Separation of metoprolol enantiomers

The efficiency of many CSPs increases dramatically when liquid eluents are replaced with sub- or supercritical fluids. During a comparison of LC and SFC performed with a Chiralcel OD CSP, Lynam and Nicolas reported that the number of theoretical plates obtained was three to five times higher in SFC than in LC [26]. The separation of metoprolol enantiomers by LC and SFC on a Chiralcel OD CSP is illustrated in Fig. 12-2. Although impressive selectivity is achieved by both techniques, resolution is higher in SFC (R = 12.7) than in LC (R = 4.8), and the higher flowrate in SFC reduces the analysis time. The increased efficiency of SFC also improves peak symmetry. 

Fig. 12-2. Separation of metoprolol enantiomers by LC and SFC on a Chiralcel OD CSP. Chromatographic conditions for LC 20% 2-propanol in hexane, with 0.1 % diethylamine, 0.5 mL min f Chromatographic conditions for SFC 20 % methanol with 0.5 % isopropylamine in carbon dioxide, 2.0 mL min 15 MPa, 30 °C.

Chiral resolution was also achieved by means of CE chromatographic techniques, with an enantioselective stationary phase, as reported by Li and Lloyd (1993), who used a,-acid glycoprotein as stationary phase packed in fused silica capillaries of 50 mm i.d. These authors reported the optimization (by varying pH, electrolyte, and organic modifier concentration in the mobile phase) of the separation of the enantiomers of hexobarbital, pentobarbital, isofosfamide, cyclophosphamide, diisopyramide, metoprolol, oxprenolol, al-prenolol, and propranolol. 

Leloux, M.S. Rapid chiral separation of metoprolol in plasma—appUcation to the pharmacokinet-ics/pharmacodynamics of metoprolol enantiomers in the conscious goat. Biomed.Chromatogr., 1992, 6, 99-105... 

Balmer, K. Persson, A. Lagerstrom, P.-O. Persson, B.-A. Schill, G. Liquid chromatographic separation of the enantiomers of metoprolol and its alpha-hydroxy metabolite on Chiralcel OD for determination in plasma and urine. J.Chromatogr., 1991, 553, 391-397 [plasma urine human dog extracted metabolites fluorescence detection chiral column temp 35 column temp 25 LOD 10 nM]... 

Figure 1. Separation of the enantiomers of p-receptor blockers as oxazolidin-2-one derivatives. Column 18-m glass capillary column with XE-60-L-valine-R- phenylethylamide. Peaks 1, dechlorobupranolol 2, toliprolol 3, demethylbupranolol 4, alprenolol 5, bupranolol 6, 2-chlorbupranolol 7, isopropylbu-pranolol 8, oxprenolol 9, penbutolol 10, metoprolol [92].

Lucic, B., Radulovic, D., Vujic, Z. and Agbada, D., Direct separation of the enantiomers of (-l-)-metoprolol tartrate on impregnated TLC plates with D-(—)-tartaric acid as a chiral selector, J. Planar Chromatogr, 18, 294—299, 2005. 

Also, D-(—)-tartaric acid has been used as a CMPA for the separation of ( )-metoprolol tartarate on silica gel plates preimpregnated with the mobile phase (ethanobwater, 70 30, v/v) containing D-(-)-tartaric acid as a chiral selector. The results of experiments performed with different concentrations of D-(-)-tartaric acid (5.8,11.6, and 23 mmol/1) revealed that the best resolution of the metoprolol tartarate enantiomers was achieved with 11.6 mmol/1 d-(-(-tartaric acid in both the mobile phase and the impregnation solution at 25 2" C. It has been assumed that tartaric acid (p Tai 2.93) dissolved in excess of ethanol could react with ethanol forming monoethyltartrate, which might play a role of a real chiral selector in this separation system [43]. The structures of CSA, ZGP, and monoethyltartrate as counterions are presented in Figure 6.3. 

