Validated HPLC and HPTLC Method for Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Bulk Drug and Formulation

Two methods are described for simultaneous determination of amlodipine besylate and olmesartan medoxomil in formulation. The first method was based on the HPTLC separation of two drugs on Merck HPTLC aluminium sheets of silica gel 60 F₂₅₄ using n-butanol: acetic acid: water (5:1:0.1, v/v/v) as the mobile phase. The second method was based on the HPLC separation of the two drugs on the RP-PerfectSil-100 ODS-3–C₁₈ column from MZ-Analysetechnik GmbH, Germany and acetonitrile/0.03 M ammonium acetate buffer (pH = 3) in a ratio of 55:45 as the mobile phase. Both methods have been applied to formulation without interference of excipients of formulation.     

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog

Nitrosylcobalamin (NO-Cbl), a novel vitamin B₁₂ analog and anti-tumor agent, functions as a biologic ‘Trojan horse’, utilizing the vitamin B₁₂ transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. A stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an Ascentis^® RP-Amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min^?1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. Additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B₁₂ therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.

     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.     

Notiz zur densitometrischen Bestimmung von aromatischen Verbindungen durch Nitrierung auf HPTLC-Fertigplatten Kieselgel 60 F254

Zusammenfassung Verbindungen, die einen aromatischen Ring enthalten und unter kurzwelligem UV-Licht auf einer HPTLC-Fertigplatte Kieselgel 60 F₂₅₄ keine Absorption aufweisen, lassen sich durch Einwirkung nitroser Gase in Nitroverbindungen überführen, die unter kurzwelligem UV-Licht visuell nachweisbar sind. Nach der Chromatographie wird die HPTLC-Fertigplatte Kieselgel 60 F₂₅₄ im Trockenschrank erhitzt (15 min bei 160° C) und anschließend in heißem Zustand der Einwirkung von nitrosen Gasen (aus Salpetersäure 100%) ausgesetzt. Einwirkungszeit zur qualitativen Beurteilung der nitrierten Flecken: 3 min, zur quantitativen densitometrischen Messung: 10 min. Im Falle von Ephedrinhydrochlorid betrug die relative Standardabweichung 1,75%.

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Note on the densitometric determination of aromatic compounds by nitration on pre-coated HPTLC plates Silica gel 60 F₂₅₄

Summary Compounds containing an aromatic ring and not exhibiting any absorption on a pre-coated HPTLC plate Silica gel 60 F₂₅₄ under short-wave UV light may be converted by nitrous gases into nitro compounds which are visually detectable under short-wave UV light. After chromatography the HPTLC plate Silica gel 60 Fe₂₅₄ is heated in a drying chamber (15 min at 160° C), and then exposed while still hot to nitrous gases (from 100% nitric acid). Exposure time for qualitative assessment of the nitrated spots: 3 min, and for quantitative densitometric measurement: 10 min. The relative standard deviation was 1.75% in the case of ephedrine hydrochloride.

     

The Online Morphology Control and Dynamic Studies on Improving Vitamin B<Subscript>12</Subscript> Production by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis> with Online Capacitance and Specific Oxygen Consumption Rate

The relationship between the morphological character of Pseudomonas denitrificans and vitamin B₁₂ synthesis based on real-time capacitance measurement and online specific oxygen consumption rate (Q _O2) control was established for enhancing vitamin B₁₂ production. Results demonstrated that the threshold Q _O2 value lower than 2.0 mmol/gDCW/l would greatly stimulate the state transfer from the cell number growth phase to the cell elongation phase and promote rapid vitamin B₁₂ biosynthesis, while the vitamin B₁₂ biosynthesis rate could also be inhibited when the rate of cell’s length-to-width ratio (ratio-LW) was higher than 10:1. Furthermore, the optimal morphology controlling strategy was achieved based on online Q _O2 control, which increases the appropriate active cell numbers at the former phase, and then control the elongation of ratio-LW no more than 10:1 at the vitamin B₁₂ biosynthesis phase. The maximal vitamin B₁₂ production reached 239.7 mg/l at 168 h, which was improved by 14.7 % compared with the control (208 mg/l). This online controlling strategy would be effectively applied for improving industrial vitamin B₁₂ fermentation.     

