Clopidogrel is a new-type thiophene and pyrazine derivative and has the anti-platelet aggregation and anti-thrombosis functions. Clopidogrel can be rapidly absorbed and metabolized by human body after it is taken orally. Its main metabolite is clopidogrel acid. Its active metabolite is a kind of mercaptan derivative generated from the hydrolyzed oxygenated chemicals which are formed by clopidogrel, and it is difficult to detect this mercaptan derivative because it is extremely unstable.
This article establishes the pretreatment method for clopidogrel active metabolite in the plasma by derivatization method on the basis of Cleanert C2 SPE cartridge as a reference for the researches on the human bioequivalence of clopidogrel.
Table 1 Information of Aimed Compound
Clopidogrel active metabolite
Clopidogrel active metabolite derivative
Materials and Instruments
Cleanert C2 cartridge (150 mg/3 mL); SPE-M08 Positive Pressure SPE Device; Unisol C18(2) (2.1 ´ 50 mm, 5 μm, 110 Å); 2-bromine-3‘-methoxyaceto-phenone (MPB).
After blood sampling, add 25 μL 500 mM of MPB dissolved in acetonitrile to each blood sample immediately, slightly mix them (leave the samples stand still for 10 min), and then keep centrifuging the samples at 3000rpm for 30 min to separate plasma. Store plasma samples at -80 °C for further detection.
Sample pretreatment: Accurately transfer 400 μL of plasma to a tube and add 550 μL of formic acid (concentration: 1%). Then oscillate the tube on the vortex mixer to mix the substances uniformly.
Activization: Pretreat Cleanert C2 columns with 300μL of methanol and 300 μL of pure water in turn.
Sample loading: Transfer the plasma mixture to the pretreated Cleanert C2 columns.
Flushing: Flush the columns with 500 μL of formic acid (concentration: 1%) and 500 μL 50 mM of ammonium acetate solution.
Elution: Elute the columns with 150 μL of methanol and 100 μL 50 mM of ammonium acetate in turn.
Analyze the eluate by LC-MS/MS method.
Chromatographic columns: Venusil MP C18 (2) (2.1 ´ 50 mm, 5 μm, 100 Å)
Flow rate: 200μL/min;
Mobile phase: formic acid solution (concentration: 0.1%)/acetonitrile (55/45, v/v)
Column temperature: 30 °C; sample size: 3 μL
Scanning mode: Normal electrospray ionization mode
Collecting mode: Multiple-reaction monitoring (MRM)
Table 2 MS Parameters
Results and Discussion
Perform the clopidogrel active metabolite adding experiment to the plasma samples, and the standard addition recovery rates are higher than 93%, and the LOQ of this method for the aimed compound is low to 0.5 ng/mL.
Figure 1 Chromatogram of MP-AM Standard Solution (concentration: 30 ng/mL)
Figure 2 Chromatogram of Plasma
Figure 3 Chromatogram of Plasma Added with Standard Solution
In this method, clopidogrel active metabolite in the plasma is extracted with Cleanert C2 cartridge and analyzed, and results show the sample matrix has no obvious interference on the analyte and the recovery rates are higher than 93%. This method applies to the daily monitoring and analysis of clopidogrel active metabolite in the plasma.
150 mg/3 mL
Unisol C18 (2)
2.1 × 50 mm, 5 μm, 110 Å
SPE-M08 Positive Pressure SPE Device
NV24A-II Nitrogen Evaporator
1.5 mL vials
Screw neck vials, 12 × 32 mm
Caps and Septa
Screw neck cap, center hole; red silicone/white PTEE septa, slitted