Iraqi J Pharm Sci, Vol.31(1) 2022 Determination of genistein by coupling reaction DOI: https://doi.org/10.31351/vol31iss1pp278-284 278 Flow Injection Spectrophotometric Technique for Determining of Genistein in Pure and Supplements Formulations Through Diazotization Coupling Reaction Farqid Faraj Muhammed*, Sadeem Subhi Abed*,1 *Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq. Abstract Genistein (GEN) is the major isoflavone found in soybeans, has a number of cardiovascular health benefits, postmenopausal syndrome and osteoporosis. A direct flow injection analysis method for estimation of GEN in pure and supplements formulation was suggested. This system is based on a diazotization coupling reaction between procaine penicillin (PR) and GEN in an alkaline medium. The formed orange dye has a maximum absorption at 416 nm. Calibration curve was constructed over different GEN concentrations with a linearity range of 10-100 µg/mL and a detection limit of 1.51 μg/mL. For the FIA technique, all analytical factors were analyzed and optimized. The established method was successfully used to determine GEN in the supplement formulations. Keywords: Genistein, Flow injection analysis, Spectrophotometry, Procaine penicillin, Diazotization coupling reaction. في المستحضرات النقية والمكمالت من خالل تفاعل تقنية طيف الحقن الجرياني لتقدير الجينيستين ازوتة واالزدواج 1*، و سديم صبحي عبد *فرقد فرج محمد قسم الكيمياء ، كلية العلوم ، جامعة بغداد ، بغداد ، العراق . * الخالصة موجود في فول الصويا بصورة رئيسية وله العديد من الفوائد الطبية لكثير من االمراض كأمراض الجينيستين هو مركب ايسوفاليفون الجينيستين القلب واألوعية الدموية وهشاشة العظام ومتالزمة ما بعد انقطاع الطمث . تم اقتراح طريقة تحليل حقن التدفق الجرياني المباشر لتقدير نية . تعتمد طريقة التقدير على تفاعل األقتران واألزوتة بين البروكائين بنسلين مادة دوائية تم استعمالها كاشف بصورته النقية وفي المكمالت الصيدال لتراكيز نانومتر . تم قياس األمتصاصية 416عند طول موجي صبغة ذات لون برتقالي وتقاس في التفاعل والجينيستين في وسط قاعدي . حيث تنتج ميكروغرام / مل . تم تقييم وتحسين جميع المتغيرات التحليلية 1.51ميكروغرام/ مل وكانت حدود الكشف 100-10مختلفة من الجينيستين المستخدمة في تطبيق الطريقة المحددة بنجاح. الكلمات المفتاحية: Introduction Flavonoids are a type of secondary metabolism molecule that occurs naturally in the world of plants. They are regarded as a quality indication for fruits and medicinal plants, and hence an important component to consider in the development of agricultural and manufactured products. Many publications have been written throughout the years about the extraction and identification of flavonoids (1,2 ) .Genistein (Figure 1) is an isoflavone that acts as a phytoestrogen and an angiogenesis inhibitor. The chemical name comes from the fact that it was initially isolated in 1899 from the dyer's broom, Genista tinctoria. The structure of the chemical was determined to be identical to that of prunetol in 1926. Genistein was quantified by spectral methods (3-6), mass spectrometry (MS) with high performance liquid chromatography (HPLC)(7), UV or electrochemical detection(8–16), gas chromatography-mass spectrometry (17), LC-MS method(18). Figure 1. Genistein 1Corresponding author E-mail: sadeem.s@sc.uobaghdad.edu.iq Received:7 /8/ 2021 Accepted:15 /11 /2021 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31iss1pp278-284 Iraqi J Pharm Sci, Vol.31(1) 2022 Determination of genistein by coupling reaction 279 The analysis method used in the present work is flow injection analysis. It's done by inserting a sample plug into a moving carrier stream (19,20). Detection methods include spectrophotometry, fluorescence spectroscopy, atomic absorption spectroscopy, mass spectrometry, and other experimental methods. FIA techniques have evolved into a wide range of applications. Flow injection's application to real-world tests benefits from automated sample processing, good repeatability, adaptability to micro-miniaturization, chemical containment, waste reduction, and reagent economy in a system that runs at microliter levels (21,22). Experimental Work Apparatus A digital spectrophotometer Shimadzu mini UV- VIS 1240 was used for absorbance equipped with flow cell with 50 µl internal volume and 1 cm of bath length. It was with a peristaltic pump (Shennchen, China) equipped with flexible vinyl tubes of an inner diameter of 0.5 to transfer the solutions. Injection valve from Knauer, Germany was used to provide appropriate injection volumes of standard solutions in addition a reaction coil (RC) made of Teflon with a 0.5 mm inner diameter was used. The manifold with two channels was used to determine GEN spectrophotometric ally . The FIA manifold used was depicted in Figure.