Performance Thin Layer Chromatographic Method Research Articles

Correction to: A validated high performance thin layer chromatography method for the simultaneous estimation of berberine, berbamine, palmatine, magnoflorine and jatrorrhizine from Berberis aristata

Correction to: A validated high performance thin layer chromatography method for the simultaneous estimation of berberine, berbamine, palmatine, magnoflorine and jatrorrhizine from Berberis aristata

A validated HPTLC method for the simultaneous determination of seasonal alterations of two antihypertensive monoterpenoid indole alkaloids in Rauvolfia species from northern India

The genus Rauvolfia belongs to the family Apocyanaceae, and is native to the tropical countries of the world. Six species of Rauvolfia have been reported from India, however, only two species viz. R. serpentina and R. tetraphylla grow in the upper Gangetic plains. The monoterpenoid indole alkaloids reserpine and ajmalicine are the major phytochemicals reported from the roots of the above plants and are well known for their potential antihypertensive activity. Presently, R. serpentina has become critically endangered due to its overexploitation. Therefore, this study was designed to develop a validated High Performance Thin Layer Chromatographic densitometric method for determination of reserpine and ajmalicine in R. serpentina and R. tetraphylla in different seasons. Plant materials were collected in March, June, September and December to determine the optimal harvesting time. The densitometric method was validated in terms of instrument precision, range, linearity, specificity, limit of detection and limit of quantitation. Reproducibility of the peak of reserpine and ajmalicine was achieved at Rf 0.56, and 0.79 respectively using optimised mobile phase toluene– ethyl acetate-diethyl amine (7:2:0.6 v/v/v). Our study concluded that maximum level of ajmalicine was found in March while that of reserpine was observed in September. Harvesting of R. tetraphylla at the optimal season may reduce the overexploitation of the endangered species R. serpentina.

Application of DoE-Based Analytical QRM to Development of the Multipurpose RP-HPLC Method for Estimation of Multiple FDC Products of Telmisartan Using Enhanced AQbD Approach.

Telmisartan is an antihypertensive drug and several FDC products of telmisartan are available in the market for the treatment of hypertension. The multipurpose reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated as economical and eco-friendly alternative to numerous published chromatography methods for synchronous estimation of FDC of telmisartan to save time, cost and solvent for analysis. The analytical quality risk management was initiated with identification of potential method parameters using cause-effect diagram followed by assessment of their risk by risk priority number ranking and filtering method. The risk of critical method parameters was controlled by their optimization using design of experiments-based full-factorial design. The method operable design ranges was identified for high-risk method parameters, and control strategy was set for mitigation of their risk throughout the life cycle of the developed RP-HPLC method. The RP-HPLC method was validated as per the ICH Q2 (R1) guideline. The RP-HPLC method was applied for simultaneous estimation of seven FDC products of telmisartan, and results were found compliance with labeled claim. The developed RP-HPLC method is fulfilling requirements of numerous RP-HPLC and high performance thin layer chromatography methods for the said estimation. It can be used as a multipurpose RP-HPLC method for quality control of FDC products of telmisartan in the pharmaceutical industry to save solvent, cost and time of analysis.

Lacidipine: review of analytical methods developed for pharmaceutical dosage forms and biological fluids.

Lacidipine (LAC) is a calcium antagonist used in the treatment of hypertension. It is a lipophilic drug containing dihydropyridine ring that is responsible for the activity. This review article gives an overview of various analytical techniques proposed for the determination of LAC in pharmaceutical dosage forms, in pure form, in biological fluids and to determine characteristics of LAC in modified release dosage forms. Ultra violet/visible spectrophotometric, spectroflourimetric, high performance liquid chromatography, high performance thin layer chromatography, electro-analytical, bioanalytical and miscellaneous methods such as microbiological assay, X-ray diffraction, differential scanning calorimetry, were discussed. Various parameters such assystem suitability, selectivity, linearity, precision, accuracy, limit of detection, limit of quantification and robustness have been discussed for the employed methods.

