Analytical Techniqes for Clopidogrel Bisulphate: Review

 

Shweta Sinha*, Jitendra Yadav, Dolly Dewangan, Kamraj, Krity Gupta, Manish Kumar Sahu,

Parimal Verma, Prashant Kumar Sahu, Aakanksha Sinha, S. J. Daharwal

University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G).

 

ABSTRACT:

P2Y12 is a purinergic G protein-coupled (GPCR) receptor that binds to adenosine diphosphate (ADP). Thromboembolisms and other clotting diseases can be treated by targeting the P2Y12 receptor, which is involved in platelet aggregation. The morbidity and mortality outcomes, such as cardiovascular death, recurrent myocardial infarction (MI), and stroke at 30 days following percutaneous coronary intervention (PCI), have improved with the use of clopidogrel (CLO). One such medication that selectively and permanently blocks the P2Y12 subtype of the ADP receptor is CLO. This paper provides a thorough explanation of different analytical techniques that have been published for the determination of CLO and its combined forms in biological matrices and pharmaceuticals. The review focuses on both fundamental and sophisticated methods used to estimate CLO. The review shows that the technique of HPLC with UV detection was commonly used, even if various analytical methods were used for the CLO assay.

 

 

INTRODUCTION:

A thienopyridine class inhibitor of P2Y12 adenosine 5′-diphosphate (ADP) platelet receptors, clopidogrel is used to prevent blood clots in peripheral vascular disease, cerebrovascular disease, and coronary artery disease. Cytochrome P450 and the CYP2C19 enzyme in the liver activate the pro-drug of carboxyl clopidogrel, clopidogrel. The active metabolite enters the platelet ADP receptor and forms a disulfide bridge, with an elimination half-life of roughly 7-8 hours.¹ It is methyl (+) -(S)-α-(2-chlorophenyl)-6,7-dihydrothieno in chemical terms [3,2-c]. (1:1) pyridine-5 (4H)-acetate sulfate. Clopidogrel bisulphate's molecular weight is 419.9 and its empirical formula is C₁₆H₁₆CINO₂S•H₂SO₄. ²

 

Clopidogrel is the thienopyridine with the most clinical experience, having been used in antiplatelet therapy since 1998. Clopidogrel (Plavix®) became to the second-highest selling medication globally by 2011. With generics hitting the market, its use was predicted to increase even further.³

 

Physicochemical Properties:

Clopidogrel bisulphate is a powder that ranges from white to off-white. At pH 1, it dissolves readily in water, but at neutral pH, it is nearly insoluble. Additionally, it dissolves easily in methanol, rather sparingly in methylene chloride, and is nearly insoluble in ethyl ether.

 

Clopidogrel's pKa is 4.56 0.20, according to calculations. The melting point is 183 C. The involved chemical structure of clopidogrel bisulphate includes a thieno[3,2-c] pyridine ring system, a methyl ester group, a chlorophenyl group, and a sulfate salt that is created when sulfuric acid is added.⁴

 

Mechanism of Action:

Clopidogrel inhibits the platelet P2Y12 adenosine diphosphate receptor irreversibly. Platelet aggregation is decreased when this receptor is inhibited because it stops the glycoprotein IIb/IIIa receptor complex from being activated downstream. Clopidogrel is an inert prodrug that needs to be activated enzymatically using a two-step bioactivation process involving several CYP enzymes, such as CYP2C19 and CYP3A4. These enzymes' genetic variations may affect how well a treatment works. One or both CYP2C19 enzyme alleles are the genetic variant associated with clopidogrel that is most frequently mentioned. Clopidogrel cannot be efficiently metabolized by patients with any loss of function allele, making it impossible to suppress platelet activity.⁵

 

Pharmacokinetics:

The prodrug clopidogrel bisulphate is rapidly absorbed when taken orally; peak plasma concentrations are attained about an hour after a single 75 mg dose. Its significant first-pass hepatic metabolism results in an absolute bioavailability of about 50%. Approximately 94–98% of the medication and its primary inactive metabolite are bound to plasma proteins, which are broadly dispersed throughout the tissues. The liver metabolizes clopidogrel primarily in two ways: approximately 15% of the absorbed dose is oxidatively activated by cytochrome P450 enzymes, mainly CYP2C19, with contributions from CYP3A4, CYP1A2, and CYP2B6, to produce a short-lived but pharmacologically active thiol metabolite, and approximately 85% of the dose is hydrolyzed by esterases into an inactive carboxylic acid derivative. [6] By binding to platelet P2Y₁₂ ADP receptors irreversibly, this active form prevents platelet aggregation for the duration of the platelet (about 7–10 days). The parent compound's elimination half-life is roughly six hours, whereas the inactive metabolite's is roughly eight hours. After taking a radiolabeled medication orally, within five days, over half of the dose is eliminated in the urine and a comparable amount in the feces. Genetic variations and pharmacological interactions that impact this enzyme can result in varying treatment responses because it depends on CYP2C19 for activation. The parent drug's pharmacodynamic action lasts for several days after stopping, despite its quick clearance from the bloodstream. This is because irreversible receptor inhibition causes the drug to continue to have antiplatelet effect.⁷

 

Figure 1: Chemical Structure of Clopidogrel Bisulphate

 

Pharmacodynamics:

The active metabolite that prevents platelet aggregation is produced when CYP450 enzymes metabolize clopidogrel. Clopidogrel's active metabolite specifically prevents adenosine diphosphate (ADP) from attaching to its platelet P2Y12 receptor and from activating the glycoprotein GPIIb/IIIa complex, which further prevents platelet aggregation. This action cannot be reversed. As a result, platelets exposed to the active metabolite of clopidogrel suffer for the remaining 7–10 days of their existence. Blocking the amplification of platelet activation by released ADP also inhibits platelet aggregation caused by agonists other than ADP. [8]

 

Methods of analysis used in pharmaceutical analysis:

Several analytical methods, such as UV/Visible Spectrophotometry (UV) HPLC, or high-performance liquid chromatography. UPLC, or ultra-high-performance liquid chromatography. HPTLC, or high-performance thin-layer chromatography, Bio-analytical research. MS, or mass spectrometry Nuclear Magnetic Resonance (NMR). Infrared Fourier Transform (FTIR) spectroscopy   were found in the literature for the determination of clopidogrel bisulphate in pharmaceutical formulations, bulk, and biological samples. [2]

 

Table 1: Development of analytical methods by using an UV spectrophotometer

S. No	Drug	Method / instrument model	solvent	wavelength	Linearity Range	R square	Reference
1.	Clopidogrel bisulfate	UV visible spectrophotometer	0.002% methanol	203 nm	1 – 2.6 ug/ml	0.9929	[9]
2.	Amlodipine besylate and clopidogrel bisulfate	UV spectrophotometer	methanol	Amlodipine besylate -360 Clopidogrel bisulfate - 270	5 – 45 ug/ml	< 1	[10]
3.	Clopdogrel bisulfate	Spectrophotometer	Ethyl ether and double distilled water	407 nm	80 and 115 ug/ml	0.9919 and 0.9949	[11]
4.	Clopidogrl bisulfate	UV Spectrophotometry	Methanol: acetonitrile	202 nm	1.25 – 25 ug/ml		[12]
5.	Clopidogrel bisulfate	Zero order, D1, D2, D3 Spectrophotometry	0.1N HCl	Zero order-270nm D1- 279.3nm D2-269.3nm D3 – 275.6 nm	16.8to 420ug/ml	0.999	[13]
6.	Rozuastatin and clopdogrel	UV spectrophotometry	Methanol	240nm	50-150ug/ml	0.999	[14]
7.	Clopidogrel bisulfate	UV spectrophotometry	Distilled water (pH 1)	222nm	40 – 70ug/ml	1	[15]
8.	Metoprolol succinate and clopidogrel bisulfate	1st order derivative and Zero order	0.1N HCl	Clp-222.22nm Metoprolol 223nm Zero -245.76nm (Metoprolol Succinate) 276.13 (clp)	5 – 25 ug/ml (MS and CLOP)	-	[16]
9.	Clopidogrel and irbesarten	UV spectrophotometer	Methanol	220nm[clp] 250[IRB]	10 -50 ug/ml	0.9996[clp] and 0.9998 [IRB]	[17]
10.	Clopidogrel bisulfate	UV spectrophotometer	Methanol and 0.1N HCl	217 nm	2.5 – 20 ug/ml	0.997	[18]
11.	Clopidogrel bisulfate	2nd order derivative	0.1 N HCl	207nm	10 – 35 ug/ml	0.9991	[19 ]
12.	Clopidogrel bisulfate	UV spectrophotometric	0.1 N HCl	220nm	25 – 50 ug/ml	0.999	[20]
13.	Clopidogrel bisulfate and aspirin	First order derivative	Methanol and 0.1N HCl	232.5nm(clp) 211.3nm (aspirin)	5 – 25 ug/ml	0.9862 (clp) 0.9914 (aspirin)	[21]
14.	Clopidogrel bisulphate and acetyl salicylic acid	First order derivative	Ethanol	229.11nm (aspirin) 252.74nm (clp)	4 – 24 ug/ml	0.9998 (aspirin) 0.9999 (clp)	[22]
15.	Clopidogrel bisulfate and rivoroxaban	(i)First order derivative (ii) Ratio derivative (iii) absorbance ratio derivative	Methanol	(i)289nm (RIV) 249.5 (CLP) (ii) 256nm (RIV) 214.5NM(CLP) (iii)249 (RIV) 232(CLP)	2 – 20 ug/ml (RIV) 5 – 60 ug/ml (clp)	0.9999	[23]

