Metabolite Analysis, Isolation and Purity Assessment Using Various Liquid Chromatographic Techniques Combined With Radioactivity Detection

Journal of Chromatographic Science, Vol. 40, November/December 2002 Metabolite Analysis, Isolation and Purity Assessment Using Various Liquid Chromatographic Techniques Combined With Radioactivity Detection Gy. Morovján1, B. Dalmadi-Kiss1, I. Klebovich1, and E. Mincsovics2 1EGIS Pharmaceuticals Ltd., P.O. Box 100, H-1475 Budapest, Hungary and 2OPLC-NIT Engineering Co. Ltd., Andor str. 60, H-1119 Budapest, Hungary Abstract During the discovery of metabolic routes of a drug candidate, radioactively labeled substances are administered. This study reports the multidimensional application of over-pressured layer chromatography (OPLC) and high-performance liquid chromatography (HPLC) coupled with online or off-line nondestructive radioactivity detection methods in metabolism studies. Among these methods, digital autoradiography and flow-cell radioactivity detectors (RD) using solid scintillators are used. In this study, the hyphenation of OPLC with RD is reported. The application of the OPLC–RD technique is demonstrated on a metabolism study as well as the multidimensional chromatographic selectivity using normal-phase OPLC for the separation in the first dimension, followed by reversed-phase HPLC–RD, which provide additional selectivity to the separation. Information regarding the identity of radiolabeled metabolites and data obtained from spectroscopic methods could be advantageously used during structure elucidation. Introduction Discovery of the metabolic pathways of an investigational drug in animals and man is a complex process. Quantitation and isolation of drug metabolites in different biological matrices require special instrumentation, clean-up techniques, and a strategy combining available analytical and preparative methods according to their advantages (1–7). For in vivo or in vitro biotransformation investigations, drugs labeled radioactively using 3H or 14C isotopes provide the possibility to track and quantitatively analyze the metabolites in complex biological matrices using separation techniques that are coupled to radioactive detection methods (8–10). Although mass spectrometry (MS) can be powerfully used for quantitative analysis and structure elucidation, its application is difficult in the quantitative assay of an unknown metabolite because MS responses strongly depend on structure-related ionization efficiency. In contrast to MS, radioactively labeled compounds always have the same response on a radioactivity detection system. Although a higher level of radioactivity provides a higher response, practical reasons dictate that the radioactive dose should be reduced to the lowest amount that provides adequate sensitivity. This also leads to the requirement of the use of instrumentation that is capable of detecting the low intensity of radioactivity. For the investigation of a metabolic pattern, sample preparation and separation methods have to be developed that provide adequate sensitivity and selectivity for forthcoming quantitative assay and spectroscopic studies for structure elucidation. In most cases, the initial steps of analysis include sample preparation and chromatographic separation. For the monitoring of these activities, liquid scintillation counting (LSC) could be used (11). During the optimization of separation method, column and layer chromatographic techniques with gradient and isocratic elution on reversed- and normal-phase sorbents are most commonly evaluated and the separation scheme is established. During the analysis of complex biological matrices (such as feces or tissues) the necessary purification of the analyte usually could not be achieved using one separation technique. In such cases, a combination of analytical methods with different selectivities should be used, thus providing a further dimension of increased selectivity. The hyphenation of various separation techniques with selective or specific detection methods is an often required and emphasized objective during the development of analytical techniques for analytes that are present in complex matrices and at low concentrations. Hyphenated techniques include online methods that are capable of detecting compounds emerging from the separation system (in real time) and off-line techniques that collect defined aliquots of the sample fractions and the detection takes place separated in space and time. Online hyphenated methods include MS and Fourier-transform infrared spectrometry (FTIR) coupled to gas chromatography and overpressure layer chromatography (OPLC) (12). Successful applications include the hyphenation of high-performance liquid chromatography (HPLC) to atomic absorption spectrometry, inductively coupled plasma–MS and nuclear magnetic resonance spectroscopy (NMR). Thin layer chromatography (TLC) with its simplicity for the Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. 1 Journal of Chromatographic Science, Vol. 40, November/December 2002 separation of nonvolatile compounds has also been successfully coupled to a range of spectroscopic (FTIR, MS, and NMR) (13–15) and other detection techniques, such as flame ionization detection (16). OPLC is a recently developed, versatile, economic, and instrumental layer chromatographic method. It allows the development of the chromatogram to be the length of the plate or overrun using isocratic or gradient elution. Sample application is performed as spots (up to 18) or to increase sample load in semipreparative separations as band. In most cases method development and transfer of existing TLC method could be easily performed using the OPLC approach. In previous reports, various off-line and online radioactivity detection methods have been described. In situ detection of spots originating from labeled compounds were performed by digital autoradiography (DAR). Spots eluted or removed from the plate were analyzed for radioactivity by LSC and structure elucidation was performed by MS and NMR (17–21). An off-line approach to isolate metabolites by micro-HPLC used fraction collection onto the wells of specially prepared microplates coated with solid scintillator, which allowed 12 samples to be simultaneously counted at the same time (10). In this study, the recently developed technique of online hyphenation of OPLC and radioactivity detection was presented during a metabolism study of a 14C-labeled investigational drug. In this technique, a radioactivity detector equipped with solid scintillator was coupled to the outlet of a personal OPLC system. This setup enabled the continuous monitoring of the effluent from the OPLC separation as well as fraction collection. After elution, the high-resolution layer was examined by DAR to prove the absence of radioactive spots, thus avoiding the loss of metabolites (23). Strongly retained, noneluted components can be detected on the plate by DAR. Despite good intrinsic separation power of OPLC, the method could not be exploit (...truncated)