J. Biochem. Biophys. Methods 48 2001. 175188 www.elsevier.

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Studies on the effect of alcohols on the chiral discrimination mechanisms of amylose stationary phase on the enantioseparation of nebivolol by HPLC
Hassan Y. Aboul-Enein) , Imran Ali
Pharmaceutical Analysis Laboratory, Biological and Medical Research Department (MBC-03), King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh-11211, Saudi Arabia

Abstract The chiral recognition mechanism of amylose CSPs has been described by achieving the enantiomeric resolution of ".-nebivolol on Chiralpak AD and Chiralpak AD-RH columns with methanol, ethanol, 1-propanol, 2-propanol, 1-butanol as mobile phases at different flow rates. The energies of interactions of methanol, ethanol, 1-propanol, 2-propanol and 1-butanol with both phases were calculated. The q.-RRRS enantiomer eluted first when using methanol, ethanol and 1-propanol, while the elution order was reversed when using 2-propanol and 1-butanol as the mobile phases. It has been concluded that the reversal elution order observed was due in part to the chiral cavities on the amylose CSP which were responsible for the bondings of different magnitude between chiral stationary phase and enantiomers, which are influenced with the type of alcohol used as mobile phase on the conformation of the 3,5-dimethyl phenyl carbamate moiety on the pyranose ring system of the amylose. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Chiral recognition mechanism; Resolution; Enantiomers; Nebivolol; Energy of interaction; Amylose tris 3,5-dimethylphenyl carbamate.; Effect of alcohols

1. Introduction The chiral resolution of racemates is an important area in the field of analytical chemistry and enantioseparations. Chromatography has become the powerful tool and

Corresponding author. Tel.: q 966-1-442-7859; fax: q 966-1-442-7858. E-mail address: enein@kfshrc.edu.sa H.Y. Aboul-Enein..

0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X 0 1 . 0 0 1 4 8 - 8

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most practical technique of enantiomeric resolution of a wide variety of racemic pharmaceuticals and drugs. A search of literature indicates that various chiral stationary phases have been developed for this purpose w17x. The understanding of chiral recognition mechanism at molecular level is of great importance in the field of chiral chromatography. Further search of literature reveals that some approaches have been done to find out the chiral recognition mechanism of different chiral stationary phases CSPs.. Attempts have been made by different workers to discuss the chiral recognition mechanism by NMR w813x and computational methods w11,1422x. The chiral recognition mechanisms have been developed for cyclodextrins w11,13x, Pirkle type w1417x and polysaccharides CSPs w2123x. Various rational models of interactions between CSPs and enantiomers have been proposed. The interaction energies between CSPs and enantiomers have been calculated by quantum mechanical calculations and the chiral recognition mechanisms have been proposed based on these calculations and molecular simulation dynamics w11,1418,20x. Among the various CSPs, the polysaccharide-based CSPs, i.e. derivatized cellulose and amylose CSPs, are considered efficient chiral stationary phases due to their wide range of applications in the field of chiral chromatography w2430x. Although attempts have been made to predict the chiral recognition mechanism of these stationary phases at molecular level, the exact mechanism is still not known. NMR spectroscopy is the

Fig. 1. The stereochemical formulae of q.-RRRS and y.-SSSR enantiomers of nebivolol.

