Developing a Method for Measuring Amlodipine Levels in Cat Plasma

1. Developing a Method for Measuring Amlodipine Levels in Cat Plasma By:Preston Bull Advisors: Mike Gallagher, Dr. Webster

2. Amlodipine Medical Uses • Amlodipine is a calcium channel blocker. • It is used to treat chest pains and decrease blood pressure.

3. Why Do We Want to Measure Amlodipine in Cat Plasma? • Looking at drug blood levels to determine the effectiveness of two different dosing routes: • Oral drug delivery • Transdermal drug delivery; i.e. gel or ointment

4. Objectives - Should we use peak area of amlodipine or the area ratio with internal standard desipramine? • Which clean up method works the best? • Centricon Filters 3,000 molecular weight (MW) exclusion or 30,000 MW exclusion • Rinse the Centricon filters with buffer or 20% ethanol to improve the recovery • Ethyl solid phase extract (SPE) columns

5. HPLC • We used the HPLC in our project to measure peak area based on UV absorbance at 240 wavelength. • The HPLC column separates the drug and the internal standard from the background materials.

6. HPLC system

7. Standard Curve of Area • Linear relationship concentration and peak area.

8. Amlodipine Concentration Versus Peak Area

9. Amlodipine Concentration Versus Area Ratio with Internal Standard Desipramine

10. Introduction to Centricon Filters • A centricon filter is a filter which allows molecules up to a certain size and smaller to pass through. Such as a 3,000 centricon filter allows molecules up to 3,000 in size to pass through. • The big molecules in cat plasma would stay on the filter and the drug would pass through.

11. Centricon Filter

12. Method for Centricon Filters • Added one mL of phosphate buffer with or without amlodipine and desipramine to membrane filter. • Centrifuged samples for 20 min at 3,500 g and/or 7,500 g for 90 minutes. • Rinsed membrane of some filters with buffer or 20% ethanol and re-centrifuged sample at 7,500 g for 90 minutes. • Concentrated all samples by blowing off liquid with air and residue was taken-up into 1 mL of mobile phase and injected on the HPLC.

13. Centrifuge

14. Results from 3,000 MW exclusions with buffer

15. Conclusions from 3,000 MW exclusions • The size of filter is so small that the centrifugation takes too long with just buffer – 7,500 g for 90 minutes. • The recover of the internal standard, desipramine, is less than the drug, amlodipine. • Desipramine must stick to the membrane • This cleanup procedure works better by measuring the amlodipine peak area and not using an internal standard • The recover of Amlodipine is best after rinsing with 20% ethanol in water.

16. Results from 30,000 MW Exclusions With Buffer

17. Conclusions from 30,000 MW Exclusions With Buffer • Centrifugation could be done for 20 minutes at 3,500 g. • 90% recover of the internal standard desipramine under all conditions • 81% recover of amlodipine with the 20% ethanol rinse. • Both the direct amlodipine measurement and the internal standard method would work with the 20% ethanol rinse.

18. Results and conclusions from 30,000 MW Exclusions With Plasma • Plasma slows down the filtration process so that the centrifugation takes too long. > 90 minutes at 7,500 g • No amlodipine or desipramine was seen in the HPLC • The drug and internal standard were not released from the plasma. • Addition of 20% ethanol to the plasma clogged the filter.

19. Introduction to SPE Column • Solid phase extraction SPE • Column separates chemicals based on how well they stick to the column and how well they like the different solutions. • This allows us to separate the drug from the sample.

20. SPE device

21. Method for SPE Column • Using columns calls for specific measurements. • First you have to condition them with ACN, 2.5% ammonia, H2O, and phosphate buffer. • Secondly you load your sample on the column. • Rinsed columns with 20% ACN in water, followed by ACN. • Lastly you have to wash the columns with ACN and 2.5% ammonia at exact measurements to remove the drugs. • Concentrated all samples by blowing off liquid with air and residue was taken-up into 1 mL of mobile phase and injected on the HPLC.

22. Results from SPE Column

23. Conclusions from SPE Column • Cat Plasma really slows down your SPE columns. • We had good recovery from both amlodipine and desipramine. • Area ratio would be the best way to measure amlodipine from cat plasma.

24. Overall Conclusions • The SPE column worked the best when extracting amlodipine from cat plasma. • Our best method for quantitating amlodipine was using area ratio with the internal standard desipramine. • More work needs to be done to get more consistent results.