The Stability of Some Drugs and Poisons in Putrefying Human Liver Tissues

ORIGINAL PAPERS

The Stability of Some Drugs and Poisons in Putrefying Human Liver Tissues HM STEVENS

Central Research Establishment, Home Office Forensic Science Service, Aldermaston, Reading, Berkshire, United Kingdom RG74PN Abstract The behaviour of 56 drugs and drug-related compounds containing various molecular structures was studied in putrefying human liver macerates over different periods of time. Molecular structures which were prone to putrefactive decomposition included those in which oxygen was bonded to nitrogen but not to carbon or sulphur, as in nitro groups, N-oxides and oximes; where sulphur was bonded as a "thiono" atom (C=S or P=S), or formed part of a heterocyclic ring; and where there was an amino-phenol structure present, i.e. O H and NH2 on the same aryl nucleus. The stability of other structures indicated the general resistance of: carbon bonds with oxygen and nitrogen, nitrogen bonds with hydrogen, and sulphur bonds with oxygen. Putrefaction, which destroyed labile structures in 3 days to 14 days, was greatly enhanced by blowflyborne bacteria, and this helped to explain the reports of the long-term stability of certain drugs in buried corpses which had been insulated from blowfly contact by interment. Key Words: Drug Stability; Putrefaction. Journal of the Forensic Science Society 1984; 24 :577-589 Revised version received 5 June 1984 Introduction The analysis of putrefying tissues for drugs and poisons often presents particular problems to the toxicologist. These problems arise from two main sources, namely, the occurrence of endogenously-produced interfering substances, and the potential destruction by the putrefactive processes of a drug or poison present in the tissues. The first of thesc has been previously addressed in reports from this laboratory [I, 21. Publications concerning the stability of drugs during the putrefaction of tissues are comparatively rare. Most have dealt with barbiturates, and there is general agreement that these drugs are stable in decaying tissues. This is 577

supported by the results of work on pentobarbitone in decaying liver tissue [3]. More recently Shvaikova and others [4] have noted the preservation of barbituric acid derivatives in decaying post-mortem liver samples, and Dunnett and colleagues [5] recovered butobarbitone from soil beneath a skeleton. There is, however, a report of a fall in level of pentobarbitone in stored dogs' blood during putrefaction [6], but this may not be putrefactively induced. Levine and colleagues [7] found that three benzodiazepine compounds were unstable in blood which was stored in corked tubes. Weinig [8] reported the successful detection of three barbiturates, carbromal, some "vegetable" alkaloids, parathion, and two halogenated compounds (bromoform and chloroform) in exhumed corpses after burial periods of months or years. In contrast to this, the author of the present work noted a total loss of chlorpromazine from case-liver tissues containing 100 pglg of the drug when stored in closed vessels at 15°C and at 29°C over a period of 53 days. However, the concurrent storage of tissues containing amitriptyline and imipramine resulted in no loss of these drugs from the tissues. A similar observation of the instability of chlorpromazine in human tissues over a 3 to 6 month period at room temperature was made by Coutselinis and colleagues [9]. These differences in stability suggested that a useful purpose would be served by studying the behaviour of different chemical structures, as represented by drugs and poisons, in putrefying visceral material. The main aim of the present investigation was to ascertain which drugs were stable, and which ones were prone to decomposition during putrefaction. In the latter case an estimate was made of the time of persistence of the drugs in the tissues before they ceased to be recognized by initial screening and assay procedures.

Experimental methods and results The preparation of samples for putrefaction Case samples of human liver tissue obtained from an operational forensic science laboratory and which had been analysed and found to be free of drugs, were macerated in bulk with an equal weight of water, and the total macerate was divided into portions of 100 g. Appropriate quantities of the drugs or compounds to be studied were dissolved in a suitable solvent (water, methanol or ethanol) to produce solutions containing approximately 10 mglml. The solutions were then assayed accurately by UV spectrophotometry, or by colorimetry, and aliquots added to separate portions of liver macerate, i.e. one drug to each 100 g portion of macerate, to give concentrations of the order of 100-200 pg druglg of macerate. The mixtures were then finely remacerated to ensure thorough distribution of the drugs in

the tissue sample. A 10 g sample of each drug-containing macerate was withdrawn for initial assay (day O), and the remaining macerates placed in a bucket covered by a lid, which was raised approximately 1cm at one point to permit the access of blow-flies. The bucket was then placed in an enclosure with a roof (an open bicycle shed) to afford some protection against the weather. Samples (10 g) of each macerate were withdrawn at pre-determined intervals after homogenizing each mixture just prior to sampling.