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powerful and main technique for revealing the chiral recognition mechanism but the polysaccharide-based CSPs are soluble in the spectroscopic solvents such as tetrahydrofuran, acetone and pyridine. These solvents interact with the carbamate moieties, which is considered an essential adsorption sites for chiral recognition, of the polysaccharidebased CSPs and hence, the chiral recognition cannot be studied using these solvents. However, recently, Yashima, et al. w12x were able to study the chiral recognition mechanism of cellulose tris 4-trimethyl silyl phenyl. carbamate by NMR . Amylose tris 3,5-dimethyl phenyl. carbamate is a semi synthetic polymer which contain a polymeric chain of derivatized D-q. glucose residues in a-1,4 linkage. These chains lie side by side in a helical fashion. The three dimensional structures of celluloseand amylose-based CSPs were determined and compared using computational chemistry w31x. Vogt and Zugenmaier w32x reported that the possible structures were 3r2 helical chain conformation for cellulose tris phenyl carbamate. and 4r1 helical chain conformation for amylose tris phenyl carbamate.. The amylose CSP is more helical in nature and has well-defined cavities, making it considerably different from the corresponding cellulose analogue, which appear to be more linear and rigid in nature. In view of the problem associated with the experimental determination of chiral recognition mechanism as discussed above, we have carried out some HPLC experiments on amylose tris 3,5-dimethylphenyl carbamate. CSPs and attempts have been made to explain the chiral recognition mechanism by these CSPs. We have selected nebivolol Fig. 1. to carry out this study as it is the newly developed b-adrenergic agent with superior vasodilating properties w3336x. The purpose of this study is to explain the chiral recognition mechanisms involved, to resolve ".-nebivolol on amylose tris 3,5-dimethylphenyl carbamate. and to study the effect of alcohols on chiral discrimination involved in the resolution of nebivolol enantiomers.

2. Experimental 2.1. Chemicals and reagents The individual nebivolol enantiomers namely q.-RRRS Product No. R85547. and y.-SSSR Product No. R85548. and their racemic mixture Product No. R67555. were obtained as gifts by Janssen Research Foundation, Beerse, Belgium. Methanol, 2-propanol and 1-butanol of HPLC grade were purchased from Fisher Scientific Fairlawn, NJ, USA.. The absolute ethanol was obtained from E. Merck Darmstadt, Germany.. 1-Propanol was supplied by BDH, London, UK. 2.2. Apparatus All the HPLC experiments were performed on a HPLC system consisting of Waters solvent delivery pump model 510., Waters injector model WISP 710B., Waters tunable absorbance detector model 484. and Waters integrator of Waters model 740.. The columns used were Chiralpak AD 25 = 0.46 cm I.D., particle size 10 m m. and

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Chiralpak AD-RH 15 = 0.46 cm. I.D., particle size, 5 m m. and were obtained from Daicel Chemical, Tokyo, Japan. 2.3. Analysis The stock solutions 0.1 mgrml. of the racemic nebivolol and its enantiomers were prepared in absolute ethanol. Twenty microliter of each of the solutions were injected on to a HPLC system described above. The mobile phases used in this study were methanol, ethanol, 1-propanol, 2-propanol and 1-butanol. The mobile phases were filtered and degassed before use. The flow rates of the mobile phases used were 0.5, 1.0 and 1.5 mlrmin, respectively, for all the alcohols except for 1-butanol where the flow rate was 0.1 mlrmin. The chart speed was kept at 0.1 cmrmin. The detection of nebivolol was achieved at 220 nm. All the experiments were carried out at 23 " 18C. To study the effect of ethanol and 2-propanol ratios on enantiomeric resolution of ".nebivolol, different percentages of ethanol and 2-propanol were used at a flow rate of 1.0 mlrmin. on Chiralpak AD-RH Chiralpak AD columns. To determine the energy of interaction of alcohols with stationary phases, the chromatograms of methanol, ethanol, 1-propanol, 2-propanol and 1-butanol were also recorded using n-hexane 1.0 mlrmin.. as the mobile phases on both Chiralpak AD and Chiralpak AD-RH columns. The n-hexane was used as the mobile phase in both the stationary phases because the polarity of the n-hexane is zero and it is supposed that there is no interaction between hexane and the chiral stationary phases. The dead time t 0 . of both the columns were determined by injecting 20 m l of air using n-hexane as the mobile phase at a flow rate of 1.0 mlrmin with detection at 220 nm. The chromatographic parameters such as capacity factor kX ., separation factor a . and resolution factor Rs. were calculated.

