BIOMEDICAL CHROMATOGRAPHY, VOL. 6,177-182 (1992)

Multimodal Liquid Chromatographic Separation Methods in the Study of Ethimizol Metabolism in Man

Ladislav Soltks Institute of Experimental Pharmacology, Slovak Academy of Sciences, CS-84216 Bratislava, Czechoslovakia

The metabolism of ethimizol in the human body has been investigated. Focus was on the detection and demonstration of the regioselective pathway of metabolic demethylation of ethimizol by determining the presence of the corresponding metabolites in blood, saliva and urine. Isolation, purification and identification of the metabolites present in the biological samples was achieved by applying a combination of the following methods: solid phase extraction, high performance liquid chromatography, high performance thin layer chromatography, nuclear magnetic resonance and mass spectrometry. The suggested chemical structures were definitely estab- lished by comparing the physicochemical characteristics of the ethimizol metabolites obtained from the individual biological fluids with the characteristics of synthetized authentic derivatives.

INTRODUCTION

Ethimizol (I, Fig. 1) (Vinogradova et al., 1961), 1- ethyl - N,N' - dimethyl-1H-imidazole-4 5dicarboxamide (ethymisole; [a-99-31, Chemical Abstracts Index Guide, 1987), a xanthine-related drug, is grouped with memory stimulating agents (Dambinova and Shabanov, 1981; Borodkin and Zaitsev, 1982; Mashkovski, 1982; Borodkin and Zaitsev, 1984; Borodkin et al., 1984). On parenteral administration ethimizol acts primarily as a central respiratory analep- tic; its long-term oral intake provides typical anti- inflammatory and antiallergic effects (Borodkin et al., 1964; Ryzhenkov et al . , 1967; Strukov, 1973; Mashkovskii, 1977). For intravenous or intramuscular administration of ethimizol the therapeutic dose given in 1.5% saline solution is 0.6-1.0 mg/kg body weight, administered 1-2 times per day. The oral dosage is 3-4 100 mg tablets per day.

At the Institute of Experimental Pharmacology of the Slovak Academy of Sciences, systematic research into the pharmacokinetics of ethimizol started in the early 1980s (Soltks et al., 1983a; Trnovec et al., 1985a). The aim of the pilot study was to assess the time dependence of the distribution of the labelled prep- aration, [2-14C]-I (Usaevich and Vekshina, 1977), in different organs of small experimental animals (rat, mouse) by using the combination of simple liquid extraction with subsequent liquid scintillation spectrometry. In appropriate cases, liquid extraction/ isolation by solid sorbents was supplemented by the separation of extracted compounds by thin layer chro- matography (TLC) (Piotrovskiy et al., 1986; Trnovec et al., 1987).

Along with the determination of the pharmaco- kinetic parameters of ethimizol and the study of its fate in experimental animals, the investigation was broad- ened to include ethimizol pharmacokinetics in healthy volunteers (Soltks et al., 1983b, 1983c; Trnovec et af., 1985b; Piotrovskiy et al., 1986). The concentration of intravenously or orally administered ethimizol was

0269-3879/92/040177-06 $08.00 0 1992 by John Wiley L Sons, Ltd

determined in blood serum and/or saliva. The treat- ment of biological material involved an isolation/ extraction step by means of solid sorbents followed by the analysis of the isolated sample usinghigh perfor- mance liquid chromatography (HPLC) (SoltCs et al., 1983d). The results of these experiments led to the assumption that ethimizol, administered to man either intravenously or orally in tablet form, was subject to rapid biotransformation. The relatively short terminal half-life time of ethimizol in healthy volunteers (34.1- 97.2 min), low bioavailability of the tablets (3.6- 22.2%) (Trnovec et al., 198Sb), as well as other param- eters (Piotrovskiy et a/ . , 1986; Trnovec et al. , 1987; Bezek et al., 1990; Kukan ef al., 1990a, 1990b) indicated the liver to be the main site of ethimizol metabolism.

Due to the fact that HPLC of the isolates revealed peaks with retention times different from that found for ethimizol, the research was extended to investigate and describe ethimizol metabolism in the human body. We investigated pooled serum samples obtained in trials

M3 Mo Figure 1. Chemical structures of ethimizol and of the derivatives M,, M3 and M,.

