INTRODUCTION:

Propranolol binds reversibly with ß₁- and ß₂-adrenoreceptors; both receptors have membrane stabilizing activity. Propranolol leads to the reduction of heart rate and cardiac output, initially the hypotensive effect is delayed because of peripheral vasoconstriction. The AV nodal conduction time and the AV refractoriness are prolonged. Blood flow decreases in most vascular territories. Propranolol also increases plasma renin activity and suppresses lipolysis. In the plasma the triglycerides increase, whereas the HDL cholesterol decreases slightly. The bond between Propranolol and ß₂-receptors causes an increase of the airway resistance and the inhibition of glycogenolysis and gluconeogenesis.¹

Fig no.1: Structure of Propronolol

Hydralazine is a vasodilator used to treat severe hypertension, congestive heart failure, myocardial infarction, and preeclampsia. It reduces peripheral resistance directly by relaxing the smooth muscle cell layer in arterial vessels. The precise mechanism of action remains unknown but potentially involves an altered Ca²⁺ balance in vascular smooth muscle cells, whereby inhibition of Ca²⁺ release from the sarcoplasmic reticulum prevents contraction mediated by Ca²⁺-dependent ATPases, kinases, or ion channels.^2,3

Fig no.2: Structure of Hydralazine

Literature survey reveals that few spectrophotometric and chromatographic methods were reported for estimation of Propranolol and Hydralazine in single and combination with other drugs. Therefore an attempt has been made to develop and validate simple, precise and accurate RP-HPLC method for simultaneous estimation of Propranolol and Hydralazine in combined dosage form^4-19.

MATERIALS AND METHODS:

Instrumentation and Materials

The liquid chromatographic system consists of Shimadzu LC Solutions- 20 AD UFLC with UV-VIS detector, binary pump and septum injector valve with 20 μl fixed loop. The analytes were monitored at 293 nm. Chromatographic analysis was performed on Phenomenex Luna C18 ODS column having 250 mm× 4.6 mm i.d. and particle size 5 μm.

Materials used:

Working standards of Propranolol and Hydralazine were procured as a gift sample by D.K. Pharma Chem. Pvt. Ltd. (Maharashtra, India). The commercial formulation [Carbetazine], Nicholas Piramal India ltd Mumbai, Maharashtra] with label claim 40 mg/25mg per tablet was purchased from local pharmacy. Water HPLC grade was purchased from E. Merck (India) Ltd., Mumbai. HPLC grade methanol was purchased from E. Merck (India) Ltd., Mumbai, India. AR Grade Orthophosphoric acid was obtained from S.D. Fine Chemicals Ltd., Mumbai, India.

Chromatographic Conditions

The Phenomnex Luna C₁₈ column ODS (250 x 4.6mm, 5µm) equilibrated with mobile phase Methanol and Water in the ratio of 40:60 (0.01% O-phosphoric acid) was used and the flow rate was maintained at 1.0 mL/min. Detection wavelength with UV detector at 293 nm, and the injection volume was 20 μL and run time was kept 10 min.

Preparation of Mobile phase

A mixture of Methanol: Water (0.01% O-phosphoric acid) in the ratio of 40:60 v/v was prepared and used as the mobile phase.

Preparation of standard solution

About 100 mg of Propranolol and 60 mg of Hydralazine were weighed and transferred into a 100 mL volumetric flask containing 25 mL of water. The solution was stirred for 5 minutes and made up with a further quantity of the mobile phase to get 1mg/mL and 0.6 mg/mL Propranolol and Hydralazine respectively. This solution was further diluted to get required concentrations during study.

Preparation of sample solution

Propranolol and Hydralazine combined dosage form (Carbetazine 40/25mg) was purchased from the Local market. Drug equivalent to 10 mg of Propranolol and Hydralazine tablet powder transferred into 10 ml volumetric flask dissolved in 7 ml of mobile phase. The contents of the flask were sonicated for about 20 min for complete solubility of the drug and the volume was made up to 10 mL with mobile phase. Then the mixture was filtered through 0.45μ membrane filter. The mean peak areas of the drugs were calculated and the drug content in the formulation was calculated.

