Thin-layer Chromatographic Method Research Articles

Sustainable and efficient monitoring of tryptophan and tyrosine serum levels: a green HPTLC method as a biomarker for type 2 diabetes

In recent years, there has been considerable interest in using amino acids like tryptophan (Trp) and tyrosine (Tyr) as biomarkers for various diseases, including type 2 diabetes mellitus (T2D). In diseases like T2D, the metabolism of Trp and Tyr is altered. The activity of enzymes involved in Trp metabolism increases, leading to a decrease in its serum level. On the other hand, the serum level of Tyr increases due to the suppressed activity of its metabolizing enzymes. These observations suggest that Trp and Tyr metabolism may play a crucial role in the pathophysiology of type 2 diabetes. Our study highlights the potential utility of Trp and Tyr as biomarkers for the early detection, prognosis, and monitoring of this metabolic disorder. Given these observations, we aimed to develop a high-performance thin-layer chromatographic (HPTLC) method that is sensitive, selective, rapid, and environmentally friendly for estimating the concentrations of Trp and Tyr in biological fluids, particularly serum samples. To evaluate the method, we performed analysis using serum samples from controlled and streptozotocin-induced diabetic rats. Our main objective was to develop a method that is sensitive and selective for precisely determining Trp and Tyr serum levels, which could serve as potential biomarkers for T2D. Fluorescence and absorption modes were employed for densitometry scanning. We assessed the precision and high separation efficiency of the chromatographic system by calculating parameters such as separation and resolution factors, number of theoretical plates, and height equivalent to theoretical plates. To evaluate the environmental impact of our proposed method, we employed the AGREE (Analytical GREEnness metric) and GAPI (Green Analytical Procedure Index) greenness assessment tools. The results confirmed that our method is environmentally friendly and exhibits superior eco-friendliness and greenness compared to other reported methods.Graphical

A high-performance thin-layer chromatography densitometric method for the separation of isomeric ceftriaxone in powder for injection formulation

The aim of this study was to develop and validate a High-Performance Thin Layer Chromatographic (HPTLC) method for simultaneous determination of ceftriaxone and ceftriaxone e-isomer in powder for injection formulation. Ceftriaxone sodium injection is an antibiotic that used globally. It has Z/E geometrical conformation, in which ceftriaxone sodium and 3 ene-isomer have Z- conformation while (E)-isomer has E- conformation and the potential toxicity of ceftriaxone (E)-isomer has been reported. Thus, to safeguard the public health, a simple and easy to use, rapid and reliable method was developed for qualitative and quantitative determination of ceftriaxone sodium and its (E)–isomer. Samples were applied on HPTLC glass plates precoated with silica gel 60F254 by using Linomat semi-auto sampler. Separation was carried out using acetone, triethyl amine, water, chloroform and ethyl acetate as a mobile phase in different ratios. The Rf values of separated compounds were 0.51 ± 0.01 and 0.62 ± 0.01 for ceftriaxone sodium and ceftriaxone (E)-isomer respectively. The method was validated by studying Specificity, Linearity, Accuracy, Precision, Robustness, Limit of Detection (LOD) and Limit of Quantification (LOQ) and Solution stability. The developed method was successfully, sensitive, simple, precise, accurate, robust and applicable for the simultaneous determination of ceftriaxone sodium and ceftriaxone (E)-isomer in powder for injection formulation.

