Simultaneous enantioseparation and sensitive detection of eight β-blockers using capillary...

Jack ZhengShahab A. Shamsi

Department of Chemistry,Center of Biotechnology andDrug Design,Georgia State University,Atlanta, GA, USA

Received November 28, 2005Revised January 28, 2006Accepted February 3, 2006

Research Article

Simultaneous enantioseparation andsensitive detection of eight �-blockers usingcapillary electrochromatography-electrospray ionization-mass spectrometry

The feasibility of using vancomycin chiral stationary phase (CSP) and polar organiceluent is investigated for simultaneous enantioseparation of eight b-blockers usingCEC coupled to ESI mass spectrometric detection (ESI-MS). The internally taperedcapillaries were utilized to pack CEC-MS columns. As compared to externally taperedcolumns, the use of internally tapered columns demonstrated enhanced stability, dur-ability, and reproducibility. A mixture containing methanol/ACN/acetic acid/triethyla-mine at 70:30:1.6:0.2 v/v/v/v was considered as optimum mobile phase since it pro-vided a good compromise between resolution and analysis time. As expected, sheathliquid and ESI-MS parameters mainly influenced the detection sensitivity. Interestingly,structural information of b-blockers was available by varying the MS fragmentor volt-age using in-house CID in the scan mode. In order to maximize the chiral/achiral res-olution, various column-coupling approaches using teicoplanin as complementaryCSP to vancomycin were tested. Several changes in the elution order of b-blockerswere observed using multimodal CSPs with some improvement in chiral or achiralresolution. The quantitative aspects of the CEC-MS method were demonstrated usingR- and S-talinolol as internal standards. The calibration curves of b-blockers showedgood linearity in the range of 3–600 mM. The enantiomer of b-blockers at a concentra-tion of 30 nM was detectable. Furthermore, both 0.1 and 1% of the S-enantiomer couldbe precisely quantified in the presence of 99.9 and 99% of the R-isomer of b-blocker.

Keywords: b-Blockers / Capillary electrochromatography / Electrospray ionizationmass spectrometry / Multimodal chiral stationary phase / Simultaneous enantio-separation DOI 10.1002/elps.200500874

1 Introduction

Simultaneous enantioseparation is a separation anddetermination of two or more pairs of enantiomers in onechromatographic run. There are several advantages toobtain simultaneous enantioseparation [1]: (i) A universalenantiomeric assay for structurally similar chiral com-pounds, using identical chiral column/selector as well as

mobile phase, eliminates the need for developing individ-ual assays for each compound. Therefore, both operationcost and analysis time could be significantly reduced; (ii)enantiomeric drugs and their chiral/achiral metabolites orintermediates can be simultaneously resolved. Forexample, (6)-propranolol can be enantioselectivelyhydrolyzed via catalysis of cytochrome P450 to produce4- and 5-hydroxylated metabolites [2, 3]; (iii) physico-chemical properties, such as capacity factor, can be cor-related with hydrophobicity (log P), and pKa’s could bedetermined for a combinatorial mixture of chiral drugs inhigh-throughput fashion. Thus, the implications of simul-taneous enantioseparation using “a selective stationaryphase with a wide chiral window” are far-reaching andcould have a large impact on how chiral assays can beperformed in the near future [1, 4–15].

Correspondence: Professor Shahab A. Shamsi, Department ofChemistry, Center of Biotechnology and Drug Design, P.O. Box4098, Georgia State University, Atlanta, GA, 30302–4098, USAE-mail: [email protected]: 11-404-651-2751

Abbreviations: CSP, chiral stationary phase; HOAc, acetic acid;MeOH, methanol; NH4OAc, ammonium acetate; TEA, triethylamine

Electrophoresis 2006, 27, 2139–2151 2139

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

2140 J. Zheng and S. A. Shamsi Electrophoresis 2006, 27, 2139–2151

Despite great demands for simultaneous enantiosepara-tion, its method development could be very challengingsince both chiral/achiral resolutions as well as high effi-ciency are desired. Currently, the most frequently appliedchromatographic methods for simultaneous enantio-separation include HPLC [4–6, 15] and CZE [7–14]. How-ever, in the case of HPLC with UV-detection, poorseparation efficiency reduces peak capacity, which hin-ders the application of simultaneous enantioseparation.The use of neutral or charged CD is potentially attractivefor chiral separation, but due to the absence of “real”stationary phase, somewhat lower achiral selectivity be-tween structurally similar chiral analogs with poor loadingcapacity is observed. Chiral CEC has demonstratedmuch higher efficiency and enantiomeric resolution thancapillary LC [16]. Hence, chiral CEC, in particular with thehyphenation to MS detection [17–19], could overcomethe aforementioned demerits of the HPLC and CZEmethods.