Development of Validated Stability-indicating High Performance Thin Layer Chromatography Method for Estimation of Rabeprazole Sodium and Aceclofenac in Bulk Drug

Objective: To develop a validated stability-indicating high performance thin layer chromatography method for the estimation of Rabeprazole Sodium (RZL) and Aceclofenac (ACF) in bulk drugs.
 Methods: A high performance thin layer chromatographic (HPTLC) method has been developed for the separation of RZL & ACF on plates precoated with aluminium back silica gel 60 F254. Different mobile phases were used on trial and error basis for separation of two drugs. The final mobile phase selected for analysis was toluene: ethyl acetate: methanol: acetic acid: ammonia in the ration of 6:4:1:0.2:0.1 (v/v). Both the drugs showed maximum absorbance at 279 nm which was selected as the detection wavelength throughout the experimental work. Developed method was validated as per ICH guidelines. Forced degradation of drugs was carried out under various stress conditions and HPTLC method was used for analysing the stability of drugs.
 Results: HPTLC method was successfully developed for separation of RZL and ACF with clear separation of bands of the drugs. Method validation after assessment of various parameters indicated low % RSD within an acceptable limit of < 2.0 and the stability studies indicated the satisfactory separation of both the drugs from that of degraded products with considerable % recovery profile.
 Conclusion: The developed method is rapid, reliable, precise, and reproducible and demonstrates the suitability of the method for stability determination of rabeprazole and aceclofenac.

Stability Indicating Simultaneous Estimation of Cetirizine Hydrochloride and Phenylephrine Hydrochloride in Combined tablet formulation by using HPTLC- Densitometric method

The new simple, precise, rapid, selective, high performance thin layer chromatography method has been developed for simultaneous estimation of cetirizine hydrochloride and phenylephrine hydrochloride in its combined solid dosage form. The HPTLC method was performed on precoated silica gel G60 F254 plates with, Toluene: Methanol: Ammonia (8: 2: 1, v/v/v) as optimum mobile phase. The plates were developed in 6.00× 0.30mm at room temperature. The developed plates were scanned and quantified at their absorption at 224 nm for CET and PHE respectively. The drugs were satisfactorily resolved with Rf = 0.36± 0.001 for CET and 0.65±0.004 for PHE. Calibration plot was linear between 100-600 ng/ spot for CET and 200-1200 ng/ spot for PHE. This HPTLC method is sensitive, economical, less time consuming than other chromatographic procedures. It can become important tool for analysis of combined solid dosage from. In addition to this results acid exposure, base exposure, oxidative exposure, neutral exposure, photolysis and dry heat exposure is carried out.

Determination of the effect of germination on the folate content of the seeds of some legumes using HPTLC-mass spectrometry-multivariate image analysis

Legumes are the main sources of folates which are not synthesized in the human body. The five folate species: 5-methyl tetrahydrofolate, tetrahydrofolate, pteroyl glutamate, 5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate were quantitatively determined in legumes seeds and sprouts by a newly developed and validated high performance thin layer chromatography method. High resolution plate imaging hyphenated to mass spectrometry was exploited for fingerprint analysis of tested samples. Results indicated that germination of all seeds resulted in a 2.5–4 fold increase in the content of total folates as well as the individual vitamers. The total amount of folate reached a maximum on the fifth day in the case of black-eyed peas (861 μg/100 g Fresh Weight), white beans (755 μg/100 g FW) and brown lentils (681 μg/100 g FW). 5-CH3-H4 folate was found to be the most dominating folate species reaching its maximum content in day 5 sprouts of black-eyed peas (490 μg/100 g FW).

Application of the thin-layer chromatography method for analysis of plant antioxidant activity

Plants are a rich source of effective non-toxic biologically active substances. Various physicochemical methods of analysis are used for evaluation of plant antioxidant activity. Composition of ethanol extracts of red, yellow and white onion husks, dried rosemary, basil, and chaga were studied by high performance thin layer chromatography (HPTLC) method. The antioxidant activity of the obtained fractions on a chromatographic plate was assessed by subsequent DPPH screening. The extracts red and yellow onion husk and rosemary demonstrated the highest antioxidant activity, variability of the qualitative composition and similarity of antioxidant profiles, while extract of white onion husks did not contain any antioxidant classes. Intensive spots with Rf of 0.13-0.97 were observed along the whole chromatogram track corresponding to red onion husks. It was also found that all tested extract, excepting white onion husk and chaga, contained spots with varying degrees of intensity in the Rf range of 0.96-0.98, which corresponded quercetin Rf value.

