Document Type : Original Research Article


Department of Chemistry, College of science, university of Baghdad, Baghdad, Iraq


In this work, we used normal flow injection analysis for determining two drugs of Quercetindehydrate symbolized as QE and Diosmin symbolized as DSN. The aim was to develop a simple, rapid, less expensive, linear, precise and accurate technique using p-Methylaminophenol Sulfate (MFS) in the presence of an oxidative agent of Sodium per iodate in aqueous medium. After the experimental conditions were passed, ideal conditions were adopted for this work, and the detection of the highest absorption of the two drugs was reached. Detected at the Maximum wavelength, we recorded them (QE= 641 nm and DSN = 665 nm). We studied beaver each drug by flow injection technique chosen concentration rang from (2-160) mg .ml-1 for QE and DSN with MFS reagent in presence of oxidative agent draw calibration graph which gave a linear relationship with correlation coefficients of 0.9994 and 0.9989, respectively. The sample response time for each drug was 20 seconds for QE and 19 seconds for DSN and the detection limit for the proposed approach was (1.4×10-5) for QE and DSN, depending on trace drug concentration. The proposed approach was used to study and determine the two suggested medications in injections and tablets with great effectiveness.

Graphical Abstract

Determination of diosmin and quercetindihydrate through charge transfer interaction with 4-methylamino phenol and normal flow injection system with spectrophotometric detection



Several studies on the mutagenicity of quercetin dehydrate have been reported. Quercetindihydrate (QE) (Figure 1) is chemically known as 4 H-1 -Benzopyran-4-one-2(3,4dihydroxyphenyl)- 3,5,7-trihydroxydihydrate, molecular formula of C15H10O7 2H2O, with formula Wt: 338.26, soluble in ethanol, and insoluble in water. QE is a natural pigment and one of the most abundant antioxidants in the diet, helping body human combat free radical damage. QE belongs to a group of plant compounds called flavonoids [1,2]. Flavonoids have been reported to contain a large spectrum of pharmacological properties. The concentration of flavonoids is reported as QE in spectroscopic methods, which is used as a standard for calibration curves [3]. Various methods have been devised for the dedication of QE in different samples of planet like excessive-overall performance liquid chromatography, capillary electrophoresis, UV spectrophotometry at 269nm [4], dispersive liquid–liquid microextraction and copied spectrophotometry. QE is determined via complexion reaction with numerous dissimilar inorganic reagents add to drug [5]. A sensitive approach has been devised based on charge transfer of a compound generated between QE and (4-methylamino)-phenol sulfate (MFS) reagent in the presence of sodium periodate, which is easier and more suited than the reference spectrophotometric methods.

The second choice is Diosmin (DSN), which is a bioflavonoid, semi-synthetic drug, (3,5,7-trihydroxy-4 methoxyflavone-7-rutinoside). Diosmin Chemical formula C28H32O15 is yellow-brown to brown powder Figure (2), with molecular mass: 608.54, insoluble in water, and soluble in DMSO and NaOH. Maximum absorption of DSN recorded in UV spectroscopy is at 268nm. Few analytical techniques have been proposed or used for determination of the drug like voltammetry [6], HPLC [7,8], TLC [9,10] or FTIR [11,12]. Flow injection analysis and spectrophotometric methods are sensitive, suitable and comparatively inexpensive. For this project, spectrophotometric technique was used based on the response of drugs by (4-methylamino)-phenol sulfate (MFS) reagent by charge transfer interaction.

The reagent (4-methylamino)-phenolsulfate (MFS) is the chemical name for the metol reagent. Chemical formula C7H9NO 12 H2SO4 is the standard formula. Molecular mass of 172.19 g/mol as sulphate purity, composition, and substance codes for compound sulphate are commonly used. Its function and applications are also commonly utilized. Figure 3 shows the chemical structure [13]:

Charge-transfer interaction of pharmaceutics forms with (4-methylamino)-phenol sulfate (MFS) reagent is an important in biochemical and bio electrochemical energy transfer process [14]. Formation of stable charge-transfer (CT) compounds that results from the reaction between acceptors and drugs or biological compound [15]. The new charge-transfer interaction of drugs with (4-methylamino)-phenol sulfate (MFS) reagent has been structurally characterized via normal flow injection analysis. In this study, two pure standard solutions of pharmaceutics forms (QE and DSN) were used to delve into interaction by charge transfer with p-Methylaminophenol Sulfate (MFS) in the presence of oxidative agent of Sodium per iodate in aqueous medium.

Experimental work

Chemicals and apparatus


Shimadzu spectrophotometer UV/VIS 260 digital double beam (Japan), with the use of 1cm path length and 50 μL volume flow cell was applied. A peristaltic pump and an injection valve were prepared form Germany, Knauer. Flexible vinyl and Teflon tubing (0.5 mm) were used for various projects.

