1. INTRODUCTION:

Method development and optimization in liquid chromatography is still an attractive field of research for the researchers and attracts also a lot of interest from practical analysts. Complex mixtures or samples required systematic method development involving accurate modeling of the retention behavior of the analyte.

Among all, the liquid chromatographic methods, the reversed phase systems based on modified silica offers the highest probability of successful results. However, a large number of (system) variables (parameters) affect the selectivity and the resolution.

Many methods reported for determination of Sulphadoxine and Pyrimethamine in pharmaceutical dosage form as well as stability indicating methods.^6-9

Proposed method was innovative simple robust and cost effective by taking Tolterodine as an internal standard.

1. Sulfadoxine:

Fig. No 1: Chemical structure of Sulfadoxine

2. Pyrimethamine:

Fig No. 2: Chemical structure of Atenolol

3. Tolterodine:

Fig No 3: Chemical structure of Tolterodine

2. MATERIALS AND METHODS:

2.1. Apparatus and Equipments:

HPLC instrumentation consisting of pump PU-2080 plus (JASCO, Tokyo, Japan), with Rheodyne manual loop injector 7725i (injection loop capacity 20𝜇L) was used. Detection was carried out using UV-2075 detector (JASCO, Tokyo, Japan). Data acquisition was done by Borwin chromatography software version 1.5 (JASCO, Tokyo, Japan). All calculations were performed using Microsoft Excel 2010 (Microsoft Corporation).

2.2. Reagents and Chemicals:

Pharmaceutical grade Sulfadoxine, Pyrimethamine was supplied as a gift sample from Ipca laboratories pvt ltd Andheri, Mumbai. Methanols used in analysis were of HPLC grade and all other chemicals and reagents were of analytical grade and were purchased from SD Fine Chemicals, Mumbai, India. Double distilled water used was freshly prepared by Double Distillation Assembly (Borosil, Mumbai, India) and further used in analysis after filtering through 0.45μ membrane filter papers purchased from Millipore (India) Pvt. Ltd., Bengaluru, India. Sulfadoxine and Pyrimethamine purches from local medical store (label claim 500mg and 25mg/tablet).

2.3. Chromatographic Conditions.

All chromatographic separations were carried out on Phenomenex Hyper Clone C 18 column (250 × 4.6mm, 5𝜇), using mobile phase comprising methanol: Phosphate buffer 60:40% v/v. The flow rate was kept constant throughout analysis at 1.0mL/min and eluent was detected at 276nm by UV- detector.

2.4. Standard Preparation:

Standard solution were prepared by dissolving 10mg of Sulfadoxine, 10mg of Pyrimethamine and 10mg of Tolterodine into a 100mL clean dry volumetric flask, add about 75mL of methanol and sonicate to dissolve it completely and make volume up to the mark with the diluent. Pipette 1mL of the above stock solution for Pyrimethamine and Sulfadoxine and 5ml for Tolterodine into a 10mL volumetric flask and dilute up to the mark with diluents to prepare 10ppm of Pyrimethamine and Sulfadoxine and 50ppm of Tolterodine.

Method Validation:

System suitability:

A Standard solution of of Sulphadoxine, Pyrimethamine and Tolterodine working standard was prepared as per procedure and was injected six times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms obtained by calculating the %RSD of retention time, tailing factor, theoretical plates and peak areas from six replicate injections are within range and results were shown in Table No.1 -3.

Linearity:

Linearity is the ability of the method to elicit test results that are proportional to concentration of the analyte in the sample.

It was found to be in the range of 2 to 12μg/ml for Sulfadoxine and 4 to 24μg/ml for Pyrimethamine in which 50μg/ml of Tolterodine can be added each time. The calibration graph was plotted, and the drug was found to be linear with a correlation coefficient (r²) of 0.995 were shown in Fig. 5 and 6

Accuracy and Precision:

It is the closeness of test results obtained by the method to the true value. It was determined by percent recovery of the standard API to the blank and It is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. It was determined by studying repeatability, intra-day and inter-day precision of method.^1-5 The average recovery of the analyte of 80%, 100% and 120% solution. The amount found (mg) and %RSD was calculated and were shown in Table No.7-9

Specificity:

Specificity is the ability to measure unequivocally the desired analyte in the presence of components such as excipients and impurities. Here, dextrose was used as excipient. Three solution of 10μg/mL was injected and one excipient solution as a blank injected and compare the chromatogram with the standard solution of Sulfadoxine and Pyrimethamine were shown in Fig. No.7

Robustness:

It is the capacity of the method to remain unaffected by small but deliberate variations in method parameters.^3,4,8 The analysis was performed by slightly changing the wavelength (220nm and 228nm), mobile phase composition (65:35 and 55:45%v/v) and flow rate (0.8 and 1.2mL/min). The variables are shown in Table No.10

