INTRODUCTION:

The empirical formula for clopidogrel bisulphate is C₁₆H₁₆CINO₂S•H₂SO₄, and its molecular weight is 419.9. Chemically speaking, it is methyl (+) -(S)-α-(2-chlorophenyl)-6, 7-dihydrothieno [3,2-c]. Pyridine-5 (4H)-acetate sulphate (1:1). As seen in Figure 1. (1)

Having been used in antiplatelet therapy since 1998, clopidogrel is the thienopyridine with the greatest clinical experience. By 2011, clopidogrel (Plavix®) was the second-best-selling drug in the world. Its use was expected to rise even more with the introduction of generics. (2)

Clopidogrel is used to prevent blood clots in peripheral vascular illness, cerebrovascular disease, and coronary artery disease. It is a thienopyridine class inhibitor of P2Y12 adenosine 5′-diphosphate (ADP) platelet receptors. The pro-drug of carboxyl clopidogrel, clopidogrel, is activated by the liver's CYP2C19 enzyme and cytochrome P450. The active metabolite has an elimination half-life of about seven to eight hours after entering the platelet ADP receptor and forming a disulfide bond. (3)

The platelet P2Y12 adenosine diphosphate receptor is permanently inhibited by clopidogrel. Inhibiting this receptor prevents the glycoprotein IIb/IIIa receptor complex from being activated downstream, which reduces platelet aggregation. A two-step bioactivation process involving many CYP enzymes, including CYP2C19 and CYP3A4, is required to activate clopidogrel, an inert prodrug. (4, 5, 6) The effectiveness of a treatment may be impacted by genetic differences in these enzymes. The most common genetic variable linked to clopidogrel is one or both CYP2C19 enzyme alleles. Patients with any loss of function allele are unable to effectively metabolize clopidogrel, which prevents platelet activity from being suppressed. (7)

The powder form of clopidogrel bisulphate is white to off-white in color. (8) At pH 1, It is almost insoluble at neutral pH, although it dissolves easily in water. It also dissolves readily in methanol, sparingly in methylene chloride, and almost insoluble in ethyl ether. (9, 10, 11)

The calculated pKa for clopidogrel is 4.56_+ 0.20. 183C is the melting point. A thieno[3,2-c] pyridine ring system, a methyl ester group, a chlorophenyl group, and a sulphate salt that is produced when sulfuric acid is introduced comprise the involved chemical structure of clopidogrel bisulphate. At pH 7.4, the clopidogrel bisulfate partition coefficient in octanol/water has been determined to be around 3.9 (8).

Because of their versatility, ease of use, and affordability in analysis, spectrophotometric methods such as absorbance and area under the curve (AUC) measurement are highly prized. Research labs and pharmaceutical manufacturers can meet their analytical needs with UV/Visible spectrophotometers, which are a useful and affordable substitute for more complicated systems like UHPLC, HPLC, and LC-MS/MS. (12)

Fig.1- Chemical structure of clopidogrel bisulphate

EXPERIMENT:

Spectrophotometric instrument:

The device was a UV-1800 dual-beam Shimadzu UV-visible spectrophotometer. While the reference and test solutions were kept in 1-cm quartz cells, their absorption spectra were measured between 200 and 400 nm.

Chemical compounds and Reagents:

0.05N. The solvent selected was an acetic acid solution made from acetic acid () in double-distilled water. The Store of College provided the powdered clopidogrel bisulphate and methanol (Merck Pvt. Ltd., India).

Pharmaceutical sample:

Ajanta Pharma Limited manufactures Aclizac 75 (Clopidogrel uncoated tablets), which contain 75mg of Clopidogrel bisulphate.

Preparation of standard stock solution and working solution:

Clopidogrel bisulphate's standard stock solutions (1 mg/ml=1000µg/ml) and working solutions (0.1 mg/ml=100µg/ml) were made in a solution of 0.05 N acetic acid. After dissolving 100mg of clopidogrel bisulphate in likely 5ml of methanol, add 0.05N acetic acid solutions to a volumetric flask to get the volume up to 100ml (stock solution). To create the working solution, pipette out 10ml of the stock solution into a 100ml volumetric flask containing 0.05N acetic acid.

Spectral characteristics of Clopidogrel bisulphate:

10mL volumetric flasks were filled with precisely measured aliquots of 50µg/mL of clopidogrel bisulphate, and the volumes were adjusted using 0.05N acetic acid. The resulting solutions' UV absorption spectra were scanned at a medium scanning speed over a wavelength range of 200–400nm, obtaining absorbance values between 0.00 and 4.00.

