Development and Validation of RP-HPLC Method for Simultaneous Estimation of Hydrochlorothiazide and Bisoprolol Fumarate in Pharmaceutical Dosage Form

Bisoprolol fumarate Chemically referred to as 1-(propan-2-ylamino)-3-[4-(2-propan-2-yloxyethoxymethyl)phenoxy] propan-2-ol, bisoprolol is a member of the beta blocker class of medications, which is mostly used to treat cardiovascular disorders. With very little effect on ?2-adrenergic receptors, bisoprolol specifically inhibits ?1-adrenergic receptors in the heart. Its selectivity for ?1-adrenergic receptors may be due to the two substituents in the para position of the benzene ring . Angina pectoris, congestive heart failure, myocardial infarction, and mild to moderate hypertension are all treated with bisoprolol.

Bisoprolol Structure
       
           
       
 Hydrochlorothiazide is a member of the thiazide diuretic medication class. Thiazide diuretics assist prevent the body from retaining fluid by reducing the amount of electrolytes that are reabsorption from the renal tubules

Chemically speaking, 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-ulfonamide is the name given to hydrochlorothiazide (Figure 2). Treatments for congestive heart failure, diabetic insipidus, renal tubular acidosis, hypertension, hypoparathyroidism, osteoporosis, and kidney stone prophylaxis sometimes involve the use of hydrochlorothiazide.

HCTZ structure

In addition to treating hypertension, the low-dose combination of bisoprolol and hydrochlorothiazide helps prevent strokes, heart attacks, and kidney issues.^9-10. When one medication is insufficient to manage blood pressure, this combination is employed. When these two medications are used together, the dosage of each medication can be decreased, lowering the likelihood of adverse effects. The dosage formulations containing bisoprolol and hydrochlorothiazide are becoming a common drug in cardiovascular therapy because of the aforementioned benefits. RP-HPLC technique  using a photodiode array detector for simultaneous quantitative measurement of hydrochlorothiazide and bisoprolol in tablet and bulk dose forms. The process that was established was validated in compliance with ICH recommendations

EXPERIMENTALS

Instrumentation

The Agilent 1260 Infinity was the HPLC apparatus used. Both the chromatographic separation and quantification were performed using an Agilent 5TC C18 analytical column (250 mm × 4.6 mm), which was maintained at room temperature. The tablet sample, drug standard solutions, and mobile phase were filtered using a Whatman filter paper before being fed into the HPLC apparatus.
Drugs, Chemicals and Reagents

Merck Specialities private limited, located in Aurangabad, India, provided gift samples of the reference standards for hydrochlorothiazide and bisoprolol fumarate. Research Lab Limited (Mumbai, India) provided TEA of HPLC quality. and acetonitrile,  Methanol which is provided by Research Lab Limited (Mumbai, India). Water of HPLC grade was used for the entire analysis. Bisobis H tablets were advertised as containing 6.25 mg of hydrochlorothiazide and 2.5 mg of bisoprolol fumarate

Chromatographic conditions

Mobile phase : 0.2% TEA: Acetonitrile: Methanol (55:20:25 v/v\v)

Flow rate : 1mL/min

Detection wavelength : 225 nm

Column temperature : Room temperature

Injection volume : 20 ?L

Run time : 12 min

Detector: PDA detector

STANDARD DRUG SOLUTION

Precisely weigh out 10 milligrams of Bisoprolol Fumarate and 25 mg of Hydrochlorothiazide, and then transfer the mixture into a 100 ml volumetric flask. Transfer 40 milliliters of iso-propyl alcohol and 60 milliliters of water (serving as a diluent) into the volumetric flask containing the Bisoprolol Fumarate and Hydrochlorothiazide. To filter the mixture, use filter paper No. 41.Raise the final volume with the water to the mark on the volumetric flask. Transfer 5 milliliters of the previously made solution into a second 100 milliliter volumetric flask using a pipette. The volumetric flask should be filled with water until the designated volume is attained.

TABLET SAMPLE SOLUTION

Weigh each of the ten tablets to find their average weight. Crush the tablets into a fine powder. A 100 ml volumetric flask is filled with the equivalent of 25 mg of Hydrochlorothiazide after the tablet powder has been weighed. Strain the mixture containing the tablet powder through filter paper No. 41 to remove any impurities or solid particles. After the solution was filtered, 5 ml was pipetted out and transferred to another 100 ml volumetric flask. The volumetric flask should be filled with water until the designated volume is attained. When the solution is prepared, introduce it into the HPLC device for analysis.

