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Supercritical fluid chromatography tandem-column …

Supercritical fluid chromatography tandem-column
method development in pharmaceutical sciences
for a mixture of four stereoisomers

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  • 1. Barnhart, Gahm, Tomas, Notari, Semin, Cheetham 619Wesley W. Barnhart Supercritical fluid chromatography tandem-columnKyung H. GahmSam Thomas method development in pharmaceutical sciencesSteve Notari for a mixture of four stereoisomersDavid SeminJanet Cheetham A tandem-column method using Chiralpak AD-H and Chiralcel OD-H columns wasDiscovery Analytical Sciences, achieved for baseline separation of a mixture of chiral pharmaceutical compoundsMolecular Structure, Amgen Inc., (i. e., four stereoisomers) via supercritical fluid chromatography (SFC) with a mobileThousand Oaks, CA 91320, USA phase consisting of 90% liquid carbon dioxide and 10% ethanol:isopropanol (50 : 50 v/v). On the contrary, this mixture (mixture A) could not be baseline separated by SFC conditions explored with individual Chiralpak AD-H and Chiralcel OD-H columns. The effects of various mobile phases on elution order, capacity factor, selectivity, and resolution were determined with mixture A on the individual aforementioned columns to develop the tandem-column method. Key Words: Supercritical fluid chromatography; Tandem-column; Diastereomer; Received: January 4, 2005; revised: February 15, 2005; accepted: February 24, 2005 DOI 10.1002/jssc.2005000051 Introduction can be overcome is to utilize tandem columns to obtain a desired single-method separation. In this paper, an analy-Supercritical fluid chromatography (SFC) is an important tical SFC method was developed by employing the cou-analytical and preparative tool used in the pharmaceutical pling of Chiralpak AD-H and Chiralcel OD-H columns toindustry. Separation of chiral pharmaceutical compounds separate a mixture of pharmaceutical compounds (i.e.,is an ever-increasing chromatographic field [1], and chiral- four stereoisomers); this separation was not achievedity is an important factor in drug development [2 – 5] due to using a single-column method. By understanding the elu-the chiral specificity of biological processes [6]. Separa- tion order and effects of various mobile phases on thetion of chiral compounds is vital because the activity of separation of the compounds by Chiralpak AD-H andeach enantiomer must be explored; it is possible that one Chiralcel OD-H columns separately, the development ofenantiomer may be inactive, an antagonist, or toxic [5 – 7]. the tandem-column method was made possible. A single-A classic example is thalidomide. Originally marketed as a method separation is more desirable than multiple meth-sedative, thalidomide was a racemic mixture; one enantio- ods, since it is less time consuming and cumbersome, Original Papermer was a sedative, while the other was teratogenic [8, 9]. requiring no change of column or mobile phase conditionsSFC was the new and exciting chiral chromatographic between the analyses of different compounds.topic in the early to mid 1980s [10, 11] when it was demon- The use of coupled columns to separate compounds chro-strated to be a technique capable of separating enantio- matographically is not a novel concept [19, 22 – 27]; themers [12]. The use of SFC for the separation of enantio- difficulty, however, is the determination of the correctmers has been one of its most successful applica- combination of columns given the large number of station-tions [13]. Though the technique is over 20 years old, its ary phases available on the market. According to San-potential is continuing to be explored. SFC is a popular dra [23], the selectivity and efficiency of a single columnchiral separation technique due to the inherent advan- often does not provide adequate separation of a giventages of using liquid CO2 in the mobile phase [1, 3, 11, mixture of compounds. Recently, Phinney et al. [24]14 – 21]. coupled achiral and chiral columns to separate a mixtureThe development of a successful and facile single-column of achiral and chiral compounds.analytical chiral separation method is not always achieved For our study, however, two chiral columns were coupleddue to limited in-house column selections or time restric- for the purpose of efficiently separating chiral compoundstions imposed by the project cycle times of early discovery (a mixture of four stereoisomers). The initial choice tochemistry projects. One way in which these limitations screen the Chiralpak AD-H and Chiralcel OD-H columns was based on past successful experiences with these col-Correspondence: Wesley W. Barnhart, Discovery AnalyticalSciences, Molecular Structure, Amgen Inc., Thousand Oaks, CA umns; Chiralpak AD and Chiralcel OD columns have been91320, USA. Phone: +1 805 447 2055. Fax: +1 805 480 3015. shown to be very successful and widely used in chiralE-mail: chromatography [1, 28, 29]. In addition to separating fourJ. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  • 2. 620 Barnhart, Gahm, Tomas, Notari, Semin, Cheethamstereoisomers in a single method analytically, the mixture variable wavelength detector, and a waste containmentwas also separated preparatively. As a proof of concept, vessel. The injector was a Modular Digital Pump (Modelapproximately 1 mg of the four-stereoisomer mixture was XL3000) from Cavro Scientific Instruments Inc. (Sunny-separated to baseline. The two coupled columns, though vale, CA, USA). The software used in the purification wasslightly cumbersome, did fit neatly into the column oven Berger SFC ProNTo v. 1.5.305.15.heater and allowed the separation of all four stereo-isomers in a single method. 2.4 Chiral packed columns The analytical chiral packed columns were Chiralpak AD-2 Experimental H and Chiralcel OD-H. Columns were purchased from Chiral Technologies (Exton, PA, USA). The columns are2.1 Materials referred to as AD-H and OD-H throughout the paper.The SFC-grade carbon dioxide was obtained from BOC Dimensions of the columns were 15064.6 mm ID withGases (Murray Hill, NJ, USA). Methanol (MeOH), ethanol 5 lm particle size.(EtOH), and isopropanol (IsOH) were HPLC-grade fromMallinckrodt Baker (Muskegon, MI, USA). The 1-propanol For preparative SFC, the chiral packed columns consisted(nPrOH) and 1-butanol (nBuOH) were purchased from of Chiralpak AD-H (250621 mm, 5 lm) and ChiralcelSigma-Aldrich (St. Louis, MO, USA). All aforementioned OD-H (250621 mm, 5 lm) columns. Columns were pur-solvents were of analytical grade. chased from Chiral Technologies (Exton, PA, USA). The preparative columns are referred to as prep-ADH andThe compounds were synthesized in-house and dis- prep-ODH throughout the paper.solved in methanol for analysis. The compound mixtureconsisted of four stereoisomers and will be referred to 2.5 Analytical analysis conditions andhereafter as mixture A. Individual stereoisomers of mix- calculationsture A were obtained by multi-step purification prior to thedevelopment of the single-step, tandem-column prepara- For single-column analytical analyses, the mobile phasetive method. The single-step purification method was consisted of 90% liquid CO2 and 10% organic modifier.developed for the possibility of future purifications and to Organic modifiers were MeOH, EtOH, IsOH, nPrOH, andprovide a more efficient purification process than the nBuOH. For tandem-column analyses, the primary col-multi-step method. umn order was AD-H and OD-H, respectively. These col- umns were connected by a two-inch piece of stainless2.2 Analytical SFC instrumentation steel tubing. Organic modifiers were EtOH, IsOH, and EtOH:IsOH (50 : 50 v/v). Methods were isocratic with aThe analytical SFC instrument was a Berger SFC unit flow rate of 3.0 mL/min. Column oven and nozzle tem-(Mettler-Toledo Autochem, Newark, DE, USA) with an perature were 408C, and the outlet pressure was 120 bar.FCM1200 flow control module, a dual pump control mod-ule, a TCM2100 thermal column module (temperature is Retention times used for calculating capacity factor andcontrolled from 7 – 1508C), a column selection valve cap- resolution were obtained from the chromatographic dataable of switching between six columns, and a solvent con- generated via MassLynx v. 4.0 SP1. Void volume wastrol valve permitting selection of up to six modifiers. The estimated by using the retention time of the peak thatSFC was equipped with an Agilent 1100 photodiode array resulted from the change in refractive index from the injec-detector with a high-pressure flow cell (Agilent Technolo- tion solvent. Peak widths were measured manually.gies, Palo Alto, CA, USA). The autosampler/injector wasa CTC LC Mini PAL from Leap Technologies (Carrboro, 2.6 Preparative analysis conditionsNC, USA). A Waters (Milford, MA, USA) ZQ benchtop sin- The mobile phase consisted of 90% liquid CO and 10% 2gle quadrupole mass spectrometer with an atmospheric EtOH:IsOH (50 : 50 v/v). The method was isocratic with apressure chemical ionization (APCI) source was coupled flow rate of 55 mL/min. Column oven and nozzle tempera-to the SFC. The software packages used in the analyses ture were 408C, and the outlet pressure was 120 bar.were Berger MassWare v. 4.01 and MassLynx v. 4.0 SP1. 