Anal. Chem. 2007, 79, 6013-6019
Combination of HPLC “Inverted Chirality Columns Approach” and MS/MS Detection for Extreme Enantiomeric Excess Determination Even in Absence of Reference Samples. Application to Camptothecin Derivatives Elena Badaloni,† Walter Cabri,† Alessia Ciogli,‡ Roberto Deias,† Francesco Gasparrini,*,‡ Fabrizio Giorgi,† Aristide Vigevani,† and Claudio Villani‡
Analytical Chemistry Department, R&D, Sigma-Tau S.p.A., Via Pontina Km 30.400, 00040 Pomezia, Rome, Italy, and Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita` “La Sapienza” P.le A. Moro 5, 000185 Rome, Italy
An original, extremely sensitive and selective HPLC-MS/ MS technique for the identification and determination of the minor enantiomer in nonracemic mixtures, even when only one enantiomer is available as reference, is described. The method is based on the so-called “inverted chirality columns approach” (ICCA) and consists of the use of chiral stationary phases (CSPs) available in both enantiomeric forms: in fact, inversion of the elution order for a pair of enantiomers is observed in response to the change in column chirality. This offers two key advantages: first, it is possible to demonstrate the potential enantioselectivity of the system by generating a virtual racemate, and second, it permits the choosing of the right column chirality for trace determination. Combination with MS/MS detection affords high specificity allowing not only high sensitivity (down to 0.0025% of the minor enantiomer) but also unequivocal peak identification in complex mixtures. Applications to semisynthetic derivatives of camptothecin, endowed with antitumor activity, are reported. Moreover, applicability of ICCA is not limited to this class of molecules but generates universal support. Its use might also be extended to other classes of compounds by using other CSPs, available in both enantiomeric forms. Chiral molecules are currently at the forefront of strategies for the development of safer, more effective, drugs.1 For example, chiral compounds currently account for at least 50% of sales and are expected to grow to 80% within 5-10 years, based on compounds now in the drug pipeline.2 Furthermore, in 2004, all the top 10 drugs were chiral. These data reflect the recognition that opposite enantiomers can have quite different pharmacological * To whom correspondence should be addressed. E-mail: francesco.
[email protected]. † Sigma-Tau S.p.A. ‡ Universita ` “La Sapienza”. (1) Chirality in Drug Research; Francotte, E., Lindner, W., Eds.; Wiley-VCH, Verlag GmbH & Co. KGaA: Weinheim, Germany, 2006. (2) Rouhi, A. M. Chem. Eng. News 2003, 81, 45-55. 10.1021/ac070776j CCC: $37.00 Published on Web 06/29/2007
© 2007 American Chemical Society
effects, as well as pressure from increasing regulatory concerns. In addition, the synthesis of single enantiomers is becoming ever more practical due to ongoing advances in stereoselective synthetic procedures3 and enantioselective SMB (simulated moving bed) chromatography.4 All these factors contribute to the need for new analytical methods to characterize chiral molecules more efficiently,5,6 by using smaller amounts of sample, providing data more quickly, or eliminating the need for additional synthetic work. For these reasons, hyphenated techniques, which are defined as the online combination of a separation technique and a spectroscopic detection system providing structural information, are becoming a widespread tool mainly in the field of medicinal chemistry. In particular, in the enantioselective HPLC of drugs, especially those coming from a natural source, an important task is the enantiomeric excess (ee) determination on highly enriched and complex mixtures when reference samples of the minor enantiomer, or of the racemate, are not easily available due to expensive or very complex preparation procedures. To the best of our knowledge, there are only two examples of enantiomer identification that do not require the preparation of individual enantiomers or of the racemic mixture. The first is a study aimed at identifying enantiomeric pairs, isomeric configurations, meso compounds and assigning the absolute configuration of the 10 stereoisomers of atracurium besylate7 through HPLC directly coupled with nuclear magnetic resonance (HPLC-NMR) and circular dichroism (HPLC-CD). The second, a novel method by Goss et al.,8 allowed the identification and characterization of an enantiomeric impurity by HPLC-UV-OR (ultraviolet-optical (3) Rouhi, A. M. Chem. Eng. News 2004, 82, 47-62. (4) Chiral Separation Techniques. A Practical Approach, 3rd ed.; Subramanian, G., Ed.; Wiley-VCH: Weinheim, Germany, 2006. (5) Ward, T. J. Anal. Chem. 2006, 78, 3947-3956. (6) Ward, T. J.; Hamburg, D. M. Anal. Chem. 2004, 76, 4635-4644. (7) Mistry, N.; Roberts, A. D.; Tranter, G. E.; Francis, P.; Barylski, I.; Ismail, I. M.; Nicholson, J. K.; Lindon, J. C. Anal. Chem. 1999, 71, 2838-2843. (8) Goss, C. A.; Morgan, D. G.; Harbol, K. L.; Holmes, T. J.; Cook, J. J. Chromatogr., A 2000, 878, 35-43.
