Determination of Sulfasalazine in Sulfasalazine Tablets Using Silver Nanoparticles

1. Introduction

    Silver nanoparticles, because of their physical  and  chemical  properties,  are extensively used in many areas such as optics [1], microelectronics [2], clinical diagnosis [3], sensors and so forth. The surface Plasmon resonance of silver nanoparticles which is due to the surface plasmon oscillation modes of conduction electrons which are coupled via the surface to electromagnetic radiations [4], make it ideal for a vast variety of applications.

    The  use  of  these  nanoparticles  in determination and detection of clinical and environmentally important compounds is recently of concern. Recently, determination of trace chlorine dioxide based on decrease in surface   Plasmon   resonance   of   silver nanoparticles is reported [5]. In another study, resonance light scattering (RLS) quenching induced by adsorption of nucleic acid on the surface of silver nanoparticles becomes the basis for the detection and determination of RNA and DNA [6, 7]. However, nano- analytical chemistry using silver nano-particles is extensively seen in the literature [8-10]. Some of them are based on silver reduction by the analyte. Others are based on quenchingnAg RLS.

     Sulfasalazine is a synthetic drug and a combination of antibiotic (sulfapyridine) and an anti-inflammatory agent (5-aminosalysilic acid) [11] which are extensively used in the treatment of inflammatory bowel diseases such as rheumatoid arthritis, Crohn’s disease and ulcerative colitis [12-14]. Post marketing surveillance study (PMS) which is performed in every  country to monitor the safety of pharmaceuticals their release on the market covers an important part of the pharmaceutical researches. Different methods of PMS study are  reported  in  the  literatures.  In  the pharmacopeia, a variety of methods are presented for different pharmaceutical forms of a certain drug. Although these methods are approved and addressed in the assay of sulfasalazine still new, accurate, and precise methods with higher number of analysis per time is required.

     The objective of this study is to introduce a precise and accurate method for the assay of sulfasalazine contents of tablets based on nanotechnology. To our knowledge, it is the first report on the analysis of sulfasalazine contents of tablets using silver nanoparticles.

2. Material & methods

2.1. Reagents

    All reagents used in this study were from analytical reagent grade. Phosphoric acid, sodium hydroxide, sodium citrate, silver nitrate, and methanol all were purchased from Merck (Germany). Pure sulphasalazine powder and sodium dodecylsulphate (SDS) were  obtained from Fulka. Sulphasalazine tablets (Mehrdaru Company, Iran) with different batch numbers were prepared from local pharmacies. De-ionized water was used throughout the study.

2.2. Instruments

    RLS  and  fluorescence  spectra  were recorded using a RF-5301PC Shimadzue spectroflourimeter (Japan). IKA-WERKE mechanical stirrer (Germany) and VS1902MF (Vision corporation) water Circulator (Korea) were used to control stirring speed and temperature during nano-silver preparation. Particle size of as prepared nano silver was determined using Scatteroscope1 nanoparticle size analyzer, Qudix Corporation (Seoul, Korea). All pH were recorded using 920- WTW pH meter (Germany).

2.3. Silver nanoparticles preparations

   Silver nanoparticles were prepared via chemical  reduction  method  previously described with slight modification [6]. To briefly describe, 0.0158 g silver nitrate was placed  in a 250 ml beaker, 40 ml distilled water was added and incubated at 80 ˚C in a  water circulator. Two ml of 1% sodium citrate solution was slowly added to the solution while stirring. The rate of citrate transfer was controlled so that its total volume was added to the beaker in about 30 min. The resulting suspension was transferred to a 50 ml volumetric flask and diluted to the mark with distilled water. This solution (2.0 × 10-4 g.ml-1 nano-silver) was stored in the refrigerator at 4 ˚C.

 Figure 1. Intensity distribution view of PSA analysis of synthesized silver nano particles.

2.4. Sample preparation

    Sulfasalazine Stock solution (250 µg.ml-1): 0.0255 g of pure sulfasalazine powder were dissolved in 30 ml methanol and diluted to 100 ml using distilled water. Working solutions were prepared by step by step dilution of this stock solution in the desired ranges (0.01 to 100 µg.ml-1).

     Tablet preparation for sulfasalazine assay: tablet solutions were prepared as described in USP [15]. Shortly, twenty tablets from each batch number were completely grinded into the  powder. About 0.17 g of this powder, equivalent to 150 mg sulfasalazine, was transferred to a 100 ml conical flask, and 50 ml of 0.1 N sodium hydroxide was added and completely mixed. The resulted solution was filtered and the first 20 ml of filtrate were discarded. The filtrate was used for the determination of sulfasalazine contents by both USP method [16] and the proposed method  after  dilution  to  appropriate concentrations with appropriate solvents of each method. Concentration of sulfasalazine in the filtrate is suggested to be 1500 µg.ml-1 according to the lable.

Table1. Method validation parameters.

^aConcentration in mg.ml^-1; ^bbetween days; ^wwithin days.

Figure 2. Effect of sodium dodecylsulphate (SDS) concentration on the change in SPR intensity of silver nano particles in the presence of 40 μg.ml-1 sulfasalazine.