Received 7 July 1991 Accepted (revised) 13 December 1991

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with healthy volunteers who had ingested cthimizol. During the trial saliva and urine were also collected within the time interval of 6 h after tablet ingestion.

The aim of the prescnt report is to give a detailed description of the experimental procedure used to determine the chemical structure of ethimizol metabo- lites circulating in blood, discharged into saliva and excreted in urine in man. A combination of liquid chromatographic separation methods was used, namely HPLC [normal phase (NP) and reverse phase (RP)] and high performance thin layer chromatography (HPTLC), which wcre supplemented by physico- chemical identification methods, i.e. nuclear magnetic resonance (NMR) and mass spectrometry (MS).

EXPERIMENTAL

Drugs and chemicals. Ethimizol tablets and the derivatives M,, M3 and Mo (Fig. 1) were supplied by the Institute of Experimental Medicine, Academy of Medical Sciences, St Petersburg, USSR. Water, methanol, 2-propano1, acetoni- trile, chloroform, deuterated chloroform, dichloromethane and 1,2-dichloroethane were of analytical (p.a.) or higher grade (UV, HPLC).

Biological trial. Six healthy volunteers, after an overnight fast, ingested ethimizol in tablet form in a dose of 2 mg/kg body weight. Each subject was instructed not to take any drugs for at least two weeks preceding the trial and not to take tea, coffee or any food containing xanthine derivatives during the last two days before the trial. They were all fasting during the trial. Blood samples were drawn from the cephalic vein at selected time intervals. Saliva and urine were collected within 6 h after ethimizol intake. Blood serum, obtained by centrifugation, as well as samples of saliva and urine were kept frozen at -20 “C until analysis. For pharmacokinetic purposes, ethimizol concentration was determined in serum aliquots. l h e rest of the samples were pooled (50mL) and used in the study of ethirnizol metabolism.

Isolation of ethimizol metabolites from serum. Five mL serum was applied onto thc preconditioned filling of a Sep-Pak C,, cartridge (Waters Associates Inc., Milford, MA. USA). The cartridge was preconditionediactivated by rinsing with 5 mL methanol followed by 5 mL distilled water. After the serum sample had run through the cartridge 1 mL distilled water was applied. Retained components of the serum sample were eluted with 2.5 mL acetonitrile. The pooled acetonitrile eluate, obtained from 10 procedures (10 X 5 mL serum), was concentrated by evaporating at 40°C with N2.

Isolation of ethimizol metabolites from saliva and urine. One end of the Sep-Pak CIS cartridge was connected to a water vacuum pump. By immersing the opposite cartridge end, consecutively 5 rnL methanol, 5 mL distillcd water, 200 mL of the biological sample (saliva or urine, from which particulate material had been removed by centrifugation) and, at the sequence end, 1 mL distilled water were gradually sucked through the cartridge filling. After disconnection from the vacuum source, a preconditioned (5 mL CH,OH, 5 mL H,O) Sep-Pak silica cartridge was attached to the outlet of the Sep-Pak CIS cartridge. The components of the biological sample were eluted from the Sep-Pak CI8 cartridge through

the Sep-Pak silica cartridge with 10 mL acetonitrile. The acetonitrile eluate was concentrated by evaporating at 40 “C with N,.

NP-HPLC. The dry residue of the acetonitrile eluate, obtained by processing 50 mL serum, was digested/extracted with chloroform. After centrifugation the extract (total volume 0.1 mL) was injected (10 x 10 pL) into a stainless steel chromatographic column (25 cm x 4.6 mm id . ) packed with LiChrosorb SI-100, sorbent particle size 5 ym (Merck, Darrnstadt, Germany). The mobile phase, an isocratic quar- ternary mixture, was generated from n-heptane, dichloro- methane, and a methanolic solution of triethylamine [CH30H : (C2H&N (95 : 5, v/v)] by a ternary proportional valve built in the low pressure part of an SP 8000 apparatus (Spectra-Physics, Santa Clara, CA, USA). Heptane and dichloromethane were continually “degassed” with helium. The final mixture, n-C,H,(,: CH2C12 : CH,OH : (C2H5)1N (85:10:4.75:0.25, vlv), was pumped at a constant flow rate of 1.2 mL/min. Separations were carried out at laboratory temperature. The effluent was monitored at 254 nm by an SP 770 spectrophotometer (Spectra-Physics). Two significantly absorbing fractions (see Fig. 2) were accumulated from 10 consecutive NP-HPLC separations. The eluate fractions were dried by evaporating at 40 “C with N2.

