INTRODUCTION:

Antihypertensive monotherapy, although commonly used, does not address the multifactorial nature of hypertension as a disease with many pathways. Using more than one drug makes more therapeutic sense because combination agents cover more than one pathway.

The use of low-dose combination antihypertensive agents is a good contemporary strategy. The combination of Lisinopril (5mg) a long acting angiotensin converting enzyme inhibitor and Amlodipine (5mg) a long acting 1,4-dihydropyridine calcium channel blocker, is well established and is widely used in patients suffering from essential hypertension.

Lisinopril (LSN) is chemically (2S)-1-[(2S)-6-amino-2-{[(1S)-1-carboxy-3- phenyl propyl] amino} hexanoyl] pyrrolidine- 2-carboxylic acid1,2¹. Amlodipine (AML) chemically is 2-[(2-Aminoethoxy)-methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5- pyridinedicarboxylic acid 3-ethyl 5-methyl ester benzene sulfonate². Both the drugs are official in IP, BP and USP. The combination tablet is not yet official in any of the pharmacopoeia. Hence, there is no pharmacopoeial method for simultaneous estimation of these drugs in tablet dosage form. However number of spectrophotometric^3-5, HPLC^6-16, LC-MS/MS¹⁷, and HPTLC¹⁸, methods are reported in the literature for estimation of AML, both individually as well as in combination with other drugs. LSN is also reported to be estimated, alone or in combination with other drugs, using Spectrohpotomtery¹⁹, Spectrofluorimetry²⁰, HPLC^21-24 and HPTLC^25-26. Few HPLC^27-29 and HPTLC³⁰ methods have been reported for simultaneous determination of AML and LSN.

The objective of the present work was to develop a economical, sensitive and less time consuming HPTLC method for simultaneous determination of AML and LSN in pharmaceutical formulation as an alternative to the reported HPLC/HPTLC methods. The proposed method is optimized and validated as per the international conference on harmonization (ICH) guidelines³¹.

MATERIAL AND METHODS:

Chemicals and reagents:

The standard of Lisinopril and amlodipine was supplied by Genpharma International Pvt. Ltd. Pune. Fixed-dose combination tablet AMLOVAS^™-L contains 5mg Lisinopril and 5mg of Amlodipine was purchased from local market. All chemical and reagents were of analytical grade and were purchased from Merck Chemicals, Mumbai, India.

Instrumentation:

The sample were spotted in the form of bands of 6mm width with a 100µL sample syringe (Hamilton,Bonaduz, Switzerland) on silica gel precoated aluminum 60F₂₅₄plates, (10×10cm with 250mm thickness; E Merk,Darmstadt, Germany) using a CAMAG Linomat 5 (Muttenz,Switzerland) sample applicator. The plates were prewashed with methanol. A constant application rate of 0.1mL/s was used, and the space between two bands was 5.6mm. The slit dimension was 5 ×0.45mm, and the scanning speed was 10mm/s. Linear ascending development was carried out in an HPTLC twin-trough glass chamber (CAMAG) saturated with mobile phase vapour. The optimized chamber saturation time was 20 min at room temperature the length of each chromatogram run was 8cm. Following the development, the HPTLC plates were dried in a current of air using an air dryer. Densitometric scanning was performed using a CAMAG TLC Scanner 3 in the reflectance-absorbance mode at 215nm for both AML and LSN operated by WINCATS software (Version 1.4.4.6337, CAMAG). The radiation source used was the deuterium lamp emitting a continuous uv spectrum between 190 to 800nm. Concentrations of the compound chromatographed were determined from the intensity of the diffused light. Evaluation was based on peak areas with linear regression.

Preparation of standard and stock solution:

Standard stock solution A: Accurately weighed quantity of AML (13.9mg, eq.to 10mg of AML) and LSN (10.0 mg) was transferred to 10.0ml volumetric flasks, dissolved and diluted up to mark with methanol.(Concentration: 1000μg/ml AML, 1000μg/ml LSN).

