Analytical Method Development and Validation for the Simultaneous Estimation of Sulfadiazine and Pyrimethamine by RP-HPLC Method

Introduction

Sulfadiazine is 4-amino-N-(pyrimidin-2-yl) benzene1-sulfonamide and belongs to the category of antiinfective agents. Pyrimethamine is 5-(4-chlorophenyl)- 6-ethylpyrimidine-2,4-diamine belonging to the category of anti-malarial and anti-infective agents. Sulfadiazine acts by inhibiting bacterial enzyme dihydropteroate synthetase which is essential for folic acid synthesis and is used to treat infections of second and third degree burns.¹ The drug Pyrimethamine acts by plasmodial form of Dihydrofolate reductase it stopping this parasite from reproducing once it is in the blood stream.^2-4

Chemicals and Reagents

Sulfadiazine and Pyrimethamine were obtained as gift samples from AURUBINDO labs Pvt. Ltd, Hyderabad. We used HPLC grade acetonitrile, water and GR grade KH2 PO4 and orthophosphoric acid.^5-6

Instrumentation

A HPLC (Alliance, Water 2695) with UV/VIS Detector/ PDA detector, UV (lab India, UV 3000 series) and Zodiac C18 250 mm × 4.6 mm × 5 µm column were used. The HPLC system was equipped with Empower software for data processing.⁷

Chromatographic Condition

The mobile phase containing 70:30 methanol:phosphate buffer was found to resolve Sulfadiazine and Pyrimethamine. Orthophosphoric acid was used for pH adjustment of buffer to 4.0. The mobile phase was filtered through 0.45 nylon filter and then ultrasonicated for 30 min. The flow rate was set to 1.0 mL/min. The drug shows good absorbance at 240 nm, which was selected as wavelength for further analysis.^8-9

Preparation of Mobile Phase

A mixture of methanol 700 mL (70%) and 300 mL of buffer (HPLC grade-70%) was prepared and degassed in ultrasonic water bath for 5 minutes. It was then filtered through 0.45 µ filter under vacuum filtration.¹⁰

Preparation of Sample Solution

8 mg of Sulfadiazine and 12.5 mg Pyrimethamine tablet powder were accurately weighed and transferred into a 10 mL clean dry volumetric flask. About 7 mL of diluent was added and sonicated to dissolve completely and volume was made up to the mark with the same solvent (Stock solution). Further 0.6 mL of the above stock solution was pipetted into a 10 mL volumetric flask and diluted up to the mark with diluent.¹¹

Preparation of Standard Stock Solutions

Accurately weighed 8 mg Sulfadiazine and 12.5 mg Pyrimethamine were transferred to 10 mL volumetric flasks, 3/4th of diluents were added and sonicated for 10 minutes. Flasks were made up with diluents and labeled as standard stock solution (5000µg/ml of Sulfadiazine and Pyrimethamine)¹²

Preparation of Standard Working Solutions (100% solution)

1 mL of Sulfadiazine and Pyrimethamine from each stock solution were pipetted out and taken into a 10 mL volumetric flask and made up with diluent (500 µg/mL of Sulfadiazine and Pyrimethamine). 

Preparation of Stock Solutions

Five tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to one tablet was transferred into a 10 mL volumetric flask. 5 mL of diluents were added and sonicated for 25 min; further the volume was made up with diluent and filtered through HPLC filters (5000 µg/mL of Sulfadiazine and Pyrimethamine).¹³

Preparation of Sample Working Solutions (100% solution)

1 mL of filtered sample stock solution was transferred to 10 mL volumetric flask and made up with diluent (500 µg/mL of Sulfadiazine and Pyrimethamine).

Preparation of buffer

0.1% OPA Buffer: 1 mL of Perchloric acid was diluted to 1000 mL with HPLC grade water

Method Development

The detection wavelength was selected by dissolving the drug in mobile phase to get a concentration of 10 µg/ mL for individual and mixed standards. The resulting solution was scanned in U.V range from 200-400 nm. The overlay spectrum of Sulfadiazine and Pyrimethamine was obtained and the isobestic point of Sulfadiazine and Pyrimethamine showed absorbance’s maxima at 240 nm. Chromatographic method development was optimized by various parameters both in API and pharmaceutical dosage form as shown in Figure 1.¹⁴

The chromatographic conditions were optimized by preparing mobile phase 70:30 methanol: phosphate buffer with flow rate 1 mL/min and was run for 10 minutes by selecting column Zodiac silca RP C18 4.5×100 mm 3.0 µm of ambient temperature.¹⁵

The retention time of Sulfadiazine and Pyrimethamine was found to be 2.170 mins and 7.280 mins respectively. Resolution was noted to be 8.67. The % purity Sulfadiazine and Pyrimethamine in pharmaceutical dosage form was found to be 99.1 and 98.2% respectively as shown in Table 1 and Figure 2.

Validation Report Specificity

The system suitability for specificity was carried out to determine whether there is any interference of any impurities in retention time of analytical peak.

Linearity

The linearity study was performed for the concentrations of 25 ppm to 150 ppm for Pyrimethamine and 16 ppm to 80 ppm for Sulfadiazine and the results are depicted in Table 2.  

Accuracy

The accuracy study was performed for 50%, 100% and 150% Sulfadiazine and Pyrimethamine. The % recovery was found to be 101.4% and 101.7% (Table 3).

Precision (Repeatability)

The precision study was performed for five injections of Sulfadiazine and Pyrimethamine. Each standard injection was injected into chromatographic system. The intermediate precision study was performed for five injections and the results are shown in Table 4.

LOD and LOQ

The LOD was performed for Sulfadiazine and Pyrimethamine and was found to be 2.17 and 0.0372 respectively. The LOQ was performed for Sulfadiazine and Pyrimethamine which was found to be 6.60 and 0.112 respectively (Figure 3).

Robustness

The robustness was performed for the flow rate variations from 0.4 mL/min to 0.6 mL/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Sulfadiazine and Pyrimethamine. It can be concluded that the variation in flow rate affected the method significantly (Table 5).

Degradation studies

Oxidation:

To 1 mL stock solution of Sulfadiazine and Pyrimethamine, 1 mL of 20% hydrogen peroxide (H2 O2 ) was added separately. The solutions were kept for 30 min at 600 C. For HPLC study, the resultant solution to obtain 500 µg/mL solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample as shown in Table 6.

Acid Degradation studies:

To 1 mL stock solution of Sulfadiazine and Pyrimethamine, 1 mL of 2N was added to resultant hydrochloric acid. The resultant solution was diluted to obtain 500 µg/mL solution and 10 microl litre is subjected to the stability of the sample.

Conclusion

It can be concluded that the proposed RPHPLC method was accurate, precise, sensitive, specific, robust and reproducible for the simultaneous analysis of Sulfadiazine and Pyrimethamine with less tailing factor and also economical. Zodiac sil C18 column (4.6×150 mm) 5µ, flow rate was 1 mL/min, mobile phase ratio was (70:30 v/v) methanol:phosphate buffer (KH2 PO4 and K2 HPO4 ), phosphate pH 3 (pH was adjusted with orthophosphoric acid), detection wavelength was 240 nm.

Consent and Ethical Approval

It is not applicable.

Competing Interest

Authors have declared that no completing interests exists