Method Validation

System Suitability

A functional standard solution containing Hydrochlorothiazide and Bisoprolol Fumarate was prepared in accordance with protocol, and it was introduced to the HPLC system five times. The system suitability characteristics were evaluated using standard chromatograms, where the percentage RSD of the retention duration, tailing factor, theoretical plates, and peak regions from five replicate injections were computed.

Specificity

The capacity to distinguish the target analyte from other components in the sample is known as specificity. The procedure's specificity was covered by the connection of the chromatograms for the standard, sample, blank, and placebo.

Accuracy

The accuracy of the procedure was measured by applying the traditional addition recovery strategy. Three different concentrations of a standard stock solution containing known amounts of bisoprolol fumarate and hydrochlorothiazide were added to the previously examined sample solutions: 80%, 100%, and 120%. Three examinations of the solutions at each level were conducted while revisiting these alternatives. A percentage of the recovery was used to determine and report the accuracy.

Precision

The precision of the analytical process is defined as the degree of agreement between multiple measurements obtained from repeated samplings of the same homogenous material under prescribed conditions. Precision can be seen in both system and method implementation.

Linearity and Range

Inject six reference solutions containing hydrochlorothiazide and bisoprolol fumarate to show the assay's linearity. A correlation coefficient of 0.99 was found. A plot of linearity was produced.

Robustness

The inclusion of mobile phases minus and plus is an intentional modification to the procedure. Moreover, the RSD percentage is computed.

Solution Stability

Standard and sample solutions were tested for 24 hours at room temperature to show the stability of the solutions during analysis.

RESULT AND DISCUSSION

The acceptance requirements are satisfied by the system suitability metrics. Make sure the system is appropriate before beginning any analytical testing, including HPLC analysis.(Table1).
The comparison between the chromatogram of the standard solution and the sample solution containing hydrochlorothiazide and bisoprolol fumarate is evident. (Figures 3, 4, and 5) The main peak's retention time is unaffected by the gap.(Tables 2 and 3)

A favorable relationship with a correlation of 0.99 can be established by adjusting the area between the drug and the drug concentration. (Tables 4 and 5) (Figures 6 and 7) Repeated investigations' findings indicated that the cure rate varied from 95% to 103%. The fact that excipients don't obstruct the analyte analysis suggests that this method was chosen (Table 6&7).The method's precision %RSD values are less than 2%, suggesting that it has good precision for the simultaneous analysis of bisoprolol fumarate and hydrochlorothiazide.(Tables 8 and 9) The findings highlight the significance of the approach by proving that minute variations in temperature and composition of the mobile phase have no effect on the drug's peak area.(Tables 10 and 11) At room temperature, samples and reference solutions remain stable for a whole day.(Tables 12 and 13) In Conclusion, we can say that the quantitative determination of hydrochlorothiazide and bisoprolol fumarate in formulation using the approved RP HPLC method is accurate, precise, affordable, and easy to use. The method's conformity with ICH requirements was confirmed. As a result, the technique may be used successfully to the regular examination of tablets containing bisoprolol fumarate and hydrochlorothiazide.

ACKNOWLEDGEMENT

The authors express their gratitude to Merck Specialities Aurangabad for donating gift samples of Hydrochlorothiazide and Bisoprolol Fumarate

Conflict of interest

The authors claim they don't have any competing interests.

REFERENCE

1. D. J. Pandya, c. n. patel and khushbu patel Indian J Pharm Sci  2024;86(2):683-69
2. Beckett AH, Stenlake JB (1988) Practical pharmaceutical chemistry, Part-2, 4th (Edn.),
3. Lough WJ, Wainer IW, (1995) High performance liquid chromatography: fundamental principles and practice. cRc press.
4. Ali BH. Pharmacological, Therapeutic and Toxicological Properties of Furazolidone: Some Recent Research. Vet Res Commun. 1999;23(6):343-60.
5. Kale AA, Rasane AB, Bhatiyani AK. Int J Pharm Tech 2012;4(3):4805-13.

Table No.1: System suitability for of Hydrochlorothiazide and Bisoprolol fumarate

       
           
       
The acceptance requirements are satisfied by the system suitability metrics. Make sure the system is appropriate before beginning any analytical testing, including HPLC analysis.