High-performance 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.

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 method: 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.

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.

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.

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.

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.

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.

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.

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%).

High-performance thin-layer chromatography and high-performance thin-layer chromatography–mass spectrometry methods for the analysis of phenolic acids

High-performance thin-layer chromatography (HPTLC) and HPTLC–mass spectrometry (MS)/(MSn) methods for analyses of phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) were developed. Separation was performed on HPTLC silica gel plates with and without fluorescent indicator (F254) in a saturated twin-trough chamber using n-hexane–ethyl acetate–formic acid (12:8:2, v/v) as the developing solvent. The developed HPTLC method is also suitable for the preliminary screening of some flavonoids (flavone, apigenin, luteolin, chrysin, quercetin, myricetin, kaempferide, kaempferol, hesperetin, naringenin, pinocembrin), although some interferences of phenolic acids with flavonoids were observed. The effect of pre-development on the HPTLC analysis of phenolic acids on the detection by densitometry and mass spectrometry was also explored. Pre-devel...

High performance thin-layer chromatography–mass spectrometry enables reliable analysis of physalins in different plant parts of Physalis alkekengi L.

We developed first HPTLC and HPTLC–MS/MS methods which enable characterization of structurally similar and complex biologically active compounds – physalins – from crude extracts of Chinese lantern (Physalis alkekengi L.). Separation on HPTLC silica gel plates developed with ethyl acetate–toluene–formic acid (7:3:0.2, v/v) enabled densitometric screening of physalins in absorption and, after post-chromatographic derivatization with sulfuric acid reagent, also in fluorescence mode. Compared to existing (U)HPLC methods, in this case TLC provides an alternative selectivity, better sensitivity and higher resolution, which was exemplified by the separation of physalin L standard and its impurity, identified as 2,3,25,27-tetrahydrophysalin A. Strong ion suppression caused by the developing solvent additive – formic acid – was efficiently solved by two successive plate pre-developments with methanol–formic acid (9:1, v/v) and methanol. This significantly improved the sensitivity of HPTLC–MS/MS method, but also required a slightly modified developing solvent ethyl acetate–toluene–formic acid (6:4:0.2, v/v). Simultaneous hyphenation of HPTLC with a triple quadrupole and an ion trap mass analyzer enabled a reliable and straightforward non-targeted characterization of physalins from the same chromatographic zone (band) and determination of physalin types. The performance of developed HPTLC-densitometric and HPTLC–MS/MS methods was demonstrated by the analysis of physalins from the aqueous extracts, prepared by an optimized fast and simple extraction method under reflux. Variations in physalin profiles and abundances in different parts of P. alkekengi L. harvested at different stages of maturity were observed. This indicates that not all parts of the plant, or plant as a whole, are appropriate for specific medicinal applications. Husks are proposed as the most suitable plant part for P. alkekengi L. quality control, because they exhibited the most obvious MS2 fingerprints of physalins with minimal interferences.

High performance thin-layer chromatography–mass spectrometry of Japanese knotweed flavan-3-ols and proanthocyanidins on silica gel plates

On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol–formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MSn methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6′-O-malonyl)-hexoside).

Determination of ranolazine in tablet formulations by high-performance thin-layer chromatography—mass spectrometry using reflectance scanning densitometry

A new quantitative densitometric high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the analysis of ranolazine (RZ) both in bulk and formulations. RZ was separated and identified on silica gel 60 F254 HPTLC plates with butanol—acetic acid—water (6:2:2 v/v) as the mobile phase. Densitometric quantification was performed at λ = 270 nm by reflectance scanning which facilitated well-resolved band for the main drug (RF 0.56 ± 0.02). Response to RZ was a linear function of concentration in the range of 100–400 ng, with a correlation coefficient, slope, and intercept of 0.99912 ± 0.00017, 8.684 ± 0.582, and 492.147 ± 2.67, respectively. The minimum amount of RZ that could be authentically detected and quantified was 14.90 and 49.67 ng band−1, respectively. Additionally, the peak identities as well as the purities were confirmed by mass spectrometry. The electrospray ionization (ESI+) mass spectra showed the [M + H]+ ion for RZ detected at m/z 428.3 being acquired directly ...

