Sensitive High-performance Thin-layer Chromatography Research Articles

Development and validation of a high-performance thin layer chromatography method for the simultaneous determination of amoxicillin and clavulanic acid combinations in tablet dosage forms

A simple and sensitive high-performance thin layer chromatography (HPTLC) method was developed and validated as per the International Council for Harmonization (ICH) guidelines for the simultaneous determination of amoxicillin (AMX) and clavulanic acid (CLA) combinations in tablet formulations. Chromatography was performed on precoated glass plates with normal phase silica gel 60 F254. The mobile phase was acetone:ethyl acetate:glacial acetic acid:water (11:9:4:2 (v/v)). The plate was scanned at a wavelength of 428 nm after derivatization with ninhydrin. The validation of the method revealed that the linearity range lies between 400 and 1200 ng/band for AMX and 100–300 ng/band for CLA, with coefficients of determination of 0.9997 and 0.9966, respectively. Recoveries in standard addition accuracy studies were 100.3 % for AMX and 96.75 % for CLA. The limit of detection (LOD) and limit of quantitation (LOQ) of the developed method are 20.3 ng/band and 61.6 ng/band for AMX and 18.5 ng/band and 56.2 ng/band for CLA, respectively. The new, novel high-performance thin layer chromatography (HPTLC) method that was successfully developed in this study was applied for the simultaneous determination of AMX and CLA in their fixed-dose tablet dosage forms obtained from retail pharmacies and offered comparable results with the official British Pharmacopoeial high-performance liquid chromatography (HPLC) method.

Simultaneous Estimation of Rhein and Aloe-Emodin in Traditional and Ultrasound-Based Extracts of Rheum palmatum L. (Rhubarb) Using Sustainable Reverse-Phase and Conventional Normal-Phase HPTLC Methods

The greenness indices of literature analytical procedures for the simultaneous measurement of rhein and aloe-emodin have not been determined. As a consequence, the first goal of this study was to design and validate a sensitive and sustainable reverse-phase high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of rhein and aloe-emodin in a traditional extract (TE) and ultrasound-based extract (UBE) of commercial Rhubarb and Rhubarb plant extracts in comparison to the conventional normal-phase HPTLC method. The second goal was to determine the greenness indices for both methods using the AGREE approach. For the sustainable reverse-phase HPTLC approach, the method was linear in the 50–1000 ng/spot range for rhein and 25–1000 ng/spot range for aloe-emodin. However, for the conventional normal-phase HPTLC approach, the method was linear in the 50–600 ng/spot range for rhein and 100–600 ng/spot range for aloe-emodin. The limit of detection (LOD) for rhein and aloe-emodin was 16.81 ng/spot and 8.49 ng/spot, respectively, using the sustainable analytical method. However, the LOD for rhein and aloe-emodin was 18.53 ng/spot and 39.42 ng/spot, respectively, using the conventional analytical method. For the simultaneous determination of rhein and aloe-emodin, the sustainable analytical method was more sensitive, accurate, precise, and robust than the conventional analytical method. The amount of rhein and aloe-emodin was higher in the UBE of commercial Rhubarb and Rhubarb plant extract over their TE. For the simultaneous quantification of rhein and aloe-emodin in the TE and UBE of marketed Rhubarb and Rhubarb plant extract, the sustainable analytical method was superior to the conventional analytical method. The AGREE index for the sustainable reverse-phase and conventional normal-phase HPTLC methods was determined to be 0.78 and 0.49, respectively, indicating an excellent greenness profile of the sustainable reverse-phase HPTLC method over the conventional normal-phase HPTLC approach. The sustainable analytical method was found to be superior to the conventional analytical method based on these results.

