Research J. Pharm. and Tech.

16(12): December 2023

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Development and Validation of a New HPLC Method for the Estimation of

Sulfamoxole in Bulk and Tablet Formulations
Kamala Karuna Moparthy1, Venkata Nadh Ratnakaram2*, Giri Prasad Gorumutchu3
1
Department of Chemistry, Government College for Women (A), Guntur - 522001, India.
2
Industrial Chemical Product Development and Analysis Centre, Department of Chemistry,
GITAM School of Science, GITAM Deemed to be University – Bengaluru Campus, Karnataka – 561203, India.
3
Department of Chemistry, A.G & S.G.S College Vuyyuru, Krishna, District - 521165, India.
*Corresponding Author E-mail: [email protected]
ABSTRACT:
To quantify the sulfamoxole content in bulk as well as tablet formulation, a new isocratic RP-HPLC method was
developed and then validated. Kromasil C18 column with a dimension of 250 X 4.6mm was used which was
filled with a particle size of 5μ. A mixture containing CH3OH, CH3CN and H2O in the volume ratio of 55, 30
and 15 was used as a mobile phase at room temperature with an optimized flow rate of 1ml/min. UV detector
was set at 241nm for sulfamoxole determination. The run time of the current method is ten minutes.
Accomplished the forced degradation studies to understand the stability indicating nature of the current method.
As per the current ICH guides (Q2R1), validation of the method was conducted.
KEYWORDS: Sulfamoxole, RP-HPLC, Method development, Validation.

INTRODUCTION: Literature study reveals that different methods were

Sulfamoxole is a prominent synthetic antibiotic. It proposed for the estimation of sulfamoxole viz.,
restrains dihydropteroate synthetase, an essential spectrophotometric, electroanalytical, and
bacterial enzyme to process PABA (para-aminobenzoic chromatographic.
acid), where PABA is vital for folic acid synthesis.
Hence, sulfamoxole hinders bacterial growth by Raghuveer et al, Reddyet al and Mohamed 3-5 estimated
inhibiting the synthesis of folic acid. Vulnerable the sulfamoxole in pharmaceutical dosage forms by
infections are treated with sulfamoxole either along with using a suitable chromogen (4-dimethylamino
trimethoprim or alone1. It is available both in suspension cinnamaldehyde; NaNO2/HCl; p-benzoquinone) to
and tablet dosage form. Chemically it is a sulfonamide convert it to a chromophore with absorption maximum
and hasbenzenesulfonamide moiety(Fig. 1). 4-amino-N- in visible region. A tri-positive copper complex was
(4,5-dimethyl-1,3-oxazol-2-yl)benzene sulfonamide is used in alkaline medium to determine the sulfamoxole
its IUPAC name. C11H13N3O3S is the molecular formula by Upadhyay et al6.
with a water solubility of 1680mg/L (at 20°C)2.
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.
Fig. 1: Chemical structure of sulfamoxole
A scalable RP-HPLC method was developed by SIELC
Technologies using a mobile phase of MeCN/H2O/
Received on 17.01.2023 Modified on 30.05.2023 H3PO4 (20/80/0.1) and Newcrom R1 HPLC column for
Accepted on 22.11.2023 © RJPT All right reserved the preparative separation of sulfamoxole9. The methods
Research J. Pharm. and Tech 2023; 16(12):5617-5623. can be extended as MS compatible by replacing the
DOI: 10.52711/0974-360X.2023.00908
phosphoric acid with formic acid.
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Liquid chromatographic techniques were adopted for RESULTS AND DISCUSSIONS:

simultaneous determination of sulfamoxole along with Method Development:
other sulfonamides in biological samples10-15. CH3OH/ To quantify thesulfamoxole, the wavelength of detector
5mM aq. CH3COONH4 and 0.1% HCOOH was used in was fixed at 24nm, taking into account of the observed
gradient mode in LC-MS/MS, to determine sulfamoxole absorption maxima (Fig.-2).
in chicken, fish muscle and eggs12.

