Thin-layer Chromatographic Separation Research Articles

Separation and characterization of the antioxidant active component from Maillard reaction products in xylose-lysine system

ABSTRACT Maillard reaction products (MRPs) play a crucial role in preserving food quality due to their antioxidant capacity. In this study, we isolated and characterized the antioxidant components from MRPs using a xylose-lysine system under optimal conditions. Methylene chloride and ethyl acetate were employed for sequential extractions, and the ethyl acetate phase underwent thin-layer chromatography and silica column separation. The identified compound, 3-hydroxy-4-methyl-2(5 H)-furanone (3H4MFN), was analyzed using FT-IR, GC-MS, and NMR, revealing its volatile nature with a chemical formula of C5H6O3. Further activity analysis demonstrated that 3H4MFN exhibited higher or comparable antioxidant activity to vitamin C (VC) and vitamin B6 (VB6). This newly discovered compound shows potential as a natural antioxidant in cosmetics, food, and nutraceutical applications.

Characterization and Identification of Novel Steroids from Nauclea pobeguinii Leaves

Nauclea pobeguinii is a plant in the Rubiaceae family endemic to the swamp forest region of the world. Its extract is widely used in traditional medicine for the treatment of a wide variety of ailments such as malarial, Jaundice, gonorrhea, fever, and stomach discomfort. While other parts of the plant have been examined for the bioactive principles responsible for the medicinal properties, limited information is available in the literature as regards the leaves, hence this study. N. pobeguinii leaves were collected, air-dried, and pulverized. The pulverized sample was extracted with solvents (n-hexane, ethyl acetate, and ethanol) of varying polarity to obtain the crude extracts. Repeated column and thin layer chromatographic separation of the crude extracts afforded three compounds, which were characterized by their IR, 1H, 13C-NMR, and 2D-NMR spectral data. A comparison of the data with literature confirmed the compounds to be 2-hydroxylstigmastane acetate (1), Ergosta-5,6-epoxy-22-en-3-yl-acetate (2), and β-sitosterol (3). Compounds 1 and 2 are novel to N. pobeguinii.

Development and Validation of Chromatographic Methods for Simultaneous Estimation of Aspirin and Pantoprazole Sodium in Pure and Mixture Form.

This technique is employed for the simultaneous quantification of aspirin (ASP) and pantoprazole sodium in both pure powder form and formulations. The high performance liquid chromatography (HPLC) method uses a C-18 column (250mm × 4.6mm, 5μm) with a mobile phase consisting of 0.05M disodium hydrogen phosphate and methanol in a 40:60% v/v ratio. The flow rate is maintained at 1.0mL/min. On a layer of silica gel 60F254 with an aluminum backing, the high performance thin layer chromatography (HPTLC) separation was carried out with ethyl acetate and methanol (8: 1.5v/v) as the mobile phase. With a mean recovery of 100.54% and 99.55% for ASP and PNT, respectively, quantification was accomplished using the HPLC method with UV detection at 286nm over the concentration range of 0.1-0.6g/mL for PNT and 0.4-2.4g/mL for ASP. With a mean recovery of 99.44% and 99.01% for ASP and PNT, respectively, quantification was achieved using the HPTLC method with UV detection at 298nm over the concentration range of 400-2400ng/spot for ASP and 100-600ng/spot for PNT, respectively. The methods can be used for the simultaneous determination of ASP and PNT in pure powder form and formulations as they are simple, accurate and sensitive.

Colorimetric analysis platform based on thin layer chromatography for monitoring gluten cross-contamination in food industry

Monitoring of the accidental presence of gluten (Glu), resulting from cross-contamination, is imperative in different industries, in particular food industry. The objective of this study was the development of an analytical platform utilizing thin-layer chromatography (TLC) with colorimetric read-out for making binary (yes/no) decisions on surfaces and/or point of these industries. The composition of the extractive phase was optimized with commercial products used in cleaning processing lines. Subsequently, an exploration of TLC separation and detection was undertaken. CN-modified nanosilica plates and 30:70 acetonitrile:water were used to achieve a selective signal for Glu residues. The study of the detection performance showed that both spectroscopic measurement and image analysis were resulted in satisfactory results for quantitate analysis (RSD = 5 %, LOD = 0.12 mg). The practical application of the proposed methodology on surfaces of the food processing lines. This work demonstrated the operational feasibility in detecting gluten cross-contaminations within the food processing industry.

