Forensic Science Znternutionul 37 (19881243-243 Elsevier Scientific Publishers Ireland Ltd.

243

ISOLATION OF ALKALOIDS AND GLYCOSIDES FROM TISSUE FOLLOWING ENZYMIC DIGESTION

V. SHANKAR, C. DAMODARAN’ and P. CHANDRA SEKHARAN

Forensic Sciences Department, Madras 600 004 (Zndti

(Received July 13th. 19871 (Revision received October 29th, 19871 (Accepted November 24th. 19871

Summary

Four ensymic digestion methods have been evaluated for their efficiency in releaaing certain alkaloids and glycosides from spiked liver tissue. Enzymic digestion gives better recoveries of all the plant poisons studied than those obtained by conventional methods. A flow diagram for the enzymic methods of drug isolation and quantitation by HPLC is presented. Enzymic digestion for the release of glycosides is reported for the first time. It is concluded that papain digestion is the most suitable method for the analysis of broad spectrum of compounds of forensic and clinical importance.

Key words: Enzymic digestion: Toxicology; Alkaloids; Cardiac glycosides

Introduction

The first step in a toxicological analysis of animal or human body tissue is often the production of a deproteinized filtrate. The remarkable binding of xenobiotics and metabolites to body proteins however prevents effective isolation of drugs from the biological matrix. The drug-protein complex is usually broken by harsh hydrolytic conditions employing acid or alkali, but such procedures are known to affect the recovery of the compound as well. The milder enzymatic digestion method of Osselton et al. [l] employing the proteolytic enzyme subtilisin Carlsberg proved to be a promising alternative method applicable to all classes of drugs [2,3]. In the search for more effective alternative enzymatic methods applicable to both acidic and basic drugs we had earlier observed the greater efficiency of papain and neutrase over others [4]. The present study forms an extension of our work and evaluates the efficiency of enzymic digestion for the release of certain alkaloids and glycosides of forensic and clinical importance from liver tissue.

*To whom correspondence should be sent.

0379-07381381$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

Materials and Methods

Stock standard solution in methanol was prepared at a concentration of 0.1 mglml for digitoxin, digoxin, gitoxin (Sigma Chemical Co., U.S.A.), strychnine and brucine (our collection). Working standards were prepared by diluting the stock solutions to give a concentration of 10 pglml.

Crystalline subtilisin-A, trypsin, liquid neutrase (Nova Industri A/S, Denmark) and papain (Loba Chemie, Bombay) were used for enzymic digestion. Methanol, acetone, acetonitrile (Fluka AG, Switzerland), diethyl ether and water (Spectrochem, Bombay) used were HPLC grade. All other chemicals and solvents used were analytical grade.

Sample preparation Liver tissue was obtained from freshly slaughtered sheep and if necessary

stored at - 2OOC. Portions of the tissue (5 + 0.1 g wet wt.) were finely minced in a Virtis homogenizer and blended with 10 ml of water in 50-ml conical flasks. The tissue homogenates were then spiked with the drug(s) at a concentration of 1 pg/g tissue. After gentle swirling the flasks were incubated at 37OC for 1 h followed by extraction as described below.

Conventional method for alhxzloids To the spiked homogenate 50 ml of acetone and 3 ml of 0.1 N HCl were

added, mixed well and filtered through a Whatman No. 1 filter paper into a 250-ml round-bottom flask. The filtrate was then evaporated under reduced pressure to remove acetone. The remaining acid layer was quantitatively transferred to a 250-ml separating funnel using several aliquots of diethyl ether and extracted. The aqueous phase after separation was transferred to a clean separating funnel and the ether phase extracted with another 5-ml portion of 0.1 N HC1. The aqueous fractions were combined and extracted with two 25ml ~01s. of dichloromethane. The organic phase was discarded while the aqueous fraction was made alkaline by the addition of ammonium carbonate until effervescence ceased and extracted with two 25-ml portions of dichloromethane. The organic phase was separated, pooled, passed through anhydrous sodium sulphate and evaporated to dryness over a water bath at 60°C. The residue was reconstituted in 100 4 of methanol prior to injection of 10-d aliquots into the HPLC.

Conventional method for glycosides The spiked homogenate wsa diluted to 25 ml with phosphate buffer (pH

6.5) and extracted four times with 50 ml portions of dichloromethane contain- ing 10% 1-butanol. The dichloromethane layer was separated, pooled, passed through anhydrous sodium sulphate and evaporated over a water bath at 60°C. The residue was reconstituted in 100 4 of methanol prior to injection of 10-A aliquots into the HPLC.

