Forensic Science International, 40 (1989) 45-55 Elsevier Sc.ientific Publishers Ireland Ltd.

45

BARBITURATE ANALYSIS IN TISSUE BY ENZYMIC DIGESTION AND HXGH-PERFORMANCE LIQUID CHROMATOGRAPHY

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

Forensic Sciences Department, Madras-600 006 (Zndti

(Received March 14th, 1988) (Revision received April 22nd, 1988) (Accepted ,4pril28th, 1988)

Summary

The relative efficiencies of four enzymic digestion procedures and two conventional methods in releasing barbiturates from spiked liver tissue have been compared. The recoveries of these acidic drugs obtained by using papain and neutrase (neutral proteinase from Bacillus subtilis) digestion methods reported now for the first time are higher than those by other enzymatic and conventional methods. These observations are similar to our earlier data on the applicability of these two enzymes to the release of some basic drugs from tissue. Papain digestion gives the maximum recovery of drugs as monitored by HPLC and is the method of choice. The enzymic digestion procedures are relatively simple, inexpensive and yield reproducible results. Analytical procedures for use in routine forensic toxicological analysis are presented.

Key words: Barbiturates; Enzymic digestion; High-performance liquid chromatography; Toxi- cology

Introduction

Analytical toxicology deals with the identification and quantitation of drugs and metabolites in body fluids and tissues. Unlike a clinical toxicologist who is interested in the analysis of known drugs and metabolites in body fluids, a forensic toxicologist has to screen for the presence of drugs, if any, a.nd in addition determine their concentration in the biospecimens provided. While the former can employ a specific method best suited for a particular compound the latter has to rely on methods that are suitable for a wide range of compounds. Although specific drugs may be suspected in cer- tain cases, analytical procedures to screen for a wide variety of drugs are required. Efficient extraction of the compound(s) of interest is important prior to identification and quantitation. In the analysis of autopsy samples a single extraction procedure that can effectively release all classes of drugs from biolLogica1 matrix is much sought after.

*To whom correspondence should be sent.

0379-0738/891$03.50 0 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

46

Conventional tissue extraction procedures generally aim at breaking down the proteins in the tissue matrix by either precipitation or acid hydrolysis to release the bound drugs. Precipitation procedures generally give low recoveries [l-3] and acid hydrolysis is not suitable for certain drugs like benzodiazepines [4] and diphenhydramine [5]. The subtilisin digestion method of Osselton et al. [4] by leading to enhanced recovery of drugs from tissues promised greater efficiency and overcame some of the problems like emulsion formation and low recovery associated with conventional procedures. Other enzymes such as trypsin [6,7] and /3-glucuronidase [6,8] have been studied as alternatives to the subtilisin method but were found to be less effective. In our earlier work on basic drugs [9] we established the greater efficiency of enzymic digestion methods using papain and neutrase over other reported methods. In order to ascertain the applicability of the enzymic methods to the analysis of acidic drugs as well, the present study was taken up on the analysis of barbiturates added to liver tissue in vitro.

Materials and Methods

Standards All barbiturates used in this study were obtained from May & Baker

(Essex, U.K.). Free acid forms of amylobarbitone and butobarbitone, calcium salt of cyclobarbitone and sodium salts of pentobarbitone, phenobarbitone, quinalbarbitone and thiopentone were used. Stock standard solution in meth- anol was prepared at. a concentration of 0.1 mglml for each of the drugs. Working standards were obtained by diluting the stock solutions to give a concentration of 10 rg/ml.

Enzymes Crystalline subtilisin-A (29.9 AU/g), trypsin (PTN, salt free; 6.0 AU/g) and

liquid neutrase (0.5 AU/g; density 1.25 g/ml) were obtained from Novo Industri A/S (Denmark); papain (purified from papaya latex) was obtained from Loba Chemie Indo Austranal Co. (Bombay, India). Dried papaya latex was obtained from Sigma Chemical Co. (MO, U.S.A.). Fresh papaya latex was col- lected locally from the green fruits of Carica papaya and allowed to solidify before use. Crystalline papain was obtained from Centron Research Labora- tories (Bombay, India).