Effect of relative humidity on the thin-layer chromatographic separation of E 472 emulsifiers

E 472 emulsifiers are organic acid esters of mono- and diacylglycerides. These emulsifiers are categorized into six subgroups (E 472a-f) based on the specific acid component, e.g., acetic acid, lactic acid, citric acid, tartaric acid, or mixtures of tartaric and acetic acid. The present study investigated the effect of relative humidity (RH) on the thin-layer chromatographic separation of four E 472 emulsifier subgroups, addressing practical concerns due to RH variations between seasons and within laboratories. Three RH-setting techniques affecting plate activity and chamber climate were assessed using saturated salt and aqueous sulfuric acid solutions. Aqueous sulfuric acid solutions for RH adjustment resulted in decreased hRF values, while no trends in hRF value changes were observed for saturated salt solutions. Unlike RH itself, the RH setting technique led to substantial changes in the chromatographic fingerprint. Thus, the choice of the RH setting method should be tailored to the specific analyte and analysis goal. Despite variations in chromatographic fingerprints between the three RH setting techniques, the fingerprint remained consistent within the same technique across the tested RH range.

Quantitative saccharide release of hydrothermally treated flours by validated salivary/pancreatic on-surface amylolysis (nanoGIT) and high-performance thin-layer chromatography

The susceptibility of hydrothermally treated flour products to amylolysis was studied. The human salivary α-amylase and porcine pancreatin enzyme mixture containing α-amylase were used on-surface to investigate the release of glucose, maltose, and maltotriose. On the same adsorbent surface (all-in-one), their high-performance thin-layer chromatography separation and detection via selective chemical derivatization was performed. For the first time, the all-in-one nanoGIT system was studied quantitatively and validated for the simulated static oral and intestinal on-surface amylolysis of ten different hydrothermally treated flours and soluble starch. Differences were detected in the digestibility of refined and whole flours from wheat, spelt, and rye as well as from einkorn, amaranth, emmer, and oat. Amaranth released the lowest amount of saccharides and spelt the highest in both oral and intestinal digestion systems. The results suggest that consumption of whole grain products may be beneficial because of their lower saccharide release, with particular attention to rye.

THIN LAYER CHROMATOGRAPHIC TECHNIQUES FOR DETECTION OF SUDAN DYES IN PALM OIL

Sudan dyes I, II, III & IV are synthetic, oil-soluble, red-coloured azo dyes. These dyes are suspected genotoxic carcinogens and have been used in the adulteration of palm oil products. Many experimental studies on animal specimen have confirmed the formation of tumour due to the presence of different Sudan dyes in food products. In this study, a Simple Thin Layer Chromatographic method is described for the qualitative detection of Sudan-I, II, III & IV in palm oil gotten from different markets. The study includes extraction of sample, preparation of Thin Layer Chromatography (TLC) plates, Separation, detection and visualization of dyes on TLC plates. The dyes were extracted with solvent Acetonitrile. Sample extracts were decanted and analysed by simple thin layer chromatography in hexane, chloroform and acetic acid solvent system. The chromatograms of samples and standards were stained with Iron (ii) chloride stain and further evaluated in term of Rf values. Out of the five samples analysed the Rf values of the colouring spots from the three samples L2, L3 and L4, matched the standards (S2). It is therefore, concluded that samples L2, L3 and L4 contain Sudan IV dye. Without using any derivation, the limit of detection (18mg/L for S2 (Sudan IV dye) and 10mg/L for SI (Sudan III dye) was determined by visually determining if a spot was visible or not at a certain concentration of standard dye.

Chemical and cytotoxicity profiles of 11 pink pepper (Schinus spp.) samples via non-targeted hyphenated high-performance thin-layer chromatography

