiral drugs, lacosamide (novel antiepileptic drug) and colchicine (antiuremic drug), commercialized as pure enantiomers. A capillary column lab-packed with an amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase was used in a lab-assembled nano-LC system. Lacosamide and colchicine enantiomers were separated in less than 8.0 and 9.0 min, respectively, with resolution values of 1.6 and 2.3, using 20 nL of sample and 1.8 µL of mobile phase per analysis. The analytical characteristics of the proposed methodology were evaluated according to the International Council for Harmonisation (ICH) guidelines, showing good analytical performance with good recoveries (97–98% and 100–103%) and precision values (relative standard deviation (RSD) <10.5 and <3.0%) for lacosamide and colchicine enantiomers, respectively. LODs were 1.7 and 2.0 µg/mL for (S)- and (R)-lacosamide, respectively, and 1.0 µg/mL for both colchicine enantiomers. Additionally, the developed methodology enabled to detect a 0.1% of the enantiomeric impurities, fulfilling the ICH regulation requirements. The method was applied to the determination of lacosamide and colchicine enantiomers in different pharmaceutical formulations to ensure their quality control. The content of the enantiomeric impurities was below a 0.1% and the amount of (R)-lacosamide and (S)-colchicine agreed with their labeled contents. Full article

Nowadays, marine microalgae are recognized to be a considerably novel and rich origin of bioactive moieties utilized in the sectors of nutraceuticals and pharmaceuticals. In the present study, Nannochloropsis oculata extract (AME) was associated with a wide variety of pharmacological studies such as in vitro antioxidant, antibacterial, and antifungal and anticancer activity (MDA-MB-231) in cancer cells through in vitro models. In the study, the chemical composition and structure of the bioactive compounds found in the AME extract were studied using the LC-MS technique. The results of the anticancer activity showed a decrease in the percentage of cell viability of the MDA-MB-231 cells in a concentration- and time-dependent manner (400 μg/mL at 24 h, 300 μg/mL at 48 h, and 200 μg/mL at 72 h). We have also observed morphological changes in the cells that could be associated with treatment with AME extract. Our observation of the AME extract-treated MDA-MB231 cells under light microscopy showed that when the concentration increased, the number of cells began to decrease. As far as LC-MS analysis is concerned, it showed the presence of the bioactive molecules was terpenoids along with carotenoids, polyphenolic and fatty acids. The result revealed that the AME extract exhibited noteworthy in vitro free radical scavenging potential, with an IC₅₀ value of 52.10 ± 0.85 µg/L in DPPH assay, 122.84 ± 2.32 µg/mL in H₂O₂ assay and, 96.95 ± 1.68 µg/mL in ABTS assay. The activity was found to be highly significant against bacteria (Gram-positive and negative) and moderately significant against fungal strain with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)/minimum fungicidal concentration (MFC) values between 15.63 and 500 µg/mL. Full article

Synthetic cathinones have become very popular recreational drugs. Therefore, determining them in biological samples is now a matter of concern. In recent years, different methods that have been developed can determine these drugs at low-concentration levels. In general, liquid chromatography mass spectrometry detection plays an important role in these methods and the trend is to use low-resolution and high-resolution mass spectrometry. In this article, for the first time, we compare these two analyzers using an Orbitrap and a triple quadrupole mass spectrometer in order to determine a group of synthetic cathinones in urine and oral fluid samples. For this comparison, we evaluated and compared different parameters: Method detection and quantification limits, linearity, apparent recoveries, matrix effect, repeatability (intra-day), reproducibility (inter-day), and accuracy. Similar results were obtained for the two analyzers for the apparent recoveries and matrix effect. However, triple quadrupole showed higher sensitivity compared to Orbitrap for both urine and oral fluid samples. The quantification limits in urine and the detection limits in saliva were two times lower for triple quadrupole. Finally, when blind samples were analyzed to study the accuracy, similar results were obtained for both analyzers. Full article

Agricultural water is closely linked to surface and ground water as well as soil; hence, ensuring its safety is an important endeavor. We used the “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) method to analyze multi-residue pesticides in agricultural water by using a combined-sorbent-based clean-up procedure. Among the various sorbents examined, clean-up using ENVI-Carb combined with a primary secondary amine sorbent delivered the highest recovery of multi-residue pesticides (>93.9%). While the developed method showed satisfactory linearity (R² > 0.9991), precision, and specificity, recovery was low for pyrazolate (29.1%) and thidiazuron (59.2%). The limits of detection and quantification for the 55 pesticides targeted in this study were in 0.02–3.0 μg L⁻¹ and 0.1–9.9 μg L⁻¹, respectively. The developed method was used to identify and quantify multi-residue pesticides during sample analysis. The results suggest that the QuEChERS method employing a combination of ENVI-Carb and another sorbent can be applied for the effective analysis of multi-residue pesticides in agricultural water. Full article

