INTRODUCTION:

Cilnidipine (CIL) (Fig. 1) is a novel, dihydropyridine class of calcium-channel blocker. It has been classified as a fourth-generation calcium-channel blocker based on its actions on sympathetic neurotransmitter release. Cilnidipine reduces BP by acting on the calcium channels (L-type) of blood vessels and blocking the incoming calcium and suppressing the contraction of blood vessels. It also works on the N-type calcium channel located at the end of the sympathetic nerve.^1,2,3

Figure 1: Chemical structure of Cilnidipine

Olmesartan (OLM) (Fig. 2) is a specific angiotensin II type 1 (AT1) receptor antagonist. It selectively inhibits the binding of angiotensin II to AT1, which is found in many tissues such as vascular smooth muscle and the adrenal glands. This effectively inhibits the AT1-mediated vasoconstrictive and aldosterone-secreting effects of angiotensin II (via the renin-angiotensin-aldosterone system) and results in a decrease in vascular resistance and blood pressure. Olmesartan is selective for AT1 and has a 12,500 times greater affinity for AT1 than the AT2 receptor.^1,2,3

Figure 2: Chemical structure of Olmesartan medoxomil

CILIDIN-O 20 tablet is a combination of olmesartan and cilnidipine which effectively lowers blood pressure. They work by relaxing the blood vessels and making the heart more efficient at pumping blood throughout the body.^1,2,3

Literature survey reveals that various analytical methods like UV^4-8 and RP-HPLC^9-13 have been reported for the analysis of cilnidipine and olmesartan either alone or in combination.

HPLC methods uses expensive instrumentation, large amount of expensive organic solvents and are time consuming. There has been a renewed interest in UV spectroscopic methods as they offer advantages of being rapid, simple and offer cost benefits. Though UV spectroscopic analysis for the drug combination has been reported previously, it employed methanol as solvent, which is expensive. Hence it was thought worthwhile to develop and validate a new and simple cost effective UV spectrophotometric method for simultaneous estimation of cilnidipine and olmesartan employing the absorption correction principle and apply the same in the estimation of the drugs in their marketed formulation.

MATERIALS AND METHODOLOGY:

Chemicals and Reagents:

Cilnidipine was obtained as a gift sample from ACME generics, Baddi, Himachal Pradesh, India. Olmesartan medoxomil was obtained as a gift sample from Unichem Laboratories Ltd., Goa, India. The tablet CILIDIN-O 20: containing 10mg cilnidipine and 20mg olmesartan, was procured from the local pharmacy. Distilled water and methanol used were of spectroscopic grade.

Instrumentation:

An UV/Visible Spectrophotometer of Shimadzu make model UV -1800 was used for analysis.

Choice of Diluent:

Stock solution of the drugs were prepared in methanol. The choice of diluent was arrived at by using various proportions of methanol and water i.e. (80:20, v/v), (60:40, v/v), (50:50, v/v), (40:60, v/v) and sonicating for 5 minutes. The absorbance values of solution prepared by taking 0.1mL of stock and diluted to 25mL using the prepared diluent were recorded. The proportion allowing the lowest amount of methanol and giving a stable absorbance reading was selected.

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

About 100mg of cilnidipine was accurately weighed and transferred into 100mL volumetric flask and dissolved with sufficient volume of methanol. Volume was adjusted to 100mL with methanol to get concentration of 1000μg/mL.

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

About 100mg of olmesartan medoxomil was accurately weighed and transferred into 100mL volumetric flask and dissolved with sufficient volume of methanol. Volume was adjusted to 100mL with methanol to get concentration of 1000μg/mL.

Preparation of working standard solution of cilnidipine (10μg/mL):

The working standard solution of cilnidipine was prepared by transferring 0.25mL of cilnidipine stock solution to 25mL volumetric flask and volume was made up to the mark with the diluent to obtain a concentration of 10µg/mL.

Preparation of working standard solution of olmesartan (10μg/mL):

The working standard solution of olmesartan was prepared by transferring 0.25mL of olmesartan stock solution to 25mL volumetric flask and then volume was made up to the mark with the diluent to obtain a concentration of 10µg/mL.