(2). Figure 2. Schematic of FIA manifold for estimated GEN . P: peristaltic pump, F.C: Flow cell, R.C: Reaction coil and W: waste. Reagents and solutions Standard GEN 200 µg/ml : Dissolving 0.02 (no. of mol) g of pure GEN in 10 ml 0.5M of NaOH in a small beaker, then transferring the solution to a 100 ml volumetric flask and completing the volume with distilled water to the mark . Diazotized Reagent solution: A solution of PR 0.003M was freshly prepared by dissolving 0.1712 g (no. of mol) of PR in 25 mL ethanol in beaker and cooling in ice bath. After that,3 ml,1M of HCl was added to it and stirring with cooling , then 0.0207(no. of mol) g of sodium nitrite (Merck ) (0.003M) was added with constant stirring. Then transferring the solution to a 100 mL volumetric flask and completing the volume by distilled water to the mark. Sodium hydroxide (BDH, England) 1M : In a 250 ml volumetric flask, dissolve 10 g of NaOH with distilled water and completed the volume to the mark by distilled water. Hydrochloride acid (BDH , England) 1M : In a 500 mL volumetric flask, dilute 43.7 mL of 11.44 M concentrated hydrochloric acid with distilled water. Preparation solutions of GEN pharmaceutical forms: (200 µg/ml) an appropriate number of capsules (10 capsules) were emptied and weighted, and the average weight of one capsule's content was selected, and accurately weighted to equate to 0.02 g of GEN and dissolved in 10 ml NaOH 0.5M. Then transferred to a 100 ml volumetric flask and completed to the mark with distilled water. Serial dilutions were done to prepare the working solutions. General FIA procedures Working solutions for the process were made from GEN stock solutions ranging from 10 to 100 g.ml-1. A 100µl portion of GEN was injected into a stream of 0.003M procaine solution, then the solution is mixed with 0.1M NaOH in 50cm reaction coil. at a total flow rate of 1.9 mL.min-1. At 416 nm, the orange dye absorption was measured. Results and Discussion Preliminary studies were indicated that procain penicillin (1 mL of 0.003M) coupled with GEN (1 mL of 200 µg .ml-1)in alkaline medium NaOH (1 mL of 0.1M) and formed orange dye can be detected spectrophotometrically. The spectra of colored complex formed are shown in Figure. 3. Figure 3. A: Absorption spectrum of azo dye formed when 20 µg.ml-1 of GEN coupled with procaine penicillin in alkaline medium, B: Blank. Iraqi J Pharm Sci, Vol.31(1) 2022 Determination of genistein by coupling reaction 280 After chemical and physical variables have been optimized the calibration graph was performed to test the linearity of GEN concentration (triplicate injections). Two stages were necessary to complete the diazotization coupling reaction. PR was initially converted to a diazo compound by reacting with nitrous acid (NaNO2/HCl). The diazonium salt was then coupled with GEN in para position of a poly phenolic molecule, producing orange dye in a basic medium in the second step, The phenolic nature of GEN resulted in fast coupling with diazotized reagent, as shown in Scheme 1. Scheme 1. Proposed mechanism for diazotization coupling reaction of GEN with Procaine Optimization of chemical parameters The chemical and hydrodynamic parameters that could affect the reaction and the stability of the colored formed were studied by changing one variable at a time while keeping the others constant. The experiment was conducted using 20 µg.ml-1 of GEN. The chemicals parameters, including the concentrations of reagent, medium, etc. were studied; the optimization of the concentration of (PR) was shown that when the concentration was increased up to 0.003 M, the absorbance increased. with increasing concentration, the absorbance was observed to decrease than it was in the previous concentration .therefore, 0.003 M concentration was selected for further used. The greater concentration leads to increase the blank signal as shown in Figure 4. Preliminary studied for type of alkaline medium reveals (NaOH, KOH and Na2CO3) that sodium hydroxide was the suitable base for this method. Different concentrations of NaOH were investigated in the range of (0.07 -0.9 M) and the concentration chosen was 0.1 M. The concentration greater than 0.1 M shown as an inhibitor for sensitivity of colored products, the results obtained are as shown in Figure 5. Iraqi J Pharm Sci, Vol.31(1) 2022 Determination of genistein by coupling reaction 281 Figure 4. Effect of the reagent concentration Figure 5. Effect of NaOH concentration Optimization the physical parameters Effect of total flow rate Total flow rate has a great role in FIA system, since it is use to get the best reaction time and has a directed effect on sampling frequency. At higher flow rate the dispersion and reagent consumption were increased , therefore ,the absorbance and sensitivity decreased with increased flow rate. In this study, a flow rate range 0.45-4.95 mL.min-1 was used, and the optimum value was 1.9 mL.min-1.The current study indicated that the absorbance was decreased above 1.9 mL.min-1 flow rate because of dispersion and dilution . The result obtained from flow rate is shown in Figure (6. a). The effect of the reaction coil length To enhance the sensitivity of the colored reaction and increase mixing of the reactants, the effect of reaction coil lengths was investigated .The dispersion of reactant zone probably increase with increasing the reaction coil length. Different reaction coil lengths were studied in the range of 25 to 150 cm, and the coil length of 50 cm gave the maximum absorbance, therefore