Standard stock solution B : Accurately weighed quantity of AML (20.8mg, eq.to 15mg of AML) and LSN (15.0 mg) was transferred to 25.0ml volumetric flasks, dissolved and diluted up to mark with methanol. (Concentration: 600μg/ml AML, 600μg/ml LSN).

Optimization of the HPTLC method:

HPTLC procedure was optimized with a view to develop assay method for simultaneous estmation of Amlodipine besylate and Lisinopril in pharmaceutical formulation. The standard stock solution B (5µL), containing AML and LSN 600µg/ml each, sample was spotted on TLC plate and run in different solvent system. Initially, n-butanol, ethyl acetate, toluene, methanol and ammonia were tried in different ratios to obtain acceptable Rf value with well separated bands. To ensure compact bands and good peak shape the chamber saturation was also varied from 10 minutes to 30 minutes. After observing the peak shape and separation of two drugs, the chamber saturation time was decided. The plates were developed to a distance of 8 cm; which takes approximately 20 min for complete development of TLC plate.

Method Validation:

Validation of the optimized HPTLC method was carried out with respect to the following parameters.

Linearity and range:

From the standard stock solution A, containing 1000µg/mL of AML and LSN each, 1 to 6µL solution was spotted on TLC plate (n=6). The plates were then developed under the optimized chromatographic conditions mentioned above under instrumentation. Peak areas were plotted against the corresponding concentration to obtain the linearity curves.

Limit of Detection and Quantification:

The limits of detection (LOD) and quantification(LOQ) were separately determined based on the calibration curves. The standard deviation of the y-intercepts and slope of the regression lines were used. The formula used to calculate LOD and LOQ were LOD = 3.3σ/S and LOQ = 10σ/S.

Precision:

The precision of the method was verified by intraday precision studies were performed by analysis of three different concentration (2,000, 6,000, 10,000ng/band) for both AML and LSN for six times on the same day. The inter-day precision of the method was evaluated, at three different levels in similar manner as under intra-day precision, by performing the analysis on three consecutive days.

Specificity:

The specificity of the method was determined by analyzing standard drug and sample. The band for AML and LSN in sample was confirmed by comparing the R_f and spectra of the band with that of standards. The peak purity of AML and LSN were assessed by comparing the spectra at three different levels, i.e. peak start, peak apex, and peak end position of the band.

Robustness of the method:

Robustness of the method was evaluated by introducing small changes in the method parameters such as mobile phase composition, mobile phase volume, duration of chamber saturation with mobile phase, spotting to development time variation (15, 20, 25 min), and stability of solution (20 min,1 hr,3hr,8hr and 24 hr). The effect on R_f values and peak shape of AML and LSN was examined.

Accuracy:

Accuracy of the method was determined by using standard addition method where known amounts of AML and LSN pure drugs corresponding to 80, 100, 120 % of label claim was added to the pre-analysed tablet powder. The samples were then treated and analyzed in similar manner as discussed under analysis of marketed formulation under optimized chromatographic conditions discussed above.

Analysis of marketed formulation:

To determined content of AML and LSN in conventional tablet (AMLOVAS™-L label claim 5 mg of Lisinopril and amlodipine besylate equivalent to 5 mg amlodipine), twenty tablet were weighed and crushed to obtain fine powder. The average weigh of tablet was calculated, tablet powered equivalent to 15 mg of LSN was transferred in 25.0ml volumetric flask dissolved in methanol and the content was kept in ultrasonicator for 5 min., the volume was then made up to the mark with methanol. The solution was then mixed and filtered through 0.2µ membrane filter. The filtrate (6 bands) and standard stock solution A (2 bands), 5µl each, was applied to TLC plate which was developed under the optimized chromatographic conditions. Six tablet power samples were analyzed in similar manner. The possibility of excipients interference with the analysis was examined.

