Application Note AN 107-01-C
Monitoring the Metabolism of Radioactively Labelled Drug Candidates by OPLC and Radioactive Detection
A
A thorough understanding of the metabolism of a drug candidate is a critical part of the development of a new pharmaceutical preparation. A common technique in bstract these investigations is the use of radioactively labeled drug candidates, as the information that is obtained can be extremely valuable in the study of the metabolism of the compound. Chromatographic methods with radioactivity detectors are commonly used to determine the presence and concentration of various radioactively-labeled metabolites as they are capable of detecting a broad range of metabolites at trace levels from a complex matrix such as urine or serum. In this application note, we describe the use of Optimum Performance Laminar Chromatography™ (OPLC) with radioactivity detection and demonstrate that it is a very powerful technique for assisting in the study of the metabolism of drug candidates in body fluids.
Introduction to OPLC
• On-line and off-line detection allows for monitoring of strongly retained compounds. • The ability to separate a large number of samples in a single run • Ease of scale up for semi-preparative separations A broad range of commonly used stationary and mobile phases is available for OPLC. The separation can be effected on the basis of either normal phase or reverse phase chromatography with a broad range of sorbents. An important benefit of OPLC is that the analyst can monitor the eluted compounds on an off-line or online basis with a radioactivity detector. • Off-line detection: the separation of the metabolites is stopped before the most rapidly eluted compound has traveled to the far edge of the bed. The bed is then scanned with a digital autoradiograph. • On-line detection: the separation is allowed to proceed in the same manner as in HPLC, with the eluent flowing through a flow cell in a radioactivity detector.
Case Study In a recent study, the metabolism of a 14C-labelled compound was studied by OPLC and HPLC. Urine samples from individuals who took the drug were purified by reverse phase solid phase extraction. The extracted material was applied to a HTSorbTM Silica OPLC plate and then separated by a butanol:water:ethyl acetate (4:1:1) mobile phase with a Bionisis™ OPLC 50.
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Figure 1: On-line and Off-line OPLC Separation of a urine sample via HTSorb™ 20 x 20 cm plate, 0.20 mm layer silica gel. - Top Figure: Flow–thru cell radioactivity Detector - Bottom Figure: Digital Autoradiograph.
The HPLC separation to determine peak purity was performed using a LiChroCart 4-4 Purospher RP-18 end capped guard column and a LiChroCart 250-4 Purospher RP-18 column on a Hewlett-Packard 1090 Series II/M HPLC system 1090. Figure 1 shows the separation using both on-column and off-column radioactivity detection of the sample. The upper chromatogram shows the separation using on-line detection (Berthold LB-506 Radioactivity detector with a 150 µL flow cell), while the lower chromatogram shows the separation using off-line detection with digital radiography (EG&G Berthold Model 287 autoradiograph). The correlation of the peaks in the two chromatograms is indicated by arrows. The purity of the peak indicated by the asterisk on Figure 2 was determined by reverse phase HPLC.
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Optimum Performance Laminar Chromatography (OPLC) is a modern liquid chromatographic technique in which the sample is separated using a flat sorbent bed and a mobile phase rather than a column. The sorbent plate is pressurized and excellent resolution can be obtained. The technique provides a number of benefits including:
Deramciclane Monitoring of the metabolism of Deramciclane (a new anxiolitic compound) was studied in dog’s plasma samples. 3H and 14C labeled compounds were synthetized, and plasma samples were collected for the 3H pharmacokinetic evaluation of the parent compound and its N-desmethylmetabolite, and urine samples were collected for 14C study of other metabolites. Pooled plasma samples (1-8 hr) and urine samples (0-24 hr, 24-48 hr, 48-72 hr) were collected, and then purified via RP-18 SPE cartridges.
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The radiochemical purity of the drug is indicated in Figure 3. A typical separation is shown in Figure 4. In addition, a 2 dimensional OPLC separation was performed. In this mode, a separation is performed, then the sorbent is rotated 90° and the same solvent is used to provide additional separation (Figure 5).
Figure 2: HPLC Separation of Collected peak indicated by *in Figure 1 Stationary Phase RP-18, (5 µm). Detection: Flow Thru Radioactivity Detector.
Figure 3: The Chemical Structure of deramciclane fumarate. The asterisks indicate the labeling positions: * = 3H, # = 14C.
Conclusions OPLC with radiochemical detection combines the efficiency and selectivity of liquid chromatography with the specificity of radioactivity detection. Off-line detection is a powerful benefit of the technique, as it permits the monitoring of strongly retained compounds, which is not possible when HPLC is used. An additional benefit is that several samples can be separated simultaneously. Once separated, the OPLC fractions can be isolated and then further separated with a different mobile phase. An additional benefit of OPLC is that the separation of interest can frequently be performed with significantly less mobile phase, and relatively inexpensive sorbent, compared to HPLC.
Figure 4: Monitoring the Radiochemical Purity of 3H (top chromatogram, eluent: n-butanol:acetic acid:water [4:1:1]) and 14C (bottom chromatogram, eluent: acetonitrile:n-butanol:acetic acid:water [44:30:7:19]). Stationary phase: Silica Gel 60.
References 1) Mincsovics E, Kiss BD, Morovjan G, Nemes KB, Klebovich I. A New Tool in Metabolism ResearchCombination of OPLC On-Line Radioactivity Detection with HPLC-RD Technique, J. Planar Chrom., 14 312-317 (2001). 2) Szunyog J, Mincsovics E, Hazai I and Klebovich, I. A New Tool in Planar Chromatography: Combination of OPLC and DAR for Fast Separation and Detection of Metabolites in Biological Samples, J. Planar Chrom., 11 25-29 (1998).
Figure 5: OPLC-DAR after 2-Dimensional separation of 0-24 h of urine sample after 10 mg/kg oral dosing of 14C deramciclane fumarate. Stationary phase: Silica Gel 60. Eluent: ethyl-acetate:ethanol: triethylamine [50:50:4].
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