INTRODUCTION:

Sulfamoxole is a prominent synthetic antibiotic. It restrains dihydropteroate synthetase, an essential bacterial enzyme to process PABA (para-aminobenzoic acid), where PABA is vital for folic acid synthesis. Hence, sulfamoxole hinders bacterial growth by inhibiting the synthesis of folic acid. Vulnerable infections are treated with sulfamoxole either along with trimethoprim or alone¹. It is available both in suspension and tablet dosage form. Chemically it is a sulfonamide and hasbenzenesulfonamide moiety(Fig. 1). 4-amino-N-(4,5-dimethyl-1,3-oxazol-2-yl)benzene sulfonamide is its IUPAC name. C₁₁H₁₃N₃O₃S is the molecular formula with a water solubility of 1680mg/L (at 20°C)².

Fig. 1: Chemical structure of sulfamoxole

Literature study reveals that different methods were proposed for the estimation of sulfamoxole viz., spectrophotometric, electroanalytical, and chromatographic.

Raghuveer et al, Reddyet al and Mohamed^3-5 estimated the sulfamoxole in pharmaceutical dosage forms by using a suitable chromogen (4-dimethylamino cinnamaldehyde; NaNO₂/HCl; p-benzoquinone) to convert it to a chromophore with absorption maximum in visible region. A tri-positive copper complex was used in alkaline medium to determine the sulfamoxole by Upadhyay et al⁶.

Voltametric quantification of sulfamoxole was done by Khan and Jain⁷ by using a novel sensor which was prepared by modifying glassy carbon electrode with a synthesized nanocomposite material (polypyrrole/TiO₂) having a highly conductivity. Chloro coulometric method was used to determine sulphamoxole⁸.

A scalable RP-HPLC method was developed by SIELC Technologies using a mobile phase of MeCN/H₂O/ H₃PO₄ (20/80/0.1) and Newcrom R1 HPLC column for the preparative separation of sulfamoxole⁹. The methods can be extended as MS compatible by replacing the phosphoric acid with formic acid.

Liquid chromatographic techniques were adopted for simultaneous determination of sulfamoxole along with other sulfonamides in biological samples^10-15. CH₃OH/ 5mM aq. CH₃COONH₄ and 0.1% HCOOH was used in gradient mode in LC-MS/MS, to determine sulfamoxole in chicken, fish muscle and eggs¹².

As the previous chromatographic methodsadopted either expensive LCMS^12,16or gradient mode HPLC^17,18, the present study is aimed at development of a simple isocratic RP-HPLC method for the estimation of sulfamoxole in bulk and formulations.

MATERIALS AND METHODS:

Instrumentation:

PEAK LC 7000 HPLC chromatogram equipped with PEAK 7000 delivery system was used. A switch (77251) containing Rheodyne manual sample injector was fitted in the chromatogram. A sample was injected into the 20 μl loop consisting of Rheodyne injector. Regarding the optimized analysis conditions, engaged the Autochro-3000 software. Weighing was done using DENVER (SI234) model electronic balance. l_max value was determined with UV 2301 model spectrophotometer. Chemicals of analytical grade and HPLC grade solvents were employed.

Preparation of Solutions (stock, working standard and sample solutions):

Sulfamoxole (10mg) was totally dissolved in methanol taken in a 10ml volumetric flask to get a stock solution of concentration 1000µg/ml. By diluting one ml of the above stock solution with the same solvent in a 10ml volumetric flask, working standard solution was obtained.Sulfuno (500mg) is the commercial tablet formulation of sulfamoxole drug. A fine powder of these tablets which contains 10mg of sulfamoxoleis dissolved in 10ml of the above-mentionedmobile phase in a 10ml volumetric flask. Filtered this solution using a filter paper made up of nylon membrane and then diluted with the same mobile phase to a concentration of 1000µg/ml.

Forced Degradation:

These studies were carried out by comparing the degradants in the chromatogram with the initial values after incubating the sulfamoxolestandard in diverse conditions of degradation like exposing the sample for 24hr to the lab or UV light (Normal and UV light degradation), keeping sample in oven at 80°C up to 24hr (Thermal degradation), hydrolyzing 100mg sulfamoxolein 20mL of decinormal HCl/NaOH solution for 24/48 hours (Acid/Base degradation) and oxidizing 100mg sulfamoxoleto 20mL of 3% H₂O₂ for 24hr (Hydrogen Peroxideoxidation).

