High-performance Thin-layer Chromatography Densitometry Research Articles

The improvement of routine drug quality control analysis of some antihypertensive drugs using high-performance thin-layer chromatography densitometry method with greenness profile evaluation.

The common antihypertensive drugs are B-blockers and diuretics. For the determination of beta-blocker medicines (bisoprolol fumarate and carvedilol) and diuretic drug (Furosemide), new and accurate chromatographic method has been developed. The separation was achieved using a developing system that includes chloroform:methanol:ethyl acetate:ammonia (6:2:2:0.2 by volume) as a mobile phase and the bands were detected at 240nm. The concentration ranges were 5-25, 1-7, and 1-3.5μg/band for bisoprolol fumarate, carvedilol, and furosemide, respectively. This chromatographic approach is the first methodology for simultaneously determining bisoprolol fumarate, carvedilol, and furosemide in their pure forms and in their pharmaceutical dosage forms. The advantages of using known analytical procedures are their simplicity, speed, cost effectiveness, lack of laboriousness, and ability to save time as the three tablets are determined in one step and can be used for routine analysis of the investigated combinations in quality control laboratories. According to International Conference of Harmonization guidelines, the established procedures have been validated, and the results were statistically compared to those obtained by the reported reversed-phase-high-performance liquid chromatography methods using Student's t-test and F-test, with no significant difference between them, indicating that the proposed methods can be used for routine drug quality control analysis.

ANTI-INFLAMMATORY STUDY (IN-VITRO AND IN-VIVO) AND HPTLC DENSITOMERIC DETERMINATION OF QUERCETIN FROM METHANOLIC LEAF EXTRACT OF HOTTUYNIA CORDATA THUNB. FROM MIZORAM, INDIA

Objective: This study aimed to investigate the anti-inflammatory activity of the methanolic leaf extract of Houttuynia cordata Thunb. from Mizoram, India, using in-vitro and in-vivo models. In addition, the study also aimed to determine the quercetin content of the methanolic leaf extract using high-performance thin-layer chromatography (HPTLC) densitometry. Methods: The methanolic leaf extract of H. cordata was prepared using successive Soxhlet extraction, and its anti-inflammatory activity was evaluated using in-vitro models such as inhibition of protein denaturation and inhibition of proteinase activity. The in-vivo anti-inflammatory activity of the extract was evaluated using carrageenan-induced paw edema in rats. The quercetin content of the extract was determined using HPTLC densitometry. Results: The methanolic leaf extract of H. cordata showed significant anti-inflammatory activity in both in-vitro and in-vivo models. The extract exhibited a dose-dependent inhibition of protein denaturation and proteinase activity and also significantly reduced paw edema in the rat model. The IC50 values of egg albumin and bovine serum albumin denaturation studies were 317.365 μg/mL and 198.918 μg/mL, respectively. The results showed that the methanolic leaf extract of the plant was 200 mg/kg b.w. and 400 mg/kg b.w., which considerably reduced carrageenan-induced paw edema, and their effects were comparable to those of the standard diclofenac sodium. The HPTLC densitometry analysis revealed the presence of quercetin in the extract, with a quercetin content of 21.8 ng/μL. Conclusion: The methanolic leaf extract of H. cordata Thunb. from Mizoram, India demonstrated significant anti-inflammatory activity in both in-vitro and in-vivo models. The presence of quercetin in the extract may contribute to its anti-inflammatory properties. These findings provide scientific evidence for the traditional use of H. cordata as an anti-inflammatory agent.