Correct assignment of lipophilic dye mixtures? A case study for high-performance thin-layer chromatography–mass spectrometry and performance data for the TLC–MS Interface

The TLC–MS Interface, the successor of the ChromeXtract, has been available for elution head-based coupling of high-performance thin-layer chromatography with mass spectrometry (HPTLC–MS) since 2009, and is meanwhile widespread in use, mainly for compound confirmation. Until now, quantitative performance data has not been reported in detail and thus were investigated in this study. The performance data of HPTLC–electrospray ionization (ESI)-MS via the TLC–MS Interface showed good mean precisions (%RSD, n=5) for 6 dyes in a commercially available dye mixture investigated at two different concentrations (7.3% for the 1:8 dilution, and 10.1% for the 1:16 diluted) in a selected worst case scenario. The respective mean precisions of absorbance measurements were ≤1.3%. For calibrations by HPTLC–ESI-MS, the mean determination coefficient was 0.9975 for the 6 dyes (versus 0.9997 for absorbance measurement). HPTLC–MS analysis revealed the incorrect assignment of components in two commercially available dye mixtures. Using an additional software (MassWorks) that delivered a 100 times increased mass accuracy, the proposal of molecular formulae was shown to be obtainable under certain conditions with a low resolution single quadrupole mass spectrometer and in the case of helpful information such as the double bond equivalents. This enabled the identification of the incorrectly assigned unknown dyes and clearly demonstrated the benefit of using HPTLC–MS for zone confirmation.

HPTLC-MS analysis of melamine in milk: standardization and validation

Melamine containing large amount of nitrogen may be deliberately added to milk in order to increase the protein content for economic adulteration in food stuffs. High performance thin layer chromatography-mass spectrometry (HPTLC-MS)-based method of quantification of melamine has been developed using Silica gel 60 F254s HPTLC plates, with optimized mobile phase iso-propanol/dichloromethane/water, 5:2.5:3, v/v/v in a twin trough chamber saturated (5 min) at pH 6.8. Validation was carried out by testing its specificity, linearity (1–50 μg), accuracy (95.908–98.369%), inter- and intraday precision (1.578 and 1.670%), respectively, limits of detection (2.688 ng) and quantification (8.146 ng). HPTLC in combination with MS has been found suitable for routine analysis of melamine added as adulterant to market-purchased milk samples.

BACKGROUND MASS SIGNALS IN TLC/HPTLC–ESI-MS AND PRACTICAL ADVICES FOR USE OF THE TLC-MS INTERFACE

Among the different thin-layer and high-performance thin-layer chromatography mass spectrometry (TLC/HPTLC-MS) approaches, the elution head-based TLC-MS Interface is increasingly used due to its good detectability, reliability, and versatility in its application. The influence of different solvents, either used for prewashing or chromatography, on the mass signal background was studied. It was shown that the use of acidic solvents can cause intense background signals (acid sodium clusters) and ion suppression in the analyte mass spectra. Using a mixture of methanol–water 3:1 (v/v) for plate prewashing, background mass signals could be reduced to a minimum for both ionization modes. The TLC/HPTLC-MS background mass signals obtained from different plates with the green manganese activated zinc silicate fluorescence indicator F254, and without, did not show substantial differences. As sodium is ubiquitary in solvent, tubing, glassware, adsorbent, and so forth, the sodium adduct was often formed and observed, whereby the extent of formation was different depending on the plate type. Recommendations for plate handling for use in TLC/HPTLC-MS are provided, as well as practical advices with regard to integration of the inline filter, various sorts of pressure increase, and leakage using the TLC-MS Interface.

Determination of Antidiabetic Polysaccharides ofOcimum basilicumSeeds Indigenous to Xinjiang of China by High-Performance Thin-Layer Chromatography-UV/Vis-Mass Spectrometry

Summary The seeds of Ocimum basilicum have traditionally been used for the prevention and treatment of a number of diseases in Xinjiang of China. In this study, six polysaccharide extracts were isolated from the seeds of O. basilicum by sequential extraction and purified. After methanolysis, the monosaccharide compositions of the six polysaccharide extracts were analyzed by high-performance thinlayer chromatography (HPTLC) on HPTLC plates silica gel 60 with a mixture of isopropyl acetate–ethyl acetate–methanol–water (5:4:1:0.1, v/v). After derivatization with the aniline diphenylamine o-phosphoric acid reagent, densitometric quantitation was performed by absorbance measurement at 370 or 630 nm. The results revealed that the polysaccharides in O. basilicum seeds consisted primarily of fructose (hRF 80), glucuronic acid (hRF 58), galacturonic acid (hRF 51), rhamnose (hRF 40), xylose (hRF 25), arabinose (hRF 18), and galactose (hR F 9). Xylose, glucuronic acid, and fructose were the three major components found and account for 45, 31, and 21%, respectively. All extracts contained uronic acids, ranged 3 to 24%. An unknown monomeric unit above glucuronic acid was characterized by mass spectrometry (MS) to be a hexuronic acid, and HPTLC–MS proved to be a well suited method for characterization of polysaccharide-based biopolymers and assignment of its monomers. The polysaccharide extracts (aqueous cold, aqueous hot, acidic, and alkaline) showed inhibitor activities of protein tyrosine phosphatase 1B in vitro with half maximal inhibitory concentration (IC50) values of 8.2, 2.2, 70.9, and 0.8 µg mL –1 , respectively. For the first time, a molecular basis was provided to explain the hypoglycemic effect of the seeds of O. basilicum that has been used as antidiabetic adjuvant in traditional Chinese medicine.