RESULTS AND DISCUSSIONS:

Method Development:

To quantify thesulfamoxole, the wavelength of detector was fixed at 24nm, taking into account of the observed absorption maxima (Fig.-2).

Fig.2: Absorption spectrum of sulfamoxole

In order to obtain the satisfactory system suitability conditions, optimized the isocratic elution parameters viz., composition of mobile phase, column, flow rate, until the appearance of acceptable peak. Figure 3 represents the demonstrative chromatograms involved in the development of method. A flow rate of 1.0mL min^–1 along with Kromasil, C18 column was used in these trials and having a dimension of 250 X 4.6mm, 5μ. The mobile phase was a mixture of Water: Methanol (50:50; 30:70 and 70:30 v/v for trials a, b and c), Methanol: Acetonitrile (75:25v/v for trial-d) and Methanol: Acetonitrile: Water (65:30:5 and 55:30:15v/v/v for trials –e and optimized condition) (Figure-3). Listed out the optimized conditions in Table-1 after fine-tuning the conditions. Kromasil C18 HPLC column was chosen in the present study as it contains the silica-based packing with low levels of alkali metals (like Ca, Mg, Na, K etc), its silica surface interacts with the sulfamoxole which is polar in nature and having free amine groups. To estimate the sulfamoxole, mobile phase consists high volume of cost-effective methanol with lower volume ratio of acetonitrile is used in the present study compared to the methods suggested by Minghui Meng et al¹⁷ and Rong-jie Fu et al¹⁹. Figure-4. displays the acquired standard chromatogram having peaks with nice resolution, good symmetryand sharpness. The elution time and run time for sulfamoxole in the present study under the optimized conditions are 4.283 and 10 minutes respectively.The retention time in the present study is 4.283 minutes which is lower compared to Abdulqawi Numan and Neil D. Danielson (2004)¹⁶, Forti et al¹² and Minghui Meng et al¹⁷ but higher compared to Abdulqawi Numan et al²⁰.

Table 1: Chromatography conditions of the method

Parameter	Condition
Mode of separation	Isocratic
Column	Kromasil, C18 column (250 X 4.6mm, 5μ)
Mobile phase	Methanol: Acetonitrile: Water 55:30:15 (v/v/v)
Flow rate	1.0mL min^–1
Injection volume	20μL
Sample temperature	Ambient
Column temperature	Ambient
Run time	10min
Detector wavelength	241nm
Diluents	Mobile phase

System Suitability and Specificity:

In order to ensure the suitability of the current method for the anticipated application, a system suitability test was conducted. System suitability parameters at optimized conditions for the current method were noticed within the acceptable criteria and are compiled in Table-2. The satisfactory tabulated values of system suitability parameters indicate the substantiated performance of the system. In order to verify the interference from blank, placebo and degradation products, a specificity study was performed. At the retention time of sulfamoxole, neither diluent nor placebo have shown interference. Less than two tailing factor value and more than 2000 as the number of theoretical plates for the sulfamoxole peak in the HPLC chromatogram confirms the system suitability.

To verify the existence of any interference from either degradation or potential impurities at the sulfamoxole retention time, a specificity study was performed. At the retention time of 4.283 minutes for sulfamoxole, the absence of peak interference from blank indicates the specificity of the current method.

(i)

(ii)

(iii)

(iv)

(v)

(vi)

Fig.3. Chromatograms of Method Development Trials

Fig.4: Chromatogram of a standard solution of sulfamoxole under the optimized conditions

Table 2: System suitability parameters at optimized conditions

S. No.	Parameter	Parameter values
1	Peak area (µV*Sec)	145822
2	Retention time (min)	4.283
3	USP tailing factor	1.21
4	USP plate count	4685
5	API Concentration (μg/ml)	20
6	SD of the area	371.6
7	%SD of the area	0.256

Stability Indicating Studies:

Carried out the forced degradation study with a purpose of understanding the specificity and stability-indicating nature of the proposed method. As a part of degradation studies, chromatograms were recorded under diverse conditions of stress (Fig.-5). All degradant peaks are well separated from each other and also from sulfamoxolepeak in each degradation condition. Hence, no interference was found from its degradation products. Based on the data, it can be concluded that the proposed method is a specific and stability indicating method. Table 3 shows the stress induced degradation results of sulfamoxole. Sulfamoxole has lower susceptibility towards oxidation, alkali and thermal degradation conditions, whereas its sensitivity towards photolytic (light/UV) and acid degradation conditions is evident from the high degradation levels (Table-3). These results signify the suitability of this method for routine quality control analysis as it is established to be selective.

(i)

(ii)

(iii)

(iv)

(v)

(vi)

Fig.-5: Chromatograms of sulfamoxoleunder Stress Conditions (i) Normal Light (ii) UV Light (iii) Thermal (iv) Acid (v) Base (vi) Hydrogen Peroxide

Table 3: Results of stress study data of Sulfamoxole

Parameter	% Assay of degraded sample (A)	*% Degradation w.r.t. control sample(B)**	Stress conditions
Control sample (No Degradation)	99.95	------	No Exposure
Acid degradation	91.40	8.56	1mL of 0.5 N HCl for 12 hr at room temperature
Base degradation	95.88	4.07	1mL of 0.5 N NaOH for 12 hr at room temperature
Oxidation	94.41	5.54	2mL of 10% H₂O₂ for 1 hr at room temperature
Thermal degradation	94.07	5.88	70°C for 48 hrs
Photolytic degradation (light)	92.92	7.02	Exposed to 1.2 Million lux hours of light
Photolytic degradation (UV)	92.29	7.67	200-Watt hours/square meter
B= (99.95– A)/ 99.95100

Validation of Method:

The present guidelines (Q2R1, ICH 2005)²¹ were followed to validate the above proposed optimized method for determination of Sulfamoxole

Linearity:

Injected different concentrations (5 to 30mg/mL) of sulfamoxole to evaluate the linearity and observed linearity in constructed calibration curve (Figure-6). Plotted the area under the curve against drug concentration to construct the analytical curve. The equation of regression was found to be y=7495.8x+4775.3 with a coefficient of correlation of 0.9992.

Figure 6. Calibration plot for Sulfamoxole

Accuracy:

The addition of known quantity of drug to the sample, followed by the recovery percentage represents accuracy, the vital parameter in the analytical methodology. Repeatability evaluates the analytical method’s accuracy. Carried out the accuracy study with 50%, 100%, and 150% recovery levels with six replicate preparations and then injecting in chromatograph. Table-2 shows the results consisting of the calculated % recovery from the chromatographic peak area at each recovery level. The recovery percentage was found to be 98.59 to 101.30 and % RSD values range between 0.222 and 0.978 (Table-4). As recovery results are within the acceptable limits, recovery values pertaining to the analyte are within the limit. Hence, it indicates the accuracy of the method which was proposed to estimate sulfamoxole.

Precision:

The precision indicates the results variability in recurrent analyses of the sulfamoxole sample following duplicate experimental settings. To validate the current method, six replicates were used to conduct the studies for both precisions i.e., method and intermediate precisions (MP and IP). Different conditions (instrument /column/analyst) were used to perform the intermediate precision. 0.248 and 0.366 were the calculated % RSD values for MP and IP respectively, which are within the limits of permissibility prescribed by ICH (Table-5). It indicates the precision as high degree, and the method is rugged. 0.248 and 0.366 were the %RSD values for method and intermediate precisions respectively. Higher than 99% values in assay suggests that the method has good precision (Table-6).

Table 5. Results of Precision (Method and Intermediate) Study

S. No.	% Assay
S. No.	M.P.	I.P.
1	99.33	99.89
2	99.39	98.92
3	99.83	99.92
4	99.25	99.41
5	99.76	99.87
6	99.18	99.39
Mean	99.46	99.56
Std. Dev	0.247	0.364
%RSD (n=6)	0.248	0.366
M.P.: Method Precision; I.P.: Intermediate Precision *at 20 μg mL–1

Robustness:

The key parameters (Mobile phase composition and wavelength) were modified thoughtfully and then evaluated their impact on the method to understand the robustness of the method using a standard solution of 20.0 μg/mlof sulfamoxole.Retention time, theoretical plates, tailing factor and peak area were not affected appreciably by the intentional deviation of experimental parameter values (Table -7). The organic solvent composition was varied ±5%. The observed change in peak area was within limits i.e., less than 2.0% and it indicates the robustness of the current method.