Quantitative Estimation of Quercetin of Some Selected Edible Plants of West Bengal by High-performance Thin-layer Chromatography Densitometry Method

ABSTRACT Introduction: Quercetin is a polyphenol, widely found in most edible fruits and vegetables. This plant pigment provides cardiovascular, anti-inflammatory, antidiabetic, asthma, and many more health benefits. Objective: The aim of this study is to quantify the quercetin by high-performance thin-layer chromatography (HPTLC) method from the methanolic leaf extracts of three edible plants of Bengal, viz., Nyctanthes arbor-tristis L., Oxalis corniculata L., and Polygonum plebeium R.Br. Materials and Methods: HPTLC was carried out on aluminum-backed plate precoated with silica gel 60 F254, using the solvent system of toluene, ethyl acetate, formic acid, and methanol (5.5:3:1:0.5 v/v/v/v). The method validation was done according to the International Conference on Harmonization guidelines in terms of linearity, limit of detection, limit of quantification, accuracy, precision, and specificity. Results: The desired compound was well separated using the solvent system toluene, ethyl acetate, formic acid, and methanol (5.5:3:1:0.5 v/v/v/v) which gave a sharp and well-defined band with Rf 0.60 for quercetin. The calibration curves showed a good linear correlation coefficient (R 2 = 0.9774). The linearity range was found to be 20–100 (μg/spot). Among the selected plants, the highest amount of quercetin was found in the leaf extracts of O. corniculata L. (220.035 ± 0.06 mg/100 g) and the lowest for N. arbor-tristis L. (31.253 ± 0.99 mg/100 g). Conclusion: The proposed HPTLC method for quercetin estimation was found to be simple, cost-effective, precise, and accurate and can be used for the quality control of plant material. O. corniculata L. was found to be a potent source of quercetin as it possesses a decent amount of quercetin.

Guesstimate of thymoquinone diversity in Nigella sativa L. genotypes and elite varieties collected from Indian states using HPTLC technique.

Thymoquinone is a valuable metabolite derived from the Nigella sativa L. seeds and has a variety of therapeutic properties. Thymoquinone was estimated using n-hexane:ethyl acetate (8:2, v/v) green solvent system and computed at a wavelength of 254 nm using the high-performance thin-layer chromatography densitometry method in distinct varieties and genotypes congregated from different geographical regions. Genotype Ajmer Nigella-13 has the paramount thymoquinone content (247.60 µg/100 mg seed) followed by Ajmer Nigella 19 (244.5 µg/100 mg seed), while the lowest amount of thymoquinone was recorded in the genotype Ajmer Nigella-6 (42.88 µg/100 mg seed). The hierarchical cluster analysis found that the collected genotypes and elite varieties were classified into four broad clusters, and the identified chemotypes with elevated thymoquinone proportion were positioned in cluster D. Significant genotypic variation in thymoquinone content is available, that can be used in exploiting pharmaceutical applications of N. sativa L. as well as a breeding programme for specific metabolite improvement perspective.

Ecofriendly chromatographic estimation of spasmolytic pharmaceutical mixture along with official toxic impurity, 3,5-dichloroaniline: Complete green profile appraisal.

Nowadays, Green Analytical Chemistry is widely applied to provide various analytical methods with eco-friendly procedures employing the least toxic, harmful reagents on humans and the environment without affecting the efficacy of the determination. Accordingly, two eco-friendly, accurate, and reliable high-performance thin-layer chromatography-densitometry and high-performance liquid chromatographic methods were established for the determination and separation of two antispasmodic drugs, namely phloroglucinol and trimethylphloroglucinol in their pure, combined dosage form along with phloroglucinol toxic impurity, 3,5-dichloroaniline. For high-performance thin-layer chromatography-densitometry, efficient separation was developed via utilizing the stationary phase of high-performance thin-layer chromatography silica gel 60 F254 plates and developing a system comprising of ethyl acetate-butanol-ammonia in the ratio of 8.0:2.0:0.2, by volume and scanning of the developed bands at 210.0nm. The subsequent method is isocratic high-performance liquid chromatography with diode array detection in which separation was successively attained using XTerra RP-C18 (250 × 4.6mm, 5μm) column as stationary phase and methanol-10.0mM phosphate buffer, pH 3.7±0.1 as mobile phase in the ratio of 75.0:25.0, v/v at flow rate 1.0ml/min and scanning at 220.0nm. The developed liquid chromatography methods were validated according to the International Council for Harmonization guidelines, and all results acknowledged their efficacy. Additionally, the proposed methods worked well for assessing the cited drugs in binary combined commercially available pharmaceutical formulation. The greenness profile of the present methods was assessed and estimated using various assessment tools, namely; Green Analytical Procedure Index, analytical eco-scale method, National Environmental Method Index in addition to Analytical GREEnness tool to evaluate the greenness of the provided methods with a statistical comparison between them to assess the more green ones.