Due to the lack of ecofriendly/green reversed-phase high-performance thin-layer chromatography (RP-HPTLC) methods for trans-anethole (TAL) and its simplicity over routine analytical techniques, there was a necessity to establish a suitable HPTLC methodology for the quantitative analysis of TAL. Therefore, the first objective of this research was to develop an accurate, rapid and green RP-HPTLC densitometry methodology for the quantitative analysis of TAL in essential oil, traditional and ultrasound-assisted extracts of Foeniculum vulgare Mill and commercial formulations. The second objective was to compare the traditional method of extraction of TAL with its ultrasound-assisted method of extraction. The chromatogram of TAL from essential oil and traditional and ultrasound-assisted extracts of fennel and commercial formulations was verified by recoding its single spectra at R_f = 0.31 ± 0.01 in comparison to standard TAL. The proposed analytical methodology has been found to be superior in terms of linearity, accuracy and precision compared to most of the reported analytical methods for TAL analysis. The amount of TAL in the essential oil of fennel was recorded as 8.82 mg per g of oil. The content of TAL in traditional extracts of fennel, formulation 1 (dietary supplement 1) and formulation 2 (dietary supplement 2), was recorded as 6.44, 4.88 and 4.48 mg per g, respectively. The amount of TAL in ultrasound-assisted extracts of fennel, formulation 1 and formulation 2, was recorded as 8.34, 6.46 and 5.81 mg per g, respectively. The ultrasound method of extraction of TAL was found to be better than the traditional method of extraction. The results of validation studies and phytochemical analysis showed that the proposed methodology could be efficiently utilized for the quantification of TAL in the wide range of products having TAL as a component. Full article

In this study, we investigated and validated a method for the quantitative analysis of ethoxyquin in paprika seasonings and sauces, which are frequently consumed in South Korea. Using this analytical method, we were able to confirm the presence of ethoxyquin under optimized high-performance liquid chromatography/photodiode array (HPLC-PDA) and liquid chromatography with tandem mass spectrometry conditions. Additionally, the antioxidant activity of ethoxyquin was assessed using the American Spice Trade Association values and its residual levels in paprika seasoning samples. The HPLC-PDA limits of detection and quantification for ethoxyquin were found to be 0.26 and 0.79 μg/mL, and 0.33 to 1.00 μg/mL, and the recoveries of ethoxyquin ranged from 86.75 to 101.70%, with relative standard deviations ranging from 0.31% to 3.59%. These results indicated that the analytical method in this study should be appropriate for the quantitative analysis of ethoxyquin in paprika seasoning and sauce matrices. Full article

Chondroitin sulphates belong to a group of naturally occurring glycosaminoglycans and play a role in many physiological processes including ageing and the effects of various diseases. Research into chondroitin sulphates has found that the most important analytes are 4- and 6-sulphated disaccharides. We developed an HPLC method for the separation and quantification of underivatized chondroitin/dermatan sulphates—unsaturated disaccharides (4- and 6-sulphated disaccharides). This method is based on the separation of disaccharides by amido as well as amino columns under acidic conditions. These columns enabled the successful separation of 4- and 6-sulphated disaccharides using 50 (amido column) and 25 mmol/L (amino column) phosphate buffer, pH 4.25 (detection at 230 nm), at retention times of less than 10 min. The limit of quantification was 0.5 μg/mL. The applicability of this method was demonstrated through analysis of unsaturated disaccharides produced from the enzymatic digestion of chondroitin/dermatan sulphates of the solubilized extracellular matrix produced from porcine urinary bladder and human umbilical cord. Full article

(1) Background: The present investigation studies the optimization of extraction, quantification, and cytokine inhibitory effects bakuchiol (BKL) in Psoralea coryfolia Linn. (2) Methods: The seeds of Psoralea coryfolia cleaned, dried, and powdered. Different separation methods maceration, reflux, Soxhlet, and ultrasonic assisted extraction (UAE) were employed for the isolation of BKL by five pure solvents. The quantity of BKL was measured by high-performance liquid chromatography (HPLC) method to determine the highest yield percentage. The effect of optimized BKL was then tested in an animal model of sepsis induced by lipopolysaccharides (LPS). (3) Results: The UAE method was found to be the best among tested separation methods and yielded highest percentage of BKL in petroleum ether extract. Septic rats showed a significant elevation in levels of biochemical markers like AST, ALT, ALP, BIL, SCr, and BUN in plasma. Proinflammatory cytokines (TNF-α and IL-1) levels were also increased in LPS-induced animals. BKL has been found to significantly reverse these elevated levels as compared to the LPS-induced animals. (4) Conclusion: The present results suggest that BKL has positive effects when administered in animals with pathogenic shock by decreasing the circulating levels of biomarkers. Further studies are necessary to explore the clinical implications of such findings. Full article