Dec 4, 2014 - Positive. Clopidogrel metabolite. 308.2â 198.1. 67. 23. 10. 10. Positive. Ticlopidine. (Internal standard). 264.0â154.0. 52. 24. 10. 10. Positive.
Clinrescon 3 rd-4th December 2014, Lucknow, India
Yarra Durga Prasad1 , Yashpal S. Chhonker1, 4, C. P Pandey2, 3, Tulsankar Sachin Laxman1, 4, Hardik Chandasana1, 4, V. S. Narain3, Madhu Dikshit 2, Rabi S. Bhatta 1, 4 1Pharmacokinetics &
Metabolism Div., 2Pharmacology Div., CSIR-Central Drug Research Institute, Lucknow-226031(India) Cardiology, King George’s Medical University, Lucknow- 226001 (India) 4Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, Rafi Marg, New Delhi-110 001 (India) 3Department of
Tab.2: Accuracy and precision of ASA and SA Concentration (ng/mL)
Metabolite of Aspirin
Aspirin LLOQ
LQC
MQC
HQC
LLOQ
(Salicylic acid) LQC MQC
1.56
6.25
200
800
1.56
6.25
200
800
Theoretical
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL
HQC
%Bias intra-assay
7.45
–5.49
–3.10
7.45
–5.59
3.49
–4.23
8.90
%Bias inter-assay
5.67
–6.39
7.30
–2.79
2.73
7.12
–2.94
–1.99
% RSD intra-assay
6.49
11.43
6.78
4.50
3.93
7.19
5.20
3.41
% RSD inter-assay
6.30
3.91
8.23
4.73
8.30
6.90
9.83
3.45
Tab.3: Accuracy and precision of CLP and CLPM
Experimental
O
OH
Aspirin and its metabolite Clopidogrel and its metabolite
Column: Concentration range: MRM Condition:
MQC
HQC
LLOQ
LQC
MQC
HQC
Theoretical
0.048 ng/mL
0.195 ng/mL
6.25 ng/mL
25 ng/mL
0.39 ng/mL
1.56 ng/mL
50 ng/mL
200 ng/mL
%Bias intra-assay
4.12
4.10
5.23
–3.90
–5.14
4.66
2.45
–4.63
%Bias inter-assay
–3.56
4.89
–3.63
–4.39
4.43
–4.34
–2.53
7.30
% RSD intra-assay
4.79
6.23
2.81
5.50
7.94
7.94
3.73
4.47
% RSD inter-assay
4.4
8.17
7.56
4.59
5.25
6.72
4.84
7.70
OH
Salicylic acid (SA)
O
O H S
Metabolite
Cl
N
OH N
Cl O
O
S
Carboxylic acid metabolite of Clopidogrel (CLPM)
Clopidogrel
Acetonitrile: Ammonium acetate buffer(3.5 mM, pH 3.5) 90:10%, v/v
Flow Rate:
Metabolite
S HO
O
Mobile Phase:
O
Aspirin
Shimadzu UFLC coupled with API 4000 Q-Trap mass spectrometer (Applied Biosystems)
Analytes :
LQC
OH
O
Instrument :
Metabolite of Clopidogrel (CLPM)
LLOQ
O
O
Method conditions
Clopidogrel
Concentration (ng/mL)
Fig.1. Chemical structures of analytes
0.75 mL/min with isocratic elution Waters Symmetry Shield (5 µm, 150 x 4.6 mm id) 1.56 – 800 ng/mL for Aspirin and its metabolite 0.048 –25 ng/mL for Clopidogrel 0.39–200 ng/mL for its metabolite Continuous polarity switch (+/-)
Fig.3. Representative MRM ion-chromatograms
Clinical study Human volunteers: Male Indian patients with acute coronary syndrome (67 volunteers) Route: Oral Formulation: Tablet Dose: Duration:
Fig.2. Proposed fragmentation pattern and MS/MS Product ion spectra
Tab.1: MRM transitions & compound parameters
75 mg Aspirin + 75 mg Clopidogrel Single tablet for at least 10 days
Bio-sample:
Plasma
Sample Analysis:
LC-MS/MS assay
Approval number:
XLIV ECM/A-P1
Sample processing Protein precipitation (0.1% formic acid in acetonitrile) followed by liquid-liquid extraction with diethyl ether and ethyl acetate 70:30, v/v.
Analyte
MRM transitions Q1→Q3
Declustering Potential (V)
Collision Engergy (V)
Entrance potential (V)
Collision cell exit potential (V)
Aspirin
178.8→137.2
–18
–10
–10
–10
137.0→93.0
–20
–18
–8
–8
Negative
163.2→119.0
–43
–12
–8
–10
Negative
Aspirin metabolite (Salicylic acid) Acetyl benzoic acid (Internal standard) Clopidogrel Clopidogrel metabolite Ticlopidine (Internal standard)
MS/MS polarity mode Negative
322.1→212.1
52
24
10
10
Positive
308.2→ 198.1
67
23
10
10
Positive
264.0→154.0
52
24
10
10
Positive
Fig.4 Variability in plasma concentration of (a) SA and (b) CLPM in patients receiving the same dose of ASA and CLP
Conclusion • Simultaneous monitoring of Aspirin and Clopidogrel along with their metabolites in human plasma was developed and validated for the first time. • Enables the identification of patients at increased risk of ASA and CLP by characterizing their metabolites concentration
