Thin-layer Chromatography Mass Spectrometry Research Articles

Quantitation of Usnic acid from three Usnea species, its commercial formulations Using High-Performance Thin-Layer Chromatography-Mass Spectrometry (HPTLC-MS) and their metabolite profiling using UPLC-QTof-MSE.

The genus Usnea (Parmeliaceae; lichenized Ascomycetes) is pale grayish-green fruticose lichens which grow as leafless mini-shrubs and comprise about 360 species. Most of the Usnea species are edible and is utilized in preparation of traditional foods as well as in medicines to combat wide range of ailments. The goal of this work was to quantify usnic acid in three Usnea spp. [Usnea ghattensis (UG), Usnea orientalis (UO) and Usnea undulata (UU)] using HPTLC-MS and chemical profiling of acetone extracts using UPLC-QTof-MSE resulted in the identification of sixteen compounds based on their MS/MS fragmentation patterns. Hyphenated techniques, HPTLC-MS and UPLC-QTof-MSE have been proposed to quantify usnic acid and analysis of metabolites in the crude extracts qualitatively. This method allowed tentative characterization of metabolites from Usnea spp. The quantification study showed the excellent linearity of the usnic acid at 0.25-1 µg/band with a correlation coefficient r 2>0.99, and LOD, LOQ was found to be 51.7 and 156.6 ng/band, respectively. Further, UPLC-QTof-MSE analysis of crude extract led identification of lichen substances through their exact molecular masses and MS/MS fragmentation studies. The present study summarizes HPTLC method for quantification of usnic acid in three different Usnea spp. Along with two herbal formulations containing Usnea spp. as the ingredient and developed method was validated as per the ICH guidelines and further UPLC-QTof-MSE analysis provides characterization of the sixteen different secondary metabolites based on their mass fragmentation studies. Rapid HPTLC method for quantification of usnic acid in three different Usnea spp. along with two herbal formulations and metabolite profiling using UPLC-QTof-MSE.

Secondary metabolites with antimicrobial activity produced by thermophilic bacteria from a high-altitude hydrothermal system.

Thermophilic microorganisms possess several adaptations to thrive in high temperature, which is reflected as biosynthesis of proteins and thermostable molecules, isolation and culture represent a great methodological challenge, therefore High throughput sequencing enables screening of the whole bacterial genome for functional potential, providing rapid and cost-effective information to guide targeted cultures for the identification and characterization of novel natural products. In this study, we isolated two thermophilic bacterial strains corresponding to Bacillus LB7 and Streptomyces LB8, from the microbial mats in the Atacama Desert. By combining genome mining, targeted cultures and biochemical characterization, we aimed to identify their capacity to synthesize bioactive compounds with antimicrobial properties. Additionally, we determined the capability to produce bioactive compounds under controlled in vitro assays and detected by determining their masses by Thin-Layer Chromatography/Mass Spectrometry (TLC/MS). Overall, both isolates can produce antimicrobial (e.g., Myxalamide C by-product) and antioxidants (e.g. Dihydroxymandelic Acid, Amide biotine and Flavone by-products) compounds. Bacillus LB7 strain possesses a more diverse repertoire with 51.95% of total metabolites unmatched, while Streptomyces LB8 favors mainly antioxidants, but has over 70% of unclassified compounds, highlighting the necessity to study and elucidate the structure of novel compounds. Based on these results, we postulate that the uncultured or rare cultured thermophiles inhabiting high-altitude hydrothermal ecosystems in the Atacama Desert offer a promising opportunity to the study of novel microbial bioactive compounds.