Development and Validation of HPTLC Method for Simultaneous Estimation of Berberine, Gallic Acid and Ursolic Acid in a Polyherbal Blend

A sensitive high-performance thin-layer chromatography method was developed for simultaneous estimation of berberine, gallic acid and ursolic acid in a polyherbal blend and validated as per ICH guidelines. Polyherb-al blend was prepared using widely recommended herbal plants for platelet augmentation activity viz. Carica papaya, Berberis aristata, Ocimum Sanctum, and Tinospora Cordifolia. The optimized separation was ob-tained with TLC aluminum plates pre-coated with silica gel G60 F254 as a stationary phase and a solvent sys-tem containing Toluene : Ethyl acetate : Methanol : Formic acid (3:3:0.2:0.1 v/v/v/v). Berberine and gallic ac-id were found to demonstrate linearity in the range of 1μg/band – 6μg/band and ursolic acid in the range of 20 μg/band – 100 μg/band with the regression coefficient in acceptable limits. The method was also found to be specific and precise one. The accuracy of the developed method at 80 %, 100 % and 120 % levels was found to be within limits and % RSD was found to be less than 2. LOD and LOQ of all three standards were also determined. Blend was also quantified for the amount of berberine, gallic acid and ursolic acid presence and was found to be 37.8 μg, 46.1 μg and 108 μg per mg, respectively.

A rapid and highly sensitive stability-indicating high-performance thin-layer chromatography technique for the determination of tedizolid phosphate with a classical univariate calibration

A rapid and highly sensitive stability-indicating high-performance thin-layer chromatography technique for the determination of tedizolid phosphate with a classical univariate calibration

Development of a specific and sensitive high-performance thin-layer chromatography assay method for the determination of linagliptin in tablet dosage form

Development of a specific and sensitive high-performance thin-layer chromatography assay method for the determination of linagliptin in tablet dosage form

Characterization of herbal teas containing lime flowers – Tiliae flos by HPTLC method with chemometric analysis

Linden trees are a source of food products called lime flowers (Tiliae flos), traditionally used in the form of infusion for the treatment of feverish colds and coughs. Lime flowers should include flowers of Tilia cordata Mill, T.x europaea L., and T. platyphyllos Scop. or a mixture of these. The aim of current research was to establish a fast, sensitive HPTLC (high-performance thin-layer chromatography) method that would allow the differentiation of material obtained from five species of lime occurring in Europe. The fingerprints for distinguishing these species were established, as well as a key for identification based on a visual evaluation of chromatograms. The results obtained were also subjected to chemometric analyses. It was shown that each species contains characteristic compounds i.e. linarin that can be used for their identification. The method developed can, in theory, be introduced for the quality control or authentication of linden flowers on the European market.

Simultaneous determination of lupeol and β-sitosterol by high-performance thin-layer chromatographic method in Crataeva nurvala Buch-Ham. stem bark

Crataeva nurvala Buch-Ham. (Capparidaceae) is an important medicinal plant, the bark of which is extensively used in Ayurveda as a remedy for kidney stones, urinary tract infections, prostate-related disorders, and benign prostatic hypertrophy. In this study, a sensitive and reliable high-performance thin-layer chromatography (HPTLC) method has been developed for the simultaneous quantification of two bioactive markers, lupeol and β-sitosterol, in the stem bark of C. nurvala using precoated silica gel G 60 F254 HPTLC plates as the stationary phase and toluene–methanol (9.6:0.4, v/v) as the mobile phase. Detection and quantification were carried out by densitometric scanning at 545 nm after derivatization using 1% anisaldehyde–sulfuric acid reagent. The method was validated for linearity, inter-day and intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The linear regression analysis data for the calibration plots showed a good linear relationship (r2 = 0.987–0.990) in the concentration range of 2 to 6 μg for lupeol and β-sitosterol, both with respect to areas. The proposed method provides good resolution of lupeol and β-sitosterol from other constituents present in the stem bark of C. nurvala. It was found to be simple, accurate, precise, and reproducible and would potentially be a useful tool for routine analysis of C. nurvala.