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Figure 1. Chemical structures of camptothecin (1) and gimatecan (2).
rotation) combined with HPLC-MS (mass spectrometry). However, neither paper reports quantitative data on highly enriched samples. We describe an innovative, extremely sensitive and selective method for the identification and accurate quantitation of the minor enantiomer present in trace without requiring its preparation or even that of the racemic mixture. In particular, we used the so-called “inverted chirality column approach” (ICCA), which is based on the possibility of column switching between chiral stationary phases (CSPs) having the same bound selector but with opposite configuration.9 This technique makes it possible to identify the minor enantiomer in samples with high ee, even if the racemate or the minor enantiomer is not available for calibration. Moreover, the enantiomeric trace analysis is easily performed by choosing the CSP which allows the trace enantiomer to elute first, thus enabling a more accurate quantitation by peak area integration. Enantiomer elution order is one of the most important topics in the field of enantioselective separations, and it becomes a major variable in direct separations of trace enantiomeric impurities.10,11 Several recently reviewed12 studies dealt with the reversal of enantiomer elution order. A trace enantiomer can be eluted before or after the major enantiomer it contaminates simply by switching the configuration of the chiral selector13,14 or by selecting various kinds of CSPs.15,16 It has been widely demonstrated that the sensitivity and the reproducibility of the determination are superior for the first eluting enantiomer.17 In the pharmaceutical field, samples are often constituted by complex mixtures of compounds in which an unambiguous peak identification by the described technique may be difficult. To (9) Cancelliere, G.; D’Acquarica, I.; Gasparrini, F.; Maggini, M.; Misiti, D.; Villani, C. J. Sep. Sci. 2006, 29, 770-781 and references therein. (10) Gasparrini, F.; Misiti, D.; Villani, C. Chirality 1992, 4, 447-458. (11) Gasparrini, F.; Misiti, D.; Villani, C.; La Torre, F. J. Chromatogr. 1991, 539, 25-36. (12) Okamoto, M. J. Parm. Biomed. Anal. 2002, 27, 401-407. (13) Gasparrini, F.; Misiti, D.; Pierini, M.; Villani, C. J. Chromatogr., A 1996, 724, 79-90. (14) Gasparrini, F.; D’Acquarica, I.; Villani, C.; Cimarelli, C.; Palmieri, G. Biomed. Chromatogr. 1997, 11, 317-320. (15) Doyle, T.; Wainer, J. W. J. High Resolut. Chromatogr. Chromatogr. Commun. 1984, 7, 38-40. (16) Dyas, A. M.; Fell, A. F.; Robinson, M. L. Chirality 1990, 2, 120-123. (17) Perry, J. A.; Rateike, J. D.; Szczerba, T. J. J. Chromatogr. 1987, 389, 5764.
