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.