Investigating the Anti-adipogenic Potential of Xanthium strumarium Linn. Leaves Fractions: HPTLC-MS Characterization, Enzymatic Analysis, and 3T3-L1 Adipocyte Differentiation Assay in Oxidative Stress-modulated Adipogenesis Cascade

Background Molecular pathogenesis of obesity is initiated by cellular oxidative stress along with increased adipogenesis. For the search of better alternatives of synthetic molecules in terms of their adverse events, naturally, existing compounds are now a research focus. In this context, Xanthium strumarium ( X. strumarium) Linn. exhibited anti-diabetic activities and proved to have efficacy in neutralizing very near pathological targets to obesity. Purpose This study is aimed toward bioactivity-guided fraction isolation of methanolic leaves extract, where bioactivity refers to anti-adipogenesis and anti-oxidant efficacy in in vitro enzymatic and three-day transfer, inoculum 3×105 cells (3T3-L1) adipocyte differentiation assay. Materials and Methods Collection and drying of the raw leaves followed by cold extraction in methanol and flash chromatography for bioactive fraction isolation. 3T3-L1 cell line was utilized for adipocyte differentiation assay of those and on the other hand in vitro pancreatic lipase, α-glucosidase, and α-amylase enzymatic assays were analyzed. Based on exhibited activities, selected fractions are further characterized by high-performance thin-layer chromatography-mass spectrometry (HPTLC-MS). Results The most active fractions from these assays underwent characterization via HPTLC-MS. Fractions 2 and 3 exhibited potent activities in all enzymatic assays (α-glucosidase IC50: 2.48 ± 0.015, 2.84 ± 0.030 µg/mL; α-amylase IC50: 1.98 ± 0.050, 1.79 ± 0.045 µg/mL; pancreatic lipase IC50: 3.16 ± 0.030, 3.18 ± 0.040 µg/mL) and displayed significant inhibition of adipocyte differentiation in 3T3-L1 cell line. These fractions further identified through HPTLC-MS, contained compounds like β-sitosterol and quercetin, affirming their potential bioactivity. Conclusion The study underscores the potential of X. strumarium Linn. fractions as promising natural alternatives for combating obesity-related pathways, highlighting their significance in developing future anti-obesity therapeutics.

Application of high-performance thin layer chromatography mass spectrometry (HPTLC MS) in foodomics authenticity

BackgroundChemometric analysis is also a powerful tool that can be used in the preliminary stages of analytical methods optimization, but is also efficient in data processing. These new approaches may be the key to the analysis of proteins, carbohydrate, sphignolipids, polyphenols in different food components. Although there are challenges in identifying and annotating those compounds due to the limited availability of analytical standards and structural diversity. Scope and approachChromatograms are a useful tool for learning more about the chemical make-up of the food being tested. This information can occasionally be implicit or presented in a subtle way. Therefore, to extract it, chemometric tools and data mining techniques are needed. A chromatogram's fingerprint allows for the possibility of performing both identity and quality testing on food. This viewpoint aims to offer a current assessment of chromatographic fingerprinting technique in the area of food authentication. Additionally, this methodology's limitations, absence from official analytical methods, and future directions are discussed. Novel techniques have been employed in the past few decades, ranging from high-pressure thin liquid chromatography (HPTLC) to mass spectrometry (MS) and other spectroscopic methods. In the last decade research developments, and the application of analytical methods in qualitative and quantitative studies of food components and their adulterants is reviewed in the present work. High-pressure Thin liquid chromatography (HPTLC) is hyphenated with high resolution mass spectrometry is particularly addressed due to its applicability in the targeted/untargeted metabolomic analysis of all food components. Key findings and conclusionsIn the current study, the HPTLC-MS technique was used to examine food components and food adulteration. The HPTLC-MS hyphenated techniques were found to be an extremely helpful method for the detection of food adulterants as well as the various food components, such as protein, carbohydrates, lipids, essential oils, and flavonoids, which were found to be effective as medicinally.

A validated bioanalytical method for the simultaneous estimation of telmisartan and gallic acid in rat plasma samples by high-performance thin-layer chromatography-mass spectrometry: Application to an oral pharmacokinetic study.

