High-performance Thin-layer Chromatography Quantification Research Articles

Quality control in pharmaceutical analysis by diode-array thin-layer chromatography: quantification of trimethoprim and sulfamethoxazole as a case study

The combination of trimethoprim and sulfamethoxazole as a fixed-dose combination in the ratio 1:5 is known as cotrimoxazole. It is used as antibiotic to treat a variety of bacterial infections. Cotrimoxazole is part of the World Health Organization’s list of essential medicines. Cotrimoxazole is an example of a drug that was partially unavailable in Germany during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the Ukraine war. The dependency on foreign sources of medicines is well known in Europe and resulted in the Pharmaceutical Strategy for Europe 2020, a strategy concept “will support the competitiveness and innovative capacity of the EU’s pharmaceutical industry”. High-performance thin-layer chromatography (HPTLC) is a cost-effective method for quantifying pharmaceutically active compounds. Diode-array detection (DAD) in conjunction with HPTLC can simultaneously detect ultraviolet‒visible (UV‒VIS) and fluorescence spectra directly from the plate. Visualization as a contour plot helps to identify the optimal wavelengths for compound quantification and reduce uncertainty in the determination. The quantification of trimethoprim and sulfamethoxazole is presented in a case study that highlights the key aspects for HPTLC quantification of pharmaceutical fixed-dose combinations with minimal uncertainty. HPTLC‒DAD allows quantification of trimethoprim and sulfamethoxazole with a required relative standard deviation of less than 2.5%.

In-vitro anti-urolithiatic activity and simultaneous HPTLC quantification of berberine and palmatine in standardized extract of Thalictrum foliolosum DC.

Thalictrum foliolosum DC. (Ranunculaceae) is a perennial plant distributed worldwide. In India it is used as a diuretic, anti-periodic, febrifuge, jaundice and in rheumatism. The present study was aimed to evaluate in-vitro anti-urolithiatic, anti-oxidant, anti-inflammatory activity along with concurrent quantification of berberine and palmatine through high performance thin layer chromatography (HPTLC). Anti-urolithiatic potential was evaluated via a number of different steps viz. nucleation, aggregation, whole urine assays and through kinetic study. The IC 50 value of nucleation and aggregation was found to be 114.314 and 67.625 µg/mL. Anti-oxidant activity was evaluated by DPPH and total antioxidant capacity with IC 50 value 54.96 µg/mL and 11.35 mg/ g anti-oxidant capacity. Anti-inflammatory potential was evaluated through inhibition of protein denaturation and antiproteinase activity, the IC 50 values were 94.61 and 60.51 µg/mL. Separation of berberine and palmatine was done on an aluminum TLC plate precoated with silica gel 60 GF 254 (10 × 10 cm), with mobile phase containing n-propanol-water-formic acid (90–10–1 v/v). Berberine and palmatine were identified at R f 0.44 ± 0.05 and 0.29 ± 0.06. Densitometric analysis was carried out at 350 nm for both the biomarkers in absorbance-reflectance mode. Berberine and palmatine concentrations were found to be 1.155 ± 0.035 and 0.111 ± 0.008 in plant on% dry weight basis. The result indicates that T. foliolosum has significant antioxidant and anti-inflammatory activity, and it is a potential candidate for phytotherapy against urolithiasis because of its ability to inhibit CaOx crystallization. Quantification of biomarkers will aid in quality standardization of raw material. • In-vitro anti-urolithiatic potential of Thalictrum foliolosum DC. root was evaluated. • In-vitro antioxidant and anti-inflammatory activity was also done to support the anti-urolithiatic potential of plant. • Two biomarkers berberine and palmatine were quantified through HPTLC.

Plicosepalus acacia Extract and Its Major Constituents, Methyl Gallate and Quercetin, Potentiate Therapeutic Angiogenesis in Diabetic Hind Limb Ischemia: HPTLC Quantification and LC-MS/MS Metabolic Profiling.

