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VOL. 1 ISSUE 1, 2015; 8 – 14;
rimidines, which are essential for DNA synthesis and cell multiplication. This leads to the failure of nuclear divi- sion. Several RP-HPLC4,5,6,9-12 , Spectrophotometric meth-ods1-3,7 & HPTLC Methods8 have been reported for simul- TION OF SULPHADOXINE
taneous estimation of sulphadoxine and pyrimethamine, AND PYRIMETHAMINE BY
literature survey reveals that there is a need for develop-ment of economic method for estimation of these drugs USING RP-HPLC IN BULK
in combination.

A HPLC (Alliance 2695) with Open labs software, Photo
Diode Array (PDA) detector, Isocratic pump, Electronic K S Nataraj*, K Chaitanya, K Alekhya, B. Surendra Babu, K Gnananath weighing Balance (Sartorius) ,pH meter (polomon) , Ultra Sonicator (Citizen, Digital Ultrasonic Cleaner), Milli Pore (Merck) and HPLC Column - Xbridge (100x4.6 mm ID) 5 5Shri Vishnu College of Pharmacy, Bhimavaram, West Godavari District, Andhra Pradesh, India, Pin -534202. Materials and Reagents:
Methanol (HPLC Grade), Acetonitrile (HPLC Grade), Wa-
ter (Milli Q Grade),Ortho phosphoric acid (AR Grade), Date Received: 6th May 2015; Date Accepted: 15th Aug potassium dihydrogen phosphate (AR Grade), pure drug 2015; Date Published: 18th Aug 2015 samples of sulphadoxine and pyrimethamine obtained as gift samples from Spectrum Laboratory and marketted formulations procured from local market. Abstract: A simple, sensitive and precise RP-HPLC me-
thod for simultaneous estimation of Sulphadoxine and Preparation of Buffer:
Pyrimethamine has been developed and validated for Weigh accurately 2.72 g of Potassium dihydrogen ortho- determination of these in bulk and commercial tablet do- sage form. The compounds were well separated isocrati- 2PO4) into a 1000ml beaker. Add about 900ml of milli-Q water and sonicate and make up the final cally on a ODS column using a mobile phase consisting of volume. Add 0.6ml of Triethylamine then adjust the solu- buffer (pH 3.2) & Acetonitrile (70:30) with a flow rate of tion to pH 3.20 with dilute Ortho Phosphoric Acid solu- 1.0 ml/min using PDA detector. Retention time for Sul- tion to get 0.02M KH phadoxine and Pyrimethamine were found to be 4.1 & 5.8 respectively. The study showed that the reverse phased Preparation of Mobile phase:
liquid chromatography is sensitive and selective for de- Buffer and Acetonitrile are taken in 70:30 % v/v and soni- tecting Sulphadoxine and Pyrimethamine using a single mobile phase. The developed method was validated and was found to be in accordance with the International Con- DILUENT: METHANOL IS USED AS A DILUENT.
ference on Harmonization (ICH) guidelines for analytical Preparation of Blank:
method validation: Q2B. Diluent is used as blank. Preparation of Standard Stock solution:
Sulphadoxine is chemically 4-amino-N-(5,6-dimethoxy Preparation of Sulphadoxine Standard Stock solution:
pyrimidin-4-yl)benzene sulphonamide and is a antima- Accurately weigh and transfer 500mg Sulphadoxine into larial sulphonamide drug . It competitively inhibits dihy- 100ml volumetric flask, add few ml diluent and sonicate dropteroate synthetase interfering with folate synthesis. to dissolve. Then it is diluted to desired volume with di- It acts by increasing oxygen in blood. luent. This gives us a standard stock solution of 5mg/ml Pyrimethamine is chemically 5-(4-chlorophenyl)-6-ethyl pyrimidine-2,4-diamine and is a antimalarial drug acts by Preparation of Pyrimethamine Standard Stock solution:
inhibiting the dihydro folate reductase of plasmodium Accurately weigh and transfer 25 mg Pyrimethamine into and thereby blocks the biosynthesis of purines and py- Nataraj KS et al, Int J. Env Chem Chrom, Volume 1, Issue 1, 2015; 8 - 14 Available Online at 100ml volumetric flask, add few ml diluent and sonicate Method Precision:
to dissolve. Then it is diluted to the desired volume with Method precision was determined by injecting six differ- diluent. This gives us a standard stock solution of ent sample solutions of same concentration which are 0.25mg/ml (250µg/ml). prepared separately. 50µL each of the above solution was injected six times and Preparation of Standard solution:
the chromatograms were recorded for the same. Pipette 2 ml of Sulphadoxine standard stock solution and The chromatograms were recorded as shown in Fig .4 and 2 ml of Pyrimethamine standard stock solution into a 10 results are shown in Table-4. ml volumetric flask and dilute to volume with diluent which gives 1000µg/ml Sulphadoxine and 50µg/ml Pyri- Accuracy:
A study of recovery was conducted from about 50%, Preparation of Sample Stock solution:
100%and 150% of the initial assay concentration. 5 tablets were weighed and crushed into powder, and Sample solutions were prepared in triplicate for each level transferred into a 100 ml volumetric flask, 70lL of diluent and analyzed as per test method. The individual % re- was added and sonicated for 25 min, further the volume covery, %average recovery and % RSD for recovery at was made up to the mark with diluent and filtered. From each level were calculated and the results are found to be the filtered solution 0.4ml was pipetted out into a 10 ml within limit. The chromatograms were recorded as shown volumetric flask and made up to 10ml. in Fig .5 and the results are shown in Table-5. System Suitability:
Standard solutions were injected six times as per test me- The Ruggedness was determined by using the data ob- thod and chromatograms and results of system suitability tained by the analysis performed by two different ana- were shown in Fig.1 and Table-1 respectively. lysts. Each analyst prepared 6 samples of the same batch and the results obtained. Chromatograms were recorded as shown in the Fig.6 and The tailing factor for Sulphadoxine and Pyrimethamine results are shown in Table-6. peaks should be not more than 2.0 from the chromato- gram of standard solution. Robustness:
The Relative standard deviation of Sulphadoxine and Py- Robustness of the method was checked by changing flow rimethamine peak areas from five replicate injections of and temperature, and standard was able to give system standard solution should be not more than 2.0%. suitability parameters within the limits, which indicates that the method is robust results are shown in Table 7 and Linearity:
The linearity of a procedure is its ability (within a given range) to obtain test results proportional to the concentra- tion (amount) of analyte in the sample. The calibration Specificity shall be established by demonstrating that the plot is as shown in Figure – 2. procedure is unaffected by presence of interference at the retention times of Sulphadoxine and Pyrimethamine with Precision:
respect to mobile phase, diluent, and place- The precision of an analytical procedure expresses the bo.Chromatograms were recorded . closeness of agreement between a series of measurement obtained from multiple sampling of the same homogen- Limit of Detection and Limit of Quantification:
ous sample under the prescribed conditions. Precision of LOD and LOQ were calculated from standard an analytical procedure is usually expressed the variance, deviation of response from precision and slope standard deviation of coefficient of variation of a series of System Precision:
System precision was determined by injecting 50µL of the σ is standard deviation from response standard solution for six times and the chromatograms S is slope from calibration curve were recorded for the same. The chromatograms were recorded as shown in Fig.3 and The LOD for this method was found to be 63.92µg/mL for results are shown in the Table-3. SDX and 4.25µg/mL for PYR respectively. The LOQ for this method was found to be 193.70µg/ml for Sulphadox- Nataraj KS et al, Int J. Env Chem Chrom, Volume 1, Issue 1, 2015; 8 - 14 Available Online at ine and12.87µg/ml for Pyrimethamine respectively. were recorded and shown in fig.11 and the % assay re-
sults were shown in Table-11
Assay of Pharmaceutical Formulation:
Results and Discussion
The proposed validated method was successfully applied System Suitability:
to determine in their tablet dosage form. Chromatograms Fig .1: Chromatogram for System Suitability
Table: 1 System Suitability


Fig: 2 Linearity graph of Sulphadoxine
Nataraj KS et al, Int J. Env Chem Chrom, Volume 1, Issue 1, 2015; 8 - 14 Available Online at Fig 2: Linearity graph of Pyrimethamine

System Precision:
Table: 3 Results of System Precision for Sulphadoxine and Pyrimethamine indicate the summary.