A novel and cost-effective high-performance thin-layer chromatography (HPTLC) method, combined with densitometric quantification, was developed for the biomedical analysis of telmisartan (TEL) and gallic acid (GA). Recent research indicates that when used in combination, these compounds offer improved therapeutic efficacy for the treatment of cardiovascular diseases with reduced side effects. The study focused on the simultaneous quantification and pharmacokinetic analysis of drugs in rat plasma. The separation was conducted using HPTLC silica gel 60 F254 plates with dimensions of 20 × 10 cm and a thickness of 0.2 mm. The mobile phase used for separation consisted of a mixture of ethyl acetate, methanol, chloroform, and acetic acid in the ratio of 4:2:2:0.2 (v/v). GA and TEL were analyzed using ultraviolet detection at specific wavelengths, with GA at 280 nm and TEL at 296 nm. Peak purity was assessed through spectral correlation analysis using Vision CATS software. The method underwent validation following the guidelines of the US Food and Drug Administration (US FDA). Calibration plots demonstrated linearity in the concentration range of 200-1200 ng/spot, with high correlation coefficients (R2 ). The retention factors (Rf ) were 0.67 for TEL and 0.60 for GA. The identity of the separated compounds was further confirmed using MS, with GA having a mass-to-charge ratio (m/z) of 168.9 in negative mode and TEL with m/z 515.2 in positive mode. In the pharmacokinetic study, the maximum peak plasma concentration (Cmax ) for GA was 899.7 ng/mL, and for TEL, it was 1013 ng/mL. The time to reach maximum concentration (Tmax ) was 2 h for GA and 6 h for TEL. This simultaneous qualitative and quantitative determination of the drugs in an oral pharmacokinetic study involving Wistar rats can serve as a valuable tool for future investigations into pharmacokinetic interactions, quality control, and routine analysis of these drugs, both in their pure forms and in novel formulations.

Green analytical chemistry-driven response surface modeling to stability-indicating chromatographic method for estimation of cilnidipine and application of high-performance thin-layer chromatography-mass spectrometry for characterization of degradation products.

Cilnidipine is a calcium channel blocker that is used to treat cardiac diseases such as angina and high blood pressure. Several column and planar chromatographic methods for estimating cilnidipine in pharmaceutical dosage forms have been documented. However, these methoddevelopments have been carried out employing organic solvents such as acetonitrile, methanol, toluene, chloroform, and others as mobile phase components or as sample pretreatment diluents. These organic solvents are neurotoxic and teratogenic to humans and aquatic animals, according to International Council for Harmonization Q3C (R8) recommendations. According to the green analytical chemistry approach, such organic solvents should be reduced or removed during the development of chromatographic methods for environmental protection and the safety of human and aquatic animal life. As a result, the stability-indicating chromatographic estimation of cilnidipine was performed utilizing less toxic organic solvents. To prevent organic solvent waste during method development, mobile-phase optimization was performed using the design of experiment-based response surface modeling. Cilnidipine has been subjected to hydrolysis, oxidation, photolysis, and dry-heat decomposition to determine its stability. The greenness profiles of the suggested and published chromatographic methods were examined using the national environment method index, analytical greenness calculator, green analytical procedure index software, and eco-scale assessment tool.

Validated simultaneous high-performance thin-layer chromatography‒mass spectrometry method for analysis of citalopram prochlorperazine, midazolam, and chlorodiazepoxide in urine for forensic analysis