Force degradation study:

Forced degradation of drug product was carried out under thermolytic, photolytic, acid/base hydrolytic and oxidative stressed conditions as per the ICH guidelines. Amount of tablet powder equivalent to about 15mg of LSN was individually transferred to five different 25.0 ml volumetric flask. To flask 1.0ml of methanol was added followed by addition of 3.0ml of 0.1 M HCl, 0.1 M NaOH and 3% H₂O_2.The contents of flask were heated in water bath for 3 hrs at 80^OC. For thermal stress, tablet powder sample was placed in a controlled temperature oven at 80^OCfor 24 h. For photolytic stress, tablet powder sample was exposed to UV radiation at 254nm for 24 h. After stipulated time interval all the flask were removed, cooled to room temperature and then the tablet sample were treated and analyzed in similar manner as described under analysis of marketed formulation.

RESULT AND DISCUSSION:

Among the different solvent combination tried in mobile phase for effective separation of AML and LSN, the mobile phase containing a mixture of N-butanol, methanol and ammonia in the ratio of 4:4:1 v/v/v was found to be optimum. The Rf values for both drugs was well within the acceptable range (Rf - 0.2-0.8), for LSN and AML the Rf values were 0.27 ±0.02 and 0.62 ± 0.02, respectively. The chromatogram obtained under optimized chromatographic conditions is depicted in Fig. 1.

Figure 1: Densitogram of Lisinopril (Rf- 0.27) and Amlodipine besylate (Rf-0.62) in Tablet Formulation

Linearity:

Linear relationship were observed by plotting drug concentration against peak area for each compound. Amlodipine and lisinopril showed linear response in concentration range of 1000-6000ng/band. The corresponding linear regression equation was y=1.143x + 321.0 for Lisinopril and y=1.021x + 508.7 for Amlodipine besylate with correlation coefficient (R²) of 0.998 for Lisinopril and 0.999 for Amlodipine besylate, respectively. The linearity curves for LSN and AML are depicted in Fig. 2 and 3, respectively.

Figure 2. Linearity curve for LSN

Figure 3: Linearity curve for AML

LOD and LOQ:

The Limit of detection and Limit of quantitation for both the drugs by proposed HPTLC method were calculated using 3σ/S and 10σ/S equations, respectively. Where σ is the standard deviation of Y intercept and S is the slope of calibration curve. The LOD and LOQ was found to be 1.437ng/band and 4.821ng/band for AML and 2.602ng/band and 7.686ng/band for LSN as shown in Table No. 1. In reported HPTLC²⁰ method LOD and LOQ was found to be 83.45ng/band and 252.87ng/band for LSN and 54.21ng/band and 164.28ng/band. The LOD and LOQ values for LSN and AML were lower than the reported method indicating the improved sensitivity of the developed method over reported HPTLC method.

Precision:

The repeatability and inter-day precision of the developed method is expressed in terms of percent relative standard deviation of percent label claim. In intra-day precision the percent RSD for LSN and AML was found to be 0.246 and 0.831. In inter-day precision the percent RSD for LSN and AML was found to be 1.293 and 1.616, respectively. The percent RSD less than 2 indicates the repeatability and reproducibility of the developed method.

Specificity:

The peak purity of LSN and AML was assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the bank. Good correlation among the spectra indicated the peak purity for LSN[Correlation r(s,m) = 0.9994 and r(m,e) = 0.9997] and AML [Correlation r(s,m) = 0.9999 and r(m,e) = 0.9996]. Hence, it can be concluded that there are no impurities or degradation products migrated with the bands obtained from solution of drugs.