Choice of wavelength for analysis:

The wavelength selected was 270nm (the λ max for Clopidogrel bisulphate). Pharmacopeia value: 270nm. ⁸

Linearity for the spectrophotometric method:

10ml volumetric flasks were filled with aliquots of the 0.1mg/ml (100µg/ml) clopidogrel bisulphate working solution (1, 2, 3, 4, 5, 6, 7, 8, and 9ml); the volume was adjusted using 0.05 N acetic acid solutions. The absorbances of each solution were measured at 270nm (Pharmacopeia value: 270nm).

Analysing the formulation of commercial tablets:

Weigh twenty tablets precisely and grind them into a fine powder. Transfer an amount of the powdered material equal to 100mg of clopidogrel bisulphate, mix it with 5ml of methanol, and fill a 100ml volumetric flask with a 0.05N acetic acid solution (Solution-A). A 100ml volumetric flask should be filled with a 0.05N acetic acid solution after the solution has been filtered and 10 ml taken. Calculate the final solution's absorbance (Solution-B). (Approximately 100µg/ml Clopidogrel bisulphate at a maximum at about 270nm).

For quantitative spectrophotometric experiments, spectral analysis:

100 µg/ml of clopidogrel bisulphate was used in the spectrum analysis to quantitatively determine the specified analyte. Using methanol and 0.05 N acetic acid solutions as blank D0 (Zero-order absorbance), the produced solution was scanned across the UV/visible spectrum range, namely from 200-400nm. The maximum peak absorbance is seen at 270nm. At 244 and 281nm, the D1 (first order amplitude) spectra of clopidogrel bisulphate were detected. The range of 265 to 275nm was used to monitor the clopidogrel bisulphate AUC spectrum.

Quantification using spectrophotometry in pharmaceutical sample:

The quantification of the mentioned analyte was examined in Ultra Drugs Pvt. Ltd.'s Acizac 75. Using methanol and 0.05 N acetic acid solutions as blank AUC spectra of clopidogrel bisulphate, the produced solution was scanned across the UV/visible spectrum region, specifically from 200–400nm. At 270nm, D0 (Zero-order absorbance) has the highest peak absorbance. At 244 and 281nm, the D1 (first order amplitude) spectra of clopidogrel bisulphate were detected.

VALIDATION OF A QUANTITATIVE STUDY:

In order to validate a spectrophotometric experiment, the data must be carefully examined to ascertain their accuracy, precision, and suitability for the intended research goals. In order to ensure the efficacy of test procedures and spectrophotometric analysis for clopidogrel bisulphate analysis in pharmaceutical matrices, validation becomes a recognized standard.
As a result, the proposed experiment has supported validation in compliance with the Q2(R1) guideline established by the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), focusing on criteria such as accuracy, precision (including intra-day, inter-day, and repeatability assessments), specificity, selectivity, and sensitivity. (13)

Linearity:

By comparing the absorbance of the clopidogrel bisulphate standard concentration (10–100µg/ml) at 270 nm to methanol and 0.05 N acetic acid as a blank, the linearity was determined. The calibration curve was then developed using concentration against absorbance. For standard concentrations of clopidogrel bisulphate, a regression equation and correlation coefficient were calculated.

Limit of detection and limit of quantification:

The limit of detection (LOD) is the lowest analyte concentration that an analytical technique can reliably distinguish from background levels.

LOD = 3.3 σ/S

LOQ = 10 σ/S

Were the formulae used in this study to calculate LOD and LOQ based on the response's standard deviation and the slope of the related curve.

Where σ is the sample's signal to noise ratio standard deviation and S is the slope of the specified calibration graphs.

The limit of quantification, or LOQ, is the lowest concentration of the standard curve that can be measured with a reasonable degree of precision, accuracy, and variability. (14)

Precision:

Ten duplicates of the Clopidogrel bisulphate levels of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100µg/ml were measured. The precision of the experiments was assessed for % assay, SD, and % RSD to ensure precision in the results and enable the acceptance criteria for the precision (intra-day and inter-day), i.e., <2%.

Recovery study or Accuracy:

The degree of agreement between the value found and the value identified as the true value was used to describe the analytical procedure's accuracy
Clopidogrel bisulphate was used in recovery tests using the standard addition method at three different concentrations: 50% (10µg/ml), 100% (20µg/ml), and 150% (30µg/ml) of the test concentration. The percentage recoveries were obtained using the measured concentrations.

