Authors:Débora Arruda Frommenwiler, Valeria Maire-Widmer, Roy Upton, Judith Nichols, Günther Heubl and Eike Reich
The herbal drug licorice root may be derived from the plant species Glycyrrhiza glabra L., Glycyrrhiza uralensis Fisch, and/or Glycyrrhiza inflata Bat. which are morphologically, chemically, pharma-cologically, and toxicologically similar. However, if an ingredient of a dietary supplement is identified as a certain species and labeled as such on the product, appropriate analytical methodologies are required to assure the authenticity. Using high-performance thin-layer chromatography (HPTLC), we were able to distinguish clearly between G. glabra and G. uralensis, the most commonly used species, which allowed us to check the corresponding label claims of twenty-six dietary supplements. Two samples of G. inflata Bat., which were available for the study, were not distinguished from G. glabra by this method. Our investigation revealed that five of the twenty-eight samples made a wrong label claim. The HPTLC results were confirmed by deoxyribonucleic acid (DNA) barcoding. For the quantitative analysis of the marker 18β-glycyrrhizic acid in licorice root, we modified our HPTLC method for base-line separation of the peaks which guaranteed accurate results. Moreover, the new method is also capable to identify and distinguish both species of licorice. The quantitative HPTLC results were in accordance with the data obtained by high-performance liquid chromatography (HPLC) following the United States Pharmacopeia (USP) method on licorice root. In addition, we used two DNA candidate barcodes (internal transcribed spacer [ITS] and psbA‒trnH intergenic spacer) for species identification.
Authors:Mathias Reisberg, Norbert Arnold, Daniel Bisrat, Kaleab Asres, Reinhard H.H. Neubert and Birgit Dräger
Glycosylceramides (GlyCers) are precursors of ceramides (Cers) that are major components of the outer layer of human skin, the stratum corneum. A Cer deficiency is associated with skin diseases such as psoriasis and atopic dermatitis and can be treated with Cer-containing semisolid formulations. Plants may serve as alternative sources for expensive semisynthetic Cer production. Since the GlyCer contents of plants vary widely, there is a need to develop a rapid, simple, selective, and precise method for GlyCer quantification in plants. In the present study, an effective and validated automated multiple development‒high-performance thin-layer chromatography (AMD‒HPTLC) method has been developed for GlyCer quantification in 9 different plant materials. An 18-step gradient elution program (n-hexane, chloroform, ethyl acetate, methanol) led to a clear separation of bands from complex matrices and allowed densitometric analysis for quantification purposes. Apple pomace and wheat germs yielded 26.8 and 39.5 mg of GlyCer per 100 g plant material, respectively, while the yields of coffee grounds were below the limit of quantification. The GlyCer contents of the seeds of six Fabaceae species, namely, Albizia grandibracteata, Albizia gummifera, Albizia lebbeck, Albizia schimperiana, Acacia etbaica, and Robinia pseudoacacia, ranged from 9.4 to 23.1 mg per 100 g plant material. GlyCers were separated by preparative thin-layer chromatography (TLC) and identified by offline high-performance liquid chromatography–mass spectrometry (HPLC–MS). Intact GlyCers were detected in the Fabaceae species for the first time. A simple AMD–HPTLC screening and quantification technique for GlyCers was developed, which may serve as a tool in searching plant GlyCers for a possible “phyto”-Cer production.
Authors:Uppuluri Venkata Mallavadhani, Yagnambhatla Aparna, Satyabrata Mohapatra and Dhanunjay Vithalrao Mane
In view of extensive applications in the Indian Ayurvedic system of medicine and the increased demand for both as single ingredient and as a major component in multiherbal formulations of Boerhavia diffusa, a rapid, sensitive and reproducible high-performance thin-layer chromatography (HPTLC) method has been developed for its quantitative evaluation with respect to its major bio-active marker, eupalitin galactoside. For the separation of the marker compound, HPTLC silica gel F254 pre-coated plates were used with n-butanol‒acetic acid‒water (8:1:1) as the mobile phase. The method was validated for limit of detection, limit of quantification, linearity, specificity, precession, and recovery. The results indicate that the bio-active marker was present in 0.075% in B. diffusa whole plant. Surprisingly, the marker compound could not be detected even in traces in two of the B. diffusa commercial formulations such as punarnava mandur and artin capsules, which contain more than ten herbal and herbo-mineral ingredients. This might be due to the loss of the marker compound during their manufacturing process or different plant parts used in the two test samples. The developed method can be used successfully for the quality control and quality assurance of B. diffusa formulations, where the whole plant is used.
Authors:Jin-Peng Yuan, Zhao-Jie Cui, Chuan-Ge Cheng, Xiao-Li Wang, Shan-Shan Wang, Xin-Li Song and Fu-Wei Li
A rapid and simple analytical method based on matrix solid-phase dispersion combined with liquid chromatography coupled with tandem mass spectrometry has been developed by using bamboo charcoal as a dispersive adsorbent to simultaneously determine tetrabromobisphenol A (TBBPA) and hexabromocyclododecane diastereoisomers (HBCDs) in soil. The factors influencing the performance of the proposed method were investigated and optimized in detail, and the matrix effects were evaluated. Under optimum conditions, the proposed method showed good linearity within the range of 0.8–80 ng g−1 and limits of detection of 4–75 pg g−1 (S/N = 3). The satisfactory recoveries of TBBPA ranging from 72.8% to 92.5% and HBCDs ranging from 76.8% to 102.2% were obtained with relatively standard deviation (RSD) ranging from 3.4% to 9.8%. The proposed method has been successfully applied to analyze TBBPA and HBCDs in actual soil samples from the Yellow River Delta in China.
Authors:Astitva Anand, Mohinder Singh Dahiya and Rakhi Agarwal
Benzene is an omnipresent liquid in industries. The occupational exposure to benzene leads to the urinary excretion of benzene metabolites, viz., phenol, pyrocatechol, hydroquinone, and trihydroxybenzene, due to its biotransformation. These metabolites are phenolic in nature and considered as immediate biomarkers of benzene exposure. The present work includes the separation and determination of urinary phenolic benzene metabolites by coupling two different techniques. Thin-layer chromatography (TLC) was used as the separation technique to get individually separated spots of all four metabolites, which were further quantified by ultraviolet (UV)–visible spectroscopy at 765 nm. For the development of the separated spots on TLC plate and determination of metabolites by UV–visible spectroscopic method, alkaline Folin‒Ciocalteau reagent was used. Folin‒Ciocalteau reagent is having wide applications for phenol determination and gives blue color with almost all types of phenols. The colored solutions were measured against the blank disk taken from the developed spots on plastic TLC plate. Based on the obtained results, a simple, rapid, and sensitive method for the quantitation of urinary phenolic benzene metabolites has been developed and validated according to the International Conference on Harmonization (ICH) guidelines. The validated method was efficaciously applied to cigarette smokers and petrol station workers, and it was found that the method has favorable application in the routine analysis of urine samples of benzene-exposed population.
Secondary metabolites are an integral part of the plant kingdom which plays a vital role in development, growth, and defense. These are also very helpful for humans against different disorders. Phenols due to their strong antioxidant activity have a central role in conferring different types of biological activities. The present study aims at the simultaneous determination of chlorogenic acid and caffeic acid in the methanolic extracts of leaf samples of Siegesbeckia orientalis L. Simultaneous elution of both compounds has been attained by using toluene‒ethyl acetate‒formic acid‒methanol (6:6:1.6:0.6 v/v) as the mobile phase, and densitometric evaluation was done at 366 nm. The linear regression data for the calibration curve of both compounds show good linear relationship in the concentration range of 200–600 ng. The proposed method has been validated in terms of limit of detection, limit of quantification, specificity, precision, and accuracy studies. The present high-performance thin-layer chromatographic method has been applied for the quantification of both compounds in the various accessions of S. orientalis collected from different localities of North India. Maximum amount of chlorogenic acid was found in the Taluka accession, while caffeic acid was found in the Rarhi accession collected from Uttarakhand state.