The hyphenation of chiral CEC to MS can provide severalbenefits. First, the submicroliter flow rate of CEC is idealfor ESI source, which in turns prevents ion-source con-tamination by HPLC mobile phase or CZE chiral selector.Even certain nonvolatile buffers or sheath liquid at lowconcentration might be employed without fouling of theESI source [20]. Second, MS detection arguably providesthe highest sensitivity among all detection methodsreported to date for CEC. Finally, MS is able to provideboth molecular mass and structural information. In par-ticular, the characterization of unknown compounds pro-vides very useful hints for structural elucidation. Thus,CEC-MS is able to provide identification and quantitationof all eluting enantiomers as well as their chiral metabo-lites in a single run. Despite these benefits offered bychiral CEC-MS, it is surprising to note that none of theearlier works in this area [20, 21] explored the possibilityof simultaneous enantioseparation.

Recently, our group reported chiral CEC-ESI-MS assay of(6)-warfarin in human plasma [22]. Although the use ofexternally tapered capillary significantly improved the re-producibility of retention time, its fragile tip caused severeproblem for CEC-MS [23]. Thus, one goal in this work wasto improve robustness of chiral CEC-MS using internallytapered columns fabricated by a novel procedure [23].In this study, simultaneous enantioseparation of eightb-blockers was performed on internally tapered capil-laries packed with vancomycin chiral stationary phase(CSP). Several mobile phase, sheath liquid, and ESI spraychamber parameters were optimized to achieve highresolution with sensitive MS detection. Moreover, theeffect of complementary enantioseparation was eval-uated using vancomycin and teicoplanin as well as multi-

modal CSPs. Finally, the quantitation aspects of thedeveloped method were evaluated, including determina-tion of enantiomeric impurity at levels of 1% as well as0.1%.

2 Materials and methods

2.1 Standards and chemicals

The 3-mm vancomycin and teicoplanin CSPs were dona-ted by Advanced Separation Technologies (Whippany,NJ, USA). Racemic mixtures and single enantiomers ofb-blockers (Fig. 1) were obtained from Aldrich (Milwau-kee, WI, USA), except (6)-carteolol and (6)-talinololwhich were donated by BetaChem (Leawood, KS, USA)and AWD Pharma (Dresden, Germany), respectively. TheHPLC-grade ACN and methanol (MeOH) were purchasedfrom Fisher (Springfield, NJ, USA). HOAc, ammoniumacetate (NH4OAc), triethylamine (TEA), and sodium chlo-ride (NaCl) were supplied by Aldrich. Water used in all ofthe experiments was purified by a Barnstead Nanopure IIWater System (Dubuque, IA, USA).

2.2 CEC column fabrication

The fused-silica capillaries (od 363 mm, id 75 mm),obtained from Polymicro Technologies (Phoenix, AZ,USA) were used to pack both CEC-UV and CEC-MS col-umns. The internally tapered column is fabricated bycarefully heating the end of a capillary using a microtorch.First, a diamond cutter is utilized to generate a clean cutat one end of a 1-m-long fused-silica capillary. Then, theaforementioned capillary end is heated to ,8007C. Afterthe polyimide coating of the column end is burned off, thecapillary end is moved quickly in and out of the flamecenter until the taper is gradually formed over a period of30–60 s. To prevent deterioration of tapered end, thecapillary is rotated during the heating process. Comparedto the published fabrication method [24], our proceduredoes not allow the taper to close the internal channelcompletely. Hence, the internal tapered capillary with id7–10 mm can be produced reproducibly without furthersanding. Finally, these internally tapered capillaries werepacked with CSP using the same procedure reportedelsewhere [22, 25]. A typical CEC-MS column consists of60 cm packed and 3 cm unpacked segment.

2.3 CEC-UV and CEC-ESI-MS instrumentation

The CEC-UV experiments were carried out with an AgilentCE system (Palo Alto, CA, USA) interfaced to a diodearray detector (DAD). The CEC-ESI-MS experiments were


Electrophoresis 2006, 27, 2139–2151 CE and CEC 2141

Figure 1. Chemical structures of eight single chiral center b-blockers (the asterisk (*) denotes chiral center).

carried out with the same CE instrument interfaced to asingle quadrupole mass spectrometer, Agilent 1100 se-ries MSD. An Agilent 1100 series HPLC pump equippedwith a 1:100 splitter was used to deliver the sheath liquid.The Agilent Chemstation and CE-MS add-on softwarewere used for instrument control and data analysis.

