INTRODUCTION:

Cilnidipine (CIL): a calcium channel blocker is used as an antihypertensive agent. It inhibits the influx of Ca²⁺ which causes the vasodilation and decreases the heart rate and also decreases cardiac contraction in heart¹.Nebivolol (NBV) is a selective β1 receptor antagonist. It blocks the β1 receptor which reverses the effect of epinephrine, lowering the heart rate and blood pressure. It has mild vasodilating effect due to interaction with L-arginine/ nitric oxide pathway¹.

Literature survey reveals that various chromatographic methods like HPTLC, RP-HPLC, LC-MS and spectroscopic methods have been reported for analysis of CIL and NBV either alone or in combination^2-16.

It was observed that the reported UV simultaneous equations method employed methanol as diluent. Spectroscopic grade methanol is an expensive organic solvent. It would be worthwhile to reduce its consumption so as to bring down the cost of analysis. UV methods are advantageous in the methods being rapid and hence in recent times there is renewed interest in development of UV spectroscopic methods. No method has been reported for estimation of CIL and NBV hydrochloride in combined dosage form by Absorbance correction method and Q absorption ratio method. Hence an attempt was made to develop two novel UV spectrophotometric methods for simultaneous estimation of CIL and NBV hydrochloride in pharmaceutical formulation.

MATERIALS AND METHODS:

Chemicals and Reagents:

Active pharmaceutical ingredients, Cilnidipine (CIL) and Nebivolol hydrochloride (NBV) were obtained as gift samples from ACME Generics, Baddiand Glenmark Pharmaceuticals Ltd, Goa, respectively. The tablets containing nebivolol hydrochloride 5mg and cilnidipine 10 mg were procured from local pharmacy sold under brand name LN βeta 5. Distilled water and methanol of spectroscopic grade was used throughout the experiment.

Instrumentation and Equipment:

A double beam UV Spectrophotometer of Shimadzu make, model UV 1800 with UV probe software was used for analysis.

Methods:

Choice of diluent:

Stock solution of the drugs was prepared in methanol. The choice of diluent was arrived at by using various proportions of methanol and water i.e. (60:40, v/v), (50:50, v/v), (40:60, v/v). The absorbance values of solution prepared by taking 0.1mL of stock and diluted to 25mL using the prepared diluent was monitored. The proportion allowing the lowest amount of methanol and giving a stable absorbance reading was selected.

Preparation of solutions:

Preparation of stock solution of drugs (1000µg/mL):

About 100mg of the drugs were accurately weighed and transferred into 100mL volumetric flasks and dissolved with sufficient volume of methanol. Volume was made up to 100mL with methanol to get concentration of 1000 μg/mL.

Preparation of working standard solution of cilnidipine (200µg/mL):

Working standard solution of cilnidipinewas prepared by taking 5mL of stock solution of cilnidipine and diluting it to 25mL using diluent to get concentration of 200μg/mL.

Preparation of working standard solution of nebivolol hydrochloride (100 µg/mL):

Working standard solution of nebivolol hydrochloride was prepared by taking 2.5mL of stock solution of nebivolol hydrochloride and diluting it to 25 mL using diluent to get concentration of 100μg/mL.

Choice of wavelength:

The working standard solutions of the drugs were scanned in the UV region 200nm to 400nm using diluent as blank. Using the peak pick function, the λ max of the drugs was obtained. The spectra were then overlain to obtain the Isosbestic point. The wavelengths for analysis for the two UV methods were then finalised as follows:

· For absorbance correction method: λ1 (λmax of CIL where NBV shows nil absorbance) and λ2 (λmax of NBV where both drugs show satisfactory absorbance).

· For Q absorption ratio method: λ1 (Isosbestic point) and λ2 (λmax of drug having longer wavelength).

Determination of absorptivity(a):

For determining absorptivity of the drugs by the two UV methods, aliquots of stock solution of CIL and NBV were delivered into a series of 25ml volumetric flasks and volume was made up with diluent to attain concentrations within the Beer Lambert’s range. Absorbance of the solutions was recorded at predetermined wavelengths and absorptivity values for CIL and NBV were calculated using the following formula:

a = A / (b × c)

where, a = absorptivity, A = absorbance, b = pathlength, c = concentration (g/1000mL).