Authors:Abhishek Niranjan, Nem Kumar Ngpoore, Naushi Anis, Anil Kumar, Alok Lehri, Pramod Arvind Shirke and Shri Krishna Tewari
Fresh pods of Moringa oleifera with nutraceutical importance are widely consumed in food commodities as vegetables. It is nutritious and it also has several biological activities. In the present study, a simple, rapid, cost-effective, and sensitive high-performance thin-layer chromatography (HPTLC) method was applied for the simultaneous determination of six phenolic compounds, viz., gallic (phenolic acid), p-coumaric, caffeic acid (hydroxycinnamic acid), chlorogenic acid (cinnamic acid derivative), quercetin and kaempferol (flavonols) in flowers, pods, leaves, twigs, and seeds of M. oleifera. Simultaneous separation and quantification of compounds were achieved on HPTLC pre-coated silica gel 60 F254 aluminum plates using the mobile phase toluene–ethyl acetate–formic acid (14:10:1). Densitometric determination was carried out at λmax 282 nm. The calibration curves were linear, ranged between 0.984 and 0.998; the limit of detection and quantification ranged between 110.8 ng mL−1 and 142.3 ng mL−1, and 301.6 ng μL−1 and 410.8 ng μL−1; and recovery ranged between 96.2% and 97.9%. The validated method was successively used to analyze the above compounds in the plant parts of M. oleifera. The amount of the total phenolic content and specific phenolic compounds ranged from 4.86 mg g−1 (gallic acid equivalent [GAE]) to 14.79 mg g−1 (GAE) and 0.007% quercetin (flower and flower with pods) to 0.099% gallic acid (pods of 15 days). This study reveals that the presence of specific phenolic compounds in M. oleifera shall be a good source for the isolation of the above-mentioned compounds for industrial use.
Authors:Hanan M. Al-Yousef, Tawfeq A. Alhowiriny, Perwez Alam, Nasir A. Siddiqui, Atallah F. Ahmed, Jawaher H. Al-Qahtani and Musarat Amina
In this study, we have developed a validated high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of two phenolic biomarkers, protocatechuic acid (compound 1) and quercetin 4ʹ-O-β-d-glucopyranoside (compound 2) in antimicrobial and antioxidant active A. cepa ethyl acetate extract (ACEAE). The quantitative analysis was carried out on normal-phase HPTLC (glass-backed silica gel 60 F254) plates with solvents toluene, ethyl acetate, and formic acid in the ratio of 3:6:1, v/v (as the mobile phase). Well-resolved, compact, and intense peaks of compound 1 (RF = 0.56 ± 0.001) and compound 2 (RF = 0.05 ± 0.001) were found at λmax = 275 nm. The linear regression equation / r2 for compound 1 (Y = 8.89X + 250.71 / 0.994) and compound 2 (Y = 6.64X + 209.34 / 0.998) in the concentration range of 100–700 ng spot−1 indicated good linear relationship. The low values of percent relative standard deviation (%RSD) for intra-day / inter-day precision of compound 1 (1.14–1.26 / 1.08–1.23) and compound 2 (0.97–1.18 / 0.93–1.16) suggested that the method is precise. The (%) recoveries for compound 1 / compound 2 were found as 98.07–99.55 / 98.20–99.89 which confirms the good accuracy of the proposed method. The quantities (%w/w) of compound 1 / compound 2 in ACEAE were found as 18.84% / 13.64% of the dried weight of the extract. In vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed the promising free radical scavenging activity of ACEAE (69.00 ± 2.99%) and compound 2 (63.86 ± 2.02%) which were comparable to ascorbic acid tested at 400 μg mL−1. ACEAE was found to be highly active against all tested bacterial strains, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; however, Candida albicans was found to be susceptible to both compound 2 and ACEAE. The presence of compound 2 in high quantity in the ethyl acetate fractions of A. cepa peel (ACEAE) validated its antimicrobial and antioxidant property. The above developed HPTLC method can be further employed in the analysis of these markers in marketed formulations and in the quality control of herbal drugs.
Authors:Haya I. Al-Johar, Hadir M. Maher, Nourah Z. Al-Zoman, Dlayel J. Al-Shammary and Hessa Al-Showiman
A stability-indicating capillary electrophoresis method coupled to a diode array detector (DAD) was developed and validated for the simultaneous determination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) in combined tablets. This proposed method utilized a fused silica capillary (effective length, 62 cm; internal diameter [ID], 75 μm) and a background electrolyte (BGE) consisting of phosphate solution (pH 9.5, 50 mM). The separation was achieved at a voltage of 25 kV and a temperature of 21 °C using paracetamol as an internal standard. The described method was linear over the range of 5–200 μg/mL for both drugs (r = 0.9992). Intra- and inter-day relative standard deviation (RSD) (n = 9) was 0.41%. The limits of detection for FTC and TDF were 1.25 and 1.00 μg/mL, respectively. The average percentage recoveries of FTC and TDF from their tablet formulations were 99.66 ± 0.73 and 99.48 ± 0.33, respectively. The two drugs were subjected to thermal, photolytic, hydrolytic, and oxidative stress conditions, and then the stressed samples were analyzed by the proposed method. Degradation products produced as a result of stress studies did not interfere with the detection of FTC and TDF. The assay can thus be considered stability indicating.
Authors:Qianqian Xing, Qing Fu, Yu Jin and Xinmiao Liang
The off-line two-dimensional supercritical fluid chromatography (SFC)–ultrahigh-performance liquid chromatography (UHPLC) was selected to separate the triterpene saponins from Panax notoginseng. The separation by SFC was performed on an Atlantis® HILIC silica column. Methanol was selected as a modifier, and the most time-saving gradient was developed. The decrease of the column temperature and the increase of the back pressure could shorten the retention time but had no effect on the separation selectivity. Then, the back pressure, column temperature, and flow rate were set as 131 bar, 45 °C, and 4.0 mL min−1, respectively. The retention behavior of the saponins from P. notoginseng was different between SFC and reversed-phase liquid chromatography (RPLC), which facilitated to construct an off-line SFC/RPLC–mass spectrometry (MS) system. In first dimension, a total of eight fractions were collected under SFC and further analyzed by RPLC–MS in second dimension. The result indicated that the retention behavior of triterpene saponins was mainly controlled by the hydrogen bonding interactions which were affected by the number and types of sugars, as well as the aglycone in the structure of triterpene saponins. Thus, the presence of “clustering effect” under SFC was observed, namely, one SFC peak always contained several saponins with same number of sugars and similar structure of aglycone. The clustering effect of triterpene saponins promised SFC to be used as first dimension to complete the preliminary crude separation in the two-dimensional liquid chromatography.
Cadmium accumulation in soils causes ecological, biological and human health risks. Previous studies have shown that reductions in the shoot height and fresh biomass of ryegrass (Lolium perenne L.) are a sensitive indicator of the cadmium pollution level in soils.