Drug breakthroughs and development have been a great blessing to mankind. Problem arises when these are used in non-medical context such as use for recreational purposes, using drugs outside the label directions, and use with malicious intent as found in homicidal cases. Young generations get trapped into vicious cycle of drug addiction that has deleterious effects on their mind and body. Drugs including psychomotor drugs, opioids, narcotics, inhalants, stimulants, and central nervous system depressants are easily accessible. Forensic science plays a crucial role in investigation pertaining to drug abuse and overdose. Complex biological and non-biological matrices are submitted to forensic science laboratories for analysis and interpretation of the effects of drugs/toxicants/poisons on human body and environment. The aim of this study was to develop a method for the determination of various prescription drugs in the urine using high-performance thin-layer chromatography‒mass spectrometry (HPTLC‒MS) technique. New combination of solvent systems was tried for the simultaneous detection of frequently used prescription drugs in urine. The method was developed and validated using HPTLC‒MS for the quantitative estimation of benzodiazepines, anti-depressants, and tranquilizers in urine using a common solvent system for all the drugs. Mobile phase cyclohexane‒toluene‒diethylamine (7:1.5:1.5, V/V) with one drop strong ammonia solution was used as mobile phase. Method validation was carried out according to the International Council of Harmonization guidelines. The present HPTLC‒MS method being simple, sensitive, precise, and accurate can be used for the detection and quantitative estimation of benzodiazepines, anti-depressants, and tranquilizers in biological specimens.

Validated High-Performance Thin-Layer Chromatography-Mass Spectrometry Method and Stability Study of Linalool in the Volatile Oil of the Rhizomes of Homalomena aromatica Schott.

Homalomena aromatica is a herb of tremendous ethnomedicinal importance to various communities residing in northeast India. In this study, a high-performance thin-layer chromatography-based densitometric method was developed for identification, quantification and stability study of linalool. Mass spectrometry was hyphenated to HPTLC for streamlining the method. The stability of linalool was studied by analyzing the effect of acid, base, UV, sunlight, thermal stress and H2O2 on linalool. The chromatographic plates were developed to a height of 70mm in toluene:ethyl acetate solvent system at a ratio of 9.5:0.5 and visualized with p-anisaldehyde reagent. The developed method was found to be precise, accurate and reproducible according to International Conference on Harmonization guidelines, and compact bands of linalool were observed at Rf of 0.351 ± 0.001. The content of linalool in the volatile oil of H. aromatica was found to be 58% v/v. By application of the hyphenated MS technique, linalool was identified at m/z 137, (M + H)+. It was observed that acidic pH has the highest effect on linalool with a percentage degradation of 65. The developed method can be used in the analysis and quality control of herbal materials and volatile oils containing linalool and quality control of rhizomes of H. aromatica.

Evaluation of cardioprotective potential of isolated swerchirin against the isoproterenol-induced cardiotoxicity in wistar albino rats

Background: Cardiotoxicity is one of the emerging health-care issues worldwide and mortality due to this is increasing exponentially. Oxidative stress, myocardial inflammation, and damage to the cardiac membrane are major causative attributes for this toxicity. Isoproterenol (ISO)-induced cardiotoxicity is a well-established and accepted model to estimate innovative cardioprotective agents, as it exhibits several morphological and biochemical aberrations in the myocardium of rat that is comparable with those detected in clinical practice. Objectives: In the present study, we explored the cardioprotective effect of swerchirin (SW) extracted from Swertia chirayita in the ISO model. Materials and Methods: SW was isolated from S. chirayita, characterized by high-performance thin-layer chromatography-mass spectrometry, 1H-nuclear magnetic resonance (NMR), 13C-NMR, and high-performance liquid chromatography techniques. The in-vitro study was performed for 2,2-Diphenyl-1-picrylhydrazyl and nitric oxide scavenging activity to explore the antioxidant capacity. In silico study was performed to ascertain the binding affinity of SW with antioxidant enzymes. The in-vivo study was performed to explore the cardioprotective activity in the ISO-induced cardiotoxic model. Results: The in vitro studies showed the significant antioxidant potential of SW whereas the in silico study revealed its effective binding into the catalytic pocket domain of superoxide dismutase and catalase. The in vivo study showed a significant improvement in antioxidant enzymes and a reduction in serum glutamic-oxaloacetate transaminase and lipid profile. Further, SW also significantly reversed the histopathological aberrations in a dose-dependent manner. Conclusion: Findings of the study showed a significant cardioprotective effect of SW against ISO-induced cardiac damage.