Estimation of Alprazolam and Sertraline in Pure Powder and Tablet Formulations by High-Performance Liquid Chromatography and High-Performance Thin-Layer Chromatography

Abstract

This article describes validated high-performance liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous estimation of alprazolam (ALZ) and sertraline (SER) in pure powder and tablet formulation. The HPLC separation was achieved on a Nucleosil C18 column (150 mm long, 4.6 mm i.d., and 5-µm particle size) using acetonitrile and phosphate buffer (50 + 50 v/v), pH 5.5, as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60 F₂₅₄ using acetone/toluene/ammonia (6.0:3.0:1.0, v/v/v) as the mobile phase. Quantification with the HPLC method was achieved with ultraviolet (UV) detection at 230 nm over the concentration range 3–18 µg/mL for both drugs with mean recovery of 101.86 ± 0.21 and 100.57 ± 0.31% for ALZ and SER, respectively. Quantification in HPTLC was achieved with UV detection at 230 nm over the concentration range of 400–1400 ng/spot for both drugs with mean recoveries of 101.32 ± 0.15 and 100.38 ± 0.51% for ALZ and SER, respectively. These methods are rapid, simple, precise, sensitive, and are applicable for the simultaneous determination of ALZ and SER in pure powder and formulations.     

Determination of Oxazepam in Pharmaceutical Formulation by HPTLC UV-Densitometric and UV-Derivative Spectrophotometry Methods

Abstract

Methods for determination of oxazepam in pharmaceutical formulation by derivative ultraviolet (UV) spectrophotometry as well as high-performance thin-layer chromatography (HPTLC) UV densitometry were described. For UV-derivative spectrophotometry, some derivatives and wavelengths may be recommended for routine quality control of the drug of interest. On the other hand, HPTLC provided good results, but only when the calibration curve was estimated using nonlinear regression analysis. The HPTLC method was developed with silica F₂₅₄ plates, a mobile phase of benzene/ethanol (5:1, v/v), and densitometric detection at 204 nm receiving R _f  = 0.47. Developed methods were validated and found to be sufficiently precise and reproducible for established conditions.     

Determination of Water Soluble Vitamins in Laboratory Animal Feeds by Micellar Electrokinetic Chromatography

Seven water-soluble vitamins: thiamine hydrochloride (B₁), riboflavin hydrochloride (B₂), pyridoxine hydrochloride (B₆), calcium pantotenate (B₅), ascorbic acid (C), folic acid (B₉), and biotin (H, B₇) were separated using the micellar electrokinetic chromatographic (MEKC) method in a single run with the electrolyte solution consisting of 100 mM H₃BO₃, 5 mM Na₂B₄O₇, and 30 mM sodium dodecylsulphate (SDS). The separation was achieved within 8 min. and the detection was performed at 200 nm. The calibration graphs plotted with six concentrations of each vitamin were linear with the determination coefficient r > 0.999. The method was applied for quantification of vitamins B₁, B₂, B₅, B₆, B₉, C, and H in novel feed for laboratory animals. The sample preparation involved solid-phase extraction of the vitamins, used prior to the MEKC analysis. The attained precision level was good with the recoveries between 98.4% and 105.6%. The simplicity of the procedure should make it highly useful for feed quality control in husbandry and the animal feed industry.     

Study on the chromatographic behavior of water-soluble vitamins on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase by HPLC

In this paper, the chromatographic behavior of some water-soluble vitamins was studied on a new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS, 5 μm particle size, the bonded amount 0.071 mmol g⁻¹) by using vitamin standards as probes for HPLC. The comparative study of the separation of these compounds was done by using CABS and ODS as stationary phases under the same chromatographic conditions. The better separation of six vitamins including: B₁, B₂, B₆, B₁₂, C, and nicotinic acid (B₅), on CABS can be achieved by using isocratic mode with methanol-phosphate buffer (25:75, (v/v)) as mobile phase within 20 min. The results show that the calix[8]arene-bonded phase exhibits high selectivity for water-soluble vitamins. We found that the elution order of B₂ (12.08 min) and B₁₂ (16.42 min) on CABS was very different from that of B₁₂ (7.76 min) and B₂ (18.47 min) on ODS, which indicate that different retention mechanisms exist in the chromatographic processes of the two stationary phases. According to the chromatographic data, it can be concluded that various chromatographic retention mechanisms are responsible for the separation of above compounds on CABS, such as hydrophobic interaction, hydrogen bonding interaction, and π-π interaction. The new packing has two advantages over ODS. On one hand, the polar and ionized analytes, such as C and B₅, exhibited stronger affinities to CABS because of hydrogen bonding interaction. On the other hand, the retention of B₂ and B₁₂ became shorter on CABS with weaker hydrophobicity in comparison with ODS. The new material exhibits the promising application in the separation of water-soluble vitamins.     

