Validation of assay for bulk clopidogrel and for some tablet forms by reverse-phase high-performance liquid chromatography

Peer review under responsibility of Taibah University

Figures & data

Fig. 1 Chemical structure of clopidogrel.

Fig. 2 Calibration curve for clopidogrel.

Fig. 3 RP-HPLC chromatogram of clopidogrel standard drug.

Fig. 4 RP-HPLC chromatogram of Clopilet^® 75 tablet.

Fig. 5 RP-HPLC chromatogram of Plavix^® 300 tablet.

Table 1 Results of optimization of the method.

Column	Mobile phase	Flow rate (mL/min)	Wavelength (nm)	Observation	Result
Microbondapak C18, 5 μm, 50 mm × 4.6 mm	Methanol only	0.5	254	No peak	Method rejected
Phenomenex reverse-phase C18, Luna 5 μm, 250 mm × 4.6 mm	Acetonitrile only	0.8	254	Resolution 1.0 but not satisfactory	Method rejected
Microbondapak C18, 5 μm, 50 mm × 4.6 mm	Acetonitrile:phosphate (70:30) buffer (pH 7.8)	1	270	Poor resolution (1.2)	Method rejected
Phenomenex reverse-phase C18, Luna 5 μm, 250 mm × 4.6 mm	Acetronitrile:phosphate (60:40) buffer (pH 5.0)	1	270	Poor resolution (1.1)	Method rejected
Develosil ODS HG-5 reverse-phase C18, 5 μm, 15 cm × 4.6 mm	Acetonitrile:phosphate (65:35) buffer (pH 2.85)	1	225	Good resolution (2.3) and sharp peak	Method accepted

Table 2 Recovery of the method.

Amount of drug added (μg) to analyte	Recovery from formulation
	Mean amount (μg) (n = 6)	Mean recovery (%)	RSD (%)	Bias (%)
16	15.95	99.71	0.36	−0.31
20	20.07	100.03	0.08	0.03
24	23.99	99.99	0.01	−0.04

Table 3 Intra- and inter-assay variation.

Observed concentration of clopidogrel (μg/mL)
Intra-day			Inter-day
Time	Mean (n = 6)	RSD (%)	Day	Mean (n = 6)	RSD (%)
09:00	40.01	0.06	1	40.03	0.06
13:00	39.99	0.06	2	40.02	0.06
17:00	40.02	0.06	3	40.03	0.06

Table 4 Robustness of proposed assay.

Change in parameter	RSD (%) (n = 6)
Flow (mL/min)
0.9	0.11
1.1	0.06
Detection wavelength (nm)
227	0.09
223	0.22
Acetonitrile:phosphate buffer
67:33	0.09
63:37	0.06

Table 5 Assay of clopidogrel tablets.

Formulation	Labelled amount (mg)	Amount found (mg)	Assay ± RSD (%)
Clopilet^® 75	75	75.01	100.004 ± 0.03
Plavix^® 300	300	299.88	99.96 ± 0.06