Method Validation¹⁷:

Linearity and range:

For determining linearity by the two UV methods, aliquots of stock solution of CIL and NBV were delivered into a series of 25ml volumetric flasks and volume was made up with diluent to attain concentrations ranging from 4 – 100μg/mL.Absorbance of the solutions was recorded at predetermined wavelengths and calibration curves were plotted of absorbance v/s concentration. The linear regression equations and correlation coefficients (r²) were determined for the drugs at the predetermined wavelengths and the Beer-Lambert’s range was established.

Precision:

Repeatability:

From the working standard solutions of each drug, 2.5 mL was withdrawn and transferred to six 25mL volumetric flasks and volume was made up to the mark with the diluent. Absorbance of these solutions was measured against the blank at the predetermined wavelengths. The amount of the drugs and %RSD was calculated by both the UV methods.

Intra-day precision:

Intraday precision was carried out by performing assay procedure three times on the same day in triplicate. The % assay and %RSD was calculated by both the methods.

Inter-day precision:

Inter day precision was carried out by repeating assay procedure on three different days in triplicate. The % assay and % RSD was calculated by both the methods.

Accuracy:

Accuracy was established by performing recovery studies by using preanalysed sample solution. The accuracy of the method was performed at three concentration levels in triplicate (80%, 100% and 120%) and the % recovery was determined.

Limit of Detection and Quantitation:

LOD and LOQ were calculated using the slope and standard deviation response of calibration curves of drugs at chosen wavelengths. The values were calculated using following mathematical formulas:

LOD = 3.3 σ/S

LOQ = 10 σ/S

where, σ – Standard deviation of response (Y intercept), S – Slope of calibration curve

Robustness:

Robustness of developed method was determined by performing assay procedure on marketed tablet formulation by employing the following deliberate variations:

· Change of UV spectrophotometer instrument.

· Change of analyst.

· Change of proportion of diluent i.e. methanol and water (35:65, v/v).

Assay of marketed tablet formulation:

For preparation of sample solution for assay, 10 tablets of LN βeta 5 (tablets containing NBV 5 mg and CIL 10 mg per tablet) were accurately weighed and finely powdered in a glass mortar and pestle. Tablet powder equivalent to 5mg of NBV and 10mg of CIL was weighed and transferred to 50mL volumetric flask and about 35mL of methanol was added and sonicated for 10 minutes. The volume was made up to the mark with methanol. The resulting solution was filtered through Whatman filter paper no. 1. From above filtrate, 2.5mL was pipetted out into a 25mL volumetric flask and volume was made up to mark with diluent. The absorbance of the resulting solution was recorded at predetermined wavelengths. Percent purity of the marketed formulation was calculated by both the methods.

Formula used for calculation of concentration of drug by:

· Absorbance Correction Method:

As per Beer-Lambert’s law:

A = abc

where, A = absorbance, a = absorptivity, b = pathlength, c = concentration

In absorbance correction method, at λ₁, since only one drug (X) shows absorbance the concentration of the drug (X) can be calculated as:

c_X = A₁/ ax₁ x b

where, c_{X =} Concentration of drug X,A₁ = Absorbance of sample solution at λ₁,

ax₁ = Absorptivity of drug X at λ₁, b = Pathlength (1cm).

At λ₂, both the drugs (X and Y) show absorbance and the concentration of the drug (Y) can be calculated as:

A₂ = A_X + A_Y

A₂ = (ax₂ x c_Xx b) + (ay₂ x c_Y x b)

c_Y = [A₂ – (ax₂ x c_Y)] / ay₂

where, c_{X =} Concentration of drug X,c_Y
= Concentration of drug Y,

A₂ = Absorbance of sample solution at λ₂,ax₂ = Absorptivity of drug X at λ₂,

ay₂ = Absorptivity of drug Y at λ₂,b = Pathlength (1cm).