Spectrophotometric,potentiometric, and conductometric studies of binary complex formation between copper(II) and three forms of vitamin B6 in aqueous solutions

This article reports the detailed study concerning the mode of binding of three forms of vitamin B₆, pyridoxamine (pm), pyridoxine (pn), and pyridoxal (pl), with Cu(II) in aqueous solutions using three independent methods: potentiometry, conductometry, and UV–vis spectroscopy. The stability constants of complexes formed between copper(II) and vitamin B₆ were investigated by potentiometric titration in 0.1 M KNO₃ ionic medium at 25 °C. While drawing the relations between molar conductance and the ratio of metal to ligand concentrations, different types of lines were obtained indicating the formation of 1 : 1 and 1 : 2 stoichiometric compounds. The stability constants have been determined using EQUID and CVEQUID computer programs and the obtained results were in agreement. The relatively high values of stability constants of Cu(II)-vitamin B₆ complexes obtained from three independent methods in comparison to those with other competing cations suggest that the complexes studied are relatively stable in aqueous solutions.     

Improved Vitamin B12 Fermentation Process by Adding Rotenone to Regulate the Metabolism of Pseudomonas denitrificans

Our previous research had revealed that the dissolved oxygen limitation was more favorable for vitamin B₁₂ fermentation, due to its inducement to the increased glycolytic flux in Pseudomonas denitrificans. In this paper, a novel strategy was implemented to further investigate the metabolic characteristics of P. denitrificans under different oxygen supply levels, by exogenously adding rotenone (a respiratory chain inhibitor interfering with the oxygen consumption) to the fermentation broths. Compared to the fermentation process without rotenone treatment, it was observed that 5 mg/L rotenone treatment could significantly strengthen the glycolytic flux of P. denitrificans via activating the key glycolytic enzymes (phosphofructokinase and pyruvate kinase), resulting in the accelerated generations of anterior precursors (glutamate and 5-aminolevulinic acid) for vitamin B₁₂ biosynthesis. Although 5 mg/L rotenone treatment had a negative effect on cell growth of P. denitrificans, the vitamin B₁₂ yield was increased from 48.28?±?0.62 mg/L to 54.70?±?0.45 mg/L, which further proved that an increased glycolytic flux in P. denitrificans was a consequence of higher vitamin B₁₂ production.     

Determination of Veterinary Pharmaceuticals in Production Wastewater by HPTLC-Videodensitometry

An SPE-HPTLC method for simultaneous identification and quantification of seven pharmaceuticals in production wastewater was optimized and validated. The studied compounds were enrofloxacine, oxytetracycline, trimethoprim, sulfamethazine, sulfadiazine, sulfaguanidine and penicillin G/procaine. The method involves solid-phase extraction on hydrophilic-lipophilic balance cartridges with methanol and HPTLC analysis of extracts on CN modified chromatographic plates followed by videodensitometry at 254 and 366 nm. Optimization of chromatographic separation was performed by systematic variation of the mobile phase composition using genetic algorithm approach and the optimum mobile phase composition for TLC separation was 0.05 M H₂C₂O₄:methanol = 0.81:0.19 (v/v). Linearity of the method was demonstrated in the ranges from 1.5 to 15.0 μg L⁻¹ for enrofloxacine, 100–500 μg L⁻¹ for oxytetracycline, 150–600 μg L⁻¹ for trimethoprim, 300–1100 μg L⁻¹ for sulfaguanidine and 100–400 μg L⁻¹ for sulfamethazine, sulfadiazine and penicillin G/procaine with coefficients of determination higher than 0.991. Mean recoveries ranged from 74.6 to 117.1% and 55.1 to 108.0% for wellspring water and production wastewater, respectively. Only sulfaguanidine showed lower results. The described method has been applied to the determination of pharmaceuticals in wastewater samples from pharmaceutical industry.     

Chemiluminescent Determination of Vitamin B12 Using Charge Coupled Device (CCD)

A method was developed for the determination of vitamin B₁₂ based on the enhancement of cobalt (II) on the chemiluminescence (CL) reaction between luminol and percarbonate (powerful source of hydrogen peroxide). The release of cobalt (II) from the vitamin B₁₂ was reached by a simple and fast microwave digestion (20 s microwave digestion time and a mix of nitric acid and hydrogen peroxide). A charge coupled device (CCD) photodetector, directly connected to the cell, coupled with a simple continuous flow system was used to obtain the full spectral characteristics of cobalt (II) catalyzed luminol-percarbonate reaction.

