Development of new UV–vis spectroscopic microwave-assisted method for determination of glucose in pharmaceutical samples

    Development of New UV–vis Spectroscopic Microwave-Assisted Method for Determination of Glucose in Pharmaceutical Samples Fazal Mabood, Z. Hussain, H. Haq, M.B. Arian, R. Boqu´e, K.M. Khan, K. Hussain, F. Jabeen, J. Hussain, M. Ahmed, A. Alharasi, Z. Naureen, H. Hussain, A. Khan, S. Perveen PII: DOI: Reference:

S1386-1425(15)30193-1 doi: 10.1016/j.saa.2015.08.035 SAA 14045

To appear in: Received date: Revised date: Accepted date:

15 June 2015 9 August 2015 16 August 2015

Please cite this article as: Fazal Mabood, Z. Hussain, H. Haq, M.B. Arian, R. Boqu´e, K.M. Khan, K. Hussain, F. Jabeen, J. Hussain, M. Ahmed, A. Alharasi, Z. Naureen, H. Hussain, A. Khan, S. Perveen, Development of New UV–vis Spectroscopic MicrowaveAssisted Method for Determination of Glucose in Pharmaceutical Samples, (2015), doi: 10.1016/j.saa.2015.08.035

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ACCEPTED MANUSCRIPT Development of New UV-Vis Spectroscopic Microwave-Assisted Method for Determination of Glucose in Pharmaceutical Samples

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Fazal Mabood*a,, Z. Hussain*b, H. Haqb, M. B. Arianb, R. Boquéc, K.M. Khand, K. Hussainb, F. Jabeene, J. Hussaina, M. Ahmede, A. Alharasia, Z. Naureena, H. Hussaina, A. Khang and S.

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Perveenf,

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a) Department of Biological Sciences & Chemistry, College of Arts and Sciences, University of Nizwa, Sultanate of Oman, ([email protected], [email protected] )

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b) Department of Chemistry, Abdul Wali Khan University, KPK, Pakistan c) Department of Analytical Chemistry and Organic Chemistry, Universitat Rovira i Virgili,

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Tarragona, Spain

d) H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan

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e) Department of Chemistry, University of Malakand, KPK, Pakistan

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75280, Pakistan

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f) PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-

g) Department of Chemistry, UNICAMP, Brasil Abstract

A new UV-Visible spectroscopic method assisted with microwave for the determination of glucose in pharmaceutical formulations was developed. In this study glucose solutions were oxidized by ammonium molybdate in the presence of microwave energy and reacted with aniline to produce a colored solution. Optimum conditions of the reaction including wavelength, temperature, pH of the medium and relative concentration ratio of the reactants were investigated. It was found that the optimal wavelength for the reaction is 610nm, the optimal reaction time is 80 seconds, the optimal reaction temperature is 160 C°, the optimal reaction pH is 4, and the optimal concentration ratio aniline/ammonium molybdate solution was found to be 1:1. The limits of detection and quantification of the method are 0.82 and 2.75 ppm for glucose solution, respectively. The use of microwaves improved the speed of the method while the use of aniline improved the sensitivity of the method by shifting the wavelength.

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Keywords Glucose analysis, Microwave synthesizer, UV-vis spectrophotometry, Carbohydrates, Aniline,

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Introduction

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1.

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molybdenum blue complex, microwave assisted synthesis

Glucose is the most abundantly found biomolecule and is a building block of many important

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biopolymers including starch and cellulose. Glucose is the source of quickly available energy in humans. Due to its diverse roles in biological systems the analysis of glucose is very important. Its analysis may help in the diagnosis of diseases, control of the physiological functions of the

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brain, muscles, drug delivery and formulations [1-7]. Glucose is determined by exploiting its reaction with copper II in terms of the mass of copper I oxide [21]. Glucose is also determined

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by its ability to reduce ferricyanide to ferrocyanide and allowing the reduced Fe2+ to react with

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[tripyridyl-s-triazine (TPTZ)] to give an intensely coloured Fe(TPTZ) complex [8]. The method has been tested on model carbohydrates and other compounds, and also on natural samples of

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coastal and oceanic waters. This indirect determination is very sensitive for the measurement of dissolved carbohydrates in sea water due to the intense color of the resulting complex of iron II. Literature reports indicate that it was determined in terms of the concentration of hydrogen peroxide or glucose oxidase [9,10]. Almost all the analytical methods including spectrophotometry, voltammetry and amperometry are based on the chemical reactions or products of the reactions of glucose [11-16]. Determination of the glucose through its direct reaction is one of the attractive options for the selectivity and sensitivity. Reactivity of the glucose and many other chemicals can be enhanced by the used of microwave radiations as source of energy. It has been observed that a variety of reactions may take place faster under microwave power [17-19]. This is due to the volumetric heating and transfer of energy under microwave radiations [20]. Microwave-assisted reactions may also give improved yield with better selectivity [18,19]. This work was carried out by profiting from the speed and selectivity of microwave reactions with the aim to develop a faster and specific spectrophotometric method for the analysis of glucose in pharmaceutical products. It is based on the microwave-assisted

ACCEPTED MANUSCRIPT reduction of the molybdenum by glucose. Then, the resulting colored solution is used for the spectrophotometric analysis of glucose.

