Short Communications 10383 Carrier Displacement Chromatography for the Identification of Deprenyl and Its Metabolites

H. Kalbsz Department of Pharmacology, Semmelweis University of Medicine, Budapest, NagyvArad t& 4, Hungary, 1089

Key Words: Thin-layer displacement chromatography Carrier displacement Deprenyl metabolites

The displacement mode of development is undergoing a renaissance in the preparative scale separation of closely related compounds [1,2]. The present communication describes a typical analytical application of displacement chromatography on thin-layer plates and suggests the use of this technique for the identification of metabolites.

Deprenyl (N-methyl-N-propargyI-(2-phenyl-l -methyl)ethylam- monium HCI) is a highly selective inhibitor of B-type monoamine oxidase [3]. On injection into rats, deprenyl yields a series of metabolites [4], which are secreted together with the unaltered deprenyl in the urine. The analysis of metabolites was greatly facilitated by the use of radiolabeled deprenyl; however, we have not been able to purchase radioactively labeled standards for identification of the metabolites. Thus, we compared the unlabeled standards and the radiolabeled metabolites at various concentrations for detection purposes. This approach, together with thesimilarityof Rfand k' (inTLCand HPLC, respectively), tend toincurthe possibilityof errorasthe elution modeofdevelopment does not rule out the joint presence of two or more components in a single peak without any visible deformation.

The identification of unaltered deprenyl and propargylanara was difficult because they had similar Rf values in TLC (Table l), while other phenylalkylamines were well separated.

Table 1

TLC characteristics of various compounds.

We have now developed a variant of two-dimensional thin-layer chromatography for the separation of deprenyl and its metabol- ites by using the carrier displacement mode of development in the 2nd dimension.

In our experiments, rats were injected with radiolabeled deprenyl (5 mglkg, i.p.), urine was collected for 24 hours and the diethyl ether extract of the urine (at pH 9.5) was used for further separation. Samples of the extracts were spotted at 3 cm from bothsidesofthe plate,togetherwiththe standardsof the tentative metabolites. Individual spots of each standard and urine sample extract were applied over the front line at 3 cm from the edge of the plate (Fig. 1). The first run was carried out using chloroform/ methanol/borate buffer (pH 6.5) (2:2:1) in the elution mode of development on silica plate.The plate wasdried and adye mixture supplied by Camag (Muttenz, Switzerland) containing Sudan blackwas spotted at the start line of the 2nd dimensional develop- ment. Then, the 2nd run was performed in chloroform containing 5% (v/v) triethanolamine as displacer. The standard substances and all metabolites of deprenyl containing a phenylalkylamine partial structure without a hydroxy substituent were displaced in the 2nd dimension. In addition to the separation, the individual components were bordered by the lines of spacer dye substances. If any metabolite corresponding to the standards is

Rf X 100 in the developing systems

Compounds chloroformlmethanoV butanollacetic acid/ tert-butanollammonial phenollwater borate buffer pH 6.5 water (3:l:l) waterlmethanol (8:2)

(7:5:1) (20:1:4:2)

Phenylethylamine 71 67 35 50 Tyramine 69 64 25 39 Dopamine 60 57 00 26 Phenylalanine 83 65 23 53 DOPA 79 56 02 21 Amphetamine 21 61 48 19 Methamphetamine 64 63 33 69 Deprenyl 85 63 85 72 Propargylanara 92 70 83 75

Silica gel plates (Merck Darmstadt, Germany) were used. The detection was performed by UV illumination of developed plates and by visualization with ninhydrin reagent and subsequent heating. 20 pg of each amine and 1 pg of phenylalanine or DOPA were spotted.

0 1983 Dr. Alfred Huethig Publishers Journal of High Resolution Chromatography & Chromatography Communications 49

Short Communications 10383

0 0

0 0

I-- I Y I

c---, 1 3 2 I u u u u 1st run

- L 3 2 1

2 nd run 1 - - Jr So

- Figure 1

Two-dimensional thin-layer chromatogram of urine extract. Spots to be separated: UrSa: sample of urine extract + amphetamine + meth- amphetamine + deprenyl + propargylanara; u: sample of urine extract; 1) propargylanara; 2) deprenyl; 3) methamphetamine; 4) amphetamine; fr) front of development.

The 1st run was performed in chloroform/methanol/borate buffer (pH6.5) (751). After drying the plate, the dye mixture was spotted in the start line of the 2nd run and the displacement mode of develop- ment was carried out using 5% triethanolamine in chloroform. Spots and lines were observed visually as well as under UV light at 254 nm.

f

Y .. - 4 3 2 1 1st run u u u u

4 3 2 1 -

2 nc run 1 - Ur So

- Figure 2

Autoradiograph of the separation shown in Figure 1.

a) propargylanara; b) deprenyl; c) methamphetamine; d) amphetamine; e, f, and g) unknown metabolites of deprenyl.

present in the urine as a metabolite of deprenyl, i.e. is present in radiolabeled form, the specific shape of the displaced standards will be radioactive and detectable by autoradiography.

The spots of amphetamine, methamphetamine, propargylanara, and deprenyl were found to show radioactivity (Fig. 2), i.e. to occur in the secreted urine.

As displacement chromatography strongly decreases the possibility of the joint presence of different compounds in the same spot, this method of identification is more reliable than those hitherto used for separation of deprenyl and its derivatives.

Acknowledgment

The author expresses his personal thanks to Or. Janchen for the supply of Test Mixture II which was used in the experiments as the dye mixture.

References

[I] Cs. Horvath, A. Nahum, and J. H. Frenz, J. Chromatogr. 218 (1981) 365.

[2] H. Kalaszand Cs. Horvath, J. Chromatogr. 215 (1981) 295.

[3] J. Knoll and K. Magyar, Adv. Biochem. Pharmacol. 5 (1972) 393.

[4] G. P. Reynolds, J. 0. Elsworth, K. Blau, M. Sandler, J. A. Lees, and G. M. Stern, Br. J. El. Pharmacol. 6 (1978) 542.

MS received: November 8.1982

Addendum The following information should have been supplied on publication of the paper “Concentration Profiles and Break- through Curves in Non-Linear Chromatography” by A. s. Said et al., HRC & CC 5 (1982) 674.

This paperwill be published in A. Frigerio, editor, “Chromatography and Mass Spectrometry in Biochemical Sciences, No. 2 (Analytical Chemistry Symposia Series, Vol. 14)” Elsevier, Amsterdam, Oxford, New York, 1983, pp. 165-1 78.

Corrigenda

The following corrections should be made in the paper “End- capping of HPLC Packing Derived from Silica Gel” by C. Dewaele, P. Mussche, and M. Verzele, HRC & CC 5 (1 982) 61 6:

In Table 1 and in the caption of Fig. 1 the words naphthalene and nitronaphthalene have to be switched.

50 VOL. 6, JANUARY 1983 Journal of High Resolution Chromatography & Chromatography Communications