U.V. Detection wavelength

The spectra of diluted solutions of the Propranolol and Hydralazine were recorded separately on UV spectrophotometer. The spectra of the both Propranolol and Hydralazine were showed that a balanced wavelength was found to be 293 nm.

METHOD VALIDATION

The developed analytical method was validated as per ICH guidelines for the parameters like specificity, linearity, accuracy, precision, robustness and system suitability.

Specificity

The specificity of the proposed method was determined by comparing the results obtained by running the standard solution and placebo solution with standard.

Linearity and Range

The linearity was determined for Propranolol and Hydralazine six different concentrations were analyzed and calibration curve was constructed by plotting mean response factor against the respective concentration. The method was evaluated by determination of the correlation coefficient and intercept value. Linearity in the concentration ranges of Propranolol for 10- 60 µg/mL and Hydralazine for 6-36 µg/mL respectively.

Precision

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the homogenous sample under the prescribed conditions.

Accuracy

Recovery assessment was obtained by using standard addition technique which was by adding known quantities of pure standards at three different levels in 50%, 100% and 150% to the pre analysed sample formulation. From the amount of drug found, amount of drug recovered and percentage recovery were calculated which sense to conformation that the proposed method was accurate.

Robustness

The robustness was determined by injecting triplicate injections of standard and three-sample solutions in single at each different condition with respect to control condition. Robustness of the method was checked by varying the instrumental conditions; flow rate (±10%mL/min) and temperature (±5˚C). Sample solution was injected in each condition.

System Suitability Parameter

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. Following system suitability test parameters were established.

Detection of limits (LOD and LOQ):

LOD & LOQ is the lowest amount of analyte in a sample that can be detect but not necessarily quantities as an exact value under the stated, experimental conclusions. The detection limit is usually expressed as the concentration of analyte. The standard deviation and response of the slope- LOD=3.3 * standard deviation (σ)/ s. The quantitation limit of an analytical procedure is the lowest amount of an analyte of a sample, which can be quantitatively determined with suitable precision and accuracy. The standard deviation and response of the slope- LOQ=10* standard deviation (σ)/ s.

RESULTS AND DISCUSSION:

Specificity

The comparison of the data of the drug solution before spiking and the spiked drug solution revealed that there was no significant interference of placebo with the recovery of Propranolol and Hydralazine, inferring that the method was specific. The results were shown in table no.1.

System suitability

The specificity of this method was determined by complete separation of Propranolol and Hydralazine. The tailing factor for peaks of Propranolol and Hydralazine was less than 2% and resolution was satisfactory. The average retention time for Propranolol and Hydralazine were found to be 2.76 and 4.69 min respectively, for six replicates. The peaks obtained for Propranolol and Hydralazine were sharp and have clear baseline separation. The system suitability parameters are given in Table no.2.

Linearity and Range

The method was found to be linear. In the linearity study, regression equation and correlation coefficient of Propranolol and Hydralazine were found to be r² =0.999. Linearity data was shown in table no.3.

Precision

The percentage RSD of Propranolol and Hydralazine were found to be less than 2%. The precision results were shown in table no.4.

Accuracy

The mean recovery of Propranolol was 99.67% and Hydralazine was 100.03%. The limit for mean recovery is 98-102%. Thus the method was found to be accurate. The results were shown in table no.5.

Robustness

This method is robust for the analysis of Propranolol and Hydralazine were within the specified range of deviations in the experimental conditions. The results were shown in table no.6 and 7.

Assay

The percent purity of Propranolol was 101.41% and Hydralazine was 101.23%.

Detection of limits (LOD&LOQ):

For propranolol LOD value 0.099µg/ml and LOQ value 0.300µg/ml was obtained. Hydralazine LOD value is 0.093µg/ml and LOQ value 0.281µg/ml was obtained respectively.