Specificity and Selectivity:

To ensure that pharmaceutical matrix components would not interfere with the spectrum analysis of clopidogrel bisulphate, research was done on AUC, D0, and D1. To find any potential interference, the spectra of clopidogrel bisulphate standards, which range from 10 to 100µg/ml, were compared to the back drop of pharmaceutical matrices. The selectivity of the analysis was confirmed by studying the average percentage recovery and percentage RSD from six various determinations at a concentration of 20µg/ml.

Repeatability:

In order to prepare a concentration of clopidogrel bisulphate (60µg/mL) for repeatability test, 6mL of standard stock solution was put into a 10mL volumetric flask, and the volume was adjusted using the solvent system. Six analyses of the solution were conducted at the same time on the same day. After recording the absorbance, the mean, standard deviation (SD), and percentage relative standard deviation (%RSD) were measured.

RESULT AND DISCUSSION:

Optimization of spectrophotometric experiment:

To develop an accurate and reliable quantitative technique for clopidogrel bisulphate, the spectrophotometer parameters were methodically improved by assessing the zero-order (D0), first-order (D1) derivative, and AUC value. It dissolves easily in water at pH 1, but it is almost insoluble at neutral pH. It also dissolves readily in methanol, sparingly in methylene chloride, and almost insoluble in ethyl ether. (15, 16) In order to fully dissolve the pharmaceutical substance, methanol was utilized as the core solvent.

However, acetic acid was used as a diluent to generate the working standard and sample solutions since it produced a constant pH environment and did not affect the absorbance of clopidogrel bisulphate. Using a Shimadzu UV spectrophotometer (UV-1800), the solution was scanned between 200 and 400 nm, and the greatest absorbance was found at 270 nm. The zero order spectra showed the most favorable linearity and absorbance characteristics among the derivative orders examined (D0 and D1). As a result, it was chosen as another method validation. In order to provide clopidogrel bisulphate with clear spectra, good stability, and repeatable absorbance values, the optimized solvent system used acetic acid solution as the diluent and methanol as the solubilizing agent.

Table 1. Observation for primarily testing of clopidogrel bisulphate

Solubility profile of Clopidogrel bisulphate
Freely Soluble	Sparingly soluble	Insoluble
Methanol Water (pH 1)	Methylene chloride	Ethyl ether
Methanol Water (pH 1)		Water (neutral pH)
0.1 N HCl		Urea
0.05 N HCl		0.1 N NaOH

Spectral characteristics of clopidogrel bisulphate:

The wavelength maximum for clopidogrel bisulphate was found in the spectra at 270 nm, and this λ max was used for clopidogrel bisulphate estimation.

(a)

(b)

(c)

(d)

Figure 2. (a) D º, (b) Dº AUC, (c) D1 (244 nm), and (d) D1 (281 nm) The UV Clopidogrel bisulphate experiment using a visible spectrophotometer at a concentration of 100µg/ml.

Table 2. Analysis determination of clopidogrel bisulphate: spectrophotometric experiments at 270 nm (n=6)

S. N.	Conc. Of Clopidogrel bisulphate (µg/ml)	ml of solution – (B) taken in 10ml volumetric flask	(λ max 270nm) Abs. mean ± S.D.	(275nm-265nm) D° AUC ± S.D.	(244nm) 1^st order derivative ± S. D.	(281nm) 1^st order derivative ± S. D.	Abs. mean / mean conc. = Absorptive
1.	10	1ml	0.0182±0.001	0.16336±0.01	-0.0112±0.0008	-0.001±0.000	0.0018
2.	20	2ml	0.0366±0.002	0.33052±0.01	-0.0216±0.0008	-0.002±0.000	0.0018
3.	30	3ml	0.0522±0.002	0.47428±0.02	-0.0322±0.0017	-0.003±0.000	0.0017
4.	40	4ml	0.0702±0.001	0.63848±0.01	-0.0408±0.0019	-0.004±0.000	0.0017
5.	50	5ml	0.0914±0.003	0.82908±0.035	-0.0528±0.0013	0.0054±0.0005	0.0018
6.	60	6ml	0.11±0.001	1.00004±0.01	-0.0638±0.0022	-0.0066±0.0005	0.0018
7.	70	7ml	0.128±0.002	1.15804±0.01	-0.0742±0.0020	0.0076±0.0005	0.0018
8.	80	8ml	0.1486±0.000	1.33974±0.00	-0.0856±0.0016	-0.0086±0.0005	0.0018
9.	90	9ml	0.1668±0.001	1.5069±0.01	-0.0964±0.0026	-0.0096±0.0005	0.0018
10.	100	10ml	0.1884±0.004	1.69638±0.06	-0.1072±0.0010	-0.0106±0.0005	0.0018

Absorptive mean± S.D.