Reagents Phosphate buffer (pH 7.41 was prepared as follows: (al 17.7 g of dibasic

sodium phosphate was dissolved in water and made up to 1 1; (bl 15.6 of monobasic sodium phosphate was dissolved in water and made up to 1 1; 19 ml of (a) and 81 ml of (bl were mixed and diluted 1:l with water to give a buffer of pH 7.4. Phosphate buffer (pH 11.41 for tissue extraction was prepared by adjusting 0.1 M dibasic sodium phosphate to pH 11.4 with 40%

47

sodium hydroxide. Tris buffer (pH 7.41 was prepared by adjusting a 1 M solu- tion of the free base to pH 7.4 with concentrated hydrochloric acid. Methanol (Fluka AG, Switzerland), diethyl ether and water (Spectrochem Pvt. Ltd., Bombay) used were HPLC grade. All other chemicals and solvents used were analytical grade.

Instrumentation The high-performance liquid chromatograph (Pye Unicam PU 4800 video

chromatographl consisted of dual pumps (PU 40101, stirrer mixer, an injector valve (Hheodyne 71251 equipped with a 20-d sample loop and variable wavelength UV detector (PU 40201 set at 240 nm. The analytical column was a reversed-phase Partisil 10 ODS (Whatman Inc., U.K.1 250 mm x 4.6 mm i.d. The eluent consisted of aqueous sodium dihydrogen phosphate (0.1 Mllmeth- anol (60 :40, v/v) adjusted to pH 8.5 with concentrated sodium hydroxide solution, filtered through a 0.45 m membrane filter (Whatman Inc., U.K.1 and degassed before use. A flow rate of 2 ml/min was used. The column was washed with methanol/water (50 : 50, v/v) at the end of each working day [lo].

Enzymic digestion Liver tissue was obtained from freshly slaughtered sheep and if necessary

stored at - 20°C. Portions of the tissue (5 f 0.1 g wet wt.1 were finely minced in a Virtis homogeniser 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 incu- bated at 37OC for 1 h, followed by dilution to 25 ml with suitable buffer as shown in the flow chart (Fig. 11. Appropriate quantities of the enzymes were then added to the spiked homogenates and incubated in temperature con- trolled shaking water bath at the optimal temperature(s) as in Fig. 1. After completion of enzyme incubation, sodium tungstate solution (25% w/v; 2.0 ml) and sulphuric acid (5% v/v; 20 ml) were added to each flask [ll]. The flasks were heated by immersion in a boiling water bath for 10 min after which the contents were filtered while hot through a Whatman No. 3 filter paper. The filtrates were cooled and adjusted to pH 6.0 with phosphate buffer pH 11.4 [12] prio:r to extraction with 2 x 30 ml portions of diethyl ether. The ether extracts were pooled, passed through anhydrous sodium sulphate and evapo- rated to dryness over a water bath at 60°C. The residues were reconstituted in 100 fi of methanol prior to injection of lO+l aliquots into the HPLC.

Tungsta te precipitation method To the spiked tissue homogenate (5 g tissue in 25 ml water), sodium

tungstate solution (25W w/v, 20 ml) and sulphuric acid (5% v/v, 20 ml) were added. The mixture was heated by immersion in a boiling water bath for 10 min and then filtered while hot. The filtrate was cooled and extracted as for the enzyme digests.