Thin layer chromatography-mass spectrometry bioautographic identification of free radical scavenging compounds and metabolomic profile of Carica papaya linn. fruit and seeds using high-performance thin-layer chromatography, gas chromatography-mass spectrometry and ultra-performance liquid chromatography-mass spectrometry

Objectives: Carica papaya Linn. a member of the Caricaceae family, is a tropical fruit, rich in various secondary metabolites owing to its antioxidant, anti-inflammatory, anti-diabetic, and antihelmintic properties. The study was carried out to investigate the metabolic profiling and free radical scavenging compounds in C. papaya fruit's pulp, peel and seeds through thin-layer chromatography-mass spectrometry (TLC-MS) bioautography. Methodology: Aqueous, hydroalcoholic and alcoholic extracts of fruit pulp and peel were prepared along with the hydroalcoholic and hexane extract of seeds. These were subjected to total phenol, flavonoid and free radical scavenging estimation. Qualitative and quantitative high-performance thin-layer chromatography analysis of the best extracts was performed followed by TLC-MS bio autography assay for the detection of free radical scavenging compounds. Results: C. papaya peel was noted to contain the highest phenol and flavonoid content, but the seeds showed better free radical scavenging activity. The hydroalcoholic extracts of pulp, peel, and seeds examined through TLC-bioautography showed the presence of chlorogenic acid, ellagic acid, quercetin, β-sitosterol, linoleic acid, and iso-oleic acid as potent free radical scavenging compounds. Liquid chromatography-mass spectrometry analysis showed the presence of β-carotene, lycopene, and β-cryptoxanthin in pulp and peel along with other carotenoids and benzyl isothiocyanate, linoleic acid, oleic acid, and methyl palmitate were the major compounds detected in seeds through gas chromatography-mass spectrometry analysis. Conclusion: This study has revealed that C. papaya fruit and seeds possess potent free radical scavenging compounds. Seeds which make up the waste material may be utilized in cosmetic industries as they signify rich antioxidants.

HPTLC–MS method for the determination of benzodiazepines in urine samples

Misuse and abuse of prescription drugs has become a global health problem in the last few years as the illicit drug cartels have grown exponentially. The non-medical use of prescribed medications, such as benzodiazepines, synthetic opioids, stimulants and tranquilizers, has increased rapidly due to their easy availability. Nowadays benzodiazepines are not only misused/abused, but also play a significant part in facilitating various crimes such as robbery, date rapes, sexual harassment, which has made its forensic examination important and critical. Considering the gravity of these crimes, an attempt has been made to develop and validate simultaneous quantification method for seven benzodiazepines (clonazepam, lorazepam, alprazolam, diazepam, flurazepam, oxazepam and nitrazepam) using high-performance thin-layer chromatography–mass spectrometry (HPTLC–MS) from urine. Quantitative analysis of clonazepam, lorazepam, alprazolam, diazepam, flurazepam, oxazepam and nitrazepam was carried out using HPTLC–MS. MS operating in selective ion-monitoring mode was attempted for the accurate identification of the drugs under study. Small-volume liquid extraction technique using diethyl ether–chloroform (80:20, v/v) was used for the extraction of urine. Pre-coated HPTLC (silica gel G 60 F254) plates were developed using the mobile-phase chloroform–glacial acetic acid (9:1, v/v). The screening of HPTLC plates was carried out by ultraviolet light, and the m/z ratio of drugs was obtained by extracting spots from plates using a TLC–MS interface. Method validation was carried out according to the International Council for Harmonisation guidelines. The current HPTLC–MS method is simple, sensitive, precise and accurate, and it can be used for the detection and quantitative estimation of benzodiazepines in urine.