Table 6: Precision Studies System Suitability Parameters

System suitability parameter	M.P.*	I.P.*
USP tailing factor	1.11	1.00
USP plate count	4624	4719
Retention time (min)	4.289	4.306
Peak area	145070	145233
SD of area	371.6	548.5
% RSD of area	0.256	0.378
* from six standard injections

LOD and LOC:

The lowest possible detectable and quantifiable concentrations of sulfamoxolewith the acceptable criteria of 3 and 10 for S/N ratio using the current method were respectively observed as 0.164 µg/mland 0.496µg/ml (Table -8). Applicability of the current method on a widespread range of concentrations can be understood from lower values of LOD and LOQ.

Pharmaceutical formulation analysis:

As the recovery values of sulfamoxole is good (Table 9), determined the amount of sulfamoxole in the tablet formulation following the above method. In addition tospectrophotometers^22-25, HPLC instruments are at affordable price to the laboratories of developing countries^26-31, hence, now-a-days HPLC method can be used in quality control laboratories of these countries. Therefore, the current method is applied for sulfamoxole determination in tablet formulations as per the existing ICH guidelines.

CONCLUSIONS:

Based on this study, it can be concluded that the HPLC method development and its validation in the present study is simple and run time is short. And also, can be successfully applied in quality control laboratories for the quantitative determination as well as stability indicating studies of sulfamoxole in API and tablet dosage formulation.

CONFLICT OF INTEREST:

Nil.

REFERENCES:

1. Dylewski J, Nsanze H, D'Costa L, Slaney L, Ronald A. Trimethoprim sulphamoxole in the treatment of chancroid. Comparison of two single dose treatment regimens with a five day regimen. Journal of Antimicrobial Chemotherapy. 1985; 16(1): 103-9. https://doi.org/10.1093/jac/16.1.103.

2. https://pubchem.ncbi.nlm.nih.gov/compound/Sulfamoxole.

3. Raghuveer S, Raju IR, Vatsa DK, Srivastava CM. Colorimetric Determination OfSulfamoxole In Pharmaceutical Dosage Forms. Eastern Pharmacist. 1993; 36: 119-119.

4. Reddy NR, Prabhavathy K, Chakravarthy IE. Spectrophotometric estimation of sulfamoxole in pharmaceutical preparations. Acta Ciencia Indica Chemistry. 2003; 29(1): 43-4.

5. Mohamed AM, Askal HF, Saleh GA. Use of p-benzoquinone for the spectrophotometric determination of certain sulphonamides. Journal of Pharmaceutical and Biomedical Analysis. 1991; 9(7): 531-8.

6. Upadhyay RP, Pathak AK, Mishra RN. Determination of silversulfadiazine and sulfamoxol by tri-positive coppercomplex in alkaline medium. Acta Ciencia Indica Chemistry. 2007; 33(1): 75.

7. Khan AL, Jain R. Polypyrrole/titanium dioxide nanocomposite sensor for the electrocatalytic quantification of sulfamoxole. Ionics. 2018; 24(8): 2473-88. https://doi.org/10.1007/s11581-017-2365-6.

8. Nikolić K, Medenica M. Coulometric determination of sulphisomidine, sulphamethoxydiazine and sulphamoxole. Journal of Pharmaceutical and Biomedical Analysis. 1991; 9(2): 199-201. https://doi.org/10.1016/0731-7085(91)80146-Z.

9. https://www.sielc.com/separation-of-sulfamoxole-on-newcrom-c18-hplc-column.html.

10. Potter RA, Burns BG, Riet JM, North DH, Darvesh R. Simultaneous determination of 17 sulfonamides and the potentiators ormetoprim and trimethoprim in salmon muscle by liquid chromatography with tandem mass spectrometry detection. Journal of AOAC International. 2007; 90(1): 343-8. https://doi.org/10.1093/jaoac/90.1.343.