2.4 CEC-UV and CEC-ESI-MS conditions

The mobile phase or sheath liquid was degassed for30 min and filtered with a 0.45 mm PTFE membrane beforeuse. The column was preconditioned using the mobilephase before CEC run. The injection was performedelectrokinetically at 6 kV for 8 s in all cases unless other-wise mentioned. For CEC-UV, separation voltage was setat 20 kV, employing a voltage ramp of 3 kV/s. During theseparation an external pressure of 12 bar was applied toboth inlet and outlet buffer vials. For CEC-ESI-MS,separation voltage was set at 25 kV, employing a voltageramp of 3 kV/s. During the separation, a 12-bar externalpressure was applied at inlet vial to suppress bubble for-mation. The following conditions were used unless other-wise stated: mobile phase, MeOH/ACN/HOAc/TEA(70:30:1.6:0.2 v/v/v/v); sheath liquid, MeOH/H2O (90:10v/v) containing 50 mM NH4OAc, flow rate, 5.0 mL/min; cap-illary voltage, 13000 V; fragmentor voltage, 80 V; dryinggas flow rate, 5 L/min; drying gas temperature, 1307C;nebulizer pressure, 4 psi. The positive selective ion mon-itoring (SIM) mode was set with m/z 250.0 (alprenolol),

267.0 (atenolol), 293.0 (carteolol), 268.0 (metoprolol),266.0 (oxprenolol), 249.0 (pindolol), 260.0 (propranolol),and 364.0 (talinolol).

2.5 Preparation of standard analytes

All b-blockers were dissolved in MeOH at a concentration30 mM and stored at 2207C. Linearity was checked bythree replicate injections of mixtures containing (6)-ate-nolol, (6)-metoprolol, (6)-oxprenolol, and (6)-proprano-lol, over the range of 3.0–600 mM, each containing300 mM of (6)-talinolol as internal standard.

2.6 Calculations

Chiral resolution (Rs), selectivity (a), and separation effi-ciency (N) of enantiomers were calculated with AgilentChemstation software (V 9.0). All chromatograms shownwere smoothed with a factor of 0.1 min. The noise levelwas determined using six times of SD of the linearregression of the baseline drift for a selected time rangebetween 10 and 20 min. The S/N was obtained as theratio of peak height over noise level. The R- and S-enan-tiomer calibration curves of four representative b-block-ers were obtained by plotting the peak area ratio of therespective b-blocker enantiomer to the internal standard(R- or S-talinolol) versus concentration. To assess linear-ity, the line of best fit was determined by least squareregression.



Figure 2. Electropherograms (a–c) and plot (d) showing effects of packed column length for simul-taneous enantioseparation of eight b-blockers. Achiral resolution (Rs) for four critical regions arenamed as A, B, C, and D. Conditions: (a) 60, (b) 30, (c) 90 cm packed-bed length, 75 mm (id) capillarytapered internal (ca. 10 mm id) and packed with 3 mm vancomycin CSP; mobile phase, MeOH/ACN/HOAc/TEA 70:30:0.4:0.2 v/v/v/v; sheath liquid, 50 mM NH4OAc in CH3OH–H2O (90:10 v/v), deliveredat a flow rate of 5 mL/min. For the other CEC-ESI-MS conditions see Section 2.

3 Results and discussion

3.1 Preliminary studies

Macrocyclic glycopeptide CSPs, including vancomycinand teicoplanin, have demonstrated broad enantioselec-tivity for basic compounds (e.g. b-blockers) with polarorganic eluent [16, 26–29]. To validate enantioselectivityof vancomycin CSP, a test mixture containing (6)-oxpre-nolol and (6)-metoprolol was separated using polarorganic phase with CEC-UV (data not shown). An opti-mum mobile phase, consisting of MeOH/ACN/HOAc/TEA

70:30:0.4:0.2 v/v/v/v, was found to provide a good com-promise between achiral/chiral resolution and analysistime.

3.2 Elution order of �-blockers

Simultaneous separation of eight b-blockers was firstconducted on a 60-cm-long vancomycin column. Asshown in Fig. 2a, these b-blockers eluted in the follow-ing order: oxprenolol , alprenolol , pindolol , metopro-lol , propranolol , talinolol , atenolol , carteolol. Clearly,this order is not in accordance to log P values as listed in



Fig. 1. It is believed that multiple intermolecular interac-tions (e.g. steric interactions, hydrogen bonding, dipoleinteraction, ion exchange, and p-p interaction) areinvolved in chiral/achiral recognition with polar organicmode using vancomycin CSP [30]. Several trends in elu-tion order and chiral selectivity of b-blockers could begeneralized. First, the steric effects influence both chiraland achiral separations on vancomycin CSP. For exam-ple, para-substituted b-blockers (e.g. carteolol, atenolol,and metoprolol) are retained much longer than ortho-substituted b-blockers (e.g. oxprenolol and alprenolol).Because vancomycin CSP contains three inclusion cav-ities, para-substituted b-blockers with linear geometrycould reach deeper into these cavities as compared toortho-substituted b-blockers with V-shaped geometry.Consequently, somewhat lower efficiencies wereobserved for the former compared to the latter set ofb-blockers. Further comparison showed that the lowestresolution and selectivity was obtained for talinolol enan-tiomers (Fig. 2a), which is probably related to the increasein size of substitution of the cyclohexyl urea group. Fur-ther investigation to correlate elution order with the inter-actions between analytes and CSP using molecularmodeling and NMR is underway. It is worth mentioningthat CEC-UV analysis of multicomponent mixture requirestime-consuming spiking to identify each component.However, in CEC-MS, the elution order for each b-blockerwas easily determined using the extract ion mode (Fig. 2ainset) with no spiking needed.