New HPTLC methods for analysis of major bioactive compounds in mate (Ilex paraguariensis) tea

New HPTLC (high-performance thin-layer chromatography) methods to evaluate major bioactive compounds in mate (Ilex paraguariensis) tea were developed. Identification of compounds was based on HPTLC-MS (high-performance thin-layer chromatography–mass spectrometry), parallel to other techniques. Quantitative evaluation of the main methylxanthines and phenolic compounds in this species was accomplished by two HPTLC methods which can be performed consecutively on one single plate. A solvent system for the separation of the identified saponins is also described for qualitative evaluations. The methods for quantification were validated in terms of: robustness; LOD and LOQ (limits of detection and quantification); repeatability; intra-day and inter-day variation; ruggedness; and accuracy. The methods have proven to be suitable for evaluation of mate tea aqueous and methanolic extracts, with the advantages of: high-throughput; reduced need of plates and application procedures; and high sensitivity – low LODs and LOQs were achieved. Aqueous extracts of nineteen mate teas of different type, geographical origin and age were evaluated in terms of methylxanthines, phenolic compounds and pH. By means of principal component analysis, distinctive characteristics between mate teas could be observed.

Open-source add-on kit for automation of zone elution in planar chromatography.

High-throughput capacity, maximal efficiency and automation are missing key features for thin-layer chromatography/high-performance thin-layer chromatography-mass spectrometry (TLC/HPTLC-MS) hyphenations. So far, commercial interfaces have been operated without an automated positioning system. Hence, a 3D-printed, fully automated, open-source add-on user interface was built for elution-head-based TLC/HPTLC-MS. Precise plate movement, batch-wise zone elution, and cleaning processes were automated and synchronized with MS data acquisition. The elution head cleaning was made adjustable in intensity and interval, and remaining elution solvent or particles were thoroughly blown out of the MS transfer line. An adjustable short gas beam across the elution zone was integrated to avoid contamination or leakage by released layer particles, or zone distortion by residual eluent flow when lifting the elution head. By clicking on zones on the chromatogram, these were selected for consecutive automated positioning below the elution head, sealing and elution. Mean spatial deviations of the positioning on 294 target zones were determined to be 160 μm for track-wise and 190 μm for randomized zone positioning order. Reproducibility of the elution of butyl paraben zones (5.4%, 10 ng/band, n = 70) and its quantitative performance (R2 = 0.992-0.999, 5-50 ng/band) were proven. The stand-alone electronic system design and the compact footprint were achieved by a minimalistic positioning system and internally installed valves. The open-source OC_manager software opened new perspectives in terms of combining MS data evaluation with previous HPTLC results. The successfully demonstrated TLC/HPTLC-MS automation presents a highly efficient, user-independent hyphenation for a broad range of application fields.

Effect-directed profiling and identification of bioactive metabolites from field, in vitro-grown and acclimatized Musa spp. accessions using high-performance thin-layer chromatography-mass spectrometry

Bananas and plantains (Musa spp.) are used as nutritious foods, and at the same time, are a source of phytoconstituents for the pharmaceutical industry. As biological activities of especially the pulp and peel of Musa spp. have been documented, this study investigated the variation in the secondary metabolite profiles of the leaves from field, in vitro-grown and acclimatized accessions. The genetic fidelity of the diverse accessions was assessed using diversity array technology sequencing. It showed that the in vitro-grown accessions were true-to-type with the field samples. The antioxidant and anticholinesterase activities of the samples from different culture systems (field and in vitro) were evaluated by UV-spectrophotometry and compared to high-performance thin-layer chromatography-effect-directed analysis (HPTLC-EDA). The latter was applied for the first time for effect-directed profiling of the polar and medium polar sample components via different biochemical and biological assays. Compound zones showed acetyl-/butylrylcholinesterase inhibition (zones 1–4), α-/β-glucosidase inhibition (zones 1 and 2) as well as antioxidative (zones 1–3) and antimicrobial (zone 4) activities. Structures were preliminary assigned by HPTLC-HRMS. The HPTLC was effective for bioactivity-guided characterization of the bioactive constituents in Musa spp. accessions. Accumulation of useful metabolites, especially compounds with antioxidant and anticholinesterase properties, was higher in samples from in vitro system. This validated the use of plant tissue culturing as an alternative method for large scale production of plant material and supply of bioactive constituents.