Extraction and estimation of the drugs in the macerates The extraction and estimation of the level of each drug in the macerate samples were carried out by the toxicological methods described in a separate Appendix (50 pp), copies of which may be obtained from the Publishers' Editorial office. It is important to note that exactly the same analytical procedure was used for a given drug throughout any sequence of analyses during a putrefactive investigation. The A:; values used in the UV assays of extracted drugs were those reported by Clarke [lo]. The TLC system used for the clean-up of visceral extracts was, unless otherwise stated, Merck silica 60F254 pre-coated plates (obtained from Anderman & Co Ltd, London) and a mobile phase of chloroform-ethanol (95 : 5 vlv).

General procedure and factors affecting the analyses Following the release and extraction of the drug from the macerate, UV spectrophotometry was initially used as a means of assay. This was usually trouble-free during the early stages of putrefaction (i.e. up to the 15th day) but after this time the production of endogenous compounds tended to distort the curves. As soon as this occurred colorimetric assay was used, but if this failed or was not feasible, the extract was purified on a TLC plate and the drug extracted from the plate for assay. Visceral extracts were also assayed by an HPLC method when necessary. This general procedure was found to be adequate to indicate a trend within a reasonable period of time (7-14 days), i.e. whether the drug under study was stable or unstable to the putrefactive process. The main endogenous basic compounds encountered were tyramine, appearing from the 7th day onwards, and tryptamine, sometimes present from the 15th day, but usually appearing by the 21st day [I]. Tyramine was not a problem, except in morphine assays, as it was easily removed from extracts by washing them with sodium hydroxide. Tryptamine caused serious distortion of the UV curves, and required to be separated from the drug using the TLC system, where it remained on the starting-line of the plate. Wherever possible, (such as in the extraction of neutral or very weakly-basic drugs), the solvent extract was washed with dilute mineral acid 579

(0.2 N) and caustic alkali solutions (2N) prior to assay. This effectively removed endogenous materials. In the later stages of putrefaction (>30days), 2-phenyl-ethylamine and 3-methyl-indole were encountered, but these also remained near the starting-line in the TLC system used for cleaning the extracts. If putrefaction had been induced by inoculation and incubation (see below) the endogenous bases appeared earlier, tryptamine appearing at about the 7th day. Endogenous acidic compounds[2] did not pose as serious a problem, for they were removed by alkaline washes, or possessed very low A:; values.

Effect of ambient temperature This did not appear to influence directly the putrefactive process to any significant extent, provided that the temperature permitted the activity of egg-laying flies (see below). Effect of pies The presence of egg-laying blowflies appeared to be the main factor in promoting putrefaction. In the absence of these flies the speed of putrefactive decomposition was considerably reduced but the inoculation of the tissues with a mixed animal and human faecal slurry, or with tissue residues which had been exposed to blowfly activity, restored impetus to the process. The effects of exposing samples of tissues containing three putrefactivelylabile drugs to flies in an open bucket over a period of 18 days, compared with samples of the same tissues in a closed bucket (which prevented the access to flies), are shown in Table 1. The tissue levels of labile drugs generally fell sharply in the first 14 days, sometimes reaching zero before, or shortly after, the hatching of blowfly eggs (4th-8th day). If a faecal or other inoculation was used, the level fell significantly shortly after the inoculation was made, i.e. within 3 or 4 days. In contrast to this, the tissue concentrations of stable drugs tended to rise, especially in the early stages, presumably due to loss of water, and then to remain steady even up to 11 weeks' putrefaction in some instances.

TABLE 1. Effects on the stability of three putrefactively-labile drugs of exposure to flies, compared with protection from flies Compound

Clonazepam Nitrazepam Flupenthixol

Initial tissue level (pglg) 20 18.5 24

Day 12 tissue levels Open to Closed to flies flies

* 14 Nil *24

13 15 30

Day 18 tissue levels Open to Closed to flies flies 3.5 8.5 Not tried

* These values are probably falsely high, owing to the greater evaporation of water from the tissues in the open bucket.

The decomposition products of some labile drugs could be recognised, e.g. in the reduction of aryl nitro and N-oxide structures, and from bendrofluazide. However products have not been identified from labile sulphur-containing compounds.