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overcome these limitations we adopted a combination of the ICCA with the highly sensitive and selective ion trap mass spectrometric detection. This technique offers multistage mass spectrometry (MSn) capability, making it ideally suited for simultaneous separation and identification purposes, especially for chiral impurities.18 Individual isomers can often be distinguished and identified by their fragmentation patterns in an HPLC-MS/MS experiment. In particular, enantiomers will provide identical MS and MS/MS spectra, whereas diastereomers will typically give different patterns. As an example of application, we report the ee determination of gimatecan,19 2, and other camptothecin derivatives (Figure 1). (S)-Camptothecin (CPT), 1, is a natural alkaloid extracted from the Chinese tree Camptotheca acuminata,20 displaying potent anticancer activity. (S)-Gimatecan is a semisynthetic derivative of CPT with promising antitumor activity, currently in phase II clinical investigation. For this class of products, the racemic material can be obtained only by a multistep, time-consuming total synthesis.21 The hyphenated technique presented herein allows the unequivocal identification and quantitative determination of the trace enantiomer in the absence of the reference sample, with very high sensitivity and accuracy in a wide range of linearity. EXPERIMENTAL SECTION Instrumentation. Experiments were carried out on an HPLC system by Thermo (San Jose, CA) which consisted of a Surveyor MS micropump, a Surveyor AS autosampler equipped with a Rheodyne model injector and 20 µL loop, a Surveyor PDA photodiode array detector coupled to a Thermo Finnigan LCQ Deca XP Plus ion trap mass spectrometer equipped with an orthogonal APCI ion source. Chromatographic data were collected and processed using the Thermo Xcalibur Chromatography Manager software, version 1.2. Online chiroptical detection was obtained with a Jasco 995 UV/CD detector (Tokyo, Japan), and (18) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry; VCH: New York, 1998. (19) Penco, S.; Merlini, L.; Zunino, F.; Carminati, P. WO 00/53607, September 3, 1999. (20) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. J. Am. Chem. Soc. 1966, 94, 3888. (21) Blagg, B. S. J.; Boger, D. L. Tetrahedron 2002, 58, 6343-6349 and references therein.
Table 1. Selectivity: Analysis of Chromatographic Parameters (See Figures 4-6) CSP configuration
sample
peak name
Rt(min)
k′
R
R
Af
N
n.a.
1.68 2.07
6700 6200
2.95
1.70
6600
2.14
6250
1.54 2.29 1.67 1.94 1.86
6750 5800 7500 6600 11000
n.a.
1.36 1.72
7100 6600
3.00
1.46
7500
1.84
6900
(R,R) (S,S)
(S)-CPT (S)-CPT
(S)-CPT (S)-CPT
14.17 16.52
4.15 5.00
(S,S)a
simulated (R,S)-CPT
(R)-CPT
14.17
4.15
1.20
(S)-CPT
16.52
5.00
(0.02b
14.19 16.44 14.31 16.75 31.50
4.16 4.98 4.15 5.02 4.70
1.20
3.00
1.21
3.29
(R,R)
(R,S)-CPT
(S,S) + (R,R)c
(R,S)-CPT
(S)-CPT (R)-CPT (R)-CPT (S)-CPT (R)-CPT + (S)-CPT
(S,S)
(R,S)-CPT
(R,R) (S,S)
(S)-gimatecan (S)-gimatecan
(S)-gimatecan (S)-gimatecan
6.10 6.96
1.22 1.50
(S,S)
simulated (R,S)-gimatecan
(R)-gimatecan
6.09
1.22
1.23
(S)-gimatecan
6.94
1.50
(0.02b
n.a.
n.a.
n.a.: not applicable a The elution order is simulated on the CSP with the indicated configuration. b Mean of 10 determinations. c Data obtained on tandem column system. V0 of tandem system does not consider the dead volume of union between the two columns.
Table 2. Enantiomeric Excess Determination of Different CPT Derivatives R1
R2
R3
analyte
ee% found
(S)-CPT 99.916-99.995a (S)-gimatecan 99.946-99.995a (S)-gimatecans >99.995 Z isomer CH(OCH3)2 H H (S)-ST2337 99.722 CHdN-OtBu H CH2OEt (S)-ST2910 99.937 CHdN-OtBu OH H (S)-CPT212 99.348
H H CHdN-OtBu H CHdN-OtBu H
a
H H H
R 1.20 1.23 1.27 1.30 1.15 1.26
Data obtained for several batches.