Choice of wavelength for absorption correction method:

The working standard solution of cilnidipine and olmesartan were scanned in the UV region 200 nm to 400 nm using diluent as blank. The two spectra were overlaid. From the overlain spectra, the two wavelengths for the absorption correction method were selected as follows: ʎ₁ absorbance due to cilnidipine only (olmesartan shows zero absorbance) and ʎ₂absorbance due to both drugs.

Determination of absorptivity:

For determining absorptivity of the drugs, aliquots of stock solution of cilnidipine and olmesartan were delivered into a series of 25mL volumetric flasks and volume was made up with diluent to attain concentrations from 10 to 100µg/mL. Absorbance of the solutions were recorded at predetermined wavelengths and absorptivity was calculated using the following formula:

a = A / b c

where,

a = absorptivity

A = absorbance

b = pathlength

c = concentration (g/1000mL)

Method Validation:

Linearity and range:

For determining linearity and range, aliquots of stock solution of cilnidipine and olmesartan were used to prepare serial dilutions having concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100µg/mL. The absorbance of these solutions was recorded at ʎ₁and ʎ₂. The calibration curve of Absorbance v/s Concentration (µg/mL) was plotted and the absorptivity, linear regression equation and correlation coefficients (r²) were determined.

Acceptance Criteria:

Correlation Coefficient (r²) ≥ 0.999

Precision:

The precision of the method was established by performing repeatability, intraday and intermediate precision studies.

Repeatability:

The repeatability of the method was established by performing the assay of the formulation six times. The amount of each drug present in the tablet formulation was calculated and results were reported in terms of % purity and relative standard deviation (% RSD).

Acceptance Criteria:

% Assay = 90 - 110 % and % RSD ≤ 2.

Intermediate Precision:

The Intra-day and Inter-Day Precision studies were performed by analysing the tablet formulation three times on same day and on three consecutive days respectively. The amount of each drug present in the tablet formulation was calculated and results were reported in terms of % purity and relative standard deviation (% RSD).

Acceptance Criteria:

% Assay = 90 - 110% and % RSD ≤ 2.

Accuracy:

The accuracy was determined by conducting % recovery studies on preanalysed formulation by adding the standard solution at three levels (80%, 100% and 120%) in triplicate. Nine 25mL volumetric flask were taken. To each flask, tablet powder equivalent to 10mg cilnidipine was added. To the first set of three flasks 0.8mL of cilnidipine and 1.6mL of olmesartan standard solution were added. To the second set of three flasks 1mL of cilnidipine and 2mL of olmesartan standard solution were added. To the third set of three flasks 1.2mL of cilnidipine and 2.4mL of olmesartan standard solution were added. Around 15mL methanol was added to all flasks and the solution was sonicated for 10 minutes. The volume was made up to the mark using methanol, mixed and then filtered through a Whatman filter paper (No.1). About 2.5mL of filtrate from each flask was further diluted to 25mL using diluent. The absorbance of the solutions were recorded at ʎ₁ and ʎ₂ and % recovery calculated.

Acceptance criteria:

% Recovery = 95%-105%

Limit of detection (LOD) and Limit of quantitation (LOQ):

The LOD and LOQ for cilnidipine and olmesartan were determined from calibration curve by calculating the signal to noise ratio (S/N). Following formulae were used:

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

where,

σ = the standard deviation of the response

S = slope of the calibration curve

Robustness:

Robustness was analysed by deliberately varying the following experimental conditions: change of UV spectrophotometer instrument, change of analyst and diluent ratio by ± 5%.

Analysis of tablet formulation:

The average weight of ten tablets was calculated after weighing. The tablets were powdered and tablet powder equivalent to 10mg of cilnidipine was weighed and transferred to a 25mL volumetric flask. Around 15mL methanol was added and sonicated for 10 minutes. The solution was made up to the mark using methanol and filtered through a Whatman filter paper (No.1). Further dilutions were made to obtain the final concentration of 15µg/mL of cilnidipine and 30µg/mL of olmesartan. The absorbance of the solution was recorded at ʎ₁ and ʎ₂. The amount of each drug present in the tablet formulation was calculated using the absorption correction principle and % purity calculated.