Quantification of piperine in different varieties of Piper nigrum by a validated high-performance thin-layer chromatography‒densitometry method

Quantification of piperine in different varieties of Piper nigrum by a validated high-performance thin-layer chromatography‒densitometry method

High performance thin layer chromatography-densitometry method based on picosecond laser-induced fluorescence for the analysis of thioridazine and its photoproducts

A densitometry method based on steady-state and time-resolved fluorescence assessments for thioridazine and its photoproducts applied on HPTLC plates has been developed. The excitation source was a picosecond diode laser emitting at 375 nm. This method was used for the analysis of the photoproducts resulted from thioridazine irradiation with 266 nm nanosecond-pulsed laser. The validation of the developed method was performed for thioridazine in terms of linearity, precision, limits of detection and quantification. Furthermore, analysis of the photoproducts of irradiated thioridazine was performed by steady-state and time-resolved fluorescence. The fluorescence spectra and fluorescence lifetime of each photoproduct were obtained and the horizontal chromatograms of fluorescence maxima were generated.

Development and Validation of Two Novel Chromatographic Methods: HPTLC and HPLC for Determination of Bromhexine Hydrochloride in Presence of Its Two Impurities.

Two simple, sensitive and validated chromatographic methods were developed for quantitative determination of bromhexine hydrochloride (BHX) in presence of its major impurities, impurity B (IMB) and impurity C (IMC), as specified by British Pharmacopoeia. First method (I) was high-performance thin layer chromatography-densitometry at which the chromatographic separation was performed using silica gel plates and developing system consisted of hexane:acetone:ammonia solution (9:0.5:0.08, by volume) with ultraviolet scanning at 240nm and linearity was achieved in the ranges of 0.40-10.00, 0.20-2.00 and 0.20-2.00μg/band of BHX, IMB and IMC, respectively. Also, second chromatographic method (II) was high-performance liquid chromatography (HPLC) where the separation was carried out on C18 column at isocratic mode at flow rate 1.5mL/min. The mobile phase consisted of methanol:water (90:10, v/v) adjusted to pH2.5 with O-phosphoric acid and temperature was adjusted to 40°C. The scanning wavelength was 240nm. The chromatographic run time was 6min. Linearity of this method was achieved in the ranges of 4.00-40.00, 0.20-10.00 and 0.50-10.00μg/mL for BHX, IMB and IMC, respectively. The validation of these chromatographic methods was made according to International Conference on Harmonization guidelines. These methods were successfully applied for determination of BHX in its pharmaceutical formulation. Also, statistical comparison was attained between the developed methods and the reported HPLC method using Student's t-test and F-test, and the obtained results showed that there was not any significant difference between them concerning with accuracy and precision.

Simultaneous quantification of vasicine and vasicinone in different parts of Justicia adhatoda using high-performance thin-layer chromatography‒densitometry: comparison of different extraction techniques and solvent systems

Simultaneous quantification of vasicine and vasicinone in different parts of Justicia adhatoda using high-performance thin-layer chromatography‒densitometry: comparison of different extraction techniques and solvent systems

Direct Quantification of Lignin in Liquors by High Performance Thin Layer Chromatography-Densitometry and Multivariate Calibration

A method for lignin quantification is presented that is based on densitometry and multivariate calibration by partial least squares (PLS). Densitometry removes interferences otherwise caused by und...

