Thin-layer Chromatographic Separation Research Articles

Development of a Thin-Layer Chromatography-Enzymatic Test Combination Method for the Isolation of α-Glucosidase Inhibitors From Thymelaea hirsuta.

A rapid, easy and simple method for the isolation and purification of α-glucosidase inhibitors of the ethyl acetate extract of Thymelaea hirsuta (EaTh) by a combination of thin layer chromatography (TLC) and enzymatic test has been developed. EaTh was demonstrated previously a potent α-glucosidase inhibitory effect. In this study, we developed a simple TLC-enzymatic test (TLC/EZ) combination to isolate α-glucosidase inhibitors present in EaTh.EaTh was extracted by Soxhlet from Thymelaea hirsuta (T. hirsuta). The EaTh was separated on a silica gel column and then on a TLC plate. After TLC separation, the TLC/EZ combination method was applied. α-glucosidase inhibitors were detected directly in the TLC plate using the glucose oxidase peroxidase method (GOD-POD). A good detection of active compounds was obtained in the TLC favoring the TLC/EZ method. Active compounds were then characterized using high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. The main α-glucosidase inhibitors present in EaTh have a molecular ion [M + H]+ at m/z = 543. This proposed method is suitable for a reliable isolation and purification of α-glucosidase inhibitors present in EaTh. It could be proposed as an interesting alternative of the classical method for the isolation and purification of α-glucosidase inhibitors in plant extracts.

Implementation of different separation techniques for resolving ternary mixture of Paracetamol, Pseudoephedrine Hydrochloride and Chlorpheniramine maleate with further quantification

Abstract Due to the wide applicability of separation techniques that rely on the property of differential migration in pharmaceutical formulations analysis, different analytical strategies have been proposed to resolve mixtures of multi-components pharmaceuticals. Three separation methods were developed and validated for the simultaneous determination of Paracetamol (PAR), Pseudoephedrine HCl (PSE) and Chlorpheniramine maleate (CHP). The first method is a thin-layer chromatographic (TLC) separation, followed by densitometric measurement. The separation was carried out on aluminium sheet of silica gel 60 F254 using ethanol:chloroform:ammonia (1:7:0.4, by volume) as the mobile phase. Determination of PAR, PSE and CHP was successfully applied over the concentration ranges of 3–25 µg/band, 0.5–10 µg/band and 0.1–6 µg/band, respectively. The second method is HPLC separation that was achieved on C18 column using the mobile phase acetonitrile:phosphate buffer pH 5 (10:90, v/v) at a flow rate 1 mL min−1. PAR, PSE and CHP were determined by HPLC in concentration ranges of 5–400 μg mL−1, 2–40 μg mL−1 and 0.5–16 μg mL−1, respectively. The third method is a capillary electrophoresis (CE) separation. The electrophoretic separation was achieved using 20 mM phosphate buffer (pH 6.5) at 20 kV. The linearity was reached over concentration ranges of 30–250 μg mL−1, 5–50 μg mL−1 and 0.8–20 μg mL−1 for PAR, PSE and CHP, respectively. The developed methods were validated with respect to linearity, precision, accuracy and system suitability. The proposed methods were successfully applied for bulk powder and dosage form analysis with RSD of precision <2%. Moreover, statistical comparison with the official methods confirms the methods' validity.

New solvent systems to separate some estrogenically active compounds by high-performance thin-layer chromatography (HPTLC)

Two solvent mixtures for high-performance thin-layer chromatographic (HPTLC) separation of some compounds showing estrogenic activity in the yeast estrogen screen (YES) assay are presented. The new method, planar yeast estrogen screen (pYES) combines the YES assay and a chromatographic separation on silica gel HPTLC plates with the performance of the YES assay. For separation, the analytes were applied bandwise to HPTLC plates (10 × 20 cm) with fluorescent dye (Merck, Germany). The plates were developed in a vertical developing chamber after 30 min of chamber saturation over a separation distance of 70 mm, using cyclohexane‒methyl-ethyl ketone (2:1, V/V) or cyclohexane‒CPME (3:2, V/V) as solvents. Both solvents allow separation of estriol, daidzein, genistein, 17β-estradiol, 17α-ethinyl estradiol, estrone, 4-nonylphenol and bis(2-ethylhexyl) phthalate.

