Degradation Profiling of Lisinopril and Hydrochlorothiazide by RP- HPLC method with QbD Approach

 

Mr. Kalleshvar P. Jatte*, R. D. Chakole, M. S. Charde

Post Graduate Department of Pharmaceutical Chemistry, Government College of Pharmacy,

Vidyanagar, Karad, Dist.: Satara, Pin- 415124, Maharashtra, India.

 

ABSTRACT:

RP-HPLC method was developed for the estimation of Lisinopril and Hydrochlorothiazide in tablet dosage form with the help of Quality by Design (QbD) approaches. In this method concentration of each drug was obtained by using the absorptivity values calculated for drug wavelength 226.0 nm and solving the equation. The RP-HPLC method was performed C18-(100mm x 4.6 mm,)2.5 μm particle size in gradient mode, and the sample was analysed using methanol 45.0 ml and 55.0 ml (pH 3.3 0.05% OPA with TEA) as a mobile phase at a flow rate of 0.8 ml/min and detection at nm. By the retention time for Lisinopril and Hydrochlorothiazide found 3.39 and 4.59 min respectively. Validation related the method is specific, rapid, accurate, precise, reliable, and reproducible. Calibration plots by both HPLC were linear over the 5-25 and 12.5-62.5 μg/ml for Lisinopril and Hydrochlorothiazide respectively, and recoveries from tablet dosage form were between 99.02 and 100.00 %. The method can be used for routine of the quality control in pharmaceuticals. The degradation profiling of Lisinopril and Hydrochlorothiazide were also carried out.

 

 

 

1. INTRODUCTION:

Investigative chemistry deals with quantitative analysis of composition of substances and complex materials in various matrices by measuring a physical or chemical property of a distinctive constituent of the drug tester. Investigative methods are classified according to the property of the analyte measured¹.

 

The pharmaceutical analysis is one of the most vital fields in investigative chemistry. Modern investigative chemistry is the backbone instrumental analysis. There are so many diverse type of instruments used today that, it seems like a puzzling array of acronyms rather than a unified field of study.² The investigative methods should be accurate as required and not as accurate as possible.³ Investigative methods are classified into instrumental and chemical method.⁴

 

1.1 Method development in HPLC:^5,17

Method development and optimization in liquid chromatography is still an attractive field of research for theoreticians. Multifaceted mixtures or testers required systematic method development involving accurate modelling of the retention performance of the analyte. Among all, the liquid chromatographic methods, the reversed segment systems based on modified silica offers the highest probability of successful results. However, a large number of (system) variables (parameters) affect the selectivity and the resolution.

 

1.2 Validation of Methods:^6,7,8

Method validation is the process use to confirm that the investigative procedure in use for a specific test is suitable for its intended use. Results from process validation can be used to judge the quality, reliability and constancy of results; it is avital part of any good investigative practice. The USP has printed precise procedures for method validation for compound evaluation.is USP defines eight steps for validation.

 

1.3 QbD Approach:

Analytical methods play an important role supporting implementation of QbD in process pharmaceutical development and development and manufacturing. QbD is defined as a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding based on sound science and quality risk management‖. In alignment with the approach proposed in the draft fad (food and drug administration) guidance for process validation, a three-stage approach can be applied to method validation...

 

Stage 1: Method design: define method requirements and conditions and identify critical controls.

 

Stage 2: Method qualification: confirm that the method is capable of meeting its design Intent.

 

Stage 3: Continued method verification: Gain ongoing assurance to ensure that the method remains in a state of control during routine use. A critical function of stage 1 is the design of an analytical target profile (ATP) for the method. To design the ATP, it is necessary to determine the characteristics that will be indicators of method performance for its intended use.¹¹

 

1.4 Force Degradation:

Forced degradation or accelerated degradation is a procedure whereby the natural degradation rate of a product or material is amplified by the application of an additional stress. Forced degradation studies are castoff to recognize reactions which might be fall to worsen a treated merchandise. Typically lead before final formulation, forced degradation observes external stresses to rapidly shade material solidities. Longer term storage tests are usually used to amount similar properties when final formulations are involved because of the stringent FDA regulations. These tests are generally more expensive (since of the time involved) than forced degradation which is therefore used for quick selection and exclusion tests.