Table no.1: Specificity data

S.No	Peak Name	Observation
S.No	Peak Name	Hydralazine	Propranolol
1	Blank	Nil	Nil
2	Placebo	Nil	Nil
3	Standard	Rt – 2.76 min	Rt – 4.69 min

Fig no.3: Blank Chromatogram

Fig no.4: Specificity data of Propranolol and Hydralazine

Table no.2: Results for system suitability

Sl.no.	Characteristic	Propranolol	Hydralazine
1	Capacity factor	4.89	2.46
2	Tailing factor	1.335	1.113
3	Theoretical plates	5907	5883
4	Resolution	7.607

Table no.3: Linearity results

Propranolol		Hydralazine
Concentration (µg/ml)	Peak area	Concentration (µg/ml)	Peak area
10	497012	6	94561
20	974982	12	191013
30	1494291	18	284610
40	2005879	24	378531
50	2498475	30	470567
60	2987407	36	563601

Table no.4: Precision results

	Propranolol		Hydralazine
S.no	Intra day	Inter day	Intra day	Interday
1	1469998	1491984	369998	380571
2	1470128	1490324	370501	379185
3	1472508	1489584	369897	378974
3	1469958	1490984	370109	381214
5	1468368	1488896	369897	379958
6	1471929	1493984	371003	381013
Avg	1470481.5	1490959	370234.2	380152.5
STDEV	1503.438	1830.393	438.431	938.630
%RSD	0.102	0.123	0.118	0.247

Fig no.5: Calibration curve of Propranolol

Fig no.6: Calibration curve of Hydralazine

Table no.5: Accuracy results

Propranolol				Hydralazine
Spiked conc	Amount added (µg/ml)	Amount found (µg/ml)	%mean recovery	Spiked conc	Amount added (µg/ml)	Amount found (µg/ml)	%mean recovery
20	20.01	19.81	99.38	12	12.00	11.96	99.99
		20.02				12.00
		19.82				12.04
40	40.02	40.01	99.94	24	24.01	24.03	100.09
		39.94				24.01
		40.04				24.06
60	60.03	59.83	99.70	36	36.02	35.99	100.09
		60.08				36.03
		59.83				36.05

Table no.6: Robustness results for Propranolol

S.no	Control	Flow rate (±10%)		Temperature (± 5^oC)
S.no	Control	0.9ml/min	1.1ml/min	30^oC	40^oC
1	1479987	1474821	1484107	1474821	1486482
2	1485012	1469581	1494219	1469581	1479084
3	1488741	1480112	1491234	1485112	1484986
Mean	1484580	1474838	1489853	1476505	1483517
SD	3586.83676	4299.28	4242.076	6451.307	3193.779
%RSD	0.24	0.29	0.28	0.43	0.21

Table no.7: Robustness results for Hydralazine

S.no	Control	Flow rate (±10%)		Temperature((± 5^oC
S.no	Control	0.9ml/min	1.1ml/min	30^oC	40^oC
1	379987	374821	404107	374821	386482
2	385012	379581	394219	379581	379084
3	388741	380112	401234	385112	384986
Mean	384580	378171.3	399853.3	379838	383517.3
SD	3586.837	2378.941	4153.137	4205.212	3193.779
%RSD	0.93	0.62	1.03	1.10	0.83

CONCLUSION:

The proposed method was simple, sensitive and isocratic separation was done and it can be used for simultaneous analysis Propranolol and Hydralazine in bulk samples and its marketed dosage form. This study results shows high efficiency and good baseline factor when compared previous article. It compliance with ICH guidelines parameters and shows good reproducible under quality control conditions.

ACKNOWLEDGEMENTS:

I would like to thank D.K. Pharma Chem. Pvt. Ltd. (Maharashtra, India) for providing working standards as a gift sample. I also extend my thanks to Director, JNTUA-OTPRI for providing necessary facilities for carrying out this research work.

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Received on 17.09.2018 Accepted on 01.11.2018

Asian J. Pharm. Ana. 2019; 9(1): 37-42.

DOI: 10.5958/2231-5675.2019.00009.7