Development and Validation of Analytical Method for Simultaneous Estimation of Ranitidine and Bismuth Subsalicylate by RP-HPLC

V. Sowmya, M D. Rahman Ghori , Dr. A. Ganesh, Dr. M. Venkataramana, Dr. N. Swathi,

doi:10.5281/zenodo.17233411

Research Paper | Open Access
Volume 03 | Issue 09 | Article Id IJPS/250309336

Development and Validation of Analytical Method for Simultaneous Estimation of Ranitidine and Bismuth Subsalicylate by RP-HPLC
V. Sowmya* M D. Rahman Ghori Dr. A. Ganesh Dr. M. Venkataramana Dr. N. Swathi
Surabhi Dayakar Rao College of Pharmacy, Kimmanaguda, Siddipet, India 502103

Abstract

A simple, precise, and accurate Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Ranitidine and Bismuth Subsalicylate in bulk drug substances and pharmaceutical formulations. Chromatographic separation was achieved using a Phenomenex Luna C18 column (4.6 × 150 mm, 5?mm) maintained at a column temperature of 45°C. The mobile phase consisted of Methanol and Water in a ratio of 80:20 v/v, delivered at a flow rate of 1.0 mL/min. Detection was performed at a wavelength of 230 nm, with an injection volume of 10 µL and a total run time of 10 minutes. Both analytes were well resolved with sharp and symmetrical peaks and showed satisfactory retention times. The method was validated according to ICH guidelines, and the results confirmed the method's linearity, precision, accuracy, specificity, and robustness.

Keywords

RP-HPLC, Ranitidine and Bismuth Subsalicylate, Phenomenex Luna C18 column, simultaneous estimation, validation, ICH guidelines, specificity.

Introduction

Chromatography is a procedure that is used for separating a complex mixture into its individual particular fractions or components. It is a separation technique and the separated compounds can be identified by using any analytical technique like UV-visible, Infrared, Mass spectroscopy, NMR etc. "Chromato” “graph” derives its name from two words as chromo means color and graph means to write i.e. color bands are formed in the procedure which are measured or analyzed. These color bands are formed due to the separation of individual compounds. Analytical chemistry deals with methods for identification, separation, and quantification of the chemical components of natural and artificial materials.

HPLC is the method of choice for checking peak purity of new chemical moieties, reaction monitoring and evaluating new formulations. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products. In the modern pharmaceutical industry, high-performance liquid chromatography (HPLC) is the major and integral analytical tool applied in all stages of drug discovery, development, and production. The Goal of HPLC method is to try & separate, quantify the main drug, any reaction im-purities, synthetic intermediates and any degradation products. HPLC principle is the solution of sample is injected into a column of porous material (stationary phase) and liquid phase (mobile phase) is pumped at higher pressure through the column. The principle of separation followed is the adsorption of solute on stationary phase based on its affinity towards stationary phase^(15-20):

Main features of HPLC:

High resolution
Small diameter, Stainless steel, Glass column
Rapid analysis
Relatively higher mobile phase pressure
Controlled flow rate of mobile phase

Classification of HPLC can be done as:

Preparative HPLC and analytical HPLC (based on scale of operation)
Affinity chromatography, adsorption chromatography, size exclusion chromatography, ion exchange chromatography, chiral phase chromatography (based on principle of separation)
Gradient separation and isocratic separation, (based on elution technique)
Normal phase chromatography and reverse phase chromatography (based on modes of operation).

Buffer Concentration^(1-2):

Generally, a buffer concentration of 10-50 mM is adequate for small molecules. Generally, no more than 50% organic should be used with a buffer.

Column selection^(4-5) :

The heart of a HPLC system is the column. Changing column will have the greatest effect on the resolution of analytes during method development. Generally, modern reverse phase HPLC columns are made by packing the column housing with spherical silica gel beads which are coated with the hydrophobic stationary phase.

Mobile phase:

The mobile phase effects resolution, selectivity and efficiency. In reverse phase chromatography, the mobile phase consists of an aqueous buffer and a non-UV active water miscible organic solvent. The effect of the organic and aqueous phase and the proportions in which they are mixed will affect the analysis of the drug molecule.

EXPERIMENTAL METHODS^(17,18):

INSTRUMENTS USED

Table 1: Instruments used

Sr. No.	Instruments And Glass wares	Model
1	HPLC	WATERS Alliance 2695 separation module, Software: Empower 2, 996 PDA detector.
2	pH meter	Lab India
3	Weighing machine	Sartorius
4	Volumetric flasks	Borosil
5	Pipettes and Burettes	Borosil
6	Beakers	Borosil
7	Digital ultra sonicator	Labman

CHEMICALS USED:

Table 2: chemicals used

Sr. No.	Chemical	Brand names
1	Ranitidine	Provided by Sura Pharma labs
2	Bismuth Subsalicylate	Provided by Sura Pharma labs
3	Water and Methanol for HPLC	LICHROSOLV (MERCK)
4	Acetonitrile for HPLC	Merck

HPLC METHOD DEVELOPMENT^(21-24):

Preparation of standard solution:

Accurately weigh and transfer 10 mg of Ranitidine and Bismuth Subsalicylate working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.

Further pipette 2.25ml of the above Ranitidine and 0.45ml of the Bismuth Subsalicylate stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

Mobile Phase Optimization:

Initially the mobile phase tried was Methanol: Water, Acetonitrile: Water with varying proportions. Finally, the mobile phase was optimized to Methanol and water in proportion 80:20 v/v respectively.

VALIDATION

PREPARATION OF MOBILE PHASE:

Preparation of mobile phase:

Accurately measured 800ml (80%) of Methanol and 200ml of Water (20%) were mixed and degassed in a digital ultrasonicate for 10 minutes and then filtered through 0.45 µ filter under vacuum filtration.

RESULTS AND DISCUSSION

Trails

Trail 1:

Column : Symmetry C18 (4.6×250mm) 5µ
Column temperature : Ambient
Wavelength : 230nm
Mobile phase ratio : Methanol: Water (95:5 v/v)
Flow rate : 1ml/min
Injection volume : 20µl
Run time : 10minutes

Figure 1: chromatogram for trail 1

Table 3: peak results for trail 1

Sr. No.	Peak Name	R_t	Area	Height	USP Tailing	USP Plate count
	Ranitidine	2.678	215471	38742	1.6	745

Observation:

In this trial it shows less plate count and improper separation of two peaks in the chromatogram. so, it required more trials to obtain good peaks.

Trail 2:

Column : ODS C18 (4.6×250mm) 5µ
Column temperature : 30?C
Wavelength : 230nm
Mobile phase ratio : Methanol: Water (85:15 v/v)
Flow rate : 0.5ml/min
Injection volume : 10µl
Run time : 8minutes

Figure 2: Chromatogram for trail 2

Table 4: Peak results for trail 2

Sr. No	Peak name	R_t	Area	Height	USP Tailing	USP plate count
1	Ranitidine	5.623	46523	29816	1.8	1937
2	Bismuth Subsalicylate	6.571	5366	1947	1.6	1183

Observation:

In this trail it shows improper separation of two peaks, shows less plate count and improper baseline in the chromatogram. It’s required more trails to get optimized peaks.

Trail 3:

Column : ODS C18 (4.6×250mm) 5µ
Column temperature : 30?C
Wavelength : 230nm
Mobile phase ratio : Methanol: Water (80:20 v/v)
Flow rate : 1ml/min
Injection volume : 10µl
Run time : 8minutes

Figure 3- chromatogram for trail 3

Table 5: - peak results for trail 3

Sr. No	Peak name	R_t	Area	Height	USP Tailing	USP plate count
1	Ranitidine	2.970	38261	17463	2.4	284
2	Bismuth Subsalicylate	3.669	6452	6482	1.9	645

Observation:

From the above chromatogram it was observed that it shows less plate count and more tailing, improper separation of two sample peaks in the chromatogram. More trails required to get optimized chromatogram.

Trial 4:

Column : Phenomenex Luna C18 (4.6 x 150mm, 5mm)
Column temperature : 45?C
Wavelength : 230nm
Mobile phase ratio : Methanol and water (80:20 v/v)
Flow rate : 1ml/min
Injection volume : 10µl
Run time : 10 minutes

Figure 4- chromatogram for trail 4

Table 6: - peak results for trail 4

Sr. No	Name	RT	Area	Height	USP Tailing	USP Plate Count	USP Resolution
1	Ranitidine	3.212	2391746	39726	1.2	9028
2	Bismuth Subsalicylate	5.385	194627	8497	1.1	7398	7.4

Observation:

This trial shows improper separation sample peaks, baseline and show very less plate count in the chromatogram. So, it’s required more trials to obtain good peaks.

Optimized chromatogram (Standard):

Table 7: Optimized Chromatogram (Standard)

Sr. No.	Name	RT	Area	Height	USP Tailing	USP Plate Count	USP Resolution
1	Ranitidine	3.212	2391746	39726	1.2	9028
2	Bismuth Subsalicylate	5.385	194627	8497	1.1	7398	7.4

Optimized Chromatogram (Sample)

Table 8: Optimized Chromatogram (Sample)

Sr. No	Name	RT	Area	Height	USP Tailing	USP Plate Count	USP Resolution
1	Ranitidine	3.214	2381649	391846	1.2	9472
2	Bismuth Subsalicylate	5.389	191057	8104	1.1	8936	7.5

VALIDATION

System suitability:

Table 9: Results of system suitability for Ranitidine

Sr. No	Peak Name	RT	Area (µV*sec)	Height (µV)	USP Plate Count	USP Tailing
1	Ranitidine	3.218	2391746	394171	8952	1.2
2	Ranitidine	3.221	2391647	381946	9561	1.2
3	Ranitidine	3.210	2381647	391746	6572	1.2
4	Ranitidine	3.214	2385631	386562	6452	1.2
5	Ranitidine	3.212	2385635	389164	7452	1.2
Mean			2387261
Std. Dev.			4363.771
% RSD			0.182794

Table 10: Results of system suitability for Bismuth Subsalicylate

Sr. No	Peak Name	RT	*Area (µVsec)**	Height (µV)	USP Plate Count	USP Tailing
1	Bismuth Subsalicylate	5.390	198362	7917	5272	1.1
2	Bismuth Subsalicylate	5.394	197486	7486	6291	1.1
3	Bismuth Subsalicylate	5.382	198354	7859	6184	1.1
4	Bismuth Subsalicylate	5.389	197352	7926	7145	1.1
5	Bismuth Subsalicylate	5.385	198453	7946	6946	1.1
Mean			198001.4
Std. Dev.			535.1774
% RSD			0.27029

Assay (Standard):

Ranitidine

Sr. No	Name	RT	Area	Height	USP Tailing	USP Plate Count
1	Ranitidine	3.124	2397162	397161	1.2	9472
2	Ranitidine	3.218	2394721	389173	1.2	9745

Bismuth Subsalicylate

Sr. No	Name	RT	Area	Height	USP Tailing	USP Plate Count
1	Bismuth Subsalicylate	5.389	198462	7811	1.1	8492
2	Bismuth Subsalicylate	5.390	198472	8193	1.1	8916

Assay (Sample):

Ranitidine

Sr.No	Name	RT	Area	Height	USP Tailing	USP Plate Count
1	Ranitidine	3.221	2391741	381612	1.2	9472
2	Ranitidine	3.214	2389166	391746	1.2	8927
3	Ranitidine	3.218	2361731	381634	1.2	9017

Bismuth Subsalicylate

Sr. No	Name	RT	Area	Height	USP Tailing	USP Plate Count	Resolution
1	Bismuth Subsalicylate	5.394	198641	8174	1.1	9284	7.18
2	Bismuth Subsalicylate	5.389	196547	8942	1.1	8974	7.44
3	Bismuth Subsalicylate	5.390	194027	7294	1.1	9017	7.38

LINEARITY

Ranitidine

Concentration mg/ml	Average Peak Area
00	00
5	909889
10	1583641
15	2395378
20	3185089
25	3943725

Bismuth Subsalicylate

Concentration mg/ml	Average Peak Area
00	00
15	61953
30	130213
45	198697
60	267002
75	321658

REPEATABILITY

Table 11: Results of repeatability for Ranitidine:

Sr. No	Peak name	Retention time	*Area (µVsec)**	Height (µV)	USP Plate Count	USP Tailing
1	Ranitidine	3.212	2397164	381741	8155	1.2
2	Ranitidine	3.214	2391741	371742	9174	1.2
3	Ranitidine	3.210	2371846	391746	7154	1.2
4	Ranitidine	3.221	2361748	391847	9917	1.2
5	Ranitidine	3.218	2371649	384622	9247	1.2
Mean			2378830
Std.dev			14958
%RSD			0.628797

Table 12: Results of repeatability for Bismuth Subsalicylate:

Sr. No	Peak name	Retention time	*Area (µVsec)**	Height (µV)	USP Plate Count	USP Tailing
1	Bismuth Subsalicylate	5.385	198464	7291	6274	1.1
2	Bismuth Subsalicylate	5.389	193643	7219	6592	1.1
3	Bismuth Subsalicylate	5.382	196462	7194	6028	1.1
4	Bismuth Subsalicylate	5.394	194644	8174	6927	1.1
5	Bismuth Subsalicylate	5.390	198464	8653	5920	1.1
Mean			196335.4
Std.dev			2190.191
%RSD			1.115536

ACCURACY:

The accuracy results for Ranitidine

%Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	1217218	112.5	112.4	99.6	99.3
100%	2397141	225	225	100
150%	3514547	337.5	332.5	98.5

The accuracy results for Bismuth Subsalicylate

%Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	98598.67	22.5	22.4	99.9	99.6
100%	198359.7	45	45	100
150%	291512.3	67.5	66.8	99

ROBUSTNESS

Ranitidine

Table 13: Results for Robustness

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Actual Flow rate of 1.0mL/min	2391746	3.202	9028	1.2
Less Flow rate of 0.9mL/min	2371831	3.218	7381	1.2
More Flow rate of 1.1mL/min	2218319	3.212	9311	1.1
Less organic phase (about 5 % decrease in organic phase)	2294821	3.210	7462	1.2
More organic phase (about 5 % Increase in organic phase)	2394811	3.212	6817	1.1

Bismuth Subsalicylate

Table 14: Results for Robustness

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Actual Flow rate of 1.1mL/min	194627	5.410	7398	1.1
Less Flow rate of 0.9mL/min	183738	5.390	6883	1.1
More Flow rate of 0.8mL/min	198373	5.385	9917	1.2

CONCLUSION

A precise and effective Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Ranitidine and Bismuth Subsalicylate in bulk and pharmaceutical dosage forms. The chromatographic separation was achieved using a Phenomenex Luna C18 column (4.6 × 150 mm, 5?μm), maintained at a column temperature of 45°C.

The optimized mobile phase consisted of Methanol and Water in a ratio of 80:20 v/v, delivered at a flow rate of 1.0 mL/min. Detection was carried out at a wavelength of 230 nm, with an injection volume of 10 µL and a total run time of 10 minutes.

Both Ranitidine and Bismuth Subsalicylate were effectively resolved under these conditions, with sharp, well-defined peaks and acceptable retention times. The method was validated as per ICH guidelines, and parameters such as linearity, precision, accuracy, robustness, specificity, and system suitability were evaluated. The results were found to be within the standard acceptable range, confirming the method's suitability for routine analysis.

The developed RP-HPLC method is simple, rapid, accurate, and robust for the simultaneous estimation of Ranitidine and Bismuth Subsalicylate in both bulk and pharmaceutical formulations. The method offers good resolution and reproducibility within a 10-minute run time, making it ideal for routine quality control analysis. Successful validation in accordance with ICH guidelines confirms the reliability and applicability of the method in pharmaceutical laboratories.

ACKNOWLEDGEMENT

The author is thankful to M. Venkataramana Principal of Surabhi Dayakar Rao College of pharmacy for providing necessary facilities to carry out this entire research work.

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