High-performance Thin-layer Liquid Chromatography Research Articles

Development of quality control parameters for two Bhutanese medicinal plants (Aster flaccidus Bunge and Aster diplostephioides (DC.) Benth. ex C.B.Clarke) using traditional and modern pharmacognostical platforms

Bhutan's scholarly traditional medical system is called Bhutanese Sowa Rigpa medicine (BSM). It was integrated with the modern healthcare system in 1967. Over 200 medicinal plants are used to produce more than 100 poly-ingredient medicinal formulations. Although BSM is supported by well-documented principles, pharmacopoeias, diagnostic procedures, treatment regimens, and traditional quality assurance systems, modern quality control parameters have become essential to distinguish closely related species and prevent contamination from exogenous impurities. This study aims to establish reliable analytical methods and quality control parameters for Aster flaccidus Bunge and Aster diplostephioides (DC.) Benth. ex C.B. Clarke used as ingredients in the BMS poly-ingredient medicinal formulations. Furthermore, their reported phytochemicals and biological activities are also discussed in this study. Standard pharmacognostic techniques, including macroscopical and microscopical examinations of crude drugs, were employed to establish the quality control parameters for two Aster species. The physicochemical limits were determined as per the World Health Organization (WHO)-recommended guidelines and methods described in the Thai herbal pharmacopoeia. A high-performance thin-layer liquid chromatography (HPTLC) was used to develop a comparative chromatogram/phytochemical fingerprint for the crude extracts obtained from two Aster species. A literature review was conducted to record their isolated phytochemicals and biological activities. Two Aster species possess macro- and microscopic features such as colour, appearance, and shape. Physicochemical analysis of crude drugs from two Aster species including HPTLC fingerprinting of their methanol crude extracts also yielded adequate data to differentiate and confirm two Aster species before adding them to the BSM poly-ingredient medicinal formulations. From the literature review, only A. flaccidus was found to be studied for its phytochemical constituents, whereby 11 pure compounds were isolated from aerial parts and roots. The current study revealed distinct species-specific distinguishing features, including ecological adaptation, micromorphology, anatomy, physicochemical values, HPTLC chromatograms. These parameters can be used to authenticate the species identity and prevent adulterations, thereby improving the quality and safety of BSM formulations.

Toward Effects of Hydrophobicity on Biosurfactant Production by Bacillus subtilis Isolates from Crude-Oil-Exposed Environments

Background: Due to their structural features, biosurfactants reveal promising physicochemical properties, making them interesting for various applications in different fields, such as the food, cosmetics, agriculture, and bioremediation sectors. In particular, the bioproduction of surfactin, one of the most potent microbially synthesized biosurfactant molecules, is of great interest. However, since the wild-type productivities are comparably low, stimulatory environmental conditions have to be identified for improved bioproduction This study aims to find a correlation between the hydrophobicity and production of the biosurfactant surfactin by B. subtilis isolates from crude-oil-contaminated soil and water. Methods: The surfactin production yield was characterized in adapted batch cultivations using high-performance thin-layer liquid chromatography (HPTLC). Defined hydrophobic environmental conditions were achieved by supplementation with hexadecane or polystyrene beads, and the effects on biosurfactant production were measured. Adaptations at the protein level were analyzed using mass spectrometry measurements. Results: The correlation between hydrophobicity and surfactin production was characterized using Bacillus subtilis strains ZH1 and P7 isolated from crude-oil-contaminated soil and water. Since these isolates show the biodegradation of crude oil and hexadecane as hydrophobic substrates, respectively, a first-time approach, using polystyrene beads, was applied to provide a hydrophobic environment. Interestingly, contrary to popular opinion, reduced biosurfactant production was determined. Using mass spectrometric approaches, the physiological effects of co-cultivation and the cellular response at the protein level were investigated, resulting in altered quantities of stress proteins and proteins involved in the carbon metabolism counter to polystyrene beads. Conclusions: Contrary to common opinion, increasing hydrophobicity does not have a stimulating effect, and even reduces the effect on the bioproduction of surfactin as the main biosurfactant using selected B. subtilis strains.

Analytical Methodologies for the Estimation of Acyclovir as Key Members of Anti-viral Agent: Two Decades in Review

Abstract: Herpes simplex virus (HSV) is a viral infection that primarily targets oral and genital organs in humans. Acyclovir is a widely prescribed anti-viral agent used in the infection caused by herpes simplex virus (HSV). This article emphasizes several analytical techniques, including spectrophotometry, High-performance liquid chromatography, High-performance thin-layer liquid chromatography, Ultra performance liquid chromatography, and Liquid chromatography/Mass detection for the quantification of acyclovir in different matrices like biological fluids and Pharmaceutical formulation. In the proposed work, numerous methods for different techniques were extracted from various databases such as Science Direct, Springer, PubMed, SCOPUS, Web of Science, etc. According to the recommendation from the internal conference on harmonization, this review describes how to determine the presence of utilizing acyclovir in different analytical techniques alone or in combination with another drug.