Plicosepalus acacia (Fam. Loranthaceae) has been reported to possess hypoglycemic, antioxidant, antimicrobial, and anti-inflammatory effects. Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis revealed the presence of a high content of polyphenolic compounds that are attributed to the therapeutic effects of the crude extract. In addition, methyl gallate and quercetin were detected as major phytomedicinal agents at concentrations of 1.7% and 0.062 g%, respectively, using high-performance thin layer chromatography (HPTLC). The present study investigated the effect of the P. acacia extract and its isolated compounds, methyl gallate and quercetin, on hind limb ischemia induced in type 1 diabetic rats. Histopathological examination revealed that treatment with P. acacia extract, methyl gallate, and quercetin decreased degenerative changes and inflammation in the ischemic muscle. Further biochemical assessment of the hind limb tissue showed decreased oxidative stress, increased levels of nitric oxide and endothelial nitric oxide synthase (eNOS), and enhancement of the levels of heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) in the groups treated with methyl gallate and quercetin. Expression levels of hypoxia inducible factor-1 alpha (HIF-1α), VEGF, fibroblast growth factor-2 (FGF-2), and miR-146a were upregulated in the muscle tissue of methyl gallate- and quercetin-treated groups along with downregulation of nuclear factor kappa B (NF-κB). In conclusion, P. acacia extract and its isolated compounds, methyl gallate and quercetin, mediated therapeutic angiogenesis in diabetic hind limb ischemia.

Compounds from Vernonia arborea Buch.-Ham. Inhibit Microbes that Impair Wound Healing

Aims: To identify the antimicrobial potency of the leaf fractions of Vernonia arborea against selected wound microbes viz., Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Stenotrophomonas maltophilia.
 Background: Wound healing is often delayed due to the presence of polymicrobial load, that have to be abolished to facilitate the healing process. A major class of antimicrobial phytocompound reported to occur in Vernonia arborea species include sesquiterpenes. Reports on the wound healing potency of V. arborea in wound models of Wistar rats however did not report antimicrobial activity of the aqueous or methanolic extracts.
 Methodology: The column fractions of the hexane leaf extract were tested against the selected strains by agar well diffusion assay and the zone of inhibition confirmed with TLC bioautography at specific Rf. The minimum inhibitory concentration (MIC) of the bioactive fractions was identified using resazurin microtiter assay (REMA) and the minimum bactericidal concentration (MBC) was determined. HPTLC quantification was also performed.
 Results: Out of the 30 pooled fractions, six showed antimicrobial potency against all the five tested wound microbes. The minimum inhibitory concentrations of these fractions were determined, ranging from 15.62 µg/mL to 500 µg/mL for the different microbes. Quantitative High-Performance Thin Layer Chromatography (HPTLC) revealed two compounds (a and b) in the bioactive fraction10 with yields of 633 mg (63%) and 97 mg (9.7%) per gram of the extract.
 Conclusion: The findings suggest the potential use of the bioactive compound in chronic infectious wound management therapy.

Thalictrum foliolosum DC: An unexplored medicinal herb from north western Himalayas with potential against fungal pathogens and scavenger of reactive oxygen species

The aim of the present study was to evaluate the antifungal, antioxidant activity and quantification of berberine in different solvent extracts of leaves of Thalictrum foliolosum DC. Cold maceration method was employed for the preparation of extracts using solvents of different polarities (petroleum ether, chloroform, and methanol). Total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, and antifungal activity of T. foliolosum extracts were investigated using various in vitro assays. A high-performance thin-layer chromatography (HPTLC) method was used for the quantification of berberine. In silico studies confirmed the results of in vitro assays and showed that berberine showed a good binding affinity with cytochrome P450 14 alpha-sterol demethylase (CYP51) (antifungal receptor protein) and human peroxiredoxin 5 (antioxidant receptor). TPC and TFC were highest in methanolic extract. HPTLC quantification of berberine showed a higher amount of berberine in chloroform extract followed by methanol extract). Chloroform extract showed the highest antifungal activity as indicated by a diameter of zone of inhibition and lower minimum inhibitory concentration (MIC) value against fungal strains [Candida albicans (ATCC90028), C. albicans (MTCC277) and Saccharomyces cerevisiae (H1068)]. Among three extracts, maximum antioxidant activity was shown by chloroform extract as evident from the lowest half-maximal inhibitory concentration (IC50) value with DPPH and FRAP assay. Molecular docking study also showed that berberine interacts strongly with cytochrome P450 14 alpha-sterol demethylase (CYP51) and human peroxiredoxin 5 proteins. Thus, T. foliolosum leaves can be exploited as an alternative source of berberine, with potent antioxidant and antifungal activity.