11. Fang GZ, He JX, Wang S. Multiwalled carbon nanotubes as sorbent for on-line coupling of solid-phase extraction to high-performance liquid chromatography for simultaneous determination of 10 sulfonamides in eggs and pork. Journal of Chromatography A. 2006; 1127(1-2): 12-7. https://doi.org/10.1016/j.chroma.2006.06.024.

12. Forti AF, Multari M, Di Stefano L, Scortichini G. Determination of some sulfonamides and trimethoprim in chicken, fish muscle and eggs by liquid chromatography-tandem mass spectrometry. VeterinariaItaliana. 2004; 40(2): 11-21.

13. Zheng Y, Fan L, Dong Y, Li D, Zhao L, Yuan X, Wang L, Zhao S. Determination of sulfonamide residues in livestock and poultry manure using carbon nanotube extraction combined with UPLC-MS/MS. Food Analytical Methods. 2021; 14(4): 641-52. https://doi.org/10.1007/s12161-020-01910-4.

14. Chen R, Yang Y, Qu B, Li Y, Lu Y, Tian L, Shen W, Ramakrishna S. Rapid determination of sulfonamide residues in pork by surface-modified hydrophilic electrospun nanofibrous membrane solid-phase extraction combined with ultra-performance liquid chromatography. Analytical and Bioanalytical Chemistry. 2016; 408(20): 5499-511. https://doi.org/10.1007/s00216-016-9648-z.

15. Fedorova G, Nebesky V, Randak T, Grabic R. Simultaneous determination of 32 antibiotics in aquaculture products using LC-MS/MS. Chemical Papers. 2014; 68(1): 29-36. https://doi.org/10.2478/s11696-013-0428-3.

16. Numan A, Danielson ND, Characterization of sulfonamides by flow injection and liquid chromatography-electrospray ionization-mass spectrometry after online photoderivatization. Journal of Chromatographic Science. 2004; 42(10): 509-515. https://doi.org/10.1093/chromsci/42.10.509

17. Meng M, He Z, Xu Y, Wang L, Peng Y, Liu X, Simultaneous extraction and determination of antibiotics in soils using a method based on quick, easy, cheap, effective, rugged, and safe extraction and liquid chromatography with tandem mass spectrometry. Journal of Separation Science. 2017; 40(16): 3214-3220. https://doi.org/10.1002/jssc.201700128

18. Jansen LJ, Bolck YJ, Rademaker J, Zuidema T, Berendsen BJ, The analysis of tetracyclines, quinolones, macrolides, lincosamides, pleuromutilins, and sulfonamides in chicken feathers using UHPLC-MS/MS in order to monitor antibiotic use in the poultry sector. Analytical and Bioanalytical Chemistry, 2017; 409(21): 4927-4941. https://doi.org/10.1007/s00216-017-0445-0

19. Rong-jie FU, Sun JL, Yun-qing LI, Agilent Technologies Shanghai Co. Ltd Analyze Quinolone Residues in Milk with an Agilent Poroshell 120, 4 μm Column, 2014. www. agilent.com/chem.

20. Numan AA, Spectrophotometric determination of diaminopyrimidines with p-benzoquinone and on-line photochemical derivatization of sulfa drugs and indoles using liquid chromatography/mass spectrometry. PhD Dissertation, 2001, Miami University.

21. Guideline, ICH Harmonised Tripartite. Validation of Analytical Procedures: Text and Methodology. Q2 (R1) 1.20 (2005): 05.

22. Gorumutchu GP, Ratnakaram VN. Determination of mianserine using Fe3+-phenanthroline by visible spectrophotometry. Research Journal of Pharmacy and Technology. 2019; 12(1): 209-12. DOI: 10.5958/0974-360X.2019.00038.6

23. Polawar, PV, Shivhare, UD, Bhusari, KP, Mathur, VB, Development and validation of spectrophotometric method of analysis for fexofenadine HCl.. Research Journal of Pharmacy and Technology. 2008; 1(4): 539-40.