Accuracy:

The accuracy of the proposed method was assessed by using standard addition method at three different levels viz. 80 %, 100 % and 120 %. The average percent recovery for AML and LSN was found to be 99.94 % and 100.29 %, respectively, indicating that there is no interference from the excipients used in tablet formulaton. Result of recovery studies are shown in Table No.2

Robustness of the method:

Robustness of the proposed method was assessed by deliberately varying the method parameters like mobile phase volume, mobile phase composition, chamber saturation time, time from spotting to development, and stability of solution. The effect of these variation on Rf value, peak area and resolution of peaks was assessed. The percent RSD for peak area of both the drugs under varied method parameters was less than 2. The resolution between LSN and AML peak was not significantly affected as there was no significant change in the Rf value of the drug peaks. The Rf value was within ±0.05 Rf units of standard values. The results of robustness studies are shown in Table No. 3. Hence, the developed method is robust for simultaneous determination of LSN and AML in fixed dose combination tablet.

Forced Degradation Studies:

In forced degradation studies, both LSN and AML were found to degrade under acidic (0.1M HCl), and oxidative (3% H₂O₂) stress conditions. However, both the drugs LSN and AML were stable under Alkaline (0.1 M NaOH), Neutral, Thermal (60⁰C for 24 h) and Photo-degradation (exposure to UV-Light at 254nm) stress conditions. The results of stress degradation studies are shown in Table No. 4 and the typical densitogram obtained under different stress conditions are depicted Fig. No. 4-9. The developed method was able selective quantitate LSN and AML in presence of degradation products confirming the stability power of the method.

Analysis of Marketed Formulation:

The percent label claim estimated by proposed HPTLC method for LSN and AML was found to be 100.24% and 99.94%, respectively. The results of analysis of marketed formulation are shown in Table No. 5.

CONCLUSION:

The developed HPTLC technique is compared with the reported HPLC method (Table No. 6) for estimation of Lisinopril and Amlodipie in fixed dose combination tablets. The results obtained by proposed HPTLC are comparable with that of the reported HPLC method. The proposed HPTLC method has distinct advantages over reported HPLC method like the amount of solvent required for analysis is very less, cost of HPTLC plate is very less as compared to the HPLC column, there is no need to filter and degas the solvents in HPTLC as necessary in HPLC, solvents of AR grade can be used instead of spectroscopic grade solvents, the chromatographic run is completed in 30 minutes (including chamber saturation time), no possibility of interference from previous analysis as fresh stationery phase and mobile phase is used for each analysis, different batches table samples can be analyzed in single run. Hence, the proposed HPTLC technique is economical and less time consuming than reported HPLC method. The proposed HPTLC method is more sensitive as compared to the reported HPLC method as the LOD and LOQ values are lower that the values obtained in reported HPLC method. Also, the results of method validation, carried out as per ICH guidelines, indicate that the method is accurate, precise, selective and reproducible.. Hence, the developed HPTLC method can be preferred as alternative to the reported methods for routine quality control of pharmaceutical formulations containing Lisinopril and Amlodipine.

ACKNOWLEDGMENT:

The author are grateful to Dr. Datta Meghe College of Pharmacy, Salod(H), and Dr. D. Y. Institute of Pharmaceutical Sciences and Research, Pune, India for providing a facilities to carry the experiment, as well as Gen Pharma Int. Pvt. Ltd, Pune for providing gift sample of Amlodipine besylate and Lisinopril.

Table 1: Summary of Validation Parameter

Parameter	LSN	AML
Linearity range (ng/band)	1000-6000	1000-6000
Correlation coefficient	0.998	0.999
Limit of detection(ng/band)	2.602	1.437
Limit of quantitation(ng/band)	7.686	4.821
Precision (% RSD) Intraday Inter day	0.246 1.293	0.831 1.616
Robustness	Robust	Robust
Specificity	0.9991	0.9996

Fig. 4: Typical HPTLC Chromatogram of Acid (0.1 M) Treated Tablet Sample

Fig. 4: Typical HPTLC Chromatogram of Alkali (0.1 M) Treated Tablet Sample

Fig. 4: Typical HPTLC Chromatogram of Oxide (3 % H₂O₂) Treated Tablet Sample

Fig. 4: Typical HPTLC Chromatogram of Tablet Sample Exposed to Neutral Hydrolysis

Fig. 4: Typical HPTLC Chromatogram of Heat (0.1 M) Treated Tablet Sample

Fig. 4: Typical HPTLC Chromatogram Tablet Sample Exposed to UV Radiations

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31. International Conference on Hormonization:ICH Q1A(R₂) Stability testing of new drug substance and products, February 2003, 1-10.

