Enantiomeric Separation of Beta-Blockers by High Performance Liquid Chromatography An Undergraduate Analytical Chemistry Experiment Chieu D. ran' and Michael Dotlich Marquene University, Milwaukee, WI 53233 The analysis of chiral drugs has become a subject of
imnortance in science as well as in technolom. The -ereater p o p u l a r i t ~ o rrather demand is based on the fact that of
1327 totallv svnthetic drugs that currently are marketed worldwide,-528 are chiral and capable of existing as two or more ootical isomers ( I ) . Verv often, only one f o m of enantiomeri is pharmacological~yactive. The other or others can reverse or otherwise limit the effect of the desired enantiomer r 1-7,. For examplo, propranolol that is one of the most widely used beta-blockers i.e., a compound thnt inhibits the action of the adrenergic agents and reduces the force of the heart muscle contraction and the heart rate) exits in two enantiomeric fonns. The enantiomer that is most active in correcting ventricular arrhythmias is much less active a s a beta-blocker (8).One of the isomers of labeto101 is a n effective beta-blocker while another is thought However, in spite of this knowlto be an alnha-blocker (8). edge, only'61 of the 528 chiral synthetic drugs are marketed a s single enantiomers, while the other 467 are sold a s racemater. It is, thus, hardly surprising thnt the pharmaceutical industry needs efft,cti\.e a n a l ~ ~ i cand a l preparative chiral separ&ion methods. High performance liquid chromatography (HPLC) seems to be the instrument of choice for chiral separation because of its efficiency, speed, wide applicability, and reproducibility. Various approaches have been made in the last few years to use the HPLC for optical resolution of racemic mixtures. Perhaps the most notable one is based on the use of cyclodextrin solid stationary phase (2-6). I n t h i s method, chiral separation is due to the diastereomeric interactions between the optically active, doughnut-shaped cvclodextrin and the two enantiomeric f o m s ofthe anal* ( 5 4 . In fact, it is now possible to perform quantitatively o ~ t i c a resolution l on a number of raremates l,.y HPLC (2-
6.
The purpose of this experiment is to familiarize the students with the principle and application of chiral separation bv HPLC throueh the use of a cvclodextrin bonded chiral stitionary phase. I t will be acc~mplishedby the initial use of this cvclodextrin column for the direct enantiomeric separations of standard solutions containing either racemic mixture or enantiomer of chemically pure beta adrenergic blocking agents (e.g., propranolol, mitoprolol, and atenolol). Knowledge gained from this study will be used for a subsequent expe>ment that aims to determine not only the chemical structure but also the stereochemistry of the active ingredient of actual beta-blocker drugs (e.g., Inderal, Tenonnin, and Lopressor). Experimental A Shimadzu isocratic pump (Model LC-6A) was used to deliver the eluent. The sample was injected into the system through a Rheodyne's Model 7125 sample injector valve equipped with a 20-@ loop. The chiral column used was a 250 x 4.6 mm ID !Scyclodextrin bonded CM 5-pm
narticles stainless steel column (Advance Se~arationTechhologies, Whippany, NJ, Cyclobond I). A ~ G m a d z umodel SPD-6Avariable wavelength W detector and a Shimadzu model CAX Chromatopac integrator-recorder were used to detect (at 254 nm) and record the chromatogram. The mobile phase used in this experiment was a mixture of 95:5:0.3:0.2 (vlv) acetonitrile:methano1:acetic acid:triethylamine. It was prepared by initially mixing 475 mL of acetonitrile with 25 mL of methanol in a l-L volumetric flask. The mixture was filtered with a membrane filter and dewssed with vacuum aspirator. Acetic acid (1.5 mL) and 1;L of triethylamine su&equently were added to the degassed mixture. The acid and amine were added after degassing the acetonitri1e:methanol mixture because 1. the amine has relatively high vapor pressure. Therefore,
if it is added before degassing, its concentration after de-
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eassine as that~ before"the deeassine. " ~mav ,not ~ he the ~same~ 2. As n,ill he explamedin the next section, theenant~~me-se-
Iertiwty resolution is strongly dependent on the relntive amount of the acid ond omme in the mablle phase The mobile phase mixture was thoroughly mixed-and introduced into the column with a flow rate of 1mlimin. DL-Atenololand metoprolol were purchased from Sigma Chemical Company (St. Louis, MO). R- and S-propranolol were bought from Aldrich Chemical Company (Milwaukee, WI). Standard solutions containing 100 ppm of either the racemic mixture or the pure enantiomer of these compounds were prepared by dissolving the appropriate compound into the mobile phase. Three beta-blocker drugs were used in this experiment. They were (their generic names are given in parentheses): Inderal LA 120 mg (propranolol hydrochloride), Tenonnin 50 mg (atenolol), and Lopressor 100 mg (metoprolol tartrate). Results and Discussion The chiral column that has been stored in a 90:lO (vlv) acetonitri1e:water mixture required a t least 1 h to be equilibrated with the mobile phase used in this experiment. Therefore, in order to finish the experiment in a n usual 4-h allocated laboratory time, the mobile phase should be prepared a s soon and a s quickly a s possible and equilibrate the column with it. To ascertain that the column is equilibrated, two chromatograms for the same standard solution were obtained. The retention time on both chromatoerams should be the same if the column is equilibrated. ?he chromatograms of four compounds, namely S-propranolol, R-propranolol, DL-atenolol,and DLmetaprolol, were obtained by injecting 20pL of each standard solution onto the equilibrated column. Optimal condition for the chemical and enantiomeric separations of these compounds were determined by varying the combi'Author to whom correspondence should be addressed. Volume 72 Number 1 January 1995