24. Kaadi, AB, Raju, SA, Manjunath, S, Darak, V, Development and validation of visible spectrophotometric methods for determination of Cefditoren Pivoxil in pharmaceutical formulations. Research Journal of Pharmacy and Technology. 2011; 4: 1269-73.

25. Gorumutchu GP, Ratnakaram VN. Extractive spectrophotometric determination of ulipristal acetate using naphthol blue black. Research Journal of Pharmacy and Technology. 2019; 12(3): 1347-52. DOI: 10.5958/0974-360X.2019.00226.9.

26. Thete, PG, Saudagar, RB, Analytical Method Development and Validation for the Determination of Ivabradine HCl by RP-HPLC in bulk and Pharmaceutical Dosage form. Asian Journal of Pharmacy and Technology, 2019; 9(2): 89-92. DOI: 10.5958/2231-5713.2019.00015.1

27. Fahelelbom, K, Al-Tabakha, MM, Eissa, NA, Obaid, DEE, Sayed, S, Development and Validation of an RP-HPLC Analytical Method for Determination of Lisinopril in Full and Split Tablets. Research Journal of Pharmacy and Technology. 2020; 13(6): 2647-52. DOI: 10.5958/0974-360X.2020.00470.9

28. Vaja, MD, Patel, RR, Patel, BD, Chaudhary, AB, Development and Validation of RP-HPLC Method for Estimation of Lurasidone and its impurities Lurasidone 1 and Lurasidone 8. Research Journal of Pharmacy and Technology, 2022; 15(11): 4999-5004.DOI: 10.52711/0974-360X.2022.00840

29. Ghante, MR, Tangade, RB, Sawant, SD, Kulkarni, PD,Bhusari, VK, Development and Validation of stability indicating RP-HPLC method for the estimation of Ertugliflozin by forced degradation studies. Research Journal of Pharmacy and Technology. 2022; 15(11): 4945-49.DOI: 10.52711/0974-360X.2022.00831

30. Sivagami, B, LM S.K, Development and Validation for the Simultaneous Estimation of Rilpivirine and Dolutegravir in Bulk and Pharmaceutical Dosage Forms by RP-HPLC Method. Research Journal of Pharmacy and Technology. 2022; 15(11): 5302-6. DOI: 10.52711/0974-360X.2022.00893

31. Satyanarayana, L, Naidu, SV, Rao, MN, Ayyanna, C, Kumar, A, The Estimation of Raltigravir in Tablet dosage form by RP-HPLC. Asian Journal of Pharmaceutical Analysis. 2011; 1(3):56-8.

Received on 17.01.2023 Modified on 30.05.2023

Research J. Pharm. and Tech 2023; 16(12):5617-5623.

DOI: 10.52711/0974-360X.2023.00908

Level of recovery (%)	Added Amount(µg mL^–1)	Recovered Amount (µg mL^–1)	% Recovered	Statistical evaluation
50	15	15.13	100.86	%Mean	101.11
	15	15.17	101.16	SD	0.225
	15	15.20	101.30	%RSD	0.222
100	20	19.97	99.86	%Mean	99.59
	20	20.03	100.17	SD	0.745
	20	19.75	98.75	%RSD	0.748
150	25	24.65	98.59	%Mean	99.70
	25	25.09	100.34	SD	0.969
	25	25.05	100.18	%RSD	0.971

Altered Parameter	Actual condition	Altered condition	RT (Min)	Theor plates	Tail factor	Peak area	% Change in peak area
Control Condition	NA	---	4.283	4685	1.21	145822	-----
Mobile phase ratio -Methanol: Acetonitrile: Water (v/v/v)*	55:30:15	50:35:15	4.300	4578	1.15	144961	0.590
Mobile phase ratio -Methanol: Acetonitrile: Water (v/v/v)*	55:30:15	65:25:15	4.283	4762	0.91	143872	1.337
Wave length (nm)	241	239	4.317	4798	1.19	145972	0.103
Wave length (nm)	241	243	4.300	4921	1.19	145872	0.034

Parameter	LOD	LOQ
Results	0.164 µg/ml	0.496 µg/ml

S.No	Brand Name	Form	Dosage	Amount Prepared	Amount Found	% Assay
1	Sulfuno	Tablet	500 mg	20µg/ml	19.92 µg/ml	99.59