IntroductionPink pepper is a worldwide used spice that corresponds to the berries of two species, Schinus terebinthifolia Raddi or S. molle L. (Anacardiaceae). Toxic and allergic reactions by ingestion or contact with these plants were reported, and classical in vitro studies have highlighted the cytotoxic properties of apolar extracts from the fruits.ObjectivesPerform a non-targeted screening of 11 pink pepper samples for the detection and identification of individual cytotoxic substances.MethodsAfter reversed-phase high-performance thin-layer chromatography (RP-HPTLC) separation of the extracts and multi-imaging (UV/Vis/FLD), cytotoxic compounds were detected by bioluminescence reduction from luciferase reporter cells (HEK 293 T-CMV-ELuc) applied directly on the adsorbent surface, followed by elution of detected cytotoxic substance into atmospheric-pressure chemical ionization high-resolution mass spectrometry (APCI-HRMS).ResultsSeparations for mid-polar and non-polar fruit extracts demonstrated the selectivity of the method to different substance classes. One cytotoxic substance zone was tentatively assigned as moronic acid, a pentacyclic triterpenoid acid.ConclusionThe developed non-targeted hyphenated RP-HPTLC–UV/Vis/FLD–bioluminescent cytotoxicity bioassay–FIA–APCI-HRMS method was successfully demonstrated for cytotoxicity screening (bioprofiling) and respective cytotoxin assignment.

Separation of Bio-active Compounds and in vitro Antibacterial Potency of Callistemon linearis Floral Extract with Mechanism of Action

Abstract: Introduction: Infection caused by bacteria is treated with antibiotics which caused several side effects in human and animals. This problem enforced scientist to work out plant originated compounds for treatment of several disease caused by morbific bacteria. Objectives: Present work revealed isolation of bioactive phytocompounds from different fractions of Callistemon linearis flower ethanol extract by chromatographic technique and determination of their antibacterial potency against selected bacteria with mode of action in vitro. Materials and Methods: Antibacterial potency of four different fractions of ethanol extract were done quantitatively via Agar well diffusion method along with determination of Minimum Inhibitory Concentration, and Minimum Bactericidal Concentrations. Further, phytochemical analysis, Fourier Transform Infrared spectroscopic analysis, thin Layer Chromatography separation and Gas Chromatography Mass Spectroscopy analysis of bioactive fractions were done simultaneously to assess the presence of bioactive phytocompounds responsible for the antibacterial potency of the fractions. Time-kill assay, Nucleic acid and protein leakage assay, and FESEM study were employed to determine the mode of activity of the isolated active phytocompounds on the tested bacteria. Results: Maximum antibacterial activity was observed in NHEX of ethanol extract against Escherichia coli (MTCC 739). Chromatographic separation of NHEX detected several bioactive compounds. Assessment of Time-kill assay in treated and untreated bacterial strains determined that bioactive compound of NHEX was bactericidal in nature. Nucleic acid and protein leakage assay of bacterial cells with isolated active compound showed cell damages and FESEM images strongly ascertained this data. Conclusion: The C. linearis floral floral extract possesses antibacterial activities against selected pathogens and causes cellular damages. Keywords: Callistemon linearis, Antibacterial activity, FT-IR, GC-MS analysis, Bioactive compounds, Mechanism of action.

Winemaking by-products as a sustainable source of antioxidant and functional compounds

In the last years, the international guidelines encourage the reduction of food waste or processing by-products to promote a circular economy approach. Winemaking represents one of the sectors with the highest by-products generation, with several potential negative impact on the environment. Winemaking by-products are mainly used to produce distillates or fertilizers, but the interest in grape pomaceas a potential source of phenolic compounds has considerably increased. The aim of this study was the application ofin vitromethods for the characterization of the phenolic fraction of winemaking by-products from different red grape varieties, and, in parallel, the evaluation of their antioxidant activity. The methods were: 1) Folin-Cocalteau's assay for the quantification of total polyphenol content; 2) the vanillin assay for the assessment of total flavan-3-ols (proanthocyanidins) content; 3) the pH differential method for the quantification of total anthocyanin content; 4) DPPH assay for the measurement of total antioxidant activity; 5) High Performance Thin Layer Chromatography for separation of phenolic substances and assessment of their antioxidant activity. Although a significant differences among varieties, grape pomace, particularly when containing seeds, are generally a good source of polyphenols with a significant antioxidant activity supporting itsuse in formulating healthy products.

Isolation and Identification of β-sitosterol, 7-hydroxystigmast-22-en-3,6-dione and 3β, 24(S)-dihydroxycholesta-5, 25-diene-7-one from stem bark of Nauclea pobeguinii

The stem bark of the Nauclea pobeguinii was collected, air-dried, and pulverized and was extracted with solvent of varying polarity (n-hexane, ethyl acetate, and ethanol) to obtain the crude extracts. Silica gel column and thin layer chromatographic separation afforded three compounds whose structures were elucidated as β-sitosterol (1), 7-hydroxystigmast-22-en-3.6-dione (2), and 3β, 24(S)-dihydroxycholesta-5, 25-dien-7-one (3) by analysis of their chemical and spectral characteristic from 1D and 2DNMR, FTIR and by comparing of data with those reported in the literature.