Aqueous control solutions of drugs at pH -7.4 Aqueous solutions of all the labile drugs were made in water at pH -7.4, and then stored for the same period as that required for the maximum amount of decomposition of each drug in the liver macerates. This enabled a comparison to be made between the amount of chemical decomposition, and the degree of putrefactive degradation for each drug. In Table 2 the findings of this study are summarized. Labile drugs are indicated, together with the percentage loss at specified times where known. Most of the macerate samples contained an added drug, but some were case-samples (grouped separately in the Table) when the material became available for experimental purposes.

Identified products from some labile compounds A N B (2-amino-5-nitro-benzophenone). This yielded 2,5-diaminobenzophenone by reduction of the nitro-group. The product was sparingly soluble in ether and required extraction by methyl acetate. Its Rf value (-0.25) on Merck silica plates (60F254) using chloroform as the eluent provided a good separation from ANB (Rf -0.7). The product was identified using the mass-spectral data of Ardrey and colleagues [ I l l . Bendrofluazide. The product from this compound was shown by mass spectrometry, using accurate mass determination, to have an empirical formula C7H8N3o4S2F3 The absence of the tropilium ion (C,H,CH,+) indicated that metabolic rupture of the thiadiazine ring had occurred at the 3-position, yielding a disulphamoyl product with the structure:

This product possessed UV maxima in sodium hydroxide (2N) of 262 nm and 315 nm, and gave a blue fluorescent spot of Rf -0.2 in the TLC system.

Chlordiazepoxide and demoxepam. These compounds rapidly lost the oxygen atom from the N-oxide group yielding desoxy-products possessing a molecular ion of 16 amu less than the parent drug. Their molecular weights were 283.8 for the chlordiazepoxide product, and 270-7 for the demoxepam product which was identified as desmethyldiazepam. In the TLC system the desoxy-product of chlordiazepoxide possessed an Rf value (-0.55) similar to 581

TABLE 2. Results of analyses of putrefying human liver macerates containing compounds stable (S) and labile (L) to putrefaction Compound

Experimental samplesa p-Aminobenzoic acid (S) 2-Amino-5-chlor-benzophenone (ACB) (S) 2-Amino-5-nitro-benzophenone (ANB) (L) bo-Aminophenol (L) 'p-Aminophenol (L) bp-Aminophenol (L) p-Aminosalicyclic acid (L) Amitriptyline (S) Amitriptyline (S) 'Bendrofluazide (L) 'Bendrofluazide (L) bButalamine (S) Carbamazepine (S) bChlordiazepoxide (L) Chlormethiazole (L) Chlorpromazine (L) Chlorpromazine (L) Clemastine (S) Clonazepam (L) Demoxepam (L) Desmetryne (S) Dextropropoxyphene (S) Diazepam (S) Diazepam (S) bDimethylglyoxime (L) Dothiepin (L) Doxepin (S) Ethionamide (L) bFlupenthixol (L) Flurazepam (S) bHydrochlorothiazide (S) Imipramine (S) Lorazepam (S) dMalathion (L) Methaqualone (S) 2-Methylamino-5-chlorobenzophenone (MACB) (S) bMetronidazole (L) Morphine (S) Morphine (S) Nicotine (S) Nifuroxime (L) Nifuroxime (L) Nitrazepam (L) Nitrazepam (L) Nitrofurazone (L)

Initial level Macerate Water (pglg) (pglml)

Losses expressed as a % of initial level Macerate Water

Nil(37 days) Nil(18 days)

ND ND

72 75 79 58 Nil (77 days) Nil (22 days) 100 100 Nil(11 days) Nil(13 days) 100 84 100 100 Nil (25 days) 60 100 Nil(18 days) Nil(22 days) Nil(22 days) Nil(77 days) 100 100 Nil(33 days) 100 100 Nil(33 days) '12(10 days) Nil(22 days) Nil(18 days) 100 Nil(33 days)

15-42' 55" 55' Nil ND ND 25 25 ND ND Nil ND 42 30 ND 4 Nil ND ND ND ND Nil Nil ND 22 Nil ND 11 ND ND 20 ND

Nil(18 days) 100 Nil(29 days) Nil(28 days) Nil(22 days) 100 100 100 71 100

ND Nil ND ND ND ND 4 15 Nil 5

Period of loss (days)