Steuer et al. demonstrated the use of supercritical fluid chromatography in the separation of enantiomers of 1,2 amino alcohols, namely pindolol, metoprolol, oxprenolol, propranolol, and DPT 201-106 using ionpairing modifiers [21]. The mobile phase consisted of carbon dioxide mixed with acetonitrile containing triethylamine as a counterion and /V-benzo-xycarbonylglycyl-L-proline as a chiral counterion. They found that the ca-... 

MIPs used as chiral stationary phases in o-CEC, p-CEC as well as in rod-CEC have shown high selectivity but relatively low efficiency. Most of the reported enantiomer separations on these phases were performed without pressurization of the flow system. Only Schweitz et al. described on the enantiomer separation of propranolol and metoprolol (print molecule R-propranolol or S-metoprolol) [57] and ropivacaine, mepivacaine and bupivacaine (print molecule S-ropivacaine) [58] by... 

Pure enantiomer imprinting of L-phenylalanine anilide, (/ )-propranolol, S)-metoprolol and (50-ropivacaine has been undertaken and these MIP capillaries have been used in the CEC mode for enantiomer separations [39-41,60-62,70,71] (Table 16.1). Baseline separations for the enantiomers of phenylalanine (Fig. 16.7) and for propranolol and metoprolol could be carried out in less than 2 min. (Fig. 16.5). A propranolol column was shown to be able to resolve several other j8-blockers, including prenalterol, atenolol, pindolol, etc. (Fig. 16.8) [41] and the ropi-... 

The separation of the metoprolol enantiomers was performed on a i-acid-glycoprotein column (100 mm x 4 mm ID) (ChromTech AB, Stockholm, Sweden) with a mobile phase of 0.25% 2-propanol in 20 mmol/1 phosphate buffer (pH=7) at a flow-rate of 0.8 ml/min. 

Figure 5. Peaks of the (+)- and (-)-metoprolol enantiomers after chiral separation, phase-system switching and moving belt LC/MS. Conditions see text.

Whereas chloroform [722, 723] and 1,2-dichloroethane [733, 739] have been used extensively as mobile phase modifiers in the separation of enantiomers, they can also be used as the major mobile phase constituent. For example, chloroform-and DCM-based mobile phases were used to separate the enantiomers of the W-3,5-dinitrobenzoyl-iJf-amino phosphonates of metoprolol, oxprenolol, propanolol, pronethalol, pindolol, and bufriralol [796]. An (/ )-Af-(3,5-dinitrobenzoyl)phenylgly-... 

The on-line combination of LC-LC-MS has been investigated for a number of reasons. In addition to the general prospects of multidimensional separation techniques, especially enhanced selectivity, there was special interest in the ability to perform LC-LC with two different mobile-phase compositions. In this way, it should be possible to avoid problems with mobile-phase incompatibility due to the use of nonvolatile mobile-phase constituents. A good example of this approach is the determination of enantiomers of p-blockers in plasma samples, described by Edholm and co-workers. Racemic mixtures of a p-blocker like metoprolol can be separated on a ai-acid glycoprotein column. However, the chromatography requires the use of a 20 mM phosphate buffer (pH 7) in the mobile phase, which is not compatible with on-line LC-MS. Therefore, the chiral column was coupled via a set of two trapping columns to a common reversed-phase LC column. After separation, the two enantiomers were sepa-... 