Design of a Safety Device Ultra Violet Light for Mercury Identification in Whitening Cream with Thin Layer Chromatography Method Using Camera OV7670 Based on Arduino Uno

Mercury is also called “air raksa” or hydrargyrum which is a chemical element with the symbol Hg and belongs to the heavy metal group with a liquid form and silvery color. Mercury is a heavy metal that is harmful to the skin when added to cosmetics, especially creams on the face, even in small concentrations because it is toxic. Identification of Mercury in the laboratory using the thin layer chromatography method with the help of ultra violet light causes medical personnel to be vulnerable to exposure to ultra violet rays which have a negative impact on eye and skin health. The purpose of this research is to design a safety to minimize exposure to ultra violet rays when identify mercury. OV76070 which can reduce direct contact with ultra violet light identifies the present of Mercury in the whitening cream will be carried out using a thin layer chromatography separation method with the help ultra violet 254nm-366nm. With the help of ultra violet lamps 254nm-366nm, and it is hoped that Mercury inspection using the OV7670 camera will be more efficient and accurate. The tool used in this research is the OV7670 camera with a power of 640 – 460 pixels (VGA) to take photo on thin layer chromatography plate, ultra violet lamps 254nm- 366nm. Overall the design of this tool is controlled by the Arduino Uno microcontroller. Image from the OV7670 camera can be monitored at the Liquid Crystal Display in the form of the appearance fluorescence spot indicating the presence of Mercury in the whitening cream sample.

HPTLC method for effective separation of L-glutamic acid and GABA present in brain tissue extracts

L-glutamic acid and ϒ-aminobutyric acid (GABA) show similar physicochemical properties, so separating them by thin-layer chromatography (TLC) is difficult. TLC is currently a widely used method because of its simplicity, flexibility, and low cost compared to liquid or gas chromatography. This paper shows the TLC separation of [3H]glutamic acid from [3H]GABA formed by incubating the former with the membrane or the cytosolic fraction obtained from the locus coeruleous on the brain. Such separation was achieved with a better resolution than the literature reported. Radiolabeled compounds were extracted from the tissue and seeded on an HPTLC plate. In addition, placed the L-glutamic acid and GABA standards in different lanes. The plates were developed in a solvent system of butan-1-ol, acetic acid, and water = 5:3:2 (v:v), containing 0.4% ninhydrin as a derivatizing agent. HPTLC was used to obtain better resolving power, faster development times, less sample diffusion, and lower solvent consumption than TLC. Results showed that the L-glutamic acid and GABA standards were adequately separated, with a resolution of 2.2. Moreover, the studied tissue transformed 9% of the radiolabeled glutamate into GABA. The L-glutamic acid was converted into a fluorescent tautomer from Schiff's base, detectable on a λ=302 nm. However, when the HPTLC dried, the tautomer was identified as GABA for its Rf=0.32, and the luminescence disappeared. The resolution between both components was excellent (Rs=2.2). In conclusion, it was possible to develop a reproducible method from the data of this study, which could attract the interest of researchers.

Evidence for Cardiac Glycosides in Foliage of Colorado Potato Beetle-Resistant Solanum okadae.

Leptinotarsa decemlineata, the Colorado potato beetle (CPB), is a herbivore that primarily feeds on Solanum foliage and is a global pest of the potato agricultural industry. Potato breeding through cross-hybridization with CPB-resistant wild relatives is used for genetic improvement. The wild species Solanum okadae was demonstrated to deter CPB feeding in choice and no choice feeding assays. Liquid chromatography-mass spectrometry (LC-MS) was used for comparative metabolite profiling between S. okadae and CPB-susceptible domesticated potato variety, Solanum tuberosum cv. Shepody. Major foliar metabolites detected were steroidal glycoalkaloids (SGAs) with tomatine and dehydrotomatine produced in S. okadae and solanine and chaconine in S. tuberosum cv. Shepody. Cardiac glycosides were also detected in the foliar metabolite profile of S. okadae but not S. tuberosum cv. Shepody. This class of plant compounds have known insecticidal activity through inhibition of animal Na+/K+ ATPase. Thin-layer chromatography (TLC) separation of foliar extracts also provided evidence for cardiac glycosides in S. okadae. Cardiac glycosides are known inhibitors of Na+/K+ ATPase, and foliar extracts from S. okadae (OKA15), but not S. tuberosum cv. Shepody, were able to inhibit the Na+/K+ ATPase of CPB. These findings suggest a novel mechanism of plant resistance against CPB involving production of cardiac glycosides in S. okadae.