Role of TLC to ensure manifestation of individual ingredients in herbal formulation

Herbal medicine is gaining its popularity day by day and gradually streaming toward integration into the mainstream healthcare systems. Therefore, standardization of herbal formulations is essential in order to assess quality of the drugs. Two commonly used paradigms, compound and pattern-based approaches, were performed to ensure quality and hence product consistency. A rapid, sensitive, and reproducible high-performance thin-layer chromatography (HPTLC) method has been developed for one of the major bioactive markers, piperine. The plates were developed using the mobile phase with toluene–ethyl acetate–formic acid–methanol (3:3:0.8:0.2, v/v). The method was validated for limit of detection, limit of quantification, linearity, specificity, precession, and recovery. The results indicate that the bioactive compound was present in 2.64% w/w in the fruit of Piper nigrum Linn. and 0.253% in a herbal formulation. Each ingredient was analyzed vis-a-vis the herbal formulation and the reflectance spectrum of the matching component(s) to ensure the presence of each ingredient in the herbal formulation. The individual component adding up has also resulted similar fingerprint pattern as that of herbal formulation. Thus, the TLC method could be a rapid tool for the delineation of each individual ingredient in the finished products for authenticity, efficacy, and for batch-to-batch consistency.

A simple, rapid and sensitive high-performance thin-layer chromatographic method for the simultaneous estimation of berberine and 5-fluorouracil in rabbit plasma

The purpose of the present study was to develop a simple, rapid and sensitive high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of 5-fluorouracil (5FU) and berberine (BER) in rabbit plasma. BER and 5-FU are currently under extensive studies for their synergistic effects in cancer chemotherapy. The HPTLC method was developed using TLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. A mixture of toluene:methanol:ethyl acetate:formic acid (6:2:6:1, v/v) was used as the solvent system for development and detection was carried out at 266 nm. The method was designed to obtain the most optimum retardation factors (Rf) with best resolution. The Rf values of BER and 5-FU were 0.28 ± 0.02 and 0.57 ± 0.02, respectively. The plasma extraction was optimized using ethyl acetate. The method was validated in compliance with the USFDA guideline for bioanalytical method validation. The method was found to be linear in the range of 20 ng/spot to 300 ng/spot with R2 of 0.9964 for 5-FU and 0.9943 for BER. The lower limit for quantification (LLOQ) of BER and 5-FU in rabbit plasma were obtained as 29 ng/spot and 26 ng/spot, respectively. The stability of BER and 5-FU in plasma were confirmed during freeze-thaw cycles (−20 °C), on bench for 5 h and long term for 21 days. The proposed method was validated statistically and was precise, accurate and sensitive for the estimation of BER and 5-FU in rabbit plasma. This method can be extended for therapeutic drug monitoring and pharmacokinetic studies in rabbits.

Optimization and Validation of HPTLC Method for Estimation of Ulipristal Acetate in Presence of Its Forced Degradation Products.

Ulipristal acetate (UPA) is used as emergency contraceptive and for uterine fibroids. No validated method has been reported to estimate UPA in presence of its degradation products. Therefore it is mandatory to develop method which can accurately measure it in presence of impurity. A simple and sensitive high-performance thin-layer chromatography (HPTLC) method was developed for the estimation of UPA. Pre-coated silica gel 60F254 TLC plates were as stationary phase and ethyl acetate:toluene:glacial acetic acid (4:7:0.3, v/v/v) was used as mobile phase. Drug was subjected to acid and alkali hydrolysis, oxidation, photo degradation and thermal degradation to study its degradation behavior. UPA eluted with Rf value 0.38±0.02. The method was found to be linear in the concentration range of 400-3,600ng/band. Limit of detection and limit of quantitation were found to be 72.7786ng/band and 220.5412ng/band, respectively. The % recovery of the proposed method was found to be 100.05-100.65%. The proposed method was specific to measure UPA in presence of degradants. The method was found to be accurate, precise, robust and can be useful for routine analysis of formulations containing UPA in presence of its degradation products.