data was collected using Borwin software (Jasco, Europe). The columns used were (S,S)- and (R,R)-DACH-DNB (250 mm × 4.6 mm i.d.), 5 µm particle size by Regis Technologies (Morton Grove, IL). Chemicals and Reagents. The samples of purified (S)-CPT and (R,S)-CPT were purchased from Boehringer Ingelheim Pharma KG, Germany. (S)-Gimatecan batches were supplied by Sigma-Tau (Pomezia, Rome, Italy). The other semisynthetic derivatives of CPT ((S)-ST2337, (S)-CPT-212, (S)-ST2910, (S)gimatecansZ isomer, see Table 2) were prepared in Sigma-Tau R&D laboratories (Pomezia, Italy). HPLC-grade n-hexane, methanol, and dichloromethane (stabilized with ethanol) were purchased from Merck (Darmstadt, Germany). Chromatographic Procedures. HPLC separations were performed by using both (S,S)- and (R,R)-DACH-DNB columns at 30 °C. For CPT, a mixture of n-hexane/dichloromethane (50/ 50, v/v, with 1% of methanol added) was used as mobile phase, whereas for gimatecan, a mixture of n-hexane/dichloromethane (40/60 v/v, with 1% of methanol added) was employed. The flow rate was 1.0 mL/min in both cases. Under these conditions, the minor enantiomers [(R)-CPT and (R)-gimatecan] were eluted first on the column with (S,S)-configuration; injection volumes of appropriate solutions were 20 µL. UV detection was performed at
370 nm for a single-wavelength measurement, and the UV spectra were recorded in a range of 200-400 nm by the PDA detector, whereas CD spectra were recorded in a range of 220-420 nm, and the CD HPLC traces were acquired at 370 nm for both compounds. MS detection was performed by an atmospheric pressure chemical ionization interface in positive polarity (+APCI); the sheath and auxiliary gases (high-purity nitrogen) were 80 and 10 (arbitrary units), respectively. MS parameters were optimized to the following: APCI vaporizer temp 450 °C, corona discharge current 5.00 mA, tube lens offset 30.0 V, source voltage 4.5 kV and current 80 µA, capillary voltage 15 V at 250 °C. The acquisition was operated in positive ion mode, and identification and quantitation were based on selected reaction monitoring (SRM) detection; in particular, the scan range for the parent scan was 250-500 atomic mass units (amu), each scan consisting of three microscans with a maximum ion injection time of 50 ms, whereas the SRM scan consisted of one microscan and a 50 ms injection time, the precursor isolation window was set at 2 amu and collision energy at 30%. Sample Preparation. Samples of (S)-CPT and (R,S)-CPT were dissolved in dichloromethane at a concentration of about 0.4 mg/ mL and 0.8 mg/mL, respectively. Gimatecan was dissolved in dichloromethane at a concentration of 0.09 mg/mL. Validation. The following criteria were used to evaluate the method: sensitivity, linearity, accuracy, and precision. The sensitivity of the method was evaluated by determining the limit of detection (LOD) and quantitation (LOQ). LOD and LOQ were defined as the concentrations with a signal-to-noise ratio of at least 3 and 9, respectively. The linearity of the method was investigated by calculation of the regression line by the least-squares method and expressed by the correlation coefficient (R2). Linearity was evaluated for CPT by consecutive racemic standard enrichments to natural (S)-CPT to cover a range between 0.079 µg/mL and 0.4 mg/mL corresponding to 0.017% of spiked (R)-enantiomer up to 100% of (S)-enantiomer concentration. For gimatecan, linearity was checked on different (S)-enantiomer solutions at eight Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
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concentration levels in a range from 0.0044 to 88 µg/mL (5 orders of magnitude). Accuracy and precision were assessed throughout the entire range for both CPT and gimatecan. Accuracy was determined by comparison of the mean result for three analyses of the spiked amount of (R)-enantiomer for CPT, whereas for gimatecan it was calculated considering the nominal concentration value of the (S)enantiomer solutions. In both cases, accuracy was expressed as the percent recovery of the analyte with respect to the theoretical value. Precision was expressed as RSD %. Safety Considerations. Special care was taken in the handling of CPT and derivatives because of the carcinogenic nature of the compounds. All samples were sequestered and disposed of according to the material safety