Acceptance Criteria:

% Assay = 90% - 110%

Calculations:

Calculations by Absorbance Correction method:

As per Beer-Lambert’s law: A = abc

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

Calculation of concentration of cilnidipine at λ₁:

Since olmesartan does not absorb at λ₁, concentration of cilnidipine was directly estimated at λ₁(365nm)

C_CIL=A₁/a_x1x b………………………………………..(1)

Calculation of absorbance of cilnidipine at λ_{2 :}

Since cilnidipine and olmesartan both absorb at λ₂, the resulting absorbance is the outcome of absorbance due to both the drugs.

A=A_CIL+A_OLM………………………………………..(2)

Absorbance of cilnidipine at λ₂was calculated as shown in the equation 3 below.

A_CILatλ2= a_x2 x C_CIL x b……………........................... (3)

Calculation of absorbance of olmesartan at λ₂ corrected for cilnidipine:

The absorbance due to olmesartan at λ₂ was calculated by subtracting the absorbance of cilnidipine at λ₂from the total absorbance at λ₂as shown in the equation 4 below.

A_OLM=A-A_CILatλ2 ……………………………………..(4)

Calculation of concentration of Olmesartan:

The concentration of olmesartan was calculated from the corrected absorbance of olmesartan at λ₂as shown in the equation 5.

C_OLM=A_OLM/a_y2……………………………………… (5)

where,

λ₁= Wavelength at which cilnidipine only absorbs,

λ₂= Wavelength at which both cilnidipine and olmesartan absorb,

C_CIL = Concentration of cilnidipine,

C_OLM = Concentration of olmesartan,

A = Total absorbance at λ_2,

A_CIL = Absorbance. of cilnidipine at λ₁,

A_OLM = Absorbance of olmesartan at λ₂,

ax₁ = Absorptivity of cilnidipine at λ₁,

ax₂ = Absorptivity of cilnidipine at λ₂,

ay₂ = Absorptivity of olmesartan at λ_2.

RESULTS AND DISCUSSION:

Choice of Diluent:

Among the various proportions of water and methanol tried as diluent i.e. (80:20, v/v), (60:40, v/v), (50:50, v/v), (40:60, v/v), the proportion of 80:20, v/v of water and methanol gave stable readings with least use for amount of methanol. Hence, water and methanol in the proportion of 80:20, v/v was chosen as diluent for further analysis.

Selection of analytical wavelength:

The working standard solutions of cilnidipine and olmesartan were scanned in the UV region 200 nm to 400 nm using diluent as blank and ʎ_maxwas recorded. The ʎ_max of cilnidipine and olmesartan was found to be 243 nm and 257 nm respectively. The study of overlain spectra of cilnidipine and olmesartan medoxomil (Fig. 3) showed that, cilnidipine absorbed at 365 nm but olmesartan showed zero absorbance while at 257 nm, both the drugs showed absorbance. Hence, 365 nm and 257 nm were selected as wavelengths for analysis (λ_{1 and}λ₂respectively). Cilnidipine was directly estimated at 365 nm and olmesartan was estimated at 257 nm after calculating the corrected absorbance.

Fig. 3: Overlain spectra of cilnidipine and Olmesartan

Determination of absorptivity:

The solutions were prepared as per the methodology and were scanned at the selected wavelengths. The absorptivity values are shown in the table 1 and 2.

Table 1: Absorptivity values for cilnidipine at 257 nm and 365 nm

Sr. No	Concentration (µg/mL)	Absorbance at 257 nm	Absorptivity ax₁ (L g^-1 cm^-1) at 257 nm	Absorbance at 365nm	Absorptivity ax₂ (L g^-1 cm^-1) at 365 nm
1	10	0.432	43.20	0.189	18.90
2	20	0.762	38.10	0.450	22.22
3	30	1.041	34.70	0.650	21.66
4	40	1.327	33.17	0.876	21.90
5	50	1.626	32.52	1.080	21.60
6	60	1.942	32.37	1.319	21.98
7	70	2.296	32.80	1.512	21.60
8	80	2.619	32.74	1.713	21.40
9	90	2.876	31.95	1.875	20.80
10	100	3.199	31.99	2.102	21.02
AVERAGE			34.34		21.31

The mean absorptivity values of Cilnidipine were obtained as: 34.34 L g^-1 cm^-1 at 257 nm and 21.31 L g^-1 cm^-1 at 365 nm.