Simple and Accurate HPTLC-Densitometric Method for Quantification of Delafloxacin (A Novel Fluoroquinolone Antibiotic) in Plasma Samples: Application to Pharmacokinetic Study in Rats.

Delafloxacin (DLX) is a recently-approved fluoroquinolone antibiotic, which is recommended for the treatment of “acute bacterial skin and skin structure infections”. A thorough literature survey revealed only a single published method for the estimation of DLX using UPLC-MS/MS technique in biological samples. There is no high-performance thin-layer chromatography (HPTLC) method has been reported for the estimation of DLX in dosage forms and/or biological samples. Therefore, a selective, sensitive, rapid and validated HPTLC-densitometry technique has been used for the estimation of DLX in human plasma for the first time. HPTLC quantification of DLX and internal standard (IS; gatifloxacin) was carried out on glass coated silica gel 60 F254 HPTLC plates using the ternary mixture of ethyl acetate:methanol:ammonia solution 5:4:2 (%, v/v/v) as the mobile phase. Densitometric detection was done at 344 nm. The Rf values were recorded as 0.43 and 0.27 for the DLX and the IS, respectively. The linearity range of DLX was obtained as 16–400 ng/band. A simple protein precipitation method was used for the extraction of analyte from plasma using methanol. The proposed HPTLC technique was validated for “linearity, accuracy, precision, and robustness”. The proposed HPTLC technique was successfully utilized for the assessment of pharmacokinetic profile of DLX in rats after oral administration. After oral administration, the peak plasma concentration of DLX was obtained as 194.19 ng/ml in 1 h. The proposed HPTLC method could be applied in study of pharmacokinetic profile and therapeutic drug monitoring of DLX in clinical practice.

Simultaneous microwave assisted extraction and HPTLC quantification of mangiferin, amarogentin, and swertiamarin in Swertia species from Western Himalayas

Swertia is one of the most widely traded genus of 150 species, many of which are used in Ayurvedic, Chinese, Tibetan and traditional medicine. Swertia herb is mostly traded and exported in dried or in extract forms. In this present study, a microwave-assisted extraction (MAE) method coupled with simultaneous quantification of swertiamarin, amarogentin, and mangiferin from different species of Swertia using high performance thin layer chromatography (HPTLC) was developed. A three-level factor Box-Behken statistical design was used for optimization, where microwave power, irradiation time, and solvent-to-solid ratio were used as independent variables whereas swertiamarin, amarogentin and mangiferin were used as the dependent variables. Densitometric-HPTLC method was developed for the simultaneous estimation of swertiamarin, amarogentin, and mangiferin using mobile phase of ethyl acetate: acetic acid glacial: formic acid: water (10:1.2:1.2:2.7 v/v/v/v) and stationary phase as silica gel F254 HPTLC plate. In this study, five different Swertia species viz. S. angustifolia, S. cordata, S. chirata, S. paniculata, and S. nervosa collected from Western Himalayan, India were used to screen the elite Swertia species. Optimal extraction conditions to achieve the maximum yield of swertiamarin, amarogentin, and mangiferin were found to be 5.86%, 0.710%, and 4.93% respectively which were very close to the predicted values of 5.916%, 0.711%, and 5.084% respectively by keeping microwave power (530 W), time (4 min), and solvent-to-solid (30 mL). Densitogram patterns obtained from all the test samples and standard compounds revealed that the peaks corresponding to Rf 0.57, 0.64, and 0.74 were meant for swertiamarin, mangiferin, and amarogentin respectively. HPTLC studied showed that S. paniculata and S. angustifolia contained high content of bioactive compounds and could be used as a substitute for S. chirata, whereas S. cordata and S. nervosa could not be recommended as substitutes for S. chirata. Thus, the newly developed optimized MAE and HPTLC-densitometry method for simultaneous quantification of three bioactive marker compounds in five species of Swertia can be effectively implemented for quality control of herbal formulations.