3. Results and discussion The chromatographic parameters, capacity factor kX ., separation factor a . and resolution factor Rs. for the resolved q.-RRRS and y.-SSSR nebivolol enantiomers on normal and reversed phases are given in Tables 1 and 2, respectively. The resolved enantiomers were identified by running the chromatograms for the individual enantiomers, i.e. q.-RRRS and y.-SSSR enantiomers under the same chromatographic conditions. Tables 1 and 2 show that the enantiomers of nebivolol have been resolved on both Chiralpak AD and Chiralpak AD-RH columns using ethanol, 1-propanol and 2-propanol as mobile phases at different flow rates. Although the base line resolution of racemic nebivolol was achieved using ethanol, 1-propanol and 2-propanol at all the reported flow rates Tables 1 and 2., the best resolution may be considered at 0.5 mlrmin. flow rate on both Chiralpak AD and Chiralpak AD-RH columns except in the case of 2-propanol on Chiralpak AD-RH where the best resolution was achieved at 1.5 mlrmin flow rate Fig. 2.. To optimize the resolution, a variation in the chromatographic parameters was carried out. Methanol and 1-butanol were also tried but a partial resolution could be only achieved on both of normal and reversed phases. The various

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Table 1 X The chromatographic parameters, capacity factor k ., separation factor a . and resolution factor Rs. for enantiomeric resolution of ".-nebivolol on Chiralpak AD stationary phase Flow rate mlrmin. MeOH 0.5 1.0 1.5 EtOH 0.5 1.0 1.5 1-PrOH 0.5 1.0 1.5 2-PrOH 0.5 1.0 1.5 1-BuOH 0.1 y.-SSSR 3.16 q.-RRRS 3.34 1.06 0.22 y.-SSSR 1.53 1.36 1.40 q.-RRRS 6.32 5.96 5.85 4.13 4.38 4.18 2.45 2.36 2.53 q.-RRRS 4.22 2.10 1.09 y.-SSSR 5.84 3.03 1.86 1.38 1.44 1.71 1.71 1.45 1.21 q.-RRRS 4.57 4.33 4.18 y.-SSSR 6.32 6.06 5.77 1.38 1.40 1.38 2.63 2.28 1.40 q.-RRRS 1.06 4.88 2.04 y.-SSSR 1.95 3.21 3.38 1.84 1.71 1.66 0.96 0.50 0.41 k1
X

k2

Rs

ratios of the reported alcohols itself and with other solvents such as n-hexane normal phase mode., acetonitrile reversed phase mode. in the presence of diethylamine were tried but no improved resolution was observed. Since only partial resolution was
Table 2 X The chromatographic parameters, capacity factor k ., separation factor a . and resolution factor Rs. for enantiomeric resolution of ".-nebivolol on Chiralpak AD-RH stationary phase Flow rate mlrmin. EtOH 0.5 1.0 1.5 1-PrOH 0.5 1.0 1.5 q.-RRRS 8.36 2.61 1.10 y.-SSSR 11.5 3.83 1.75 1.38 1.47 1.59 1.76 1.20 1.10 q.-RRRS 5.65 3.00 2.60 y.-SSSR 7.94 4.27 3.75 1.41 1.42 1.44 1.73 1.10 1.15 k1
X

k2

Rs

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Fig. 2. Chromatograms of resolved enantiomers of ".-nebivolol on I: Chiralpak AD with a. ethanol 0.5 mlrmin., b. 1-propanol 0.5 mlrmin. and c. 2-propanol 1.5 mlrmin. and II: Chiralpak AD-RH with d. ethanol 0.5 mlrmin. and e. 1-propanol 1.5 mlrmin..

achieved when using methanol and 1-butanol as mobile phase, it may be concluded that the enantiomeric resolution of nebivolol is controlled by both polarities and viscosities of the alcohols. Accordingly, the polarities and viscosities of ethanol, 1-propanol and 2-propanol seem suitable for the resolution of enantiomers of nebivolol. Generally, the values of a for the resolved enantiomers increased with the increase of flow rates. It is due to the fact that the retention of enantiomers decreases by increasing the flow rates. It is of interest to note that the a values for the enantiomeric resolution in all cases using ethanol and 1-propanol at all flow rates. are greater on reversed stationary phase than the normal stationary phase. However, the differences of the a values for ethanol and 1-propanol at different flow rates is greater in normal phase condition than in the reversed stationary phase which may be due that the normal