Sustainable liquid chromatographic determination and purity assessment of a possible add-on triple-action over-the-counter pharmaceutical combination in COVID-19

Nowadays, all researchers are focused on combating the pandemic COVID-19. According to recent statistics, most patients are managed at home. An over-the-counter (OTC) triple action formula containing paracetamol (PAR), aspirin (ASP), and diphenhydramine (DIPH) is widely prescribed for pain, fever and as night-time sleep aid. For COVID-19 patients, this combination is now suggested as part of symptomatic therapy and prophylaxis. In this work, two simple liquid chromatographic approaches were designed for simultaneous determination of PAR, ASP, and DIPH in Excedrin® PM caplets, beside three specified official toxic impurities, namely, p-aminophenol, p-nitrophenol, and salicylic acid. The first method comprised high-performance thin-layer chromatographic separation coupled with densitometric quantification, on silica gel HPTLC 60 F254 aluminium sheets as the stationary phase, ethyl acetate–methanol-aqueous ammonium hydroxide (10.0: 2.0: 0.1, by volume) as the developing system and scanning was performed at 210.0 nm. The second one is a high-performance liquid chromatography coupled with diode array detector. Successful separation of the six components was performed on XTerra C18 column with isocratic elution of mobile phase 0.1% triethylamine acidified water: methanol (70:30, v/v) adjusted with o-phosphoric acid to pH 3.0 and methanol (90:10, v/v) with flow rate programming and detection at 210.0 nm. Validation of the proposed methods was performed according to ICH guidelines. Both methods were successfully used for quality control of the cited drugs in their marketed formulation. Moreover, the in-vitro release study was monitored using the proposed HPLC-DAD method. The greenness profile of the proposed methods was assessed and comparatively evaluated through various assessment tools, specifically; the analytical eco-scale system, national environmental method index (NEMI), green analytical procedure index (GAPI) and analytical greenness (AGREE) metric.

Proximate analysis, HPTLC finger print analysis and multi spectrometric analysis of Strychnos nux-vomica nuts.

In alternative medicine, plants pay a major role. Some plants are known for their poisonous nature but still have some importance in the herbal drug industry for their medicinal value. Strychnos nux-vomica is one such plant. Its nuts are called as poison nut due to the presence of alkaloids. Both the nut and its minerals are having medicinal properties and hence the present study was indented to understand the nature of primary metabolites and multi elemental composition. The nuts of S.nux-vomica were procured, authenticated, powdered and subjected to proximate analysis parameters, visualization of thin layer chromatographic separation (TLC) and finger print profiling through high performance thin layer chromatographic (HPTLC); surface morphology by scanning electron microscopy, energy dispersive X-ray analysis, X-ray fluorescence spectrometry, X-ray photoelectron spectrometry, powder X-ray diffractometry and inductively coupled plasma optical emission spectrometry. In HPTLC, 7 spots each under 254nm, 366nm, derivatization with vanillin sulphuric acid (VSR) reagent appeared and 2 spots with Dragendorff's reagent. In HPTLC, 12 peaks at 254nm, 9 peaks at 366nm, 7 peaks at 520nm after derivatization with VSR reagent detected. Elements such as potassium, calcium, magnesium, chlorine, aluminium, iron, manganese, sodium, nickel, phosphorus, copper, zinc, sulphur and silicon were identified. PXRD revealed that the presence of potassium chloride, calcite and dolomite as major elemental composition. The presence of all the above elements has vital roles on human physiology. Potassium, calcium, chlorine, aluminium, nickel, phosphorus, sulphur and silicon are reported for the first time in this study.

Non-target bioanalytical eight-dimensional hyphenation including bioassay, heart-cut trapping, online desalting, orthogonal separations and mass spectrometry

It is still a challenge to discover and identify individual bioactive compounds directly in multicomponent mixtures. Current workflows are too tedious for routine use. Hence, the hyphenation of separation and detection techniques is a powerful tool to maximize the information obtained by a single sample run. A robust eight-dimensional (8D) hyphenation was developed. Orthogonal separations, biological assay detection, analyte trapping, desalting, and physico-chemical detections were arranged in the following order, i.e. 1) normal phase high-performance thin-layer chromatography (NP-HPTLC) separation, 2) Vis detection, 3) UV detection, 4) fluorescence detection (FLD), 5) bioassay for effect-directed analysis (EDA), 6) heart-cut trapping/desalting/elution to reversed phase high-performance liquid chromatography (RP-HPLC) separation, 7) photodiode array (PDA) and 8) mass spectrometry (MS) detection. For the first time, the hyphenation exploited online analyte trapping to desalt the eluted bioactive zone from the plate containing highly salted bioassay media. Subsequent valve switching guided the trapped analyte(s) to the main column, followed by multiple detection. As proof-of-principle, cinnamon samples were analyzed by NP-HPTLC−UV/Vis/FLD−EDA−RP-HPLC−PDA−MS, whereby a bioactive zone was separated into two distinct peaks detected by PDA and MS to be 2-methoxy cinnamaldehyde and cinnamaldehyde. The developed 8D hyphenation is applicable for routine, allowing the non-target high-throughput screening of complex samples for individual bioactive compounds.