Novel sublingual tablets of Atorvastatin calcium/Trimetazidine hydrochloride combination; HPTLC quantification, in vitro formulation and characterization

BackgroundIschemic heart disorders and accumulation of lipids in blood vessels could contribute to angina pectoris. Therefore, the aim of this study was to formulate sublingual tablets containing a novel combination of Atorvastatin calcium (ATOR) and Trimetazidine HCl (TMZ) for efficient treatment of coronary heart disorders. MethodsThe dissolution rate of water-insoluble ATOR was enhanced via complexation with sulfobutyl ether-β-cyclodextrin (SBE-β-CD) and addition of soluplus as a polymeric solubilizer excipient. The solubilized ATOR and TMZ were compressed into a sublingual tablets by direct compression technique and evaluated for their tableting characteristics. In addition, a new validated method based on High Performance Thin Layer Chromatography (HPTLC) was developed for simultaneous determination of both drugs in pure forms and sublingual tablets. ResultsThe developed HPTLC method showed LODs of 0.056 and 0.013 μg/band and LOQs of 0.17, 0.040 μg/band for TMZ and ATOR, respectively and proved to be linear, accurate, precise and robust. The optimum formulation containing mixture of superdisintegrants; Ac-Di-Sol and crospovidone (4.8% w/w, each) showed the shortest disintegration time (65 s) and enhanced release profiles of both drugs. ConclusionsThe prepared sublingual tablets combining ATOR and TMZ will be a promising dosage form for coronary heart disease patients with an instant action and improved patient compliance.

Simultaneous HPTLC quantification of three caffeoylquinic acids in Pluchea indica leaves and their commercial products in Thailand

In Thai traditional medicine, Pluchea indica (L.) Less., Asteraceae, leaf has been widely used for the treatment of diabetes mellitus, tumors, hypertension, cystitis, and wounds. P. indica herbal tea is commercially available in Thailand as a health-promoting drink. The study was conducted to develop and validate a high-performance thin-layer chromatography (HPTLC) method for the quantitative analysis of chlorogenic acid, 3,4-O-dicaffeoylquinic acid, and 3,5-O-dicaffeoylquinic acid in P. indica leaf extract and their commercial products in Thailand. The method was validated according to ICH guidelines. The proposed HPTLC method showed acceptable validation parameters. The content of chlorogenic acid, 3,4-O-dicaffeoylquinic acid, and 3,5-O-dicaffeoylquinic acid in P. indica leaves from seven different provinces in Thailand was in the range of not detectable −1.94±0.02%w/w, 0.71±0.01–1.89±0.05%w/w, and 1.00±0.01–2.18±0.03%w/w, respectively, while in the commercial products, it was in the range of 0.59±0.03–2.17±0.05%w/w, 0.53±0.04–3.77±0.03%w/w, and 0.88±0.05–4.72±0.10%w/w, respectively. The results indicated that plantation of P. indica in coastal saline land would be beneficial as it would increase the concentration of its active compounds and improve its medicinal quality. The developed high-performance thin-layer chromatography could be used as a rapid, reliable, less demanding, and cost-effective analytical method.

