Sensitive High-performance Thin-layer Chromatographic Method Research Articles

Quantification of Suvorexant in Human Urine Using a Validated HPTLC Bioanalytical Method.

Suvorexant (SUV) is a new sedative/hypnotic medicine that is recommended to treat insomnia. It is an important medicine from a forensic point of view due to its sedative/hypnotic and depressant effects. To the best of our knowledge, high-performance thin-layer chromatography (HPTLC) bioanalytical methods have not been published to measure SUV in human urine and pharmaceutical samples. Accordingly, this study was designed and validated a sensitive and rapid bioanalytical HPTLC method to determine SUV in human urine samples for the very first time. The densitometric measurement of SUV and the internal standard (IS; sildenafil) was performed on glass-coated silica gel normal-phase-60F254S TLC plates using a mixture of chloroform and methanol (97.5:2.5 v/v) as the eluent system. Both the SUV and IS were detected at a wavelength of 254 nm. Both analytes were extracted using the protein precipitation technique utilizing methanol as the solvent. For the IS and SUV, the Rf values were 0.09 and 0.45, respectively. The proposed bioanalytical method for SUV was linear in the 50-1600 ng/band range. The current bioanalytical technique was linear, precise (% RSD = 3.28-4.20), accurate (% recovery = 97.58-103.80), robust (% recovery = 95.31-102.34 and % RSD = 2.81-3.15), rapid, and sensitive (LOD = 3.73 ng/band and LOQ = 11.20 ng/band). These findings suggested that the current bioanalytical method can be regularly used to determine SUV in wide varieties of urine samples.

Cytochrome P450 3A4-mediated pharmacokinetic interaction study between tadalafil and canagliflozin using high-performance thin-layer chromatography.

Diabetes is a known risk factor for sexual dysfunction in men; diabetic men have an increased risk of erectile dysfunction compared to non-diabetic men. Canagliflozin is one of the common antidiabetic drugs that is readily used in the treatment of type-2 diabetes. Concomitantly phosphodiesterase 5 inhibitors, such as tadalafil, can be given to the patient to alleviate erectile dysfunction. Canagliflozin is reported to be one of the cytochrome P450 3A4 enzyme inhibitors, that might seriously influence blood concentration levels of tadalafil but there is no study till now, discussing this interaction. Therefore, a fast, simple, and sensitive high-performance thin-layer chromatographic method was developed, validated, and applied for the simultaneous determination of tadalafil and canagliflozin in spiked and real human plasma. The limit of detection for tadalafil was 0.14ng/band and for canagliflozin was 0.16ng/band. The limit of quantitation value for tadalafil was 0.43ng/band and for canagliflozin was 0.47ng/band. Tadalafil and canagliflozin were determined simultaneously in real human plasma using the described procedure and the method was applied for in vivo pharmacokinetic drug interaction study between the studied drugs, which proved significant interaction between them when administered simultaneously.

Determination of Gefitinib Using Routine and Greener Stability-Indicating HPTLC Methods: A Comparative Evaluation of Validation Parameters

“High-performance thin-layer chromatography (HPTLC)” methods for gefitinib (GFT) estimation are scarce in the literature. In addition, greener analytical techniques for GFT estimation are also lacking in the literature. Accordingly, an attempt was undertaken to invent and validate a sensitive and greener normal-phase HPTLC method for GFT analysis in commercial tablets in comparison to the routine normal-phase HPTLC method. The greenness index for both methods was assessed using “Analytical GREENness (AGREE)” methodology. GFT detection was carried out using both methods at 332.0 nm. In the 30–700 ng/band and 20–1400 ng/band ranges, the routine and greener HPTLC assays were linear for GFT estimation. The greener HPTLC method was highly sensitive, more accurate, more precise, and more robust than the routine HPTLC assay for GFT estimation. Both methods were able to detect GFT in the presence of its degradation products, suggesting the stability-indicating property of both methods. The assay of GFT in commercial tablets was 92.45% and 99.74% using the routine and greener HPTLC assays, respectively. The AGREE index for routine and greener analytical assays was predicted to be 0.44 and 0.77, respectively, indicating the excellent greenness index of the greener HPTLC assay over the routine HPTLC assay. The greener HPTLC assay is considered superior to the routine HPTLC assay 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, sensitive, and greener stability-indicating normal-phase HPTLC method with univariate calibration for the estimation of chlorhexidine acetate in its commercial formulations