Method Precision:

Table: 4 Results of Method Precision for Sulphadoxine and Pyrimethamine


Table: 5 Accuracy results for Sulphadoxine and Pyrimethamine
% Recovery
Nataraj KS et al, Int J. Env Chem Chrom, Volume 1, Issue 1, 2015; 8 - 14 Available Online at
6. Results of Ruggedness for Sulphadoxine and Pyrimethamine:

Robustness: Change in flow rate(± 0.2 ml/min):

Table: 7 Results of Robustness for Sulphadoxine and Pyrimethamine

Change in Column temperature (± 50C):

Table: 8 Results of Robustness for Sulphadoxine and Pyrimethamine
Change in the mobile phase concentration:

Table: 9 Results of Robustness for Sulphadoxine and Pyrimethamine
Mobile Phase Concentration (65:35) Mobile Phase Concentration (75:25) Specificity:
No interference of blank and placebo with the peaks of
Sulphadoxine and Pyrimethamine indicates the specificity
of the developed method.
Nataraj KS et al, Int J. Env Chem Chrom, Volume 1, Issue 1, 2015; 8 - 14 Available Online at found to be precise, selective, sensitive and economical. The present study describes the method development and validation for simultaneous estimation of Sulphadoxine and Pyrimethamine tablet dosage form by RP-HPLC. The dosage form was analyzed XBridge column (100 x 4.6 mm) using phosphate buffer of analysis pH (3.2) and Ace-tonitrile in an isocratic programme with flow rate 1.0ml/min and PDA detection was performed at a wave-length of 224nm. The retention times observed as 4.1 min for Sulphadoxine and 5.8 min for Pyrimethamine. The Fig:10.1 Chromatogram for Specificity (Blank)
linearity for detector response was observed in the con- centration range of 250 to 1500µg/ml of the test concentra-tion and the correlation coefficient (r2) for calibration curve was found to be 0.999 for Sulphadoxine and con-centration range of 12.5 to 75µg/ml of the test concentra-tion and the correlation coefficient (r2) for calibration curve was found to be 0.999 for Pyrimethamine. The re-sults of the recovery studies between 50 to 150% were in the range of 98.57 to 100.77% for Sulphadoxine and 99.35 to 100.34% for Pyrimethamine indicating accuracy of the method and good recovery of the drug. The %RSD for the Fig : 10.2 Chromatogram for Specificity (placebo)
tablet analysis is less than 1 which is indicating high de- gree of precision. The results of the robustness study indi- Limit of Detection and Limit of Quantification:
cates that the method is robust and is unaffected by small The LOD for this method was found to be 63.92µg/mL for variations in the chromatographic conditions. The LOD SDX and 4.25µg/mL for PYR respectively. The LOQ for and LOQ were calculated and found to be 63.92µg/mL this method was found to be 193.70µg/mL for SDX and 193.70µg/mL for Sulphadoxine and 4.25µg/mL and 12.87µg/mL for PYR respectively. 12.87µg/mL for Pyrimethamine respectively. The assay of the sample shows the drug is present within the specifica- Assay of Pharmaceutical Formulation:
tion limit of the formulation indicates the purity of the formulation. Hence, it can be concluded that the devel- oped RP-HPLC method is accurate, precise, rapid and
selective and can be employed successfully for the estima-
tion of Sulphadoxine and Pyrimethamine tablet dosage