3 Results and discussion2.3 Preparative instrumentationThe preparative SFC was a Berger Multigram II from Met- 3.1 Single column analyses of mixture Atler-Toledo Autochem (Newark, DE, USA). The compo- To gain a better understanding of the effects of organicnents were the Separator Control Module (SCM)-2500, modifiers (i. e., MeOH, EtOH, IsOH, nPrOH, and nBuOH)Electronics Control Module (ECM)-2500, CO2 solvent on the separation of the four stereoisomers, mixture Adelivery module, modifier solvent delivery module, direct was screened overnight with the OD-H and AD-H columnsexpansion probe chiller, ventilated collection cabinet, UV separately (Figure 1 and Figure 2, respectively). TheJ. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  • 3. SFC tandem-column method development 621Figure 1. Analytical chromatograms of mixture A with the OD-H column. The mobile phase consisted of 90% liquid CO2 and 10%of either MeOH, EtOH, IsOH, nPrOH, or nBuOH at a flow rate of 3.0 mL/min. Column temperature and outlet pressure were408C and 120 bar, respectively. Peaks 1 and 4 are an enantiomeric pair (S,R and R,S), and Peaks 2 and 3 are an enantiomericpair (S,S and R,R). (Peak number indicates and correlates the order of elution with the OD-H column.)goal was to develop a single method to separate all four with EtOH and nBuOH used as the organic modifer. Over-stereoisomers. No single-column method, however, all, however, the elution order was not predictable withyielded baseline separation of all four stereoisomers. respect to the various organic modifiers that were studied.Therefore, the individual stereoisomers (designated as A closer look at individual peaks and their elution orderPeaks 1 through 4) were screened by OD-H (Figure 1) with respect to modifier (Figure 2) indicated three differentand AD-H (Figure 2) columns to determine the identity of cases. One such case was the elution order of Peak 2. Itsthe peaks noted in the screening of mixture A. The stereo- elution order remained the most varied throughout the usechemistry of the stereoisomers represented by Peaks 1, of the various modifiers. When MeOH was used as the2, 3, and 4 are S,R; S,S; R,R; and R,S, respectively. The modifier, Peak 2 eluted first. With IsOH as the modifier,peak number is based on the peak order found with the though, Peak 2 eluted last. Only with EtOH and nBuOH (2OD-H column, since the peak order remained constant out of 5 modifiers) did Peak 2 have a consistent elutionregardless of the modifier. order. The second case is the elution of Peak 1. Peak 1Considering the OD-H column results (Figure 1), no remained most consistent with its elution order. Exceptchange in peak order was observed throughout the use of when MeOH was used as the modifier, Peak 1 eluted firstthe various mobile phases. Overall, adequate separation (4 out of 5 modifiers). Lastly, the elution order of Peaks 3of all four components was not achieved with the OD-H and 4 remained the same for three of the five modifierscolumn. Further exploration with IsOH via the OD-H col- (MeOH, EtOH, and nBuOH). Overall, as observed withumn was performed, but baseline separation of Peaks 2 the OD-H column, no single AD-H method provided ade-and 3 was not achieved. Decreasing the amount of IsOH quate separation of the four the mobile phase did not improve separation.Figure 2 clearly shows the elution order varied for Peaks 1 3.2 Capacity factor (k 9) of individual peaks ofthrough 4 via the AD-H column with the use of different mixture A for OD-H and AD-H columnsorganic modifiers in the mobile phase. The only two ana- The capacity factor (k 9) for the four individual stereo-lyses that yielded the same order of elution (1-2-4-3) were isomers was calculated and plotted (Figure 3). WhenJ. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  • 4. 