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2. Materials and methods

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2.1 Reagents:

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Pharmaceutical grade D-glucose purchased from Unilever Pakistan Food limited (Pakistan). Ammonium molybdate tetrahydrate and aniline were purchased from RDH Laboratory

prior treatment. Solutions preparation:

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2.2

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Chemikalien GMBH (Germany). These chemicals were used without further purifications or

100 mL of the 0.1 M aqueous solution of D-glucose was prepared by dissolving 1.8 g of the D-

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glucose in distilled water. 100 mL of the stock solution of 0.1 M aniline was prepared by

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dissolving its 0.93 mL in distilled water. The stock solution of 0.01 M ammonium molybdate was prepared by dissolving 1.27 g ammonium molybdate in 100 mL of distilled water. Apparatus:

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UV-visible spectrophotometer by Perkin Elmer (USA) was used for analysis. The reaction was carried out using CEM Microwave synthesizer (USA). 2.4 Procedure/Method:

500 µL of 0.1M solution of glucose and 500 µL of the 0.1M solution of aniline were mixed with 500 µL of 0.01 M ammonium molybdate in the microwave transparent tube and diluted up to 2 mL. The reaction was carried out in microwave synthesizer for microwave-assisted synthesis of glucosyl amine and then it was converted to colored complex with molybdenum. Microwave synthesizer was operated in the range of 100-160 °C using 100 watt microwave power according to the requirement. No set value was assigned to the pressure and the system was allowed to adjust in its permissible default value. This value of pressure was 150 psi which was never obtained The maximum pressure value in all these experiments was found to be 138 psi. The mixture of glucose, aniline and ammonium molybdate was exposed to the microwave power as

ACCEPTED MANUSCRIPT per requirement of the experiment. The resulting product was diluted after microwave treatment up to 10 mL. The blank contained the same amount of aniline and ammonium molybdate except glucose and it was diluted up to 10 mL. The absorbance of the blank as well as standard

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solutions were measured in the range of 370-800 nm.

3.1 Investigation of the optimum wavelength

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3. Results and discussion

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Microwave-assisted reaction of the glucose with ammonium molybdate gives a bluish solution. The absorption profile of this solution was scanned for investigation of the optimal absorption

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wavelength using UV- Visible spectrophotometer in the range of 370 to 800 nm. The spectrum is shown in Figure 1.

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1.2 0.8 0.6

0.4 0.2 0

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Absorbance

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wavelength (nm)

Figure 1. UV-Vis spectrum of the product of the reaction As it can be seen from Figure 1, the optimal wavelength is 610 nm having maximum absorbance. This result is different from the earlier reported work on spectrophotometric determination using the molybdenum blue method, which reported optimal values at 700, and 850 nm [21]. Unlike the previous work [21] the plot shows only one prominent peak at 610 nm, which indicates differences in the nature of the species produced by this microwave-assisted reaction of glucose with aniline and ammonium molybdate. Literature reports indicate the reaction of aniline with

ACCEPTED MANUSCRIPT molybdate and formation of a polymeric composite material [23-25]. However, the reaction is believed to form complex other than polymers of aniline due to the solubility of the product. This

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reaction gives a bluish solution, which indicates the change in oxidation state of the molybdenum. Investigation of the optimum reaction time

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Glucose was determined from the absorbance of the colored solution produced by the microwave-

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assisted reaction of glucose with aniline in the presence of ammonium molybdate. The absorbance of this solution depends upon the concentration of product, which varies with the

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reaction time. The optimal reaction time of this study was investigated in a set of ten experiments by varying the time of reaction and keeping the rest constant. The reaction time was varied in the

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range of 30 to 100 sec. These reactions were carried under identical temperature at 160 °C and 100 watts microwave power. Each of the experiments was conducted in triplicate and the average

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was reported in Figure 2. In each experiment the absorbance was measured at 610 nm.

Figure 2. Optimization of the reaction time It can be seen from figure 2 that the absorbance increases with the reaction time until equilibrium. At equilibrium the graph reaches a steady state. It was found that the equilibrium is established after irradiation of the sample for 80 sec. 3.3 Temperature optimization

ACCEPTED MANUSCRIPT Microwave-assisted reaction of glucose with aniline and ammonium molybdate also depends on the temperature of the reaction mixture. Temperature affects both the rate and yield of the reaction. The effect of temperature on the concentration of the absorbing species obtained by the

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microwave reaction of glucose with aniline and ammonium molybdate was investigated in a set of eight experiments and in the range of temperature from 100 to 200 °C. The temperature of the

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aqueous solution in this range was obtained by using minimum volume of the solution in a

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spacious tube, which gives high temperature due to the increase pressure [26]. All the experiments were conducted in triplicate and using 0.66 mL of glucose, aniline and ammonium

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molybdate each having a concentration of 0.01 M. All samples were irradiated for 80 sec at 100

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watt power. Results of this study are shown in Figure 3.