Till date, there is no report on high-performance thin-layer chromatography (HPTLC) analysis of chlorhexidine (CHD) or its salts like CHD acetate (CHD-A), CHD gluconate, and CHD hydrochloride. Therefore, a rapid, sensitive, and greener normal-phase HPTLC method has been reported for the analysis of CHD-A in its four different commercial dosage forms. The quaternary combination of ethyl acetate: ethanol: water: formic acid (75:10:10:5, v v v v−1) was optimized as the green solvent system/mobile phase for CHD-A analysis. The CHD-A detection was carried out at 264 nm. The greener normal-phase HPTLC assay was linear in the range of 10–2000 ng band−1. In addition, the proposed method was accurate, precise, sensitive, and selective for CHD-A analysis. The greener normal-phase HPTLC method was able to detect CHD-A in the presence of its degradation products, suggesting the stability-indicating property of this method. The content of CHD-A in commercial products A, B, C, and D was detected as 0.18, 2.02, 1.46, and 0.19% w w−1, respectively. The greenness scale for the greener normal-phase HPTLC assay was estimated as 0.88 utilizing “analytical GREEnness (AGREE)” calculator, suggested the greener nature of the normal-phase HPTLC assay. Overall, these data suggested that the greener normal-phase HPTLC method can be successfully used for the determination of CHD-A in its commercial products.

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.

Enrichment of alliin in different in vitro grown tissues of Allium sativum through CdCl2 elicitation as revealed by high performance thin layer chromatography (HPTLC)

Plant cell tissue and organ culture techniques provide an efficient way for the biosynthesis of high-value secondary metabolites in which elicitation has been recognized to be a potent approach in enriching the bioactive compounds. In this study, an effort was made to assess the influence of Cadmium chloride (CdCl2) on alliin yield of different in vitro grown tissues such as callus, somatic embryos, leaves and roots of Allium sativum. The tissues were cultured in MS amended with various levels of CdCl2 [control (0 mM), T1 (0.05 mM), T2 (0.1 mM), T3 (0.15 mM) and T4 (0.2 mM)] and the yield of alliin was quantified by using high performance thin layer chromatography (HPTLC). Biochemical analyses revealed that sample tissues under increasing CdCl2 conditions accumulated higher levels of sugar, protein and proline with maximum content of sugar (19.61 mg g−1 FW), protein (6.01 mg g−1 FW) and proline (12.19 mg g−1 FW) in leaves at T3 after 4 days of elicitor treatment. Changes within the activities of varied antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) was assayed as the addition of CdCl2 in the medium causes stress. We noted that with an elevated level of CdCl2, antioxidant enzyme activity increased linearly with the highest activity observed in leaves at T4 of 6 days of elicitation (4.97 EU min−1 mg−1 protein CAT, 6.30 EU min−1 mg−1 protein SOD and 1.125 EU min−1 mg−1 protein APX). Increased activities of enzymes are involved in negating the cellular stress triggered due to the overproduction of ROS. The yield of alliin in response to varying levels of CdCl2 was estimated by using a simple, rapid, and sensitive HPTLC method. The best separation was achieved using butanol:propanol:acetic acid:water (3:1:1:1) as mobile phase. For analysis of alliin, the plates were scanned at 550 nm after spraying with ninhydrin and a good resolution was achieved with Rf value of 0.66. In our study, the alliin yield enhanced in all tested cultures with the elicitor level up to T3. In leaves, maximum accumulation of alliin (3.37 μg g−1 dry wt.) was noted on 0.15 mM (T3) CdCl2 added medium. Our data indicated the increase of alliin in CdCl2 added cultures, which suggests that the use of elicitors as a potential strategy for augmenting secondary metabolite synthesis.