High-performance thin-layer chromatography marker-based standardization of Piperine, Asiaticoside, and Withanolide-A in the developed polyherbal formulation and in vitro evaluation of acetylcholinesterase inhibition

Background: Preparation of highly standardized polyherbal formulation with its chief active chemical constituents supported by therapeutic efficacy in vitro is a valuable approach in the field of pharmaceutical sciences. Objective: The present work aims to develop the high-performance thin-layer chromatography (HPTLC) marker-based standardization of polyherbal formulation using Piperine, Asiaticoside, and Withanolide-A and in vitro acetylcholinesterase inhibition activity. Materials and Methods: For successful standardization, the HPTLC quantification of Piperine, Asiaticoside, and Withanolide-A was carried out. Suitable solvent systems were optimized to achieve the better resolution of the marker compounds, extracts, and sample formulation. The reproducibility of the methods was also confirmed by repeating the procedure twice. The identity of the bands in the sample formulation was confirmed by comparing the Rf value with those of their respective reference standards. In vitro acetylcholinesterase inhibition assay was done on the different crude extracts as well as tablet formulation. Results: HPTLC quantification of formulation for Piperine content showed 1.97% content w/w, whereas Piper longum extract showed 2.44% w/w. Similarly, Asiaticoside showed 0.71% in formulation and in Centella asiatica extract, it was 1.33% w/w. Contrary to the above, Withanolide-A content was 2.12% w/w in formulation and in Withania somnifera extract, it was only 0.81% w/w. In vitro acetylcholinesterase inhibition assay exhibited significant inhibition with IC50 value of 70 μg for tablet formulation. Conclusion: The presence of Piperine, Asiaticoside, and Withanolide-A in formulation was identified by rapid HPTLC quantification method. The method is very precise, reproducible, and accurate. The developed method can be used to quantify the marker compounds in the formulations available in the market. The formulation also evaluated for its acetylcholinesterase inhibition assay and showed significant activity. Hence, it could be used in the treatment of memory-related disorders such as Alzheimer's disease.

Quantification of the secondary metabolites by HPTLC, analgesic and antipyretic activity evaluation of Ficus racemosa L. leaves

There have been no reports of simultaneous quantification of kaempferol and quercetin in Ficus racemosa L. Objective of the present work included to perform extraction using petroleum ether (PEE) and ethanol (EE), to perform preliminary phytochemical test, quantitative estimation of phytochemicals and simultaneous estimation of kaempferol and quercetin in Ficus racemosa L. using high performance thin layer chromatography (HPTLC) method and to assess the analgesic and antipyretic activity of the extracts. PEE and EE obtained from cold maceration technique. Extracts were subjected to qualitative test and estimated quantitatively. Extracts were applied on silica gel G 60 F254 plate; solvent-Toluene: Chloroform: Acetone 4.5:4.5:1 (v/v) and scanned at 254 nm. The extracts were also subjected to analgesic activity (hot plate method and tail immersion method) and antipyretic (Brewer’s yeast induced pyrexia model and Lipopolysaccharide induced pyrexia mode) activity. Findings of preliminary phytochemical test and quantitative estimation of phytochemicals confirms the presence of flavonoids in both the extracts (PEE and EE). The amount of flavonoids in PEE and EE were found to be 24.58 ± 0.60 mg/100 g and 21.01 ± 0.58 mg/100 g respectively. HPTLC system resulted well resolved bands for quercetin (Rf 0.38) and kaempferol (Rf 0.55). The validated HPTLC method was found suitable for kaempferol and quercetin quantification in Ficus racemosa L. Hot plate reaction time response was improved in a dose-dependent manner in a group of rats treated with PEE suggesting its analgesic potential. In view of analgesic potential by tail immersion method, PEE (400 mg/kg b.w.) showed no significant difference in tail flick reaction time as shown by standard treated animal (Aspirin 100 mg/kg b.w.). The findings of antipyretic activity suggested that PEE (400 mg/kg b.w.) have no significant difference in treating pyrexia when compared with Paracetamol (100 mg/kg). PEE (400 mg/kg b.w.) was found to be most potent. It may be due to the presence of comparatively high amount of flavonoids in PEE confirmed by the preliminary phytochemical test, quantitative estimation of phytochemicals and HPTLC quantification the PEE was found to be more effective when compared to EE.