3.3 Optimization of CEC and ESI-MSparameters

The electropherogram in Fig. 2a shows that 15 out of 16enantiomers (obtained from eight b-blockers) areresolved within 60 min. However, the achiral separationbetween the b-blockers needs to be improved. Forexample, peaks 3’ and 5 (R-pindolol and S-propranolol)are coeluted. In addition, there are four regions where onlypartial achiral resolution was observed, namely peaks 1’and 2 (R-oxprenolol and R-alprenolol, region A), peaks 3and 4 (S-pindolol and S-metoprolol, region B), peaks3’ 1 5 and 4’ (R-pindolol 1 S-propranolol and R-meto-prolol, region C) as well as peaks 8 and 7’ (S-carteolol andR-atenolol). In the following section, the separation pa-rameters (i.e. packed-bed length and mobile phase com-position), were discussed first. Then, sheath liquid andspray chamber parameters were optimized.

3.3.1 Evaluation of column packed-bed length

By keeping the field strength constant on the packed bed,another two columns, one packed with 30 cm (30 cm

unpacked, separation voltage 10 kV) and the otherpacked with 90 cm (2 cm unpacked, separation voltage30 kV) were tested (Figs. 2b and c). Compared to 60 cm(Fig. 2a) or 90 cm packed bed (Fig. 2c), the 30 cm column(Fig. 2b) showed the fastest separation (,45 min) withslightly higher resolution for region A, but no resolution forregion C. However, the resolution deteriorated in bothregions C and D (Fig. 2d). Both 60 and 90 cm columnsshowed achiral resolutions for all four regions. Although90 cm packed column could be used for large-volumeinjection without overloading, the total analysis time forthis column increased substantially to ,100 min. As aresult, the 60-cm-long column was considered as theoptimum packed bed.

3.3.2 Optimization of CEC-MS mobile phase

Appropriate choice of mobile phase for CEC-MS isimportant to achieve high resolution as well as highthroughput but without compromising the ESI-MS sensi-tivity. In this experiment, the polar organic mobile phaseconsists of MeOH, ACN, HOAc, and TEA. Thus, the firstset of experiments to be discussed is related to the var-iation in volumetric ratio of these four components in themobile phase. To locate a suitable EOF marker for CEC-ESI-MS, several neutral compounds, i.e. acetone, DMSO,and thiourea, were tested. The first two compounds wereeliminated because acetone had no ESI-MS signal andDMSO disturbed the column stability by damaging the fritwhich resulted in a significant current drop after electro-kinetic injection. As a result, thiourea was chosen as theEOF marker since it provided an acceptable ESI-MSsensitivity.

3.3.2.1 Influence of mobile phase MeOH/ACNratio

In this set of experiments, the volume fraction of MeOHand ACN was varied from 90:10 to 50:50 while HOAc/TEAwas fixed at 0.4:0.2 v/v. As shown in Fig. 3d, the retentiontime of thiourea decreased by 50% when MeOH/ACNratio changed from 90:10 to 70:30 v/v. When this ratio wasfurther changed from 70:30 to 50:50 v/v, thiourea reten-tion time only decreased slightly. The enhanced EOF wasdue to higher dielectric constant-to-viscosity ratio (er/Z)using higher volume fraction of ACN [28]. Thus, theretention times of all b-blockers first decreased as theMeOH/ACN ratio changed from 90:10 to 70:30 v/v due tohigher EOF (Fig. 3a and b). However, the change inMeOH/ACN ratio from 70:30 to 50:50 v/v increasedretention of all the b-blockers (Fig. 3b and c). This is be-cause ACN and MeOH also compete for hydrogen bond-ing sites on vancomycin CSP with the analytes (b-block-



Figure 3. Electropherograms (a–c) and plots (d and e) showing effects of mobile phase MeOH/ACN ratio v/v for simulta-neous enantioseparation of eight b-blockers, (d) thiourea retention time, and (e) achiral Rs for A–D regions. Conditions:mobile phase, MeOH/ACN/HOAc/TEA v/v/v/v (a) 90:10:0.4:0.2, (b) 70:30:0.4:0.2, and (c) 50:50:0.4:0.2. Peak identificationsand other conditions are the same as in Fig. 2 or described in Section 2.

ers). Since ACN is a poorer hydrogen bonding donor thanMeOH [28], the aforementioned competition decreases atlower MeOH content. Thus, the increase in EOF appearedto be less significant than the contribution of elutropicforce by hydrogen-bonding interactions. As presented inFig. 3e, only MeOH/ACN 70:30 showed a degree of reso-lution for all A–D regions. In contrast, MeOH/ACN 90:10and 50:50 v/v provided higher resolution for one regionbut only at the expense of the other regions. Thus, MeOH/ACN 70:30 v/v was selected as the optimum because itprovided a good compromise between resolution andanalysis time.