Four soils with different types and properties were included in the experiment. In the first period of the biotest, 2 g cotton-wool pads moistened with distilled water were planted with 2 g of perennial ryegrass seeds and the seedlings were grown for 6 days. On the 7th day the cotton-wool pads containing the seedlings were placed on soils polluted with four levels of cadmium: 0, 1, 2 and 4 mg Cd kg−1 soil, added to the soil in the form of cadmium acetate. After a first nutrient-deficient period, the seedlings took up nutrients and toxic substances intensively from the soil samples. After a 14-day period of soil–plant contact the fresh biomass, dry biomass and Cd concentration of the shoots were measured, in addition to which the shoot height was measured every 2 days.
Cadmium treatment significantly reduced the shoot height and fresh weight of ryegrass in all the tested soils, and the damaging effect was proportional to the applied dose. A reduction of more than 10% in the shoot height and fresh weight were observed even at a Cd pollution level of 1 mg Cd kg−1 soil. At the highest Cd level the decrease in shoot height was more than 40% and the decrease in fresh weight more than 35% in all the soils.
The increasing level of Cd application significantly increased the Cd concentration of the shoots. More Cd was accumulated in ryegrass shoots on soils with low pH and low organic matter content.
The results indicate that the ryegrass biotest method is suitable for the characterization of Cd contamination in different soils.
Authors:András Makó, Hilda Hernádi, Gyöngyi Barna, Réka Balázs, Sándor Molnár, Viktória Labancz, Brigitta Tóth and Zsófia Bakacsi
The particle size distribution (PSD) values obtained for a soil database representing the main Hungarian soil types using the Hungarian standard (MSZ-08-0205-78) and the international standard (ISO/DIS 11277:1994) were compared with the pipette method. The relationship between these PSDs and other physical soil characteristics (upper limit of plasticity according to Arany, water vapour adsorption according to Sík) was also analysed, and a suggestion was made of how these results could be converted into each other.
Experience showed that the pre-treatments applied as part of the ISO/DIS method may change the ratio of particle size fractions: there was a significant increase in the clay content, while the silt content decreased to a lesser and the sand content to a greater extent, possibly because some of the particles remain in microaggregate form when the MSZ method is used. The results confirmed the greater accuracy of the ISO/DIS method: the clay contents measured with the ISO/DIS method exhibited stronger correlations with the upper limit of plasticity according to Arany and with hygroscopicity values than those measured with the MSZ method.
The estimated ISO/DIS fractions became much closer to the measured ones when the suggested pedotransfer functions were applied. The conversion method proved to be more reliable for the prediction of clay and sand content than for silt content. In its present form the estimation method is not suitable for replacing the ISO/DIS method, but it could be of good service in research and comparative analysis in cases where only the MSZ method can be used or where only old MSZ PSD data exist.
The present study is the first attempt on the phytochemical analysis of Lasiurus scindicus Henrard, a desert-specific plant, through high-performance thin-layer chromatography (HPTLC). This technique has been developed and validated for the quantification of stigmasterol from 48 extracts, i.e., four different plant parts with six different solvents (butanol, chloroform, ethanol, hexane, methanol, and petroleum ether) in both hot and cold macerations. This method gave the best resolution bands with toluene‒methanol‒formic acid (9:1:0.3, v/v) for 25 min as the mobile phase at retention factor (RF) of 0.27 for stigmasterol. The method was validated using the International Conference on Harmonization (ICH) guidelines in terms of precision, repeatability, and accuracy. The linearity range for stigmasterol was found to be 2–12 μg spot−1. Both hot and cold extractions with six different solvents were compared. The highest concentration of stigmasterol was found in the leaves of L. scindicus in cold hexane extract (52.07 mg g−1), stem and inflorescence in cold ethanol extract (93.81 and 59.34 mg g−1, respectively), and root in cold chloroform extracts (5.82 mg g−1). It follows in the decreasing order of stem > leaves > inflorescence > roots. The recovery value was 99.33% which shows excellent accuracy of the method. From the obtained results, it is concluded that cold extractions are best for the plant taken under investigation. HPTLC was found to be a simple, precise, accurate, and convenient method for the rapid screening of steroids in L. scindicus.
Authors:Jong-Woo Jeong, Yun-Hwan Seol, Hun-Chan Hyun, Hye-Rim Kim, Jong-Hwa Lee, Young-Dae Gong, Nam Sook Kang and Tae-Sung Koo
A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of an anticancer drug, supinoxin (RX-5902), in rat plasma. Following precipitation pretreatment using 0.1% formic acid in acetonitrile, separation was performed using a reverse phase liquid chromatography column packed with C18 (3.5 μm, 2.1 × 50 mm) along with a mobile phase of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL min−1. Detection was achieved using MS/MS by multiple reaction monitoring via an electrospray ionization source at mass/charge transitions of m/z 442.30 → 223.30 for supinoxin and m/z 430.08 → 223.20 for the internal standard DGG-200064. This method demonstrated a linear standard curve (r = 0.9980) over a supinoxin concentration range of 0.0005–1 μg mL−1, as well as intra- and inter-assay precisions below 7.08% and 13.74%, respectively, and an accuracy of 1.15–4.50%. The matrix effect, recovery, and process efficiency were 93.63%, 99.70%, and 93.33%, respectively. Thus, a sensitive and reliable LC–MS/MS method was developed and validated for the quantification of supinoxin in rat plasma. This method was successfully applied to the evaluation of pharmacokinetic studies after single intravenous and oral administration of 1 mg kg−1 supinoxin in rats.
Authors:N. H. Hoang, N. L. Huong, S.-Y. Hong and Je Won Park
A highly sensitive analytical tool for the fast quantification of irsogladine in human plasma was developed. Cleanup using a solid-phase extraction technique is a simple method for extracting both irsogladine and lamotrigine (internal standard) spiked into human plasma. The resolvable separation of both analytes through reversed-phase high-performance liquid chromatography (HPLC) was carried out within 5 min. The HPLC–electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) method, which was operated in a selected reaction monitoring mode specific to the target analytes, was verified for use in the quantification of irsogladine. The inter- and intra-day precision (relative standard deviation, RSD) of irsogladine spiked into quality control samples were <7%, and their accuracies were between 96.6% and 102.1%. The calibration curve for irsogladine spiked into human plasma was linear over the range from 1.8 to 100 ng mL−1 with lower limit of quantification at 1.8 ng mL−1. The established method was successfully applied for a bioequivalence study of irsogladine.
Authors:Eszter Draskovits, Barbara Németh-Borsányi, Pierre-Adrien Rivier and Anita Szabó
Agricultural utilisation is one of the most promising uses of sewage sludge in Hungary. Sewage sludge can be applied to agricultural fields in two ways: the injection of dewatered sewage sludge and the application of sewage sludge after composting. Vermicomposting is a special type of composting, where the organic residues are broken down by earthworms. The worms facilitate the decomposition process both by mixing the sludge and by physically degrading it. Earthworm species have various morphotypes requiring different habitats. Compost worms have great adaptability to extreme conditions and are capable of exploiting organic matter in a state of decomposition. Eisenia sp., Eudrilus eugeniae and Perionyx excavatus are important species for vermicomposting.
When examining the role and possibilities of vermicomposting, it is important to compare it with traditional composting methods.
The most important aspect of producing vermicompost is to ensure optimum environmental conditions for the earthworms, especially in terms of temperature, humidity and aeration, which requires constant attention.
An important feature of traditional composting is the thermophilic phase, during which the pathogenic organisms in sewage sludge are destroyed. The thermophilic phase is omitted during vermicomposting due to the thermal sensitivity of the earthworms, but the presence and activity of the earthworms results in similar sterility.