Voacamine: Alkaloid with its essential dimeric units to reverse tumor multidrug resistance

Search for natural substances in association with conventional chemotherapeutic drugs with a chemiosensitizing action easily accessible to the tumor mass has encouraged our studies on voacamine (VOA) and its monomeric units, voacangine and vobasine. Our previous results showed that VOA sensitized multidrug resistant (MDR) osteosarcoma cells (U-2 OS/DX) to doxorubicin (DOX) cytotoxicity. VOA, extracted by Peschiera fuchsiaefolia plant, is a bisindole alkaloid consisting of an Iboga skeleton (voacangine) directly linked to a 2-acyl indole unit (vobasine). High-performance thin-layer chromatography densitometry demonstrated the purity of VOA, voacangine and vobasine samples. Flow cytometry analysis showed that VOA, voacangine and vobasine enhanced DOX accumulation of U-2 OS/DX cells, in equally way, whereas VOA reduced more efficiently DOX efflux. Optical microscopy and clonogenic assay confirmed that VOA was more effective than voacangine and vobasine in enhancing DOX cytotoxic effect. These results showed that monomers linked together are necessary to modulate resistant phenotype of osteosarcoma cells. To complete the study, we evaluated the effect of three compounds on microtubules by confocal microscopy, suggesting that only the whole molecule depolymerizes the microtubules blocking so DOX efflux-mediated by vesicles.

Isolation and quantification of bioactive Carpaine from Carica papaya L. and its commercial formulation by HPTLC densitometry

Carpaine, the major alkaloid present in Carica papaya leaves, have recently been shown to have antiplasmodial activity. Traditionally, water decoctions of papaya leaves (boiling in water) have been used as a malaria preventative. In this paper Carpaine from the shade-dried leaves of C. papaya have been isolated, characterized, and quantified. A rapid, sensitive, and reproducible high-performance thin-layer chromatography (HPTLC) method was developed for the bioactive marker. Chloroform:methanol (70:30, v/v) was used as the mobile phase over pre-coated HPTLC silica gel F254 plates. The plates were developed to a distance of 60 mm at 23 ± 4 °C in a chamber previously saturated for 20 min. Under these conditions, the retention factor (Rf) of Carpaine was 0.59 and it was quantified at 550 nm, its wavelength of maximum absorbance after derivatization with Dragendorff’s reagent. Carpaine showed a linear function of the amount over the range 400–1200 ng per band; the correlation coefficient was 0.99451, indicating a good linear relationship between peak area and amount. The method was validated for precision, repeatability, and accuracy. The recovery percentage of Carpaine was found to be 101.56% w/w. It is found to be present 0.52% (w/w) in shade-dried leaves, 5.02% (w/w) in processed extract and 0.04% (w/w) in the Zandu Papaya tablet (commercial formulation).

Transfer of TLC screening methods to quantitative HPTLC–densitometry methods for pharmaceutical products containing amlodipine besylate, cefpodoxime proxetil, cetirizine 2HCl, diclofenac sodium, efavirenz, mefenamic acid, and atovaquone + proguanil HCl

Transfer of seven thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab protocols for screening counterfeit pharmaceutical products in the field to quantitative high-performance TLC (HPTLC)–densitometry methods was performed using a model process published previously. The developed and validated methods for tablets containing amlodipine besylate, cefpodoxime proxetil, cetirizine 2HCl, diclofenac sodium, efavirenz, mefenamic acid, and atovaquone + proguanil HCl involved the use of only relatively inexpensive and nontoxic solvents, Merck KGaA Premium Purity HPTLC silica gel 60 F254 plates, semi-automated sample and standard solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification. In addition, previously transferred HPTLC–densitometry methods for azithromycin and for cephalexin were used to analyze a new product of each drug to demonstrate the applicability of the methods.