A rapid, simple, economical and non interfering simulta-
neous method has been developed for qualitative, quan-
titative, determination and quantification of Sulphadoxine Fig:11 Chromatogram for assay of Sulphadoxine and
and Pyrimethamine simultaneously using RP-HPLC. This method was found to be far better in comparision with the existing analytical methods in various aspects. This Table:11 Results of Assay for Sulphadoxine and Pyri-
method gave good separation and resolution of the chro- methamine
matographic peaks with less retention time and good re- producibility during analysis unlike existing methods. The results are accurate and precise and have been validated and confirmed by the statistical parameters in accordance with the ICH guidelines. This method can be used for the estimation of raw materials, bulk drugs and formulations containing single drugs or in combination. Discussion:
The reliability, rapidity, simplicity, sensitivity, economy, The proposed method for the determination of Sulpha- good recovery, and precision of this HPLC method gives doxine and Pyrimethamine in tablet dosage form was its advantages over other reported HPLC methods for Nataraj KS et al, Int J. Env Chem Chrom, Volume 1, Issue 1, 2015; 8 - 14 Available Online at determination of Sulfadoxine and Pyrimethamine in Pharmacia Sinica, 2010, 1 (2): 52-60. bulk as well as pharmaceutical dosage forms. 10. Ashenafi D, Verbeek A, Hoogmartens J, Adams E. Development and validation of an LC method for the determination of Emtricitabine and related com- 1. Nagaraju P.T, Channabasavaraj K.P, Shantha Kumar P. pounds in the drug substance. J Sep Sci. 2009, 32(11): T. Development and Validation of Spectrophotome- tric Method for Estimation of Emtricitabine in Tablet 11. Kavitha K Y, Geetha G, Hariprasad R, Venkatnaraya- Dosage Form. International Journal of Chem Tech Re- na R, Subramanian G. Development and Validation of search. 2011, 3(1): 23-28. RP-HPLC Analytical Method for Simultaneous Esti- 2. Anindita Behera, Aurobinda Parida, Amit Kumar mation of Emtricitabine, Rilpivirine, Tenofovir Diso- Meher, Dannana Gowri Sankar, Swapan Kumar Moi- proxil Fumarate and its Pharmaceutical Dosage tra, Sudam Chandra Si. Development and Validation Forms. Pharmacie Globale. 2013, 4(1): 1-6. of Spectrophotometric method for determination of 12. Prashant S. Devrukhakar, RoshanBorkar, NaliniSha-
Emtricitabine and Tenofovir Disoproxil Fumarate in stri, K. V. Surendranath. A Validated Stability- Bulk and Tablet dosage form. International Journal of Indicating RP-HPLC Method for the Simultaneous Pharm Tech Research. 2011, 3(3): 1874-1882. Determination of Tenofovir, Emtricitabine, and aEfa- 3. Viswanath V, Shanmugasundaram P, Ravichandiran virenz and Statistical Approach to Determine the Ef- V. UV-Spectrophotometric Absorbance Correction fect of Variables. ISRN Chromatography 2013, 2013: Method For The Simultaneous Estimation Of Tenofo- vir Disoproxil Fumerate And Emtricitabine in com-bined tablet dosage form. International Journal of Pharm Tech Research. 2013, 5(3): 1179-1185. 4. S Dharmaraj Santhosam,V Nagalakshmi Bhavani, M Suresh. Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Emtrici-tabine and Tenofovir from Bulk and Dosage Form. In-ternational Journal of Pharmaceutical Chemistry Re-search. 2012, 1(4): 2278 – 8700. 5. Narendra Devanaboyina, Satyanarayana T, Ganga Rao B. HPLC Method Development and Validation for Simultaneous Estimation of Tenofovir and Emtri-citabine in Combined Pharmaceutical Dosage Form. International Journal of Research in Pharmaceutical and Biomedical Sciences. 2006, 3(1): 361-367. 6. DeepthiKomaroju, G Nagarjuna Reddy, K Dhana- lakshmi. Method development and Validation for Si-multaneous Estimation of Emtricitabine and Tenofo-vir Disoproxil Fumarate in Pure and Tablet Dosage Form by using RP-HPLC. International Journal of Pharma Research & Review. 2013, 2(10): 1-11. 7. Mohammad H Abdel Hay, Azza A Gazy, Rasha A
Shaalan, Heba K Ashour. Simple Spectrophotometric Methods for Determination of Tenofovir Fumarate and Emtricitabine in Bulk Powder and in Tablets. Journal of Spectroscopy. 2013, 2013: 1-7. 8. Maithilee Joshi, AP Nikalje, M Shahed, M Dehghan. HPTLC method for the simultaneous estimation of Emtricitabine and Tenofovir in tablet dosage form. Indian J Pharm Sci. 2009, 71(1): 95–97. 9. Ananda Kumar Karunakaran, Kannan Kamarajan and Vetrichelvan Thangarasu. A Validated RP - HPLC Method for Simulataneous Estimation of Emtricita-bine and Tenofovir Disoproxil Fumarate in Pure and in Tablet Dosage Form. Pelagia Research Library, Der



International Food Research Journal 23(5): 1953-1959 (2016) Anti-cholinesterase inhibitory activities of different varieties of chili peppers Nantakornsuttanan, N., Thuphairo, K., Kukreja, R.K., Charoenkiatkul, S. and *Suttisansanee, U. Institute of Nutrition, Mahidol University, Phutthamonthon 4 Rd.,Salaya, Phutthamonthon, Received: 1 July 2015