Neuroprotective effect of the marine macroalga Gelidiella acerosa: identification of active compounds through bioactivity-guided fractionation

Context Gelidiella acerosa (Forsskål) Feldmann & G. Hamel (Rhodophyta-Gelidiales) is a marine red macroalga. Our previous work found that a benzene extract of G. acerosa possesses noticeable neuroprotective activity, when evaluated through in vitro and in vivo systems.Objective Bioactive-guided fractionation and identification of active compounds by column chromatography using solvents of varying polarity.Materials and methods Fractionation was done by column chromatography, antioxidant and anticholinesterase activity was assessed by DPPH and cholinesterase inhibition assays (50–200 μg/ml), compound identification was done by LC-MS analysis, the mode of interaction of active compound was analyzed through docking studies and quantification was done by high-performance thin-layer chromatography (HPTLC) analysis.Results The results suggest that fractions F9–F13 exhibited significant (p < 0.05) antioxidant and anticholinesterase activities. Hence, these fractions were pooled together and verified for neuroprotective activity. The pooled fraction was subjected to LC-MS analysis and among all the compounds, phytol was previously reported to possess excellent neuroprotective potential. Hence, the neuroprotective potential of phytol was assessed. The results suggest that phytol showed significant (p < 0.05) antioxidant activities (25–125 μg/ml) with an IC50 value of 95.27 ± 1.65 μg/ml and cholinesterase inhibitory potential (5–25 μg/ml) with IC50 values of 2.704 ± 0.07 and 5.798 ± 0.72 μg/ml for AChE and BuChE, respectively. Molecular docking studies suggest that phytol interacts with cholinesterase through the arginine residue of the enzyme. HPTLC quantification showed that about 6.266 μg of phytol was present per mg of pooled fraction.Conclusion The study suggests that phytol might act as the key compound in contributing to the neuroprotective potential of G. acerosa.

Quantification of gallic acid in fruit and leaves of Careya arborea by High performance thin layer chromatography (HPTLC)

Objective: The present study was aimed to quantification of Gallic acid in Careya arborea (Lecythidaceae). Method: High performance thin layer chromatography (HPTLC) quantification was carried out for various extract of leaf and fruits of C. arborea. The thin layer chromatography (TLC) plate was spotted with gallic acid and fruits and leaves extract of Careya arborea. The extract of fruit and leaves shows the Gallic acid spot at Rf: value 0.68 using mobile phase ethyl acetate: toluene: formic acid (8:2:0.3, v/v). So, the gallic acid was selected as marker. The gallic acid was quantified using HPTLC technique in CFE (C. arborea fruit ethyl acetate extract), CFA (C. arborea fruit alcoholic extract), CLE (C. arborea leave ethyl acetate extract) and CLA (C. arborea alcoholic extract of leaves) of Careya arborea. Result: The percentage (w/w) amount of gallic acid was found to 2.11%, 1.2 %, 0.54% and 0.48% in CFE, CFA, CLE and CLA of C. arborea respectively. Conclusion: This HPTLC quantification method useful for standardization of fruits and leaf Careya arborea.

Qualitative and quantitative analysis of hyaluronan oligosaccharides with high performance thin layer chromatography using reagent-free derivatization on amino-modified silica and electrospray ionization-quadrupole time-of-flight mass spectrometry coupling on normal phase

Purified oligomers of hyalobiuronic acid are indispensable tools to elucidate the physiological and pathophysiological role of hyaluronan degradation by various hyaluronidase isoenzymes. Therefore, we established and validated a novel sensitive, convenient, rapid, and cost-effective high performance thin layer chromatography (HPTLC) method for the qualitative and quantitative analysis of small saturated hyaluronan oligosaccharides consisting of 2–4 hyalobiuronic acid moieties. The use of amino-modified silica as stationary phase allows a simple reagent-free in situ derivatization by heating, resulting in a very low limit of detection (7–19pmol per band, depending on the analyzed saturated oligosaccharide). By this derivatization procedure for the first time densitometric quantification of the analytes could be performed by HPTLC. The validated method showed a quantification limit of 37–71pmol per band and was proven to be superior in comparison to conventional detection of hyaluronan oligosaccharides. The analytes were identified by hyphenation of normal phase planar chromatography to mass spectrometry (TLC–MS) using electrospray ionization. As an alternative to sequential techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), the validated HPTLC quantification method can easily be automated and is applicable to the analysis of multiple samples in parallel.