48

SPIKED LIVER HOMOGENATE

(5 pg of drug to 5 g wet weight tissue in 10 ml water)

Tris buffer to 25 ml

1 M, pH 7.4

crystalline Subtilisin-A

5 mg

Phos. buffer to 25 ml

0.02 M, pH 7.4

papain (purified from papaya latex) 2.5 g + cysteine 0.005 M and EDTA 0.002 M

Phos. buffer to 25 ml

0.02 M, pH 7.4

neutrase 1 ml

Phos. buffer to 25 ml

0.02 M, pH 7.1

trypsin

5 mg

incubation 55-60% lh

incubation 37% 2h

incubation 40% lh

incubation 37% 3h

tungstate’ precipitation

filtered; filtrate adjusted to pH 6.0

ether extraction

extract evaporated to dryness and reconstituted in MeOH

HPLC

Fig. 1. Flow chart for enzymatic extraction of barbiturates from tissue.

Acid hydrolysis method Ten ,millilitres of concentrated hydrochloric acid was added to the spiked

tissue homogenate (5 g tissue in 10 ml water) and heated by immersion in a boiling water bath for 15 min. The contents were then filtered, cooled and extracted as for the enzyme digests.

Results and Discussion

The Ipercent recovery obtained for each of the seven barbiturates studied by emlploying the various digestion procedures is listed in Table 1. Calibration graphs were prepared for each of the drugs by plotting peak heights against concentrations (100 - 500 ngl of drug standards. The linearity between the concentration and peak heights was excellent with correlation coefficient (~1 values >0.994. Peaks obtained for experimental samples were quantitated by reference to the calibration graphs. Recoveries have been express’ed relative to the zero-time recoveries. (Zero-time recovery refers to that obtained immediately after spiking to fresh, untreated liver homogen- ates).

For enzymic digestion methods the optimal conditions of pH, temperature and incubation periods established by us earlier [9] were adopted as such in the present study. In the HPLC analysis of the different enzyme digests, no peaks accounting for any endogenous or conversion product interfering with the analysis of drugs in question were observed.

The enzymic digestions far excelled the conventional tungstate precipita- tion and acid hydrolysis methods for percent recovery (Table 11. Among the enzymes, papain gave the highest recoveries for all the seven drugs studied. Recoveries by the four enzymic digestion procedures were generally in the order papain > neutrase > subtilisin-A > trypsin. Digestion with neutrase, reported now for the first time for acidic drugs, gave recoveries next to papain and much higher than that obtained by using subtilisin-A. Amongst the enzymic digestion procedures trypsin gave the lowest recoveries for all the drugs. Interestingly these observations are similar to our experience with these enzymes in an earlier study with certain basic drugs [9].

With a wide range of drugs being encountered in forensic toxicological analysis the search is on for a single extraction procedure that can effectively release all classes of drugs from body fluids and tissues. The enzymilc method employing subtilisin Carlsberg promised to be a significant step forward in this direction. Employing mild hydrolytic conditions this method seemed to be particularly suitable for heat- and acid-labile drugs. Subsequent studies have focussed on the use of various other enzymes such as trypsin and 6-glucuronidase [6] protease I and protease V [7] and papain [8] apart from subtilisin [7,8,11,13,14] to the digestion of biospecimens prior to solvent extraction. All these studies however have dealt with only autopsy samples and compare recoveries obtained by an enzymic digestion method to that by a conventional one. The efficiency of an extraction procedure can be

TAB

LE 1

RE

CO

VE

RY

OF

BA

RB

ITU

RA

TES

FRO

M S

PIK

ED

LIV

ER

TIS

SUE

USI

NG

EN

ZYM

IClC

ON

VE

NTI

ON

AL

ME

THO

DS

-W

Perc

ent

reco

veqf

rf

: SD

.

Neu

traa

e Pa

pain

Su

b til

hin-

tl Tr

ypsi

n Tu

nge

tate

A

d

Am

ylob

arbi

tone

91

.0 +

1.5

98

.0 +

1.1

66

.0 f

1.

4 55

.0 +

1.4

55

.0 f

1.

4 42

.0 f

1.

3 B

utob

arbi

tone

98

.0 z

t 1.

