You work at the Norwegian Medicines Agency and must develop a method for separating and determining the levels of the three different -blockers Metoprolol, Atenolol

You work at the Norwegian Medicines Agency and must develop a method for separating and determining the levels of the three different β-blockers Metoprolol, Atenolol and Propranolol (drugs for treatment of hypertension) in rat brain extracellular fluid. The background is that a some patients who were on beta blockers experienced sleep disturbances and nightmares. The Swedish Medicines Agency suspected that the β-blockers in the body cross the blood-brain barrier (BBB) and they started a study where they gave the different β-blockers to laboratory rats then analyzed the content of beta blockers in both plasma and extracellular fluid from the brain, at a number of times. You must set up an analysis method to determine which concentration found in the cerebrospinal fluid (fairly straightforward pure solution) and which concentration found in the plasma (complex matrix) after different times after man gave the rats a mixture (in equal amounts) of the different beta blockers. Beta blockers acid constants and partition constants, for the partition between octanol and water, are:

Alprenolol with pKa = 9.5 and Log KD = 3.10

Metoprolol with pKa = 9.5 and Log KD = 1.88

Propranolol with pKa = 9.5 and Log KD = 3.56

For the structures of the beta blockers see below.

a) Suggest an HPLC method that can be used to separate the three beta blockers after sample injection of a mixture of its 3 components. Give detail like type of chromatographic column, composition of the mobile phase and whether you should have a pH buffer in the mobile phase and what pH the buffer should maintain. What detection principle do you suggest? after the column outlet? Motivate!

b) With your proposed chromatographic system in task a, which β-blocker comes out as the first peak and which one comes out as the last peak? Motivate!

c) Give a detailed account of the sample processing, separation and detection of the plasma samples and extracellular cerebrospinal fluid samples

 

Alprenolol H3C NH CH3 OH FCH Metoprolol H3C NH CH3 OH CH3 H3C Propranolol NH CH3 OH