Table 2: Absorptivity values for olmesartan at 257 nm

Sr. No	Concentration (µg/mL)	Absorbance at 257 nm	Absorptivity a_x1 (L g^-1 cm^-1) at 257 nm
1	10	0.447	44.70
2	20	0.899	44.95
3	30	1.338	44.60
4	40	1.800	45.00
5	50	2.202	44.04
6	60	2.628	43.80
7	70	3.129	44.70
8	80	3.466	43.32
9	90	3.704	41.55
10	100	3.815	38.15
AVERAGE			44.39

The mean absorptivity value for olmesartan at 257 nm was found to be 44.39 L g^-1 cm^-1.

Method Validation:

Linearity:

The calibration curves for cilnidipine and olmesartan obtained are displayed in the figures 4 and 5. The Beer Lambert’s range as seen in table 3 for cilnidipine was established from 10-100 µg/mL and that for olmesartan was established from 10-80 µg/mL. The calibration curves obtained were linear in the concentration range as the correlation coefficient (r²) was greater than 0.990.

Fig 4: Calibration curve of cilnidipine at 257 nm and 365 nm

Fig 5: Calibration curve of olmesartan at 257 nm

Table 3: Linearity data for cilnidipine and olmesartan

Sr. No	Parameter	CIL		OLM
	Wavelength	257 nm	365 nm	257 nm
1.	Linearity Range (µg/mL)	10-100	10-100	10-80
2.	Slope (m)	0.1182	0.0215	0.0303
3.	Intercept	0.0308	0.021	0.0435
	Linear regression equation	y = 0.0308x + 0.1182	y = 0.021x + 0.0215	y = 0.0435x + 0.0303
4.	Correlation coefficient (r²)	0.999	0.998	0.999

Precision:

The solutions for precision studies were prepared as per methodology and absorbance values recorded at selected wavelengths. The precision analysis results are shown in table 4.

Accuracy:

Accuracy of the method was confirmed by carrying out % recovery of standard solution of drug added at three levels (80%, 100% and 120%) to preanalysed formulation. The results of accuracy studies are shown in table 5.

Table 5: % Recovery for cilnidipine and olmesartan

Amount of sample (µg/mL)	Level of addition (%)	Amount of pure drug added (µg/mL)		Total concentration (µg/mL)		Abs at 257 nm	Abs at 365 nm	Total concentration found (µg/mL)		% Recovery		Mean recovery (%)
Amount of sample (µg/mL)	Level of addition (%)	CIL	OLM	CIL	OLM	Abs at 257 nm	Abs at 365 nm	CIL	OLM	CIL	OLM	CIL	OLM
CIL: 15 (µg/mL) OLM: 30 (µg/mL)	80	1.20	2.40	16.20	32.40	1.998	0.345	16.19	32.48	99.93	100.24	100.04	100.56
	80	1.20	2.40	16.20	32.40	1.998	0.346	16.24	32.44	100.25	101.12
	80	1.20	2.40	16.20	32.40	1.999	0.345	16.19	32.50	99.93	100.31
	100	1.50	3.00	16.50	33.00	2.037	0.352	16.52	33.11	101.12	100.32	100.04	100.43
	100	1.50	3.00	16.50	33.00	2.038	0.351	16.43	33.16	99.5	100.38
	100	1.50	3.00	16.50	33.00	2.038	0.351	16.43	33.16	99.5	100.48
	120	1.80	3.60	16.80	33.60	2.075	0.359	16.85	33.71	100.29	100.32	100.29	100.26
	120	1.80	3.60	16.80	33.60	2.074	0.359	16.85	33.68	100.29	100.23
	120	1.80	3.60	16.80	33.60	2.074	0.359	16.85	33.68	100.29	100.23

The % recovery at 80%, 100% and 120% for cilnidipine was found to be 100.04%, 100.04% and 100.29% respectively and for olmesartan was 100.56%, 100.43% and 100.26 respectively and it lies within the acceptable criteria of 95 – 105 %. Hence the developed method was found to be accurate.