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).

High-performance thin-layer chromatographic determination of trigonelline content in various extracts and different varieties of some commercial coffees available in the Saudi Arabian market

The proposed research study was undertaken to establish a simple, rapid and sensitive high-performance thin-layer chromatography (HPTLC) method for the quantification of trigonelline (TGN) in methanolic, aqueous and hydroalcoholic extracts of different brands of coffee samples with different colours due to roasting time. The proposed HPTLC methodology was carried out using glass coated silica gel 60 F254 plates. The mobile phase used was quaternary mixture of ethyl acetate:methanol:water:formic acid 5.4:2.3:1.5:0.8 (%, v/v) which furnished a compact spot of TGN at Rf 0.24 ± 0.01. The developed plate was scanned and quantified densitometrically at 267 nm. Linear regression analysis presented a good relationship between peak area and amount of TGN in the range of 100–700 ng/band. The method was validated as per the International Conference on Harmonization (ICH) recommendations. The maximum amount of TGN was recorded in roasted Arabic coffee among different brands studied. The amounts of TGN in methanolic, aqueous and hydroalcoholic extract of roasted Arabic coffee were obtained as 0.74, 0.80 and 0.79% w/w, respectively. The proposed analytical methodology will be useful to enumerate the therapeutic dose of TGN in different herbal products.

Herb-Drug Interaction of Quercetin on the Pharmacokinetics of Losartan in Rats: A High-Performance Thin-Layer Chromatography Study

Losartan potassium (LOS), used in the treatment of hypertension, is metabolized primarily by cytochrome P450. This study investigates the effect of quercetin (QU), a CYP3A4 inducer, on the pharmacokinetics of LOS in rats. A rapid, sensitive high-performance thin-layer chromatography (HPTLC) method was developed and validated for the bioanalysis of losartan using olmesartan as internal standard (IS). The salting-out assisted liquid—liquid extraction (SALLE) employing acetonitrile and MgCl2 gave high recovery of LOS (>90%). HPTLC separation, achieved on silica gel 60 F254 plates employing toluene—ethyl acetate—acetone—formic acid (4:4:1:0.5 V/V) as the mobile phase, with densitometric analysis at 240 nm, gave good linearity (50–1200 ng mL-1) with high intra-day and i nter-day precision. LOS in plasma samples was stable when stored under different stability conditions. The pharmacokinetics of LOS was found to be significantly altered when co-administered with QU: C max = 809.8 ± 4.1 at 40 min (t max) to C max = 1124.8 ± 86.6 ng mL-1 at 120 min (t max). This study indicates the chances of herb—drug interaction when LOS is co-administered with QU, leading to its increased bioavailability, potentiating its side effect/toxic manifestation. As QU is abundantly present in herbs and dietary food, patients of LOS therapy need to be cautious while concurrently consuming herbal preparations containing QU. This study also demonstrates the utility of HPTLC as an effective tool for pharmacokinetics study for the estimation of herb—drug interactions.

Development of a high-performance thin-layer chromatographic method for the simultaneous determination of newly co-formulated antiviral drugs sofosbuvir and velpatasvir in their pure forms and tablet dosage form

The discovery of new direct-acting antiviral drugs gave rise to a leap forward in the treatment of hepatitis C viral infections. For the first time since 1998, the Food and Drug Administration (FDA) approved interferon-free oral treatment paradigms. Among the new treatment regimens, the combinations of sofosbuvir/daclat-asvir and sofosbuvir/velpatasvir became ideal treatment regimens for being potent, highly tolerated, and used once daily. For that purpose, a new sensitive high-performance thin-layer chroma-tography (HPTLC) method was developed and validated for the simultaneous determination of sofosbuvir and velpatasvir. The proposed method used Merck HPTLC silica gel 60 F254 aluminum plates as the stationary phase. The mobile phase was a combinat ion of methylene chloride-methanolethyl acetate-ammonia (25%) at a ratio of 5:1:3:1 (v/v). The calibration curves were linear over a wide range of 100–2000 ng per spot for the studied drugs. The limits of detection were 30.02 and 27.60 ng per spot, and the limits of quantification were 90.06 and 82.80 ng per spot for sofosbuvir and velpatasvir, respectively. The suggested method was successfully applied for analysis of the studied drugs in their pharmaceutical dosage forms, and excellent recovery results were obtained. Being simple, fast, robust, and economic, this method is eligible for use in the routine work in pharmaceutical quality control laboratories.