Antihyperglycemic Activity of TLC Isolates from the Leaves of Aloe megalacantha Baker in Streptozotocin-Induced Diabetic Mice.

BackgroundDiabetes mellitus (DM) is a group of metabolic disorders that are characterized by hyperglycemia which results from defects in insulin release or its efficient use by the human body. Although significant progress has been made to manage DM and related complications, it remains a major global health problem. To this end, the search for new antidiabetic drugs from traditionally claimed medicinal plants is important. Aloe megalacantha Baker is an endemic plant used traditionally to treat diabetes in Ethiopia. This study aimed to investigate antidiabetic activity of isolates from the leaf of A. megalacantha Baker in streptozotocin-induced diabetic mice.MethodsThe exudate of A. megalacantha was collected by cutting the leaves and scraping the yellow sap and then dried at room temperature. The dried exudate was subjected to repeated thin layer chromatographic (TLC) separations using a mixture of solvent system to isolate the major compounds identified on the TLC plate. A single dose of streptozotocin (50 mg/kg) was injected intraperitoneally to overnight fasted mice to induce diabetes. Antidiabetic activity of TLC isolates was assessed by in vivo methods including oral glucose tolerance test, antihyperglycemic and hypoglycemic activity tests.ResultsThree major isolates were obtained from the TLC analysis of the exudate of A. megalacantha. Exudate and TLC isolates were found to be non-toxic up to a dose of 2000 mg/kg. The TLC isolates (Ia and Ib) significantly (p<0.05) reduced blood glucose levels and also increased body weight change of the diabetic mice compared with control groups.ConclusionThe present study demonstrated the ability of the exudate of A. megalacantha and its TLC isolates to significantly decrease blood glucose levels and increase body weights in mice, thus strengthening the claim of its traditional use in DM-related disorders.

Ester Group Detection of Biodiesel from Used Cooking Oil with Sulphuric and Toluene Sulphuric Acid Catalysts

Used cooking oil is potential as raw material to produce biodiesel. We discovered fatty acid ethyl esters (FAEEs) and methyl esters (FAMEs) as biodiesel content indicator from esterification and trans-esterification reactions of used cooking oil with sulphuric acid and toluene sulphuric acid as catalysts. The purpose of this study was to examine some characteristics of FAEE and FAME synthesis from used cooking oil. The FAEEs and FAMEs were detected by separation in thin layer chromatography (TLC) and Fourier Transform Infrared (FT-IR) and compared to laurate standar. The used cooking oil was produced after frying of meat chicken for seven hours in a household. The Retardation Factor (Rf) of TLC of FAME of methyl laurate was 0.36 and FAEE of ethyl laurate was 0.23. The wavenumber indicating specific functional group of =CH was 3392 cm-1, while of alcohol as ester compound was 1739.79 cm-1. The wavenumber of C-C and CO groups were 1635.64 cm-1and 1165 cm-1, respectively. These indicate the ester group in used cooking oil, which reflects the formation of bio-diesel.

Unexpected Encapsulation of Selected Polycyclic Aromatic Hydrocarbons by β-Cyclodextrin Studied Using UV-Vis Spectrophotometry, Micro-Planar Chromatography and Temperature Dependent Inclusion Chromatography