Regarding its nutrient content, vermicompost contains larger quantities of total and plant-available macroelements than conventional composts. A further advantage is the presence of the plant hormone agents excreted by earthworms.
From the environmental point of view, the ability of earthworms to accumulate heavy metals and the role of their special gut flora in the decomposition of organic pollutants could contribute to the wider use of vermicomposting to dispose of sewage sludge.
While vermicompost has many advantages, a number of obstacles need to be overcome before it can be routinely used in Hungary. Many landowners regard sewage sludge compost as hazardous waste that could contaminate their soil and crops rather than as a nutrient and soil amendment. Although numerous studies have been published on sewage sludge, the assessment of long-term effects, including the issues currently of most concern in Hungary, is still lacking.
Vermicomposting is therefore a promising, innovative technology for sewage sludge recycling. Sewage sludge and sewage sludge composts with pollutant contents greater than the limits laid down in Government Regulation 50/2001. (IV.3.) can be made suitable for agricultural use by vermicomposting.
Authors:Slavica Oljačić, Andjela Arsić, Darija Obradović, Katarina Nikolić and Danica Agbaba
The retention behavior and lipophilicity of aripiprazole and its nine impurities as well as ziprasidone and its five impurities have been examined by thin-layer chromatography using RP-18 stationary phase and different mixtures of methanol, water, and ammonia; and ethanol, water, and ammonia as the mobile phases. In both examined chromatographic systems, linear relationships were established between retention parameters and the volume fraction of the methanol and ethanol in mobile phase (r > 0.948 for methanol and r > 0.971 for ethanol). Correlation matrices obtained between experimentally obtained lipophilicity indices (RM0, m, and C0) and calculated log P values showed that, for the examined antipsychotics and their impurities, RM0 and m values are more reliable lipophilicity parameters compared to C0 values. In addition, the performed principal component analysis (PCA) has provided new information about the similarity and differences between the tested compounds as well as the experimental lipophilicity indices and calculated log P values. The experimentally obtained RM0 values and the computed molecular parameters of the examined compounds were further used for the quantitative structure—retention relationship (QSRR) study in order to determine the most important properties governing retention. The QSRR modeling was performed by use of the partial least squares regression, and predictive performances of the developed QSRR models were tested by use of the cross-validation and external test set prediction. The obtained results revealed that, apart from lipophilicity, topological descriptors and molecular weights of the tested compounds have the strongest influence on the retention behavior of the examined antipsychotics and their impurities in reversed-phase thin-layer chromatography. The predictive performance of the developed QSRR model suggests its applicability for a reliable prediction of the retention behavior of the congeners.
Authors:Łukasz Komsta, Urszula Dunin and Robert Skibiński
During our previous studies, we discovered that reversed-phase thin-layer chromatography (RP-TLC) plates can behave in typical normal-phase manner, which is not widely known nor used in the literature. Therefore, we decided to investigate this behavior more comprehensively on RP-8 plates. We used 35 model compounds, and performed chemometric screening mixture design approach for 20 popular solvents. This gave us the possibility to estimate separate retention effect for each solvent being the measure of average solvent strength. The results were compared to the previous study done on silica gel. It can be concluded that RP-8 plates can be used in typical normal-phase systems and their behavior does not differ substantially from silica gel plates.
Sample application is probably the most important and time-intensive step in high-performance thin-layer chromatography, owing to the fact that sample preparation can be reduced to a minimum. The modern automatic application devices offer a variety of application modes, which allow an exact application of small nanoliter volumes up to the application of the samples in the milliliter range. On the example of 4 parabens, the spotwise, bandwise, or area application was examined. In addition, the differences in the quantitative results were investigated when the same substance amount was applied via different volumes of respectively concentrated solutions. Another important factor in sample application is the dosage speed. This was examined for nine bandwise applications using dosage speeds between 50 and 1400 nL s−1 and three spotwise applications using speeds of 10, 20, and 50 nL s−1. A further point in the investigations was the band length of the application, which can decisively influence the resolution and detectability. Furthermore, it was examined whether there is a difference when the substances are applied as a mixture solution or as individual solutions via so-called overspotting, which means that the individual substance solutions are applied to exactly the same position. In order to investigate matrix effects on the application form, volume, speed, and band length, a honey sample which was spiked with 5-hydroxymethylfurfural was tested under the abovementioned conditions.
Thin-layer chromatography (TLC) is a simple, cost-effective, and robust analytical technique, very useful in various different application fields like, e.g., fast screening and quality control of pharmaceuticals, phytochemicals, and alimentary products. Its versatility to a large extent owes to the seminal works of the late Professor Edward Soczewiński and, in the first instance, to the rationale implemented to liquid chromatography by the Soczewiński—Wachtmeister and the Snyder—Soczewiński equations. Among the most challenging applications of TLC, one can name the enantioresolution of the racemic and scalemic mixtures and a statement that the chiral TLC in this particular respect outperforms the instrumentally more advanced chromatographic techniques is far from being an exaggeration. In the course of the past ca. 12 years, the authors of this paper have extensively investigated — mostly by means of chiral TLC — the enantioresolution of the low molecular weight carboxylic acids, to discover by sheer luck that all of them spontaneously undergo an oscillatory process, i.e., chiral conversion. Later, they collected abundant experimental evidence that the oscillatory chiral conversion is accompanied by the oscillatory condensation. Herewith, we highlight applications of the chiral TLC to demonstrate the dynamic phenomenon of the spontaneous oscillatory chiral conversion with the low molecular weight carboxylic acids.
Salicylic acid and its derivatives, such as acetylsalicylic acid, are commonly used non-steroidal anti-inflammatory agents. In the present work, different sorbents as the stationary phases and also various manners of detection using well known and new visualizing reagents have been tested for the separation and detection of the two compounds by adsorption and also partition thin-layer chromatography (TLC) with densitometry. Densitometric and spectrodensitometric analysis was used to evaluate the detectability of the examined compounds using the newly developed TLC procedures. Of all the applied manners of detection, the most universal to distinguish acetylsalicylic acid from salicylic acid in normal- as well as in reversed-phase TLC system is the use of methanolic solution of FeCl3 and CoCl2. The densitograms obtained under these conditions show symmetric and also by adsorption well separated TLC peaks of both compounds. The shape of all absorption bands of acetylsalicylic acid (ASA) and 2-hydroxybenzoic acid (SA) recorded using a scanning densitometer in the range of 200–700 nm is regular. In the case of adsorption TLC performed on chromatographic plates precoated with silica gel 60 F254 and silica gel 60, a very effective spray reagent was also Janus blue. The satisfactory results of detection on silica gel 60 plates assure the use of 1% NaOH as spray reagent followed by heating at 90°C for 60 min. The results performed in the present work confirm the utility of TLC in combination with densitometry in the qualitative screening and identification of SA and ASA. The most efficient manners of detection described in this work can be helpful in the quality control of acetylsalicylic acid, e.g., in monitoring the synthesis reaction of acetylsalicylic acid from salicylic acid as well as in the quality control of ASA in commercially available products.
