Simultaneous Estimation of Sulfamethoxazole and Pyrimethamine in Bulk and Combined Tablet

Dosage Form

Saravanan. R1, Bharani Pandilla1, Vijayageetha. R1, Kavitha. M1, Santhosh kumar. R1
1
Department of Pharmaceutical Analysis, C. L. Baid Metha College of Pharmacy, Thorapakkam,

Chennai-600097.

ABSTRACT

A simple, accurate, precise, rapid, selective and reproducible RP-UPLC method was developed for

simultaneous estimation of Sulfamethoxazole (SMX) and Pyrimethamine (PYM) in the

pharmaceutical dosage form. Chromatographic separation was carried out using Acquity UPLC

(BEH C18 1.7μm, 2.1x50 mm) column and mobile phase consists of phosphate buffer: acetonitrile

(60:40 V/V; pH 6.8). The flow rate was 0.5 mL/min and detection was set at 220 nm in UV

detector. Retention time of SMX and PYM were 1.61 min and 3.02 min respectively. The method

shows good linearity over the concentration range of 100-600 μg/mL SMX and 5-30 μg/mL PYM.

The correlation coefficients for the calibration curve of sulfamethoxazole and pyrimethamine was

found to be 0.999 and 0.999 respectively. The developed method was validated according to ICH

guidelines.

Keywords: RP-UPLC, Sulfamethoxazole (SMX), Pyrimethamine (PYM), Validation.

INTRODUCTION

Ultra Performance Liquid Chromatography (UPLC) is a relatively new modern technique

which gives a new direction for liquid chromatography and it is applicable for particle having less

than 2 µm in diameter to acquire better resolution, speed and sensitivity as compared with High-

Performance Liquid Chromatography (HPLC). It uses fine particles and saves time and reduces

solvent consumption. The UPLC system reduces analysis time up to nine times comparing to the

conventional system using 5 μm particle packed analytical columns. In UPLC the separation is

performed under tremendous pressures (up to 100 MPa is possible), but it has no negative
impact on analytical column as well as other components of chromatographic system. Separation

efficiency remains maintained and also it is even improved more1-4.

Malaria is an infectious disease commonly found in the tropical countries. Eukaryotic

plasmodium parasites (mainly Plasmodium falciparum and Plasmodium vivax) are the root cause of

this deadly disease. The mode of transmission includes the bite of anopheles mosquitoes (Mojab,

2012; Dua et al., 1998)5. Sulfamethoxazole is an antibacterial drug which has been used to the

treatment of various bacterial infections in humans and other species. It is the sulfonamide drug

most commonly used by combination with trimethoprim for the treatment of urinary tract

infections or with pyrimethamine for the treatment of Chloroquine-resistant Plasmodium

falciparum malaria. Sulfamethoxazole abbreviated SMX. Synonyms, 4- Amino-N-(5-methyl-3-

isoxazolyl) benzenesulfonamide; N1-(5-Methylisoxazol-3-yl) sulfanilamide, Molecular formula

C10H11N3O3S and structural formula shown in figure 1. It is slightly soluble in water (0.5 g/L)

and benzene, slightly soluble in chloroform, diethylether, Isopropanol and soluble in ethanol and

methanol, Melting point is 167 oC (Gennaro, 1995, Rudy et al., 1973 and Budavari, 2000)6.

Pyrimethamine (PYM), an antimalarial drug, works by blocking the biosynthesis of

pyrimidines and purines, which plays an important role in DNA synthesis and cell multiplication.

This is achieved by inhibiting the dihydrofolatereductase of plasmodia. It is chemically 5-(4-Chloro

phenyl)-6-ethylpyrimidine-2,4-diyl diamine and structural formula shown in figure 2. (Meena and

Sandhya, 2013)7.

The literature study reveals that there are numerous analytical methods reported for

quantification of SMX and PYM. The study includes UV spectrophotometry 8-16, HPLC17-27, LC-

MS/MS28 and HPTLC29. However, no methods were reported for the simultaneous estimation of

SMX and PYM using UPLC till now.

MATERIALS AND METHODS:

Chemicals and reagents:

private limited, Pondichery. Tablet used for analysis, P-KALFIN (label claim: 500mg of

sulfamethoxazole and 25mg of Pyremethamine) were purchased from the local pharmacy in

Chennai. HPLC grade acetonitrile and water were purchased from Merck. potassium dihydrogen

phosphate & orthophosphoric acid were procured from Ranken.

Instrumentation:

Chromatography was performed on UPLC Agilent technology-1200 infinity series withhigh

speed auto sampler containing PDA detector. The chromatographic separation was achieved by

using Acquity UPLC (BEH C18 1.7μm, 2.1x50 mm) column. Data acquisition and integration were

performed using open lab CHEMSTATION software.

Chromatographic conditions:

The chromatographic separation was achieved using Acquity UPLC (BEH C 18 1.7μm,

2.1x50 mm) column with isocratic elution of mobile phase consists of mixture of phosphate buffer

at pH 6.8 and acetonitrile (60:40 V/V) at a flow rate of 0.5 mL/min. The volume of sample solution

injected was 20 μL and the total run time was 5mins. UV detection was done at 220 nm. The eluent

was monitored using UV detector at a wavelength of 220 nm.