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.

A validated HPTLC method for quantitative analysis of morin in Maclura cochinchinensis heartwood

ObjectiveThe study was conducted to develop and validate a high performance thin-layer chromatography (HPTLC)-densitometric method for the quantitative analysis of morin in Maclura cochinchinensis heartwood collected from different locations in Thailand. MethodsHPTLC analysis was performed on an aluminium sheet of silica gel 60 F254 using toluene: ethyl acetate: formic acid (36:12:7, volume percent) as a mobile phase. The densitometric scanning was performed at the wavelength 410 nm. HPTLC method was validated according to ICH guideline. ResultsThe proposed HPTLC method showed acceptable validation parameters. The content of morin in M. cochinchinensis heartwood collected from eight different provinces in Thailand were in the ranges of 1.53%−2.73%. ConclusionThe simple and sensitive HPTLC method was successfully developed and validated for determination of morin in M. cochinchinensis heartwood. The proposed HPTLC method was found to be simple, fast and inexpensive, and can be used for the routine quality control of raw materials.

DEVELOPMENT AND VALIDATION OF HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF NEBIVOLOL HYDROCHLORIDE AND CILNIDIPINE

Objective: The proposed method describes method development and validation of nebivolol hydrochloride and cilnidipine in combined pharmaceutical tablet dosage form by high-performance thin-layer chromatography (HPTLC) having adequate specificity, sensitivity, and reproducibility.
 Methods: Nebivolol hydrochloride and cilnidipine drug combination is used for the treatment of hypertension. Precoated aluminum plates with silica gel 60 F254 (E-Merck, Germany) were used for the chromatographic separation which was carried using chloroform:glacial acetic acid:methanol, in 8.5:1:0.5 (v/v/v) as a mobile phase. HPTLC separation of two drugs was carried by densitometric measurement at 270 nm.
 Reults: The drugs were satisfactorily resolved with retardation factor values of 0.0.29±0.008 and 0.69±0.007 for nebivolol hydrochloride and cilnidipine, respectively. The method was found to be linear in the range of 100–1000 ng/spot and 50–500 ng/spot for nebivolol hydrochloride and cilnidipine, respectively. The correlation coefficient was found to be 0.989 and 0.996 for nebivolol hydrochloride and cilnidipine, respectively. Limit of detection and limit of quantitation values were 16.395 ng/band and 49.681 ng/band and 31.788ng/band and 96.328 ng/band, respectively. The mean recovery was found to be 100.570–101.936 and 100.269–101.333 for nebivolol hydrochloride and cilnidipine, respectively. The intra- and inter-day precision was found to be within the limit.
 Conclusion: A simple, accurate, precise, and sensitive HPTLC method has been developed and validated in combined pharmaceutical tablet dosage form for simultaneous estimation of nebivolol hydrochloride and cilnidipine.

A comparative study for the extraction methods and solvent selection for isolation, quantitative estimation and validation of ursolic acid in the leaves of Lantana camara by HPTLC method

To select the best organic solvent and ideal extraction technique for isolation and quantitative estimation of ursolic acid from the leaves of Lantana camara (L. camara) by high performance thin layer chromatography (HPTLC). A sensitive, fast and reproducible HPTLC method was developed and validated for estimation of ursolic acid from the leaves of L. camara. Different leaves extracts of L. camara were prepared through different extraction methods (maceration, reflux, soxhlet and ultrasound assisted extraction technique) by using four different solvents of varying polarity such as ethanol, methanol, acetone and chloroform. The maximum % extract yield was obtained in methanol, so we select methonolic extract for HPTLC evaluation. The calibration plot of standard ursolic acid with respect to peak area showed an tremendous linear relationship in the concentration range of 1000–4000 ng/spot with a correlation coefficient r2 = 0.994. The separation was completed on precoated silica gel aluminium plates 60F254 using Toluene: Acetone: Formic Acid (7.8:2.2:0.15) as mobile phase which gives a compact spot of ursolic acid (Rf value 0.49). For the proposed method (Limit of Detection) LOD (561.85 ng/spot), (Limit of Quantification) LOQ (1702.58 ng/spt), recovery of drug (99.02–101.43%) and precision (0.4303–2.1576% RSD) was found to be satisfactory. It can be concluded on the basis of HPTLC results that methanol was found to be the best solvent and Soxhlet was found to be an ideal extraction technique for the isolation of ursolic acid from L. camara leaves for routine analysis.

