INTRODUCTION:

Quinapril is chemically known as (3S)-2-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino}propanoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Its chemical formula and molar mass were C₂₅H₃₀N₂O₅ and 438.5161 g/mol¹. Quinapril is a prodrug that belongs to the angiotensin-converting enzyme (ACE) inhibitor class of medications. It is metabolized to quinaprilat (quinapril diacid) following oral administration^2,3. Quinaprilat is a competitive inhibitor of ACE, the enzyme responsible for the conversion of angiotensin I (ATI) to angiotensin II (ATII)^4,5. ATII regulates blood pressure and is a key component of the renin-angiotensin-aldosterone system (RAAS). Quinapril may be used to treat essential hypertension and congestive heart failure⁶. The structure of quinapril⁷ is given in the fig.1

Tolcapone:

Tolcapone is chemically known as5-(4-methylbenzoyl)-3-nitrobenzene-1,2-diol. Its chemical formula and molar mass were C₁₄H₁₁NO₅ and 273.2408 g/mol. Tolcapone is a drug that inhibits the enzyme catechol-O-methyl transferase (COMT). It is used in the treatment of Parkinson’s disease as an adjunct to levodopa/carbidopa medication. It is a yellow, odourless, non-hygroscopic, crystalline compound⁸. Tolcapone is associated with a risk of hepatotoxicity. The structure of Tolcopone is shown in the fig.2.

Fig 2: Structure of Tolcopone

MATERIALS AND METHODS:

Chemicals and reagents:

The Samples of Quinapril And Hydrochlorothiazide Drugs were obtained as gift samples from Chandra labs, Hyd. Quinapril And Hydrochlorothiazide Drugs (lable claims) were obtained from Obtained from local pharmacy.

Table 1: Instruments used

UV-Visible Spectrophotometer	Nicolet evolution 100
HPLC	Shimadzu(LC 20 AT VP)
HPLC	Agilent 1200 series
Ultra sonicator	Citizen, Digital Ultrasonic Cleaner
pH meter	Global digital
Electronic balance	Shimadzu
Syringe	Hamilton
HPLC Column	INERTSILcolumn,C18(150x4.6 ID) 5µm

Table 2: Reagents used

Water	HPLC Grade
Methanol	HPLC Grade
Potassium Dihydrogen ortho Phosphate	AR Grade
Acetonitrile	HPLC Grade
Ammonium acetate	AR Grade
Tetra Hydro Furan	AR Grade

Preparation of Mobile Phase:

A mixture of 40 volumes of mixed phosphate buffer (KH₂PO4 +K₂HPO4) and 60 volumes of acetonitrile were prepared. The mobile phase was sonicated for 10min to remove gases and filtered through 0.45µ membrane filter for degassing of mobile phase.

Preparation of Phosphate buffer :

Weigh 1.62gms of KH₂PO₄and 0.3gms of K₂HPO₄ were weighed and dissolved in 550ml of water and volume was made up to 550ml with water. Adjust the pH to 6.5 using ortho phosphoric acid. The buffer was filtered through 0.45µ filters to remove all fine particles and gases.

Determination of Working Wavelength (λmax):

In simultaneous estimation of two drugs isobestic wavelength is used. Isobestic point is the wavelength where the molar absorptivity is the same for two substances that are interconvertible. So this wavelength is used in simultaneous estimation to estimate both drugs accurately.

Preparation of standard stock solution of Quinapril:

50mg of Quinapril was weighed and transferred in to 500ml volumetric flask and dissolved in methanol and then make up to the mark with methanol and prepare 10 µg/ml of solution by diluting 1ml to 10ml with methanol.

Preparation of standard stock solution of Tolcapone:

50mg of Tolcapone was weighed in to 500ml volumetric flask and dissolved in Methanol and then dilute up to the mark with methanol and prepare 10 µg/ml of solution by diluting 1ml to 10ml with methanol.

Method validation:

Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. Validation parameters a) Specificity / Selectivity b) Accuracy c) Precision d) Linearity and Range e) Limit of Detection f) Limit of Quantitation g) Robustness h) Ruggedness i) System Suitability.

RESULTS AND DISCUSSION:

Linearity and range:

Preparation of mixed standard solution:

10mg of Quinapril and 10mg of Tolcapone is weighed in 10ml of volumetric flask and dissolve in 10ml of mobile phase and make up the volume with mobile phase. The correlation coefficient for linear curve obtained between concentration vs. Area for standard preparations of Quinapril and Tolcapone is 0.995 and 0.999. The relationship between the concentration and area of Quinapril and Tolcapone is linear in the range examined since all points lie in a straight line and the correlation coefficient is well within limits. The results are given in the table 3 and table 4 and in the fig 3 and 4.