Enzymatic Production of Chitooligosaccharide Using a GH Family 46 Chitosanase from Paenibacillus elgii and Its Antioxidant Activity

Chitooligosaccharide (COS), a natural antioxidant, is a hydrolysis product of chitosan created using enzymatic or chemical methods. COS has received considerable attention recently, making its efficient bioproduction of great value. This study investigated the optimal conditions for the enzymatic method using a GH family 46 chitosanase from Paenibacillus elgii TKU051 to prepare COS based on the response surface methodology (RSM). The results showed optimal values for chitosan hydrolysis, such as a pH of 5.5, an incubation temperature of 58.3 °C, an [E]/[S] ratio of 118.494 (U/g), and an incubation time of 6.821 h. Under the optimal conditions, the highest reducing sugar level (per substrate, w/w) of the chitosan hydrolysis process that could be reached was 690.587 mg/g. The composition of the obtained COS was analyzed using the thin-layer chromatography (TLC) method, yielding (GlcN)2 and (GlcN)3 as the products. The ascorbic acid equivalent antioxidant capacity (AEAC) of the obtained COS was found to be 1246 mg/100 g (via a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay) and 3673 mg/100 g (via an ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical-scavenging assay). This green and efficient bioproduction method may possess excellent potential for application in bioactive COS preparation.

Stability-indicating high-performance thin-layer chromatography method development and validation for the simultaneous estimation of azithromycin, fluconazole, and secnidazole in active pharmaceutical ingredient and its tablet dosage form

Stability-indicating high-performance thin-layer chromatography method development and validation for the simultaneous estimation of azithromycin, fluconazole, and secnidazole in active pharmaceutical ingredient and its tablet dosage form

Validated stability-indicating instrumental thin-layer chromatography method for the quantification of alizarin from Ayurvedic formulations

Validated stability-indicating instrumental thin-layer chromatography method for the quantification of alizarin from Ayurvedic formulations

Validated high performance thin layer chromatographic method for simultaneous quantification of betulinic acid, β-sitosterol and lupeol in fruits, leaves, root bark and stem bark of Dillenia indica Linn

AbstractDillenia indica is one of the important medicinal plants and is extremely popular in many systems of medicine including ayurvedic, homeopathic, and siddha. Traditionally, different parts of this plant have been used to treat cancer, wound healing, diabetes, diarrhea, bone fractures, abdominal pains, cuts and burns. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous estimation of three pharmaceutically active compounds i.e., betulinic acid (BE), β-sitosterol (BT) and lupeol (LP) in fruits, leaves, root bark and stem bark of D. indica. Standards and plant samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene:methanol:chloroform (8:1:1, v/v/v) as a mobile phase which resulted in better separation of compounds. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BE, BT and LP standards and those of plant samples were found to be 0.38 ± 0.01, 0.54 ± 0.01 and 0.65 ± 0.02 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BE, BT and LP, the linear regression data for the calibration plots revealed a satisfactory linear association with correlation coefficients (r2) = 0.9736, 0.9989 and 0.9957, respectively. Linear ranges for BE, BT and LP were 2,000–6,000 ng/band, 200–1,000 ng/band, and 200–600 ng/band respectively. Accuracy of the method was assessed by recovery study conducted at three different levels with the average recovery of 99.19, 99.69 and 100.95% for BE, BT and LP, respectively. The results exhibited the highest content of BE (0.148 ± 0.01%), BT (0.031 ± 0.01%) and LP (0.047 ± 0.01%) in stem bark. The developed method will be useful for routine and quality control analysis of fruits, leaves, root bark and stem bark of D. indica species.

Validation of chromatographic methods, TLC and HPLC, to quantify known radiochemical impurities and the radiochemical purity the radiopharmaceutical [177Lu]Lu-PSMA I&T

The Energy and Nuclear Research Institute (IPEN) is studying the production of the radiopharmaceutical [177Lu]Lu-PSMA I&T, in accordance with the good manufacturing practices recommended by the National Health Surveillance Agency (ANVISA), to be used in the therapy of prostate cancer. This work aims to validate chromatographic methods, Thin-Layer Chromatographic (TLC) and high-performance liquid chromatography (HPLC), to determine the radiochemical purity (RCP) of the product. The entire validation process of this work was based on ANVISA's RDC 166, 2017 and the Guide 10, version 1, 2017, guided the statistical treatments adopted. With the selectivity study we can observe that the presence of impurities or excess excipients in the sample does not interfere with the quantification of the product. The proposed methods were linear with linear correlation coefficients (r) above 0.99. The precision and repeatability presented relative standard deviation values lower than specified (RSD < 5 %). The small controlled variations in the method suggested for the robustness test also did not affect the radiochemical purity of the product. In view of the results and in accordance with the criteria established by the National Health Surveillance Agency (ANVISA), the two chromatographic methods were validated in accordance with RDC 166, 2017, proving to be selective, precise, linear and robust. The validation of TLC and HPLC methods enables their application in the batch release routine of the new radiopharmaceutical at Radiopharmacy Center of IPEN.