Development of a highly sensitive high-performance thin-layer chromatography method for the screening and simultaneous determination of sofosbuvir, daclatasvir, and ledipasvir in their pure forms and their different pharmaceutical formulations.

The combination of sofosbuvir and daclatasvir or sofosbuvir and ledipasvir is now widely used as an ideal treatment for hepatitis C virus infection. For this purpose, a simple, sensitive, accurate, economic, and precise high-performance thin-layer chromatography was developed and validated for the determination of sofosbuvir, daclatasvir, and ledipasvir in their pure form as well as their different pharmaceutical products. The method used Merck high-performance thin-layer chromatography aluminum plates precoated with silica gel 60 F254 as a stationary phase and mobile phase consisting of methylene chloride/methanol/ethyl acetate/ammonia (25%) (6:1:4:1, v/v/v/v). This system was found to give compact symmetric peaks of sofosbuvir, daclatasvir, and ledipasvir with retardation factors of 0.27 ± 0.01, 0.50 ± 0.007, and 0.68 ± 0.008, respectively. The densitometric scanner was set at 275nm using a deuterium lamp. The calibration curves were linear over the range of 100-3000ng/spot for sofosbuvir, and daclatasvir, and range of 50-3000ng/spot for ledipasvir. The detection limits were 22.5, 31.90, and 15.80 for sofosbuvir, daclatasvir, and ledipasvir. The quantitation limits were 67.50, 95.60, and 47.50 for sofosbuvir, daclatasvir, and ledipasvir. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines and the results were acceptable.

HiGh-performance thin-layer chromatography method for the simultaneous determination of vulgarin and epivulgarin biomarkers in Artemisia judaica L.

Novel, simple, and sensitive high-performance thin-layer chromatography (HPTLC) with fluorescence detection method has been successfully established and validated for the simultaneous determination of vulgarin and epivulgarin in different collections of Artemisia judaica. HPTLC method was carried out using glass plates coated with silica gel 60 F254 using petroleum ether‒acetone (7:3, v/v) as the mobile phase. After development, the plates were scanned and quantified densitometrically at 224 nm for both vulgarin and epivulgarin. The two compounds’ peaks from A. judaica were identified by comparing their single spot at RF = 0.30 ± 0.02 and RF = 0.36 ± 0.01, respectively, with those of vulgarin and epivulgarin. Linear regression analysis revealed a good linear relationship between peak area and amount of vulgarin and epivulgarin in the range of 100–700 ng band−1 for both compounds. The method was validated, in accordance with the International Conference on Harmonization (ICH) guidelines, for precision, acc...

Chromatographic Methods for Quantitative Determination of Ampicillin, Dicloxacillin and Their Impurity 6-Aminopenicillanic Acid.

Two accurate, precise and sensitive high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) methods were developed for assay of ampicillin (AMP) and dicloxacillin (DX) in the presence of their impurity, 6-aminopenicillanic acid (APA). Method (A) is HPTLC method; using silica gel HPTLC F254 plates as a stationary phase with methanol: chloroform: acetic acid (1:9: 0.2, by volume) as a developing system. All the bands were scanned at 220 nm. Method (B) is reversed phase- HPLC which depended on isocratic elution using C18 column and mobile phase consisting of acetonitrile: water (60:40, v/v), pH adjusted to 4 with orthophosphoric acid, at a flow rate of 1 mL min-1 and ultraviolet detection at 240 nm. The proposed methods were validated as per ICH guidelines and their linearity was evident in the ranges of 0.5-2 μg band-1, 0.4-2 μg band-1 and 0.2-1.2 μg band-1 for method (A) and 5-40 μg mL-1, 5-40 μg mL-1 and 2-16 μg mL-1 for method (B) for AMP, DX and APA, respectively. The proposed methods were successfully used for assay of AMP and DX in pure form and in pharmaceutical formulation where no interference from the excipients was detected.