245

I

Spiked liver homosenate (5 ye drug to 5 g wet eight tissue

in 10 ml water)

I I I Trig buffer t:, 25 ml

lM, pH 7.4 I

CrystalliW subtilisin-A

5 md

incubation 5 5-60% 1 h

Phos. buffer Phos. buffer Phos. buffer to 25 ml to 25 ml to 25 ml

O.O2M, pH 7.4 O.O2M, pH 7.4 O.O2M, pH 7.4 I I I

papain neutrase (nurified from

trypsin 1 ml 5

‘papaya latex > 2.5 g or cryst-

alline papain 10 mg + cysteine 0.005M and EDTA 0.002M

I incubation incubation incubation

37°C 2 h :O? PP

Alkaloids Glycosides I

acidified and extracted with dichlorometbane

I aqueous phase made alkaline

I extracted with dichloromethane

I extract evaporated and reconstituted in MeOH

ad justed to pH’6.5

1 extracted with dichloro- methane containit@ 10butanol (10s V/V)

I extract evaporated and reconstituted in MeOH

I I

I HPLC

Fig. 1. Flow diagram for the isolation of alkaloids and glycosides by enzymic digestion methods.

Enzymic digestion For the enzymic digestion methods the spiked tissue homogenates were

diluted with appropriate buffer and incubated after the addition of enzyme(s) as shown in Fig. 1. After incubation the homogenates were processed for extraction as in the conventional methods above.

A high-performance liquid chromatograph (Pye Unicam PU 4800 video chromatograph) consisting of dual pumps (PU 4010), stirrer mixer, an injector valve (Rheodyne 7125) equipped with a 20-d sample loop and variable

246

wavelength UV detector (PU 4020) was used for quantitation. For alkaloids the analytical column was a Partisil 10 Silica (Whatman Inc., UK) 250 x 9.4 mm i.d. The eluent was a mixture of 1 N ammonium nitrate solution, 2 N ammonium hydroxide and methanol (1:2:27) at a flow rate of 3 ml/min. The eluate was monitored at 254 nm. For glycosides the analytical column was a reversed-phase Partisil 10 ODS (Whatman Inc., UK) 250 x 4.6 mm i.d. The eluent was 40% acetonitrile in water at a flow rate of 2.2 ml/min and the UV detector was set at 220 nm.

Results and Discussion

The percent recovery obtained for each of the five compounds studied by employing the various enzymic digestion/conventional procedures is listed in Table 1. Calibration graphs were prepared for each of the drugs by injecting known concentrations (100-500 ng) of standards into the HPLC. The linearity between the concentration and peak heights was excellent with correlation coefficient (~1 values > 0.997. Peaks obtained for experimental samples were quantitated by reference to the calibration graphs. Recoveries have been expressed relative to the zero-time recoveries (considered 100%1) for each of the drugs. For this purpose, the absolute recovery of the drugs from fresh untreated liver homogenates (extracted immediately after drug spiking-zero time) was taken as zero-time recovery.

For enzymic digestion methods the optimal conditions of pH, temperature and incubation period established by us earlier [4] were adopted as such in the present study. For quantitation of alkaloids by HPLC the method of Stevens et al. [5] and for glycosides the method of Castle [S] with slight modification were used. In the HPLC analysis of the different enzyme digests no peaks accounting for any endogenous or conversion product interfering with the analysis of drugs were observed.

TABLE 1

RECOVERY OF ALKALOIDS AND GLYCOSIDES FROM SPIKED LIVER TISSUE USING ENZYMIClCONVENTIONAL METHODS

compou?ui % Recoverya k S.D.

Neutmse Papain Sub tilisin-A Trypsin Conventional

Brucine 88.8 f 1.3 95.9 + 1.9 90.9 * 1.3 78.7 f 1.7 70.7 k 2.6 Strychnine 82.8 + 1.7 92.9 f 1.2 82.8 + 1.8 80.8 * 2.1 75.7 f 2.3 Digitoxin 89.3 2 1.3 92.3 f 1.7 89.3 k 2.5 81.2 f 2.1 76.1 2 2.8 Digoxin 92.9 + 2.4 85.8 2 1.9 85.8 f 2.1 85.8 f 1.7 80.8 f 3.1 Gitoxin 81.2 f 2.1 84.2 f 1.9 78.1 f 2.6 75.1 -t 2.2 71.0 f 3.6

’ All values are mean of two determinations. Figures represent values calculated against the zero time recovery (considered 100%) from fresh, untreated liver homogenates.

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Enzymatic digestion evidently gives greater recovery of the alkaloid and glycosides studied than the conventional non-enzymatic method (Table 11. Among the enzymes papain gave the highest recovery of brucine, strychnine, digitoxin and gitoxin while neutrase gave the highest recovery of digoxin. Digestion with neutrase reported now for the first time for alkaloids and glycosides gave recoveries comparable to those with subtilisin-A. Incidentally it may be noted that subtilisin-A has been applied for the first time to the release of glycosides from tissue. Trypsin offered the lowest recovery among the enzymes tested but nevertheless higher than by the conventional method. These observations are similar to our experience with these enzymes in an earlier study with certain basic drugs [4]. Stevens et al. [5] have studied four conventional methods of deproteination for the extraction of alkaloids from spiked tissue and reported aluminium chloride deproteination to be the most effective. However the percent recovery of strychnine from spiked liver tissue by that method was only 38%. Instead, in the present study, for the conventional method we adopted the procedure of Hunter and Creekmur [7] and observed the percent recovery of brucine and strychnine as 70.7 and 75.7, respectively (Table 11, with the absolute zero-time recovery being 99% in either case.