622 Barnhart, Gahm, Tomas, Notari, Semin, CheethamFigure 2. Analytical chromatograms of mixture A with the AD-H column. The mobile phase consisted of 90% liquid CO2 and 10%of either MeOH, EtOH, IsOH, nPrOH, or nBuOH at a flow rate of 3.0 mL/min. Column temperature and outlet pressure were408C and 120 bar, respectively. Peaks 1 and 4 are an enantiomeric pair (S,R and R,S), and Peaks 2 and 3 are an enantiomericpair (S,S and R,R). (Peak number indicates and correlates the order of elution with the OD-H column.)comparing k 9 values for Peaks 1 through 4 for both theOD-H and AD-H columns, it appears that there was aclear difference between columns in the trend found for k 9.The k 9 values of the AD-H column with nBuOH (7.2, 9.4,11.5, and 10.0 for Peaks 1, 2, 3, and 4, respectively) weregreater than the OD-H column with nBuOH (4.5, 5.8, 6.1,and 8.3 for Peaks 1, 2, 3, and 4, respectively). However,IsOH provided the largest k 9 values (i. e., 12.7 and 22.0 forPeak 4 via AD-H and OD-H, respectively) with both col-umns.All four stereoisomers showed the same trend with theOD-H column. The k 9 increased from MeOH to IsOH, withthe maximum value (8.0, 11.2, 13.3, and 22.0 for Peaks 1,2, 3, and 4, respectively) found using IsOH as the modi-fier. Unlike the AD-H column results, Peaks 2 and 3 main-tained the most similar k 9 values between the use of thevarious organic modifiers with the OD-H column.For the AD-H column, all of the peaks did not follow thesame pattern with respect to the k 9 values; however, simi-larities did emerge. Peaks 1 and 3 had very similar pat-terns of k 9 vs. modifier (Figure 3). Also, Peaks 2 and 4 Figure 3. Capacity factor (k9) of all four peaks with variousshowed similar trends and k 9 values. Peaks 2, 3, and 4 mobile phase components utilizing OD-H and AD-H col-show very similar results from IsOH through nBuOH as umns. Liquid CO2 is 90% of the mobile phase, with thethe modifier. Both the pattern and k 9 values are very simi- remaining 10% as MeOH, EtOH, IsOH, nPrOH, or nBuOH.J. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  • 5. SFC tandem-column method development 623 3.4 Analytical scale AD-H-OD-H tandem column analyses of mixture A Individual AD-H and OD-H columns did not provide an adequate method for the baseline separation of all four stereoisomers. The individual column results previously discussed, however, indicated the possibility of separat- ing all four stereoiosomers if the AD-H and OD-H columns were coupled. Organic modifiers were chosen based on the results of the individual AD-H and OD-H column screening. When considering tandem-column LC, the retention time noted for a particular analyte is the sum of the retention times of the analyte for the individual col- umns; this is independent of the column order [25]. It was by adding the peak retention times for the individual AD-H and OD-H columns (with EtOH and IsOH as the organic modifiers) that there appeared the possibility of resolving all four stereoisomers through the coupling of the two col- umns. The tandem-column methods explored utilized mobile phases that consisted of 90% liquid CO2 and 10% EtOH, IsOH, or EtOH : IsOH (50 : 50 v/v) (Figure 5). Looking atFigure 4. Resolution of Peaks 1 & 4 (D) and 2 & 3 (F) utilizingdifferent mobile phase components (MeOH, EtOH, IsOH, the tandem-column separation with EtOH and IsOH indivi-nPrOH, and nBuOH) with the OD-H and AD-H columns. dually, it was predicted that a combination of the twoLiquid CO2 is 90% of the mobile phase, with the modifier would provide the desired separation. A combination of(MeOH, EtOH, IsOH, nPrOH, or nBuOH) as the remaining EtOH and IsOH (EtOH : IsOH (50 : 50 v/v)) was then used10%. to achieve the separation of the four stereoisomers with an analysis time of less than 15 min. Since baseline sep-lar, indicating the AD-H is a column that had very similar aration was already achieved with the 50 : 50 mixture,affinities for all three peaks with IsOH, nPrOH, and further exploration of EtOH : IsOH combinations fornBuOH as the modifiers. method optimization was not conducted. Also, reversing the order of the tandem-columns did not result in a notable difference in the separation of the stereoisomers.3.3 Resolution (R ) of enantiomeric pairs of By utilizing the separation characteristics of individual mixture A for OD-H and AD-H columns Chiralpak AD-H and Chiralcel OD-H columns, it was pos- sible to obtain baseline separation of all four of the stereo-The resolution (R) for the enantiomeric pairs was calcu- isomers through the coupling of the two columns; this waslated and plotted (Figure 4). The resolution