 

Table 2: Development of analytical methods by using the HPLC technique

S. No	Drug	Mo. phase	Stationary phase	Flow rate	Wavelength	Retention time	Reference
1.	Clopidogrel bisulphate	Potassium dihydrogen orthophosphate buffer: acetonitrile 32:68 (PH 4)	C18	1 ml/ min	220nm	3.847min.	[24]
2.	Clopidorel bisulphate and aspirin	Acetonitrile: potassium Dihydrogen phosphate buffer: methanol 50:30:20	_	1.5 ml/ min.	240nm	7.47 min. (clp) 2.2 min (aspirin)	[25]
3.	Clopidogrel and aspirin	Acetonitrile: methanol: phosphate buffer 3 (50:7:43)	C18	1 min/ml	240nm	2.40min (aspirin) 9.27(clp)	[26]
4.	Aspirin, atorvastatin clopidogrel bisulphate	Acetonitrile: phosphate buffer PH3 with o phosphoric acid (50:50v/v)	Inertsil ODS Analytical column	1.2ml/ min.	235nm	1.89(ASP) 6.6(ATR) 19.8(CLO)	[27]
5.	Clopidogrel bisulphate	(920 ml:50 :0.3) n Hexane, ethanol, isopropyl alcohol	Chiral cel OD-H Column	0.9ml/min	240nm	20.8min.	[28]
6.	Clopidogrl bisulphate	Acetonitrile: acetic acid (85:15)	C18	1ml/min	220nm	7.3min	[29]
7.	Clopidogrel bisulphate and aspirin	0.3% ortho phosphoric acid (v/v) acetonitrile (40:60v/v)	C18	1ml/min	226nm	6.6 min(clo) 8.4min (aspirin)	[30]
8.	Clopidogrel bisulphate and rivaroxaban	Buffer (0.05M KH2PO4 Ph4): methanol (30:70v/v) .	BDS hipersil C18	1ml/min	220nm	2.39min. (CLO) 4.04 min.(rivoroxaban)	[31]
9.	Clopidogrel bisulphate	Buffer 3, orthophosphoric acid and methanol(20:80)	C18	1ml/min	240nm	4.388min	[32]
10.	Clopidogrel bisulphate Acetyl salicylic acid and atorvastatin	0.01N KH2 PO4 Buffer: acetonitrile: methanol (20:40:40)	HDS Hipersil C18	0.8ml/ min	220nm (CLO) 230nm (Acetyl Salicylic acid and 244nm (ATR) 220nm.	-	[33]
11.	Atorvastatin calcium and clopdogrel bisulphate	Acetonitrile and0.01M Potassium dihydrogen phosphate (75:25) v/v 6.1pH	C18	1ml/min	240nm	3.5min (ATR) 10.75Min (CLO)	[34]
12.	Rozuastatin and clopidogrel bisulphate	Methanol: water 3pH (80:20)	C18	1ml/min	240nm	0.8 min	[35]
13.	Amlodipine besylate and clopidogrel bisulphate	Methanol: Water (70:30)	C18	1ml/min	238nm	3.85min (amlodipine) 7.41min (CLO)	[36]
14.	Clopidogrel bisulphate	Acetonitrile methanol water (45:45:10)	C18	0.9ml/min	230nm	3.1min	[37]
15.	Rosuvastatin andclopidogrel bisulphate	Methanol: Water (80:20)	C18	1ml/min	240nm	3.483(ROSU)4.983(CLO)	[38]
16.	Clopidogrel bisulphate	Acetonitrile methanol phosphate buffer 0.1 M (80:10:10)	C18	0.9ml/min	240nm	5.21min	[39]
17.	Clopidogrel bisulphate	Acetonitrile: OPA Orto phosphoric acid buffer (50:50v/v)	Altima C18	1ml/min	240nm	-	[40]
18.	Atorvastatin calcium and clopidogrel bisulphate	OPA buffer: CAN (70:30, V/V)	Zodiac C18	1ml/min	241nm	5.8 min (ATOR) 3.5min (CLO)	[41]
19.	Aspirin and clopidogrel	Buffer solution 0.3% O Phosphoric acid: acetonitrile (65:35) v/v	Phenomax gemini C18	1mi/min	266nm	-	[42]
20.	Clopidogrel and acetylsalicylic acid	(A) (5:95 V/V methanol and 1 g/L solution of sodium octane sulfonate monohydrate 2.5 pH with dil. Phosphoric acid (B) (5:95v/v methanol and acetonitrile)	Luna C18	1ml/min	220nm	-	[43]
21.	Aspirin, rozuvastatin and clopidogrel bisulphate	Water 2.51 with 0.1% (v/v) O phosphoric acid: acetonitrile (50:50)	C18	1ml/min	237nm	4.3min (ASP) 7.6min (ROSU) 16.6 min (CLO)	[44]
22.	Amlodipine besylate and clopidogrel bisulphate	Acetonitrile and phosphate buffe (3.4g of potassium dihydrogen phosphate in 1000ml of dis. Water 3 pH with ortho phosphoric acid (60:40v/v)	C18	1ml/min	250nm	Amlodipine (4.2min) CLO (9.1 min)	[45]
23.	Aspirin and clopidogrel bisulphate	55:45 V/V phosphate buffer and acetonitrile	C18	-	235nm	-	[46]