This research communication significantly extends our previous studies focusing on the temperature effects related to the unexpected chromatographic behavior of 1-acenaphthenol in the presence of native β-cyclodextrin (β-CD) additive, working under thin-layer chromatographic (TLC) conditions. We have applied complementary and orthogonal techniques including (i) temperature-controlled ultraviolet-visible (UV-VIS) spectroscopy, (ii) thermostated microplanar high-performance chromatography (micro-HPTLC) and (iii) temperature-dependent inclusion chromatography based on high-performance liquid chromatography (HPLC) to investigate the retention behavior of related host molecules. Particularly, various symmetric and asymmetric molecules were tested, such as: naphthalene and its derivatives including acenaphthylene, acenaphthene and selected dimethynaphthalenes: 1,8-DMN, 1,5-DMN, 2,3-DMN and 2,6-DMN. Reported raw experimental data, particularly performed in liquid phase and detected by UV-Vis spectrophotometry, may suggest that solubility changes of the supramolecular complexes studied and differences in total analysis time between TLC and HPLC separation can trigger strong retention of target components in planar chromatographic systems. This was also supported by principal component analysis (PCA) of the multi-source data obtained. It is hoped that the reported analyses enable the adjustment of phenomenological models describing liquid chromatography retention and the solubility behavior of low-molecular mass guest molecules, controlled by supramolecular interactions with selected macrocycles. It should be noted that the reported phenomenon, specifically supramolecular complexes precipitation, may have a number of practical applications. This can be used to improve the efficiency and selectivity of planar and/or microfluidic systems. On the other hand, precipitation via host-guest interactions may be applied for highly selective water purification technological processes that will be designed for the removal of given organic micropollutants.

Profiles of nutritional, bioactive compounds and cytotoxic activity of Dwarf date palm (Phoenix loureiroi) fruit

The wild edible fruits are basic food sources and these contain potential dietary compounds which provide a healthy diet. Such wild edible fruits play an important role in supplementing the diet and livelihood of rural/tribal communities. Thus, the present study determined the nutritive values of Phoenix loureiroi fruit by screening the phytochemical compounds, organic acids and polyphenolics along with the evaluation of cytotoxic activity. The nutritional composition revealed high contents of total proteins (36.30 mg BSAE/g dry fruit), total free amino acids (6.15 mg LE/g dry fruit), reducing sugars (85.40 mg GE/g dry fruit), vitamin E (2.50 mg TE/g dry fruit) and sodium (1.26 mg/g dry fruit) at Rutab stage of maturation. Anti-nutritional compositions of fruit were found to be chymotrypsin inhibitors (3.31 CIU/mg protein), trypsin inhibitors (0.01 TIU/mg protein) and phytate (0.42 g phytate/100 g dry fruit) contents. High-performance thin-layer chromatography (HPTLC) separation demonstrated that fruit contains a high amount of essential (tryptophan, 41.30 mg/g dry fruit) and non-essential (tyrosine, 25.70 mg/g dry fruit) amino acids. The total phenolics concentration was found to be 49.68 mg GAE/100 g dry fruit. Liquid chromatography analyses revealed that the fruit contained high amounts of fructose (0.12 mg/g dry fruit), ascorbic acid (2.80 mg/g dry fruit), catechin (3.78 mg/g dry fruit) and succinic acid (1.57 mg/g dry fruit). The bioactive compounds of fruit extract inhibited the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC50 = 8.51 µg/mL). The cell viability value (65.87%) on fibroblast L929 cell lines indicates fruit extract was safe up to 1000 µg/mL concentration. The results revealed that the P. loureiroi fruit was rich in nutraceutical compounds and could be used for the development of safe natural products.

Antibacterial activity of selected plants extract against pathogenic bacteria and detection of phytochemicals

Aim: The aim of the present study was to assess the antibacterial activity of selected plants extract against Gram- positive and Gram- negative pathogenic bacteria. Methodology: Extracts of neem leaf (Azadirachta indica), rose petal (Rosa indica), amla fruit (Emblica officinalis) and clove buds (Syzygium aromaticum) were prepared in different solvents. Antibacterial activity against selected microorganisms was assayed using agar cup diffusion method, thereby measuring the zone of inhibition. The extracts with higher zone of inhibition were run on Thin Layer Chromatography (TLC) plates. The presence of phytochemicals was detected using iodine fumigation method followed by contact bioautography. Results: The extracts of neem leaf, rose petal, amla fruit and clove bud prepared in solvents like acetone, ethanol and ethyl acetate showed higher antibacterial effect against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium in agar cup diffusion assay. TLC separation of the extract of plant products showed dark brown bands of phytochemicals on silica-gel G 60 plate. Contact bioautography results showed maximum antibacterial activity against Klebsiella pneumoniae. Interpretation: The results of the present study provide scientific evidence for the traditional uses of Azadirachta indica, Rosa indica, Emblica officinalis and Syzygium aromaticum.