Influence of microorganism and plant oils on the structure of mannosylerythritol lipid (MEL) biosurfactants revealed by a novel thin layer chromatography mass spectrometry method.

Mannosylerythritol lipids (MEL) are microbial glycolipid biosurfactants with great potential for application in cosmetics and household detergents. In current biotechnological processes, they are produced by basidiomycetous fungi, the Ustilaginaceae, as a complex mixture of different chemical structures. It was the aim of this paper to study the influence of producer organisms and substrates on the resulting MEL structures with a novel high-resolution HPTLC-MALDI-TOF method. Given the seven different microbes and four plant oils, our analysis revealed that the product concentrations varied strongly between organisms, while they were similar for the different substrates. Coconut oil presented an exception, since only one organism was able to synthesize MEL from this substrate in considerable yields. Analysis by GC-FID further showed that the chain length pattern of hydrophobic fatty acid side-chains was very specific for individual organisms, while substrates had only a minor influence on the chain length. Our novel HPTLC-MALDI-TOF combination method finally demonstrated the presence of multiple MEL sub-variants with differing acetylation and fatty acid chain lengths. It also revealed the production of a more hydrophilic biosurfactant mannosylmannitol lipid(MML) as a side-product in certain fungi. Overall, it was concluded that the pattern of produced biosurfactant structures are mainly governed by producer organisms rather than substrates.

Densitometry and indirect normal‐phase HPTLC–ESI–MS for separation and quantitation of drugs and their glucuronide metabolites from plasma

The present work describes novel methods using densitometry and indirect or off-line high performance thin-layer chromatography-mass spectrometry (HPTLC-MS) for the simultaneous detection and quantification of asenapine, propranolol and telmisartan and their phase II glucuronide metabolites. After chromatographic separation of the drugs and their metabolites the analytes were scraped, extracted in methanol and concentrated prior to mass spectrometric analysis. Different combinations of toluene and methanol-ethanol-n-butanol-iso-propanol were tested for analyte separation and the best results were obtained using toluene-methanol-ammonia (6.9:3.0:0.1, v/v/v) as the elution solvent. All of the drug-metabolite pairs were separated with a homologous retardation factor difference of ≥22. The conventional densitometric approach was also studied and the method performances were compared. Both of the approaches were validated following the International Conference on Harmonization guidelines, and applied to spiked human plasma samples. The major advantage of the TLC-MS approach is that it can provide much lower limits of detection (1.98-5.83 pg/band) and limit of quantitation (5.97-17.63 pg/band) with good precision (˂3.0% coefficient of variation) compared with TLC-densitometry. The proposed indirect HPTLC-MS method is simple yet effective and has tremendous potential in the separation and quantitation of drugs and their metabolites from biological samples, especially for clinical studies.

Identification of genotoxic transformation products by effect-directed analysis with high-performance thin-layer chromatography and non-target screening

The w ide spread us e of the phar maceut ical met form in for diabete s therapy has led to finding its way into many surface waters up to the μg L-1 range and subsequently into different water treatment processes. In this study, metformin was treated with hypochlorite on the laboratory scale, and the resulting transformation products were investigated with the umu assay in a microtiter plate, where a genotoxic effect was detected. For the characterization of this genotoxic effect, the sample was separated using high-performance thin-layer chromatography (HPTLC), and 29 zones over the whole retardation area were extracted from the HPTLC plate with the thin-layer chromatography—mass spectrometry (TLC—MS) Interface. Then, the umu assay was performed again with each extracted zone, such that the genotoxic effect in the sample could be assigned to a certain zone. By the measurement of this effective zone with high-performance liquid chromatography with high-resolution mass spectrometry and by performing a non-target screening, the effective substance could be identified as a cyclic dehydro-1,2,4-triazole derivate with an intense yellow color. This substance formerly was found by Armbruster et al. (Water Research, 2015), which is a major transformation product of chlorine-treated metformin.