data sheet (MSDS) and SigmaTau S.p.A. hazardous handling and waste policy. RESULTS AND DISCUSSION “Inverted Chirality Columns Approach” (ICCA). Method development for a direct chromatographic analysis of a given chiral analyte commonly starts with a screening stage in which several CSPs are tested for their ability to resolve the enantiomers of interest. Once a CSP has been selected, method optimization is carried out to adjust critical parameters such as retention and selectivity. At this stage, either the racemate or the individual enantiomers must be available for method calibration and development. In cases where these conditions cannot be fulfilled, i.e., the chiral analyte is available only as a (supposedly) single enantiomer, method development is seriously hampered for two reasons: (1) it is impossible to determine whether a given CSP will resolve the enantiomers under investigation and (2) it is impossible to positively identify the trace enantiomer in real samples of the single enantiomer analyte, as additional impurities will often accompany the major component. A potential solution to this problem is offered by the ICCA that can be exploited with CSPs based on totally synthetic selectors available in both the enantiomeric versions. This approach is based on the inversion of the elution order for a couple of enantiomers that is always observed in response to the change of “column chirality”.9 Thus, it is expected that a single enantiomer analyte will have different retention times on two columns having identical selectors with opposite configurations, provided the system (CSP-analyte-eluent) shows some degree of enantioselectivity under the chosen experimental conditions. If enantioselectivity is observed in this way, the minor peak tracking problem is also solved because the elution times of the two enantiomers (major and trace components) on a single column are equivalent to the elution times of a single enantiomer on the two “enantiomeric” columns (Figure 2). However, a stringent requisite for the validity of the approach is the exact chemical and physical equivalence of the “inverted chirality columns” that must differ only in the selector configuration (see ahead). When analyzing a racemic mixture on stationary phases with bonded selectors having opposite chirality [CSP(+) and CSP(-)], the same chromatographic profile is obviously observed with conventional detectors (Figure 3, top, black and gray traces); however, the inverted elution order is evident using a chiroptical 6016 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
Figure 2. Chromatograms of a nonracemic mixture run on two stationary phases with opposite chirality: CSP(+) and CSP(-), detection by UV.
Figure 3. Chromatogram of a racemic mixture run on two stationary phases with opposite chirality CSP(+) and CSP(-), with UV and CD detection.
detector that yields bisignate signals for the two resolved enantiomers (Figure 3, bottom, black and gray traces). As an additional benefit of the approach, one can choose the selector configuration that produces a chromatographic profile with the more convenient elution order, i.e., with the trace enantiomer eluting first and avoiding being hidden under the tailing edge of the major enantiomer peak. The enantiomeric trace analysis is therefore easily performed by choosing the CSP which allows the above-mentioned elution order, thus enabling a more accurate quantitation by peak area integration22 with a great increase of sensitivity.17 Control of the Chromatographic Equivalence of the Columns with Opposite Chirality. In order to obtain reliable results when determining retention times and peak area values, the two columns with opposite selector configuration must be completely equivalent both in terms of chemical (retention, selectivity) and geometrical characteristics (dimensions, particle size, packing efficiency, etc.). A simple approach is applicable to this purpose. HPLC analysis of the same solution of a racemate with both CSP(+) and CSP(-) columns must give two identical chromatograms when standard nonchiroptical detectors are used. Moreover, the same racemic mixture can be analyzed using a tandem column arrangement, whereby the two columns are connected in series via a zero dead volume union. In such case, the two enantiomers will coelute, (22) Meyer, V. R. Chirality 1995, 7, 567-571.