HPTLC. The dry isolate, obtained by treatment of 200mL saliva/urine, was digested in 10 mL chloroform. The sample extract was applied onto an HPTLC plate (No. 13727; Merck) by repetitive immersing of the concentrating zone of the plate into the chloroform solution (HPTLC Application Sheets). The plate was repeatedly developed by ascending chroma- tography: mobile phase n-heptane : 1,Zdichloroethane : 2- propanol : triethylamine (85 : 10: 4.75 : 0.25, v h ) ; detection at 254nm (Universal UV lamp 29200; Camag, Muttenz, Switzerland). Silica gel was scraped from the migration zones which had retention factor values different from those

I

3

i

LO ’ * m i n 0

Figure 2. Chromatogram (NP-HPLC) of a serum sample contain- ing the ethimizol metabolites M, and M3.

MULTIMODAL LIQUID CHROMATOGRAPHY IN DRUG METABOLISM STUDIES 179

Figure 3. Chrornatograrn (HPTLC) of a saliva sample containing the ethimizol metabolites M, and MB.

obtained from processing blank samples (cf. Figs. 3 and 4). The silica gel was extracted by methanol, which was then evaporated at 40 "C with N2.

RP-HPLC. Each dry solid sample, separated by the NP-HPLC or HPTLC method, was dissolved in water. The resulting solutions (0.1 mL) were injected after centrifuga- tion into a stainless steel chromatographic column (25 em x 4.6 mm i.d.) packed with LiChrosorb RP 8 sorbent, mean particle size 5 pm (Merck). The aqueous methanolic mobile phase was generated by an SP 8000 HPLC apparatus set to the mode of a linear gradient composed of H20:CH,0H (100:0, v/v) at sample injection, and 0: 100 at 30 min, maintained until 60 min; flow rate 0.5 mL/min. The injected sample volume was 100 pL. The effluent was moni- tored at a wavelength of 254 nm by a spectrophotometer SP 770. Each significantly absorbing fraction was collected.

The efficiency of the RP-1lPLC separation with gradient elution was verified by reinjecting the collected fractions into the above-mentioned chromatographic column. For such an analysis, however, an isocratic water-methanol mobile phase of H 2 0 : CH,OH (60: 40, v/v) was applied. The eluent flow rate was set to lmL/min; detection at 200nm. Such an RP-HPLC analysis demonstrated that each of the collected fractions contained exclusively one single component.

NMR. "C NMR spectra were accumulated at 25.04 MHz by a JEOL FX-100 spectrometer (Jeol, Tokyo, Japan). The spec- tral width was 5 kHz, the resolution 1.2Hz/point. Samples were measured as CDCI, solutions.

MS. Mass spectra were measured in the range 130-180 "C by a JEOL JMS-Dl00 spectrometer (Jeol). The ionization energy was set at 12 eV and the emission current was 300 PA.

Figure 4. Chromatogram (HPTLC) of a urine sample containing the ethirnizol metabolites M, and M3.

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RESULTS AND DISCUSSION

Treatment of biological samples by means of solid sorbent

Ethimizol is a weak base with a pK, value of 1.75 If: 0.05 (Piotrovskiy et al., 1984), and thus at physiological pH ethimizol molecules are virtually unionized. The parti- tion coefficient of the n-octanollphosphate buffer (0.067mol/L, pH 7.4) is 3.62 (Trnovec et al.. 1985b; Piotrovskiy et al., 1986). Therefore ethimizol, as the structurally related xanthines, can be isolated/extracted from biological fluids by means of solid hydrophobic sorbents (Soltks, 1992). On using both model and real biological samples containing [2-"C]-I, the recovery of extraction by octadecylsilanized (C1J silica gel "filled cartridges was found to be virtually quantitative (SoltCs et al., 1983a, 1983d; BurdatS et al., 1985; Soltks and Smri, 1987). Model testing of the recovery of M,, MI or M, derivatives, both from aqueous buffered solu- tions and from the biological fluids investigated, i.e. serum, s$va and urine, confirmed the yield values of >95% (Soltks et al., 1983~). This is why the Sep-Pak C,, cartridge is of particular advantage for achieving simultaneous trapping of the parent drug and of some of its more polar metabolites.