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stationary phase is more affected by the flow rate in comparison to the reversed stationary phase. It was found that q.-RRRS enantiomer eluted first than the y.-SSSR enantiomer when using methanol, ethanol and 1-propanol as mobile phases while the order of elution was reversed when using 2-propanol and 1-butanol as the mobile phase on Chiralpak AD column. However, the elution order has not been reversed on Chiralpak AD-RH column. This could be due to the fact that alcohols interact strongly with the normal phase than the reversed phase. The poor interaction of alcohols with reversed chiral phase stationary phase may be due to the presence of a repulsion between the alcohols alkyl chains and the alkyl chains which make the chiral stationary phase reversed in nature. of the reversed CSP. We have used the reverse elution order observed as the mean to explain the chiral recognition mechanism of the amylose

Fig. 3. The relationship between the percentage of ethanol and 2-propanol and the retention times of q. and y. enantiomers of nebivolol.

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stationary phases. To understand the chiral recognition mechanism on Chiralpak AD, the resolution of nebivolol was tried with different ratios of ethanol and 2-propanol. The resolution of nebivolol with different ratios of 1-propanol and 2-propanol was also tried but there was a detection problem. The resolution behavior of nebivolol with different ratios of ethanol and 2-propanol is given in Figs. 3 and 4. Fig. 3 shows that the q.-RRRS enantiomer eluted first using ethanol and the difference of the retention times between the two enantiomers decreases by increasing the volume of 2-propanol. There was no resolution when the mobile phase consisted of ethanol:2-propanol 77:23, vrv. was used. However, by increasing the volume of 2-propanol, the resolution of these two enantiomers occurred but with the reverse order of elution. Furthermore, the values of resolution factors Rs. were more than 1.0 using ethanol or 2-propanol, but these values decreased by using different ratios of ethanol and 2-propanol and it became zero at ethanol:2-propanol 77:23, vrv. mobile phase indicating no resolution as shown in Fig. 4. In order to explain this behavior, the energies of interaction of alcohols with

Fig. 4. The relationship between the percentage of ethanol and 2-propanol and the resolution factor Rs. of q. and y. enantiomers of nebivolol.

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the stationary phases were determined and shown in Table 3. The energies of the interactions of alcohols were calculated by the formula described by Yuasa et al. w37x. DDG s RT ln a This equation has been used to calculate the difference of the energies DDG . of interactions of the two enantiomers with chiral stationary phase but to determine the energies of interactions G . of alcohols with chiral stationary phases, we have solved this equation as follows: DDG s RT ln a or DDG s RT ln kX2rkX1 or G s RT ln kX where kX is equal to t r y t 0 .rt 0 for different alcohols. The retention times of the alcohols were determined by using the pure n-hexane hexane has zero polarity and supposed to be inert in both normal and reversed phases. as the mobile phase with 1.0 mlrmin. as the flow rate on both normal and reversed phases. To calculate the value of kX , the dead times of the columns t 0 . were determined by using the same mobile phase and by loading 20 m l of air as the air is supposed to be inert and no interaction occurred between the air and the stationary phases. Table 3 shows that the values of energies of interactions of methanol, ethanol and 1-propanol were negative in magnitude with Chiralpak AD, while the values of energy of interactions of 2-propanol and 1-butanol were positive in magnitude and increase drastically in comparison to methanol, ethanol and 1-propanol. The values of energies of interaction of these alcohols with Chiralpak AD-RH were of negative in magnitude indicating a very poor interaction of alcohols with chiral reversed stationary phase. These findings clearly indicates that the alcohols interact very strongly with the normal phase while the interaction is very poor in case of

Table 3 The energies of interactions of alcohols with Chiralpak AD and Chiralpak AD-RH stationary phases Alcohols Chiralpak AD Methanol Ethanol 1-Propanol 2-Propanol 1-Butanol Chiralpak AD-RH Methanol Ethanol 1-Propanol 2-Propanol 1-Butanol t r min. 4.56 5.60 5.70 14.23 14.57 k
X

G K Calrmol. y0.56 y0.21 y0.18 0.71 0.73

0.37 0.70 0.73 3.33 3.43

1.96 1.97 2.00 2.10 2.26

0.026 0.031 0.047 0.099 0.180

y2.15 y2.04 y1.80 y1.36 y1.00

t 0 : 3.29 and 1.91 min for Chiralpak AD and Chiralpak AD-RH stationary phases respectively.

