1. INTRODUCTION:

Clopidogrel, a compound synthesized from Ticlopidine, serves as a vital adenosine diphosphate (ADP) inhibitor, primarily employed to mitigate thrombotic events in the treatment of conditions such as myocardial infarctions, strokes, and cardiovascular diseases¹. Its mechanism of action involves the irreversible blockade of the ADP receptor P2Y12, a pivotal element in platelet aggregation and the cross-linking of platelets via fibrin. Inhibition of this receptor disrupts the glycoprotein IIb/IIIa pathway, resulting in the inhibition of platelet aggregation².

Figure 1: Structure of Clopidogrel Bisulphate

Utilizing the nasal route for the administration of clopidogrel bisulphate to access the brain is an emerging and promising strategy. Simulated nasal fluid closely mimics the ionic composition of natural nasal fluid. To enable the nasal administration of clopidogrel bisulphate, it becomes imperative to develop a UV spectroscopic method for assessing drug release and permeation within the nasal environment.

Clopidogrel bisulphate presents as a white to off-white powder, exhibiting practical insolubility in neutral water but readily dissolving in water with a pH of 1. It also demonstrates solubility in methanol, limited solubility in methylene chloride, and virtually no solubility in ethyl ether.

A comprehensive review of the literature has revealed various estimation methods for clopidogrel bisulphate, including high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS)³^,⁴. However, the existing literature lacks any method for the estimation of clopidogrel bisulphate in simulated nasal fluid. Consequently, this current investigation was undertaken to develop and validate a UV-spectrophotometric method for the analysis of clopidogrel bisulphate in simulated nasal fluid (NSF).

2. MATERIAL AND METHOD:

2.1 Material:

Clopidogrel bisulphate was acquired as a complimentary sample from Dr. Reddy's Laboratory, situated in Buddi, Himachal Pradesh, India. All other chemicals utilized in this study were of analytical grade.

2.2 Method Development:

2.2.1 Instrumentation: A spectral scan was conducted using a Shimadzu UV spectrophotometer, Model 1800 (Shimazdu 1800, Japan).

2.2.2 Preparation of simulated nasal fluid: Simulated nasal fluid (SNF) was formulated by dissolving precisely measured amounts of NaCl (7.45mg/ml), KCl (1.29mg/ml), and CaCl₂·2H₂O (0.32mg/ml) in one liter of distilled water, resulting in the creation of SNF. The pH of the solution was then adjusted to 6.5 using triethanolamine⁵.

2.3 Standard stock solution:

A standard stock solution of Clopidogrel Bisulphate was prepared in 100ml of SNF with a concentration of 10 mg/ml. Subsequently, 1ml aliquots were extracted and transferred into 100ml volumetric flasks to obtain a concentration of 100µg/ml⁶. Different standard solutions spanning concentrations from 0.5 to 3µg/ml were then derived by withdrawing various fractions from the stock solution (100µg/ml) and adjusting the volumes with SNF using 10ml volumetric flasks⁷.

3. METHOD OPTIMIZATION:

3.1 Solvent Selection and Optimization:

The selection of solvents may have a discernible impact on the characteristics and form of the spectra. When considering drug delivery via the intranasal route, it becomes imperative to quantify the drug within simulated nasal fluid.

3.2 Wavelength Selection:

The wavelength at which the highest absorbance was detected was designated as the maximum wavelength⁸.

4. VALIDATION OF METHOD:

The validation of the method was conducted in accordance with the guidelines outlined by the International Conference on Harmonization (ICH), encompassing parameters such as linearity, accuracy, precision, robustness, and sensitivity.

4.1 Linearity:

A linearity assessment was performed over a concentration range spanning from 0.5 to 3µg/ml. Standard solutions were subjected to UV spectrophotometric scanning at their respective maximum wavelengths, and a concentration versus absorbance graph was constructed⁹.

4.2 Accuracy:

Accuracy was assessed through a recovery study at three distinct levels: 50%, 100%, and 150%. Mean percentage recovery was computed based on the collected data⁹.

4.3 Precision:

The precision of the method was evaluated using both intraday and interday studies. For intraday precision, solutions of 5, 10, and 15µg/ml were analyzed three times on the same day. Interday precision involved the analysis of 5, 10, and 15µg/ml solutions over three consecutive days¹⁰.

4.4 Sensitivity:

Sensitivity was determined by calculating the limit of detection (LOD) and the limit of quantification (LOQ) using the following formulas:

LOD = 3.3 × Standard Deviation (S.D)/Slope (S) and

LOQ = 10 × S.D / S

Where S.D represents the standard deviation, and S signifies the slope¹¹.