Authors:Hariom Nagar, Jürgen Martens and Ravi Bhushan
Enantioresolution of three active pharmaceutical ingredients (APIs), namely, atenolol, betaxolol, and orciprenaline, marketed as racemic mixture, has been achieved in a direct mode using (S)-glutamic acid as chiral additive in thin-layer chromatography. Two different approaches were adopted: (1) (S)-glutamic acid was mixed in the silica gel slurry for making thin-layer plates, or (2) it was added in the mobile phase and plain plates without any chiral additive were used. Both (1) and (2) were capable of separating enantiomers of all the three racemates, but different combinations and proportions of solvents were found successful in the two cases. Good resolution was achieved in both cases, and the results are compared for these two sets of studies among themselves and with other literature reports. Iodine was used to locate the spots of the corresponding enantiomers. The detection limits for each enantiomer were found in the range of 1.4–1.9 μg (per spot).
Authors:Eva Kranjc, Alen Albreht, Irena Vovk, Vesna Glavnik and Damjan Makuc
High-performance thin-layer chromatography (HPTLC) is a powerful separation technique which is often overlooked. In this study, we comprehensively assessed the applicability, ease, and performance of HPTLC in combination with densitometry and mass spectrometry (MS) to characterize physalins — relatively polar secondary metabolites from Physalis alkekengi L. HPTLC silica gel plates were evaluated in combination with 14 developing solvents (13 published in the literature). Bonded stationary phases (HPTLC RP-18, RP-18 W, CN F254S) were also tested. Four detection reagents (sulfuric acid, anisaldehyde, 4-dimethylaminocinnamaldehyde (DMACA), and molybdatophosphoric acid) were compared to ascertain which one is the most suitable. For all chromatographic analyses, a commercial standard physalin L and a P. alkekengi L. crude extract were used. In some cases, physalin L standard appeared as two clearly resolved bands on silica plates, but only after derivatization, where sulfuric acid reagent provided the best selectivity and sensitivity. Physalin L standard impurity was found to belong to the physalin family as confirmed by HPTLC–MS/(MS) and nuclear magnetic resonance (NMR) spectroscopy. Compared to high-performance liquid chromatography (HPLC), our HPTLC method showed extremely high sensitivity for standard impurity (ca. 4% determined by NMR) as it was clearly visible on the plate during image analysis after derivatization. Unlike (ultra)-high-performance liquid chromatography ((U)HPLC), HPTLC was also able to separate physalin L standard from its impurity. We show that (HP)TLC is a suitable chromatographic technique for the analysis of physalins and can even surpass the performance of (U)HPLC, namely, due to a wide array of different developing solvents available.
Authors:Radosław Łukasz Gwarda, Wojciech Szwerc, Monika Aletańska-Kozak, Anna Klimek-Turek, Andrzej Torbicz, Adam Chomicki, Ryszard Kocjan, Dariusz Matosiuk and Tadeusz Henryk Dzido
In our previous papers, we have mentioned some specific disruption of peptide zones shape and chromatogram distortion, when using mobile phase containing ion-pairing acids. This problem is investigated here. It concerns not only some specific separation conditions but also various separation systems with silica-based adsorbents and water—alcohol mobile phases. We show that the problem results from significant amount of metallic impurities present in the adsorbents investigated. Our results prove that these impurities strongly affect the activity of free silanol groups and thus the retention of basic or amphoteric compounds and the quality of the results obtained. The standard method of washing adsorbent layer with methanol is not effective against the impurities. Washing chromatographic plates with a solution containing an acid significantly reduces the amount of metallic impurities in the adsorbent, resulting in the reduction/elimination of these adverse effects. However, it also leads to the increase of heterogeneity of acidic groups activity and deterioration of separation system efficiency. Therefore, removing metal ions from the adsorbent may not always be advantageous. Avoiding of use of strong ion-pairing acids is also problematic and not always possible. Thus, the production of high-purity silica of homogenous activity seems to be the best and the most reliable solution of the problem described.
Authors:Lucyna Lewandowska, Elżbieta Włodarczyk, Bożena Fenert, Aleksandra Kaleniecka and Paweł K. Zarzycki
This experimental work deals with preliminary studies concerning combined planar electrophoresis–electrochromatography system involving stationary phases composed of various natural materials applied for the separation of selected colorants. The main goal of the presented experiment is to investigate the key parameters (applied voltage, electrolyte composition, pH) and separation protocol setup (stationary phase/connection strips type and geometry) enabling the fast prototyping of simple analytical systems for the fractionation and/or separation of low-molecular mass compounds. Four water-based electrolytes that are as simple as possible (without additional organic liquid components or modifiers), including boric acid 100 mM (pH = 4.3), 1:1 mixture of boric acid 90 mM and Tris base 90 mM (pH = 8.2), boric acid 100 mM titrated with NaOH 1 m (pH = 8.5), and formic acid 100 mM (pH = 2.4), were tested. As target analytes, two colorants, namely, methyl red and ponceau 4R, were selected. These dyes are commonly used as printing inks component or pH indicator (methyl red) and food products colorant (ponceau 4R). Electroseparation experiments were conducted using commercially available, temperature non-controlled, open-air electrophoresis box equipped with homemade support for the positioning of active separation layers and electrodes connection strips. Thirteen types of separation layers (working zone = 20 × 100 mm; total length with connection strips = 20 cm) including cellulose-based polymers (filtrating paper, office paper, chromatography paper, and two Japanese papers for aircraft paper models), potato starch on cellulose support, common thin-layer chromatography–high-performance thin-layer chromatography (TLC–HPTLC) glass-based plates coated with cellulose, silica gel 60W (wettable with water), silica gel RP-18W, and aluminum oxide as well as glass-based nutrient agar layers (0.5 and 1.0 mm, approximately) were investigated. Moreover, detailed preparation procedure for manufacturing starch layer on cellulose and agar glass plate supports is described. Conducted investigations have revealed substantial differences between the electrophoretic migration of target dyes within cellulose type layers and also in comparison to the remaining stationary phases studied. The best separation under the given analytical conditions (voltage applied ΔV = 500 V and run time 20 min) was observed for cellulose pre-coated TLC plate (Rs = 2.89) and starch layer on filtrating paper support (Rs = 2.26). Baseline separation of investigated dyes was observed for filtrating paper strip and agar 0.5-mm thick layer (Rs = 1.07 and 1.08, respectively). There was no electrophoretic mobility shift of the tested dyes on polyamide and RP-18W coated TLC–HPTLC plates, while dye short migration without separation was observed on the remaining layers. The obtained results, especially the recorded values of physicochemical parameters (the observed electric currents, migration distances, and peak resolution for different electrolytes, layer type, and thickness) create initial data set platform that can help in the further design of simple separation devices involving natural polymeric layers. Particularly, the described analytical protocol may be implemented for microfluidic paper-based analytical devices (μPADs) enabling fast and non-expensive separation of complex samples.
Authors:Magdalena Kręcisz, Monika Waksmundzka-Hajnos and Anna Oniszczuk
Natural compounds possess multiple biological properties, among others antioxidant activity. Phenolic compounds which exhibit excellent antiradical activity may act as scavengers of free radicals or prevent their formation. The main objective of the present research was the examination of the free radical scavenging activity of instant corn gruels with 0%, 1%, 3%, 5%, and 10% addition of dried cranberry fruits. The extrusion of gruels was carried out at varying rotational speeds of the plasticising system (80, 100, and 120 rpm). The study was developed using two methods: thin-layer chromatography–2,2-diphenyl-1-picrylhydrazyl (DPPH) test and spectrophotometric analysis. The findings confirm that the use of higher screw rotations during the extrusion entailed a higher antioxidant activity of the obtained extrudates. Also, a high activity was reported for the extruded corn gruels without additives. This, in turn, demonstrates an insignificant influence of the extrusion process on the biological value of the obtained products.