Development of Amla Nutri-bar: Proximate analysis, gallic acid quantification and its validation through HPTLC, and spectroscopy

BACKGROUND: “Ayush Ahara” confluences advanced food technology with Ayurveda to develop dietary products rooted in traditional medicine. Here, Amla (Indian gooseberry), a medicinal fruit well-known for its health benefits, was used to develop a novel dietary product—Amla Nutri-bar (ANB). Furthermore, this research aims to substantiate its therapeutic potential through quality control analysis. METHODS: Three batches of ANB were prepared at the pharmaceutical laboratory, All India Institute of Ayurveda, New Delhi, India. These were evaluated for sensory attributes using a 9-point hedonic scale and underwent physicochemical, phytochemical, total phenolic content, chromatographic, and spectroscopy assays. RESULTS: The sensory evaluation indicated positive feedback, with an overall acceptability score of 8, followed by taste and flavor rated at 7 Physiochemical constants included pH: 2.2, moisture: 11.48%w/w, total ash: 0.17%w/w, water-soluble extractive value: 88.4%w/w, and alcohol-soluble extractive value: 56.65%w/w. Phytochemical screening revealed that the product is rich in flavonoids, tannins, and alkaloids. Nutritional composition per 100 g had energy: 373.76 kcal, proteins: 1.61%, carbohydrates: 88.59% by wt, dietary fiber: 2.72 g, total fat: 1.44%, vitamin C: 300.11 mg, vitamin A: 71.23 pg, iron: 5.63 mg, calcium: 126.69 mg, and potassium: 103.57 mg. Total phenolic content was 4.95 mg gallic acid equivalents/g, with gallic acid quantified at 270.4 μg/100 mg employing a validated high-performance thin layer chromatography method. Fourier transform infrared spectroscopy analysis showed the presence of alcohol, phenol, and alkanes. All the tested pesticide residues were found below detection limits. CONCLUSION: ANB can be a food product that is, not delicious only but can also contribute significantly to overall health and well-being.

Green stability-indicating RP-HPTLC technique for determining croconazole hydrochloride

Abstract The objective of the proposed investigation is the development and validation of a green stability-indicating reverse-phase high-performance thin-layer chromatographic method to determine croconazole hydrochloride (CCZ). The developing system used was an 80:20 v/v mixture of acetone and water. The measurement of CCZ was done at 198 nm. With the use of the Analytical Eco-Scale (AES), ChlorTox, and Analytical GREEnness (AGREE) tools, greenness was evaluated. The linearity was demonstrated by the present method in the 25–1,200 ng/band range. The present approach was additionally reliable, accurate, sensitive, precise, and green. An exceptional greenness profile was demonstrated by the AES, total ChlorTox, and AGREE scales, which were determined to be 89, 1.08 g, and 0.82, respectively. The greenness metrics of the present method were much better than the reported high-performance liquid chromatography approach. Under acid and oxidative degradation circumstances, CCZ was shown to be unstable, while under alkaline and thermal-stress settings, it was sufficiently stable. Furthermore, the stability-indicating component determined by analytical method identified CCZ in the presence of its degradation products. Commercial CCZ cream was found to contain 0.98% w/w of CCZ. The investigation’s results suggested that CCZ in commercially available creams might be regularly examined with the help of the recommended green technology.