Determination of lutein by high-performance thin-layer chromatography using densitometry and screening of major dietary carotenoids in food supplements

The main problem in the densitometric determination of carotenoids is their rapid degradation during and immediately after chromatography, respectively. In this study, we show that 15ng of lutein, lycopene and β-carotene standards applied on C18 RP high-performance thin-layer chromatography (HPTLC) plates pre-developed with dichloromethane–methanol 1:1 (v/v) remained stable for 1h after the development of chromatogram using methanol–acetone 1:1 (v/v) with 0.1% of 2-tert-butylhydroquinone (TBHQ), which is a substantial improvement of their stability. An HPTLC quantification procedure for free lutein, with densitometry at 450nm based on the developed method described above, was established and validated. Repeatabilities of the chromatography expressed by the relative standard deviation (RSD) from 6 applications of lutein standard at 5, 15 and 25ng were 3.41, 1.33 and 1.65%, respectively. The best fit calibration curve from 5ng to 30ng of lutein was polynomial. Limit of detection (1.5ng) and limit of quantification (5ng) were the best achieved so far. With these chromatographic conditions dietary carotenoids lutein esters, lycopene, free lutein and β-carotene from food supplements were also well separated and were identified by visible absorption spectra scanned in situ and by mass spectra. Some additional developing solvents with the same type of chromatographic layer are proposed for the fast separation of lutein esters from free lutein in food supplements.

Rapid and precise estimation of biodiesel by high performance thin layer chromatography

Various sophisticated chromatographic techniques employing gas chromatography (GC) and high performance liquid chromatography (HPLC) were used to quantify biodiesel in terms of conversion percentage of the oil feedstock. These techniques are time consuming. In the present study, a rapid and reproducible technique was developed using high performance thin layer chromatography (HPTLC) for the accurate quantification of the conversion percentage of triglycerides into biodiesel (fatty acid methyl esters/FAME). The oil substrate was transesterified by a conventional process using alkali catalyst. The monoglycerides, diglycerides, unreacted triglycerides, free fatty acid and biodiesel (FAME) product were analyzed by HPTLC. An absorption maximum of the mixture of standard methyl esters was determined to be 203.5 nm by spectroscopic scan. The conversion percentage was calculated from the corresponding peak areas of the glycerides and biodiesel product, separated on thin layer chromatography using hexane, ethyl acetate and acetic acid (9:1:0.1) as mobile phase. In terms of reproducibility and precision of data and also the ease and quickness of simultaneous processing, HPTLC based analysis and quantification of biodiesel (FAME) proved to be an alternative to other conventional analytical techniques like GC and HPLC.

High-performance thin layer chromatographic quantification of bioactive psoralen and daidzein in leaves of Ficus carica L.

This study was undertaken to quantify psoralen and daidzein by high-performance thin layer chromatography (HPTLC). The methanolic extract of 10 mg mL−1 concentration solution was prepared for HPTLC quantification of psoralen and daidzein. HPTLC aluminium-backed plates coated with 0.2 mm layers of silica gel 60 F254 were used as the stationary phase. The working standard solution of psoralen and daidzein was applied along with the test sample solution by means of Camag Linomat IV sample applicator. R f values of psoralen and daidzein were found to be 0.60 and 0.88, whilst as their percentage values in methanolic extract were found to be 3.02% and 5.64% (w/w), respectively. A simple quantitative estimation method of psoralen and daidzein by HPTLC is reported that can be used for the quality control of marketed preparations containing Ficus carica. However, further study is warranted to isolate and quantify active constituents present in the leaves of F. carica by sophisticated techniques.