 

1.5 Drug Profile:

Lisinopril:

 

Figure No. 1: Chemical structure of Lisinopril⁹

 

Main structure activity:

Generic name:	Lisinopril
IUPAC Name:	(2S)-1-[(2S)-6-amino-2-[[(1S)-1-carboxy-3- phenylpropyl]amino] hexanoyl]pyrrolidine-2-carboxylic acid
Therapeutic:	ACE Inhibitors and Calcium Channel Blockers
Molecular weight:	405.5 g/mol12
Chemical formula:	C21H31N3O5
Pka:	2.5
Melting point:	145-148° C13
Absorbance:	6-60 % orally14

 

Hydrochlorothiazide:

Molecular Formula:	C7H8CLN3O4S2
IUPAC Name:	6-Chloro-1, 1-Dioxo-3, 4-Dihydro-2h-1λ6, 2, 4- Benzothiadiazine-7-Sulfonamide
Molecular Weight:	297.7 G/Mol12
Melting Point:	272-73 ° C15
Half-Life:	5.6 Hrs16

 

Figure No 2: Chemical Structure of hydrochlorothiazide¹⁰

 

2. MATERIAL AND METHODS:

2.1 Chemicals, Reagents and Instruments:

Lisinopril and Hydrochlorothiazide were obtained from R.S.I.T.C. Jalgaon. Orthophosphoric acid of HPLC grade from Avantor performance material India Ltd. Thane. Methanol and Water of HPLC grade from Merck speciality Ltd. Mumbai. Other chemicals used were of analytical grade.

The analysis of the drug was carried out on Agilent (S.K.) Gradient System UV Detector. Equipped with Reverse Phase (Agilent) C18 column (4.6mm x 250mm; 2.5µm), a SP930Dpump, a 20µl injection loop and UV730D (DAD) Absorbance detector and running Chemstation software.

 

Methods:

2.2 Preparation of standard solution:-

Preparation of std. Lisinopril solution: (Stock I)

An accurately weighed quantity, 5mg of Lisinopril (LSP) was dissolved in methanol in a 10ml volumetric flask and volume made up to 10.0ml to produce a solution of 500ug/ml. From the freshly prepared standard stock solution (500ug/ml), 0.1ml stock solution was pipetted out in 10 ml of volumetric flask and volume was made up to 10ml with mobile phase to get final concentration of 5ug/ml.

 

Preparation of std. Hydrochlorothiazide solution: (Stock II):

An accurately weighed quantity, 12.5mg of Hydrochlorothiazide (HCZ) was dissolved in methanol in 10ml volumetric flask and volume made up to 10.0ml to produce a solution of 1250ug/ml From the freshly prepared standard stock solution (1250ug/ml), 0.1ml stock solution was pipetted out in 10 ml of volumetric flask and volume was made up to 10ml with mobile phase to get final concentration 12.5ug/ml.

 

Preparation of std. Lisinopril and Hydrochlorothiazide solution: (Stock III)

From the freshly prepared standard stock solution (500 and 1250 ug/ml), 0.1 ml stock solution was pipetted out in 10 ml of volumetric flask and volume was made up to 10 ml with mobile phase to get final concentration 5 – 25 and 12.5 – 62.5 ug/ml.

 

3. RESULTS AND DISCUSSION:

3.1 Mobile phase optimization:

After the selection of suitable mobile phase, it was then optimized for its reproducibility, sensitivity and accuracy. The optimized parameters for selected method are as below.

 

The final chromatographic conditions selected were as follow:

DAD Detector Agilent (S.K) Gradient System:

Analytical column	:	(Agilent) C18 column (4.6mm x 100mm)
Injection volume	:	20µl
Flow rate	:	0.8 ml/min
Mobile phase	:	MEOH +0.1OPA (45+55 % v/v)
Detection	:	226 nm
Run Time	:	15 min

 

Figure No. 3: Chromatogram of standard Combination of Lisinopril and Hydrochlorothiazide

 