The optima experimental conditions were established: 8.0 m mol L^?1 luminol in a 0.075 mol L^?1 carbonate buffer (pH 10.0) and 0.15 mol L^?1 sodium percarbonate, in addition to others experimental parameters as 0.33 mL s^?1 flow rate and 2 s integration time, were the experimental conditions which proportionate the optimum CL emission intensity. The emission data were best fitted with a second-order calibration graph over the cobalt (II) concentration range from 4.00 to 300 µ g L^?1 (r² = 0.9990), with a detection limit of 0.42 µ g L^?1. The proposed method was successfully applied to the determination of vitamin B₁₂ in pharmaceuticals.     

Separation of 5-Lipoxygenase Metabolites Using Cyclodextrin-Modified Microemulsion Electrokinetic Chromatography and Head Column Field-Amplified Sample Stacking

A cyclodextrin-modified microemulsion electrokinetic chromatography method employing head column field-amplified sample stacking was developed for the analysis of arachidonic acid metabolites of the lipoxygenase pathways. The influence of the concentration of boric acid, the surfactant sodium dodecyl sulfate, the co-surfactant 1-butanol and the oil phase octane as well as the pH of the background electrolyte, the separation voltage and the separation temperature was studied. The optimized microemulsion consisting of 20 mM boric acid buffer, pH 9.0, 3.0 % (m/v) sodium dodecyl sulfate, 0.5 % (v/v) octane, 5.0 % (v/v) 1-butanol and 15 mM α-cyclodextrin enabled the separation of 20-hydroxy-leukotriene B₄, leukotriene B₄, 6-trans-leukotriene B₄, 6-trans-12-epi-leukotriene B₄, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 12(S)-hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid, 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid as well as the internal standard prostaglandin B₁ in <10 min employing a separation voltage of 17.5 kV at a temperature of 23 °C. A matrix peak from solid-phase extraction sample workup co-migrated with 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid affecting peak integration. The addition of 5 % (v/v) 2-propanol to the microemulsion resulted in the separation of this eicosatetraenoic acid and the matrix components at the expense of analysis time and peak resolution between the diastereomers 6-trans-leukotriene B₄ and 6-trans-12-epi-leukotriene B₄. In summary, the MEEKC method appeared to be especially suitable for the more polar arachidonic acid metabolites.     

Simultaneous Separation and Determination of Lamivudine and Zidovudine in Pharmaceutical Formulations Using the HPTLC Method

Abstract

A high-performance thin-layer chromatographic method (HPTLC) for the simultaneous determination of lamivudine and zidovudine in a binary mixture has been developed. The method developed was based on HPTLC separation of the two drugs followed by densitometric measurements of spots at 276 and 271 nm for lamivudine and zidovudine, respectively. Separation was carried out on Merck HPTLC silica-gel 60 F₂₅₄ plates, using toluene/chloroform/methanol (1:6:3 v:v) as the mobile phase. Validation of the method was performed based on The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Second-order polynomial equations were obtained for the regression line in the ranges of 250–1400 and 250–1700 ng/spot for lamivudine and zidovudine respectively. Correlation coefficient (r) values were 0.9998 for both analytes. The method provides sufficient accuracy as indicated by recovery percentages given for lamivudine and zidovudine. For system precision study, the low coefficient of variation values (<2%) for both lamivudine and zidovudine ensured reproducible performance of the instrument. In the method precision study, coefficients of variation <2% were obtained, which showed that the proposed method provides acceptable intraday and interday variation. The detection and quantification limits and were 3.06 and 9.28 ng/spot for lamivudine and 3.34 and 10.13 ng/spot for zidovudine, respectively. Parameters such as mobile-phase composition, volume of mobile phase, time from spotting to development, and time from development to scanning were employed while testing for robustness of the method, and the standard deviation of peak areas was calculated for each parameter. The low coefficient of variation values indicated the robustness of the method. Statistical manipulation did not show any significant effect of one parameter over the others on the robustness of the method.     