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reversed phase and therefore no reverse order of elution was observed in case of reversed phase. It is also interesting to observe that the interaction of methanol, ethanol and 1-propanol with Chiralpak AD is poor in comparison to the interaction of 2-propanol and 1-butanol. Therefore, 2-propanol and 1-butanol interact very strongly with normal chiral stationary phase and create a strain on the amylose chain, which resulted into the change of its configuration, i.e. the configuration of the pyranose ring of the amylose polysaccharide chain reversed. Besides, the long alkyl chains of these two alcohols create a strain on amylose stationary phase and are responsible for the reverse order of elution. It has been reported w38x that, under the normal conditions, the configuration of the glucose units of cellulose remains in the chair form with equatorial positions of hydroxyl groups, which is supposed to be the most stable configuration. In the same way, the amylose tris 3,5-dimethylphenyl carbamate. with equatorial position of carbamate moieties may be considered as the most stable configuration. The chiral recognition mechanism at a molecular level on the polysaccharide-based CSPs is not known although it has been reported that the chiral resolution by celluloseramylose-CSP is achieved through the hydrogen bonding w21x and p`p interactions w39x between the chiral stationary phase and the enantiomers. These hydrogen bondings occurred between the carbamate moieties of the celluloseramylose-CSPs and the functional groups such as hydroxyl, carbonyl, amino, etc., of enantiomers w21x. Although it is known that the chiral recognition occurred due to the different hydrogen and p`p bondings, however, the reason for the different magnitude of these bondings with enantiomers is not known. Therefore, attempts have been made to explain the chiral recognition mechanism in detail. The change in the configuration of chiral stationary phase has been explained by calculating a volume and energy factor F . for ethanol:2-propanol mobile phase at different ratios and it was calculated by the following equation. F s VE where V and E are the volume of ethanol or 2-propanol and the energy of interaction of ethanol or 2-propanol, respectively. The values of this factor for different ratios of ethanol and 2-propanol are given in Table 4. The factors F . for ethanol and 2-propanol were represented by Fet energy factor for ethanol. and Fpr energy factor for 2-propanol., respectively. Table 4 shows that the values of Fet are negative and the values of Fpr are positive, which corresponds to the different configurations of chiral stationary phase in ethanol and 2-propanol, respectively. To prove the different configuration of the chiral stationary phases in ethanol and 2-propanol, we have calculated the sum of Fet and Fpr and is given in Table 4. The Fet q Fpr . values are either positive or negative in magnitude Table 4. and the negative values correspond to the greater percentage of ethanol while the positive values are for greater percentage of 2-propanol in ethanol and 2-propanol mixture. The negative values may be correlated with the stable configuration of amylose stationary phase with the equatorial positions of 3,5-dimethylphenyl carbamate moieties. On the other hand, the positive values may be due to the less stable configuration of amylose stationary phase, i.e. with the axial positions of 3,5-dimethylphenyl carbamate moieties. The values of Fet q Fpr . decreases as the percentage of

H.Y. Aboul-Enein, I. Ali r J. Biochem. Biophys. Methods 48 (2001) 175188 Table 4 The calculated values of Fet , Fpr and Fet q Fpr . on Chiralpak AD stationary phase Volume ml. EtOH 00 50 55 60 65 70 75 77 80 85 90 95 100 2-PrOH 100 50 45 40 35 30 25 23 20 15 10 05 00 0.00 y10.50 y11.55 y12.60 y13.65 y14.70 y15.75 y16.17 y16.80 y17.85 y18.90 y19.95 y21.00 71.00 35.50 31.95 28.40 24.85 21.30 17.75 16.33 14.20 10.65 7.10 3.55 0.00 71.00 25.00 20.40 15.80 11.20 6.35 2.00 0.16 y2.60 y7.20 y11.80 y16.40 y21.00 Fet Fpr Fet q Fpr .