Chemotaxonomic differentiation of Clerodendrum species based on high-performance thin-layer chromatographic fingerprinting of key secondary metabolites and chemometric data analysis

Clerodendrum viscosum leaves are used in indigenous systems of medicines of mainland and maritime Southeast Asian countries for the treatment of fever, pain, dysentery, colic, and removal of Ascarids. The Clerodendrum species under study exhibit various phytochemical and morphological similarities. Therefore, it is very challenging to distinguish raw powdered materials used for therapeutic purposes. A validated high-performance thin-layer chromatography (HPTLC) method with 4 key markers, viz., 24β-ethylcholesta-5,22E,25-triene-3β-O-D-glucoside, clerodinin-A, 24β-ethylcholesta-5,22E,25-triene-3β-ol, and lupeol coupled with a chemometric analysis was used to distinguish 3 closely related Clerodendrum species, viz., C. inerme, C. multiforum, and C. viscosum. PRISMA approach was applied for effective HPTLC fingerprint development. The HPTLC—densitometry method was validated following the current International Conference on Harmonisation (ICH) guidelines. Taxonomic differentiation was established by fingerprint-based similarity analysis, a chemotaxonomic study using hierarchical clustering analysis (HCA), and principal component analysis (PCA) was done. HPTLC chromatogram similarity was calculated as correlation coefficient and congruence coefficient values, demonstrating poor similarities (0.26–0.86). However, PCA has resulted in 2 principal component (PC) loadings. PC1 separated C. multiforum, explaining 85.48% of variance mainly due to distribution of 2 triterpenoids. The present HPTLC method is coupled to marker-based quality determination of raw plants as well as discrimination of Clerodendrum species. Chemometric analysis based on 4 metabolites clearly establishes a practical identification of Clerodendrum species intended for therapeutic use.

Development of quantitative HPTLC–Densitometry methods for the analysis of amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide using a model process developed earlier for the transfer of TLC screening methods

A model process, previously developed in a series of studies, allows for the transfer of thin-layer chromatography (TLC) methods for qualitative screening of counterfeit drug products published in the Global Pharma Health Fund (GPHF) Minilab manual and US Food and Drug Administration (FDA) Compendium of Unofficial Methods for Screening of Pharmaceuticals by TLC to quantitative high-performance TLC (HPTLC)–densitometry methods. This article describes HPTLC–densitometry methods developed and validated according to this model process for pharmaceutical products of amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide, for which qualitative screening methods have not been published in the Minilab manual or FDA Compendium. These methods use relatively inexpensive and nontoxic “green solvents” for sample and standard solution and mobile phase preparation, Merck Premium Purity silica gel 60 F254 plates, automated standard and sample solution bandwise application, and automated densitometry for the assessment of peak purity and identity and quantification. Corresponding to the quantitative HPTLC–densitometry methods, qualitative TLC screening methods for these drug products were developed and posted online with open access as supplements to the FDA Compendium.

Quantification of herbal drug hypoxoside from the roots of South African Hypoxis hemerocallidea using cost-effective high-performance thin-layer chromatography–densitometry validated method

Hypoxis (Hypoxidaceae) consists of about 90 species of plants reported worldwide, of which 76 occur in Africa. As many as 41 species are indigenous to countries belonging to the Southern African Development Community (SADC), including South Africa. Of all the Hypoxis species, Hypoxis hemerocallidea has versatile application in traditional health care system of over 85% of South Africans and is regarded as one of the most ethnomedicinally important and most marketed species in South Africa. H. hemerocallidea corm’s water or alcoholic extract is widely used as traditional medicine for the treatment of benign hypertrophy and urinary tract infections as well as for boosting the immune system of people living with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) among others. However, the use of other parts of Hypoxis plant as medicine is vital for conservation purposes. The roots attached on the corm of H. hemerocallidea contain hypoxoside, but the roots are usually ripped off during the preparation of Hypoxis-containing traditional medicines and other herbal products. A developed and validated, affordable but reliable high-performance thin-layer chromatography (HPTLC) densitometry for the rapid and repeatable visualization and quantitative determination of hypoxoside from the roots of H. hemerocallidea was performed. After thin-layer chromatography analysis, the hypoxoside resolved and was visualized at RF of 0.30 in CHCl3–MeOH–H20 (70:30:2 v/v). The method was linear with R2 of 0.9876 over a calibration range of 0.20 × 10−4–1.80 × 10−3 mg mL−1. The limits of detection (LOD) and quantification (LOQ) were 5.08 × 10−4 and 1.65 × 10−3 mg mL−1, respectively, while the percentage recovery and the method repeatability (%RSD) were 84.10 and 4.98, respectively. The roots of H. hemerocallidea were found to contain 4.101 × 10−4 mg mL−1 of hypoxoside.