As the previous chromatographic methodsadopted either

expensive LCMS12,16 or gradient mode HPLC17,18, 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- Fig.2: Absorption spectrum of sulfamoxole
3000 software. Weighing was done using DENVER In order to obtain the satisfactory system suitability
(SI234) model electronic balance. max value was conditions, optimized the isocratic elution parameters
determined with UV 2301 model spectrophotometer. viz., composition of mobile phase, column, flow rate,
Chemicals of analytical grade and HPLC grade solvents until the appearance of acceptable peak. Figure 3
were employed. represents the demonstrative chromatograms involved in
the development of method. A flow rate of 1.0mL min–1
Preparation of Solutions (stock, working standard along with Kromasil, C18 column was used in these
and sample solutions): trials and having a dimension of 250 X 4.6mm, 5μ. The
Sulfamoxole (10mg) was totally dissolved in methanol mobile phase was a mixture of Water: Methanol (50:50;
taken in a 10ml volumetric flask to get a stock solution 30:70 and 70:30 v/v for trials a, b and c), Methanol:
of concentration 1000µg/ml. By diluting one ml of the Acetonitrile (75:25v/v for trial-d) and Methanol:
above stock solution with the same solvent in a 10ml Acetonitrile: Water (65:30:5 and 55:30:15v/v/v for trials
volumetric flask, working standard solution was –e and optimized condition) (Figure-3). Listed out the
obtained.Sulfuno (500mg) is the commercial tablet optimized conditions in Table-1 after fine-tuning the
formulation of sulfamoxole drug. A fine powder of these conditions. Kromasil C18 HPLC column was chosen in
tablets which contains 10mg of sulfamoxoleis dissolved the present study as it contains the silica-based packing
in 10ml of the above-mentionedmobile phase in a 10ml with low levels of alkali metals (like Ca, Mg, Na, K etc),
volumetric flask. Filtered this solution using a filter its silica surface interacts with the sulfamoxole which is
paper made up of nylon membrane and then diluted with polar in nature and having free amine groups. To
the same mobile phase to a concentration of 1000µg/ml. estimate the sulfamoxole, mobile phase consists high
volume of cost-effective methanol with lower volume
Forced Degradation: ratio of acetonitrile is used in the present study
These studies were carried out by comparing the compared to the methods suggested by Minghui Meng et
degradants in the chromatogram with the initial values al17 and Rong-jie Fu et al19. Figure-4. displays the
after incubating the sulfamoxolestandard in diverse acquired standard chromatogram having peaks with nice
conditions of degradation like exposing the sample for resolution, good symmetryand sharpness. The elution
24hr to the lab or UV light (Normal and UV light time and run time for sulfamoxole in the present study
degradation), keeping sample in oven at 80°C up to 24hr under the optimized conditions are 4.283 and 10 minutes
(Thermal degradation), hydrolyzing 100mg respectively.The retention time in the present study is
sulfamoxolein 20mL of decinormal HCl/NaOH solution 4.283 minutes which is lower compared to Abdulqawi
for 24/48 hours (Acid/Base degradation) and oxidizing Numan and Neil D. Danielson (2004)16, Forti et al12 and
100mg sulfamoxoleto 20mL of 3% H2O2 for 24hr Minghui Meng et al17 but higher compared to
(Hydrogen Peroxideoxidation). Abdulqawi Numan et al20.

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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:

(ii)
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. (iii)

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.

(iv)

(i)

(v)

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(i)
(vi)
Fig.3. Chromatograms of Method Development Trials

(ii)

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 (iii)
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, (iv)
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. (v)
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method for determination of Sulfamoxole

Linearity:
Injected different concentrations (5 to 30g/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.
(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 % Degradation Stress
degraded w.r.t. control conditions
sample (A) sample*(B)
Control 99.95 ------ No Exposure
sample (No
Degradatio
n)
Acid 91.40 8.56 1mL of 0.5 N
degradation HCl for 12 hr at
room
temperature Figure 6. Calibration plot for Sulfamoxole
Base 95.88 4.07 1mL of 0.5 N
degradation NaOH for 12 hr Accuracy:
at room
temperature
The addition of known quantity of drug to the sample,
Oxidation 94.41 5.54 2mL of 10% followed by the recovery percentage represents
H2O2 for 1 hr at accuracy, the vital parameter in the analytical
room methodology. Repeatability evaluates the analytical
temperature method’s accuracy. Carried out the accuracy study with
Thermal 94.07 5.88 70°C for 48 hrs
degradation 50%, 100%, and 150% recovery levels with six replicate
Photolytic 92.92 7.02 Exposed to 1.2 preparations and then injecting in chromatograph. Table-
degradation Million lux 2 shows the results consisting of the calculated %
(light) hours of light recovery from the chromatographic peak area at each
Photolytic 92.29 7.67 200-Watt
degradation hours/square
recovery level. The recovery percentage was found to be
(UV) meter 98.59 to 101.30 and % RSD values range between 0.222
*B= (99.95– A)/ 99.95*100 and 0.978 (Table-4). As recovery results are within the
acceptable limits, recovery values pertaining to the
Validation of Method: analyte are within the limit. Hence, it indicates the
The present guidelines (Q2R1, ICH 2005)21 were accuracy of the method which was proposed to estimate
followed to validate the above proposed optimized sulfamoxole.

