Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Pyrimethamine and Sulphadoxine in Bulk and Tablet Dosage Form

Article history: Received on: 24/12/2015 Revised on: 15/01/2016 Accepted on: 08/02/2016 Available online: 30/03/2016 A stability indicating simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method was developed and validated for the combined tablet formulation of pyrimethamine & sulphadoxine. Chromatographic separation was optimized by gradient HPLC on a C18 column [Inertsil Silica, 250 x 4.6 mm, 5μ] utilizing a mobile phase of potassium dihydrogen phosphate and acetonitrile taken in the ratio 70:30 at a flow rate of 1.0 ml/min with UV detection at 221nm. The retention time of pyrimethamine and sulphadoxine was 2.77 and 6.57 min respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, robustness and stress degradation studies. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated method can be recommended for the routine laboratory analysis.


INTRODUCTION
Pyrimethamine is an anti-malarial drug which inhibits the dihydrofolatereductase of plasmodia and there by blocks the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell multiplication.Sulfadoxine is a sulfa drug, often used in combination with pyrimethamine to treat malaria.Review of literature for pyrimethamine & sulphadoxine gave information regarding the various studies conducted and analytical methods established for the drugs alone, in combination and in combination with other drugs in pharmaceutical dosage forms and in biological fluids (Minzi et al., 2013, Sinnaeve et al., 2005).There are few methods reported in the literature for analysis of pyrimethamine & sulphadoxine alone or in combination with other drugs in the pure form, pharmaceuticals formulations and biological fluids by UVspectrophotometer (Onah and Odeiani, 2002, Meena and .Sandhya, 2013), HPTLC (Meena and Sandhya, 2013), Capillary zone electrophoresis (Amin et al., 2012), RP-HPLC (Green et al., 2002, Bergqvist et al., 1991;Astier et al., 1997;Bergqvist et al., 1985;SaeedArayne et al., 2010), LC-MS (Sinnaeve et al., 2005).
There was no stability indicating HPLC methods established for the simultaneous estimation of pyrimethamine & sulphadoxine in formulation.The aim of the present work is to develop a stability indicating analytical method for the combined tablet formulation of pyrimethamine & sulphadoxine.Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients.Thus validated method can be recommended for the routine laboratory analysis.

Preparation of standard solutions
Accurately weighed and transferred 10 mg of Pyrimethamine and 10 mg of Sulphadoxine working Standards into two separate 100 ml clean dry volumetric flasks, add 30ml of diluent , sonicated for 5 minutes and make up to the final volume with diluent.

Chromatographic Conditions
The HPLC system consisted of Shimadzu gradient HPLC (JAPAN) with dual λ Absorbance UV detector.The wavelength of detection as set at 221nm.Separation was carried out in gradient mode on inertsil C18 column (4.6x250mmx5µm) and the retention time of pyrimethamine and sulphadoxime was found to be 2.952 and 6.832 respectively (figure 1), using 70:30 v/v dihydrogen orthophosphate : acetonitrile as mobile phase at a flow rate of 1 ml/min.The mobile phase filtered through nylon milli pore (0.2µm) membrane filter, purchased from pall life sciences, Mumbai and degassed with Ultrasonicator prior to use.Chromatography was carried out at room temperature 25 0 c and maintains the column temperature at 32 0 c.

Preparation of Standard Solutions
Stock solutions of pyrimethamine (0.5mg/ml) and sulphadoxime (1mg/ml) were prepared in methanol.Further dilutions were carried out in 60% acetonitrile and calibration standards were prepared freshly with pyrimethamine and sulphodoxime stock solutions to give the concentrations of 5, 10, 15, 20, 25 and 30 μg/ml.

Sample Preparation (Assay)
5 tablets were weighed and calculate the average weight of each tablet then the weight equivalent transferred into a 100 mL volumetric flask, 60mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered.From the filtered solution 0.2ml was pipetted out into a 10 ml volumetric flask and made upto 10ml with diluent.

Method Validation
Method validation was performed as per the ICH guidelines Q2 (R1) Validation of Analytical Procedure.The developed method was validated for the following parameters.

Linearity
Linear concentrations of both drugs were prepared and the best fit line was calculated.Wide range calibration was determined by solutions containing 5μg/ml to 30μg/ml (table 1).Correlation coefficient was found to be 0.999 & 0.997 for Pyrimethamine & Sulphadoxine respectively (shown in fig 2&3).

