LC/MS/MS Method Development of Clonidine in Pig Tissue Samples
Rachel Sun, John Torchia, Jason Plassard
BASi, 2701 Kent Avenue, West Lafayette, IN 47906
Introduction
Clonidien is a widely used antihypertensive drug. It is an imidazoline derivative and acts as an agonist on alpha-2 receptors. Transdermal drug delivery has greater therapeutic benefit compared with oral and parenteral routes including decreased drug degradation, maintaining the therapeutic drug concentration over a long period of time, decreasing adverse reaction. Several LC-MS/MS methods have been developed and validated for quantitation of clonidine in plasma matrix.
This is the first time to measure clonidine in pig tissue samples in the range of 50 pg/mL to 50000 pg/mL.
Method
Sample preparation:
A certain volume of 50/50 acetonitrile/water was added to each tissue sample and homogenized. The added volume was recorded for calculation later.
A portion of each sample was then centrifuged and the clear solution was used for analysis. 200 µL of clear solution was mixed with 50 µL of isotope labeled internal standard and 600 µL of acetonitrile for protein precipitation. The supernatant was collected and dried under nitrogen at 40°C. The samples were reconstituted with 100 µL of 15% acetonitrile in water with 0.1% formic acid and injected onto an LC/MS/MS system using a Restek PFP analytical column with an ammonium acetate / acetonitrile / formic acid / water mobile phase.
Blank tissue samples were homogenized and supernatant was used for calibrators and QC preparation.
HPLC conditions:
Column:
Mobile Phase:
Run time:
Flow Rate: |
Restek Allure PFP column
80:20 ACN/Water with 20 mM ammonium formate, 1.0% of formic acid
4.0 minutes
0.8 ml/min
|
Tandem mass spectrometry:
Mass spectrometer:
Source:
Resolution:
|
API 4000
Turbo Ionspray
Unit/Unit
|
| Ions Monitored: |
Compound |
Precursor ion (Q1 m/z) |
Product ion (Q3 m/z) |
| |
Clonidine
Clonidine-d4 |
230.0
234.0 |
44.2
48.2 |
Product ion scan
Results
Standard Calibrator Performance
QC performance
Typical calibration curve
Typical Chromatograms
Blank Matrix
LLOQ 50.0 pg/mL
ULOQ calibrator 50.0 ng/mL
Discussion: How to improve sensitivity?
Instrument Platform
Quantum TSQ vs API 4000
TSQ, 1.0 ng/mL
API 4000, 1.0 ng/m
Mobile Phase and Column
C18 Column: not strongly retained, low organic in mobile phase, possible ion suppression from the early elute
PFP Column: strongly retained, high organic content in mobile phase, increased sensitivity.
SymmetryShield RP18 Column
50*2.1mm, 0.5 mL/min
Mobile phase: 10% ACN
SB-C18 Column
50*2.1mm, 0.5 mL/min
Mobile Phase: 5% ACN
Allure PFP Column
100*3.5mm, 0.8 ml/min
Mobile phase: 80% ACN
Conclusion
A fast LC/MS/MS method has been developed for quantitaion of clonidine at the range of 50 pg/mL to 50000 pg/mL in pig tissue samples. This method has been successfully applied in several non GLP studies.
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