· Q Absorption Ratio Method:

C_X = (Qm - Qy / Qx - Qy) X A₁/ax₁

C_Y= (Qm - Qy / Qx - Qy) X A₁/ay₁

where, C_X= Concentration of drug X,C_Y
= Concentration of drug Y,

Qm = Abs. of tablet sol. at λ₁/ Abs. of tablet sol. at λ₂,

Qx = ax₂/ ax_1,Qy = ay₂/ ay₁,

A₁ = Abs. of sample solution at λ₁, A₂ = Abs. of sample solution at λ₂,

ax₁ = Absorptivity of drug X at λ₁, ay₁ = Absorptivity of drug Y at λ₁,

ax₂= Absorptivity of drug X at λ₂, ay₂ = Absorptivity of drug Y at λ₂.

RESULTS AND DISCUSSION:

Method development:

Choice of diluent:

CILand NBV are soluble in methanol. Most of the analytical methods reported useof methanol as solvent. In the current developed method, optimum use of methanol so as to bring down the cost of analysis was attempted. Various proportions of methanol and water as diluent were tried. Mixture of methanol and water in proportion of 40:60, v/v was chosen as diluent with satisfactory results.

Choice of wavelength:

Working standard solutions of CIL and NBV when scanned in UV range 200-400 nm against blank, showed λ _max at 242.6nm and 280.6nm for the drugs respectively. The overlain spectra (fig 1) of cilnidipine and nebivolol hydrochloride showed Isosbestic points at 275nm and 292.6nm.

Fig. 1: Overlain spectra of CIL and NBV showing Isosbestic points.

The wavelengths for analysis for the two UV methods were arrived as follows:

· For absorbance correction method, λ_max of CIL 242.6 nm was chosen as λ₁, as NBV shows nil absorbance at this wavelength and 280.6nm, the λ_max of NBV was chosen as λ₂, as both the drug showed satisfactory absorbance at this wavelength.

· For Q absorption ratio method, 275nm (Isosbestic point) was selected as λ₁ and 280.6 nm ₍λ_maxof NBV) as λ₂

Determination of absorptivity:

· Absorptivity for CIL and NBV by absorbance correction method:

Solutions were prepared as per methodology and were scanned at selected wavelengths. The absorptivity values obtained for CIL were 83.59 and 17.35 L/gm/cm at 242.6 and 280.6 nm respectively. For nebivolol, the mean absorptivity was 12.64 L/gm/cm at 280.6 nm.

· Absorptivity for CIL and NBV by Q absorption ratio method:

The absorptivity values obtained for CIL were 20.79 and 17.35 L/gm/cm at 275 and 280.6 nm respectively. For nebivolol, the mean absorptivity was 10.21 and 12.64 L/gm/cm at 275 and 280.6 nm respectively.

Method Validation:

Linearity and Range:

Linearity data by Absorbance correction method:

The calibration curves obtained for cilnidipine and nebivolol are displayed in figures 2 and 3 below.

Fig 2:Calibration curves of CIL at 242.6 and 280.6nm

Fig 3: Calibration curve of NBV hydrochloride at 280.6nm

As seen above, Beer Lambert’s range was established for CIL from 4-48µg/mL, and for NBV hydrochloride from 4-100 µg/mL.

Linearity data by Q absorption ratio method:

The calibration curves obtained for the drugs are displayed in figures 4 and 5.

Fig4: Calibration curves of CIL at 275 and 280.6nm

Fig 5: Calibration curves of NBV hydrochloride at 275 and 280.6nm

As seen above, Beer Lambert’s range was established for CIL from 4-48µg/mL, and for NBV hydrochloride from 4-100µg/mL.

Precision:

Solutions for precision study were prepared as per methodology and absorbance values recorded at predetermined wavelengths. The results for repeatability, Intraday and Interday precisionby the methodsare depicted in table 1 below.

Table 1: Precision data of CIL and NBV

By absorbance correction method
Drug	Conc. (µg/mL)	Mean % assay ± SD	% RSD
Repeatability (n=6)
CIL	20	97.03 ± 0.531	0.547
NBV	10	97.13 ± 1.425	1.467
Intra-day precision (n=3)
CIL	20	96.62 ± 0.685	0.709
NBV	10	101.96 ± 1.071	1.050
Inter-day precision (n=3)
CIL	20	99.69 ± 0.405	0.407
NBV	10	107.36 ± 0.778	0.725
By Q absorption ratio method
Repeatability (n=6)
CIL	20	96.54 ± 0.374	0.387
NBV	10	97.99 ± 1.253	1.278
Intra-day precision (n=3)
CIL	20	93.49 ± 0.901	0.964
NBV	10	108.83 ± 0.295	0.271
Inter-day precision (n=3)
CIL	20	98.30 ± 0.576	0.586
NBV	10	109.99 ± 0.509	0.463

The % RSD for repeatability study, intraday and inter-day precision for CIL and NBV hydrochloride by both methods was found to be within the acceptable limits (<2%); hence the developed methodsare precise.