Rapid identification and on-site analysis by miniature mass spectrometry of chemical markers for fragrant rosewood authentication

Dalbergia odorifera T. Chen (Dalbergiae Odoriferae Lignum, DOL), renowned for its therapeutic applications in traditional Chinese medicine and its value in crafting luxury items, faces challenges due to the slow heartwood formation process and subsequent market adulteration. The proliferation of counterfeit products necessitates reliable methods for rapid, on-site authentication. This study aims to establish a rapid, green, and on-site method to identify the authenticity of DOL. A comprehensive analysis was conducted on 10 batches of DOL samples sourced from diverse locations, utilizing a miniature mass spectrometer (mini MS) equipped with a paper capillary spray (PCS) technique. Multivariate statistical approaches were employed to classify the samples and pinpoint chemical markers indicative of authenticity. Subsequent MS-guided separation and thin-layer chromatography (TLC) verified the markers' validity and assessed the greenness profile by Analytical Eco-scale (AES), Green Analytical Procedure Index (GAPI), and Analytical GREEnness (AGREE). A total of 10 batches of DOL samples detected by PCS-mini MS were classified into authentic and counterfeit, by unsupervised cluster analysis. Sativanone (m/z 301.1, VIP=6.0, p=0.000001) and 3′-O-methylviolanone (m/z 331.1, VIP=3.2, p=0.000382) were regarded as the chemical markers for the rapid identification of DOL. The results of the TLC method were consistent with this method, and the new method is greener. The application of mini MS for on-site authentication of DOL via specific chemical markers offers significant advantages, including operational simplicity, high efficiency, greenness, and accuracy. The deployment of this strategy promises to facilitate the effective regulation of DOL, ensuring authenticity and quality.

Development and Validation of a High-Performance Thin-Layer Chromatography Method for the Qualitative Analysis and Quantification of Hispidulin in Hydroethanol Extract of S. barbata D. Don

Background: Hispidulin, a flavonoid renowned for its diverse pharmacological activities, is abundant in S. barbata D. Don. Given its prevalence, it is essential to establish a dependable analytical method for quantifying hispidulin in this plant. Methods: This study introduces a sensitive and reliable high-performance thin-layer chromatographic (HPTLC) method for both qualitative and quantitative analysis of hispidulin in S. barbata D. Don. Pre-coated TLC aluminum plates were utilized, and after testing various mobile phase combinations, the most effective separation was achieved with Toluene-ethyl acetate-formic acid (5:4:1, v/v/v). The method underwent extensive validation for linearity, specification, instrument precision, precision, limits of LOD and LOQ, accuracy, and robustness. Results: The developed method demonstrated excellent linearity over a specified concentration range, with a determination coefficient (r2) of 0.988. A well-defined spot at Rf 0.7, corresponding to hispidulin, was observed. The concentration of hispidulin in the hydroethanolic extract of S. barbata D. Don was quantified as 13.02 μg/mg of extract. Conclusion: In conclusion, the HPTLC method presented in this study offers a simple, accurate, and robust tool for quantitatively analyzing hispidulin in the hydroethanol extract of S. barbata D. Don. This method is suitable for routine quality control and standardization of herbal formulations containing S. barbata D. Don, thereby contributing to the advancement of research in natural product- based pharmaceuticals.

HPTLC Densitometric Analysis for Quantification of Sennocide-B and Rhein in Leaf Extracts of Senna alexandrina Mill. and Their Commercial Formulations.