Figure 4. A. UV and CD chromatograms of (R,S)-CPT at 370 nm on (R,R)-DACH-DNB. (B) (S,S)-DACH-DNB. (C) Tandem columns (R,R)-DACH-DNB + (S,S)-DACH-DNB. Peak 1 corresponds to (S)CPT and peak 2 to (R)-CPT. (D) Online CD spectra of (R)-CPT and (S)-CPT. See the Experimental Section for detailed chromatographic conditions.
giving a single peak with retention time and theoretical plates, which are the sum of the averaged values obtained (for the racemate) on the single columns. Chromatographic Method Development. The selection of the stationary phase was dictated by both the capacity of an optimum separation between the enantiomers and the availability of columns containing CSPs with opposite configurations.9 In our case, both criteria are satisfied by the DACH-DNB stationary phase,23 which is commercially available both in the (R,R)- and (S,S)-configurations. Method Setup. As far as the mobile phase is concerned, best results for different derivatives of the CPT series were obtained by isocratic elution with mixtures of dichloromethane and nhexane in different ratios to which 1% of methanol was added (see the Experimental Section for other chromatographic details). The method was set up for an analysis of CPT for which the racemic mixture is available as reference sample. The elution order was defined for the whole CPT series and confirmed by CD spectra (Figure 4D). In particular, (S)-enantiomers (major peaks) are better retained by the stationary phase with (S,S)-configuration; consequently, this CSP was chosen for quantitative purposes, allowing the trace compounds ((R)-enantiomers) to elute first. Column equivalence was demonstrated by analyzing an (R,S)CPT sample by both columns: the corresponding chromatograms (23) Gasparrini, F.; Misiti, D.; Villani, C. Italian Patent No. 21584 A, August 29, 1989.
Figure 5. (A) Chromatograms of (S)-CPT (natural source) on (R,R)DACH-DNB. (B) Chromatograms of (S)-CPT (natural source) on (S,S)-DACH-DNB (left UV traces, right CD traces at 370 nm). (C) Computer-generated chromatogram of “virtual racemate”. See the Experimental Section for detailed chromatographic conditions.
(UV traces) were found to be superimposable (Figure 4, parts A and B). Table 1 gives detailed chromatographic parameters. Moreover, the same racemic sample run with the tandem column arrangement presented above gave a chromatogram showing the coeluting enantiomers in a single peak (UV trace) with double area and summed retention time (see above) (Figure 4C and Table 1), whereas the signal on the CD trace is missing since the enantiomers are coeluting as racemic mixture. In addition, (S)-CPT (natural source) was analyzed with both (R,R)- and (S,S)-DACH-DNB columns and correspondent chromatograms were reprocessed to give the “computer-generated” racemate (obtained by ElabChrom, a lab-made software that merges two independent chromatograms) which was compared with the real (R,S)-CPT sample run under the same conditions (Figure 5 and Table 1). The same procedure was applied to the analysis of gimatecan, for which neither the racemate nor the (R)-enantiomer is easily available as reference since total synthesis is a complex and expensive task. In particular, the position of the minor enantiomer peak identification was assessed by computer reprocessing of chromatograms obtained by running the single (S)-enantiomer on (S,S)- and (R,R)-DACH-DNB alternatively, to give the “virtual racemate” presented in Figure 6C. Table 1 reports the chromatographic parameters. Selection of the stationary phase with (S,S)configuration resulted mandatory for trace (R)-enantiomer detection: Figure 7 shows that by using the CSP with opposite chirality peak determination is impossible. Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
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Figure 8. Comparison of (S)-CPT chromatograms: UV detection (A) and MS/MS detection (B). See the Experimental Section for detailed chromatographic conditions.
Figure 6. (A) Chromatograms of (S)-gimatecan on (R,R)-DACHDNB. (B) Chromatograms of (S)-gimatecan on (S,S)-DACH-DNB (left UV traces, right CD traces at 370 nm). (C) Computer-generated chromatograms of “virtual racemate”. (D) Online CD spectrum of (S)gimatecan. See the Experimental Section for detailed chromatographic conditions.
Figure 7. Separation of (S)-gimatecan from its (R)-enantiomer with MS detection (see the Experimental Section for detailed chromatographic conditions). At low concentrations the (R)-enantiomer can be determined only by using the (S,S)-DACH-DNB column.