(4-methylamino)-phenol sulfate (MFS): or metol reagent solution (0.005 M)

The stock solution was prepared by mixing 0.8612 g of MFS reagent with M.Wt:172.19mol-1 in 10 mL ethanol with 90 mL distilled water and volume was completed to mark in volumetric flask.

Sodium per iodate NaIO4(0.02 M): Oxidizing agent was used weighing about 0.428g of NaIO4(M. wt. 213.29 g mol-1, purity99%) dissolved in 5 mL distilled H2O and the volumetric flask was completed to 100 mL.

Stock solution QE: (200 μg mL-1) was organized by dissolving 0.02g of QE in 10 mL of Ethanol and volume was completed with distilled water in 100 mL volumetric flask, stored at 8C, and used within 1 week.

A stock standard DSN: (200 μg mL-1) was prepared by dissolving 0.02 g DSN in 10 mL of 0.5 M (5%) sodium hydroxide. The solution was diluted with doubly-distilled H2O. The stock solution was stored for 2 weeks in dark place [16].

Sodium Hydroxide (0.5 M): It wasprepared via dissolving 2g of sodium hydroxide in distilled water and then completed the flask to the mark with the same solvent to100 mL.

General normal FIA procedures

Two channel manifolds were used for the spectrophotometric willpower of two drugs, solution of (4-methylamino)-phenol sulfate (MFS) reagent in 0.005 M for (2 – 160) μg mL-1QE and DSN drug prepared from stock solution. QE 200 μL portion was injected in the stream of 0.02M NaIO4 and was then joint by a stream of 0.005 M MFS reagent with a total movement rate of 2 mL/min and mixing the solutions in 150 cm reaction coil. The colored product absorbance measured at 641 nm for QE drug 200 μL portion was injected into the stream of the of 0.02 M NaIO4 and 0.005 M MFS solution with a total flow rate of 2 mL/min. The reagent was mixed in 150 cm reaction coil. The colored product absorbance was measured at 665nm. Then, FIA manifolds were used as shown in Scheme 1.

Results and discussion

Absorption spectra

Determining QE and DSN drugs have been reported by charge transfer interaction (scheme 2) with the reagent solution (0.005 M) (4-methylamino)-phenol sulphate (MFS) to give a colored products at 641 nm for QE and 665 nm for DSN in the presence of (0.02 M) an Sodium per iodate(NaIO4) oxidizing agent.

Interaction mechanism and pathway

Charge transfer interaction, an electronic transition among orbitals which can be centered on exclusive atoms, is called rate switch transition and absorption band is typically very sturdy. These transitions involve electron transfer from one part of a compound to another one. The electronic absorption spectra of all compounds in two solvents of ethanol and water were studied. The conc. of 0.0006 M to QE and 0.00033M to DSN were prepared to interact with MFS in the presence of oxidative agent sodium periodate and UVV scan ranged from 200-800 nm. Figures (5 [A and B]) exhibit mainly two bands with π → π* and n→ π* transitions due to allowed transition of the electrons between energy levels. The both drugs QE and DSN are shown two types of transition at the wavelength (λmax of 641 nm for QE and λmax 665 nm for DSN). Absorptions fall inside the observed place; they frequently produce rich colors. The interaction given in Scheme 1 was assumed. The preoccupation spectrum of the blank is assumed in Figure 4 but colored product for drugs (QE and DSN) is assumed in Figure 5. Under the interaction circumstances, the color product might be stared as a kind of charge switch interaction. The rate transfer might be supposed to be captivating location related to electron transmission after the upper most busy (π) molecular detour of phenolic multiple (tablets) to the empty bottom molecular orbital (π*) of four-methyl amino phenol. Charge-transfer transitions are diagnosed by their excessive intensity and the sensitivity in their dynamisms to solve polarity (Figures 6, 7 and 8).

Optimization of chemical parameters

The chemical variables that impact in the presentation of the future method were specified. The optimum conditions method for all QE and DSN drugs were selected based on calibration curve, reproducibility, throughput and sensitivity.

Effect of (4-methylamino)-phenol sulfate (MFS) reagent concentration

The method for each drug (QE and DSN) included taking different concentrations of (4-methylamino)-phenol sulfate (MFS) reagentin the range of (1 × 10 -3to 10 ×10 -3) M examined with test approved in 10 mL bottle covering 50 μg mL-1 of (QE and DSN) with 1 ml of NaIO4 (0.02M) for measuring absorbance in normalflow injection method. It was found that the maximum absorbance intensity was obtained at 0.005 M to QE and DSN. A decrease in absorbance occurred above these concentrations. Consequently, the concentration of 0.005 M was selected for further use as given in Figure 9.