3 10

0.0

rt 1

.4

60.5

+ 2

.0

55.0

+ 1

.4

50.0

+ 2

.0

45.0

f

1.7

Cyc

loba

rbit

one

85.0

f

1.3

95.8

rt

1.3

69.0

+ 1

.1

44.3

f

1.3

37.1

f

2.0

47.9

+ 1

.3

Pent

obar

bito

ne

75.8

zt

1.3

82.1

+ 2

.0

63.1

+ 2

.0

42.1

f

1.4

47.3

f

1.4

52.6

+ 1

.3

Phen

obar

bito

ne

74.5

f

1.4

76.5

f

1.4

56.1

+ 2

.0

38.7

+ 1

.3

30.6

+ 1

.3

’ 25

.5 f

1.

4 Q

uina

ibar

bito

ne

78.9

rt

1.4

84.2

+ 1

.2

63.1

f

1.2

47.3

+ 1

.0

50.0

+ 1

.3

42.1

+ 1

.3

Thio

pent

one

65.6

zt

1.2

85.8

f

1.6

60.6

+ 1

.6

50.5

+ 1

.2

60.6

+ 1

.3

50.5

+ 1

.0

‘AB

val

ues

are

mea

n of

tw

o de

term

inat

ions

. Fi

gure

s re

pres

ent

valu

es c

alcu

late

d ag

ains

t th

er z

ero-

tim

e re

cove

ry (

cons

ider

ed l

OO

Oh)

from

fre

sh,

untr

eate

d liv

er

hom

ogen

ates

.

51

better estimated only when the quantity of drug originally present in the specimen analyzed is known. In vitro studies are better suited for this pur- pose and hence adopted.

Several reports on the effectiveness of enzymic digestion for the release of acidilc drugs from tissue have appeared earlier [6,7,11,15,16]. While increased recoveries have been reported by some [6,7,11] others [15,16] have not observed such increase after enzymic digestion. In the present in vitro study, however, highest recoveries for all the seven barbiturates were obtainedi by the papain digestion method. It is interesting to observe that neutrase (neutral proteinase from B. subtilis) is more effective than subtilisin- A (alkabne proteinase from B. subtilis) in the release of acidic drugs from tissue and in most cases gave recoveries only slightly lower than that by papain.

Comparison of the recoveries (Table 1) for all the seven drugs by the papain method alone presents interesting facts. While butobarbitone could be recovered completely after the enzymic digestion, only 76.50/b of spiked phenobarbitone could be obtained. Recoveries for the other drugs ranged in between. these two values. It is also noteworthy that the recovery of pheno- barbitone was the lowest compared to the other drugs in all the enzymicl conventional methods employed. Taking into account the zero-time recov- eries (>95O/bl from fresh, untreated spiked liver homogenates and assuming almost similar binding of each of these drugs to the tissue matrix the differ- ential recovery of the different barbiturates is puzzling. This introduces the question of stability of the drugs concerned to the conditions employed for enzymic digestion. It is quite possible that the compounds themselves may as a result of being structurally analogous to the enzyme substrates be sus-

TABLE 2

SPECIFICITIES OF PROTEOLYTIC ENZYMES USED FOR RELEASING BARBITURATES FROM LIVER TISSUE [17]

Primary site(s) SQCOndary Site(S)

Neutrase

Papain

Subtilisin.A

Trypsin

B. subtilis

Papaya latex

B. subtilis

Pancreas

Ala- Ser- Thr- His- Lys- Arg- Leu- Aromatic- Lys- Arg-

Various

Various

Various

-

‘Refers to amino acids whose carboxyl groups form the amide bond.