Authors:Wioleta Jesionek, Barbara Majer-Dziedzic, Györgyi Horváth, Ágnes M. Móricz and Irena M. Choma
Thin-layer chromatography—direct bioautography (TLC—DB) followed by liquid chromatography—tandem mass spectrometry (LC—MS/MS) was used for screening and tentative identification of the antibacterial constituents of Salvia officinalis L. ethanol extract. Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, that is, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, luminescence gene-tagged Pseudomonas syringae pv. maculicola, and naturally luminescent marine bacterium Aliivibrio fischeri. Eight fractions with the widest antimicrobial spectrum were detected using TLC—DB, isolated by semi-preparative TLC, and subjected to LC—MS/MS analyses. Finally, five bioactive components were tentatively identified, based on their fragmentation pattern, such as salvigenin, cirsimaritin, rosmanol, carnosic acid, and 12-O-methyl carnosic acid.
Authors:Tomasz Baj, Elwira Sieniawska, Agnieszka Ludwiczuk, Jarosław Widelski, Anna Kiełtyka-Dadasiewicz, Krystyna Skalicka-Woźniak and Kazimierz Głowniak
Essential oils obtained from Origanum species are characterized by high diversity in composition. In this work, thin-layer chromatography (TLC)—fingerprinting of Origanum species was performed for the first time and enabled the discrimination of three chemotypes (carvacrol, caryophyllene oxide, and terpineol/sabinyl). Gas chromatography—mass spectrometry and statistical analyses confirmed the presence of the mentioned chemotypes and deepened the characterization of the studied essential oils. TLC—bioautography showed that thymol and carvacrol are the main antioxidant compounds in Origanum sp. essential oils (EOs).
Authors:Irena Malinowska, Marek Studzinski and Henryk Malinowski
Magnetic field can influence some processes taking part in the solid—liquid interphase area. An excellent tool for the investigation of this phenomenon is thin-layer chromatography. In this experiment, the influence of magnetic field parallel with chromatogram development direction on retention and system efficiency was investigated. The application of superconducting magnet allowed for generating the adjustable magnetic field up to about 2 T and allowed for investigation on dependence between inductivity of the field and retention changes of the chosen polyaromatic hydrocarbons. The obtained results show that the presence of magnetic field alternates the interactions among all components of a chromatographic system. Thus, in order to predict substance retention and system efficiency changes induced by the presence of the field, more parameters than the force acting on chromatographed molecule must be taken into account.
Authors:Anna Hawrył, Mirosław Hawrył, Ryszard Świeboda and Monika Waksmundzka-Hajnos
Fourteen different Achillea species were extracted using the Soxhlet apparatus with dichloromethane and then methanol as solvents. The obtained dichloromethane and methanolic extracts were analyzed using thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) to obtain fingerprint profiles. The first chromatographic system consisted of silica gel as adsorbent, a mixture of toluene—ethyl acetate—formic acid (5:4.9:0.1, v/v) as the mobile phase and anisaldehyde as the derivatization reagent. The second adsorbent was cyano (CN) silica gel with non-aqueous (2-propanol and n-heptane [5:5, v/v], both for dichloromethane and methanolic extracts) and aqueous (methanol and water [6:4, v/v] for dichloromethane extracts and methanol and water [4:6, v/v] for methanolic extracts) mobile phases. Anisaldehyde (for dichloromethane extracts) and Naturstoff (for methanolic extracts) derivatization reagents were used. The obtained chromatograms were processed to obtain selected images using the ImageJ program. The similarity between the studied Achillea species was confirmed using the chemometric methods (principal component analysis [PCA], similarity, and distance measures).
We present a two-dimensional (2D) planar chromatographic separation method for phytoestrogenic active compounds on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic extract of liquorice (Glycyrrhiza glabra) roots contains four phytoestrogenic active compounds. As solvent, in the first direction, the mix of hexane, ethyl acetate, and acetone (45:15:10, v/v) was used, and, in the second direction, that of acetone and water (15:10, v/v) was used. After separation, a modified yeast estrogen screen (YES) test was applied, using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducing the reporter gene lacZ which encodes the enzyme β-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside). The enzyme can also hydrolyse X-β-Gal (5-bromo-4-chloro-3-indoxyl-β-d-galactopyranosid) into β-galactose and 5-bromo-4-chloro-3-indoxyl. The indoxyl compound is oxidized by oxygen forming the deep-blue dye 5,5β-dibromo-4,4β-dichloro-indigo which allows to detect phytoestrogenic activity more specific in the presence of native fluorescing compounds.
Authors:Qiang Ren, Tianrui Xia, Xian-Gao Quan, Lin Ding and Hui-Yun Wang
Scutellaria barbata D. Don has been used as a traditional Chinese medicine for antitumor and anti-inflammatory. However, there were just a few investigations about S. barbata D. Don according to bioactivity-directed isolation and online identification for the chemical constituents. In this work, eight compounds were isolated from S. barbata D. Don. The three flavonoids indicated the cytotoxic activity against human leukemic Reh cell lines. In addition, the constituents of S. barbata D. Don were further characterized and identified by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The UHPLC- Q-TOF-MS method was in negative ion mode. HPLC separation was performed on a Tosoh TSK gel ODS-100V (4.6 × 150 mm, 3.0 μm) column by gradient elution using water containing 0.3% formic acid and acetonitrile as mobile phase at a flow rate of 0.8 mL min−1. A total of 18 compounds, including 4 phenolic acids and 14 flavonoids were tentatively characterized and identified by means of the retention time, accurate mass, and characteristic fragment ions.
Authors:Zhongming Han, Miaomiao Xu, Yunhe Wang, Limin Yang and Mei Han
Ionic liquid-based ultrasonic-assisted extraction (ILUAE) was successfully applied to the extraction of the four chromones (prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside, and sec-O-glucosylhamaudol) from Saposhnikovia divaricata (Radix Saposhnikoviae) for the first time. A series of l-alkyl-3-methylimidazolium ILs differing in anion and cation compositions was evaluated for extraction efficiency, and [C3MIM]Br was selected as the optimal solvent. In addition, ultrasound extraction parameters were optimized, and the chromones were directly quantified and analyzed by rapid resolution liquid chromatography-electrospray ionization/mass spectrometry (RRLC-ESI/MS). The optimal conditions were as follows: 0.4 M concentration of [C3MIM]Br, 20:1 solvent to solid ratio, and ultrasonic time, temperature, and frequency of 5 min, 40 °C, and 50 kHz, respectively. This approach obtained the highest extraction yield of 10.188 ± 0.473 mg g−1 for total chromones. Compared with regular UAE, the proposed approach exhibited a higher efficiency (61.56% increase) and shorter extraction time (nine times shorter). Also, ILUAE was an efficient, rapid, and simple sample preparation technique for extraction of chromones, and the established RRLC-DAD method could serve as a rapid and effective technique for extracting chromones from Radix Saposhnikoviae.
Continuous-flow processing in the manufacturing of modern biotherapeutics represents a great potential and could significantly improve productivity and product quality as well as reduce operating costs. Microfluidic perfusion systems are not only capable for producing therapeutic proteins but also suitable for organ-on-a-chip based drug testing and toxicology studies. Integrating modular unit operations for protein purification in the microfluidic cell culture device can lead to point-of-care therapeutic protein production. The multi-organ microfluidic platforms that integrate several organ-on-a-chip microfluidic units will help in preclinical testing of drug substances and toxicological studies by producing highly reliable preclinical pharmacokinetic data. In this perspective, the current state of the art and future trends of continuous flow systems are summarized for biopharmaceutical production and organ-on-a-chip drug testing.