Precision
The intraday precision was demonstrated by injecting six test solutions at 25 µg/ml concentration as per the test procedure (shown in table no 2&3) & recording the chromatograms of six test solutions.The % RSD of pyrimethamine and sulphodixime was found to be 0.207 and 0.324 respectively.

Intermediate Precision
Intermediate precision of the analytical method was determined by performing method precision on in three successive days by different analysts under same experimental condition.Assay of all six replicate sample preparations was determined and the mean % RSD of pyrimethamine and sulphodixime was found to be 0.259 and 0.353 respectively (shown in table no 4).

Accuracy
Accuracy of the method was established by performing recovery studies according to the ICH guidelines.Spiked samples were prepared by spiking pre-analyzed sample solutions with pure drug at three different concentration levels each in triplicate.Mean percentage recovery values at three different concentrations of the two drugs was calculated.The % recovery of Pyrimethamine (98.02-100.45%)& Sulphadoxine (99.79-100.20%) at each level was within the limits of 98% and 102% (shown in table no 5&6).
Hence, accuracy was established for the present work and the method was said to be accurate.

Limit of Detection & Quantification
In the present study, the LOD and LOQ were calculated according to the standard deviation of the response and the slope of the calibration curve i.e., 3.3σ/S and 10σ/S criterions, respectively; where σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve.The lowest possible concentration of Pyrimethamine that can be detected and quantified by the present method was found to be 0.264 µg/ml and 0.800 µg/ml respectively and that of Sulphadoxine was found to be 0.53 µg /ml and 1.62µg/ml respectively.

Robustness
Robustness of the proposed method was determined by varying various parameters, the %RSD reported was found to be less than 2 %.As the system suitability parameters for the standard and test chromatograms of Pyrimethamine & Sulphadoxine were within limits for variation in flow rate (±0.1ml) and mobile phase composition, the allowable variation in flow rate, organic solvent ratio in mobile phase composition and column temperature should be 1±0.1ml/min,65±2ml and 30±5 0 c respectively (shown in table no 7 & 8).

STRESS DEGRADATION STUDIES
Stress degradation studies were performed as per the ICH guidelinesQ1A (R2) Stability Testing of New Drug Substances and Products, using the proposed validated analytical method and the results were shown in table no 9 & 10.

Acid degradation studies
To 1ml of stock solution pyrimethamine and sulphadoxine, 1ml of 2N HCl was added and refluxed for 30min at 60 0 c.From the above solution10µl was injected into the system and the chromatograms were recorded to detect the stability of sample (figure 4).

Alkali Degradation Studies
To 1ml of stock solution of of standard drug and sample pyrimethamine and sulphadoxine, 1ml of 2N NaOH was added and refluxed for 30min at 60 °C.From the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of sample (figure 5).

Oxidation
To 1ml of stock solution of standard drug and sample of pyrimethamine and sulphadoxine, 1ml of 20% H 2 O 2 was added and refluxed for 30min at 60 0 c.From the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of sample (figure 6).

Photo Stability Studies
The photochemical stability of the drug was also studied by exposing the 25 µg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m 2 in photo stability chamber . .For HPLC study, from the above solution10 µl was injected into the system and the chromatograms were recorded to detect the stability of sample (figure 7).
Pyrimethamine and Sulphadoxine undergoes degradation in acidic, oxidation, alkaline, and UV.More degradation was found for oxidation.As per ICH guidelines peak purity angle should be less than peak purity threshold.Hence, method of the analysis of PYR and SUL in tablet dosage form shows that the degradation product doesn't interfere with the analytical determination.The stress degradation studies showed that the drug formulation containing pyrimethamine and sulphadoxine undergoes degradation in acidic, oxidation, alkaline, and UV (7.29% ,15.01%,16.13% , 7.88% , 5.51% and 12.92% , 10.72% , 7.13% , 13.32% , 6.01%).hence the proposed analytical method is also useful for the determination of pyrimethamine and sulphadoxine stability in sample of pharmaceutical dosage form.

CONCLUSION
The proposed HPLC method was found to be simple, specific, precise, accurate, rapid and economical for simultaneous estimation of pyrimethamine & sulphadoxine in bulk and tablet dosage form and was found to be suitable for the routine analysis and quality control and percentage degradation of pharmaceutical preparations containing these drugs either individually or in combination.

Table 2 :
Method Precision of Pyrimethamine.

Table 3 :
Method Precision of Sulphadoxine

Table 9 :
Results of stress degradation studies of Sulphadoxine.

Table 10 :
Results of stress degradation studies of Pyrimethamine.