RP-HPLC Method Development and Validation of Labetalol HCl for Bulk and Pharmaceutical Formulations

Introduction

Labetalol is used to control blood pressure and heart related disorders.¹ Labetalol is a combined alpha and  betaadrenoceptor blocking agent for oral and intravenous use in the treatment of hypertension, pheochromocytoma, pregnancy hypertension and hypertensive emergencies. In addition, preliminary studies indicate that labetalol may be of value in the management of ischemic heart disease. The most troublesome side effect of labetalol therapy is posture-related dizziness. While a higher dose of the medication may have adverse effects, a lower dose is associated with a moderate therapeutic effect. Quality control is crucial for pharmaceutical antihypertensive formulations.² Only a few analytical techniques have been described for estimating Labetalol, namely Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and stability indicating techniques.³ Therefore, there is a need to develop an easy, efficient and rapid analytical technique for the development and validation of labetalol in dosage forms.

^{Materials and Methods}

Labetalol HCl bulk drug has been procured from Yarrow Chem Ltd, Bangalore. Acetonitrile, methanol (HPLC graded) were obtained from Thermo Fisher Scientific, Mumbai. All other chemicals, such as disodium hydrogen phosphate, potassium dihydrogen phosphate, and solvents, were of AR (Analytical Reagent) and HPLC grade.

Instrumentation

The instrument used for experimental work was Shimadzu HPLC system (LC-20 AD) and PDA detector with Lab Solution software. UV detector Shimadzu 1800+, pH meter (lutronYK-21PH),electric weighing balance, sonicator (power sonic 405) were used for the experimental work.

Material Characterization

Melting point

The melting point of labetalol HCl was evaluated using electronic melting point apparatus and was found to be 189-190ºC. The reference melting point of Labetalol HCl was 188-190°C.⁴ The experimental melting point was found in conformity with standard melting point (188-190ºC).

Determination of absorption maxima

The absorption maximum of labetalol HCl was determined in mobile phase consisting of Acetonitrile: Phosphate buffer of pH 6.5 in a ratio 60:40. About 10 μg/mL solution was selected for this experiment and the same was prepared as per the procedure used earlier. The max absorbance at 306 nm has been utilized for UV detector in HPLC for detection of labetalol HCl.

Optimized Chromatographic Condition

Labetalol HCl (100 μg/mL) having an injection volume of 20 μL, with constant rate of flow at 1.0 mL/min, and detecting wavelength of 306 nm was employed.

Preparation of Standard Stock Solution

Labetalol HCl (10 mg) was precisely weighed in a 10 mL cleaned, dried volumetric flask (VF), to which 7 mL diluent was added. The mixture was sonicated for complete dissolution, and then the volume was made up to the mark with diluent to obtain a concentration of 1000 µg/mL. From the aforesaid standard stock, 1 mL was transferred to a 10 mL VF and diluted with diluent to obtain appropriate concentration (100 µg/mL).

Preparation of Sample Solution

Ten tablets of Labetalol HCl were weighed (1.89g) and triturated to a fine powder. A powder equivalent to 0.98 g was transferred to a 100 mL cleaned, dried VF. After adding 70 mL diluent and sonicating for about 30 minutes, the solution was diluted to the mark. About 1 mL of the supernatant solution was transferred to a 10 mL VF and diluted with diluent to the mark and the solution was filtered through 0.45 µm filter under vacuum filtration. The concentration obtained was 100 µg/mL of Labetalol HCl.

Results

Method validation

System suitability

Six-time injection of Labetalol HCl solution (100 µg/ mL) into the HPLC instrument revealed that all the six chromatograms showed reproducible results. The tailing factor was found to be 1.34 (acceptance limit < 1.5) and number of theoretical plates were found to be 2825 (acceptance limit > 2000). The percentage RSD (relative standard deviation) for retention time, area of peak, theoretical plates count and symmetry factor was identified to be 0.245, 0.9941, 0.037 and 0.383, respectively. All the parameters were inside the accepted range (%RSD < 2.0). The system suitability test findings have been presented in Table 1. The method’s suitability was thus established.

Linearity and Range

Linearity experiments helped to know the method can produce accurate results across a range of concentrations. The selected linearity levels of the labetalol HCl were 10, 20, 30, 40 and 50 µg/mL at wavelengths of 306 nm. The linear calibration plot for concentration of Labetalol HCl against peak area got straight line equa tion (Y = 12441x + 623.3). Linear regression (R2) of Labetalol HCl was found to be 0.9998 (Acceptance cri teria: not less than 0.999). 

Acceptance criteria: Correlation coefficient should be not less than 0.999.