Analytical sample processing by HPLC and HPTLC

Introduction of hydrophobized silica gels [octadecyl (C,,), octyl (C,), etc.] for HPLC resulted in a rapidly growing application for separation in ryerse phase mode, especially in biomedical research (SoltCs, 1989). However, due to the problems involved in performing reliable analysis of some groups of drugs (e.g. bases) by means of RP-HPLC, NP-HPLC is now increasingly in use again (The Supelco Reporter. 1989). One of the major advantages of working with bare silica gel is the fact that many structural isomers (e.g. Mo and M, in our study) can be separated by using this stationary phase, which by RP-HPLC can be achieved only with diffi- culty, if at all.

The NP-HPLC mode selected for separation of ethi- mizol and its metabolites in the present study required the use of an adequate mobile phase. Since its compo- sition is governed by the principles of liquid chroma- tography and by the properties of the solvents involved (Snyder, 1978), the choice of methanol as a solvent with high elution strength and of n-heptane as a liquid with practically nil elution capacity required addition of CH,Cl, to achieve a mixture of two commonly unmis- cible liquids (SP 8000 Manual). Triethylamine is fre- quently added to such a solvent mixture to induce partial passivation/modulation of the stron adsorption

1983d; Soltks and Trnovec, 1987). The addition of triethylamine provided rapid and readily reproducible separation of ethimizol (retention time 6.3 min) from its metabolites MI (10.2 min) and M, (18.9 min). Such an effective separative force of the analytical column used made it possible to inject a sample corresponding to up to 5 mL serum. Thus processing/fractionization of 50 mL of the biological sample was achieved by 10 consecutive separations only. The favourable differ-

properties of microparticulate silica gel ( i! oltCs et d.,

ence between the retention times of the separated metabolites MI and M, (cf. Fig. 2) promoted very easy and reliable separation and collection of individual fractions.

For HPTLC with silica gel as the stationary phase the four-component mixture was again used as the mobile phase. Yet in contradistinction to NP-HPLC, CHZC12 (boiling point = 40.7 "C) was substituted by 1,2- dichloroethane (84.1 "C) and CH,OH by 2-propano1, which is well miscible with n-heptane. As seen in Figs. 3 and 4, separation of individual components proved to be excellent and a sample obtained from up to 200 mL of biological material could be separated by single processing.

RP-HPLC with gradient elution is one of the most efficient methods for clean-up of substances. At the selected gradient, i.e. 100% H,O at injection, MI and M,, the metabolites to be cleaned-up, became concen- trated on the head of the analytical column filled with C, silica gel. On achieving the appropriate composition of the mobile phase, i.e. the H,O: CH,OH mixture, M, or M, was eluted as a discrete peak.

To verify the chromatographic purity of thc fraction with the M, or M? metabolite RP-HPLC was used again, but with an isocratic mobile phase of H,O : CH,OH (60: 40, v/v). The applied 200 nm wave- length of detection warranted the disclosure of all the substances present. As only the peak representing the main component appeared at any of the analyses per- formed, the fractions of MI and M3 metabolites isolated from serum, saliva and urine were submitted to physicochemical identification by NMR and MS.

Identification of analytes

By "C NMR the MI metabolite was found to be deficient of a -CH3 group in the 4-carboxamid position, originally present in the ethimizol molecule. The mass spectrum of the MI metabolite demonstrated that its molecular ion ([MI" = 194) was reduced by 14 mass units compared to the molecular ion of ethimizol ([MI'' = 210). For M,, the combination of the above- mentioned methods showed the lack of both -CH3 groups, establishing it as didesmethylethimizol. The definitive proof of the identity of the estimated chemi- cal structures was provided by comparing the physico- chemical characteristics of the ethimizol metabolites isolated from individual biological fluids with the char- acteristics of the synthetized authentic derivatives MI , M3 and Mo. Simulated isolation, purification, and iden- tification of the Mn derivative, added to the blank biological sample (serum, saliva, urine) in the concen- tration of 1 ng/mL, corroborated the finding that the in uiuo metabolic reaction of mono-N-demethylation of ethimizol was a regioselective process yielding only a metabolite identical in structure with the derivative ML .