The solvating power of an extractant is known to be increased by the addition of an alcohol. Eriksson et al. [8] have reported extraction efficiencies of 90-990/b for digoxin and its hydrolysis metabolites using dichloromethane containing 1-butanol or heptafluorobutanol. Hence in the present study, for the conventional method, we employed dichloromethane containing 1OW l- butanol for extraction and observed the absolute zero time recoveries to be greater than 98.5% for each of the three glycosides. Obviously this seems to be a well suited conventional method for the efficient extraction of glycosides. While recoveries of the glycosides from the spiked homogenates by conventional method were 27I%, enzymic digestion prior to extraction further enhanced the recovery.

No explanation seems possible however to the differential recovery of the three cardiac glycosides from tissue following enzymic digestion although zero time recoveries were almost identical for each of them. This is also true in the case of recovery obtained for the three glycosides by any one enzymic method. The only logical assumption appears to be that the drugs are bound to tissue proteins in relatively different manner. Binding of drugs to tissue matrix is much too complex; yet if we go by plasma protein-binding as a yardstick it is reasonable to expect that digoxin (which is bound only to the extent of 20-40% to plasma proteins1 [9] would be well extracted by the conventional method itself and that the enzymic digestion would only marginally increase the recovery. On the other hand digitoxin which is bound to plasma proteins to the extent of about 950/b would be extracted to a lesser extent by the non-enzymic method while greatly enhanced recoveries could be expected after enzymic digestion. A similar argument seems suitable for the alkaloids too. The results obtained (Table 11 appear to agree with the above hypothesis.

Enzymatic cleavage of the drug-protein bond is obviously responsible for the enhanced recovery; however the enzymic digestion methods require that the drugs in question are stable to the conditions employed for enzymic digestion. It appears possible that certain compounds may as a result of being structurally analogous to the enzyme substrates be susceptible to enzymatic degradation as observed by Hammond and Moffat [lo] in some drugs containing amide and ester functional groups. A study to evaluate the stability of various compounds to the conditions employed for enzymic digestion using papain and neutrase is presently underway and the results will be reported later. Stability to enzymatic action could thus also account for the observed differential recoveries of similar class of compounds.

It is evident that enzymic digestion greatly improves the recovery of drugs from biospecimens although the mechanism underlying the superiority of enzymic procedures remains to be explained in full. For fruitful application in forensic and clinical toxicology use of a single enzyme for analyzing a wide range of xenobiotics will be highly beneficial. In this respect, based on our studies [4] and the present report it is concluded that papain digestion is the best method for monitoring basic as well as acidic drugs and plant poisons.

References

1 M.D. Osselton, M.D. Hammond and P.J. Twitchett, The extraction and analysis of benzodiazepines in tissues by enzymic digestion and high-performance liquid chromatography. J. Pharm. Phannacol, 29 (1977) 460 - 462.

2 M.D. Osselton, The release of basic drugs by the enzymic digestion of tissues in cases of poisoning. J. Forensic Sci Sot., 17 (1977) 189- 194.

3 M.D. Osselton, I.C. Shaw and H.M. Stevens, Enzymic digestion of liver tissue to release barbiturates, salicylic acid and other acidic compounds in cases of human poisoning. Analyst, 103 (1978) 1160- 1164.

4 V. Shankar, C. Damodaran and P. Chandra Sekharan, Comparative evaluation of some enzymic digestion procedures in the release of basic drugs from tissue. J. Anal Toxicol, 11 (1987) 164 - 167.

5 H.M. Stevens, P. Owen and V.W. Bunker, The release of alkaloids from body tissues by protein precipitating reagents. J. Forensic Sci Sot., 17 (1977) 169- 176.

6 M.C. Castle, Isolation and quantitation of picomole quantities of digoxin, digitoxin and their metabolites by high-pressure liquid chromatography. J. Chromatogr., 116 (1975) 437- 445.

7 R.T. Hunter and R.E. Creekmur, Liquid chromatographic determination of strychnine as poison in domestic animals. J. Assoc. Off. AnaL Chem.. 67 (1984) 542-545.

8 B.M. Ericksson, L. Tekenbergs, J.O. Magnusson and L. Molin, Determination of tritiated digoxin and metabolites in urine by liquid chromatography. J. Chromatogr., 223 (1981) 401- 408.

9 A.C. Moffat ted.), Clarke’s Isolation and Identification of Drags, The Pharmaceutical Press, London, 1986, pp. 541- 544.

10 M.D. Hammond and A.C. Moffat, The stability of drugs to the conditions used in the enzymic hydrolysis of tissues using subtihsin Carlsberg. J. Forensic Sci Sot., 22 (1982) 293 -295.