Limit of Detection (LOD):

LOD is the lowest level of concentration of analyte in the sample that can be detected, though not necessarily quantitated. It is calculated to be 0.913μg/mL for Sulfadoxine and 1.29μg/mL for Pyrimethamine by using the formula,

LOD= 3.3σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

Limit of Quantitation (LOQ):

LOQ is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied.^1-5 It was calculated to be 2.769μg/mLfor Sulfadoxine and 3.93μg/mL for Pyrimethamine by using the formula,

LOQ=10σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

3. RESULTS AND DISCUSSION:

Table No 1: Result for system suitability test of Sulfadoxine

Sample Name	Retention Time (min)	Area	Plate Count	Asymmetry
Standard 1	2.925	1140860	10470	1.18
Standard 2	2.925	1143927	10476	1.18
Standard 3	2.983	1208848	10576	1.22
Standard 4	2.983	1151378	10646	1.14
Standard 5	2.925	1233716	10567	1.2
Mean		5878729
S.D		42660.12
% RSD		0.725669

Table No 2: Result for system suitability test of Pyrimethamine

Sample Name	Retention Time (min)	Area	Plate Count	Asymmetry
Standard 1	4.075	577584	6237	1.3
Standard 2	4.075	642174	6346	1.29
Standard 3	4.008	592684	5805	1.32
Standard 4	4.075	546048	5720	1.29
Standard 5	4.008	579625	5712	1.39
Mean		2938115
S.D		34980.91

Table No. 3: Result for system suitability test of Tolterodine

Sample Name	Retention Time (min)	Area	Plate Count	Asymmetry
Standard 1	6.908	302090	6550	1.39
Standard 2	6.742	315354	6848	1.48
Standard 3	6.908	315578	6776	1.43
Standard 4	6.742	312266	6637	1.5
Standard 5	6.742	321672	6335	1.44
Mean		1566960
S.D		7179.65
% RSD		0.45819

Table No. 5: Result and Statistical data of Linearity (Sulfadoxine)

Sr. No.	Concentration (µg ml^-1)	Area ratio of Drug and IS
1	2	0.731719
2	4	1.68912
3	6	2.402375
4	8	3.009254
5	10	3.73827
6	12	4.41881
	Correlation Coefficient	0.995
	Slope (m)	0.359
	Intercept (y)	0.145

Table No 6: Result and Statistical data of Linearity (Pyrimethamine)

Sr. No.	Concentration (µg ml^-1)	Area ratio of Drug and IS
1	4	0.7268
2	8	1.5237
3	12	2.2128
4	16	2.7804
5	20	3.6722
6	24	4.5369
	Correlation Coefficient	0.995
	Slope (m)	0.186
	Intercept (y)	0.030

Fig No. 5: Linearity plot of Sulfadoxine

Fig No. 6: Linearity plot of Pyrimethamine

Table No. 7: Result and Statistical data of Accuracy (Sulfadoxine)

Sr. No.	Conc. Level %	Conc. (µg/mL)		Area/Ratio of sulfadoxine+IS	Conc. Found (µg/mL)	% Recovery	% mean Recovery	% RSD
Sr. No.	Conc. Level %	Sample taken	Pure API spiked	Area/Ratio of sulfadoxine+IS	Conc. Found (µg/mL)	% Recovery	% mean Recovery	% RSD
1.	80	5	3	3.05243733	8.09871	101.2339	99.93	1.149
2.	80	5	3	3.01287727	7.98851	99.85645
3.	80	5	3	2.98685567	7.91603	98.95041
4.	100	5	5	3.70483655	9.91597	99.15979	99.944	0.680
5.	100	5	5	3.74604186	10.0307	100.3076
6.	100	5	5	3.74811236	10.0365	100.3653
7.	120	5	7	4.45612234	12.0086	100.0725	99.93407	0.732
8.	120	5	7	4.41605907	11.8971	99.1425
9.	120	5	7	4.47829791	12.0704	100.5872
					Average		99.933	0.853

Table No 8: Result and Statistical data of Accuracy (Pyrimethamine)

Sr. No.	Conc. Level %	Conc. (µg/mL)		Area/Ratio of sulfadoxine+IS	Conc. Found (µg/mL)	% Recovery	% mean Recovery	% RSD
Sr. No.	Conc. Level %	Sample taken	Pure API spiked	Area/Ratio of sulfadoxine+IS	Conc. Found (µg/mL)	% Recovery	% mean Recovery	% RSD
1.	80	5	3	1.542608	8.1323	101.2339	100.0136	1.092
2.	80	5	3	1.516442	7.99162	99.85645
3.	80	5	3	1.512626	7.97110	98.95041
4.	100	5	5	1.858846	9.83250	99.15979	99.9442	1.056
5.	100	5	5	1.839284	9.72733	100.3076
6.	100	5	5	1.87793	9.93510	100.3653
7.	120	5	7	2.224601	11.7989	100.0725	99.93407	1.245
8.	120	5	7	2.204149	11.6889	99.1425
9.	120	5	7	2.258393	11.9806	100.5872
					Average		99.96395	1.131

