Thin Layer Chromatography - The Use of Digital Autoradiography for Biological and Drug Metabolism Studies 2


Kjebovich et al. (17) demonstrated the successful use of auto-radiography to follow the metabolism of deramciclane (a new anxiolytic drug) in dogs, from urine samples analyzed by thin layer chromatography. An example of the radio chromatograms obtained are shown in figure 31. The dogs were treated both orally and intravenously with [3H]deramciclane at doses of 3 and 10 mg per kilogram respectively. Urine was collected between 10 and 12 hours, 12 and 24 hours and between 24 and 48 hours respectively.


Courtesy of J. Planar Chromatogr [ref. 7]


Figure 31. Digital Auto-Radiograms of Dog Urine Samples Taken 6 hr, 18 hr and 36 hr After Treatment with [3H]deramciclane


Samples were placed on the plate in 10 mm bands using a precision syringe and the separation was developed in a normal chamber to a distance of 16-18 cm. After development, the plates were dried and then scanned with a LB 287 type Berthold Digital Auto Radiograph in conjunction with Hewlett-Packard Vectra VL24/66 computer controlled with WinDAR software V.1.09. The instruments scope was to scan an area of 20 x 20 cm which contained a 600 x 600 wire grid, two-dimensional, position-sensitive multiwire proportional chamber. A 9 to 1 mixture of argon and methane bubbled at 5 ml/min through methylal at 2.8˚C was used as the counting gas and the potentials employed for 3H- and 14C-labeled compounds were 2040 and 1200 volts respectively. Signal analysis was carried out by measuring 5 x 360000 detector cells per sec. The separation took approximately 6 hr and, subsequently, the detection procedure took only 2.5 hr (compared with the two days required by contact auto radiography). Unfortunately, accuracy and precision were not reported but it would appear that both were better or at least as good as that obtained from contact auto-radiography. The advantage that was emphasized was the significantly reduced analysis time.