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Vol 17, Issue 3, 2024

Research Article

A SIMPLE RP-HPLC METHOD DEVELOPMENT AND VERIFICATION FOR THE QUANTITATIVE
ESTIMATION OF SODIUM BUTYRATE IN TABLETSBEMBADI MUKUND REDDY, BHEEMI REDDY ASHOK REDDY, AKUTHOTA ASHOK KUMAR*,RAVINDER KODIPYAKA, RAJESH VOOTURIFormulation AR&D, Aurigene Pharmaceutical Services Limited, Hyderabad, Telangana, India.
*Corresponding author: A Ashok Kumar; Email: ashokkumara@aurigeneservices.com
Received: 04 October 2023, Revised and Accepted: 15 November 2023

ABSTRACTObjective: To develop a simple, accurate, precise, and linear reverse-phase high-performance liquid chromatographic (RP-HPLC) method and verify
the quantitative estimation (assay) of sodium butyrate in tablets.
Methods: The optimized RP-HPLC method uses a reverse phase stationary phase which is YMC Triart C18 column, having specifications of
250×4.6 mm; 5 μm, a mobile phase composition of pH 8.0 sodium dihydrogen phosphate buffer and acetonitrile in the proportion of 92:8 v/v, flowrate of 0.5 ml/min, injection volume of 10 μL, and detection wavelength of 210 nm using a UV/PDA detector.
Results: The developed method gave sodium butyrate eluting at about 6 min. Sodium butyrate exhibited linearity in the range of 119.56–1195.6 μg/
ml. The precision was exemplified by a relative standard deviation of 0.30%. The percentage of individual recovery was found to be in the range of
97.0 and 103.0 during accuracy studies.

Conclusion: A simple, specific, accurate, precise, and linear RP-HPLC method was developed and verified for the quantitative estimation (assay) ofsodium butyrate in tablets and hence this method can be explored for the analysis of sodium butyrate in tablets in various pharmaceutical industries.
Keywords: Reverse-phase high-performance liquid chromatographic, Sodium butyrate, Analytical method development, Tablets.

© 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ajpcr.2024v17i3.49574. Journal homepage: https://innovareacademics.in/journals/index.php/ajpcr
INTRODUCTION
Sodium butyrate is the sodium salt of butyric acid (Fig. 1), which exhibits
various properties such as immune system modulator, antioxidant, andanti-inflammatory properties [1]. Furthermore, sodium butyrate is used
as a promoter of growth in milk replacer formula for young calves [2].
Only two articles exist regarding the analysis of sodium butyrate in
the literature in various matrices [3,4]. Therefore, we have focused on
developing a reverse-phase high-performance liquid chromatographic(RP-HPLC) method, using a simple isocratic elution technique, mobilephase, and diluent in a better combination and a simple sample
extraction procedure for achieving a reproducible assay result. We
present here only the analytical work developed and verified on
quantitative estimation of sodium butyrate (assay) in tablets, whoselabel claim is 600 mg. The developed analytical method is a simple anda sensitive RP-HPLC method, which can be explored for routine analysis
in various pharmaceutical industries.
METHODS

Chemicals and reagents
Analytically pure sample of sodium butyrate with purities greater than
95% was obtained from Sigma-Aldrich and tablet formulation wasprepared in our Formulation R&D laboratory, with a labeled amountof 600 mg of sodium butyrate. Acetonitrile (HPLC grade of Standardmake), sodium dihydrogen phosphate monohydrate (Merck makeEmsure), sodium hydroxide pellets (Extra pure grade), orthophosphoricacid (Finar HPLC grade), and water (MilliQ) were used for the analysis.

Instrument
HPLC analysis was performed on Agilent and waters makes HPLC’s
having UV/PDA detectors capable of setting a detection wavelengthof 210 nm. Stationary phase used in the developed RP-HPLC
method is a reverse-phase C18 column-YMC Triart C18 column ofspecifications-250×4.6 mm; 5 μm, and part number TA12S05-2546WT.
The HPLC system was controlled with “EMPOWER” software. An
electronic analytical weighing balance (0.1 mg sensitivity, Sartorius
make, ME5 model) and a sonicator (Hwashin Make, Powersonic 420model) were used for the analysis.
Selection of wavelength
The suitable wavelength for the HPLC analysis for sodium butyrate
was determined by recording the UV spectrum in the range of 200–
400 nm. The suitable wavelength selected was 210 nm considering themaximum absorbance (Fig. 2).

Chromatographic conditions
The developed RP-HPLC method uses a stationary phase which
is a reverse-phase C18 column- YMC Triart C18 column, whose
specifications are 250×4.6 mm; 5 μm, part number TA12S05-2546WT,  
a mobile phase composition of pH 8.0 sodium dihydrogen phosphatebuffer and acetonitrile in the proportion of 92:8 v/v, flow rate of
0.5 ml/min, injection volume of 10 μL, detection wavelength of 210 nm
using a UV/PDA detector, setting column temperature and sample
compartment temperature of 20°C and ambient, respectively, and runtime as 12 min.
Reagents solution preparationPreparation of dilute orthophosphoric acid: Dilute 10 mL of 85%
orthophosphoric acid to 100 mL with Milli Q water.
Preparation of sodium hydroxide solution (2N): Transfer 8.0 g ofsodium hydroxide pellets into a 100 mL volumetric flask, dissolve, anddilute to volume with Milli Q water.

Reddy et al.

Preparation of pH 8.0 Sodium dihydrogen phosphate buffer (0.025M):
Weigh about 3.45 g of sodium dihydrogen phosphate monohydrate
into 1 L of Milli Q water and dissolve by sonication. Adjust the pH to8.0 using 2N sodium hydroxide or dilute orthophosphoric acid. Filterthrough 0.45 μm nylon membrane filter. Degas by sonication for
10 min.
Mobile phase preparation
The mobile phase was prepared by mixing pH 8.0 sodium dihydrogen
phosphate buffer and acetonitrile in the proportion of 92:8 v/v,
respectively, followed by degassing in a sonicator for 10 min.
Diluent preparation
The diluent solution was prepared by mixing pH 8.0 sodium dihydrogen
phosphate buffer and acetonitrile in the proportion of 92:8 v/v,
respectively, followed by degassing in a sonicator for 10 min.
Preparation of stock and working standard solution
Weigh accurately about 60 mg of sodium butyrate standard into a clean
and dried 100 ml volumetric flask. Later add 70 mL of the diluent andsonicate to dissolve. Dilute to volume with the diluent and mix well to
get a working standard concentration of about 600 µg/mL.
Preparation of stock and working sample solution
Drop the 5 doses of tablets into a clean and dry 1000 mL volumetricflask. Add 750 mL of the diluent and then stir on a magnetic stirrer for
60 min at 1000 rpm. Later remove the magnetic bead using a magnetic
retriever and ensure to rinse the magnetic bead with about 10 mL ofdiluent into the volumetric flask, to avoid losses, and later sonicate
for 30 min (maintain temperature of water in sonicator at 25°C) withFig. 1: Structure of sodium butyrate

Asian J Pharm Clin Res, Vol 17, Issue 3, 2024, 45-48

intermittent shaking. Dilute to volume with diluent and mix well.
Centrifuge a portion of the sample solution at 3000 rpm for 10 min.
Pipette 5.0 mL of supernatant and transfer into a clean and dry 25 mL
volumetric flask, dilute to volume with diluent, and mix well.
RESULTS AND DISCUSSION

Method development
A reverse-phase HPLC method chromatographic conditions weredeveloped keeping in mind the system suitability parameters, i.e.
tailing factor (T), % RSD from six replicate injections of standard andblank interference along with simple sample extraction procedure insample preparation and run time. The optimized method developed
resulted in the elution of sodium butyrate at about 6.0 min. Figs. 3-6
represent specimen chromatograms of blank, placebo, standard, andsample solutions. The total run time was 12 min. System suitability
tests were an integral part of method development and were used toensure adequate performance of the chromatographic system, whosedetails are summarized in Tables 1a and b.

To evaluate the applicability of the developed method to a formulation,
sodium butyrate tablets were chromatographed at a concentrationof about 600 µg/ml, and it is shown in Fig. 6. The sample peak wasidentified by comparing the retention time with the standard drug
Fig. 5. To ensure the RP-HPLC method developed is specific, precise
and accurate, method was verified whose details are mentioned in thebelow section.
Calculations
The assay of sodium butyrate was calculated using the below formula.

WA1000 25 P
100
Tablets Assay, Percent Label Claim = T × S ××××AS 100
L55 100

Where,
AT is the peak area of sodium butyrate in the sample solution.
AS is the average peak area of sodium butyrate from six replicate
injections of standard solution A injected under system suitability.

Fig. 2: UV spectrum of sodium butyrate

Fig. 3: Typical chromatogram of blank solution
46

Reddy et al.

Asian J Pharm Clin Res, Vol 17, Issue 3, 2024, 45-48

Fig. 4: Typical chromatogram of the Placebo solution

Fig. 5: Typical chromatogram of the standard solution

Fig. 6: Typical chromatogram of the sample solution
WS is the weight of sodium butyrate standard taken, in mg for thepreparation of standard solution
P is the potency of sodium butyrate standard
L is the label claim of sodium butyrate in mg, 600 mg
Method verification
Assay method verification of the analytical method for sodium butyrate
in tablets was done for the parameters such as system suitability,specificity, linearity, accuracy, and precision.

Specificity (placebo interference)
To establish non-interference, blank and placebo solutions wereprepared and injected into HPLC along with standard and sample
solutions (Refer Figs. 3-6). Figures 3-6 reveal that the peak obtainedin the standard solution and sample solution was only because of thedrug as blank and placebo had no peak at the retention time of sodium
butyrate standard. Accordingly, it can be concluded that the method
developed is said to be specific.
Method precision
Method precision was determined by performing an assay of thesample by spiking API to the placebo as per the sample preparation
at 100% level. The percentage assay of each replicate, average ofthree replicates, and % RSD were calculated. The % relative standard
deviation (RSD) of the assay of sodium butyrate was not more than 2.0
and, hence, can be concluded that the method is precise by the test of

Table 1a: System suitability acceptance criteria
Parameters

Acceptance criteria

Blank interference

No peak shall be observed in the blank at
the retention time of sodium butyrate.
The tailing factor for the sodium butyrate
peak shall not be more than 2.0 from thefirst injection of the Standard solution.
The percentage RSD of the average peakarea response from six injections of sodium
butyrate shall be NMT 3.0%.

Tailing factor (T)% RSD (relativestandard deviation)

Table 1b: System suitability results
Parameters

Sodium butyrate

Blank interference
Tailing factor (T)% RSD (relative standard deviation)

Nil
1.21
0.21

repeatability and can be understood that the method gives consistently
reproducible results (Table 2).

Accuracy
Accuracy was determined by means of recovery experiments, by thedetermination of % mean recovery of the sample at two different levels
47Reddy et al.

Asian J Pharm Clin Res, Vol 17, Issue 3, 2024, 45-48

Table 2: Method Precision results
Sample no. (n)% Assay

123Average
%RSD

101.3
101.5
100.9
101.2
0.3

Table 3: Results of accuracy studies% Level% Individual Recovery% Mean recovery

100

101.3
101.5
100.9
101.0
100.4
101.1

101.2

150

100.8

Table 4: Linearity data
% LevelConcentration (g/ml)

Peak area

20
50
80
100
120
150
200
Regression/
Correlation
coefficient
Bias
Regression
equation

119.564298.910478.256597.819747.274896.7291195.639

100182252555404258501888625652752814
9982480.999980.56
y=834.25x+2828.53(100 and 150%). At each level, three determinations were performed.
Individual and percentage mean recovery were calculated as shownin Table 3. All observed data were within the required range of 97.0–
103.0, which indicates good recovery values and hence the accuracy ofthe method developed.

Fig. 7: Linearity graphaccurate method. Accordingly, it can be concluded that the developed
reverse-phase isocratic HPLC method is specific, accurate, precise,and linear, and therefore, the method can be explored for the routine
analysis of sodium butyrate in tablets.
ACKNOWLEDGMENT

The authors would thank the management of Aurigene Pharmaceutical
Services Limited, Hyderabad, for providing the necessary facilities toconduct this analytical research work and encouraging for publication.
APSL Clearance No: “APSL_P42_14/09/2023. The author Akuthota
Ashok Kumar would like to thank all the coauthors for their valuable
contribution in getting this research work completed successfully.
AUTHORS CONTRIBUTIONS

Bembadi Mukund reddy, Bheemi reddy, and Ashok reddy have conducted
the method development and method verification activities. Ravinder
kodipyaka has provided valuable suggestions during analytical methoddevelopment. Dr. Rajesh vooturi has reviewed the manuscript andprovided valuable suggestions in drafting the manuscript.
CONFLICTS OF INTERESTSNil.

FUNDING

Linearity
Standard solutions of sodium butyrate at different concentrations level(20%, 50%, 80%, 100%, 120%, 150%, and 200%) were prepared. The
calibration curve was constructed by plotting the concentration levelof the drug versus the corresponding peak area. The results showed anexcellent correlation between the peak area and concentration level ofthe drug within the concentration range (119.564–1195.639 µg/ml) for
the drug, and the results are given in Table 4 and Fig. 7. The correlation
coefficient of sodium butyrate was 0.99998. The results revealed thatthe method is linear for the quantification of sodium butyrate in theproposed range.

The complete infrastructural facilities were provided by ourorganization, Aurigene Pharmaceutical Services Limited, Hyderabad.
Furthermore, the funding was provided by our organization and thecustomer for whom this project was done.

A reverse-phase HPLC isocratic method developed was verified in termsof specificity, accuracy, precision, and linearity for the quantitativeestimation (assay) of sodium butyrate in tablets. The precision wasexemplified by a relative standard deviation of 0.3%. A good linear
relationship was observed for the drug between concentration rangesof 119.564 and 1195.639 µg/ml. Accuracy studies revealed thatmean recoveries were between 97.0 and 103.0%, an indicative of an

3.

CONCLUSIONREFERENCES
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