Canagliflozin: A review with specific focus on analytical methods in biological matrices and pharmaceuticals

Abstract Sodium-glucose transporter 2 inhibitor emerges as the latest group of oral hypoglycemic agents, which shows insulin-independent pathology and provides an upper hand to enhance renal glucose elimination. Canagliflozin (CGN) was the number one drug, approved by FDA on 29th March 2013 for the treatment of type 2 diabetes mellitus. By totting up to its glucose-lowering effects, it exhibits beneficial effects on the heart and potentially on the kidneys. The study aims to summarize various analytical techniques, such as chromatography, spectrophotometry, and hyphenated techniques, such as Liquid chromatography with tandem mass spectrometry (LC-MS/MS) and Ultra performance liquid chromatography with tandem mass spectrometer (UPLC-MS) for the analysis of CGN. In the proposed work, we have reviewed various analytical methods reported for the estimation of CGN in biological matrices and Pharmaceuticals from various databases like ScienceDirect, Springer, PubMed, Scopus, Taylor & Francis, and Web of Science for the estimation of CGN. Various analytical methods adapted were high-performance liquid chromatography, UPLC, LC-MS/MS, high-performance thin-layer liquid chromatography, Fourier-transform infrared spectroscopy, spectrofluorimetry, and UV spectrophotometry. This current review presented the determination of CGN using various analytical techniques and biological matrices either in single or in combination with other hypoglycemic agents, as per International Conference on Harmonization guidelines. Further, some future trends that can be integrated were also suggested.

Physicochemical Characterization, Phytochemical and HPTLC Fingerprinting Studies on Fruit of Couroupita Guianensis

Abstract Traditional knowledge and literature studies report that each part of a plant has tremendous medicinal values. Validation of these medicinal plants scientifically is an important criterion for the development of plant-based drugs. Couroupita guianensis (Family: Lecythidaceae) is a plant with immense medicinal properties. To authenticate its biological value, the present investigation aims to standardize the fruit of C. guianensis based on physicochemical characterization, phytochemical analysis both qualitatively and quantitatively, and high-performance thin-layer liquid chromatography (HPTLC) fingerprinting studies. Fruit pulp of C. guianensis was obtained, processed and extracted with solvents such as petroleum ether, chloroform, ethanol and hydroalcohol. Moisture content, total ash, water-soluble ash and acid-insoluble ash values were calculated. Preliminary phytochemical analysis revealed the existence of several secondary metabolites in the extracts. In addition, interpreting peaks obtained from HPTLC analysis revealed the presence of potential bioactive phytoconstituents in all the extracts. The quantitative determination proclaimed that fruit pulp was found to be rich in phenolics and flavonoids followed by tannin and saponin. Further, primary metabolites were quantified and they were found to be abundant in the fruit pulp. Henceforth, the outcome of these results provides information for assessing the quality of the sample that could help in ensuring its therapeutic efficacy. Keywords: Couroupita guianensis, HPTLC, Physicochemical characterization, Phytochemical, Fingerprinting

Pretreatment and optimization of processing conditions for extraction of oleuropein from olive leaves using central composite design

Background: The extraction methods used for isolation of biomolecule from Olive leaves show risk of residual solvent and less extraction efficiency. Hence, there is a need to develop novel techniques to encapsulate the risks headed with extraction process. Objective: The goal was to unravel the effect of novel extraction techniques on the extraction efficiency of Oleuropein from Olea europaea, a major secoiridoid resided in Olive leaf. Materials and Methods: Olive leaves were collected, authenticated, and subjected to proximate, phytochemical analysis to contemplate the source of active moiety. The precised solvent, i.e., water: glycerol (3:1%v/v) was functionalized to depict the influence of independent variable on response using central composite design. For hot blanching, the independent factors selected were treatment temperature (50°C–70°C) and duration of blanching (10–30 min) whereas the observed response is percentage extraction efficiency of Oleuropein. The hot blanched leaves were subjected to extraction by cold maceration, microwave-assisted extraction (MAE), and ultrasound-assisted extraction (UAE). The content of Oleuropein was analyzed by high-performance thin-layer liquid chromatography. Results: From the design space, the model is stable at a range of 0.002–0.80 which indicates lack of fit is very less and more curvature effects are clearly visualized with P = 5% level of significance. Maximum response was attained at a temperature of 60°C–65°C and duration of 15–20 min. Microstructural changes in leaf were observed through scanning electron microscopy studies. From the study, pretreated leaves followed by UAE result in higher yield of Oleuropein compared to MAE and maceration. Conclusion: Hot blanching technique shows a significant linear upswing in the concentration of Oleuropein when compared to direct extraction techniques. Blanching of Olive leaves causes deactivation of enzymes, and further exposure to ultrasonic waves enhances mass transfer of solvent and promotes the release of Oleuropein.