Table No. 9: Results and Statistical data of Method Precision

Injection No.	Sulfadoxine (10ppm)		Pyrimethamine (10ppm)
Injection No.	Retention Time(min)	Area	Retention Time(min)	Area
1	2.958	3.734912	4.075	1.895277
2	2.958	3.734025	4.117	1.881342
3	2.958	3.70559	4.108	1.885424
4	3.067	3.713505	4.183	1.886704
5	3.067	3.716563	4.300	1.895648
6	3.150	3.71597	4.183	1.884638
Mean		3.720094		1.882685
S.D.		0.032886		0.026177
%RSD		0.884006		1.39041

Fig No.7: Representative Chromatogram of Specificity

Table No. 10: Robustness studies by 2³Factorial Design

Parameter	Variables
Parameter	+	-
Flow rate (1mL/min)	0.2mL	0.2mL
Mobile phase (70:30%v/v)	10%	10%
Wavelength (224nm)	4nm	4nm

From the above variables, only retention time was change with respect to change in flow rate of mobile phase and mobile phase ratio. Hence, all variable of the method was robust.

4. CONCLUSION:

In conclusion, a simple, selective, sensitive and accurate stability indicating RP-HPLC method was developed and validated for the analysis of Sulfadoxine and Pyrimethamine with tolterodine as an internal standard. The retention time of Sulphadoxine, Pyrimethamine and Tolterodine were found to be 2.967, 4.058 and 6.908 respectively Linearity of Sulphadoxine and Pyrimethamine in the range of 2 to 12μg/mL and 4 to 24μg/mL respectively. The % recoveries of Sulphadoxine and Pyrimethamine were found to be in between 99.93% to 99. 96% respectively.

Further the method was found to be linear, precise, accurate and robust. The degradation studies will not carried out due to stickiness of drug to the column. Hence the proposed method can be used for the estimation of Sulfadoxine and pyrimethamine in routine analysis.

5. ACKNOWLEDGMENT:

The authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities and to Ipca Laboratories Andheri, Mumbai, for providing pharmaceutical grade Sulfadoxine and Pyrimethamine as gift sample.

6. REFERENCE:

1. Christen GD. Analytical Chemistry. 5^thed. John Wiley and Sons; 2003; 35-42, 131-132.

2. Sharma BK. Instrumental Methods of Chemical Analysis. 25^th ed. Meerut: Goel Publication Co; 1983; 3: 6.

3. Skoog DA, Holler FJ, Crouch SR. Principle of Instrumental Analysis. 6^th ed. India: Thomson Publications; 2007; 1-3: 145-147, 180.

4. ICH, Q2 (R1); Validation of analytical procedures: text and methodology. International Conference on Harmonization, Geneva; 2005; 1- 13.

5. Nash R.; Pharmaceutical Process Validation; 3^rd edition; Marcel Dekker Publication; New York; 107; 2002: 340-370.

6. K S Nataraj, K Chaitanya, K Alekhya, B. Surendra Babu, K Gnananath. Simultaneous estimation of Sulfadoxine and Pyrimethamine in Bulk and tablet dosage form. International Journal of Environmental Chemistry and Chromatography 2015(1): 8-14

7. B. Hari babu, Kasthuri Jyoti K, Satyanarayana P.V.V, Kumari Kusuma M.A validated liquid chromatographic method for the Determination of Sulfadoxine and Pyrimethamine in pharmaceutical formulation. World Journal of Pharmacy and Pharmaceutical Sciences 2014; 3(10): 1032-1042.

8. Vemula Venkata Raveendra Babu, Sharma Pankaj Kumar V, Singhvi Indrajit. HPLC method development and validation of simultaneous estimation of sulfadoxine and pyrimethamine in Tablet dosage form. International Journal of Pharma Sciences 2013; 3(4): 295-298.

9. Ambalkar S. U, Bakal R. L, Dewani A. P, Chandewar A. V. HPLC-DAD determination of Sulfadoxine, Pyrimethamine and Artisunate in a kit of Tablets, validation and application in vitro dissolution study. International Journal of Drug Formulation and Research 2013; 4(5).

Received on 30.12.2020 Revised on 27.01.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):145-150.

DOI: 10.52711/2231-5675.2021.00025

Sr. No.	Peak Name	Retention Time	Area	Resolution	Plate Count	Asymmetry
1	Sulfadoxine	2.983	1140860	0.00	10470	1.18
2	Pyrimethamine	4.075	577584	6.075	6237	1.3
	Tolterodine	6.908	302090	10.131	6550	1.39