3.2 Design of Experiments:

Preliminary Screening and Optimization Data Analysis Preliminary experiments were performed by using Taguchi screening Method to identify the critical factors and to set their levels (maximum and minimum) for the experimental design. In this step The following parameters were investigated: selection of a chromatographic Column (C8 and C18), column temperature, mobile phase (ratio of ACN: MEOH: buffer), concentration of the buffer if present In the mobile phase, buffer ph, injection volume, mode of flow (isocratic/gradient) as well as determining the ideal flow rate. Based on the results obtained from the Taguchi screening, multiple linear regression analysis (MLRA) was applied for the studied Design using Design Expert® software version 9.0.02 to fit the full second-order polynomial equations with added Interaction terms. The method chosen to optimize separation of Lisinopril and Hydrochlorothiazide with the shortest analysis time was Box–Behnken design (BBD) with three replicates at the centre point (middle level). The independent variables were investigated and their low, Medium, and high levels described in Table below the QbD trials. The evaluated Responses (dependent variables) were the no. of theoretical plates (Y1), assay (Y2), and tailing factor (Y3). Prediction of the optimum Composition was carried out using overlay plotting, brute Force method, and numeric approach of desirability function. Overlay Plot (i.e., combined contour plot) option in the software was Also embarked upon to locate the optimum composition. Within This optimal area, an optimum chromatographic condition was located by trading off different responses. The prognosis of the Optimum analytical condition was also conducted using numerical Optimization technique/with help of Design Expert software. The Box-Behnken Design Validation Fifteen runs were done, selected from grid search data, prepared as per the chosen composition(s), and evaluated for the critical Quality attributes (CQA), viz. Number of theoretical Plates (TP), Assay, and tailing factor (TF). The predicted and observed Responses were compared, and linear correlation plots were constructed Percent bias (error) was calculated with respect to the Observed responses and the residual plots were also constructed For TP, assay, and TF.

 

3.3 Method Validation:

Linearity:

Linearity of Lisinopril and Hydrochlorothiazide was observed in both methods the range of 12.5-62.5 and 5-25 ug/ml. Detection wavelength used was 226 nm.

 

Figure No. 4: Calibration curve of Lisinopril

 

Figure No. 5: Calibration curve of Hydrochlorothiazide

 

Accuracy (recovery):

Accuracy of RP-HPLC method and UV Spectrophotometric method is ascertained by recovery studies performed at different levels of concentrations (80%, 100% and 120%). The % recovery was found to be within 98-101%.

 

Table No. 1: Result of recovery data for Lisinopril and Hydrochlorothiazide

METHOD	Drug	Level (%)	Amt. taken (ug/ml)	Amt. Added (ug/ml)	Absorbance Mean* ± S.D.	Amt. recovered Mean *±S.D.	%Recovery Mean *± S.D.
RP-HPLC Method	LSN	80%	5	4	9.03±0.010	4.03±0.010	100.56±0.08
		100%	5	5	9.95±0.016	4.95±0.016	99.00±0.28
		120%	5	6	11.05±0.04	6.05±0.04	100.94±0.77
	HCZ	80%	12.5	10	22.49±0.094	9.99 ±0.089	99.87±0.89
		100%	12.5	12.5	24.96±0.106	12.46±0.106	99.71±0.84
		120%	12.5	15	27.48±0.069	14.98±0.069	99.89±0.46

 

System suitability parameters: (Repeatability):

Table No. 2: Repeatability studies on RP-HPLC for Lisinopril and Hydrochlorothiazide

METHOD	Concentration of Lisinopril and Hydrochlorothiazide (mg/ml)	Peak area	Amount found (mg)	% Amount found
HPLC LSN	20	698.12	19.78	98.90
METHOD	20	692.54
	Mean	695.33
	SD	3.94
	%RSD	0.169
	37.5	1701.649	37.70	98.71
HPLC HCZ	37.5	1695.547
METHOD	Mean	1698.60
	SD	0.12
	%RSD	0.16

 

Repeatability studies on RP-HPLC method for Lisinopril and Hydrochlorothiazide was found to be, The %RSD was less than 2%, which shows high percentage amount found in between 98% to 102% indicates the analytical method that concluded.

 

Table No. 3: Result of Intraday and Inter day Precision studies on RP-HPLC and UV method for Lisinopril and Hydrochlorothiazide

METHOD	Drug	Conc. (µg/ml)	Interday Precision		Intraday Precision
			Mean± SD	%Amt Found	Mean± SD	%Amt Found
RP- HPLC METHOD	LSN	10	9.93±0.96	99.35	9.88 ± 0.96	98.77
		15	15.12±0.24	100.81	15.17 ±0.68	101.10
		20	20.01±0.15	100.07	19.91 ±5.16	99.56
	HCZ	25	23.62 ±5.10	94.47	23.68±0.55	94.73
		37.5	37.65±0.89	100.41	37.66±1.87	100.42
		50	48.98±2.86	97.97	49.01±0.80	98.01

*Mean of each 3 reading for RP-HPLC method

 

Precision:

Intraday and Inter day Precision studies on RP-HPLC and UV method for Lisinopril and Hydrochlorothiazide which shows the high precision % amount in between 97% to 101% indicates to analytical method that concluded.