Table 2. Analysis of sulfasalazine content in the tablets with different batch numbers determined by both proposed and USP methods

2.5. Proposed method procedure

     Three ml of 2×10-4 g.ml-1 of nano silver, 2 ml of phosphate buffer pH=8, 0.4 ml of SDS (8×10-7  M) and different amounts of standards and/or appropriate amounts of tablet solution ( explained in section 2.4) were added to a 10 ml volumetric flask and the solution was made to volume using distilled water. RLS of these solutions were recorded using synchronous technique. Blank solutions were prepared  in the same way except sulfasalazine was absent.

3. Results and discussion

3.1. Silver nano particles characterization

     Silver naoparticles were characterized by observing the UV-Vis spectra in the range of 200-700 nm. As it is reported in the literatures [16, 17], surface Plasmon resonance band of nano silver is a good indicative of its particle size. So a maximum observed at 430 nm and yellow color of the solution indicates that nano-silver was prepared. Particle sizes of the synthesized nanoparticle were also determined by observing intensity distribution data obtained by particle size analyser demonstrated in Figure 1, which indicates that the mean sizes of particles are around 30 nm.

     The stability of refrigerated nAg was also evaluated by measuring the change in the absorbance of the nAg solution at 430 nm and the extent of wavelength shift with time. No shift in wavelength that is an indication of nAg aggregation and no absorbance changes within 3 months confirmed that particles are stable while keeping in the refrigerator at 4 ˚C.

3.2. Evaluation of the method

     RLS of nAg in the presence of sodium dodecylsulphate was decreased on addition of sulfasalazine. However, this decrease was proven to be proportional to sulfasalazine concentration and can be the basis of its assay. In order to optimize the response, the effect of different parameters such as the amount of nanoparticles, pH, volume and kind of buffer, SDS concentration and time on nano-silver RLS   quenching   in   the   presence   of sulfasalazine was investigated. At first it was shown that SDS increases RLS intensity of nAg compared to nAg alone. Adsorption of SDS on the surface of nAg causes more dispersion of the nanopartilces and decreased their aggregation. As the result changes in RLS intensity in the presence of sulfasalazine becomes higher. So, the effect of SDS concentration on the  signal difference was investigated. As it is obvious in Figure 2, the best concentration of SDS is in the range of 1.6×10-8 to 4.8×10-8 M.

     Figure 3 shows that the increase in pH increases the difference in RLS intensity till pH 8 while sulfasalazine is present. So, pH 8 was selected as the optimum pH. In this pH the anionic form of sulfasalazine is easily adsorbed  on the surface of nAg. Different buffer systems, namely acetate, citrate, carbonate, borate, and phosphate were tested and among them phosphate buffer showed the most reliable results. Volume of phosphate buffer solution was also optimized and the 2 ml in 10 ml volumetric flasks was showed to be adequate.

Figure 3. Effect of pH on the change in RLS intensity of silver nano particles in the presence of 40 μg.ml-1 sulfasalazine.

    Figure 4 shows that 6×10-5 g.ml-1 of silver nanoparticle produces the highest signal changes in the presence of sulfasalazine. It was indicated that after this concentration a slight decrease in  I is observed.

Figure 4. Effect of nano silver concentration on the change in RLS intensity of silver nano particles in the presence of 40 μg.ml-1 sulfasalazine.

     The effect of different waiting times after mixing all reagents was evaluated. It was concluded that signals must be recorded within 15 min. After that time, a slight decrease in the signal was observed. So the analysis time should not exceed 15 min.

     Figure 5 shows the decrease in RLS intensity  in  the  presence  of  different concentration of sulfasalazine prepared according to procedure described in section 2.5. As it is demonstrated in Figure 5, sulfasalazine does not show any emission at working conditions but nAgs RLS intensity quenches  more  with  the  increase  in sulfasalazine concentration. Adsorption of sulfasalazine on the surface prevents nAg electrons to interact with the incident light. So, this   prevention   is   increased   as   the concentration of sulfasalazine increases. Table 1 demonstrates the figure of merit of the method as a quantitative technique for the analysis of the sulfasalazine. The table also indicates good precision.

Figure 5. RLS spectra of silver nano particle+SDS and increasing concentration of sulfasalazine (15 to 60 μg.ml-1).

     The accuracy of the method was evaluated by determining sulfasalazine contents of tablets with different batch numbers by both proposed methods and those reported in USP. Table 2 illustrates that the proposed methods is reliable and can be successfully used for the assay  of  this  pharmaceutical  form  of sulfasalazine. It is to be mentioned that no interferences from tablet excipients was observed.

4. Conclusion

     As    the    results    indicated,    silver nanoparticles can be used in the determination of sulfasalazine in sulfasalazine tablets with reasonable reliability. To our knowledge, it is the first report on use of nano silver in the assay of sulfasalazine tablets.

Acknowledgements

     We acknowledge Khozestan environmental protection     agency,    Ahvaz     branch. Collaboration of their lab personnel especially Mr. Alanezhad and Miss Khaksar is gratefully acknowledged.  Our  thanks  also  go  to nanotechnology    research    center    of Jundishapur University of Medical Sciences.