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.

New, simple and sensitive HPTLC method for simultaneous determination of anti-hepatitis C sofosbuvir and ledipasvir in rabbit plasma

Sofosbuvir (SOF) and ledipasvir (LDS) represent anti-hepatitis C binary mixture. Herein, a fast high-performance thin-layer chromatography (HPTLC) method was developed, validated and applied for simultaneous determination of SOF and LDS in biological matrix. An innovative strategy was designed which based on coupling dual wavelength detection with HPTLC. This strategy enabled sensitive, specific, high sample throughput and cost-effective determination of the SOF-LDS binary mixture. The developed HPTLC procedure is based on a simple liquid–liquid extraction, enrichment of the analytes and subsequent separation with UV detection. Separations were performed on HPTLC silica gel 60 F254 aluminum plates with a mobile phase consisting of ethyl acetate–glacial acetic acid (100:5, v/v). The Rf values for SOF and LDS were 0.62 and 0.30, respectively. Dual wavelength scanning was carried out in the absorbance mode at 265 and 327 nm for SOF and LDS, respectively. The linear ranges were 40–640 and 9–144 ng/band for SOF and LDS, respectively with correlation coefficients of 0.9998. The detection limits were 10.61 and 2.54 ng/band and the quantitation limits were 32.14 and 7.70 ng/band for SOF and LDS, respectively indicating high sensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in rabbit plasma with good percentage recovery (95.68–103.26%). Validation parameters were assessed according to ICH guidelines. The proposed method represents a simple, high sample throughput and economic alternative to the already existing more complicated reported LC-MS/MS techniques. The method would afford an efficient tool for therapeutic drug monitoring and bioavailability studies of SOF and LDS.

Simultaneous quantification of Precocene I and Precocene II through high-performance thin layer chromatography validated method in Ageratum conyzoides L. germplasms from western himalayas

Background: Ageratum conyzoides L. is a traditionally used herb for various ethnoveterinary purposes. There are no earlier reports on simultaneous high-performance thin layer chromatography (HPTLC) quantification of bioactive markers. Objective: To develop a sensitive, robust, and replicable HPTLC method for simultaneous estimation of precocene I (PI) and precocene II (PII) in A. conyzoides L. collected from the Western Himalaya region of India, with an aim to understand the level of chemotypic differences arising in the intraspecific population of this ethnobotanically important plant. Materials and Methods: A sensitive HPTLC method was developed to resolve PI and PII using toluene: ethyl acetate (9.8:0.2 v/v) as mobile phase. The method was validated for selectivity, specificity, linearity, and precision as per International Conference on Harmonization guidelines. Results: Good linearity was achieved over a five-point concentration range with a correlation coefficient of 0.986 and 0.988 for PI and PII, respectively. The PI content varies in the range of 0.0016% (NAC-77) to 0.0834% (NAC-82), whereas PII was reported to be present in the range of 0.016% (NAC-85) to 0.143% (NAC-91) on a dry weight basis. A principal component analysis biplot of samples based on the content of PI and PII identified four elite chemotypes, namely, NAC-81, NAC-82, NAC-85, and NAC-91. Conclusion: The study identifies superior germplasms for commercial prospection and develops a validated method that can be used for the quality control of herbal drug/formulation using A. conyzoides as an ingredient. Abbreviations used: HPTLC: High performance thin layer chromatography, PI: Precocene I, PII: Precocene II, ICH: International conference on harmonization, SD: Standard deviation, RSD: Relative standard deviation