Table 2(contd.) Compound

Initial level Macerate Water (pglg) (pglml)

p-Nitrophenol (L) Noxythiolin (L) Obidoxime (L) Paracetamol (S) Paraquat (S) Parathion (L) Parathion (L) Pentachlorophenol (S) Perphenazine (L) Pethidine (S) Phenazone (S) Polythiazide (S) Quinine (S) Salicylamide (S) Salicylamide (S) Strychnine (S) Strychnine (S) Sulphanilamide (S) Sulphanilamide (S) Thiopentone (S) Trifluoperazine (L)

Case samples Amitriptyline (S) Chlormethiazole (L) Glutethimide (S) Methaqualone (S) Paracetamol (S) Pentazocine (S) Pethidine (S)

132 39 50 11 36 165 95

ND 48 ND ND ND ND ND

Losses expressed as a % of initial level Macerate Water 100 84 100 Nil(21 days) Nil(21 days) 100 97 Nil(21 days) 100 Nil(28 days) Nil(14 days) Nil(9 days) Nil(22 days) Nil(17 days) Nil(21 days) Nil(48 days) Nil(33 days) Nil(28 days) Nil(14 days) Nil(14 days) 100

Nil Nil 55 ND ND 4 ND ND Nil ND ND ND ND ND ND ND ND ND ND ND 8

Nil(24 days) 100 Nil(24 days) Nil(20 days) Nil(35 days) 57(28 days)' Nil(20 days)

ND Nil ND ND ND ND ND

Period of loss (days)

NA 14 NA NA NA 28 NA

" Compounds indicated were added to drug-free liver macerates, and water control. Winter experiment, where the macerate was initially inoculated from the residue of a summer putrefaction, and incubated at 25'C. This reduced the persistence time of labile compounds below the general 14-days period, and precluded the complications of flies and maggots. ' Probably due to chemical hydrolysis, as the percentage loss from aqueous solution was virtually the same. Blood only. ' Only the three aminophenols were dissolved in boiled water containing added formaldehyde because losses from water are due to oxidation (which would not occur in putrefying tissues) and depended on the efficiency of air-exclusion from the aminophenol solutions. This loss of pentazocine was not due to instability but to the consumption of the tissue by blowfly maggots. The last sample to be analyzed consisted of maggots and brown fluid only.

'

ND Experiment not done especially if drug is stable in putrefying macerate.

NA Not applicable. 583

its parent drug, while desmethyldiazepam had an Rf -0.7, and was well separated from demoxepam (-0.5). Both desoxy-products in sulphuric acid (2N) have UV peaks at around 283 nm compared with 305 nm for the parent drugs. Clonazepam and nitrazepam. With these drugs there was a rapid conversion of their 7-nitro groups to 7-amino structures which were sparingly soluble in ether, but more soluble in methyl acetate. Mass spectrometry showed them to have m/z values of 285 for the clonazepam derivative and 251 for the nitrazepam one. Both products possessed Rf values of 0.3 in the TLC system which were lower than those of the parent compounds, and both reacted with formaldehyde-sulphuric acid (60:40 v/v) [12) giving similar colours to those of their parent drugs. p-Nitrophenol. This was quickly converted to p-aminophenol which gave mass spectral peaks at m/z 80, 107 and 109. Parathion. No final products were identified; some p-nitrophenol was detected after 6 days putrefaction, but this had disappeared by the 12th day.

Discussion and conclusions Early in this study it was appreciated that rapid putrefaction of the liver tissue samples depended largely on the presence of blowflies, and for this reason the study was initially pursued from May to September of successive years. Some drugs which were labile to putrefaction were totally destroyed in macerates before deposited fly eggs had hatched, suggesting that it was the bacteria carried by the flies which were chiefly responsible for putrefaction. The same effects were induced, in the absence of flies, by inoculating the macerates with a mixture of human and animal excrement, material upon which flies alight. Later, some winter experiments were successfully carried out indoors by inoculating the macerates from a tissue residue which had been exposed to blowfly activity during the summer and then stored at 4°C until required. In view of this it is suggested that the storage of such a culture on a routine basis could assist toxicologists in the event of information being required quickly about the stability of a drug or poison being sought in decomposed post-mortem tissues. Burial of the samples was avoided, as it would have meant the exclusion of flies from a closed container. If the samples were not enclosed, or were contained in a porous wrapping, the liquid portion of the macerates would have drained into the surrounding soil. Conversely, rain water could have entered the specimens. The experimental findings of this work, given in Table 2, indicated that the destruction of labile drugs was being effected by putrefaction caused by the 584