3.3.2.2 Influence of mobile phase ionic strength(MeOH–ACN:HOAc–TEA volumetricratio)

In this set of experiments, the volume fractions of HOAcand TEA were varied from 0.2:0.1, 0.4:0.2, to 0.8:0.4,while the MeOH/ACN was fixed at 70:30 v/v. Since HOAc

and TEA can react to generate triethylammonium acetate,the increase of HOAc and TEA content will lead to theincrease in salt concentration (ionic strength) of themobile phase. As expected, EOF decreases with anincrease in HOAc/TEA v/v (Fig. 4d). This is most likely dueto a decrease in zeta potential as well as an increase inmobile phase viscosity at higher salt concentration [31].However, reverse trends were observed for the retentionof b-blockers (Figs. 4a–c), especially for later elutingb-blockers. These observations implied that negativelycharged carboxylate groups on vancomycin CSP serveas weak cation-exchange sites [32]. When mobile phaseionic strength increases, triethylammonium ions in themobile phase could compete with positively charged b-blockers; therefore, the analytes retention decreases. Inaddition, high mobile phase ionic strength also providedmuch robust current and EOF along with sample stack-ing, which in turn enhanced the detection sensitivity. TheHOAc/TEA ratio at 0.4:0.2 v/v was selected as the opti-mum since it provides a good compromise between res-olution and analysis time.



Figure 4. Electropherograms (a–c) and plots (d and e) showing effects of mobile phase ionic strength for simultaneousenantioseparation of eight b-blockers, (d) thiourea retention time, and (e) achiral Rs for A–D regions. Conditions: mobilephase, MeOH/ACN/HOAc/TEA v/v/v/v (a) 70:30:0.2:0.1, (b) 70:30:0.4:0.2, and (c) 70:30:0.8:0.4. Peak identifications andother conditions are the same as in Fig. 2 or described in Section 2.

3.3.2.3 Influence of mobile phase HOAc/TEAratio

Further experiments were directed to examine the acid/base ratio (HOAc/TEA) to reduce analysis time withoutcompromising chiral/achiral resolution. When HOAc/TEAv/v was varied from 0.2:0.2 to 1.6:0.2, the electro-pherograms shown in Figs. 5a–d were obtained. As pre-sented in Fig. 5e, EOF consistently decreased withincreasing acid/base ratio. This is related to the sup-pressed ionization of the carboxylic groups on vancomy-cin CSP. Although EOF is decreased (Fig. 5e), the reten-tion times for all eight b-blockers consistently drops(Figs. 5a–d). Since HOAc/TEA ratio could affect thecharge on both vancomycin CSP and b-blockers, theincrease of acid/base ratio seemed to predominantly de-crease the ion-exchange capacity (due to protonation ofcarboxylate groups) on vancomycin CSP [33]. On theother hand, with increasing HOAc volumetric ratio, nega-tively charged acetate ions and positively charged b-

blockers could form ion-pairs, which decreased theretention of b-blockers. By comparing the achiral resolu-tion of A–D regions, HOAc/TEA ratio at 1.6:0.2 was cho-sen since the overall resolutions of A–C regions were sig-nificantly improved, while the resolution of only region Dwas slightly deteriorated (Fig. 5f). Furthermore, the totalanalysis time significantly drops by 40% with HOAc/TEAat 1.6:0.2 v/v compared to HOAc/TEA at 0.2:0.2 v/v.Again, similar to aforementioned mobile phase ionicstrength study, higher HOAc/TEA volumetric ratio result-ed in sharper peaks due to sample stacking, which in turngreatly improved detection sensitivity.