Facile detection of carbendazim in food using TLC-SERS on diatomite thin layer chromatography.

This work aims to isolate and detect pesticide (carbendazim) residue in real food samples: orange juice and kale leaves. The combination of on-chip thin layer chromatography (TLC) and surface enhanced Raman scattering (SERS) spectroscopy was used for the separating and detecting of carbendazim (MBC) from the complex food sample. In order to achieve on-site detection of MBC from real food sample, the portable Raman spectrometer was coupled with TLC-SERS. The porous stationary phase composed of diatomite biosilica is beneficial for SERS enhancement and eluent migration. The experiments exhibited that the diatomite chip was suitable for TLC separation and has not shown SERS background and provided excellent separation efficiency, 10-8M silver colloids were appropriate for the SERS measurement on TLC chip. The food sample was directly spotted onto the diatomite chip for TLC separation without any pretreatment. The separation and detection process were finished in less than 5min, the mixture of pyrimethanil, pymetrozine and MBC could be distinguished simultaneously by TLC-SERS at one diatomite chip. The MBC in orange juice and kale were successfully detected, and a limit of detection (LOD) less than 2ppm could be achieved.

Alterations in allocation and composition of lipid classes in Euonymus fruits and seeds.

The spindle tree (Euonymus europaeus L.) is a much-branched deciduous shrub or small tree. Its fruit capsules contain seeds with remarkably high content of oil interesting for industry, especially the 3-acetyl-1,2-diacyl-sn-glycerols (AcDAG) synthesized by a specific acetyl-CoA diacylglycerol acetyltransferase. The distribution and amount of individual triacylglycerols (TAG) and especially acetyl-triacylglycerols (AcDAG) in Euonymus fruit have previously been assigned to specific tissues. Using anatomical and microscopical observations, we studied the fruit morphology, and for the first time, we identified a more detailed allocation of oil bodies in individual tissue structures. Thin layer chromatography separation of extracts from immature and mature fruits confirmed TAG and AcDAG as the most abundant lipid classes in both endosperm and embryo, followed by fatty acids and polar lipids. The abundance of fatty acids was further studied in the TAG and AcDAG fractions using gas chromatography. Data revealed particular FAs in both fractions allocated in tissue-specific manner and/or as indicators of maturation of E. europaeus seeds. While the abundance of cis-vaccenic-, linoleic as well as α-linolenic acids in the AcDAG structures generally drop with maturation in both embryo and endosperm, content of oleic acid increases. Abundance of cis-vaccenic acid in TAG was recorded in immature endosperm. For embryo, the abundance of stearic acid in AcDAG and oleic acid in TAG fraction was distinctive. Deeper understanding of underlying metabolic processes will be essential for targeted metabolic engineering and/or application for oilseed crops.

Smart TLC–densitometric methods for determination of ophthalmic ternary mixture containing chloramphenicol in the presence of its synthetic precursor: Comparative eco-scaling for greenness assessment

Green analytical methods have gained a growing interest in the field of pharmaceutical research to reduce impacts on the environment and enhance analysts’ health safety. Chloramphenicol (CHL), dexamethasone sodium phosphate (DSP) and tetrahydrozoline HCl (THZ) form an ophthalmic ternary mixture that is co-formulated for conjunctivitis treatment. In the present work, for time saving and higher sensitivity, two green thin-layer chromatography (TLC) methods were developed for the determination of this ophthalmic ternary mixture in the absence or presence of p-nitroacetophenone (PNA), a synthetic precursor of chloramphenicol. In both proposed methods, silica gel 60 F254 plates were used as the stationary phase. The mobile phase used for method (A) was ethanol‒water‒ammonia (7.0:2.5:0.5, V/V), while, for method (B), acetonitrile‒water‒ammonia (10.0:3.0:0.5, V/V) was used as the mobile phase. TLC separation was followed by quantitative determination of the aforementioned drugs at wavelengths 242.0 nm and 220.0 nm. Both methods were validated in compliance with the International Conference on Harmonisation (ICH) guidelines, where both methods were found to be reliable, reproducible, and selective. Statistical comparison of the developed methods was done with a reported high-performance liquid chromatography (HPLC) method where no significant difference was found. Analytical eco-scaling depends on penalty point which was calculated to be 92, 88 and 87 for methods A, B and the reported HPLC, respectively, suggesting that the proposed methods are eco-friendlier with penalty point scoring very high on the scale than the reported one.