Validated HPTLC Method to Estimate Silymarin in Marketed Herbal Formulation

Objectives: The objective of present research work is to develop simple, rapid and accurate high performance thin layer chromatographic method for quantitative estimation of silymarin in marketed hepatoprotective formulation. Methods: The separation was performed on silica gel 60 F254 precoated TLC aluminum plates and toluene: ethyl acetate: formic acid: methanol as mobile phase. Determination and quantitation were performed by densitometric scanning at 288 nm. Validation of method is carried out as per International Conference of Harmonization (ICH) guidelines. Results: Accuracy, precision and recovery were all within the required limits. Conclusion: In conclusion, the developed HPTLC method is precise and accurate for implementation in routine quality control. The selected marketed hepatoprotective formulation contains the sufficient amount of the active ingredient which is required for its therapeutic efficiency.

Eco-Friendly Direct GC–MS Method for Estimation of Niacin and Related Impurities Involving Pyridine in Food Supplements

Niacin is a water-soluble vitamin whose deficiency causes many disorders and diseases, including pellagra and high blood cholesterol. Herein, niacin and four common impurities, isonicotinic acid (ISO), 5-ethyl-2-methylpyridine (MP), pyridine-2, 5-dicarboxylic acid (PDC) and pyridine PYR, are simultaneously analyzed, where PYR is known as potentially nephrotoxic and hepatotoxic. The separation of a mixture using gas chromatography–mass spectroscopy (GC–MS) without any derivatization steps was the main target. Many studies have been published to study Niacin and its impurities using colorimetry and HPLC. GC–MS was selected to study the analyzed mixture owing to its known sensitivity and selectivity. In this study, a single quadrupole mass spectrometer operated in selected ion monitoring (SIM) mode at an electron ionization energy of 70 eV was applied for the quantitative analysis of Niacin. Helium was used as the carrier gas and adjusted to run through an HP-5ms (5%-phenyl)-methylpolysiloxane column. Statistical analysis proved that this method is equally effective as the previously reported method. Importantly, this study was eco-friendly as compared to the reported high performance thin layer chromatographic method (HPTLC). Finally, this study provides a new and valid eco-friendly method analysis to determine the concentration of niacin and its common impurities at very low concentration. Conclusion: A simple, rapid accurate and green GC–MS analytical method was developed and validated to determine niacin and its related official impurities.

Simultaneous Quantification of Quercetin and Syringic Acid in Methanolic Extract of Leucas lavandulifolia by using Validated HPTLCDensitometric Method

A simple, sensitive and reliable High Performance Thin Layer Chromatography (HPTLC) method was developed for the simultaneous quantification of bioactive compounds i.e quercetin and syringic acid in Leucas lavandulifolia. Methanolic extract used on HPTLC silica gel 60 F254, 10×10 (Mark) plate for the separation and quantification of bioactive compounds by using toluene: ethyl acetate: formic acid (7: 2.5: 0.5 v/v) as mobile phase. The used mobile system gives well-resolved bands of quercetin (Rf value 0.32 ± 0.06) and syringic acid (Rf value 0.38 ± 0.05) in the extract of L. lavandulifolia. The correlation coefficient was found to be 0.987 and 0.997 with standard deviation of 667.77 and 3819.77 for quercetin and syringic acid. The accuracy of the method was confirmed by recovery studies at different levels using the standard addition method. Precision studies (both inter day and intraday) are within the standard limit, RSD (%) is less than 3%. The average recovery of quercetin and syringic acid was 100.02% and 100.13% suggesting the accurateness of the method. The quantification of quercetin and syringic carried out by using a densitometric absorption mode at 275 nm and 370 nm respectively. The developed method was validated and found to be specific, linear and accurate with precision and accuracy in the concentration range of 2.0–6.0 μg/spot for quercetin and syringic acid. Hence, simple, reproducible and selective HPTLC method has been developed for the estimation of the identified bioactive markers which may be utilized as a tool for the proper authentication and standardization of L. lavandulifolia.

Estimation of Adrographolide in Andrographis paniculata Herb, Extracts and Dosage forms

ABSTRACT Andrographolide is an anti-anti-inflammatory, antipyretic, anti-viral, immunostimulatory,anti-hyperglycemic, and anti-oxidant agent. High performance liquid chromatographic(HPLC) and high performance thin layer chromatographic (HPTLC) methods for quantitativedetermination of andrographolide were developed and validated. The methods showed satisfactorylinearity, precision, good recovery and appropriate limits of detection (LOD) and quantification(LOQ). The content of andrographolide was determined and the results obtained by HPLCand HPTLC methods were in good agreement. The methods developed are suitable for the qualitycontrol applications in Andrographis paniculata plant, extracts and dosage forms.