The leaf extract of Senna alexandrina Mill., a plant from the Fabaceae family, contains bioactive anthraquinones sennoside-B and rhein, traditionally used in Indian medicine for various health issues. However, standardizing the quality of S. alexandrina and its commercial products is challenging. To address this, we established two high-performance thin-layer chromatography (HPTLC) methods for accurate analysis of these biomarkers in S. alexandrina leaf extract and its commercial formulations. For sennoside-B, a mobile phase combination of solvent for example, butanol, water, and glacial acetic acid (6:3.5:0.5, v/v/v) was used, with detection at 254 nm, producing spots at an Rf value of 0.37 ± 0.006. The method showed linearity between 100 to 2000 ng/spot (R² = 0.9983 ± 0.0017), with a limit of detection (LoD) of 22.84 ± 0.554 ng/band and a limit of quantitation (LoQ) of 69.22 ± 0.859 ng/band. For rhein, a mobile phase combination of solvent toluene, ethyl acetate, and glacial acetic acid in the ratio (7:2.5:0.5, v/v/v) was utilized and was also detected at 254 nm. The obtained spots had Rf value of 0.67 ± 0.004. The linearity was confirmed within the same range (R² = 0.9985 ± 0.0005), with an LoD of 15.49 ± 0.645 ng/spot and an LoQ of 46.94 ± 1.172 ng/spot. Herein, the methods have followed the guidelines of ICH (Q2) R1. These developed HPTLC methods are selective, simple, sensitive, accurate, and economical, making them suitable for everyday assessment of these bioactive markers in S. alexandrina and its commercial products. This study supports the quality control of S. alexandrina, ensuring product consistency, efficacy, and safety.

Development and validation of High-Performance Thin Layer Chromatographic methods for simultaneous determination of antibacterial pharmaceutical formulations containing Clindamycin phosphate.

The main objective of this research is to establish and authenticate the High-Performance Thin-Layer Chromatographic techniques for the simultaneous assessment of Clindamycin phosphate in combination with Tretinoin (formulation-1) and Clindamycin phosphate with Adapalene (formulation-2), both of which are antimicrobial combinations. In the developed HPTLC methods, for combination-1, the optimized mobile phase was a mixture of n-Hexane: Ethyl acetate: ACN: Methanol (5:2:2:1 by volume) with observed Rf values of 0.413 and 0.787 for Clindamycin phosphate and Tretinoin, respectively. Similarly, for combination-2, the mobile phase comprised n-Hexane: Diethyl ether: Dichloromethane: Acetic acid (4:4:2:0.1 by volume) with recorded Rf values of 0.407 and 0.559 for Clindamycin phosphate and Adapalene, respectively. The developed methods are successfully validated as per the regulatory guidelines. This approach proves to be significantly time-saving and costeffective, rendering it suitable for routine analyses of the formulations containing selected antibacterial active pharmaceutical ingredients and commercial formulations.

Stability-indicating high-performance thin-layer chromatography method development and validation for dapagliflozin propanediol monohydrate, vildagliptin, and metformin hydrochloride in active pharmaceutical ingredient and its tablet dosage form

Stability-indicating high-performance thin-layer chromatography method development and validation for dapagliflozin propanediol monohydrate, vildagliptin, and metformin hydrochloride in active pharmaceutical ingredient and its tablet dosage form

An eco-friendly smartphone based HPTLC method versus conventional densitometric one for determination of Naltrexone and Bupropion

The rapid uprising technologies of smartphone applications and software introduced a new era for analytical detection techniques. It has transformed bench-top laboratory methods into simpler ones depending on cost-effective, portable, and widely accessible devices. In this work, two high performance thin layer chromatographic (HPTLC) methods were developed based on smartphone’s camera detection and either ImageJ desktop software or Color-Picker smartphone’s application as alternative techniques to conventional densitometric detection. A mixture of Naltrexone hydrochloride (NAL) and Bupropion hydrochloride (BUP) was chromatographed on HPTLC- plates using ethyl acetate, methanol, acetone, and glacial acetic acid (3:6:1:0.5, by volume) as a developing system. The developed plates were scanned at 203 nm for the densitometric analysis, then visualized by modified Dragendorff’s reagent and shot by a smartphone’s camera. The captured images were uploaded to either ImageJ software or Color-Picker application to detect the separated spots. The results derived from the three detection methods were compared over the concentration range of 0.4–24 & 0.6–18 µg/band for the densitometric method, 0.4–24 & 2–24 µg/band for ImageJ built method and 0.8–20 & 5–20 µg/band for Color Picker built method for NAL and BUP, respectively. The methods were found to be appropriate for assaying both active drug substances in pure forms and combined in marketed pharmaceutical formulations. The excellent sustainability of densitometric and ImageJ-based methods enabled also the assessment of their dosage form content uniformity. The greenness and sustainability of the methods were assessed by three metric tools, namely Green Analytical Procedure Index (GAPI), Analytical GREEnness Metric Approach (AGREE), and White Analytical Chemistry (WAC). The assessments results confirmed the sustainability and superiority of the proposed methods in terms of sample treatment, waste mount, energy consumption, cost, and number of analyzed samples per an hour.

