Qualitative and quantitative aspects of the analysis of 4-hy..:.
droxycoumarin and some of its biologically active derivatives
(dicumarol, TromexanR, warfatin, 3-(co-bromoacetyl)-4-hydroxycoumarin
and 3-(co-bromoacetyl)-4-hydroxy-7-bromocoumarin) were
studied. The coumarin derivatives can be sharply separated by
TLC on .silica gel HF254 using Eshtblausalz B for detection. Azo
compounds of coumarine derivatives show that a linear relationship
exists between spot-eluate absorbances and the amonts applied
from 10 to 30 μg. The new rapid and sensitive method was
obtained for qualitative and quantitative analysis of 4-hydroxy-
. coumarin derivatives

B. Bobarević

I. Tabaković

M. Kuleš

M. Trkovnik

English

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CROATICA CHEMICA ACTA CCA-1226 CCACAA 53 (3) 461-464 (1980) YU ISSN 0011-1643 UDC 545.844 Original Scientific Paper Qualitative and Quantitative Aspects of Using Eshtblausalz B to Visualise 4-Hydroxycoumarin and Some of its Biologically Active Derivatives on Thin-Layer Chromatographs M. Trkovnik, M. Kules, I. Tabakovic, and B. Bobarevic Laboratory of Organic Chemistry and Biochemistry, Technological Faculty, University of Banja Luka, 78000 Banja Luka, Bosnia and Hercegovina, Yugoslavia Received November 5, 1979 Qualitative and quantitative aspects of the analysis of 4-hy..:. droxycoumarin and some of its biologically active derivatives (dicumarol, TromexanR, warfatin, 3-(co-bromoacetyl)-4-hydroxycou-marin and 3-(co-bromoacetyl)-4-hydroxy-7-bromocoumarin) were studied. The coumarin derivatives can be sharply separated by TLC on .silica gel HF254 using Eshtblausalz B for detection. Azo compounds of coumarine derivatives show that a linear relation-ship exists between spot-eluate absorbances and the amonts applied from 10 to 30 µg. The new rapid and sensitive method was obtained for qualitative and quantitative analysis of 4-hydroxy-. coumarin derivatives. We believe that any new contribution which improves the existing analy-tical methods for coumarins, furocoumarins, and other derivatives of a-pyrone should be of considerable practical value. To justify this view let us remind the reader that the a-pyrone structure occurs not only in numerous physio-logically important plant constituents1, but also in many synthetic agents which produce useful effects in animals and man2• Our own interest in such compounds centers around the hydroxylated coumarins, because several of these compounds have been introduced into human therapy3• The methods currently used to detect and quantitate compounds containing the coumarin ring system are based on composite procedures including chroma-tography - especially GLC4•5 - and other methods of separation, color--forming reactions6 and colorimetry, ultraviolet and visible absorption and emission (fluorescence) spectrophotometry7, and electrochemical recording by potentiometry8 and polarography9- 11• We have reported previously12 that se-veral coumarin derivatives can be sharply separated by TLC on silica gel, and detected on chromatograms by exciting their fluorescence. This procedure has been thereafter successfully used with the parent 4-hydroxycoumarin and its 7-hydroxylated derivative, with anticoagulant drugs belonging to the 4-hydroxycoumarin series (dicumarol. TromexanR, warfarin), and with two agents producing multiple biological effects - fungicidal, insecticidal, roden-ticidal -,. 3-(w-bromoacetyl)-4-hydroxycoumarin and its 7-brominated deri-vative11. 462 M. TRKOVNIK ET AL. Meanwhile we have modified our original procedure by int roducing a different mode of visualization. Instead of exciting fluorescence we now produce visible colorations by reacting the chromatographically separated coumarin derivatives, in situ, with Echtblausalz B (EBB), a double salt con-taining 4,4'-bis(diazonium)-3,3'-dimethoxybiphenyl dichloride and ZnCl2 • The modified procedure seems to offer some advantages which will be described in the next Section. RESULTS We have summarized in Table I some of the da:ta showing the merits of EBB detection. In order to obtain the data presented, fluorescence excitation and the development of color