introduction of fly-borne faecal bacteria. Generally, losses of labile drugs from the aqueous solutions used as controls were small, which would be expected from hydrolysis. Three main criteria appear to determine the potential destruction of a compound during putrefaction, namely: the possession of readily available oxygen for anaerobic purposes; the presence of suitably bonded sulphur; and an aminophenol structure where O H and NH2 are present on the same aryl nucleus. The conditions of the first criterion are fulfilled when oxygen is bonded to nitrogen but not to carbon or sulphur. This occurs with nitro groups bonded to either an aromatic nucleus as in 2-amino-5-nitrobenzophenone, (ANB), clonazepam, nitrazepam and p-nitrophenol or to a non-aromatic structure as in metronidazole and nitrofurazone; with oximes such as dimethylglyoxime and obidoxime, and with N-oxide structures as with chlordiazepoxide and demoxepam. Nifuroxime offers two points of attack as it contains both a nitro and an oxime group, and is rapidly destroyed as such in the macerates. The oxime group may, of course, have hydrolysed or reacted with an endogenous amine. Evidence that faecal anaerobic bacteria have broken oxygen-nitrogen bonds is supported by some investigations by Ramsey and Hayward [13] who found indole in some samples of blood and urine during analysis for basic drugs. They listed several faecal anaerobes of the Clostridium group among those which are able to produce indole from tryptophan. Faecal anaerobes are likely to be present in viscera taken post-mortem from the body cavity, and this would account for the slow conversion of drugs like nitrazepam to a 7-amino-derivative in liver macerates from which flies had been excluded. The impetus restored to the speed of decomposition of drugs in macerates by inoculation with a faecal slurry also suggests that faecal bacteria are playing a major role in the putrefactive processes. Basaga and colleagues [14]found that the caecal bacteria Escherichia coli and Serratia marcescens reduce metronidazole and misonidazole (presumably the nitro group of each), and O'Brien and Morris [15]also discovered that the nitro group of chloramphenicol is reduced to an amino group by some species of Clostridium. The report by Levine and fellow workers [7] of the instability of chlordiazepoxide and nor-chlordiazepoxide in stored blood suggest that anaerobes were active on the N-oxide group; the loss of nordiazepam however may be due to a process other than that caused by an anaerobic bacterium, as, unlike the other two compounds, the blood level of nordiazepam does not reach zero from storage at 25°C. As regards the second criterion, drugs which contain sulphur in a chain bonded as a thiono-group (C==S, P=S) appear to be prone to decomposition, examples being ethionamide, malathion and noxythiolin. Compounds containing sulphur attached by single bonds in a chain (e.g. desmetryne and 585

polythiazide) appear to be stable. Malathion could only be extracted from blood after its addition; all attempts at extraction from liver macerates failed. The stability of thiopentone appears to be an anomaly, but presumably the sulphur in the 2-position is not released from the molecule for bacterial metabolism except by oxidation of the compound to pentobarbitone. This is unlikely to occur under the anaerobic conditions of putrefaction. It is also possible that the thiono group is sufficiently enolized in the barbiturate ring to reduce the electron deficiency of the double bond. Sulphur forming part of a heterocyclic ring causes some instability to putrefaction, for instance in chlormethiazole, dothiepin, flupenthixol and the phenothiazines. The destruction of these drugs usually takes place within the 14-day persistence period for labile compounds but occasionally does not occur, even with the admission of flies and/or faecal inoculation. This suggests that the bacteria required to decompose this type of compound are less widely encountered than those which break down the other structures described. With the third criterion, aminophenols like o- and p-aminophenol and p-aminosalicylic acid steadily disappeared from the macerates during putrefaction and had a half-life of about 11-12 days (4-7 days by inoculation and incubation). Structures possessing a primary aryl amine group, but which are not phenolic, such as 2-amino-5-chloro-benzophenone (ACB), p-aminobenzoic acid, and sulphanilamide, or are phenolic but do not possess such an amine group, as in pentachlorophenol and salicylamide, or have a substituted amine group like paracetamol, were all stable in the putrefying macerates. Bonds between oxygen and carbon, or oxygen and sulphur, are not broken. This confers stability to putrefaction on amides, carboxyl, esters, ether linkages, heterocyclic rings containing oxygen, hydroxyl, ketones, phenols, phenones, sulphonamides and sulphones such as in thiazides. Similarly, bonds between nitrogen and carbon, and nitrogen and hydrogen are not broken, giving stability to amides, amines, heterocyclic rings containing nitrogen, and N-methyl groups. The possibility of the occurrence of putrefactively-produced drug conjugates was appreciated when butalamine and phenazone, which were stable to putrefaction over the standard 14-day period, could not be obtained from rotting macerate material after storage at 4°C for several months, until strongly acidic deproteinising conditions were used prior to solvent extraction. This caused the butalamine yield to rise from nil using direct extraction in alkaline conditions to 12pg/g, and the phenazone yield to increase similarly from 1pg/g with alkaline extraction to 66pg/g. 586