4.5 Robustness:

Robustness was assessed by using a 2µg/ml solution. The evaluation involved altering the maximum wavelength from 242 while maintaining the concentration at 2µg/ml.

4.6 Reproducibility:

Reproducibility was determined by analyzing a working concentration of 2 µg/ml six times to assess the method's consistency and reliability¹².

5. RESULT AND DISCUSSION:

5.1 Method Optimization:

The maximum wavelength (λ max) was identified at 242 nm, as illustrated in Figure 2.

Table 1: Estimation of Maximum Wavelength

Concentration (µg/ml)	Maximum wavelength (nm)	Absorbance
100	242	0.652
100	276	0.212
100	240	0.295

Figure 2: UV-visible Spectra of Clopidogrel Bisulphate in Simulated Nasal Fluid

5.2 Method Validation:

5.2.1 Linearity: The linear regression analysis over the concentration range of 0.5 to 3µg/ml demonstrated a strong correlation in the calibration curve. The linear regression equation was determined as y = 0.3015x - 0.0636 (R² = 0.996), as depicted in Figure 3.

Figure 3: Calibration curve for Clopidogrel Bisulphate in Simulated Nasal Fluid.

5.2.2 Accuracy:

The mean percentage recovery was calculated to be 95.84%, and the detailed data is presented in Table 2.

Table 2: Result of accuracy

Recovery Level	Concentration of Drug (µg/ml)	Amount Recovered (µg/ml)	Recovery (%)	Mean Recovery (%)
50	5	4.732	94.64	95.84
100	10	9.512	95.12
150	15	14.668	97.78

5.2.3 Precision:

Precision, expressed as %RSD values, indicated a method precision when RSD values were below 2. Intraday precision values ranged from 0.142 to 0.474, while interday precision values were observed to be within the range of 0.278 to 0.354, as outlined in Table 3.

Table 3: Result of Intraday and Interday Precision

Concentration (µg/ml)	Intraday		Interday
Concentration (µg/ml)	Avg. Absorbance ± SD	% RSD	Avg. Absorbance ± SD	% RSD
5	0.102±0.0002	0.196	0.112±0.0004	0.354
10	0.232±0.0011	0.474	0.251±0.0007	0.278
15	0.422±0.0006	0.142	0.462±0.0011	0.238

5.2.4 Sensitivity:

Sensitivity, evaluated through the determination of the LOD and LOQ using the Standard Calibration Curve, revealed LOD and LOQ values for clopidogrel as 0.193 and 0.6434, respectively.

5.2.5 Robustness:

The alteration of the wavelength at 242 by ±2 was carried out, and the results are summarized in Table 4.

Table 4: Result for Robustness

Maximum wavelength	Average Absorbance	Standard Deviation	%RSD
240	0.598	0.0033	0.598
242	0.603	0.0018	0.298
244	0.576	0.0026	0.451

5.2.6 Reproducibility:

Reproducibility, assessed by analyzing a solution with a concentration of 2 µg/ml six times, yielded a %RSD value below 2%, as presented in Table 5.

Table 5: Result for Reproducibility

Concentration

Mean

absorbance

Std

Deviation

%RSD

2 µg/ml

0.512

0.00334

0.64

6. SUMMERY:

All validation parameters fell within the specified range, and a comprehensive summary is presented in Table 6.

Table 6: Summery

S. No.	Validation Parameter	Result
1	Maximum Absorption ƛ max	242nm
2	Linearity Range	0.5to 3 µg/ml
3	Standard Regression Equation	y = 0.3015x - 0.0636
4	Correlation Coefficient	R² = 0.996
5	Slope	0.3015
6	Intercept	0.0636
7	% Recovery	95.84 %
8	Interday Precision in terms of %RSD	0.142-0.474
9	Intraday Precision in terms of %RSD	0.238-0.354
10	LOD	0.193
11	LOQ	0.6434

7. CONCLUSION:

The proposed method has been devised for the quantitative estimation of Clopidogrel Bisulphate in simulated nasal fluid. The developed and validated method was observed to be straightforward, accurate, precise, sensitive, cost-effective, and reproducible. This UV spectrophotometric approach holds potential for the estimation of Clopidogrel Bisulphate in nasal formulations.

8. ACKNOWLEDGMENT:

The authors express gratitude to Mahatma Jyotiba Fuley Training Institute, Nagpur, for their financial support through the MJPRF 2021 Fellowship. Additionally, heartfelt thanks are extended to Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, for their invaluable support in the execution of this project.

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Received on 10.04.2024 Revised on 06.07.2024

Accepted on 13.09.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):815-818.

DOI: 10.52711/0974-360X.2025.00120

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