APCI-MS/MS Detection. An APCI interface was chosen instead of electrospray (ESI) mainly because normal phase solvents do not support the formation of ions, which is critical for ESI; in addition, high hexane content introduces a possible explosion hazard in the presence of the high voltage of the electrospray needle for ESI. Moreover, APCI interfaces are known 6018 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
to be well suited to detect nonpolar and medium polarity molecules with masses between 100 and 1000 Da, and to be compatible with analytical flow rates, offering a wide linear dynamic range for quantitative purposes.24 The precursor and product ions for each analyte of interest were determined by direct infusion of single analyte solutions. The mass spectra recorded in full scan acquisition mode showed the protonated molecular ion [M + H]+ as base peak, with a mass to charge ratio m/z ) 349 for CPT and m/z ) 448 for gimatecan. The fragmentation pattern obtained by the isolation of [M + H]+ ion gave an MS/MS spectrum containing a major fragment at m/z ) 305 corresponding, to [M + H - CO2]+ for CPT and a major fragment at m/z ) 392, corresponding to [M + 2H - C(CH3)3]+ for gimatecan. The ion transitions 349-305 and 448-392, for CPT and gimatecan, respectively, were chosen for the SRM acquisition mode that permitted high selective detection of the chosen transition, which is typical and specific for both enantiomers of the given compound. Figure 8 reports the comparison of typical chromatograms of CPT (natural source) obtained by UV and MS detection in SRM mode. Validation. The method was evaluated according to the criteria described in the Experimental Section. LOD values for (R)-enantiomers were determined at 5 pg injected for CPT and 0.7 pg, injected for gimatecan, whereas LOQ values were 16 pg for CPT and 2.2 pg for gimatecan. Linearity was obtained throughout the tested range with the following linear relationships and correlation coefficients (R2): y ) 3.65 × 108x + 1.57 × 108, R2 ) 0.9999 for CPT and y ) 1.97 × 108x + 1.69 × 109, R2 ) 0.9987 for gimatecan. Very good values of accuracy and precision were obtained, such as 99.8% with an RSD of 0.2% (n ) 3) for CPT and 98.2% with an RSD of 0.5% (n ) 3) for gimatecan. As can be observed, the wide dynamic linearity range obtained (5 orders of magnitude) covers at least the composition limit of 1:50 000, enabling the quantitation of the trace compound down to 0.0025% of the main compound, corresponding to ee values up to 99.995%. Analysis of Different CPT Derivatives. The method was also applied, with the appropriate modifications of mobile phase and ion transition for detection, to the analysis of different CPT derivatives with high optical purity (see the general structure in Figure 9 and summary of results in Table 2) demonstrating that the DACH-DNB column is suitable for the ee determination of the whole CPT class of compounds currently available. (24) Niessen, W. M. A.; Tinke, A. P. J. Chromatogr., A 1995, 703, 37-57.
Figure 9. General chemical structure of CPT derivatives.
CONCLUSIONS The successful application of the ICCA to the analysis of CPT, gimatecan, and other CPT semisynthetic derivatives demonstrates its selectivity and specificity, with the unique advantage of making possible an accurate determination of extreme ee values, even when only one enantiomer is available. The results are attainable only through a combination of ICCA and MS/MS detection. In fact, ICCA offers two key advantages: first, it is possible to demonstrate the potential ability of the system to separate the two enantiomers by calculating the retention and enantioselectivity factors for the virtual racemate, and second, it permits the choosing of the right column chirality that allows the minor enantiomer to elute first, thus enabling its trace determination.
On the other hand, the high selectivity obtained by APCI-MS/ MS detection, which excludes any interference from foreign components, allows an unambiguous peak identification even in highly enriched and complex mixtures, thus permitting the attaining of extreme sensitivity levels unobtainable by any other currently available analytical technique. It is noteworthy to mention that the employment of ICCA is not limited to this class of molecules but generates universal support. We expect that the method will find widespread application to other classes of compounds and CSPs that are available in both enantiomeric forms, thus permitting validation of the general approach presented here, unique in the field of chiral compounds and asymmetric synthesis. ACKNOWLEDGMENT This work was supported in part by funds of MIUR, PRIN Contract 2005037725.
Received for review April 18, 2007. Accepted May 22, 2007. AC070776J
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