were carried out, in succession, on the same chromatograms so that the results are directly comparable. Two series of chromatograms were prepared using the same stationary and mobile phases, one series with a layer of 0.25 mm, the other with 0,50 mm thickness. The layer thickness had practically no influence on the migration speed of any compound tested, as shown by the almost equal Rf values*, but it did affect the sensitivity of detection in both modes of visualizatiqn. On the other hand, the mode of visualization also influenced the detection sensitivities, except in one instance, namely with 3-(w-bromoacetyl)-7-bromo-4-hydroxy-coumarin. With all other compounds the use of EBB distinctly lowered the minimum detectable mass (m. d. m.), this increasing the detection sensitivities. Finally, the compounds tested produced different nuances of blue and could TABLE I Comparison of Fluorescence Excitation and Color Development as Mod.es of Visualization for 4-Hydroxycoumarin Derivatives on TL Chromatograms Chromatography. - Stationary phase: silica gel; I , commercial", 0.25-mm layer on aluminum (DC Alufolien Kiesel-gel 60 HF254) - II, laboratory-made, 0.5-mm layer on glass [crystalline powder HF254 (Stahl's type 60)" was used]. - Mobile phase: benzene-acetic acid-ethylmethylketone, 8 : 1 : 1 all solvents of highest purity grade". Detection. - FLU: fluorescence excited at 360 nm; EBB: spraying with a 0.25°/e solution of Echtblausalz Bb in 0.05 M NaOH, followed by drying at room tempe-rature. Positive or negative outcomes were judged by inspection. Figures represent averages from three experiments. "i:.. Rr Substituents Minimum detectable ca~ in the 4-hydroxycoumarin ring system mass/µg 'i:: s Posi- FLU EBBd C1l ;:s on I on II Position 3 en s:: tion 7 I II I II 1 0.28 0.27 H D 2.0 2.0 1.0 0.3 2 0.92 0.88 ( 4' -Hydroxycoumarin-3' -yl)methyl 0 H 1.0 0.5 0.2 0.2 3 0.23 0.22 (3'-0xo-1'-phenyl)butyl warfarin /::),. H 1.0 0.5 0.5 0 .2 4 0.42 0.41 co-Bromoacetyl x H 1.0 1.0 0.5 O.!\ 5 0.46 0.45 w-Bromoacetyl Br 2.0 2.0 2.0 2.0 6 0.15 0.15 H A OH 5.0 5.0 1.0 0.5 • All-supported silica gel, Stahl s crystalline powder, and the solvents used in preparing the mobile phase were from E. Merck, Darmstadt, w. Ger. - hFrom FLUKA AG, Buchs, Switzer-land. - • Compounds were applied to the plates in form of solutions in 96'/• ethanol, except for compd. 3 which was dissolved in acetone. - d The coloration of spots was in different shades of blue. · ESHTBLA USALZ B 463 be distinguished at a glance, which may enable rapid identification by com-parison with color standards and is an additional advantage of using EBB. Quantitative relationships that can be derived directly from chromato-grams, or after eluting the stains, are presented graphically in Figure 1. The relationship, spot-area vs. amount of compound applied, shows that the colo-red-spot areas as they result from the EBB treatment depend linearly on the amount of compound applied to the plate, and this relationship is valid up to 30 µg except for 4-hydroxy and 4,7-dihydroxycoumarins for which lineari-ties extend only to 20 µg. Similarly, the relationship, absorbance vs. ammount of compound applied, shows that a l•inear relationship exists between spot-a6 fl fl 0.5 0.4 .. "' ... E c: aJ a E ..Q '-a- 0 0.2 ~ t <:( a .. aT 0 Cl. Cl) 0 • A • 0 B 200 TOO 0 10 20 30 Amount of compound applied, µg Figure 1. Quantitative evaluation of 'l'L chromatograms prepared with some 4-h ydroxy-coumarin derivatives on 0.50 mm silica gel plates and visualized with Echtblausalz B For a description of materials and procedures see legend of Table I. Increasing amounts of each compound were applied onto the baseline of the same plate and the front of developing solvent was allowed to ascend 15 cm above this line in all runs. Points on the graphs represent aver ages f rom three experiments : , 4-hydroxycoumarin ; ~. warfarin 3-(3'-oxo-l'-phenylbutyl)-4-hydro-cycoumarin ; 0 dicumarol 3,3'-methylenebis(4-hydroxycoumarin); X , 3-(w-bromoacetyl)-4-hydro-xycoumarin : .A. 4,7-dihydroxycoumarin. A, Spot area vs. amount applied. B, Absorbance of stain eluates (5 ml of eluent per spot: CHCls for all