was selected and used in subsequent experiments(Figure 6. b). The influence of the sample volume injected Effect of injection volume was investigated with varying sample loops in the range of 75 to 200 μL. The highest absorbance was obtained at 150 µL, and this sample volume was used in subsequent experiments. The obtained result is shown in (Figure 6.c). Figure 6 . (a) The effect of total flow rate on reaction absorbance, (b) Effect of reaction coil of the absorbance, (c) Effect of the injection loop Iraqi J Pharm Sci, Vol.31(1) 2022 Determination of genistein by coupling reaction 282 Analytical characteristics The calibration curve was constructed under optimal conditions for GEN estimation (Table 1). Table 2 shows the values for the calibration curve's intercept, slope, correlation coefficient, and molar absorptivity, as well as values for analytical statistical treatments. Table 1. Summary of the analytical parameters obtained from calibration graph, which indicated good linearity, highly reproducibility and low limit of detection Accuracy and precision To determine the accuracy and precision, three different concentrations of standard solution of Genistein were investigated in five replicates. For each concentration, the accuracy and precision were performed and the relative standard deviation RSD% was obtained with relative error E%. Low values of the RSD% and E% indicated that method gave acceptable results. Table 3 shows the precision and accuracy for the suggested method. Table 2. Analytical values of the techniques suggested for determinate GEN Parameter Value Regression equation y = 0.0153 x – 0.1223 Correlation coefficient, r2 0.9989 Linearity percentage ,R2 % 99.89 Slope, b (µg mL-1) 0.0153 Intercept, a 0.1223 Linearity range (µg mL- 1) 10 - 100 Standard deviation of the slope, Sb 1.83 X 10-4 Relative standard deviation RSD% 0.7 Recovery range % 99.45 – 100.56 Molar absorptivity , Ɛ (L mol-1 cm-1) 4.135 X 103 LOQ, (µg mL-1) 2.67 LOD, (µg mL-1) 0.88 Standard deviation of the residuals, Sy/x 1.66 X 10-2 Sandell,s sensitivity (µg cm-2) 0.0654 Table 3 . The precision and accuracy Present Found Error SD% *Rec.% *RSD% 20 19.89 -0.55 0.003 99.45 0.71 40 39.93 -0.17 0.002 99.83 0.27 50 50.28 0.56 0.002 100.56 0.22 *Average of five determinations Analytical applications (GNS supplements) The current method was used to determine the amount of GEN in capsules by analyzing three different concentrations (20,40, and 50 µL mL-1) under calibration graph conditions directly. According to the obtained results shown in Table 4, the small values of calculated RSD% and E% refer to the repeatable and accurate of the suggested method. Table 4. Application of the proposed method for determining GEN in pharmaceutical formulations by using direct method Conc. GNS Alternative Medicine Solutions, Inc.USA µg mL-1 Found µg mL-1 Error% Rec.% RSD% 20 20.3 1.5 101.5 0.5 40 40.2 0.5 100.5 0.3 50 50.9 1.8 101.8 0.2 As shown in Table 5, the calculated t and F-test values did not outperform the theoretical ones .When the suggested method and the ported UV method were evaluated, the findings revealed that there was no significant difference between them. Value Parameters 20 μg mL-1 Conc. of GEN 0.003 M Conc. of PR 0.1 M Conc. of NaOH 1.9 mL.min-1 Total flow rate 150 µL Sample loop 50 cm Reaction coil Iraqi J Pharm Sci, Vol.31(1) 2022 Determination of genistein by coupling reaction 283 Table 5. The t- and F-tests were used to compare the proposed methods to the UV method Preparation form Proposed method Classical UV method Recovery % GEN ( pure) 101.00 99.23 GEN capsule 125 mg 99.27 101.34 t-calculate (t*=4.303) 0.13 F-calculate (F*=161.4) 1.44 The influence of foreign compounds was eliminated using standard addition method which applied under calibration curve conditions. Good accuracy and precision was obtained .The results are shown in Figure 7 and Table 6. Figure 7. Standard addition method for determination of 20 µg.ml-1 of GEN in supplements Table 6. Application of the proposed method for determining GEN in pharmaceutical formulations by using standard addition method . Pharmaceutical preparation Proposed method Classical UV method Conc. of GEN (µg.mL-1) *Rec.% *RSD% Conc. of GEN (µg.mL-1) *Rec.% *RSD% Present Found Present Found Genistein capsule 125 mg 15 15.1 100.7 0.5 2 2.03 101.5 1 20 19.8 99.0 0.6 3 3.02 100.7 0.7 * Average of five determinations Conclusion According to the research study, no flow injection methods for estimating GEN in medicinal preparations have been documented. The current study demonstrated that a new spectrophotometric- flow injection approach for determining GEN based on the diazotization coupling reaction using reagent PR at the microgram level was simple, fast, and robust. the suggested method has a high sensitivity, linearity, and cost effectiveness when compared to previous FIA methods and other methods such as LC-MS, HPLC, HPTLC, GCE, and HPLC-UV. The recovery values were satisfactory, indicating the suggested method's excellent accuracy and reproducibility. 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