Establishment of adventitious root cultures of Allamanda cathartica L. for the production of iridoid glycosides and its identification using HPTLC MS

This study attempts to develop a reproducible protocol for in vitro adventitious root regeneration for enhanced biomass production from nodal segments of Allamanda cathartica L., which may be of use as an alternative source for production of iridoid glycosides. Allamanda cathartica (Apocynaceae) is a medicinally rich vine, used in the treatment of human carcinoma, human immunodeficiency virus (HIV), jaundice and malaria. The roots of the plant are the main reservoir of secondary metabolites. The main active constituent in root is iridoid glycosides. The explant for root initiation was cultured in ½ strength Murashige and Skoog (MS) liquid medium augmented with different levels of Indole-3-butyric acid. 0.5 μM Indole-3-butyric acid with 4% sucrose resulted in 1.810 ± 0.049 g and 0.376 ± 0.053 g fresh and dry weight respectively. Among different levels of sodium chloride (80, 100, 120 and 150 mM) added to optimized media, 120 mM sodium chloride treatment increased the root biomass considerably compared with Indole-3-butyric acid alone and other combination, and recorded the fresh weight 2.090 ± 0.049 g and dry weight 0.400 ± 0.005 g after 12 weeks of incubation. The phytochemical investigations using high performance thin-layer liquid chromatography (HPTLC) and mass spectrometry (MS) analysis revealed concentration dependent modulation of selected iridoids in the cultures treated with sodium chloride. The protocol developed can be utilized as an alternative source for production of iridoid glycosides and the findings may be explored on industrial scale.

Reversed-Phase High-Performance Liquid Chromatography and High-Performance Thin-layer Liquid Chromatography Methods for Simultaneous Determination of Theophylline, Guaifenesin and Guaifenesin Impurity (Guaiacol) in Their Bulk Powders and in Dosage Form.

Two simple, rapid chromatographic methods were developed for the simultaneous determination of Theophylline (THP), Guaifenesin (GUI) and Guaifenesin impurity namely Guaiacol (GUA). The first method is an isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method, where separation of THP, GUI and GUA was achieved on C18 column using methanol:water (containing 0.1% triethylamine):acetonitrile (30:60:10, by volume) as a mobile phase with a flow rate of 0.7 mL /min and UV detection at 275 nm. The separation was achieved at retention times (Rt 3.76, 6.76 and 8.79 for THP, GUI and GUA, respectively). The calibration plots were linear over the concentration range of 2-25, 2-37 and 0.5-10 μg /mL for THP, GUI and GUA, respectively. The second method is high pressure thin layer liquid chromatography method, which was developed using silica gel plates 60F254 as a stationary phase with ethyl acetate:hexane:methanol:ammonia (65:35:10:2, by volume) as a developing system. The densitometric measurements were performed at 275 nm with good Rf values (0.13, 0.35 and 0.8) for THP, GUI and GUA, respectively. The calibration plots showed good correlation over the range (0.4-2 μg/band) for both THP and GUI, and (0.4-1.2 μg/band) for GUA. The two proposed methods were validated according to ICH guidelines. The results for the two methods were statistically compared to those obtained by a reported high-performance liquid chromatography method and no significant difference was found regarding accuracy and precision.

Development and validation of HPTLC method for estimation of gymnemic acid in microencapsulated antidiabetic polyherbal formulations

Summary Gymnemic acid (GA) is one of the phytoconstituents present in Gymnema sylvestre. Estimation of GA was carried out first time from microencapsulated polyherbal formulation. Microencapsulated polyherbal formulations (F1 and F2) contain various plant extracts; hence, proper resolution of GA peak in high-performance thin-layer liquid chromatography (HPTLC) analysis of F1 and F2 is the problem. Hence, HPTLC analysis method for F1 and F2 is developed and validated for quantitative determination of GA. HPTLC analysis of F1 and F2 was carried out using TLC aluminum plates precoated with silica gel 60F254 eluted with chloroform-methanol-water (6.5 mL + 4.5 mL + 1.0 mL), and densitometric analysis was carried out at 580 nm. Complete validation was performed using standard methods. This HPTLC method was found to be reproducible, accurate, and can detect GA at microgram level. The new optimized mobile phase gave good resolution of GA peak for its proper quantification in microencapsulated polyherbal formulation.