52

ceptible to enzymatic hydrolysis to some extent. Hence the presence of such susceptible groups in the structure of the compound as well as the proteoly- tic properties of the enzymes used need to be identified. Table 2 lists the proteolytic specificities of the four enzymes used in the present study and Table 3 shows the structures of the barbiturates analyzed. It appears that differences in steric hindrances attributable to the substitution groups in the barbiturates may cause distinct intramolecular reorientations thereby offer- ing differential susceptibility to enzyme action. It is of interest here to note that Hammond and Moffat [18] studied the stability of certian drugs contain- ing amide and ester functional groups to the conditions used in the enzymic digestion of tissues by subtilisin Carlsberg and reported enzymatic degrada- tion of some drugs having amide functions. They also suggested that the

TABLE 3

STRUCTURESOFBARBITURATESUSEDINTHEPRESENTSTUDY

0 :: 9

0

2 1 6

IiN 5 RI 4 %

Name

d

R, R*

Amylobarbitone

Butobarbitone

-C&-4 - CH, - CH, - CHKH,),

-C*H, -CH-CH,--CH, I

Cyclobarbitone - C*H,

Pentobarbitone -C*H, -CH-CH,-CH,-CH, I

Phenobarbitone -C*H,

CH,

0 / \ Quinalbarbitone -CH,-CH=CH, -CH-CH,-CH,-CH,

I CH,

Thiopentone” -ca, - CH - CH,- CH,CH, I CH,

‘For thiopentone the bond C = 0 at position 2 is replaced by C = S.

53

environment of the amide bond is important as far as its susceptibility to enzymatic cleavage is concerned. A similar study with respect to papain and neutrase is underway in our laboratory and the results will be reported later.

In our earlier study on basic drugs [9] as well as in the present one we used papain (Loba, Bombay) purified from papaya latex. This enzyme prepa- ration though not crystalline has been obtained by partial purification of crude pa.paya latex and was distinct from the crystalline enzyme Kentron, Bombay),. Since the crude latex is known to contain besides papain other enzymes like chymopapain and lysozyme [17], papain represents only a part of the total proteolytic activity; the partial purification obviously removes the contaminating protein and enzymes, yielding pure papain. In order to ascertain the relative efficiencies of different preparations of papain we stud- ied the recovery of cyclobarbitone from liver tissue using five different papain preparations as listed in Table 4. It was interesting to observe that the crystalline enzyme (Centron, Bombay) at a concentration of 2 mglg tissue gave recovery comparable to that obtained by using the one purified from latex (Loba, Bombay1 at a concentration of 500 mglg tissue. This is attributed to the specific activity in 2 mg of the former being similar to that in 500 mg of the la.tter. However, the lower recovery by using papain crystallized in our laboratory (Table 41 is probably due to lower specific activity in this preparation. Recovery obtained by using fresh papaya latex (our collection) was lowler than that by using dried latex (Sigma) and in fact the lowest among the five preparations evaluated (Table 41. This discrepancy seems

TABLE 4

RECOVERY OF CYCLOBARBITONE FROM SPIKED LIVER TISSUE EMPLOYING VARIOUS PREPARATIONS OF PAPAIN

E?@pe Amou& added prepratios lmg/g tissuel

Percent recovery of cyclobarbitone f 5lD.0

(1) Fresh p,apaya latex (our collection)

(2) Dried papaya latex (S:igma)

(3) Papain purified from pajpaya latex (Loba, Bombay)

(4) Crystalline papai+ (5) Crystalline papain

(Centron, Bombay)

500 64.5 f 2.1

500 87.0 2 2.4

500 95.8 f 1.3

2 82.4 + 1.6 2 93.5 f 1.8

‘All values are mean of two determinations and figures correspond to recoveries obtained rela- tive to zero-time recoveries (considered 100%) from fresh, untreated liver homogenates.

Qystalline papain isolated in our laboratory from fresh papaya latex [17].

54

reasonable since both of them were unpurified thus accounting for very low specific activity of papain and further the fresh latex was measured as wet weight and would hence contain lower quantities of active papain compared to the dry latex (Sigma). The results indicate that papain is responsible for the release of drugs during enzymic digestion and that either purified papaya latex (Loba, 500 mglg tissue) or crystalline papain (Centron, 2 mglg tissue1 can be employed.