High performance thin-layer chromatography–mass spectrometry methods on diol stationary phase for the analyses of flavan-3-ols and proanthocyanidins in invasive Japanese knotweed

We developed the first four HPTLC methods for the separation of proanthocyanidins according to degree of polymerization on HPTLC diol F254S plates. Acetonitrile, ethyl acetate, ethyl acetate–formic acid (9:0.1, v/v) and toluene–acetone–formic acid (3:6:1, v/v) were used as developing solvents and 4-dimethylaminocinnamaldehyde (DMACA) as the detection reagent. Each of these methods enables separation of standards of procyanidin dimers from procyanidin trimer (procyanidin C1) and separation of B-type dimers (procyanidins B1, B2, B3) from A-type dimers (procyanidins A1, A2). Based on these HPTLC methods we developed four new HPTLC-MS/MS methods for analyses of proanthocyanidins on HPTLC diol F254S plates and we identified B-type proanthocyanidins from monomers up to decamers in crude extracts of invasive Japanese knotweed (Fallopia japonica Houtt., Polygonum cuspidatum Sieb. & Zucc.) rhizomes. Monomers, monomer gallates, dimers, dimer gallates, dimer digallates, trimers, trimer gallates, tetramers, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes using developing solvents acetonitrile and toluene–acetone–formic acid (3:6:1, v/v). Ethyl acetate enabled separation from monomers up to hexamer gallates and ethyl acetate–formic acid (9:0.1, v/v) from monomers up to hexamers. During the five hours of stability testing of (–)-epicatechin and procyanidin B2 standards on HPTLC diol plates developed with all solvents we observed enhanced absorption at 280 nm. This was a totally unexpected phenomenon. This new discovery confirmed what we reported in our previous study on HPTLC silica gel. Enhanced absorption is influenced by the developing solvent (more than 30%), the stationary phase (up to 24%) and the light (up to 15%).

2′-Chloro-4-(1-methyl-1H-imidazol-2-yl)-2,4′-bipyridine

The compound 2′-chloro-4-(1-methyl-1H-imidazol-2-yl)-2,4′-bipyridine was obtained with a good yield by the reaction of 2-chloro-4-(1-methyl-1H-imidazol-2-yl)pyridine with (2-chloropyridin-4-yl)boronic acid and structurally characterized by nuclear magnetic resonance (1H-NMR and 13C-NMR), thin-layer chromatography–mass spectrometry (TLC–MS), HPLC, gas chromatography–mass spectrometry (GC–MS), and elemental analysis. The functionalization of the pyridine was achieved by the palladium-catalyzed Suzuki–Miyaura carbon–carbon cross-coupling reaction that afforded the target compound.

N-[4-(1-Methyl-1H-imidazol-2-yl)-2,4′-bipyridin-2′-yl]benzene-1,4-diamine

N-[4-(1-Methyl-1H-imidazol-2-yl)-2,4′-bipyridin-2′-yl]benzene-1,4-diamine was synthesized with a good yield by the reaction of 2′-chloro-4-(1-methyl-1H-imidazol-2-yl)-2,4′-bipyridine with 4-phenylenediamine. The functionalization of the pyridine was accomplished by a nucleophilic aromatic substitution (SNAr) reaction that afforded the target compound. The synthesized compound was characterized by chemical analysis, which includes nuclear magnetic resonance (NMR) (1H-NMR and 13C-NMR), Thin Layer Chromatography-Mass Spectrometry (TLC-MS), high- performance liquid chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS), and elemental analysis.