71
atenolol and metaprolol) or near base line enantimeric separation for each compound. The beta-blocker drugs listed above can be either a tablet or a capsule. To enhance their solubility in water, the active ingredients of these drugs are in the acid hydrochloride form. To separate them from the water-soluble packing materials (i.e., starch) they should initially he converted to the free base form (to decrease their solubility in water and increase their solubility in nonpolar organic solvents), and subsequently extracted from the aqueous phase to the organic (diethyl ether) phase. The extraction process was as follows: 2 mg of one of these drugs were weighed and dissolved in 10 mL of pH 10.00 aqueous solution to convert the acid hydrochloride to 30 10 the free base fonn. The basic aqueous solution was prepared by adding 1drop of 50% (wlw)NaOH aquetime, min ous solution to 250 mL of deionized water. Two milliliters of ether were added to the drug solution and the free base form of the ingredient was extracted from the aqueous phase into the ether phase by means of a separating funnel. The organic layer was removed, and the aqueous phase was extracted one more time with 2-mL ether. The two ether portions were then combined and filtered with a syringe membrane filter. The ether solution was then injected onto the column to obtain chromatogram for each drug. The chromatograms obtained for the three beta-blocker drugs (Inderal LA, Tenormin and Lopressor) are shown in the figure, parts lc, 2b, and 3b, respectively. When these chromatograms and their retention times were compared with those of the standard compounds (figure, parts l a , lb, 2a, and 3a) it is evidently clear that the active ingredient of these drugs are propranolol. atenolol, and metoprolol. The other important information that can be deduced from these chromatograms is that these drugs are sold not as optically active compounds but rather as racemic mixtures. Thc mechanism for the enantiomeric separation is still unclear at this time. Recause t h s column cantime, min time, rnin not resolve these beta-blockers under reversed Chromatograms of the standard wmpounds (la. Ib. 2a. and 3a) and actual phase condition (8)and there is no water in the mobeta-blocker drugs (lc. Zb, and 3b). bile phase used in this work, it is very unlikely that inclusion complexes is formed between cyclodextrin nations of the mobile ~ h a s (i.e.. e the concentration of aceand beta-blockers. The most likely mechanism probably is tonitrile, methanol, aEetic acid, and triethylamine). Simidue to the diastereomeric interactions between the cylar to Armstrone and co-workers (8).we also found that the clodextrin and enantiomers of the beta-blocker where the aromatic and polar moiety of the drugs enable them to capacity factors, k', can be adjusted by changing the constrongly interact with the cyclodextrin and sit on top of the centrations of methanol and acetonitrile, andlor the total cyclodextrin cavity like a lid. amount of acid and amine. The resolution, &, however, is dependent on the relative amount of acid and amine added Summary to the mobile phase. The combination that provides the We have successfully demonstrated the principle and best chemical and enantiomeric separation, and relatively practical application of chiral separation with the use of a short retention times(l1 to 12 minfor propranolol, 12 to 1i cyclodextrin bonded stationary phase. The experiment is min for metuprolol, and 30 to 40 min for atenolol~for these relatively simple, inexpensive (the most costly item is the compounds was found to be 95:5:0.3:0.2 (VIVI acetonicyclodextrin column that is about $350 but with proper trile:methanol:acet~cacid:triethvlamine. This combination was, therefore, used as a mobile phase for the subsequent Chromatographic Data on the experiment. The chromatograms of these four compounds Resolution of Beta-Blockers obtained with this mobile phase is shown in the figure, parts la, lb, 2a, and 3a. The capacitor factors (k'), the selectivity (a), and the resolution (R.) were calculated based Compounds Capacity factor Separation Resolution (Rs) on these chromatograms and the results are listed in the (Va factor(a) table. As illustrated and listed in the table, the retention propranolol 2.91 1.06 0.63 times of each compound is much different from the others. This cyclodextrin column can, therefore, be used to differaten0101 10.11 1.11 1.29 entiate one compound from others. The stereochemistry of metaprolol 3.27 1.11 1.07 each compound also can be deduced from the chromatograms because this wlumn provides either base line (for 'Capacity factor of the fist eluted enantiomen.
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72
Journal of Chemical Education
care, it can last for many injections), and fast (the whole experiment can be performed in the usual 4-h allocated laboratory time). Due to the cogmexially limited availability of the standard compounds and of the fact that we could obtain only the three actual beta-blocker drugs at the time of experiment, the present chiral separation was performed for only three drugs. However, chiral separation can be accomplished with other beta-blockers including alprenolol, cateolol, and timolol with the use of the same cyclodextrin column and the same (or with a very little vanation in the concentrations) mobile phase (8).
Acknowledgment
The authors are grateful to Daniel W. Armstrong for stimulated discussion.
i:3. E23i,~~2~h,'gd~~;$.3&,AMI, Chem, lsl)S, 175, ninze, w L. sap. bfah. is81,10,159. PUC
: ~ninze,w.~.mdh.tmng,~. $~ ~ $ , ~ ~~ ~ D ~ ~i ~ ~ ~ 2~1 3 3 ; w. can ,
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chemical ~ o c i e t ywashinan, : DC. 1987. .19,62, 2467, w.;chen,s.;chang,c.; changS.J
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