plots in Fig- not achieved with a single column. In addition, the base-ure 4 show similar patterns found for the selectivities (not line separation was only accomplished by combining twoshown), as expected. For the OD-H column, the resolution mobile phase components (EtOH and IsOH) for thepatterns of both peak pairs (Peaks 1 & 4 and 2 & 3) were coupled column method.very similar; however, Peaks 1 & 4 demonstrated muchgreater R values than those found with Peaks 2 & 3, thusindicating that the OD-H column was much better suited 3.5 Application of the analytical tandem-columnfor separating the enantiomers 1 & 4 than 2 & 3. Peaks 1 & method4 were best separated on the OD-H column using MeOH The analytical tandem-column SFC method was success-as the organic modifier. fully applied to quickly determine the results of a reactionA closer look at the AD-H results showed that each of the designed to transform a precursor into two of the fourenantiomeric pairs had much different behavior. The stereoisomers found in mixture A. Figure 6 shows themodifier that resulted in the greatest R value for enantio- chromatograms indicating the precursor, the two stereo-meric pairs 2 & 3 was MeOH. The lowest R value for enan- isomers formed, and a chromatogram of the original mix-tiomeric pairs 2 & 3 was obtained through the use of IsOH ture A. The precursor could be easily separated from theas the organic modifier. For 1 & 4, the greatest R value stereoisomers in mixture A, which would provide a quickwas observed with nPrOH as the modifier, and the lowest and facile procedure to monitor both the presence of theR value resulted from the use of MeOH as the modifier. precursor and the product of the reaction utilizing a singleJ. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  • 6. 624 Barnhart, Gahm, Tomas, Notari, Semin, CheethamFigure 5. Chromatograms of the AD-H and OD-H column coupling for the separation of the four stereoisomers. The mobilephase consisted of 90% liquid CO2 and 10% EtOH, IsOH, or EtOH : IsOH (50 : 50 v/v). The temperature and outlet pressure were408C and 120 bar, respectively. Total flow rate was 3.0 mL/min. (Peak number indicates and correlates the order of elution withthe OD-H column.)method. This was critical for a timely analysis of the single-column method. However, with the columns in tan-results of the reaction. A single column could have been dem, a single method was successfully established. Theemployed to resolve Peaks 1 and 2; however, both benefit of a single method was quickly realized when aPeaks 3 and 4 would not have been identified (i. e., reaction (that converted a precursor into two of the stereo-resolved) if they had also been formed during the reac- isomers of mixture A) was efficiently and effectively moni-tion. tored; this provided critical data in a timely manner to easily fit within the timelines provided for project support.3.6 Preparative scale AD-H-OD-H tandem-column Overall, the order of elution of the four stereoisomers with separation of mixture A the AD-H column was difficult to predict, while the order ofAfter establishing a tandem-column analytical method, elution remained constant with the OD-H column. Thisthe next step was to attempt a preparative separation. To indicates the separation mechanism on the AD-H columndemonstrate proof-of-concept, the same mobile phase for these compounds is more complicated compared tocomposition (90% liquid CO2 and 10% EtOH : IsOH the OD-H column. Since both columns contain the same(50 : 50 v/v)) was used for the preparative separation (Fig- derivative (tris-3,5-dimethylphenylcarbamate [30, 31]),ure 7) as that used on the analytical scale. The overall the behavior of the stereoisomers with the AD-H column isseparation time was 24 min. This example illustrates how most likely due to the difference in the structure of amy-a single method with coupled columns can be used to lose (AD-H) compared to cellulose (OD-H). The helicalbaseline separate four stereoisomers and facilitate the nature of amylose allows for better inclusion when com-purification process. pared to the more planar cellulose [3]. With the AD-H col- umn, different alcohol modifiers in the mobile phase can alter the chiral cavities and could modify the elution order4 Conclusions of the peaks [32]. The AD-H column may therefore allowMethod development to separate a mixture of four stereo- more diverse interactions than the OD-H column resultingisomers with AD-H or OD-H columns did not produce a in varied elution order with change of organic modifier.J. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  • 7. SFC tandem-column method development 625Figure 6. Application of the tandem-column method for the analysis of the end products of a reaction. Chromatograms of a pre-cursor, peaks 1 & 2, and mixture A are shown. The mobile phase consisted of 90% liquid CO2 and 10% EtOH : IsOH (50 : 50 v/v).The temperature and outlet pressure were 408C and 120 bar, respectively. Total flow rate was 3.0 mL/min. (Peak number indi-cates and correlates the order of elution with the OD-H column.) Figure 7. Preparative chroma- togram of 1 mg of mixture A uti- lizing the tandem-column method. The mobile phase con- sisted of 90% liquid CO2 and 10% EtOH : IsOH (50 : 50 v/v). The column oven and nozzle temperature were 408C, and the outlet pressure was 120 bar. Total flow rate was 55 mL/min. (Peak number indi- cates and correlates the order of elution with the OD-H col- umn.)An advantage of analyzing the effects of organic modifiers Referenceson elution order of the stereoisomers is to enable an effi- [1] L. Toribio, J.L. Bernal, M.J. del Nozal, J.J. Jimenez, E.M.cient purification of future mixtures of one or more of the Nieto, J. Chromatogr. A 2001, 921, 305 – 313.stereoisomers. Typically after the potency of the individual [2] Y. Zhao, G. Woo, S. Thomas, D. Semin, P. Sandra, J. Chro-stereoisomers is ascertained, further purification may only matogr. A 2003, 1003, 157 – 166.involve one or two of the stereoisomers. With the ability to [3] A. Medvedovici, P. Sandra, L. Toribio, F. David, J. Chroma-change the selectivity of the peak with the AD-H column, it togr. A 1997, 785, 159 – concluded that a scalable purification method could be [4] U. Selditz, S. Copinga, J.P. Franke, H. Wikstrom, R.A. dederived based on the current data. Zeeuw, Chirality 1996, 8, 574 – 578.J. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  • 8. 626 Barnhart, Gahm, Tomas, Notari, Semin, Cheetham [5] G. Cancelliere, I. DAcquarica, F. Gasparrini, D. Misiti, C. Vil- [20] Y.K. Ye, K.G. Lynam, R.W. Stringham, J. Chromatogr. A lani, PSTT 1999, 2, 484 – 492. 2004, 1041, 211 – 217. [6] J. Szymura-Oleksiak, J. Bojarski, H.Y. Aboul-Enein, Chiral- [21] Y. Liu, A. Berthod, C.R. Mitchell, T.L. Xiao, B. Zhang, D.W. ity 2002, 14, 417 – 435. Armstrong, J. Chromatogr. A 2002, 978, 185 – 204. [7] M.S. Villeneuve, R.J. Anderegg, J. Chromatogr. A 1998, [22] T.A. Berger, W.H. Wilson, Anal. Chem. 1993, 65, 1451 – 826, 217 – 225. 1455. [8] J.C. Reepmeyer, Chirality 1996, 8, 11 – 17. [23] K. Anton, C. Berger, Supercritical Fluid Chromatography [9] S.N. Osipov, P. Tsouker, L. Hennig, K. Burger, Tetrahedron with Packed Columns: Techniques and Applications, Marcel 2004, 60, 271 – 274. Dekker, Inc., New York 1998. [24] K.W. Phinney, L.C. Sander, S.A. Wise, Anal. Chem. 1998,[10] R.M. Smith, J. Chromatogr. A 1999, 856, 83 – 115. 70, 2331 – 2335.[11] G. Terfloth, J. Chromatogr. A 2001, 906, 301 – 307. [25] W.H. Pirkle, C.J. Welch, J. Chromatogr. A 1996, 731, 322 –[12] O. Gyllenhaal, J. Chromatogr. A 2004, 1042, 173 – 180. 326.[13] K.L. Williams, L.C. Sander, J. Chromatogr. A 1997, 785, [26] T.A. van Beek, M.S. Wintermans, J. Chromatogr. A 2001, 149 – 158. 930, 109 – 117.[14] W.C. Ripka, G. Barker, J. Krakover, DDT 2001, 6, 471 – 477. [27] Q.B. Cass, R.F. Gomes, S.A. Calafatti, J. Pedrazolli Jr., J.[15] K.G. Lynam, E.C. Nicolas, J. Pharm. Biomed. Anal. 1993, Chromatogr. A 2003, 987, 235 – 241. 11, 1197 – 1206. [28] J. Whatley, J. Chromatogr. A 1995, 697, 251 – 255.[16] M. Johannsen, J. Chromatogr. A 2001, 937, 135 – 138. [29] F. Wang, T. Dowling, D. Ellison, J. Wyvratt, J. Chromatogr.[17] K.L. Williams, L.C. Sander, S.A. Wise, Chirality 1996, 8, A 2004, 1034, 117 – 123. 325 – 331. [30] E. Lipka-Belloli, C. Len, G. Mackenzie, G. Ronco, J.-P.[18] C.J. Welch, W.R. Leonard Jr., J.O. DaSilva, M. Biba, J. Alba- Bonte, C. Vaccher, J. Chromatogr. A 2001, 943, 91 – 100. neze-Walker, D.W. Henderson, B. Laing, D.J. Mathre, [31] L. Chen, R.A. Thompson, B.D. Johnson, J.M. Wyvratt, Chir- LCGC North Am. 2005, 23(1), 16 – 29. ality 2002, 14, 393 – 399.[19] E. Lesellier, C. West, A. Tchapla, J. Chromatogr. A 2003, [32] R.M. Wenslow Jr., T. Wang, Anal. Chem. 2001, 73, 4190 – 1018, 225 – 232. 4195.J. Sep. Sci. 2005, 28, 619 – 626 i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim