Development and Validation of an RP-HPLC Method for Pamabrom in Bulk and Pharmaceutical Dosage Form

P. B. Jadhav*, S. G. Bhokare, M. N. Madibone

Srinath College of Pharmacy, Aurangabad, Maharashtra, India

*Corresponding Author E-mail: sandhya.madala@gmail.com

Abstract:

An accurate, Precise, Simple and Economical High Performance Liquid Chromatographic method for the estimation of Pamabrom in bulk form has been developed. The method so developed is Reverse Phase High Performance Liquid Chromatographic method using Primesil C18 column (Length: 250nm, Diameter: 4.6nm, Particle size: 5µ) with a simple methanol and Water (0.05 % OPA) mixed in a proportion of 80:20 v/v as mobile phase. The retention time for Pamabrom was found to be 4.48 min. The linearity for the method was observed in a concentration range of 10-50μg/mL with the correlation coefficient of 0.999. The method so developed was validated in compliance with the regulatory guidelines by using well developed Analytical method validation tool which comprises with the analytical method validation parameters like Linearity, Accuracy, Method precision, Specificity, System suitability, Robustness and Ruggedness. The results obtained were well within the acceptance criteria.

KEY WORDS: Pamabrom, RP-HPLC, Method development, Validation.

INTRODUCTION:

Pamabrom chemically, 2-amino-2-methyl-propanol 8-bromo theophylline is a weak diuretic (fig. 1). It is soluble in water and methanol, it is white crystalline powder. It is effective in treating dysmenorrhoea and premenstrual syndrome ^[¹^]. It is only official in United State Pharmacopoeia ^[²^]. Literature review reveals that various analytical methods like UV Spectrophotometry ^[³^,7,8,9], HPLC ^[⁴^,10,11,12], HPTLC ^[⁵^,13,14,15], human plasma ^[⁶^] and other analytical methods have been developed for individually and combination with other drug^[16]. However, no accurate and highly precise RP-HPLC method was developed for estimation of Pamabrom in Bulk and pharmaceutical dosage form. Hence, it was proposed to develop simple, accurate, precise and specific Stability indicating RP-HPLC method for estimation of Pamabrom in pharmaceutical dosage form without using buffer.

Fig 1: Chemical Structure of Pamabrom

MATERIALS AND METHOD:

Instrumentation:

Chromatographic separation was achieved using a C-18 column (250mm x 4.6mm i.d., 5μm particle size) of Younglin (S.K) Gradient system that is equipped with UV Detector

Materials Required:

Pamabrom pure standard was purchased from Swapnaroop drug agency (India). Methanol and Water of HPLC grade were purchased from Merck (India) and Qualigens (India) respectively. Pamabrom tablets available under the brand name Diurex Max (50mg, Alva Amco) were purchased and used. Optimized Conditions The mobile phase with methanol and Water (0.05% OPA) in the ratio of 80:20 %v/v was employed in isocratic mode at a flow rate of 1.0 ml/min. The run time was 10 mins and 20μL of the sample was injected for every run into the column. The wavelength of the UV detector was set at 277nm.

Chromatographic conditions:

A mixture of Water and methanol in the ratio of 80:20V/V was found to be the most suitable mobile phase for ideal chromatographic separation of Pamabrom. The solvent mixture was filtered through 0.45 μ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 mL/min. Injection volume was 20µL and the column was maintained at ambient temperature. The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior to the injection of the drug solution. The detection of the drug was monitored at 277 nm. The run time was set at 10min.

Preparation of Standard Stock Solution:

Accurately about 10mg of Pamabrom was weighed and transferred to a 10mL volumetric flask. 5mL of methanol was added to the flask and sonicated to dissolve it. The volume was then made up to the mark with methanol to get a standard solution of Pamabrom at a concentration of 1000μg/mL

Preparation of sample Solutions:

Working solutions for HPLC injections were prepared on daily basis. Aliquots of the standard stock solution were taken and diluted with the mobile phase to get solutions in a concentration range of 05-25μg/mL.

Linearity:

Several aliquots of standard solution of Pamabrom was taken in different 10 mL volumetric flasks and diluted up to the mark with diluents such that the final concentrations of Pamabrom were in the range of 05 to 25µg/mL. Evaluation of the drug was performed with UV detector at 277 nm, peak area was recorded for all the peaks. The correlation coefficient value of Pamabrom was 0.999. The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range indicated. The data is tabulated in table 1.

System suitability:

System suitability parameters like retention time, theoretical plates and tailing factor were calculated and compared with standard values.

Accuracy:

The recovery studies for the method were carried out by standard addition method. It was evaluated at three concentration levels (80,100 and 120%) and the percentage recoveries were calculated. The data is tabulated in Table 2.

Precision:

The precision of the method was determined by intra and inter day precision studies. This was evaluated by injecting three independent sample preparations of Pamabrom from a single formulation at three different concentration levels on the same day (Intra day) and on three different days (Inter day).The %RSD was then calculated. The data is represented in table 3.

Limit of Detection and Limit of Quantification:

The Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined based on the standard deviation of the response and the slope of the calibration curve. The sensitivity of the method was established by the LOD and the LOQ values.

Robustness:

Robustness was established by introducing small changes in the HPLC optimized conditions which include mobile phase ratio (±1) ,flow rate ratio (±0.1) and wavelength(±1). This was studied using two replicates at a concentration level of 20μg/mL of Pamabrom.