Using High Performance Thin Layer Chromatography-Densitometry to Study the Influence of the Prion [RNQ+] and Its Determinant Prion Protein Rnq1 on Yeast Lipid Profiles.

The baker’s yeast Saccharomyces cerevisiae harbors multiple prions that allow for the creation of heterogeneity within otherwise clonal cell populations. However, in many cases, the consequences of prion infection are entirely unclear. Predictions of prion-induced changes in cell physiology are complicated by pleotropic effects, and detection is often limited to relatively insensitive cell growth assays that may obscure many physiological changes. We previously showed that silica gel high performance thin-layer chromatography-densitometry (HPTLC) can be used to empirically determine prion-induced changes in lipid content in yeast. Here, we conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-glacial acetic acid (80:20:1, v/v/v) mobile phase and phosphomolybdic acid (PMA) detection reagent]; steryl esters, methyl esters, and squalene [hexane-petroleum ether-diethyl ether-glacial acetic acid (50:20:5:1, v/v/v/v) and PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5, v/v/v) and cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([RNQ+]) and prion-free ([rnq−]) cells in both stationary- and logarithmic-growth phases. We detected multiple statistically significant differences between prion-infected and prion-free cells that varied by growth phase, confirming our pr evious observations that prions exert distinct influences on cell physiology between stationary- and log-phase growth. We also found significant differences between cells expressing or lacking the Rnq1 protein which forms the [RNQ+] prion, providing new clues to the as yet unresolved normal biological function of this prion-forming protein. This investigation further emphasizes the utility of HPTLC-densitometry to empirically determine the effects of prions and other presumed innocuous gene deletions on lipid content in yeast, and we expect that additional analyses will continue to resolve the physiological effects of prion infection.

Application of High Performance Thin Layer Chromatography-Densitometry and UV- Visible Spectrophotometry for the Simultaneous Determination of Thiamine in Green Beans

Application of High Performance Thin Layer Chromatography-Densitometry and UV- Visible Spectrophotometry for the Simultaneous Determination of Thiamine in Green Beans

Development of quantitative HPTLC–densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of cefixime, cefuroxime axetil, cephalexin⋅H2O, ciprofloxacin HCl, levofloxacin, and metronidazole

Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund Minilab kit protocols for detecting fake or markedly substandard drugs in pharmaceutical products in the field to quantitative high-performance TLC (HPTLC)–densitometry methods was carried out using a model process published earlier. The developed and validated methods for tablets or capsules containing cefixime, cefuroxime axetil, cephalexin·H2O, ciprofloxacin HCl, levofloxacin, and metronidazole involved use of EMD Millipore Premium Purity silica gel 60 F254 plates, automated sample and standard solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification.

Development and validation of a high-performance thin-layer chromatography—densitometry method for the quantitative estimation of morphine in the classical Ayurvedic formulation Kamini Vidrawan Ras

Kamini Vidrawan Ras is a classical Ayurvedic medicine, referred to in Bhaisjya Ratnavali, a book recognized by the Drugs and Cosmetics Act of India. Its usefulness has been stated in erectile dysfunction, impotence, and premature ejaculation. Opium is the major ingredient of formulation which is highly addictive as it contains narcotic alkaloids (morphine, codeine, and thebaine). Opium is a natural product; hence, the morphine content varies from 4–21%. The aim of this study was to develop a simple, precise, rapid, and reliable high-performance thin-layer chromatography— densitometry method for the quantitative estimation of morphine in the tablets of the said Ayurvedic medicines. Aluminum-backed silica gel F254 (20 cm × 10 cm) was used as the stationary phase and ethyl acetate, methanol, and ammonia (85:10:5, v/v) as the mobile phase. The RF value for morphine was 0.36, and the quantitative evaluation of the bands over plates was performed in the reflectance—absorbance mode at 280 nm. The regression anal...