Simultaneous quantification of six phenolic compounds in various parts of Moringa oleifera Lam. using high-performance thin-layer chromatography

Fresh pods of Moringa oleifera with nutraceutical importance are widely consumed in food commodities as vegetables. It is nutritious and it also has several biological activities. In the present study, a simple, rapid, cost-effective, and sensitive high-performance thin-layer chromatography (HPTLC) method was applied for the simultaneous determination of six phenolic compounds, viz., gallic (phenolic acid), p-coumaric, caffeic acid (hydroxycinnamic acid), chlorogenic acid (cinnamic acid derivative), quercetin and kaempferol (flavonols) in flowers, pods, leaves, twigs, and seeds of M. oleifera. Simultaneous separation and quantification of compounds were achieved on HPTLC pre-coated silica gel 60 F254 aluminum plates using the mobile phase toluene–ethyl acetate–formic acid (14:10:1). Densitometric determination was carried out at λmax 282 nm. The calibration curves were linear, ranged between 0.984 and 0.998; the limit of detection and quantification ranged between 110.8 ng mL−1 and 142.3 ng mL−1, and 301....

Stress-induced analysis of piperine variability in different marketed black pepper powders and seeds by a validated high-performance thin-layer chromatography method

In this article, a simple and sensitive stress-induced high-performance thin-layer chromatography (HPTLC) method is proposed for the quantitative analysis of piperine (a bioactive marker) in the alcoholic extracts of five different marketed powders (P1–P5) and dried seeds (P6) of black pepper (Piper nigrum). HPTLC was performed on glass-backed silica gel 60 F254 HPTLC plates with toluene, ethyl acetate, and glacial acetic acid (7:3:0.5; v/v) as the mobile phase. Scanning and quantification of the developed HPTLC plate (λ = 280 nm) furnished compact and intense spots of piperine at RF = 0.40 ± 0.01. For the proposed method, the estimated regression equation (Y) and r2 were 9.473X + 2262.23 and 0.997, respectively, in the range of 100–900 ng, the limit of detection (LOD) and limit of quantification (LOQ) were 19.63 and 59.47 ng, respectively. The quantified piperine values (% w/w) in P1, P2, P3, P4, P5, and P6 were 1.75, 2.71, 1.21, 1.67, 2.45, and 1.80, respectively. In the stress study, the treatment of p...

High-performance thin-layer chromatographic method for the estimation of benzyl isothiocyanate in Salvadora persica root extract and dental care herbal products

A novel, simple, and sensitive high-performance thin-layer chromatography (HPTLC) method has been successfully developed and validated for the quantification of benzyl isothiocyanate in Salvadora persica (Miswak or Siwak) extract and some dental care herbal products labeled to contain Siwak extract. A modified HPTLC method was carried out using glass-coated silica gel 60 F254 plates and mobile phase consisting of n-hexane—ethyl acetate (9:1) furnished compact spots at RF 0.61 ± 0.01. The developed plate was scanned and quantified densitometrically at 191 nm. Linear regression analysis revealed a good linear relationship between peak area and amount of benzyl isothiocyanate in the range of 100–600 ng band−1 (r2 = 0.9991). The method was validated in accordance with the International Conference on Harmonization (ICH) guidelines for precision, accuracy, and robustness. The proposed method will be useful to evaluate the therapeutic efficacy of Siwak extracts and dental care herbal formulations containing Siwak.