Limit of detection and Limit of quantitation:

The LOD and LOQ for cilnidipine and olmesartan were determined from the calibration curves by calculating the signal to noise ratio (S/N). The LOD and LOQ values are given in table 6. As seen from table, the method was found to be sensitive.

Table 6: LOD and LOQ data for cilnidipine and olmesartan

Parameter	CIL		OLM
Parameter	At 257 nm	At 365 nm	At 257 nm
LOD (µg/mL)	0.859	3.223	4.737
LOQ (µg/mL)	2.60	9.767	14.37

Robustness:

Robustness of the method was established by performing the assay of the formulation by deliberately introducing minor changes in experimental conditions like: varying the diluent ratio by ±5%, change of UV spectrometer instrument and change of analyst. The results of robustness study are shown in table 7.

Table 7: Robustness data for cilnidipine and olmesartan

Parameters	Absorbance		% Assay
Parameters	at 257 nm	at 365 nm	OLM	CIL
Change of UV instrument
Instrument 1	1.902	0.321	103.97	100.43
Instrument 2	1.909	0.324	104.13	101.37
Change of analyst
Analyst 1	1.902	0.321	103.97	100.43
Analyst 2	1.902	0.322	103.77	100.75
Change of diluent proportion
Water: Methanol (80:20, v/v)	1.902	0.321	103.97	100.43
Water: Methanol (85:15, v/v)	1.906	0.328	103.41	102.62

The method was found to be robust as minor, deliberate changes introduced in the experimental conditions did not significantly affect the results of analysis. Hence, the developed method for cilnidipine and olmesartan was found to be robust.

Assay of marketed formulation:

The developed method was applied for assay of cilnidipine and olmesartan in tablet formulation. The sample solution was prepared as per the methodology and the absorbance was recorded at preselected wavelengths. The results are displayed in table 8.

Table 8: Results of assay of marketed formulation

Drug

Label claim

(mg)

Amt. found

(mg)

% assay

CIL

10.07

100.75

OLM

20.76

103.80

As seen above, % assay for marketed formulation was found to be 100.75% for cilnidipine and 103.80% for olmesartan which was within the acceptance criteria (90-110%).

CONCLUSION:

A simple and novel UV absorption correction method has been developed for simultaneous analysis of cilnidipine and olmesartan in bulk and in tablet formulation. The developed UV spectroscopic method employed water : methanol (80:20, v/v) as the diluent. The method was validated following the ICH guidelines. The linearity was established over a concentration range of 10-100μg/mL for cilnidipine and 10-80μg/mL for olmesartan. The method was found to be accurate, precise, sensitive and robust. The developed method was applied for assay of cilnidipine and olmesartan in tablet formulation. The % assay in the marketed formulation was found to be 100.75% for cilnidipine and 103.8% for olmesartan which was within the acceptance criteria. Hence, the developed method can be used as a cost-effective alternative to the earlier reported UV and HPLC methods for the simultaneous estimation of the drugs both in bulk and in their combined formulation.

ACKNOWLEDGEMENT:

The authors are thankful to ACME Generics, Baddi, Himachal Pradesh, India for gift sample of Cilnidipine and Unichem Laboratories Ltd., Goa, India for gift sample of Olmesartan medoxomil.

CONFLICT OF INTEREST:

Authors do not have a conflict of interest.

REFERENCES:

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Received on 17.09.2020 Modified on 06.03.2021

Research J. Pharm.and Tech 2022; 15(2):863-869.

DOI: 10.52711/0974-360X.2022.00144

Drug	Conc. (µg/mL)	Mean % assay ± SD	% RSD
Repeatability (n=6)
CIL	10	100.49 ±0.501	0.499
OLM	20	103.77 ± 0.293	0.283
Intra-day precision (n=3)
CIL	10	100.87 ± 0.218	0.753
OLM	20	103.81 ± 0.008	0.083
Inter-day precision (n=3)
CIL	10	101.12 ± 0.760	0.215
OLM	20	103.81 ± 0.087	0.008