0.00178 ± 0.000043

Table 3. Clopidogrel bisulphate analytical analysis in pharmaceuticals using standard addition percentage recovery and spectrophotometric tests.

Conc. Of standard (µg/ml) A	Conc. Of sample (µg/ml) B	Total conc. (A+B) (µg/ml)	Abs. for mixture Std. + sample	Total amount (A+B)	Recovery of standard (µg/ml)	% Recovery of std.	Mean ± SD
10	20	30	0.051	30.37	10.37	103.7	102.56 ± 1.00
20	20	40	0.071	40.45	20.45	102.2
30	20	50	0.092	50.54	30.54	101.8

Conc. Of standard (µg/ml)	Conc. Of sample (µg/ml)	Total conc. (A+B) (µg/ml)	AUC for mixture std. + sample	Total amount (A + B)	Recovery of standard (µg/ml)	% Recovery of std.	Mean ± SD
10	20	30	0.4662	29.85	9.85	98.5	100.51 ± 1.8
20	20	40	0.6451	40.41	20.41	102.05
30	20	50	0.8342	50.30	30.30	101

Conc. Of standard (µg/ml)	Conc. Of sample (µg/ml)	Total conc. (A+B)	D¹ (281) for mixture std. + sample	Total amount (A+B)	Recovery of standard (µg/ml)	% Recovery of std.	Mean ± SD
10	20	30	-0.003	30	10	100	100 ± 0.00
20	20	40	-0.004	40	20	100
30	20	50	-0.005	50	30	100

Conc. Of standard (µg/ml)	Conc. Of sample (µg/ml)	Total conc. (A+B)	D¹ (244) for mixture std. + sample	Total amount (A+B)	Recovery of standard (µg/ml	% Recovery of std.	Mean ± SD
10	20	30	-0.032	29.81	9.81	98.1	99.36 ± 1.09
20	20	40	-0.040	40	20	100
30	20	50	-0.052	50	30	100

Table 4. Validation parameter for determining Clopidogrel bisulfate: A comparison analysis with existing literature.

Validation parameters		Linearity range (µg/ml)	Correlation coefficient	Slope	Intercept	Accuracy (mean ± SD)	Sensitivity (µg/ml)		Precision (% RSD)
Validation parameters		Linearity range (µg/ml)	Correlation coefficient	Slope	Intercept	Accuracy (mean ± SD)	LOD	LOQ	Intra day	Inter day
Spectrophotometric	D° (270nm)	10 - 100	0.9992	0.0019	-0.002	99.58 ± 0.51	1.73	5.26	0	0.45
	D° AUC	10 - 100	0.9994	0.0169	-0.0154	99.65 ± 0.65	1.95	5.91	0.34	0.86
	D¹ (244)	10 - 100	0.9996	-0.0011	0.0002	98.75 ± 0.71	3.0	9.09	1.02	1.03
	D¹(281)	10 - 100	0.9987	-0.0001	0.00004	101 ±0.61	0	0	0	0
(17)	D¹	5 - 25	0.9862	-0.0006	0.0015	99.73 ± 0.04	2	5	-	-
(18)	D° (270 nm)	16.8 – 420	0.999	0.6735	-0.007	98.7	1.39	4.62	0.97

CONCLUSION:

Clopidogrel bisulphate in bulk and tablet formulations can be analyzed using the basic, accurate, and specific procedure that has been suggested. The UV spectrophotometric method was validated in accordance with ICH recommendations. Because it uses a cheap and easily available solvent for analysis, this approach was also economical for estimating clopidogrel bisulphate in bulk and tablet forms. Because the common excipients and other additives that are usually included in the tablet formulation do not interfere with the analysis of clopidogrel bisulphate in the technique, it may be simply utilized for routine evaluation of the medicines in tablet formulation.

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Received on 08.11.2025 Revised on 05.12.2025

Accepted on 30.12.2025 Published on 27.01.2026

Available online from February 02, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(1):14-20.

DOI: 10.52711/2231-5675.2026.00003

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