Simultaneous colorimetric sensing of malachite & leucomalachite green in aquatic products based on novel ionic associate self-visualization HPTLC strips

The development of simultaneous and rapid screening of the residues of veterinary drugs and their metabolites is up to now still a challenge. In this study, a novel strategy of read-out analysis is proposed based on high-performance thin-layer chromatography (HPTLC) separation coupled with self-visualization detection of trace of leucomalachite Green (LMG) and malachite green (MG) in aquatic products. The novel self-visualization nanomaterial was successfully synthesized and subsequently fully characterized. The ionic associate MG–I3 formed on the visualization sensing interface was effectively attested by MALDI-TOF/TOF-MS for the first time. A serials of visualization reactions were conducted on the target zone surface of HPTLC after separation. Under the optimum conditions, the calibration curves of LMG and MG were obtained with good linearity in the range of 0.25–8.0 μg mL−1 and 0.125–4.0 μg mL−1, respectively. The limits of detection and limit of quantification were calculated as 1.71 and 5.19 μg kg−1 for LMG and 0.9 and 2.74 μg kg-1 for MG, respectively. Real sample analysis and validation provided satisfactory results. The proposed method has the potential to detect simultaneously, rapidly, and accurately LMG and MG in aquatic products during storage, handling, and sales.

Validated Stability-Indicating HPLC and HPTLC Methods for the Determination of Ritonavir in Bulk Powder and in Capsules

Two sensitive and reproducible methods were described for the quantitative determination of the antireteroviral drug ritonavir in the presence of its degradation products. The first method was based on high performance liquid chromatographic (HPLC) separation of the drug from its stress degradation products with the use of a reversed phase Agilent Eclipse XDB-C18 column (5 µm, 4.6 × 150 mm) and a mobile phase consisting of acetonitrile : 0.05 M phosphoric acid (55 : 45, v/v) at a flow rate of 1.0 mL/min. The retention time of the drug was found to be 4.82 ± 0.002 min. Quantification was achieved with diode array detection (DAD) at 210 nm based on peak area and a linear calibration curve in the concentration range of 1-500 µg/mL. The proposed method made use of diode array detection as a tool for peak purity and identification. The second method involved a high performance thin layer chromatographic (HPTLC) separation followed by densitometric measurement of the spots at 240 nm. The separation was carried out on Fluka TLC aluminium sheets of silica gel with fluorescent indicator (254) nm and the mobile phase was acetonitrile - water (1 : 2, v/v), adjusted to pH 5.0 using 1 M orthophosphoric acid solution . The proposed procedure gave compact spots for ritonavir (retention factor, Rf = 0.41 ± 0.014). The linear regression equation was generated by least-squares treatment of the calibration data in the range of 0.8-12.5 µg/spot. The reliability and analytical performance of the proposed methods, including linearity, range, precision, accuracy, detection and quantitation limits, were statistically validated. The proposed methods were applied to Norvir capsules and no chromatographic interference was observed. When ritonavir was subjected to stress conditions; according to ICH guidelines, the proposed methods could effectively separate the drug from its degradation products, and were thus considered as good stability-indicating procedures.

Validated Chromatographic methods for estimation of Solanine in Mahamrutyunjaya rasa

Introduction: Mahamrutyunjaya rasa is a herbomineral ayurvedic formulation used for various indications like fever, pain, cardiac arrhythmia, etc. It contains Solanum indicum as one of the ingredients having anti-inflammatory, diuretic, and diaphoretic properties. Solanine is one of the active biomarkers for Solanum indicum. The therapeutic window of Solanine is very narrow, with the LD50 value of 3-6 mg/kg body weight in humans. Objective: Estimation of such biomarkers is very significant to avoid any adverse events due to the administration of ayurvedic preparations containing Solanine. In the present study, two simple, sensitive, reliable chromatographic techniques have been developed and validated for the estimation of Solanine in Mahamrutyunjaya rasa. Materials and Methods: High-performance liquid chromatography (HPLC) separation of Solanine was performed on an reversed phase C-18 column (250 mm 9 4.6 mm ID, 5 lm particle size), with isocratic elution using a mixture of Tris buffer (10mM, pH 6.00): Acetonitrile(60:40, v/v) at a flow rate of 1 mL/min with UV detection at 218 nm for solanine. High-performance thin-layer chromatography (HPTLC) separation was done on Silica gel 60 F254 pre-coated plates using Chloroform: Methanol: Ammonia (7:3:0.5 v/v). The densitometric scanning was performed at 500 nm. The developed methods were validated for linearity, LOD, LOQ, accuracy, precision, and specificity as per ICH guidelines. Results: Solanine was eluted at 4.43 ± 0.1 min and established a linearity range of 1 – 100 μg/ml (r2 = 0.9992). In the HPTLC method, the Rf value of Solanine was 0.05 in a linearity range of 1600-4800 ng/ml. Conclusion: Reliable, rapid, simple, and sensitive chromatographic methods were developed for the quantification of Solanine in Mahamrutyunjaya Rasa.