Table 3: Linearity of Quinapril

S. No.	Conc.(µg/ml )	Area
1	50	808.453
2	75	1164.555
3	100	1471.354
4	125	1944.375
5	150	2244.008

Table 4: Linearity of Tolcapone

S. No.	Conc.(µg/ml )	Area
1	62.5	2774.562
2	93.75	4032.779
3	125	5007.437
4	156.25	6609.492
5	187.5	7528.872

Accuracy:

Accuracy of the method was determined by Recovery studies. To the formulation (pre analyzed sample), the reference standards of the drugs were added at the level of 50%, 100%, 150%. The recovery studies were carried out three times and the percentage recovery and percentage mean recovery were calculated. To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50%, 100% and 150%. From the results the percentage mean recovery of Quinapril and Tolcapone is 101.02% and 99.55% respectively.

Fig.3: Linearity graph of Quinapril

Fig.4: Linearity graph of Tolcapone

Precision:

Method precision:

Prepared sample preparations of Quinapril and Tolcapone as per test method and injected 6 times in to the column. Test results for Quinapril and Tolcapone are showing that the %RSD of Assay results are within limits. The results are shown in the table 5.

Table 5: Results for Method precision of Quinapril and Tolcapone

Quinapril			Tolcapone
S.No.	Rt	Area	S.No.	Rt	Area
1	3.827	1487.147	1	2.813	5005.745
2	3.750	1496.768	2	2.760	5044.357
3	3.750	1482.466	3	2.760	4993.823
4	3.740	1448.567	4	2.753	4890.628
5	3.827	1505.906	5	2.813	4976.613
6	3.787	1463.248	6	2.767	4997.266
avg	3.7802	1484.171	avg	2.778	4982.233
stdev	0.0397	21.855	stdev	0.028	56.942
%RSD	1.05	1.47	%RSD	1.00	1.56

Robustness:

To demonstrate the robustness of the method, prepared solution as per test method and injected at different variable conditions like using different conditions like temperature and wavelength. System suitability parameters were compared with that of method precision.

From the observation it was found that the system suitability parameters were within limit at all variable conditions.

Ruggedness:

The ruggedness of the method was studied by the determining the analyst-to-analyst variation by performing the Assay by two different analysts. From the observation the %RSD between two analysts Assay values not greater than 2.0%, hence the method was rugged.

CONCLUSION:

A simple and selective HPLC method is developed for the determination of Quinapril and Tolcapone tablet dosage forms. Chromatographic separation was achieved on a c₁₈column using mobile phase consisting of a Mixed Phosphate buffer (KH2PO4 +K2HPO4): Acetonitrile 40:60, with detection of 239 nm. Linearity was observed in the range 50 - 150 µg /ml for Quinapril (r² =0.995) and 62.5- 187.5µg /ml for Tolcapone (r² =0.999) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim.

The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.

ACKNOWLEDGEMENT:

The authors are also grateful to Chandra labs, Hyderabad for providing gift sample of quinopril and tolcapone drug.

REFERENCES:

1. Wishart DS. Jewison T. Guo AC, Wilson M and Knox C. et al The Human Metabolome Database. Nucleic Acids Res 2013; 1: 801-7 doi: 10.1093/nar/gks1065

2. Espinoza S. Managó F. Leo D. et al Role of catechol-O-methyltransferase (COMT)- dependent processes in Parkinson’s disease and L-DOPA treatment. CNS Neurol. Disord. Drug Targets 2012; 11(3): 251-63. doi: 10.2174/187152712800672436

3. Goncalves D. Alves G.Soares-Da-Silva P. Falcao A et al Bioanalytical chromatographic methods for the determination of catechol-O-methyltransferase inhibitors in rodents and human samples: a review. Anal. Chim. Acta 2012; 710: 17-32. DOI: 10.1016/j.aca.2011.10.026

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5. Wright JM. Musini VM. Gill R.et al First-line drugs for hypertension. Cochrane Database Syst Rev 2018; 4: CD001841. doi: 10.1002/14651858.CD001841.pub3

6. Long CM, Tang K. Chokshi H. Fotaki N et al Surface dissolution UV imaging for investigation of dissolution of poorly soluble drugs and their amorphous formulation. AAPS Pharm Sci Tech 2019; 20(3): 113. doi: 10.1208/s12249-019-1317-z.

7. FDA Approved Drug Products: Quinapril Oral Tablet, www.accessdata.fda.gov/drugsatfda_docs/label/2017/019885s043lbl.pdf

8. Begum MH: Analytical method development and validation for the estimation of quinapril and tolcapone using RP-HPLC. Int J of Innovative Pharmaceutical Sciences and Research 2018; 6(01): 99-113. doi: 10.13040/IJPSR.0975-8232.12(6).3375-80

Received on 03.08.2021 Modified on 03.01.2022

Research J. Pharm.and Tech 2022; 15(4):1680-1682.

DOI: 10.52711/0974-360X.2022.00281