The following chiral reagents were employed for diastereomer formation before sample application and chromatography on silica gel or silica gel G TLC plates (L)-leucine Af-carboxyanhydride for D,L-dopa-carboxyl- " C separated with ethyl acetate/formic acid/water (60 5 35) mobile phase and detected by ninhydrin [7 f 0.38 (d)/0.56 (l)] [43] Af-trifluoroacetyl-L-prolyl chloride for D,L-amphetamine separated with chloroform/methanol (197 3) and detected by sulfuric acid/formaldehyde (10 1) (Rf 0.49 (d)/0.55 (l)) [44] Af-benzyloxycarbonyl-L-prolyl chloride for D,L-methamphetamine separated with n-hexane/ethyl acetate/acetonitrile/diisopropyl ether (2 2 2 1) and detected by sulfuric acid/formaldehyde (10 1) [/ f 0.57 (l)/0.61 (d)] [44] (l/ ,2/ )-(-)-l-(4-nitrophenyl)-2-amino-1,3-propanediol (levobase) and its enantiomer dextrobase for chiral carboxylic acids separated with chloroform/ethanol/acetic acid (9 1 0.5) and detected under UV (254 nm) light R[ values 0.63 and 0.53 for 5- and / -naproxen, respectively) [45] (5)-(4-)-a-methoxyphenylacetic acid for R,S-ethyl-4-(dimethylamino)-3-hydroxybutanoate (carnitine precursor) with diethyl ether mobile phase [/ f 0.55 R)/0J9 (5)] [46] and (5)-(4-)-benoxaprofen chloride with toluene/acetone (100 10, ammonia atmosphere) mobile phase and fluorescence visualization (Zeiss KM 3 densitometer 313 nm excitation, 365 nm emission) (respective R values of R- and 5-isomers of metoprolol, oxprenolol, and propranolol were 0.24/0.28, 0.32/0.38, and 0.32/0.39) [47]. 

The mobile phases consisting of acetonitrile/methanol [16] or acetoni-trile/methanol/water [18] in different ratios with a few drops of ammonia solution were found to discriminate between two enantiomers of these three -blockers in one-dimensional (ID) ascending development mode. The mobile phase composition and values of chiral separation factor a are listed in Table 11.3. The zones of separated antipodes were detected using iodine vapor and the detection limit of both alprenolol and propranolol racemates was 2.6 ng, while that of metoprolol was 0.26 /rg [16]. The effects of temperature on the separation of these three jS-blockers were also investigated [16]. It was observed that no resolution of atenolol racemate was achieved at the temperatures higher than 15" C, as well as below 8°C. The best resolution of propranolol and metoprolol racemates was achieved at 22°C. Increase of the temperature above 22°C led to the tailing, while a decrease up to 6°C had little or no effect on the quality of resolution. 

The effects of different temperatures on the separation of all three j8-blockers using L-aspartic acid as a chiral selector were also investigated. It has been found that 17°C was the most suitable temperature for the resolution of the examined jS-blockers, providing desired mobility to the diastereomeric ion pair formed anionic species of L-aspartic acid and protonated cations of amino moieties of the corresponding )3-blockers. The presence of chiral selector in situ was established by treating the developed chromatograms with ninhydrin that produced a characteristic color with aspartic acid in both spots of the resolved enantiomers [18]. This method was very sensitive, enabling detection of 0.26 fig atenolol and 0.23 tig of each metoprolol and propranolol. 

Enantiomers of metoprolol were separated on LiChrosorb Diol HP-TLC plates with dichloromethane and A/-benzoxycarbonyl-glycyl-L-proline as chiral selectors added to the mobile phase. The experiments revealed that saturation of the chromatographic system with water has a beneficial effect on chromatographic separation and reproducibility [20]. 

A number of enantiomers (aminoglutethimide, chlorpheniramine, chlorthalidone, fluoxetine, ibuprofen, ketoprofen, methylphenidate, metoprolol, phensuximide, propranolol, suprofen and mephenytoin) were separated on a -cyclodextrin column using 40/60 to 20/80 acetonitrile/water (0.1% triethylammonium acetate pH 4.1 or 7.1) mobile phase (analyte dependent) [1550]. 

Enantiomers of (-P/—)-metoprolol tartrate were separated on silica gel 60F plates using the chiral selector d-(—)-tartaric acid (11.6 mM solution) both impregnated into the layer and as mobile phase additive. The mobile phase was ethanol/water (70 30) and development temperature was 25 C. The layer was impregnated at ambient temperature for 90 min before sample application. Separated zones were detected by viewing at 254 nm, scanning at 230 nm, and exposure to iodine vapor. Rf values were 0.65 and 0.50 for the S-(—) and R-(+) enantiomers, respectively [19]. 

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