Conclusion

Papain and neutrase are well suited for the release of acidic drugs from tissue. However papain is better due to its higher efficiency, marked stability, long shelf life and relatively low cost. Papain is hence advocated as the method of choice for handling small sample volumes to monitor therapeutic and low toxic concentrations.

Acknowledgements

Thanks are due to M/s May & Baker, Essex, U.K. for gift samples of barbiturates and to M/s Novo Industri A/S, Denmark for free trial samples of the enzymes. The financial assistance to one of us (VS) by the Bureau of Police Research and Development, New Delhi is gratefully acknowledged.

References

5

9

10

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. A.S. Curry, Poison Detection in Human Organs, Charles C. Thomas, Springfield, IL., 1976, p. 106. E.G.C. Clarke ted.), Isolation and Identifiation of Drugs in Pharmaceuticals, Body Fluids and Post-MO&em MateriuL Vol. 2, The Pharmaceutical Press. London, 1975, p. 918. M.D. Osselton, M.D. Hammond and P.J. Twitchett, The extraction and analysis of benzodi- azepines in tissues by enzymic digestion and high-performance liquid chromatography. J. Pharm. PharmacoL, 29 (1977) 460-462. B. Caddy, F. Fish, P.W. Mullen and J. Tranter, An oxidative analytical procedure for some compounds containing the diphenylmethyiidene group. J. Forensic Sci Sot., 13 (1973) 127- 136. M. Bogusz, J. Biaika and J. Gierz, Enzymatic hydrolysis of tissues before XAD-2 extraction in poisoning cases. Forensic Sci Int., 20 (1982) 27-33. M. Bogusz, J. BiaIka and J. Gierz, Enzymic digestion of biosamples as a method of sample pretreatment before XAD-2 extraction. 2. Rechtsmed, 87 (1981) 287 - 295. M. Holzbecher and H.A. Ehenberger, Simultaneous determination of diphenhydramine, methaqualone, diasepam and chlorpromazine in liver by use of enzyme digestion: A compari- son of digestion procedures. J. AnuL TokcoL, 6 (1981) 62-64. 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. It. GilI. A.A.T. Lopes and A.C. Moffat, Analysis of barbiturates in blood by high-perfor- mance liquid chromatography. J. Chromatog., 226 (1981) 117- 123.

55

11

12

13

14

15

16

17

18

M.D. Gsselton, I.C. Shaw and H.M. Stevens, Enzymic digestion of liver tissue to release barbiturates, salicyclic acid and other acidic. compounds in cases of human poisoning. Analysst. 103 (1978) 1160- 1164. S.A. Stout and C. Lindsay De Vane, Tissue assay of phenobarbital, phenytoin and p hydroxyphenytoin by high-performance liquid chromatography. J. Chromatog., 285 (1984) 500 - !i8. L.A. King and K.J. Kimber, The use of subtilisin for the release of basic drugs from tissues. Experience at the Wetherby Forensic Science Laboratory. In R.A.A. Maes fed.), Topics in Fownsic and Analytical Toxicology, Elsevier, Amsterdam, 1984, pp. 81-86. M.D. Gsselton, The release of basic drugs by the enzymic digestion of tissues in cases of poisoning. J. Forensic Sci Sot., 1’7 (1977) 189- 194. N. Dunnett and P.G. Ashton, Experience with enzymic deproteination in general toxicologi- cal case work. In J.S. Oliver (ed.1, Forensic Tozicology, Croom Helm Ltd., London, 1980, pp. 272--:!80. E. Klug, Einige erfahrungen mit dem enzymatischen anfschlu/l von lebergewebe durch subtihsin carlsberg. Z. Rechtsmed, 86 (1981) 103-107. G.E. Perlman and L. Lorand feds.), Methods in Enzymology, Vol. XIX, Proteolytic Enzymes, Academic Press, New York, 1970. M.D. Hammond and A.C. Moffat. The stability of drugs to the conditions used in the enzymic hydrolysis of tissues using subtilisin carlsberg. J. Forensic Sci Sot., 22 (1982) 293 -295.