HPTLC estimation of oroxylin A inOroxylum indicumherbal formulations

Oroxylum indicum (L.) Kurz is an important medicinal plant with a wide spectrum of medicinal properties. It is frequently used in a large number of traditional medicinal formulations. Oroxylin A is considered as one of the major bioactive compounds in O. indicum. Optimization of semi-preparative high-performance liquid chromatography (HPLC) conditions for the isolation of oroxylin A from the plant parts and development of a high-performance thin-layer liquid chromatography (HPTLC) analytical method for the quantification of oroxylin A in polyherbal formulations are reported in this study. The HPTLC method was validated in terms of its linearity, LOQ, precision, and accuracy and was demonstrated for the quantification of oroxylin A in O. indicum plant parts and some commercial “Ayurvedic polyherbal formulations”. The method was able to identify and quantify oroxylin Ain complex mixtures of phytochemicals and could be extended to the marker-based standardization of other polyherbal formulations.

Development and validation of an HPTLC-densitometric method for simultaneous analysis of lamivudine, tenofovir disoproxil fumarate, and efavirenz (LTE) in tablets

Analysis of fixed dose combination products can present daunting challenges to the analytical chemist. This paper presents a validated analytical method for simultaneous analysis of lamivudine, tenofovir disoproxil fumarate, and efavirenz using high-performance thin-layer liquid chromatography (HPTLC)-densitometric method. Separation was achieved by use of HPTLC pre coated plate with silica gel 60F254 using mobile phase containing toluene-methanol (27:6 v/v). RF values were 0.18 ± 0.02 for lamivudine, 0.33 ± 0.05 for tenofovir disoproxil fumarate, and 0.48 ± 0.02 for efavirenz, respectively. The regression line had best fit using second order polynomial function for all the three APIs (active pharmaceutical ingredients) with r2 ≥ 0.98 at the concentrations of 375–900 ng spot−1 for lamivudine, tenofovir disoproxil fumarate and 750–1800 ng spot−1 for efavirenz, respectively. Repeatability and intermediate precision had % RSD ≤ 2. The method had acceptable level of accuracy for all three APIs with mean recov...

Characterization and Dating of Blue Ballpoint Pen Inks Using Principal Component Analysis of UV–Vis Absorption Spectra, IR Spectroscopy, and HPTLC

The ink of pens and ink extracted from lines on white photocopier paper of 10 blue ballpoint pens were subjected to ultraviolet-visible (UV-Vis) spectroscopy, infrared (IR), and high-performance thin-layer liquid chromatography (HPTLC). The R(f) values and color tones of the bands separated by thin-layer chromatography (TLC) analysis used to classify the writing inks into three groups. The principal component analysis (PCA) investigates the pen responsible for a piece of writing, and how time affects spectroscopy of written ink. PCA can differentiate between pen ink and ink line indicates the influence of solvent extraction process on the results. The PCA loadings are useful in individualization of a questioned ink from a database. The PCA of ink lines extracted at different times can be used to estimate the time at which a questioned document was written. The results proved that the UV-Vis spectra are effective tool to separate blue ballpoint pen ink in most cases rather than IR and HPTLC.

Antioxidant potential of Amorphophallus paeoniifolius in relation to their phenolic content

The present study evaluates the antioxidative and radical scavenging potential of the tuber extract of Amorphophallus paeoniifolius (Dennst.) Nicolson, (Araceae). The ethanol extract of A. paeoniifolius (APE) was studied for the inhibition of lipid peroxidation estimated in terms of thiobarbituric acid reactive substances (TBARS) and the levels were reduced by 4.3% to 67.2% in a dose-dependent manner. Further, APE was analyzed for scavenging capacities based on 1,1-diphenyl-2-picrylhydrazyl-2-radical (DPPH) assay and percentage inhibition activity based on 2,2-azinobis-(-3-ethyl) benzo-thiozoline-6-sulfonate (ABTS+) and H2O2. The A. paeoniifolius extract showed a maximum of 68.6% of DPPH scavenging activity and the maximum inhibition of 74% and 67.2% in the case of ABTS and H2O2, respectively. The antioxidant efficiency and inhibition of oxidation of the extract was found to be dose-dependent at the tested concentrations of 1-50 µg/mL. High-performance thin layer liquid chromatography (HPTLC) profile of the extract suggests the presence of polyphenols such as gallic acid, resveratrol, quercetin and two unidentified compounds. The results suggest that the ethanol extract of A. paeoniifolius has a potent antioxidant activity in vitro and can be utilized as an effective and safe source of antioxidants.