Enumerate chlorogenic acid in different forms of coffee by simple high performance thin layer chromatographic method

Enumerate chlorogenic acid in different forms of coffee by simple high performance thin layer chromatographic method

Enumerate chlorogenic acid in different forms of coffee by simple high performance thin layer chromatographic method

The health benefit of coffee consumption mainly depends on chlorogenic acid (CGA), one of its major active phenolic constituent. CGA have several biological properties like anticancer, antihypertensive, hepatoprotective, neuroprotective, renoprotective, antimicrobial etc. It is assumed that different forms of coffee contains different amount of CGA. In this regards, we developed and validated a simple and cost effective chromatographic technique to identify and quantify CGA in green coffee, roasted coffee and branded instant coffee available in Indian market.

Development of High Performance Thin Layer Chromatography Method for Quantitation of Gallic Acid in Normacid Syrup: A Poly Herbal Formulation

Normacid syrup consisiting of 17 ingredients, used in the treatment of hyperacidity and gastritis. Gallic acid is one of the phytoconstituent present in Normacid syrup- a poly herbal formulation. In the present study an attempt has been made to develop a HPTLC method for quantitative estimation of gallic acid in dried fruits used in formulations. This HPTLC method was found to be reproducible, accurate, precise and detect gallic acid at nanogram level. The developed HPTLC method would be an important tool in the quality control method of poly herbal formulation. 
 Keywords: HPTLC, Polyherbal, Gallic acid, Gastritis

Research of the physical and chemical properties and methods of Red rice (food colour) determination in sausage products

In Russia, in the production of meat and sausage products, the food colour, named as Red rice, is use. Red rice is obtain by the cultivation the strains of the Monascus fungus on various carbohydrate substrates, for example rice. That Red rice may contain the mycotoxin citrinin, but neither the purity of the food colour nor the safety profile are regulated. The aim of this work was to study the physical and chemical properties of Red rice and to develop method for its determination in sausage products. The experiments were carried out on model and commercial samples of sausages. The samples were analyzed using spectrophotometry and highefficiency planar and liquid chromatography. Spectrophotometric analysis revealed differences in solubility, coloring power, spectral characteristics, composition and ratio of pigments in commercial samples of Red rice. The extraction parameters of colouring substances from sausages model samples were determined. It has been established that Red rice is extracted with chloroform, acetone, ethanol and its aqueous solutions. Petroleum ether was proposed for degreasing sausages. Red rice from sausages was extracted with acetone because it did not extract synthetic colours and carminic acid. It was found, that 90% of Red rice is extracted from samples of sausages by double extraction with ultrasonic treatment with a power of 128 W. High performance thin layer chromatography method and high performance liquid chromatography method for the Red rice identification was proposed. The content of Red rice in the extracts was determined by spectrophotometric method. The developed method for the determination of Red rice was tested on commercial samples of sausage products.

Isolation, quantification and characterisaion of alkaloids from Eulophia ochreata Lindl by HPTLC analysis

In healthcare system, the development and modernization of phytomedicine in phytochemistry play a central and important role in drug development. Isolation and purification of bioactive compounds from naturally occurring substance is of prime importance. A sensitive and reliable high performance thin layer chromatography method has been developed to estimate gallic acid in ethanolic extract of Eulophia ochreata Lindl. Ethanolic extract prepared as applied on silica gel G 60 F254plate. The plate was developed using Toluene: Ethyl acetate: Methanol: Formic acid(6:6:0.4:1.6) as a mobile phase and detection and quantification were performed by densitometric scanning at 275nm. The system was found to give well resolved bands for alcoholic extract having Rf values as 0.8 was matched with the standard Rf values like 0.82. The densitometric chromatogram of HPTLC fingerprint of the alcoholic extract, isolate and standard gallic acid was obtained. The calibration curve of gallic acid was linear over a concentration range (0.2- 2 microg/ml) with a good correlation coefficient (R2= 0.9986) and coefficient of variation as CV- 2.4663%. The method was validated for linearity, precision, specificity and it was found to be precise, reliable, and suitable. The proposed method is simple, rapid, precise and accurate. The method was suitable for qualitative and quantitative analysis of gallic acid in the alcoholic extract of Eulophia ochreata Lindl.