Authors:Daniel Blanco-Ania and Floris P. J. T. Rutjes
While continuous-flow chemistry is steadily increasing its footprint in academic research and in the manufacturing of pharmaceutical intermediates and fine chemicals, the attention for flow chemistry in educational programs is on average rather limited. This account is meant to provide a personal overview of the possibilities to address the involvement of flow chemistry in the various stages of chemical education.
Authors:K. Wróblewski, A. Petruczynik, B. Buszewski, M. Szultka-Młyńska, H. Karakuła-Juchnowicz and M. Waksmundzka-Hajnos
Vortioxetine is a new drug against major depressive disorder with high affinity for a range of different serotonergic targets in the central nervous system. Therapeutic drug monitoring is an important tool for the clinical management of patients receiving a pharmacotherapy, particularly in psychiatry. For this reason, determination of drug concentration in biological fluids is important for a rational dosage of drugs. Rapid and reliable analytical assays are also required to detect and identify drugs of toxicological importance. For analysis of vortioxetine by high-performance liquid chromatography (HPLC), no procedures for its determination in saliva have been reported and there are only a few ones for its determination in serum. A sensitive and selective highperformance liquid chromatography with diode array detector (HPLC-DAD) or mass spectrometer (HPLC-MS) method was developed for the fast quantification of vortioxetine in human saliva and serum. The determination was performed on a Synergi Polar RP column in isocratic mode under the optimal mobile phase containing 70% methanol, 20% acetate buffer at pH 3.5, 10% double distilled water, and 0.025 M L−1 diethylamine.
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of doxazosin mesylate (DOX) and finasteride (FIN) in bulk powders and pharmaceutical formulations. The compounds were separated on a Pinnacle II C18 column (250 × 4.6 mm i.d.; particle size, 5 μm) with an isocratic mobile phase at a flow rate of 1.0 mL min−1. The mobile phase was a mixture of 25 mM ammonium acetate and acetonitrile in the ratio of 50:50 %v/v. The pH of the buffer was adjusted to 4.0 ± 0.05 with glacial acetic acid. The detection was performed at 230 nm. The total chromatographic analysis time per sample was 15 min with DOX and FIN eluting at 3.9 and 7.2 min, respectively. The accuracy, precision, specificity, linearity, and sensitivity of the method were validated according to the International Conference on Harmonization (ICH) guidelines. The calibration plots were linear (r2 > 0.999) over the concentration range 24.25–291.0 μg mL−1 and 122.5–1470.0 μg mL−1 for DOX and FIN, respectively. The method was used for the simultaneous determination of DOX and FIN in capsules.
Rhizome extracts of Hedychium coronarium are widely used as phytotherapeutics. As of date, there is no documented study on the standardization of H. coronarium extract, and the following research is an effort in this direction. Coronarin D is an important bioactive compound present in H. coronarium which shows chemopreventive activity against cancer. H. coronarium extracts were assessed for coronarin D content for the first time. The extraction was checked using different solvents: n-hexane, acetone, and methanol. Coronarin D was separated on silica gel 60F254 high-performance thin-layer chromatography (HPTLC) plates by isocratic gradient method using n-hexane-ethyl acetate (80:20 v/v) as mobile phase. Densitometric quantification was performed at 231 nm in absorption mode. This method gave a well-defined peak at Rf 0.20 corresponding to coronarin D. The method was validated using International Conference on Harmonization (ICH) guidelines in terms of precision, repeatability, and accuracy. Linearity range of coronarin D was 200–1000 ng spot−1 with a correlation coefficient of R2 ± SD = 0.9987 ± 2.62% in the concentration range of 200–1000 ng spot−1. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 35 and 115 ng, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels, and the average percentage recovery was found to be 98.22 % for coronarin D. Among the different solvents, acetone produced maximum extraction efficiency of coronarin D. The proposed HPTLC method can be applied for robust identification and quantitative determination of coronarin D in H. coronarium extracts.
Authors:Biljana Petanovska-Ilievska, Lenche Velkoska-Markovska and Mirjana S. Jankulovska
Reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of sodium benzoate and potassium sorbate in beverages was developed using high speed column. The simple and rapid reverse-phase method for quantitative determination of both preservatives was established on LiChroCART® Purospher STAR RP-18e (30 mm × 4 mm; 3 μm) column, mobile phase consisted of acetonitrile-phosphate buffer (pH = 3.5) in volume ratio of 8:92 (v/v), flow rate of 1 mL min−1, ultraviolet (UV) detection at 195 nm for sodium benzoate and 260 nm for potassium sorbate, and constant column temperature at 25 °C. Linearity, precision, accuracy, limit of quantification (LOQ), and limit of detection (LOD) were tested for method validation. Linearity range for sodium benzoate was 6.04–200.27 mg L−1 (R2 = 0.999) while, for potassium benzoate (R2 = 0.999), 12.19–406.36 mg L−1. The RSD values ≤1.03% demonstrate excellent intra-day precision. LOD for sodium benzoate and potassium sorbate was 0.004 and 0.003 mg L−1, while LOQ was 0.012 and 0.009 mg L−1, respectively. This method was applied for quantitative determination of investigated preservatives in beverages which were taken from Macedonian markets.
Authors:Dong-Hyeon Ko, Ki-Won Gyak and Dong-Pyo Kim
The past three decades have seen increasing progress in the integration and process diversification of microfluidic systems for use in chemistry, biochemistry, and analysis. Here we summarize recent achievements in microreaction modules and microseparation units. We look into recent developments of microreaction systems fabricated by various 3D printing techniques for chemical synthetic applications. Moreover, we take a look at the recent achievements of newly developed microseparation technologies with enhanced separation efficiency realized by adopting single or hybrid principles as well as novel device concepts. Emerging technologies of 3D printing have potential to realize a vertically stacking the microchannels and miniaturization of bulky microreaction accessories. When the advanced microreaction systems are integrated with newly developed microseparation technologies, automated synthesis of industrial compounds, such as pharmaceuticals which need multiple types of salification chemistry, will be almost completed. Many opportunities are open to developing innovative microreaction systems with these techniques that can also be highly durable under harsh conditions.
Flow chemistry has become a vibrant area for research over the past decade. This perspective is intended to capture insights on how these advances have and will continue to impact the development and commercialization of active pharmaceutical ingredients. A series of chemistry examples from a number of pharmaceutical companies will highlight the influence of flow chemistry on this industry.
Authors:Gellért Sipos, Tamás Bihari, Dorottya Milánkovich and Ferenc Darvas
For successful deep space exploration, a vast amount of chemistry-related challenges has to be overcome. In the last two decades, flow chemistry has matured enough to take the lead in performing chemical research in space. This perspective article summarizes the state of the art of space chemistry, analyzes the suitability of flow chemistry in extraterrestrial environment, and discusses some of the challenges and opportunities in space chemistry ranging from establishing an end-to-end microfactory to asteroid mining.
In this perspective article, the use of continuous flow synthesis to prepare advanced pharmaceutical intermediates in developing economies is highlighted. Case studies are presented to suggest that cost effective local manufacture of life saving drugs, may potentially be implemented to facilitate better access to drugs to the underprivileged.