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F s VE, where F , V and E are factor, volume of alcohols and energy of interaction, respectively. Fet and Fpr are factors for ethanol and 2-propanol respectively.

ethanol increase in ethanol and 2-propanol mobile phase. The values of Fet q Fpr . change from positive to negative through a value of 0.16, which is the smallest values and equal to zero. Therefore, it may be concluded that the chiral configuration of amylose phase changed by replacing ethanol by 2-propanol but at the mixture of ethanol:2-propanol 77:23, vrv. the environment of amylose phase is 50% with equatorial and 50% with axial positions of the 3,5-dimethylphenyl carbamate. moieties. Therefore, at this mobile phase composition, the magnitude of hydrogen and p`p bondings are the same for both of the enantiomers and hence, no resolution occurred Figs. 3 and 4.. The cavities on the amylose CSPs provide the suitable site for a particular enantiomer, i.e. either for q.-RRRS or y.-SSSR. One of the two enantiomers fits more closely with greater chances of the bondings by hydrogen and p`p interactions. It may be assumed that the configuration of amylose chiral stationary phase remains in the form of chair with equatorial position of 3,5-dimethylphenyl carbamate moieties in presence of ethanol and the chiral cavities provide the suitable site for y.-SSSR enantiomer of nebivolol, i.e. the y.-SSSR enantiomer fit more closely than the q.-RRRS enantiomer. Therefore, the chances of the hydrogen and p`p interactions in case of y.-SSSR enantiomer is greater than the q.-RRRS enantiomer. Accordingly, the y.-SSSR enantiomer retained more and eluted after q.-RRRS enantiomer. On the contrary, in the presence of 2-propanol or 1-butanol, the configuration of the amylose stationary phase reversed, i.e. the 3,5-dimethylphenyl carbamate moieties are changed from equatorial to axial position. This configuration provides the better site for q.RRRS enantiomer than y.-SSSR enantiomers and hence, q.-RRRS enantiomer retained more and eluted after y.-SSSR enantiomer. The change of cellulose configuration through equatorial and axial positions in different mobile phases has been reported by Yuasa et al. w38x.

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Attempts have also been made to find out the different hydrogen bonding interactions between chiral stationary phase and nebivolol enantiomers. It has been observed that the magnitude of hydrogen bonding between y.-SSSR and chiral stationary phase with equatorial position was greater in comparison to q.-RRRS enantiomer. On the other hand, the hydrogen bondings were greater between chiral stationary phase with axial positions of 3,5-dimethylphenyl carbamate moieties. and q.-RRRS enantiomer in comparison to y.-SSSR enantiomer. Besides, we have also observed that the difference of the magnitude of the hydrogen bondings of the two enantiomers was greater in case of amylose chiral stationary phase with axial positions of 3,5-dimethylphenyl carbamate moities. This explains the greater difference of retention times of the two enantiomers on the chiral stationary phase with the axial positions of carbamate moieties. The reverse elution order on cellulose chiral stationary phases has also been reported by other workers. The high percentage of 2-propanol has reversed the order of elution of b-blockers on Chiracel OD column wcellulose tris 3,5-dimethyl phenyl. carbamatex w40x. The different enantioselectivity by different alcohols has been observed by Tang et al. w25x, Wainer et al. w41x and Wainer and Stiffin w42x. Therefore, it may be concluded that the chiral recognition mechanism of celluloseramylose phases are more or less similar in nature. In summary, it may be concluded that the configuration of the chiral cavities in the amylose tris 3,5-dimethylphenyl carbamate. is determined by the composition of mobile phase in normal phase mode while the configuration of reversed phase mode remains unchanged. The chiral recognition is not simply due only to hydrogen bondings and p`p interactions but also due to the chiral cavities with specific configuration on the stationary phases that are responsible for bondings of different magnitude between the stationary phase and enantiomers.

Acknowledgements The authors are thankful to JANSSEN Research Foundation, Beerse, Belgium for providing the nebivolol enantiomers and the racemic form used in this study. The authors would also like to thank the King Faisal Specialist Hospital and Research Centre administration for their support for the Pharmaceutical Analysis Laboratory Research Programs.

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