Thin-layer chromatographic separation of a number of bile acids with mobile phases based on surfactants

The aim of this study was the separation of cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic, and lithocholic bile acids by thin-layer chromatography (TLC) with mobile phases based on surfactants. The effect of surfactant type and concentration, the pH of the mobile phase, the type and amount of the mobile phase modifier, and the effect of temperature and type of material backed silica gel TLC layers on the investigated bile acid separation were studied. The separation was carried out on normal-phase TLC plates Sorbfil PTLC-P-A-UV-254 and Sorbfil PTLC-AF-A-UV-254. The acid zones after separation were visualized by using sulfuric acid solutions followed by heating the TLC plate and acid spots observing under 365-nm ultraviolet (UV) light. Mobile phases based on surfactants, namely, cationic cetylpyridinium chloride, anionic sodium dodecyl sulfate, and nonionic Tween-80, were used. Aliphatic alcohols 1-propanol, 1-butanol, and 1-pentanol as well as the nonionic surfactant Tween-80 were used as modifiers of the mobile phase. The optimal separation of the studied acids was achieved using the two-dimensional TLC method with sequential elution with mobile phases based on cetylpyridinium chloride, first with the addition of 1-butanol and then, perpendicular to the initial direction, with the addition of 1-pentanol. The proposed method for the bile acid separation can be used in the analysis of human biological fluids and drugs.

A rapid method for the separation of the phospholipids from the neutral lipids in plasma

The blood fraction most commonly used for the assessment of fatty acid status is the plasma or serum phospholipids, since these provide a measure of long term dietary intake. However, conventional assays of fatty acid status in human plasma and serum require labour intensive, multi-step approaches, which are impractical for high-throughput analyses. This study reports a system capable of selectively separating plasma phospholipids from other lipid classes in just a few minutes. We demonstrate that compositional analysis of the fatty acids in plasma phospholipids using our plasma spot method resulted in greater than 97% of neutral lipid standards had been eluted from the paper, whereas more than 96% of the PC remained on the paper. These results were almost identical to conventional methods involving liquid/liquid extraction and thin layer chromatography separation. Evaluation of our plasma spot fractionation and assay using plasma from 110 human subjects (75 males, 35 females), provides confirmation of significant correlations between the fatty acid measures and those obtained from conventional measures for all fatty acids (r > 0.97, P<0.0001), including the omega (n)-6 (r = 0.988, P<0.0001) and n-3 long chain polyunsaturated fatty acids (r = 0.997, P<0.0001). These results establish our newly developed plasma spot separation technique as a rapid and reliable method for the assessment of plasma phospholipid fatty acid composition.

Thin-layer chromatographic enantioseparation of atenolol and propranolol using (S)-naproxen as chiral selector: direct and indirect approaches

Enantiomeric separation of (RS)-atenolol, and (RS)-propranolol has been achieved by thin-layer chromatography (TLC) using both direct and indirect modes. (S)-naproxen, containing a carboxylic group, was utilized as the chiral selector. For direct TLC separation, (S)-naproxen was used as a chiral additive in the stationary phase in a non-covalent mode and there was no chiral additive in the mobile phase; the native enantiomers were isolated and characterized. For the indirect approach, the diastereomeric derivatives of analytes were synthesized with (S)-naproxen (as chiral derivatizing reagent) and the derivatives were then separated on achiral plates (without chiral selector); these were then isolated and characterized. The effect of the composition of the chiral selector, pH, and temperature was studied for the optimization of successful separation conditions. The developed method was validated. The limits of detection for enantiomers of atenolol and propranolol were found to be 1.2 μg mL−1 and 1.6 μg mL−1, respectively.