Optimizing current chemical processes alone does not yield the improvements required in the fine chemical and pharmaceutical industries. At least partially, a switch from batch to continuous manufacturing is needed. Cost-, time-, and atom-efficient routes frequently demand the application of high temperatures, pressures, and concentrations, and/or the use of highly reactive reagents. These chemistries often cannot be employed in conventional reactors. Costly and long alternative synthetic routes are chosen instead. The application of continuous-flow microreactors allows to access “harsh” or “hazardous” reaction conditions and, furthermore, enables entirely new transformations.
Authors:Long Wang, Yuan-Yuan Jiang, Li Zhang, Tao Wang, Rui-Wu Yang, Chun-Bang Ding, Xiao-Li Wang and Yong-Hong Zhou
A high-performance liquid chromatography (HPLC) method has been developed for the simultaneous identification and quantification of active compounds (cryptotanshinone, dihydrotanshinone I, tanshinone IIA, tanshinone I, salvianolic acid A, salvianolic acid B, protocatechuic aldehyde, and rosmarinic acid) contained in traditional Chinese folk medicine Salvia przewalskii Maxim. The herb samples (including wild, cultivated, and yin pian) from fourteen main regions were investigated. Chromatographic separation was performed on an Agilent Eclipse XDB-C18 reserved-phase column (250 mm × 4.6 mm i.d., 5 μm) using gradient elution with water-formic acid (99.9: 0.1, v/v) and acetonitrile at a flow rate of 0.8 mL min−1, an operating temperature of 30 °C, and a wavelength of 275 nm. Similarity analysis (SA), principal component analysis (PCA), and hierarchical cluster analysis (HCA) were used to analyze the data based on fingerprints. For fingerprint analysis, 27 peaks were selected as the common peaks to evaluate the similarities among different samples. The results of SA showed that the method permits to obtain desired linearity, precision, accuracy, and recovery. All samples were divided into three categories by PCA and HCA, and the concentration of the eight bioactive compounds varied significantly from different regions. It was demonstrated that chromatographic fingerprinting by HPLC combined with the simultaneous determination of eight bioactive compounds was a helpful method for the quality control of S. przewalskii.
Authors:Huba Kalász, Attila Hunyadi, Kornélia Tekes, Rafael Dolesal and Gellért Karvaly
Blood-brain penetration of 20-hydroxyecdysone 2,3;20,22-diacetonide (20DA) has been scouted using chromatographic methods. In vivo experiments were performed by treating male Wistar rats intraperitoneally (i.p.) with a dose of 50 mg kg−1 20DA. Control experiment was done by using 20-hydroxyecdysone (20E). Definite brain penetration of 20DA was found by using high-performance liquid chromatography (HPLC), while 20E does not show this type of distribution.
Authors:Jun-ichi Yoshida, Heejin Kim and Aiichiro Nagaki
This perspective article discusses the basic concept of time control by space based on flow and micro, some examples that realized extremely fast reactions which were difficult to achieve by conventional flask chemistry, and the future of this fascinating chemistry.
There are great opportunities for innovation in the drug discovery process, particularly in the lead development phase. The traditional “design–synthesize–screen” cycle has seen little innovation as a whole despite major advances at each stage, including automated purification and synthesis as well as high throughput biological screening. It could be argued that the hit-to-lead and lead optimization processes remain slow and modular with inefficient flow of information, resulting in a loss of time and money. New flow technologies may provide a promising foundation for developing a continuous integrated small molecule optimization platform that would greatly enhance hit-to-lead and lead optimization programs. Herein, we discuss major developments in integrating synthesis, purification, screening, and machine learning into a single continuous-flow platform and provide some insight into future directions of this field.
Authors:Bartholomäus Pieber, Kerry Gilmore and Peter H. Seeberger
The way organic multistep synthesis is performed is changing due to the adoption of flow chemical techniques, which has enabled the development of improved methods to make complex molecules. The modular nature of the technique provides not only access to target molecules via linear flow approaches but also for the targeting of structural cores with single systems. This perspective article summarizes the state of the art of continuous multistep synthesis and discusses the main challenges and opportunities in this area.
Photochemistry and photoredox catalysis have witnessed a remarkable comeback in the last decade. Flow chemistry has been of pivotal importance to alleviate some of the classical obstacles associated with photochemistry. Herein, we analyze some of the most exciting features provided by photo flow chemistry as well as future challenges for the field.
Authors:Victor Sebastian, Saif A. Khan and Amol A. Kulkarni
Continuous-flow synthesis of specific functional materials is now seen as a reliable synthesis approach that gives consistent product properties. This perspective article aims to survey recent work in some of the relevant areas and to identify new domains where flow synthesis of functional materials can be better than the conventional synthesis methods. It also emphasizes the need for developing high-throughput integrated synthesis and screening systems for almost all functional materials so that laboratory-scale recipes can be transformed into reliable manufacturing processes. New areas relevant to functional materials which have remained unexplored in flow synthesis are also highlighted.
Electrosynthesis is an old method currently moving again in the focus of organic synthesis. Some limitations of conventional electrosynthesis can be overcome by the use of electrochemical flow devices. This perspective indicates where the pitfalls, where the advantages and where the challenges are in implementing flow electrosynthesis as an alternative tool for the synthetic chemist.
The current state of the art of polymer synthesis in (microstructured) continuous-flow reactors is given, focusing on controlled/living polymerization methods that allow for precision polymer design. Emerging trends and the most notable developments are discussed. Especially, the field of multistep reactions and online monitoring are highlighted, which in combination may give access to fully automated high-throughput polymer synthesis reactors in the future.
We present a new simple thin-layer chromatographic method designed for determination of the main alkaloids of Chelidonium majus L. In this study, we used roots and herb of the plant collected in spring and autumn. The alkaloid fractions were prepared according to modified pharmacopeial procedure .
In our method, we performed two-step elution onto silica gel plates. The first eluent consisted of chloroform, methanol, and water mixed with 70:30:4 proportion. The second eluent comprised of toluene, ethyl acetate, and methanol with 83:15:2 proportion. The described thin-layer chromatography (TLC) system allows qualitative and quantitative determination of the following alkaloids: sanguinarine, chelerythrine, chelidonine, coptisine, and berberine. For determination of protopine, eluent with n-buthanol, acetic acid, and water in 15:1.5:4 proportion was investigated.
The dominant alkaloids observed in studied fractions were coptisine (1027.096 ± 13.367–287.474 ± 3.069 mg/100 g dry matter ± sdv) and chelidonine (1780.667 ± 263.522– 115.929 ± 14.694 mg/100 g dry matter ± sdv). The alkaloid detected in the least amount was chelerythrine (30.74 ± 7.526–1.143 ± 0.0651 mg/100 g dry matter ± sdv). The highest total amount of all alkaloids was determined in the fractions obtained from herbs in spring, and the lowest amount was detected in herbs autumn.
Additionally, we compared amounts of studied alkaloids in different parts of plants (aerial parts and roots). The plants were collected in spring and autumn.
Authors concluded that the presented method can be used as a valuable tool for screening studies on C. majus L.
Authors:Dariusz Szeremeta, Magdalena Knaś, Ewa Długosz and Mieczysław Sajewicz
The present research is focused on identification of volatile components of different commercial products containing raw herbs of Cistus incanus L. The dried herbal material was hydrodistilled, and the obtained essential oils were analyzed by means of gas chromatography with mass spectrometric detection. Alternatively, the headspace analysis of the volatile sample components was also performed. It was found out that the investigated samples of the C. incanus L. species show a wide variation in terms of quality and quantity of the respective essential oils, which might result in their variable biological activity also. In conclusion, a postulate for standardization of chemical composition of the raw plant material used in therapeutic preparations is formulated.