3.3.3 Optimization of sheath liquid and spraychamber parameters

The effects of sheath liquid and ESI-MS spray chamberparameters were also investigated. It was found thatthese parameters mainly affect detection sensitivity (data



Figure 5. Electropherograms (a–d) and plots (e and f) showing effects of mobile phase HOAc/TEA ratio v/v for simulta-neous enantioseparation of eight b-blockers, (e) thiourea retention time, and (f) achiral Rs for A-D regions. Conditions:mobile phase, MeOH/ACN/HOAc/TEA v/v/v/v (a) 70:30:0.2:0.2, (b) 70:30:0.4:0.2, (c) 70:30:0.8:0.2, and (d) 70:30:1.6:0.2.Peak identifications and other conditions are the same as in Fig. 2 or described in Section 2.

not shown). To achieve the highest detection sensitivity,the following ESI-MS settings were utilized: sheath liquid,MeOH/H2O (90:10 v/v) containing 50 mM NH4OAc, flowrate, 5.0 mL/min; capillary voltage, 13000 V; fragmentorvoltage, 80 V; drying gas flow rate, 5 L/min; drying gastemperature, 1307C; nebulizer pressure, 4 psi.

It is well known that high fragmentor voltage can causecollision of analyte ions, which provides valuable struc-tural information. Most of the CEC-MS studies to date areperformed in SIM mode mainly due to its high sensitivity.Consequently, to overcome low sensitivity of scan mode,a larger injection (applying 5 kV for 60 s) was performed.The fragmentor voltage was set at 80, 130, and 180 V. Al-though simultaneous separations of all eight b-blockerswere achieved in the scan mode, for the purpose of sim-plicity, MS spectra of three pairs of well-separated enan-tiomers (oxprenolol, alprenolol, and talinolol) are dis-cussed. As shown in Fig. 6a, when relatively lower frag-

mentor voltage of 80 V was applied, all three b-blockersshowed the highest abundance of respective protonated[M 1 H]1 molecular ions in their mass spectra. Note that afragment ion at m/z 225 (due to [M 1 H 2 C3H5]

1) isobserved in the mass spectra of oxprenolol. This sug-gests that oxprenolol has the lowest stability among threeb-blockers. When further increasing fragmentor voltageto 130 V, one more prominent fragment ion at m/z 248(due to [M 1 H 2 H2O]1) was observed for oxprenolol(Fig. 6b). At the same fragmentor voltage, two additionalfragment ions, one at m/z 226 and the other at m/z 308,were observed in the mass spectrum of talinolol. Thesecould be due to the simultaneous loss of tertiary butylgroup and cyclohexyl group (i.e. [M 1 2H 2 C4H9 2

C6H11]1) or the loss of only tertiary butyl group (i.e.

[M 1 2H 2 C4H9]1). Finally, at fragmentor voltage of

180 V, neither any distinguishable chromatographic peak,nor any molecular ions or major fragments of oxprenololand alprenolol were observed in mass spectra (Fig. 6c). In



Figure 6. Electropherograms (a–c) showing effects of fragmentor voltage upon abundance and fragmentation pattern ofb-blockers. Insets in each electropherogram of three b-blockers ((6)-alprenolol, (6)-oxprenolol, and (6)-talinolol) showtheir corresponding mass spectra. Conditions: drying gas temperature 1807C, drying gas flow rate 9 L/min, and nebulizerpressure 4 psi, fragmentor voltage (a) 80, (b) 130, and (c) 180 V; scan mode: m/z 110–400. Other conditions are the same asin Fig. 5 or described in Section 2, except electrokinetic injection performed by applying 5 kV for 60 s.

contrast, both chromatographic peaks and protonatedmolecular ion of talinolol at m/z 364 could still be easilydistinguished. Specifically in talinolol mass spectrum, therelative abundance of the molecular ion at m/z 364decreased and that of m/z 308 as well as m/z 226increased (Fig. 6c inset). This suggests that talinolol hasthe highest stability among three b-blockers.

3.4 Complementary separation usingteicoplanin and multimodal CSPs

As shown in previous sections, there were still two enan-tiomers (R-pindolol and S-propranolol) that remain unre-solved with vancomycin column even using our optimummobile phase. Structurally similar to vancomycin CSP,teicoplanin CSP is another macrocyclic CSP with fivemore stereogenic centers [30]. Recent reports show thatthis CSP has enantiomeric capability complementary to

vancomycin CSP [34]. Thus, in this section, four columnsthat included one teicoplanin CSP, two duplex-sectioncolumns and one mixed-bed column were utilized forsimultaneous separation of all eight b-blockers. Theobserved elution order, achiral and chiral selectivity, effi-ciency, resolution values are compared and discussed asfollows.

First, the column packed with teicoplanin CSP was test-ed. Compared to the vancomycin CSP column (Fig. 5d),this column showed a higher enantiomeric resolutionsand efficiency. Interestingly, different elution orders wereobserved in the two columns (Fig. 7a). For example, pro-pranolol was eluted earlier than metoprolol, and carteololwas eluted earlier than atenolol for the column packedwith only teicoplanin CSP. Overall, this teicoplanin columnwas able to resolve 15 out of 16 enantiomers, but higherresolution and selectivity was achieved at the expense oflonger analysis time (,50 min).