Table 4: Results of accuracy for Sulfamoxole

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

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Precision:
Table 6: Precision Studies System Suitability Parameters
The precision indicates the results variability in
System suitability M.P.* I.P.*
recurrent analyses of the sulfamoxole sample following parameter
duplicate experimental settings. To validate the current USP tailing factor 1.11 1.00
method, six replicates were used to conduct the studies USP plate count 4624 4719
for both precisions i.e., method and intermediate Retention time (min) 4.289 4.306
precisions (MP and IP). Different conditions (instrument Peak area 145070 145233
SD of area 371.6 548.5
/column/analyst) were used to perform the intermediate % RSD of area 0.256 0.378
precision. 0.248 and 0.366 were the calculated % RSD * from six standard injections
values for MP and IP respectively, which are within the
limits of permissibility prescribed by ICH (Table-5). It LOD and LOC:
indicates the precision as high degree, and the method is The lowest possible detectable and quantifiable
rugged. 0.248 and 0.366 were the %RSD values for concentrations of sulfamoxolewith the acceptable
method and intermediate precisions respectively. Higher criteria of 3 and 10 for S/N ratio using the current
than 99% values in assay suggests that the method has method were respectively observed as 0.164 µg/mland
good precision (Table-6). 0.496µg/ml (Table -8). Applicability of the current
method on a widespread range of concentrations can be
Table 5. Results of Precision (Method and Intermediate) Study understood from lower values of LOD and LOQ.
S. No. % Assay
M.P. I.P.
1 99.33 99.89 Pharmaceutical formulation analysis:
2 99.39 98.92 As the recovery values of sulfamoxole is good (Table 9),
3 99.83 99.92 determined the amount of sulfamoxole in the tablet
4 99.25 99.41 formulation following the above method. In addition
5 99.76 99.87
6 99.18 99.39
tospectrophotometers22-25, HPLC instruments are at
Mean 99.46 99.56 affordable price to the laboratories of developing
Std. Dev 0.247 0.364 countries26-31, hence, now-a-days HPLC method can be
%RSD (n=6) 0.248 0.366 used in quality control laboratories of these countries.
M.P.: Method Precision; I.P.: Intermediate Precision Therefore, the current method is applied for sulfamoxole
*at 20 μg mL–1
determination in tablet formulations as per the existing
Robustness: ICH guidelines.
The key parameters (Mobile phase composition and
wavelength) were modified thoughtfully and then CONCLUSIONS:
evaluated their impact on the method to understand the Based on this study, it can be concluded that the HPLC
robustness of the method using a standard solution of method development and its validation in the present
20.0 μg/mlof sulfamoxole.Retention time, theoretical study is simple and run time is short. And also, can be
plates, tailing factor and peak area were not affected successfully applied in quality control laboratories for
appreciably by the intentional deviation of experimental the quantitative determination as well as stability
parameter values (Table -7). The organic solvent indicating studies of sulfamoxole in API and tablet
composition was varied ±5%. The observed change in dosage formulation.
peak area was within limits i.e., less than 2.0% and it
indicates the robustness of the current method.
Table 7: Results of robustness/ruggedness experiment of Sulfamoxole
Altered Parameter Actual Altered RT (Min) Theor Tail Peak % Change in
condition condition plates factor area peak area
Control Condition NA --- 4.283 4685 1.21 145822 -----
Mobile phase ratio -Methanol: 55:30:15 50:35:15 4.300 4578 1.15 144961 0.590
Acetonitrile: Water (v/v/v)* 65:25:15 4.283 4762 0.91 143872 1.337
Wave length (nm) 241 239 4.317 4798 1.19 145972 0.103
243 4.300 4921 1.19 145872 0.034

Table 8: Sensitivity
Parameter LOD LOQ
Results 0.164 µg/ml 0.496 µg/ml

Table 9: Pharmaceutical Formulation Assay

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

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CONFLICT OF INTEREST: https://doi.org/10.1093/chromsci/42.10.509

17. Meng M, He Z, Xu Y, Wang L, Peng Y, Liu X, Simultaneous
Nil. extraction and determination of antibiotics in soils using a method
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