compounds except 4,7-dihydroxycoumarin which was eluted with acetone) vs . amount applied; measurements were made at the wavelengths of maximum absorbance as indicated in the graph. * Two additional anticoagulants, phenprocoumon Marcumar• by Hoffmann -La Roche, Basie, Switzerland: 3-(l'-phenylpropyl)-4-hydroxycoumarin and ethyl biscoumacetate TromexanR by Geigy, Basie, Switzerland : 3,3'-carbethoxymethlene-bis(4-hydroxycoumarin), too, had practically the same Rr value on chromatograms with either, layer thickness: 0.79-0.78 and 0.54-0.54, respectively. The minimum detectable mas of both compounds was assessed by fluorescence only and was one µg. 464 M. TRKOVNIK ET AL. -eluate absorbances (at the wavelengths of maximum absorbance indicated) and the amounts applied. Again, this relationship is obeyed up to 30 µg, except, for the parent 4-hydroxycoumarin for which it only holds up to 20 µg. In conclusion: the results obtained by using EBB to detect, identify, and quantitatively determine several derivatives of 4-hydroxycoumarin separated by TL chromatography are promising and the EBB treatment may turn out to be at least as satisfactory with other derivatives from the same series. This potentially useful method requires only basic equipment as may be found in any laboratory. Acknowledgement. - This investigation was supported financially by a Grant--in-aid from the Republic's Association for Scientific Work of Bosnia and Herce-govina. REFERENCES 1. H. A. Kuznets ova, Prirodniye kumariny i furokumariny, Nauka, Leningrad 1967. chap. I, II. 2. T. 0 . S o in e, J. Pharm. SCi. 53 (1964) 231. 3. E. Renk and W. St o 11, Progr: Drug. Res. 11 (1968) 1043. 4. W. F. Deckert, J. Chromatogr. 64 (1972) 355. 5. T. Furuya and H. Ko j i ma, J. Chromatogr. 29 (1967) 382: 6. B. La k a·s, Handbuch der Kolorimetrie. Band I. Kolorimetrie in der Pharmazie, Jena 1962, p. 354. . . 7. M. E. Pere 1 son, Yu. N. Shein k er, and A. A. Savin a, Spektri i Stroenie Kumarinov, Khromanov i Ksantonov. Medicina, Moskva 1975. Chap. I, II. 8. E. P. Kem er t e 1 id z e and V. P. Georgie vs k ii, Fiziko-Khimicheskie Metodi Analiza, Nekatorih Biologicheski Aktivnih Veshchestv Rastitelnogo Proishozhdenia. Mecnierba, Tbilisi 1976., p. 172. · 9. M. Dez e 1 i c and M : Tr k o v n i k, Croat. Chem. Acta 33 (1961) 309. 10. M. D e z e 1 i c and M. T r k o v n i k, Croat. Chem. Acta 35 (1963) 43. 11. Yu. E. 0r1 o v and A. P. Prok b pen k o, Zh. Obshch. Khim. 9 (1970) 1159. 12. M. Trkovnik, ·M. Kules, I. Tabakovic, and M. Zecevic, J. Chro-matogr. 128 (1976) 227. . 13. M. Tr k o v n i k, .N. 1; i v k ii v i ·c, M. K u 1 e s, and R. D j u d j i c, Org. Prepa-rat. Proc. Internat. 10 (1978) 215. SAZETAK Kvalitativni i kvantitativni aspekt uporabe echtblausalz B za odredivanje 4-hidro-ksikumarina i njegovih bioloski aktivnih derivata na tankoslojnom kromatogramu M. Trkovnik, M. Kules, I. Tabakovic i B. Bobarevic Za svrhe kvalitativne i kvantitativne analize 4-hidroksikumarina i nekih nje-govih fizioloski aktivnih dei'ivata . kao sto SU antikoagulansi (warfarin, dikumarol, tromexan) i derivati s fungicidnim i insekticidnim djelovanjem (co-brom-3-acetil i co-brom-3-acetil-7-brom-4-hidroksikumarin) oni su bili razdvojeni tankoslojnom kromatografijom na silikagelu HF2s4 pri cemu je za detekciju uspjesno primjenjen Echtblausalz B. Azo-spojevi kumarinskih derivata sa Echtblausalz B nakon eluiranja organskim otapalima pokazuju linearnu zavisilost apsorbancije o koncentraciji kro-matografirantlh supstancija od 10- 30 µg. Na taj nacin dobivena je nova osjetljiva i brza metoda za kvantitativnu i kvalitativnu analizu derivata 4-hidroksikumarina. LABORATORIJ ZA ORGANSKU KEMIJU I BIOKEMIJU TEHNOLOSKI FAKULTET, UNIVERZITET U BANJOJ LUCI 78000 BANJA LUKA Prispjelo 5. studenog 1979 

Qualitative and Quantitative Aspects of Using Eshtblausalz B to Visualise 4-Hydroxycoumarin and Some of its Biologically Active Derivatives on Thin-Layer Chromatographs

Qualitative and Quantitative Aspects of Using Eshtblausalz B to Visualise 4-Hydroxycoumarin and Some of its Biologically Active Derivatives on Thin-Layer Chromatographs

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