Figure 7. Electropherograms(a–d) showing the effect of sin-gle vs. couple column with dif-ferent combinations of vanco-mycin and teicoplanin CSPsupon separations of eight b-blockers. Conditions: 75 mm (id)capillary internal tapered andpacked with (a) 60 cm teicopla-nin CSP, (b) 30 cm teicoplaninfollowed by 30 cm vancomycinCSP, (c) 30 cm vancomycin fol-lowed by 30 cm teicoplanin CSP,and (d) 60 cm teicoplanin–van-comycin CSP 1:1 w/w. Peakidentifications are the same asin Fig. 2. Other conditions arethe same as in Fig. 5 or de-scribed in Section 2.

Next, the two duplex columns were tested. The firstduplex column which was packed with 30 cm teicoplaninCSP followed by 30 cm vancomycin CSP from the inletend (Fig. 7b) shows slightly lower selectivity and effi-ciency than either vancomycin or teicoplanin CSPs.More specifically, this column provided lower resolutionin the D region. Furthermore, carteolol (8, 8’) was elutedearlier than atenolol (7, 7’). However, unlike teicoplaninCSP, propranolol was eluted later than metoprolol. Simi-lar elution trends were also observed on second duplex-section column which was packed with 30 cm vanco-mycin CSP followed by 30 cm teicoplanin CSP from theinlet end (Fig. 7c). As compared to the aforementionedfirst duplex column, the overall enantiomeric resolutionsof the second duplex-section column were slightly lower

(probably due to lower efficiency). Nevertheless, bothduplex columns could only resolve 14 out of 16 enantio-mers.

Finally, the performance of mixed-bed column, which wasprepared by physically mixing vancomycin CSP and tei-coplanin CSP in 1:1 w/w, was tested (Fig. 7d). Since thiscolumn provided the lowest efficiency (probably due toinhomogeneous mixing of two CSPs), achiral selectivityand resolution were reduced, which in turn led to lowerpeak capacity. Note that this column has the same elutionorder of b-blockers (Fig. 7d) as the column packed withonly teicoplanin CSP (Fig. 7a) but different from twoduplex columns (Figs. 7b and c). However, only 12 out of16 enantiomers were resolved using this column.



3.5 Quantitative analysis of �-blockerenantiomers

In our last set of experiments, we examined the quantita-tive applicability of the developed CEC-MS method. Thisincludes simultaneous setup of linear calibration curvesand measurement of the LOD for each enantiomer. Inaddition, the determination of trace amount of enantio-meric impurity was also investigated.

3.5.1 Simultaneous determination of calibrationcurves and LODs

The standard solutions of (6)-atenolol, (6)-metoprolol,(6)-oxprenolol, and (6)-propranolol at five concentrationlevels were prepared for conducting simultaneousseparation and setting up the calibration curves for indi-vidual enantiomers using (6)-talinolol as internal stand-ard. As listed in Table 1, all the calibration curves showedgood linearity (R2 = 0.9940–0.9988) over a wide con-centration range (3–600 mM).

For the determination of the LOD, standard solution con-taining 3 mM of each enantiomerof the four aforementionedb-blockers were further diluted and injected at 5 kV for 60 s.Since b-blockers were dissolved in pure MeOH, most ofinjected analytes are retarded at the head of CEC columndue to lack of ion-exchange process (i.e. on-columnstacking). Therefore, this large-volume injection improveddetection sensitivity without causing significant bandbroadening. As displayed in Table 1, each enantiomer ofthe four aforementioned b-blockers could be detected at30 nM. Notably, the detection limits of b-blockers could bepushed down remarkably by using even larger injectioncontaining lower concentration of internal standard (toavoid the overlapping of R- and S-talinolol peaks).

3.5.2 Determination of trace amount ofenantiomeric impurity

Finally, the feasibility for determination of trace amount ofenantiomeric impurity using the developed CEC-MS methodwas investigated. Although the level of quantitation at 1% issufficient to meet the regulatory requirements during the dis-covery phase, a chiral assay with an LOD of 0.1% enantio-meric impurity is mandatory for later stages of drug develop-ment [35]. Thus, 300 mM each of S-alprenolol and R-propra-nolol were spiked with 3 mM R-alprenolol and 0.3 or 3 mM (i.e.0.1 or 1%) S-propranolol, respectively. As shown in Figs.8a–c, due to high efficiency and resolution in CEC, the spikedenantiomers were well separated from the major enantio-mers, irrespective of the elution order at 1:100 and 1:1000levels. In addition, the obtained peak area ratios demon-strated good correlations with spiked concentration ratios.