Figure 3. Optimization of the reaction temperature It can be seen from Figure 3 that the absorbance of solution increases with temperature until 160 °

C and then a decrease is observed. This increase is due to the increased concentration of the

absorbing species. However, the product may pass through decomposition or other reactions after 160 °C, leading to decrease in concentration of the absorbing species. Based on the maximum absorption of solution, 160 °C was found as the optimum temperature for this microwave-assisted reaction.

3.4 Optimization of the reaction pH

ACCEPTED MANUSCRIPT Most of the reactions involving metal ions depend on the pH of the media [27]. Variation of the pH changes both the kinetics and the thermodynamics of the reaction, and it also affects the equilibrium constant. The effect of pH on the yield of this color producing reaction was

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investigated using different types of buffer solutions having pH in the range of 2-13. The pH range from 2-4 was obtained by using a buffer solution composed of HCl and glycin, pH 5 to 6

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was obtained by buffer solution of potassium hydrogen phthalate and sodium hydroxide, pH

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range 7 to 10 was controlled by the combine action of sodium hydroxide and hydrochloric acid and pH 11-13 was adjusted using sodium hydroxide solution. All pH optimization experiments

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were conducted in triplicate using 100 ppm concentration of glucose along with other reagents. The volume of both glucose and reagents was also kept the same. That was 600 µl from the

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stock. All the experiments were conducted under identical conditions, with the only variation in

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pH of the media. The results of this study are shown in Figure 4.

Figure 4. Optimization of the reaction pH It can be seen from Figure 4 that the absorbance of the colored solution changes with pH of the media. It was found that the absorbance of the solution is maximum at pH 4.

3.5 Optimization the concentration ratio of Aniline and ammonium molybdate solutions The present reaction occurs in between aniline and glucose followed by the complexation with molybdate ion. It was reported that reducing sugars form colored solutions on reaction with

ACCEPTED MANUSCRIPT molybdate ion in a two-step process and produces molybdenum blue complex. Although the present work is different from both the previous works in a number of ways still producing colored products. The evidence of difference from the molybdenum blue method is difference in

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ʎmax which is 610 nm unlike 700, 850 and 660 nm for those blue solutions [28-30]. This is also different from the earlier reported color producing reaction of the aniline which uses biphenyl

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aniline and takes place in two steps [31]. In this study glucose, aniline and ammonium molybdate

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reacts simultaneously through a microwave-assisted process. Investigation of the optimum combining ratio of these ensures the maximum sensitivity for the spectrophotometric

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determination of glucose. Each of the aniline and ammonium molybdate is the limiting reactants and the concentration of absorbing species is dependent on the combining ratio of these. This

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combining ratio was investigated in two set of experiments. In one case the concentration and volume of all other reagents except aniline were kept constant. In the second case the volume of ammonium molybdate solution was varied and all other reagents were kept constant. Results of

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the aniline optimization process are presented in Figure 5 and that of molybdate in Figure 6.

Figure 5. Optimization of the aniline concentration for the reaction It can be seen from Figure 5 that the absorbance gets a constant value when the volume of 0.01 M aniline is 600 µl. This gives 1:1 combining ratio to glucose and aniline in this microwave assisted reaction.

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Figure 6. Optimization of the ammonium molybdate concentration for the reaction

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Figure 6 shows the optimization of concentration of ammonium molybdate. It can be seen from the results that the absorbance of the solution increases with the concentration of the molybdate

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solution. However, the graph straightened parallel to the base line at 600 µl of the solution.

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Again the ratio is 1:1 for glucose and ammonium molybdate.

3.6 Investigation of the limit of detection (LOD), and the limit quantification (LOQ) for the glucose concentration

The limit of detection (LOD), is defined as 3s/m, where “s” is the standard deviation corresponding to 10 blank injections and “m” the slope of the calibration graph. The limit of quantification (LOQ) is defined as 10s/m [31]. The limits of detection (LOD), and quantification (LOQ) for the Microwave-assisted glucose, aniline and ammonium molybdate reactions were investigated by reacting various quantities of the glucose with the aniline and ammonium molybdate solutions according to the optimized ratios. The absorbance of these solutions was used for the spectrophotometric determination of glucose, which obeys Beer Lambert law. It was carried out in the range from 5 to 80 ppm concentration of glucose. The results of this study are shown in Figure 7.

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Figure 7. The limit of detection (LOD), and the limit quantification (LOQ)

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The lowest limit of detection was found 0.82 ppm, while the limit of quantification investigated

Conclusion

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was 2.75 ppm for glucose solution concentration.

Molybdenum blue method was successfully improved for the determination of glucose in the aqueous solutions. The use of microwaves improved the speed of method while the use of aniline improved the sensitivity of the method by shifting the wavelength to 610nm. This method was found sensitive to 2.75 ppm limit of quantification for glucose. It can be easily applied for the analysis of glucose in pharmaceutical formulations.

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Graphical abstract

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Highlights of the study 1. Development of New UV-Vis Spectroscopic Microwave-Assisted Method for Determination of Glucose in Pharmaceutical Samples 2. Improvement of Molybdenum blue method for determination of glucose in the aqueous solutions 3. The use of microwaves improved the speed of method while the use of aniline improved the sensitivity