Potential of an endophytic fungus Alternaria tenuissima PE2 isolated from Psidium guajava L. for the production of bioactive compounds

Searching of novel bioactive compounds from natural sources is increasing day by day for overcoming multidrug resistant microorganisms and endophytic fungi are considered as important sources in this regard. In the present study, one potent endophytic fungal isolate PE2 was isolated from the leaves of Psidium guajava L. which was identified as Alternaria tenuissima by 28S rDNA sequence homology. The isolate was found to produce thermostable, non-proteinacious antimicrobial substances. Ethyl acetate (EA) extract of the cell free supernatant (CFS) of the isolate was found effective against both Gram-positive and Gram- negative bacteria with MIC values of ∼500 µg/mL and ∼800 µg/mL, respectively. Degradations of bacterial cells upon treatment were supported by the release of intracellular materials as well as scanning electron microscopic (SEM) studies. EA extract also exhibited an adverse effect on bacterial central carbohydrate metabolic pathway enzymes viz. phospho fructokinase, isocitrate dehydrogenase and fructose-1,6-bisphosphatase. Production of inhibition zones against pathogenic fungi including human pathogenic yeast Candida albicans, inhibition of fungal radial growth and conidial germination upon treatment suggested antifungal potential of the EA extract of the isolate. SEM study revealed massive deformation of cellular integrity of C. albicans, indicated the probable effect of antimicrobial compounds. Thin layer chromatographic (TLC) separation and GC-MS analysis revealed the production of numbers of bioactive compounds by the isolate. Based on the present study, isolate Alternaria tenuissima PE2 could be considered as a promising source of natural bioactive metabolites.

Recent findings by high-performance thin-layer chromatographic separation for a comprehensive analysis of Withania somnifera by densitometry and mass spectrometry: an assessment to quality and adulteration

Recent findings by high-performance thin-layer chromatographic separation for a comprehensive analysis of Withania somnifera by densitometry and mass spectrometry: an assessment to quality and adulteration

Development and validation of TLC-densitometry method for quantification of tetraprenyltoluquinone in the stem bark hexane extract of Garcinia cowa roxb

A thin layer chromatography (TLC) method coupled with densitometry was optimized and established to quantify tetraprenyltoloquinon (TPTQ) in the hexane extract of Garcinia cowa roxb stem bark. The stationary phase was TLC plates of silica gel 60 F254, with a thickness of 250 μm, while a mobile phase was the mixture of hexane: chloroform: ethyl acetate: formic acid in the ratio of 20:70:9:1 was selected for the chromatographic system. TLC separation of the components was followed by densitometric analysis at 230 nm. The chromatogram revealed a tight spot for TPTQ at the Rf = 0.41 ± 0.03). The value of the regression equation (r2 = 0.992) demonstrated an excellent linear association between peak area and TPTQ amount in the 250–750 μg.spot-1 range. The precision, specificity, and accuracy of the approach were all validated. The 72.65 and 242.15 μg.spot−1 were found as the limit of detection and quantification, respectively. The proposed TLC method can be considered for identifying and quantifying TPTQ in Garcinia cowa Roxb bark hexane extracts.

Anti-MRSA and clot lysis activities of Pestalotiopsis microspora isolated from Dillenia pentagyna Roxb.