In uiuo metabolic pathway of ethimizol demethylation

Of the two potential structural isomers MI and M, , the exclusive presence of the metabolite MI was unequivo- cally demonstrated in blood, saliva and urine of healthy volunteers. Under in uiuo conditions the other poten- tial mono-N-demethylated ethimizol derivative, Mo ,

MULTIMODAL LTQUID CHROMATOGRAPHY IN DRUG METABOLISM STUDIES 281

I 6 41.t i o n

C l e a n - u p

R P H P L C , a r a d i r n t

Figure 5. Pathway of ethimizol metabolic demethylation in the human body.

N M H

M s

failed to be established. This finding clearly demon- strates the rather important fact that mono-N- demethylation of ethimizol is controlled enzymatically by a regioselective enzymatic reaction.

Two potential chemical reactions are most frequently involved in the process of N-dernethylation in living organisms: (1) direct demethylation, cleavage of the methyl group; (2) via an intermediate, N-hydroxy- methyl, formed by oxidation of the methyl group, from which formaldehyde is split off, with the resulting end- product-desmethyl. There are but scarce literary data providing evidence on the formation of the N-hydroxy- methyl derivative in uiuo. Our in uiuo demonstration of the generation of mono-N-hydroxymethylethimizol (M2; retention time by NP-HPLC = 14.3 mi?), an inter- mediate established in the serum of rats (SoltCs et al. , 1983d) , has considerably contributed to the under- standing of the chemistry of the metabolic deformyl- ation reaction in general, and particularly in ethimizol.

Since mono- and di-desmethylethimizol have been unequivocally determined in human body fluids after ethimizol intake, the suggested metabolic pathway of ethimizol given in Fig. 5 appears to be justified. In the rat, however, the pathway most probably involved in mono-N-demethylation of ethimizol resulting in the formation of the MI metabolite is presented in Fig. 6.

The described steps of isolation, separation and puri- fication of ethimizol metabolites are schematically depicted in Fig. 7 . The following liquid chromato- graphic techniques were used: cartridge (mode RP), HPLC or HPTLC (mode NP), HPLC (mode RP, gradient), in combination with the physicochemical identification methods of NMR and MS. The presented

W 0

1 h' 1 h' I

Figure6. The most probable pathway of the ethimizol meta- bolic mono-N-demethylation step in the rat organism.

l d e n t i f l c r t i o n

Figure 7. Scheme of a universal procedure for isolation, purifi- cation and identification of xenobiotics present in biological samples.

approach is a generally applicable procedure both in in uiuo and in uitro studies of the metabolism of xenobio- tics.

CONCLUSION

Liquid chromatography and its modifications, particu- larly high performance column (HPLC) and high per- formance thin layer (HPTLC), are considered the most powerful methods of determination of xenobiotics and their metabolites in body fluids in man. These methods complemented by the efficient physicochemical identi- fication methods of NMR and MS provided the basis of our study of ethimizol metabolism in man. The follow- ing findings were established:

(1) The primary product of ethimizol metabolic demethylation is mono-N-desmethylethimizol, i.e. the derivative M I .

(2) Cleavage of one methyl group from the ethimizol molecule proceeds under enzymatic control and is strictly regioselective.

( 3 ) The subsequent cleavage of the other methyl group yields didesmethylethimizol, i.e. the derivative M3. (4) Both the M, and M3 metabolites circulate in

blood, are discharged into saliva and are excreted in urine.

Acknowledgements

The author is indebted to Drs. Irina S. Alcksandrova and L. B. Piotrovskiy of the Institute of Expcrimental Medicine of the Academy of Medical Sciences, St Petersburg for synthesizing the authentic derivatives M,. M, and M,, The GA SAV 283 grant from the Slovak Grant Agency, Bratislava, Czechoslovakia is gratcfully acknowledged.

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