Preparation of buffer:

10mM phosphate buffer was prepared by dissolving 1.36 g of potassium dihydrogen

orthophosphate in 1000 mL distilled water. Then pH was adjusted to 6.8 with ortho phosphoric

acid and solution was fil tered through

0.45 μ nylon filter.

Preparation of mobile phase:

Mobile phase was prepared by mixing 10mM phosphate buffer: Acetonitrile in the ratio of

60:40 and filtered through 0.45μ nylon filter.

Preparation standard solution:

Standard stock preparation:

concentration 2000 μg/mL and 1000 μg/mL respectively. Working standard solution was prepared

by mixing 10 mL of SMX and 1 mL of PYM standard stock solution in 50 mL volumetric flask,

diluted with the mobile phase to the mark.

Sample preparation:

Transfer 60.15 mg of P-KALFIN formulation into a 100 mL standard flask. About 50 mL of

mobile phase was added to this standard flask and sonicated in an ultrasonic bath for 15 min and

then volume make up with same. The solution was filtered through 0.45 μm nylon syringe filter.

RESULTS AND DISCUSSION:

Method development:

Variety of mobile phase was investigated in the development of a RP-UPLC method for the

simultaneous estimation of Sulfamethoxazole (SMX) and Pyrimethamine (PYM). The system

suitability was appropriate using Acquity UPLC (BEH C18 1.7 μm, 2.1 x 50 mm) column with

isocratic elution of mobile phase consists of mixture of phosphate buffer at pH 6.8 and acetonitrile

(60:40 V/V) which results in the retention time of SMX and PYM were 1.61 min and 3.02 min

respectively.

Method validation:

The optimized method was validated as per ICH Q2 (R1) and the following parameters

were considered: system suitability, accuracy, precision, robustness, specificity, linearity, LOD

and LOQ.

System suitability:

System suitability was performed by six replicate injection of standard solution with the

concentration of 20 μg/mL of MF and 400 μg/mL of MN was injected. The parameters like

retention time, theoretical plate, resolution and peak areaare shown in the Table 1 and Figure 3.

Specificity:
 Specificity is the ability to check clearly the analyte in the presence of components which

may expect to be present. Typically, these might include impurities, degradant and matrix. There

was no interference from excipient and other component with the drug peak. So, the developed

method has been found to be specific (Figure 4).

Linearity:

The linearity of the method was performed by preparing the concentration range of 5-30

μg/mL and 100.- 600 μg/mL for SMX, PYM, from standard stock solution. Calibration curves were

constructed by plotting concentration versus area of SMX and PYM. The results are shown in

Figure 5 and 6.

Accuracy:

The accuracy was calculated by the analysis of tablet and standard at low, medium and

high concentration level. The accuracy was estimated from three replicate injections and

calculated as the μg/mL drug recovered from the drug matrix. The method is found to be accurate

and results are summarized in table 3 & 4.

Precision:

The precision of the proposed assay method was assessed by analyzing standard

solution of 400 μg/mL of SMX and 20 μg/mL of PYM for six times and calculate the % RSD.

The results of test method for precision are displayed in Table 4.

Robustness:

The robustness of a method was analysed by changing experimental, chromatographic

condition. Altering in flow rate (0.6±1 mL/min), changes in column oven temperature (40±5

°C), Changes in mobile phase buffer pH (3.5±0.2), changes in mobile phase composition and

changes in wavelength allowable limits from actual chromatographic condition. It was noted

that there was no recognizable change in mean RT and RSD and parameters fell within the

limit of ≤ 2. The theoretical plate, tailing factor, resolution was found to be good of MF and

MN. This method is robust with variability condition. The analytical condition results are
shown in Table 5.

Solution stability:

Stability of sample solution was confirmed by storing it at ambient temperature for 15 hrs. The

assay of Sulfamethoxazole and Pyrimethamine were analysed. It was found that percentage

labelled amount of Sulfamethoxazole at 5, 10 and 15 hours were 100.79, 100.54 and 100.06 %

respectively. Percentage labeled amount of Pyrimethamine at 5, 10 and 15 hours were 100.87,

100.62 and 100.05 % respectively.

CONCLUSION:

The major supremacy of the UPLC method is significant saving in run time. Based on

the study reports of the present research work, it is obvious that the developed method also had

a very short noticeable reduction in the total run time. In addition, it is a very simple and a

novel method in the midst of commercial applicability. The current developed method offers a

lot of advantages over the others like speedy acquisition of results, remarkable savings in

operational cost and short, sharp retention time with good resolution. Moreover, the results of

the validation studies indicated that the developed RP-UPLC method is simple, accurate

androbust. The Validated data by ICH guidelines also confirms the effectiveness of the

developed method for quantitative analysis of SMX and PYM in bulk and pharmaceutical

dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to Synthiya Research Lab Pvt Ltd., Pondicherry for providing

standards and all facilities throughout the research work. The authors sincerely show gratitude

to Department of pharmaceutical Analysis, C.L. Baid Metha College of Pharmacy,

Thoraipakkam, Chennai, for providing lab facilities and for the constant encouragement during
the research work carriedout.

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