 

Table 3: Development of analytical methods by using the HPTLC technique

S. No.	Drug	Mo. phase	Stationary phase	Wavelength	Reference
1.	Clopidogrel bisulphate	Carbon tetrachloride chloroform acetone (6:4:0.15) v/v/v	TLC aluminium plate precoated with silica gel 60F 254	230nm	[47]
2.	Aspirin and clopidogrel	CCl4 acetone (6:2.4) v/v	TLC aluminium plate (silica gel 60F254)	220nm	[48]
3.	Clopidogrel bisulphate	CCL4: ethyl acetate: ammonium (5:0.3:0.2)	TLC aluminium plate (silica gel 60F254)	230nm	[49]
4.	Aspirin, atorvastatin and clopidogrel bisulphate	(6.5:3.5:0.1) v/v toluene: methanol: formic acid)	TLC aluminium plate (silica gel 60 F254)	254nm	[50]
5.	Clopidogrel bisulphate	(16:2:1.5:0.5) v/v/v hexane: methanol: chloroform	TLC aluminium plate (silica gel 60F254)	254nm	[51]

 

Table 4: Analytical techniques for measuring CLOPIDOGREL BISULPHATE in biological fluids

S. No.	Drug	Model	Method	Solvent	Stationary phase	Reference
1.	Clopidogrel bisulphate	Serum-urine	Chemically modify carbon paste sensor	Dist. water	-	[52]
2.	Clopidogrel bisulphate	Serum - urine	Chemically modify carbon paste sensor	Double dist. water	-	[53]
3.	Clopidogrel bisulphate	Human plasma	UHPLC LC-MS/MS	Acetonitrile and methanol	-	[54]
4.	Clopidogrel and pantoprazole	Rat plasma	RP-HPLC	40:60 v/v (0.03M Potassium dihydrogen ortho phosphate buffer 3pH	C8 (250 * 4.6MM, 5um)	[55]
5.	Clopidogrel bisulphate	Plasma and urine	(CZE)Capillary zone electrophoresis	Filled Phosphate buffer2.5 pH with uncoated silica capillary	(35cm * 50 m I d 26.5cm) fused silica	[56]
6.	Clopidogrel Bisulphate	Wister rat plasma	HPLC	75:25%V/V (Acetonitrile ACN:0.05M potassium dihydrogen orthophosphate buffer 4.2 Ph	(250 * 4.6mm,5u) Xterra	[57]
7.	Clopidogrel and aspirin	Human plasma	UFLC	(35:65V/V) Phosphate buffer and acetonitrile	(250 * 4.6mm, 5um) phenomenex C8	[58]
8.	Clopdogrel Bisulphate	Beagle plasma	LC- MS/MS	(22:78V/V) Acetonitrile ammonium acetate (10Mm, 4.5 pH	(150 * 2.0MM, 5M) Ultron ES-OVM	[59]
9.	Clopidogrel, candesartan and atorvastatin	Human plasma	LC- MS/MS	2:8 v/v isocratic mixture of 0.1 % acetic acid and methanol	(4mm * 5 cm) 5um C18 for plasma and solution (2.1mm * 5cm)	[60]
10.	Clopidogrel Bisulphate	Human plasma	LC – MS/MS	(4:1V/V Acetonitrile and 0.1M ammonium chloride aqueous solution)	ODS	[61]
11.	Clopidogrel bisulphate	Human plasma	LC- MS/MS	0.1 % (V/V) Water and acetonitrile	(Zorbax plus C18) HPLC	[62]
12.	Clopidogrel bisulphate	Human plasma	UHPLC – MS/MS	Methanol and acetonitrile	C18	[63]
13.	Clopidogrel, aspirin and atorvastatin	Rat plasma	RP-HPLC	3. 0 pH phosphate buffer and acetonitrile	(25 * 0.46CM, 5MM) C18	[64]
14.	Clopidogrel bisulphate	PVC membrane sensor	Potentiometric	Dis. water	-	[65]
15.	Clopidogrel bisulphate	Human plasma	HPLC- MS/MS	Water / acetonitrile (60/40, v/v) [A] and ammonium acetate 250mMin water / acetonitrile (60/ 40 v/v) [B]	SAX (250 * 4.6 mm, 5um ) anion exchange	[66]
16.	Clopidogrel bisulphate	Human plasma	LC- MS/MS	(90:10, v/v) Acetonitrile: milli Q/HPLC grade water	100-5C18 Kromasil 100x 4.6mm, 5um	[67]
17.	Clopidogrel bisulphate	Human plasma	LC – tendam mass	(5:95, V/V ammonium formate / methanol)	(5mm, 150mm 4.6mm I d) C8	[68]
18.	Clopidogrel bisulphate	Human plasma	HPLC- MS/MS	(65:35, V/V) 0.04% formic acid, 3 mmol /L ammonium acetate in acetonitrile / water	Kromasi Eternity – 2.5- C18	[69]
19.	Clopidogrel bisulphate	Human plasma	HPLC- UV	(80:20v/v) methanol and phosphoric acid	RP 18 (Hypersil BDS (250x4.6mm, 5ml)	[70]

 

CONCLUSION:

The wide range and development of analytical techniques for clopidogrel bisulfate analysis are highlighted in this comprehensive review. Considering sensitivity, accuracy, cost, and sample complexity, researchers and pharmaceutical professionals can select the best method for their particular analytical goals.

 

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Received on 12.09.2025     Revised on 20.10.2025 Accepted on 08.11.2025     Published on 28.11.2025 Available online from December 31, 2025 Research J. Engineering and Tech. 2025; 16(4):171-178. DOI: 10.52711/2321-581X.2025.00018 ©A and V Publications All right reserved
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