A novel high-performance thin layer chromatography method for quantification of long chain aliphatic hydrocarbons from Cissus quadrangularis

Context: A high-performance thin layer chromatography (HPTLC) is an analytical technique, which can be used for the determination of constituents or marker components in various parts of the plants. Earlier studies have estimated phytoconstituents from the stem and other aerial plant parts of Cissus quadrangularis Linn. Estimation of hydrocarbons can also be successfully done using HPTLC technique using suitable derivatization. Aims: To develop and validate a simple and rapid method for the estimation of long chain aliphatic hydrocarbons from the leaves of C. quadrangularis using HPTLC technique. Methods: Precoated silica gel 60 F254 plates were used as stationary phase. The mobile phase used was hexane (100 %). The detection of spots was carried out using berberine sulphate as detecting reagent. Results: The method was validated in terms of linearity, sensitivity, accuracy, and precision. Linearity range was found to be 2-10 µg/mL, limit of detection 0.127 µg/mL, and limit of quantification 0.384 µg/mL. Conclusions: A novel, simple, accurate, precise and sensitive HPTLC method has been developed and validated for the estimation of long chain aliphatic hydrocarbons obtained from the leaves of C. quadrangularis Linn.

Bergenin determination in different extracts by high-performance thin-layer chromatographic densitometry.

Aim:Bergenin is an active secondary metabolite, found in Bergenia ligulata, family Saxifragaceae, which is an important medicinal plant used in the traditional system of medicine. It is distributed throughout the South and East Asia and some European countries, usually growing on high altitude in the Himalayan region and known as Pashanbhed (meaning “to break the stone”). The rhizome of B. ligulata has been used since long time in different traditional formulations of kidney and liver disorders. Due to its exhaustive use in the traditional system, it is commonly adulterated with the rhizome of other plants which do not contain its chemical marker bergenin. Hence, we developed high-performance thin-layer chromatographic (HPTLC) method for quantification of bergenin in B. ligulata which can be used for its quality control.Materials and Methods:A sensitive HPTLC method has been developed for the estimation of bergenin in different extracts of B. ligulata and its traditional formulations. Precoated HPTLC silica gel plates were used as stationary phase, and chloroform: methanol: acetic acid (8:1:1, v/v/v) was used as mobile phase.Results:The Rf value of bergenin was found to be 0.28 ± 0.03. Detection and quantification were performed by densitometry at 276 nm. The calibration plot was linear in the range of 200–5000 ng of bergenin with the correlation coefficient of (r2) 0.999, which confirms good linearity. The content of bergenin in methanolic and acetone extracts was found to be 5.51 ± 0.14 and 5.76 ± 0.16, respectively.Conclusion:The method can be applied for quality control and standardization of B. ligulata and its traditional formulations as well as for checking the presence of adulterants.

Densitometric simultaneous quantification of three naphthoquinones fromImpatiens balsaminaL. leaves by high-performance thin-layer chromatography

Impatiens balsamina belonging to family Balsaminaceae is commonly known as terda. The plant has extensive range of phytoconstituents like naphthoquinones, coumarins, glycosides, phenolic acids, flavonoids, anthocyanidins, and steroids which have effective antimicrobial, anti-anaphylaxis, anti-allergic, antipruritic, and anti-inflammatory activity. Potent naphthoquinones like lawsone, methylene-3,3′-bilawsone, and lawsone methyl ether which are present in the leaves of the plant that exhibits were proved to possess an intensive antitumor activity.Taking into account these ethnobotanical, nutritional, medicinal, and commercial values of I. balsamina, a simple, rapid, and sensitive high-performance thin-layer chromatography method for the quantification of three naphthoquinones was developed. The stationary phase used was HPTLC plates silica gel 60 F254 precoated plates (10 × 10 cm). N-butyl acetate.chloroform.glacial acetic acid (6:4:0.1) was used as mobile phase, detection at wavelength of 280 nm using Des...