The kinetics of photostabilization of cyanocobalamin in liposomal preparations

Cyanocobalamin (B₁₂) is a photosensitive vitamin, and its photodegradation to hydroxocobalamin (B_12b) in liposomes has been investigated. The values of apparent first-order rate constants (k_obs) for the photodegradation of B₁₂ in liposomes (nine preparations) are in the range of (0.52-2.24) × 10^–3 min^–1, compared to 3.21 × 10^–3 min^–1 for B₁₂ in aqueous solution (pH 5.0). The entrapment efficiency of B₁₂ in liposomes is 26.4-38.8%. The values of k_obs show a linear relation with phosphatidylcholine (PC) content in liposomes, indicating the influence of PC in inhibiting the rate of photolysis of B₁₂. The value of the bimolecular rate constant for photochemical interaction of B₁₂ and PC is 0.32 M^–1 min^–1, indicating the stabilizing effect of PC on the photolysis of B₁₂. The ratio of B₁₂ stabilization in liposomal preparations is in the range 2-6 compared to that of the unentrapped vitamin The stabilization of B₁₂ is mediated by a photoinduced charge-transfer B₁₂-PC complex that leads to the reduction of B₁₂ to B_12r, which is then oxidized to B_12b that has low susceptibility to photolysis. The extent of stabilization of B₁₂ probably depends on the degree of interaction between the two compounds under the reaction conditions, indicated by the loss of B₁₂ fluorescence.     

Determination of Vitamin B12 in Multivitamin Tablets by High Performance Liquid Chromatography

Abstract

Two procedures for separation and determination of vitamin B₁₂ in multivitamin tablets by reversed phase high performance liquid chromatography are proposed. Sample preparation is very simple: tablets are dissolved in distilled water, centrifuged and filtered. The sample solution is directly applied in the sample loop injector and chromatograms are obtained with gradient elution using water-methanol and water-acetonitrile as solvents. The peak of vitamin B₁₂ from samples of B-complex tablets is well separated with the two procedures. For multivitamin tablets, however, only the procedure with water and methanol as solvents was good for separation and quantification of vitamin B₁₂. Both procedures were verified by the standard addition method and also compared to a previously developed method using electrothermal atomic absorption spectrometry for vitamin B₁₂ determination.     

Chromatographic methods development,validation and degradation characterization of the antithyroid drug Carbimazole

The current paper reports the development and validation of stability‐indicating HPLC and HPTLC methods for the separation and quantification of main impurity and degradation product of Carbimazole. The structures of the degradation products formed under stress degradation conditions, including hydrolytic and oxidative, photolytic and thermal conditions, were characterized and confirmed by MS and IR analyses. Based on the characterization data, the obtained degradation product from hydrolytic conditions was found to be methimazole—impurity A of Carbimazole as reported by the British Pharmacopeia and the European Pharmacopeia. A stability‐indicating HPLC method was carried out using a Zorbax Eclipse Plus CN column (150 × 4.6 mm i.d, 5 μm particle size) and a mobile phase composed of acetonitrile–0.05 m KH₂PO₄ (20: 80, v/v) in isocratic elution, at a flow rate of 1 mL/min. The method was proved to be sensitive for the determination down to 0.5% of Carbimazole impurity A. Additionally, a stability‐indicating chromatographic HPTLC method was achieved using cyclohexane–ethanol (9:1, v/v) as a developing system on HPTLC plates F₂₅₄ with UV detection at 225 nm. The proposed HPLC and HPTLC methods were successfully applied to Carbimazole^® tablets with mean percentage recoveries of 100.12 and 99.73%, respectively.     

Simultaneous Determination of Quercetin, Rutin and Coumaric Acid in Flowers of Rhododendron arboreum by HPTLC1

A simple and fast method was developed for simultaneous quantitative determination of three biologically active phenolic compounds i.e. quercetin, rutin and coumaric acid in flowers of Rhododendron arboreum using high-performance thin-layer chromatography (HPTLC). The separation was performed on TLC aluminium plates precoated with silica gel RP-18 F_254S. Good separation was achieved in the mobile phase of methanol-water-formic acid (40:57:3, v/v/v) and densitometric determination of these compounds was carried out at 280 nm in reflectance/absorbance mode. The linear regression data for the calibration plots showed a good linear relationship with r = 0.9971, 0.9953 and 0.9960 for quercetin, rutin and coumaric acid, respectively. Accuracy of the method was checked by recovery study conducted at two different levels with the average recovery of 99.90%, 99.02% and 99.16% for quercetin, rutin and coumaric acid, respectively. The present method is being reported first time and may be used for routine quality control of the flowers of R. arboreum.