Leveraging Strengths of green chromatography in Developing eco-friendly HPLC and HPTLC methods for the simultaneous analysis of Lamivudine, Dolutegravir and tenofovir alafenamide

The development of green analytical methodologies is a critical challenge in the field of pharmaceutical analysis. The ternary mixture investigated in this study consists of the antiretroviral drugs Lamivudine (LMV), Tenofovir alafenamide fumarate (TAF), and Dolutegravir (DTG), which are all approved antiretroviral drugs for the treatment of HIV-1 infection. While LMV was approved in 1995, DTG and TAF were more recently approved in 2013 and 2016, respectively, as newer generation HIV medications with improved efficacy and safety profiles compared to earlier antiretroviral drugs. This study presents three eco-friendly approaches for the simultaneous determination of the ternary mixture. Two HPLC methods were applied: an organic-solvent free mixed-micellar HPLC technique was developed as (method A), using a Cyano column (150 × 4.6 mm,5.0 µm) with a mobile phase consisting of 0.03 M Brij-35, 0.09 M sodium dodecyl sulfate, and 0.02 M ammonium acetate in water, adjusted to pH 3.0. Green solvents were applied in (method B), namely, phosphate buffer 25 mM adjusted to pH 3.0 and ethanol (45:55, v/v) using an Inertsil-C18 column (250 × 4.6 mm,5.0 µm,). Peaks were detected at 258 nm, and the retention times for (method A) were 3.10, 3.95 and 5.03 min, and for (method B) were 2.53, 3.63 and 4.86 min for LMV, TAF and DTG, respectively. Additionally, a green thin-layer chromatographic (TLC) method was developed using a water-isopropanol-formic acid mobile phase (65/35/10,v/v/v) and RP-18 silica gel 60 HPTLC-plates. The drugs were successfully resolved, with retardation factor (Rf) values of 0.26, 0.54 and 0.82 for DTG, TAF, and LMV, respectively. The novel methods were validated and applied successfully in marketed dosage forms. Their greenness profiles were assessed using two recent metrics.

Rethinking Thin-Layer Chromatography for Screening Technetium-99m Radiolabeled Polymer Nanoparticles.

Thin-layer chromatography (TLC) is commonly employed to screen technetium-99m labeled polymer nanoparticle batches for unreduced pertechnetate and radio-colloidal impurities. Although this method is widely accepted, our findings applying radiolabeled PLGA/PLA-PEG nanoparticles underscore its lack of transferability between different settings and its limitations as a standalone quality control tool. While TLC profiles may appear similar for purified and radiocolloid containing nanoparticle formulations, their in vivo behavior can vary significantly, as demonstrated by discrepancies between TLC results and single-photon emission computed tomography (SPECT) and biodistribution data. This highlights the urgent need for a case-by-case evaluation of TLC methods for each specific nanoparticle type. Our study revealed that polymeric nanoparticles cannot be considered analytically uniform entities in the context of TLC analysis, emphasizing the complex interplay between nanoparticle composition, radiolabeling conditions, and subsequent biological behavior.