Table 1: Data for Linearity

CONC.	Avg.Area
5	218.87
10	446.03
15	674.41
20	907.12
25	1124.13

Table 2: Recovery Studies for Pamabrom

% Spike Level	Amount Added(μg/mL)	Peak Area	Amount Found(μg/mL)	%Recovery	Mean % Recovery
80	8	812	18.03	8.03	99.79
80	8	807.22	17.93	7.93	99.79
100	10	880.56	19.54	9.54	98.94
100	10	888.08	19.71	9.87	98.94
120	12	971.25	21.54	11.54	96.44
120	12	974.16	21.60	11.60	96.44

Table 3: Data for Precision

Sr. No.	Conc.(µg/ml)	Inter day		Intra day
Sr. No.	Conc.(µg/ml)	*Mean Area±SD**	RSD	*Mean Area±SD**	RSD
1	10	445.76±1.24	0.28	544.03±6.59	1.21
2	30	679.29±0.89	0.13	1545.30±14.27	0.92
3	50	907.00±1.49	0.16	2556.72±5.10	0.20

*Mean area of two injections.

Fig 2: Chromatogram of Pamabrom

Fig 3: Calibration Curve of Pamabrom

RESULTS AND DISCUSSION:

The proposed method was found to be simple. Linearity was observed in the concentration range of 05-25μg/mL with the regression equation y= 45.43x - 7.371 and the correlation coefficient of 0.999.System suitability parameters indicates high column efficiency with large number of theoretical plates (>2000).The tailing factor was found to be 1.4 which is does not exceed the critical value (2).The average retention time was found to be 4.483min. No interference was seen from any of the components of the pharmaceutical dosage form indicating the specificity of the method. The recovery studies were performed and the % RSD was found to be in the range 0.58 -0.17. The % RSD was found to be 1.21-0.20 for intraday and 0.28-0.16 for inter day precision studies. Thus the method was found to be accurate and precise as the %RSD was not more than 2%.The limit of detection and limit of quantification for Pamabrom were found to be 0.084μg/mL and 0.255μg/mL respectively. The RSD for the % assay of sample was calculated for each parameter in robustness and was found to be less than 2% confirming the robustness of the method.

CONCLUSION:

A validated RP-HPLC method was developed for the determination of Pamabrom in dosage form and bulk forms. As the proposed method is simple, rapid, accurate, precise and specific it can be employed for the routine analysis of Pamabrom in pharmaceutical dosage forms.

REFERENCES:

1. Drug Bank – Open data drug and drug target database; Available from: http://www.drugbank.ca/drugs/DB00218 [Last accessed on 2012 Jul 08].

2. United State Pharmacopoeia/National Formulary. 24th ed. Rockville, MD: Pharmacoepial Convention; 2000. p. 2847.

3. Padaliya H, Bhati M, Sanjaysinh V, Patel H, Pancholi SS. Development and validation of UV spectrophotometric methods for estimation of paracetamol and pamabrom in bulk and synthetic mixture by simultaneous equation method. Inventi Rapid: Pharm Anal QualAssu 2013; 1:1-4

4. Zhihong C, Wei WJ, Jianjun L, Ling-Bo Q. RP-HPLC Determination Pamabrom raw materials and Pamabrom tablets for Pamabrom content. J Zhengzhou Uni (Med Sci) 2008; 1:165-6.

5. Prajapati PP, Captain AD, Patel DS. Development and validation of HPTLC method for simultaneous determination of Paracetamol and Pamabrom in synthetic mixture. Int Res J Pharm 2012;11: 167-71.

6. Zhou L, Gu L, Wang Y, Linang J. HPLC for the determination of two constituents in compound Acetaminophen and Pamabrom tablets in human plasma. Chin J N Drug ClinRemed 2007; 3:61-5.

7. A. Weston, P.R. Brown, High Performance Liquid Chromatography, Separations in High Performance Liquid Chromatography, Instrumentation for HPLC. In: HPLC and CE – Principles and practice. Academic Press, USA, 1997, pp 1-11, 24-32.

8. Analytical chemistry. http://lingualeo.com/es_LA/jungle/analytical-chemistry-163830#/page/1 (accessed on Jan 07, 2016).

9. B. K. Sharma, Instrumental Method of Chemical Analysis, 12^thed,; Merrut Goel Publishing House, 1992 , pp 56-126.

10. Pamabrom. https://pubchem.ncbi.nlm.nih.gov/compound/Pamabrom (accessed Oct 21, 2015).

11. D. A. Skoog, F. J. Holler, S. R. Crouch, Principle of Instrumental Analysis, 6^thed.; Thomson Publications, India, 2007, pp 1-6, 145-180.

12. G. R. Chatwal, S. K. Anand, Instrumental Methods of Chemical Analysis. 5^th ed.; Himalaya Publishing House, 2002, pp 107-110.

13. G.D. Christian, Analytical Chemistry, 7^th ed.; John Wiley and Sons, 2003, pp 5-42, 131-132.

14. H. H. Willard, L. L. Merritt, J. A. Dean, F. A. Settle, Instrumental Methods of Analysis, 7^th ed.; CBS Publisher and Distributors, New Delhi, 2002, pp 617.

15. ICH Guidelines on Validation of Analytical procedure: Text and Methodology Q 2 (R1), (2011). [RJC-761/2011].

16. Indian pharmacopoeia, (1996) Vol-2. Controller of Publications, Ministry of health and family welfare (131-132), Delhi, Government of India.

Received on 04.05.2019 Modified on 03.06.2019

Research J. Science and Tech. 2019; 11(3):179-182.

DOI: 10.5958/2349-2988.2019.00027.5