 

Robustness:

Robustness Study of Lisinopril:

The changes were did flow rate (±1 ml/ min^-1), PH of mobile phase composition (±1 ml/ min^-1), and Wavelength (±1 ml/ min^-1). %RSD for peak area was calculated which should be less than 2%.the result shown in analytical method that concluded.

 

Robustness Study of Hydrochlorothiazide:

The changes were did flow rate (±1 ml/ min^-1), PH of mobile phase composition (±1 ml/ min^-1), and Wavelength (±1 ml/ min^-1). %RSD for peak area was calculated which should be less than 2%.the result shown in analytical method that concluded.

 

LOD AND LOQ:

The LOD and LOQ of Hydrochlorothiazide was found to be 0.2375 (ug/mL) and 0.7199 (ug/mL), analytical method that concluded.

 

The LOD and LOQ of Lisinopril was found to be 0.199 (ug/mL) and 0.6040 (ug/mL), analytical method that concluded.

 

3.5 Analysis of tablet formulation:

Table No. 4: Analysis of marketed formulation

Assay	Drug	Amt. Found	% Label Claim	SD	%RSD
Rp-HPLC Method	LSN	19.86	99.33	0.047	0.14
	HCZ	48.37	99.40	0.18	0.37

 

Analysis of marketed formulation were also %Label Claim was found to be 99-100% Satisfactory are concluded.

 

3.6. Degradation study:

Table No. 5: Result of Degradation Study of Lisinopril and Hydrochlorothiazide

Parameters	Drug name	Area	% Assay	% Degradation	% RSD
Acid	HCZ	1421.89	83.79	16.21	0.93
	LSN	591.67	85.05	14.95	0.48
Base	HCZ	1509.74	88.96	11.04	0.61
	LSN	594.53	85.46	14.54	0.99
Oxidative	HCZ	1523.82	89.79	10.21	0.89
	LSN	601.92	86.52	13.48	0.26
Neutral	HCZ	1649.23	97.18	2.82	0.51
	LSN	691.85	99.45	0.55	0.91

 

4. CONCLUSIONS:

Simple, rapid, accurate and precise RP-HPLC have been developed and validated for the routine analysis of Lisinopril and Hydrochlorothiazide in API and tablet dosage forms. Both methods are suitable for the simultaneous determination of Lisinopril and Hydrochlorothiazide in Single-component formulations without interference of each other. The developed methods are recommended for routine and quality control analysis of the investigated drugs in two component pharmaceutical preparations. The amount found from the proposed methods was in good agreement with the label claim of the formulation. Also the value of standard deviation and coefficient of variation calculated were satisfactorily low, indicating the suitability of the proposed methods for the routine estimation of tablet dosage forms. The developed method is carried out with the help of Qbd approach. The complete degradation profiling was also carried out according to the ICH guidelines.

 

5. REFERENCES:

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2.      Rashmin. An introduction to analytical method development for pharmaceutical formulations. Pharma info.net, 2008: 6(4): p.1.

3.      Sethi. PD. In; HPTLC Quantitative analysis of pharmaceutical formulations, 1^st ed. CBS publishers and distributors, New Delhi, 2001; preface IIV, p.3.

4.      Willard HH, Merritt LL, Dean JA, Settle FA. Instrumental Methods of Analysis. A 7^th ed. CBS Publisher and Distributors, New Delhi.p.5.

5.      Sethi PD. High Performance Liquid Chromatography Quantitative Analysis of Pharmaceutical Formulation. I^st ed. New Delhi: CBC Publication and Distributors; 2001.p.35.

6.      Sethi PD. High Performance Liquid Chromatography. Quantitative Analysis of Pharmaceutical Formulations. I^st ed. New Delhi CBS Publication and Distributors, 2001. p.116-120.

7.      Cindy Green, RAC. A Step By Step Approach to Establishing a Method Validation. Journal of Validation Technology August 2007; 13(4): p.317-323.

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9.      Pubchem.

10.   Medindia.net

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Received on 19.05.2021       Modified on 21.06.2021

Accepted on 13.07.2021   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2021; 11(4):270-274.