Development and Validation of HPTLC Method for Studying Stress Degradation of Aspirin in Fulvic Acid Inclusion Complex

A new, precise high performance thin layer chromatographic (HPTLC) method for the analysis aspirin (ASP) in inclusion complexes with HPβCD and fulvic acid (FA) was developed and validated as per ICH guidelines. A precoated silica gel aluminium plate 60F-254 and a mixture of solvents, toluene: ethylacetate: formic acid (5:4:1 v/v) were used as stationary and mobile phase, respectively. This developed method was found to give an excellent defined sharp peak at a retention factor (RF) value of 0.52 ± 0.001. The LOQ and LOD values were found 35.29 and 123.54 ng / spot, respectively. The spray dried inclusion complexes of ASP/HPβCD and ASP/FA in the molar ratio 1:1, were subjected for forced degradation under stress conditions, and a significant reduction of ASP degradation were noted in complexed ASP as compared to ASP alone.

Quantitative Determination of Aloin in Aloe vera and Its Antioxidant Activity

Aloe vera is a popular medicinal plant used widely by the cosmetic, pharmaceutical and food industries. The Aloe vera gel, which is used mostly for its positive effects on human health,contains over 75 different bioactive compounds, including aloin. A sensitive and reliable densitometric High Performance Thin Layer Chromatography method has been developed for the quantification of aloin, an anthraquinone present in Aloe vera leaves. Chromatographic analysis was performed using methanol extract of leaves of Aloe vera using solvent system ethyl acetate: Methanol: water (100:13.5:10). Detection and quantification of aloin was done by densitometric scanning at 350nm. The results of linearity range and correlation coefficient show that there was a good correlation between peak area and corresponding concentration of aloin. The method developed here in can be implemented in the analysis and routine quality control of herbal materials. Antioxidant activity of Aloe vera was also done by using DPPH method and found that Aloe vera is also a natural antioxidant.

Structural characterization of plant glucosylceramides and the corresponding ceramides by UHPLC-LTQ-Orbitrap mass spectrometry

Ceramides (CERs) play a major role in skin barrier function and direct replacement of depleted skin CERs, due to skin disorder or aging, has beneficial effects in improving skin barrier function and skin hydration. Though, plants are reliable source of CERs, absence of economical and effective method of hydrolysis to convert the dominant plant sphingolipid, glucosylceramides (GlcCERs), into CERs remains a challenge. This study aims at exploring alternative GlcCERs sources and chemical method of hydrolysis into CERs for dermal application. GlcCERs isolated from lupin bean (Lupinus albus), mung bean (Vigna radiate) and naked barley (Hordium vulgare) were identified using ultra high performance liquid chromatography hyphenated with atmospheric pressure chemical ionization - high resolution tandem mass spectrometer (UHPLC/APCI-HRMS/MS) and quantified with validated automated multiple development-high performance thin layer chromatography (AMD-HPTLC) method. Plant GlcCERs were hydrolyzed into CERs with mild acid hydrolysis (0.1 N HCl) after treating them with oxidizing agent, NaIO4, and reducing agent, NaBH4. GlcCERs with 4,8-sphingadienine, 8-sphingenine and 4-hydroxy-8-sphingenine sphingoid bases linked with C14 to C26 α-hydroxylated fatty acids (FAs) were identified. Single GlcCER (m/z 714.5520) was dominant in lupin and mung beans while five major GlcCERs species (m/z 714.5520, m/z 742.5829, m/z 770.6144, m/z 842.6719 and m/z 844.56875) were obtained from naked barley. The GlcCERs contents of the three plants were comparable. However, lupin bean contains predominantly (> 98 %) a single GlcCER (m/z 714.5520). Considering the affordability, GlcCER content and yield, lupin bean would be the preferred alternative commercial source of GlcCERs. CER species bearing 4,8-sphingadienine and 8-sphingenine sphingoid bases attached to C14 to 24 FAs were found after mild acid hydrolysis. CER species with m/z 552.4992 was the main component in the beans while CER with m/z 608.5613 was dominant in the naked barley. However, CERs with 4-hydroxy-8-sphingenine sphingoid base were not detected in UHPLC-HRMS/MS study suggesting that the method works for mainly GlcCERs carrying dihydroxy sphingoid bases. The method is economical and effective which potentiates the commercialization of plant CERs for dermal application.