Effect of oxidative agents

As given in Figure 10, in this work we used various types of oxidative agents including NaIO4, K3[Fe(CN)6],K2S2O8, FeCl3) prepared concentration 0.02M from each agent for experimental conditions to obtain the best agent. Normal flow injection analysis recorded the maximum absorbance of sodium periodate to oxidized (4-methylamino)-phenol sulfate (MFS) reagent to react with 50 μg mL-1 of each of the two drugs (Figure 11); the response concentration of oxidative agent of sodium periodate in maximum absorbance was explained at 0.02 M as the best one for QE and DSN.

Optimum conditions in this work

Effect of physical parameters for experimental method (normal flow injection analysis) was measured for two drugs (QE and DSN). The optimization conditions were realized by altering one change condition and the others at steady state. Table 1 summarizes the optimization conditions for both analyte drugs. The result of entire movement rate on the response of the method was examined in the range of (0.8 to 2) mL min-1 correspondingly (Figure 12). In each carrier solutions in normal flow injection analysis, the sign absorbance was sharp up to 2 mL min-1 for drugs; therefore, the flow rates of 2 mL min-1 were designated as optimum movement solutions for (QE and DSN) correspondingly, which will be used in the next studies. The second effect is injection sample volume (Figure 14). The volume (60 to 200 μL) was injected into the carrier stream for each drug. It can be understood that 150 μL as an injected volume logged the best response for QE and 100 μL for DSN at higher absorbance. As third effect, length of reaction coil of mixing coil length on the reply changed into enhanced in range of [0 (without reaction coil) to 250] cm. Rendering to the consequences (Figure13), lengths of 75 cm changed into recorded as top-quality period that provided the most absorbance for the colored merchandise for every QE and DSN correspondingly and may be utilized in subsequent research.

Calibration graph

This specific wavelength corresponds to the maximal absorbance value in drugs from pure standards. Thus, measuring absorbance at 641 nm provides a useful quantitative estimation of total QE content, while measuring absorbance at 665 nm provides a useful quantitative estimation of total DSN content. Therapy of the colored products gives response absorbance and plotted against the attentiveness of pharmaceutics forms contingent on the designated limits as mentioned in Table 1. Two series of drugs solutions were chosen in the range of (2-160) μg mL-1. To obtain linear relationship, we followed lambert beer law (Figure 15). Table 3 summarizes the other analytical statistical treatments calculated for the calibration curve. Dispersion of colored product was studied by using optimum conditions and the nFIA manifolds.

From the calibration curve, absorbance of pharmaceutics forms against (0-160) ppm concentration and linear equation can be drawn. It is easy to apply the statistics including the collection and interoperation of numerical data, and the estimation analytical values that are given in Table 2.

Accuracy and precision

As shown in Table 3, both drugs were assessed by injections of pure drug solution at dissimilar attentions. The precision and accuracy of the results presented in Table 3 tell us that the closeness of repeated measurement from each other means a measure of internal agreement among a set of replicate observations while accuracy shows numerical differences between an analytical result and accepted value for the measure quantity.

Stoichiometric measurements

Job's method of continuous variation [17,18] was applied to determine the measure of the measurable association amid the crops and reactants of a assumed chemical interaction in footings of their comparative relations of mass or volume of a binding by keeping the molar concentrations of reactants constant of drugs solutions with A 1.0 × 10–4 M normal solution with drugs base. A sequence of solutions was ready in the entire volume of drug and MFS substance was reserved at 10 mL for drugs and reagent, correspondingly. The absorbance was measured in normal flow injection with UV absorbance detector at the optimum wavelength for each drug color product. We explain determining drugs with reagent by the incessant differences method (Job's method). The results showed that 1:1 (drug: dye) shaped the electron charge transfer amid drugs and 4-methylaminophenol. The results obtained are represented in Figure 16.

In continuous variation method, volumes 0.5 – 4.5 mL of 0.005 M portions of MFS (VS) were diazotized using its equimolar and coupled according to analytical procedure with the corresponding complementary volume of each drug  to give a total volume of 5 ml for VS+ VR in 2 mL of 0.02M sodium periodate  and diluted to 25 mL with distilled water.

According to the difficulties of using the standard method for determination of QE and DSN in its pharmaceutical preparations, so that a standard addition method has been used for its simplicity which proves that the proposed method was applied successfully for the determination of drugs method of standard additions used to overcome the matrix effects, which cause the reduction or enhancement of analyte signal. Equal volumes of example solution are occupied and results planned as shown in Figure 17 below: 


By using normal flow injection technique, the donor-acceptor compounds between MFS reagent and (QE and DSN) were investigated. This study reported a molar of 2:1 ratio. The UV-Vis spectra demonstrated the transition in the presence of oxidizing agent and MFS π acceptors as an analytical substance for the growth method for the accurate estimation of two drugs (QE and DSN) in pharmaceutical dosage forms. The applied nFIA gives very stiffed procedure as a cheap and simple instrument. The results obtained gave RSD % ≤ 5. The proposed method used inexpensive reagent with excellent charge transfer transition. The mechanism was dependent on the interaction of drugs amino group (n-donor) with the oxidized MFS (π-acceptor) reagent to form custody transfer compound, which produced a colored product that was slow. In addition, the recommended methods were applied to the analysis of QE and DSN in dietary supplements and were compared statistically with an official UV method. The statistical analysis disclosed no noteworthy variance in correctness and exactness amid the current methods and the official method.