3.6 Reproducibility test

Both durability and day-to-day reproducibility of internallytapered columns were tested. A column packed with vanco-mycin CSP was utilized to conduct 20 injections of eightb-blockers on daily basis for four consecutive days (a total of80 injections performed on this column). Acceptable RSDvalues were found with respect to the retention times forintraday precision that ranged from 1.9 to 3.7%, while theinterday precision of retention times in 4 days ranged be-tween 2.8 and 3.2%. This suggests high durability of intern-ally tapered columnsusing our fabrication procedure. Inorderto study the batch-to-batch column reproducibility, four col-umns were utilized to conduct 20 runs of same b-blockersmixture oneachcolumn.Typical electropherograms ofall fourcolumns are overlaid in Fig. 9. The intracolumn RSD valuesfor retention times were found to range from 0.9 to 2.7% whilethe intercolumn RSD values of retention times as a mean offour columns were between 3.8 and 4.1%.

Table 1. Calibration curves, LOQ, and LOD for individual enantiomer of four b-blockers ((6)-atenolol,(6)-metoprolol, (6)-oxprenolol, and (6)-propranolol)a)

Analyte Calibration curveb) R2 Concentrationrange (mM)

LOQ (mM) LOD (nM)

S-Oxprenolol y = 0.112x – 0.04 0.9983 3–600 3.0 30R-Oxprenolol y = 0.115x – 0.1092 0.994 3–600 3.0 30S-Metoprolol y = 0.0072x – 0.0163 0.9971 3–600 3.0 30R-Metoprolol y = 0.0071x – 0.0171 0.9972 3–600 3.0 30S-Propranolol y = 0.0044x – 0.0241 0.9984 3–600 3.0 30R-Propranolol y = 0.0044x – 0.0172 0.9988 3–600 3.0 30S-Atenolol y = 0.0033x – 0.0346 0.9946 3–600 3.0 30R-Atenolol y = 0.0032x – 0.0275 0.9954 3–600 3.0 30

a) Conditions are the same as in Fig. 6 or described in Section 2, except that electrokinetic injectionwas performed by applying 5 kV for 60 s.

b) Using S-talinolol and R-talinolol as internal standards.



Figure 8. Electropherogramsshowing the analysis of thenonracemic mixtures of (a)alprenolol (300 mM S-alprenololspiked with 3 mM R-alprenolol),(b) propranolol (300 mM R-pro-pranolol spiked with 3 mM S-propranolol), and (c) propranolol(300 mM R-propranolol spikedwith 0.3 mM S-propranolol).Conditions are the same as inFig. 5 or described in Section 2,except electrokinetic injectionperformed by applying 5 kV for60 s.

Figure 9. Electropherograms (a–d) showing batch-to-batch reproducibility for the separations of eight b-blockers usingfour different columns. Conditions are the same as in Fig. 5 or described in Section 2.



4 Concluding remarks

For the first time, CEC-MS simultaneous enantiosepara-tion was demonstrated. It was found that the use ofinternally tapered column greatly enhanced the stability,durability, and reproducibility of CEC-MS. Steric interac-tion appears to have significant impact on the elutionorder of these structurally similar b-blockers. In addition,hydrogen-bonding interactions (mainly affected byMeOH/ACN v/v) and ion-exchange interactions (mostlyinfluenced by salt concentration (v/v) and HOAc/TEA(v/v)) showed significant impact on the retention andchiral resolution. Thus, the result of these retentions,chiral/achiral resolution, and selectivity trends using van-comycin CSP raised important questions that will beanswered in our forthcoming work. A mixture containingMeOH/ACN/HOAC/TEA at 70:30:1.6:0.2 v/v/v/v wasconsidered as optimum mobile phase since it provided agood trade-off between resolution and analysis time.

Sheath liquid and ESI-MS spray chamber parametersmainly influenced the detection sensitivity. By varyingfragmentor voltage under MS scan mode, the feasibility ofobtaining collision ion spectra of b-blockers weredemonstrated for the first time in chiral CEC-MS. Thismight provide a good opportunity for compound identifi-cation and structural investigation. In order to maximizethe achiral resolution, various column-coupling ap-proaches using teicoplanin as complementary CSP weretested. Certain degrees of improvement in resolution aswell as switching elution order were observed. Finally, thecalibration curves of four b-blockers were set up simulta-neously, which demonstrated good linearity in the rangeof 3–600 mM and LOD of 30 nM. In addition, thishyphenation technique was capable of distinguishingminor enantiomer (0.1–1%) in excess of major enantiomer(99–99.9%).

Financial support for this project was provided by theNational Institute of Health (Grant No. GM 62314–02). Theauthors thank Advanced Separation Technologies(Whippany, NJ) for donation of vancomycin and teicopla-nin CSP, and BetaChem (Leawood, KS) and AWD Pharma(Dresden, Germany) for providing (6)-carteolol and(6)-talinolol. J. Z. is grateful to J. T. Lee (AdvancedSeparation Technologies) for helpful discussions.

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