Accuracy:

Accuracy of the methods were confirmed by carrying out % recovery of standard solution of drug added at three levels in triplicate (80%, 100%, and 120%) to preanalysed formulation. The results obtained are depicted in table2.

Table 2: % Recovery of CIL and NBV

By absorbance correction method
Amt. of sample (μg/mL)	Level of addition (%)	% Recovery		Mean% Recovery
Amt. of sample (μg/mL)	Level of addition (%)	CIL	NBV	CIL	NBV
CIL: 20 (μg/mL) NBV: 10 (μg/mL)	80	97.52	101.05	97.55	101.27
	80	97.44	101.50
	80	97.69	101.27
	100	101.62	100.85	101.43	100.68
	100	101.37	100.45
	100	101.32	100.75
	120	98.70	101.72	98.72	102.20
	120	98.65	102.81
	120	98.81	102.09
By Q absorption ratio method
CIL: 20 (μg/mL) NBV: 10 (μg/mL)	80	95.83	104.22	96.38	103.81
	80	96.94	103.11
	80	96.38	104.11
	100	99.57	104.70	99.44	104.38
	100	98.57	105.00
	100	100.20	103.45
	120	96.81	100.45	97.66	102.40
	120	97.97	104.50
	120	98.22	102.27

The results of % recovery at 80%, 100% and 120% for CIL and NBV lie within acceptable criteria of 95 – 105 %. Hence the developed methodsare accurate.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The results for LOD and LOQ obtained are shown below in table 3.

Table 3: LOD and LOQ of CIL and NBV

By absorbance correction method
Parameter	CIL		NBV
	At 242.6 nm	At 280.6 nm	At 280.6 nm
LOD (µg/mL)	1.351	0.605	1.760
LOQ (µg/mL)	4.004	1.834	3.333
By Q absorption ratio method
Parameter	CIL		NBV
	At 275 nm	At 280.6 nm	At 275 nm	At 280.6 nm
LOD (µg/mL)	0.194	0.605	1.905	1.760
LOQ (µg/mL)	0.588	1.834	3.773	3.333

As seen from table, the developed methods for CIL and NBV hydrochloride were sensitive.

Robustness:

The results obtained forrobustness study by both absorbance correction method and Q absorption ratio method showed that deliberate changes introduced did not affect the results of analysis significantly. Hence, both the developed methods were robust.

Assay of Marketed formulation:

The results for % assay of marketed formulation for CIL and NBV hydrochloride by absorbance correction method was found to be 97% and 102% respectively, while by Q absorption ratio method it was 95% and 105% respectively, which were within the acceptance criteria (90-110 %).

CONCLUSION:

Two novel, simple and economical UV Spectroscopic methods for simultaneous analysis of CIL and NBV in bulk and in tablet formulation have been developed and validated. The developed UV spectroscopic methods employed 40% methanol as diluent for analysis. The methods were validated as per ICH guidelines. The % assay of CIL and NBV in tablet dosage form were found to be 97 % and 102 % respectively by absorbance correction method, and 95 % and 105 % respectively by Q absorption ratio method, which were within acceptable limits. Hence, the developed methods can be used as a cost-effective alternative to the earlier reported methods for the simultaneous estimation of the drugs both in bulk and in their combined formulation.

ACKNOWLEDGEMENT:

The authors are thankful to ACME Generics, Baddiand Glenmark Pharmaceuticals Ltd, Goa for the gift samples of CIL and NBV.

CONFLICT OF INTEREST

Authors do not have a conflict of interest.

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Received on 11.01.2022 Modified on 13.08.2022

Research J. Pharm. and Tech 2023; 16(7):3213-3218.

DOI: 10.52711/0974-360X.2023.00528