A High-Performance Thin-Layer Chromatographic Method for the Evaluation of Marmelosin fromAegle marmelosCorr. Extract and Its Traditional Formulation from Rat Plasma: Application to Pharmacokinetics

Marmelosin is one of the major phytochemical constituents of Aegle marmelos (L.) Corr. (Rutaceae). Till date, there are no methods reported for the estimation of marmelosin in plasma matrix using high-performance thin-layer chromatography (HPTLC) which is a versatile analytical technique promoting shorter analysis time and less consumption of solvents with efficient data acquisition and processing. This study is an attempt to develop a rapid and sensitive HPTLC method for the estimation of marmelosin from rat plasma and its application to evaluate pharmacokinetics of A. marmelos fruit pulp extract and a traditional Unani medicine Sharabat-e-Belgiri. Plasma processing involved liquid-liquid extraction with diethyl ether followed by HPTLC. Separation and determination of marmelosin were performed on silica gel 60 F254 TLC plate using toluene-ethyl acetate-glacial acetic acid as mobile phase. United States Food and Drug Administration (USFDA) guidelines for bioanalytical validation were followed to validate ...

Development of a Sensitive HPTLC Method for Quantification of Nimbolide in Azadirachta indica and Its Dosage Form

An improved and sensitive High Performance Thin-Layer Chromatography (HPTLC) method has been developed for determination of anticancer compound, nimbolide in different parts of Azadirachta indica and its dosage form. A quick and simple ultrasonication technique was used for the preparation of sample solutions. Separation of the components was achieved on precoated TLC plates by using optimized tertiary mobile phase consisting of n-hexane:ethyl acetate:acetic acid (6:4:0.2, v/v/v) with a solvent migration distance of 68 mm. Densitometric quantification was performed after derivatization of the plate with methanol-sulfuric acid reagent in reflection/absorption mode at 515 nm. A linear response of nimbolide was obtained over the range of 200-1400 ng/spot with a correlation coefficient of r(2) = 0.99968, indicating good relationship between concentration and peak area. The method sensitivity was found to be increased by performing the analysis after derivatization with methanol-sulfuric acid reagent. The limit of detection and limit of quantification were found to be 70 and 200 ng/spot, respectively. The obtained recovery range from 96.70 to 98.01% with an average of 97.46% proves excellent accuracy of the method. ICH protocol was followed for validation of the HPTLC method in terms of precision, repeatability and accuracy. The developed method was found to be highly sensitive and the mobile phase efficiently separated nimbolide from other components. The maximum content of nimbolide was found in leaves. Further, the developed HPTLC method can be applied successfully for the marker evaluation of the formulations containing A. indica.

Development of a new, rapid and sensitive HPTLC method for estimation of Milnacipran in bulk, formulation and compatibility study

A simple, sensitive and rapid high performance thin layer chromatographic (HPTLC) method has been developed and validated for quantitative determination of Milnacipran Hydrochloride (MIL) in bulk and formulations. The chromatographic development was carried out on HPTLC plates precoated with silica gel 60 F254 using a mixture of acetonitrile, water and ammonia (6:0.6:1.6) (v/v/v) as mobile phase. Detection was carried out densitometrically at 220nm. The Rf value of drug was found to be 0.63±0.02. The method was validated as per ICH guideline with respect to linearity, accuracy, precision, robustness etc. The calibration curve was found to be linear over a range of 100–1000ngμL−1 with a regression coefficient of 0.999. The accuracy was found to be very high (99.12–100.87%). %RSD values for intra-day and inter-day variation were not more than 1.43. The method has demonstrated high sensitivity and specificity. The method was applied for compatibility studies also. The method is new, simple and economic for routine estimation of MIL in bulk, preformulation studies and pharmaceutical formulation to help the industries as well as researchers for their sensitive determination of MIL rapidly at low cost in routine analysis.