Specificity

In specificity test, standard and sample chromatograms had almost virtually indistinguishable retention period.⁵ Blank chromatogram showed no interference peaks during the analysis time (10 min) near the retention time of Labetalol HCl. The approach was confirmed to be specific since none of the chromatograms under investigations showed any signs of interference.

Acceptance criteria: The chromatograms of standard and sample should be identical with near retention time. 

Accuracy

The mean percentage recovery of Labetalol HCl at three accuracy levels (80%, 100%, 120%) was found to be 101.23%, 97.64% and 100.76%, respectively. All the accuracy data were tabulated in Table 2. The accuracy experiment revealed that levels of accuracy were within the acceptance criteria (98% - 102%).

Precision⁶

Precision is measuring data closeness down the identical set of experimental conditions.⁶ Repeatability studies were compiled in Table 3.

The developed method’s precision was confirmed through repeatability as well as intermediate precision (inter and intraday) studies. Six injections of Labetalol HCl showed good repeatability as suggested by lower % RSD. The % relative standard deviation for area of the peak, retention time and symmetry factor were observed to be 0.70060, 0.518 and 0.408, respectively.

The intermediate precision of Labetalol HCl was found to be good both inter and intraday. The % RSD of peak area and retention on day 1 was found to be 0.745 and 0.0123, respectively. Similarly, % RSD of day 2 was found to be 0.668 and 0.0252, respectively. Precision data had been compiled in Table 3. (Acceptance criteria: % relative standard deviation for the results should not be more than 2.0%).

Robustness

The robustness of Labetalol HCl was evaluated by changing the rate of flow level to 0.9/1.1 mL from the actual flow rate of 1 mL.7 At 0.9 mL, the percentage relative standard deviation for area of peak, retention time and symmetry factor was found to be 0.394, 0.269 and 0.720, respectively. Similarly, the %RSD values at 1.1 mL were revealed to be 0.481, 0.174 and 1.85, respectively. The robustness findings for the impact of f low rate changes are presented in Table 4.

The robustness of Labetalol HCl was evaluated by varying the mobile phase ratio by ±10 % from the optimized ratio of 60:40. At 65:35, the percentage relative standard deviation for area of peak, retention time, and symmetry factor was revealed to be 0.346, 0.232 and 0.717, respectively. Similarly, %RSD at 55:45 was found to be 1.252, 0.148 and 0.722, respectively. Robustness findings for mobile phase ratio change studies are compiled in Table 4. (Acceptance criteria: % relative standard deviation for the results should not be more than 2.0%).

Limit of detection (LOD)

The detection limit calculated by calibration curve method was found to be 0.132 µg/mL.

Limit of quantification (LOQ)

The quantification limit calculated by calibration curve method was found to be 0.44 µg/mL.

Assay

The percentage assay of Labetalol HCl present in the marketed formulation (Labebet-100 mg) was found to be 102.34%. Table 5 demonstrates the comparison of the developed method with the published methods.

Discussion

For estimation of the Labetalol HCl drug, a simple, linear, reproducible, and precise technique has been developed, and hence it can be applied for rapid analysis.

For Labetalol HCl, the maximum absorption was noticed at 306 nm. Hence, 306 nm was selected as detecting wavelength for the proposed research. Labetalol HCl had shown well resolved peak with acetonitrile and phosphate buffer of pH 6.5 as mobile phase at the retention duration of 3.328 min, at the flow rate of 1.0 mL/min. Since the retention time is short, more samples can be analyzed, making the method suitable for processing larger samples. All the parameters for system suitability studies were within the acceptable range. The LOD and LOQ of the drug were significantly low. The results of analysis showed that % RSD values were low, thus confirming the ability of the analytical assay.

Conclusion

Labetalol HCl was estimated using a RP-HPLC technique that has been successfully developed and validated.

The main advantage of the current research is that it is efficient and has a shorter retention period compared to the reported ones, in addition to the suitable conditions used for the method development. The peaks eluted for Labetalol HCl were well resolved with retention time of 3.32 min. The established technique has been validated in accordance with ICH Q2 R1 guidelines. Thus, the established technique was considered as efficient, specific, accurate, linear, precise, robust and applied for routine analysis of Labetalol HCl in pharmaceutical formulations.

When compared to the published methods, the proposed method was precise and rapid with a lower retention time of 3.32 min. All the parameters for the optimized method were validated according to ICH Q2 R1 guidelines and were within the acceptance limits. LOD and LOQ values were comparatively low, and within a reasonable range.

Conflict of Interest

The authors have no conflict of interest regarding this investigation.

Acknowledgement

All the authors are thankful to Dr. Nirankar Nath Mishra, Professor, Centre of Applied Research and Nanotechnology (CARN), Siddaganga Institute of Technology, Tumkur for providing experimental support.