The authors thank the Department of Chemistry, College of Science, the University of Baghdad for the help provided in this work.


Subhi Abed Sadeem:


How to cite this article: Allaa Hussein Mhdi*, Sadeem Subhi Abed. Determination of diosmin and quercetindihydrate through charge transfer interaction with 4-methylamino phenol and normal flow injection system with spectrophotometric detection. Eurasian Chemical Communications, 2022, 4(11), 1115-1129. Link:


Copyright © 2022 by SPC (Sami Publishing Company) + is an open access article distributed under the Creative Commons Attribution License(CC BY)  license  (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


[1] L. Pavun, P. Đurđević, M. Jelikić-Stankov, D. Đikanović, A. Ćirić, S. Uskoković-Marković, Maced. J. Chem. Chem. Eng., 2014, 33, 209–215.[crossref], [Google Scholar], [Publisher]
[2] A.F. Almeida, G.I.A. Borge, M. Piskula, A. Tudose, L. Tudoreanu, K. Valentova, G. Williamson, C.N. Santos, Compr. Rev. Food Sci. Food Saf., 2018, 17, 714–731. [crossref], [Google Scholar], [Publisher]
[3] A. Riva, M. Ronchi, G. Petrangolini, S. Bosisio, P. Allegrin, Eur. J. Drug Metab. Pharmacokinet., 2019, 44, 169–177. [crossref], [Google Scholar], [Publisher]
[4] I.M.J. AL mashhadani, S.S. Abed, Iraqi J. Sci., 2018, 59, 635-644. [crossref], [Google Scholar], [Publisher]
[5] P.S. Sandh, S. Beg, R. Kumar, O.P. Katare, B. Singh, J. Liq. Chromatogr. Relat. Technol., 2017, 40, 506-516. [crossref], [Google Scholar], [Publisher]
[6] M. Szymański,a, D. Młynarek, A. Szymański, I. Matławskaa, Iran. J. Pharm. Res., 2016, 15, 141–148. [Google Scholar], [Publisher]
[7] E. Huwait, M. Mobashir, Biomedicines, 2022, 10, 1076.[crossref], [Google Scholar], [Publisher]
[8] Y.A. Borko, I.V. Kovalevska, O.A. Ruban, O.V. Kutova, Farm. Zh., 2022, 74-85.[crossref], [Google Scholar], [Publisher]
[9] K. Hasegawa, K. Minakata, M. Suzuki, O. Suzuki, Forensic Toxicol., 2021, 39, 311–333.[crossref], [Google Scholar], [Publisher]
[10] A.I. Foudah, P. Alam, M.K. Anwer, H.S. Yusufoglu, M.S. Abdel-Kader, F. Shakeel, Processes, 2020, 8, 817.[crossref], [Google Scholar], [Publisher]
[11] L. Hssaini, R. Razouk, Y. Bouslihim, Front. Plant Sci., 2022, 429. [crossref], [Google Scholar], [Publisher]
[12] R.N. Oliveira, M.C. Mancini, F.C.S.D. Oliveira, T.M. Passos, B. Quilty, R.M.D.S.M. Thiré, G.B. McGuinness, Matéria (Rio de Janeiro), 2016, 21, 767-779.[crossref], [Google Scholar], [Publisher]
[13] G.A. Hasan, M.H. Bakir, IOP Conf. Ser.: Mater. Sci. Eng., 2021, 1058, 012079.[crossref], [Google Scholar], [Publisher]
[14] R.J.M.L. Abdulredha, Bas. J. Sci., 2015, 33, 10-24. [Google Scholar], [Publisher]
[15] H.H. Eldaroti, S.A. Gadir, M.S. Refat, A.M.A. Adam, J. Pharm. Anal., 2014, 4, 81–95. [crossref], [Google Scholar], [Publisher]
[16] M.M. Mohammed, S.S. Abed, Int. J. Pharm. Res., 2021, 13, 1. [crossref]
[17] N.E. Boland, A.T. Stone, Environ. Chem., 2013, 10, 409-416. [crossref], [Google Scholar], [Publisher]
[18] T.J. Ugye, A. Uzairu, S.O. Idris, H.O. Kwanashie, J. Environ. Sci., Toxicol. Food Technol., 2013, 5, 42-45. [Google Scholar], [Publisher]