HPTLC method for determination of anti-hypertensive drug combination metoprolol succinate and dapagliflozin

Combination of Metoprolol Succinate and Dapagliflozin is recommended in the treatment of Hypertension. Accurate and precise thin layer chromatographic method has been developed for the determination of Metoprolol Succinate and Dapagliflozin in combination. Pre-coated silica gel G60—F254 aluminium sheet (10 × 10 cm),0.2 mm layer thickness used as stationary phase and n-Butanol: Ethyl acetate: Triethylamine (6:4:0.1 v/v/v) used as mobile phase. The method was found to be linear with a range of 200–1200 ng/band for DAP and 1000–6000 ng/band for MET with correlation coefficient (r2) 0.996 for both DAP and MET. The proposed method was validated as per ICH Q2 (R2) guideline with respect to linearity, accuracy and precision. Stress degradation was carried out to find out the intrinsic stability of the molecules. Both the drugs were highly susceptible to acid and base hydrolysis and degradation products were well separated with drug peak. The method was successfully used for determination of metoprolol succinate and dapagliflozin in combination.

Study on Preparation Technology and Quality Standard of Acne Granules

AbstractThe study aimed to optimize the preparation process of acne granules and establish their quality standards. In this work, the extraction process of Chinese herbal extract was optimized by the amount of water added, the number of decoction, the extraction time, and the soaking time with extraction yield as an evaluation index. The indexes of the acne granules such as molding rate, dissolvability, angle of repose, moisture content, and ease of preparation were evaluated. Thin-layer chromatography (TLC) was used to identify Salviae, Scutellaria baicalensis, and Indigowoad Leaf. High-performance liquid chromatography (HPLC) was used to determine the baicalin content in the granules. Based on orthogonal and single-factor experiments, the optimized extraction process of the prescription of nine medicinal materials was as follows: soaked in cold water for 2 hours, boiled three times, decocted with eight times the amount of water for 1.5 hours for the first time, and six times the amount of water for 1 hour for the second and third times. The combined extracts were concentrated to a relative density of 1.30 to 1.40 (20–30°C), and mixed evenly according to the mass ratio of extract to excipient 1:5, and dextrin: powdered sugar = 1:3. The mixture was granulated, dried, prepared into granules, and the acne granules were formed at a molding rate of 95.52% and a critical relative humidity of 82%. The spots in TLC were clear and easy to identify. The HPLC result showed that the content of baicalin was not less than 1.0 mg/g. The study provides a valuable reference for the production and preparation of the granules through optimization of the wet process and the excipient dosage. Furthermore, the established TLC method for the identification and the HPLC method for baicalin quantification laid the foundation for the quality control of the preparation in future studies.

HPTLC – densitometric method of analysis for estimation of efonidipine hydrochloride and telmisartan used in treatment of hypertension

Precise, accurate, and sensitive high performance thin layer chromatographic method has been developed and validated for simultaneous determination of efonidipine hydrochloride (EFD) and telmisartan (TEL) in a tablet dosage form which is used in treatment of hypertension. The proposed method used aluminum plates pre-coated with silica gel 60 F254 as a stationary phase and n-butanol: toluene: acetic acid (3:7:0.2, v/v/v) as a mobile phase for separation purpose. Compact spots of TEL and EFD were obtained at Rf 0.73 and 0.37, respectively. Densitometric detection was carried out at 299 nm in UV. Linear responses were shown by the detector over the range of 200–1200 ng/band for both drugs. The proposed method was validated as per ICH guidelines. The method was successfully applied for estimation of both drugs in tablet formulations. Forced degradation study was carried and efonidipine was found to be susceptible to base hydrolysis and oxidative stress degradation while TEL was stable in acid-base hydrolysis, oxidative stress, dry heat and photo stability testing conditions. The developed method can be used for the analysis of stability samples. Greenness assessment of developed method was performed using AGREE software. The method was found to be greener having greenness score of 0.69.