Received on 24.09.2020 Modified on 03.12.2020

Research J. Pharm.and Tech 2021; 14(12):6250-6256.

DOI: 10.52711/0974-360X.2021.01081

Drug	Level	Amount tablet powder taken (mg)	Amount of standard added (mg)	*Amount recovered (mg)**	% Recovery ± %RSD (n=3)
LSN	I	739.2	12	12.69	99.91 ±0.082
	II	739.2	15	15.09	100.81±0.405
	III	739.2	18	18.00	100.16±0.208
AML	I	739.2	12	11.98	99.92± 0.685
	II	739.2	15	14.99	99.91±0.085
	III	739.2	18	18.04	100.0± 0.185

Parameter	Percent Relative Standard Deviation of Peak Area*		Rf Value*
Parameter	LSN	AML	LSN	AML
Mobile phase composition (± 0.1 mL)	1.23	1.01	0.24	0.65
Total amount of mobile phase (± 1 mL)	0.51	0.15	0.29	0.60
Duration for chamber saturation (± 10 %)	1.37	0.61	0.30	0.66
Time from spotting to development
5 min	1.50	0.82	0.27	0.64
30 min	1.43	0.89	0.26	0.63
1 h	1.65	0.96	0.29	0.61
Time from development to scanning
5 min	1.13	0.97	0.25	0.63
30 min	0.94	0.88	0.28	0.66
1 h	1.07	1.02	0.31	0.64

Stress Conditions	Percent Assay of Active Substance
Stress Conditions	LSN	AML
Acidic (1 N HCl)	89.56	91.23
Alkaline 0.1 N NaOH	98.12	99.85
Oxidative (3.0 % H₂O₂₎	88.25	89.60
Neutral (Water)	100.07	99.86
Thermal (60⁰C)	100.00	99.89
Photo-degradation (UV-254 nm)	100.03	100.0

Marketed Formulation	Drug	Label claim(mg/tab)	Estimated % of label claim ± SD*
Amlovas™-L	LSN	5	100.24 ± 0.328
Amlovas™-L	AML	5	99.84 ± 0.106

Method Parameter	Proposed HPTLC Method		Reported HPLC Method
Stationery Phase	Silica Gel 60F254 (10 X 10 cm)		Phenomenex C18 (250 X 4.6 mm, 5µ)
Mobile Phase	N-butanol : methanol: ammonia (4:4:1 v/v/v)		0.02 M Phosphate buffer : methanol (75 : 25 v/v)
Linearity	1000 – 6000 ng/band for both drugs		10 – 110 µg/ml for both drugs
Drug Estimated mg/tablet	LSN- 5.01 AML- 4.99		LSN- 5.05 AML-5.03
Precision (% RSD)	Less than 2		Less than 2
Accuracy (% recovery)	LSN	AML	LSN	AML
Level- 80 %	99.91 ±0.082	99.92 ±0.685	100.54 ±0.536	100.13 ±0.853
Level- 100 %	100.81 ±0.405	99.91±0.085	100.06 ±0.626	100.57 ±0.514
Level- 120 %	100.16 ±0.208	100.0 ±0.185	100.04±0.127	100.51±0.405
Specificity	Specific		Specific
LOD and LOQ	LOD and LOQ: LSN- 2.602 and 7.686 ng/band AML- 1.437 and 4.821 ng/band		LOD and LOQ: LSN- 0.025 and 0.075µg/ml AML- 0.029 and 0.090 µg/ml
Robustness	Robust		Robust