There is an urgent call to search for novel natural compounds against developing multidrug-resistant microorganisms. The present work focuses on the characterization of a plant-associated fungus having bioactivity against methicillin-resistant Staphylococcus aureus (MRSA) strains. A fungal strain P31 was isolated from bark of Dillenia pentagyna and identified as Pestalotiopsis microspora. The maximum anti-MRSA activity was observed from extract of P31 grown in sabouraud dextrose broth. The minimum inhibitory concentrations(MIC) values of P31 extract were 14 μg/ml for methicillin-sensitive S. aureus (MSSA) and 32 μg/ml for MRSA strain, respectively. A crude P31 extract showed strong bactericidal activity by killing all treated MRSA cells within 24 h of treatment at their respective MIC value. A scanning electron microscopic study visualized morphological damage of MRSA cells. The membrane permeability of P31 extract-treated MRSA cells gradually increased which caused release of internal cytoplasmic nucleic acids, proteins and potassium ions (K+ ) from cells suggesting cell lysis or leakage from cells. A very low concentration of P31 extract was able to inhibit biofilm formed by MRSA cells. Thin layer chromatographic separation followed by gas chromatography-mass spectrometry analysis of the P31 extract revealed a number of antimicrobial compounds along with an anti-MRSA compound 2,4-di-tert-butylphenol. In addition, the P31 extract also showed in-vitro human blood clot lysis activity at various concentrations. The clot lysis activity of P31 extract was found maximum at 500 µg/ml. These findings suggest that fungal isolate P31 has potential as a source of anti-MRSA compounds useful in staph infections.

Development of a Thin-Layer Chromatography-Enzymatic Test Combination Method for the Isolation of α-Glucosidase Inhibitors From Thymelaea hirsuta.

A rapid, easy and simple method for the isolation and purification of α-glucosidase inhibitors of the ethyl acetate extract of Thymelaea hirsuta (EaTh) by a combination of thin layer chromatography (TLC) and enzymatic test has been developed. EaTh was demonstrated previously a potent α-glucosidase inhibitory effect. In this study, we developed a simple TLC-enzymatic test (TLC/EZ) combination to isolate α-glucosidase inhibitors present in EaTh.EaTh was extracted by Soxhlet from Thymelaea hirsuta (T. hirsuta). The EaTh was separated on a silica gel column and then on a TLC plate. After TLC separation, the TLC/EZ combination method was applied. α-glucosidase inhibitors were detected directly in the TLC plate using the glucose oxidase peroxidase method (GOD-POD). A good detection of active compounds was obtained in the TLC favoring the TLC/EZ method. Active compounds were then characterized using high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. The main α-glucosidase inhibitors present in EaTh have a molecular ion [M + H]+ at m/z = 543. This proposed method is suitable for a reliable isolation and purification of α-glucosidase inhibitors present in EaTh. It could be proposed as an interesting alternative of the classical method for the isolation and purification of α-glucosidase inhibitors in plant extracts.

Implementation of different separation techniques for resolving ternary mixture of Paracetamol, Pseudoephedrine Hydrochloride and Chlorpheniramine maleate with further quantification

Abstract Due to the wide applicability of separation techniques that rely on the property of differential migration in pharmaceutical formulations analysis, different analytical strategies have been proposed to resolve mixtures of multi-components pharmaceuticals. Three separation methods were developed and validated for the simultaneous determination of Paracetamol (PAR), Pseudoephedrine HCl (PSE) and Chlorpheniramine maleate (CHP). The first method is a thin-layer chromatographic (TLC) separation, followed by densitometric measurement. The separation was carried out on aluminium sheet of silica gel 60 F254 using ethanol:chloroform:ammonia (1:7:0.4, by volume) as the mobile phase. Determination of PAR, PSE and CHP was successfully applied over the concentration ranges of 3–25 µg/band, 0.5–10 µg/band and 0.1–6 µg/band, respectively. The second method is HPLC separation that was achieved on C18 column using the mobile phase acetonitrile:phosphate buffer pH 5 (10:90, v/v) at a flow rate 1 mL min−1. PAR, PSE and CHP were determined by HPLC in concentration ranges of 5–400 μg mL−1, 2–40 μg mL−1 and 0.5–16 μg mL−1, respectively. The third method is a capillary electrophoresis (CE) separation. The electrophoretic separation was achieved using 20 mM phosphate buffer (pH 6.5) at 20 kV. The linearity was reached over concentration ranges of 30–250 μg mL−1, 5–50 μg mL−1 and 0.8–20 μg mL−1 for PAR, PSE and CHP, respectively. The developed methods were validated with respect to linearity, precision, accuracy and system suitability. The proposed methods were successfully applied for bulk powder and dosage form analysis with RSD of precision <2%. Moreover, statistical comparison with the official methods confirms the methods' validity.