The reason for this phenomenon being the protein-binding of the drugs was thought to be unlikely as, if this was so, there would be rapid initial disappearance of the compounds from the macerates, followed by gradual release with time. More probably it was due to the formation of a complex from a slowly-produced endogenous substance at 4°C. This complex would be insoluble in organic solvents, but easily dissociated in hot acid. The same process could have happened with time at ambient temperatures, but the drying-out of the viscera may have arrested the formation of the complex. It is therefore recommended that any failure to extract directly a compound deemed to be stable to putrefaction should be complemented by an acidic method of tissue hydrolysis, ensuring, of course, that the drug being sought is, itself, stable to the hydrolytic method used. The use of acidic methods to hydrolyse putrid macerates which had contained putrefactively-labile drugs continued to give nil recoveries of those drugs. The instability of bendrofluazide to putrefaction appeared to be an anomaly, as none of the three criteria applied. However, it is known that diamines condense with aldehydes giving compounds of the general structure:

which can be readily hydrolysed to give back their starting materials. On this basis the hydrolysis of bendrofluazide would yield phenylacetaldehyde in addition to the disulphamoyl compound which was identified. Phenylacetaldehyde, by conversion to phenylethanol, would act as an electron-accepting source for bacterial metabolism. If this occurred it would offer an explanation for the breakdown of the drug to the disulphamoyl compound. Phenylethanol has previously been found as an endogenous product of visceral putrefaction [2] and so its presence would not be conclusive evidence of the suggested mechanism. The findings of this study support virtually all of the previous observations reported by other authors on the stability of drugs to putrefaction. Of special interest, in this respect, is the review of Weinig [8]. Most of the corpses mentioned, in which drugs were detected, were exhumed and therefore had been excluded from contact with blow-flies by burial; in addition, the drugs which were detected in them, with one exception, contained structures which were non-labile to putrefaction. The exception was parathion which, with its metabolite p-nitrophenol, was detected in considerable quantities inside the stomachs of some cadavers, one of which 5 87

had rotted without burial. In this corpse, attempts to detect parathion in the other organs were, however, unsuccessful; no figure is given for the quantity of parathion found in the stomach, and, naturally, no knowledge could be gained as to how much parathion had decomposed prior to the discovery of the cadaver. In respect of the crucial role played by rapid initial screening tests on post-mortem tissue extracts in British operational forensic science laboratories, the use of UV or colour assays complemented by a chromatographic procedure and, where necessary, mass spectrometry, was found to be a very satisfactory modus operandi in determining whether a compound was affected by tissue putrefaction. In view of the very poor yields obtained with tricyclic bases like amitriptyline and chlorpromazine with the hot hydrochloric acid method [16,17], this method was superseded later in the study by direct solvent extractions from tris-buffered macerates [18]. The poor yield from the hot hydrochloric acid method occurred mainly because the acid digests were filtered before extraction. Unfiltered digests were previously found to furnish compounds which masked UV or colour assays [19]. The tris-buffered extractions did not usually produce as high recoveries of drugs as the subtilisin-digested samples [18], but were less time-consuming, as the one-hour incubation period was avoided. Extraction with methyl acetate gave good recoveries for compounds which were sparingly soluble in ether, especially if the aqueous phase was previously saturated with sodium chloride. Complete evaporation of methyl acetate was necessary before any back extraction with aqueous sodium hydroxide was possible owing to the hydrolysis of the ester by the alkali. The successful extraction of 2,5-diaminobenzophenone (metabolite of ANB) with methyl acetate, and the poor yields of the 7-amino derivatives of clonazepam and nitrazepam using ether for extraction indicates that if such amino metabolites are being sought in tissues, methyl acetate should be used as the extracting solvent. The extraction method used for nifuroxime and noxythiolin (details contained in Appendix, available from publisher) may, it is hoped, prove useful for other neutral compounds. Depending upon some degree of solubility of these two compounds in aqueous media they were removed from the fatty residues obtained from the solvent-extracted macerates by triturating the residues with aqueous sulphuric acid (2N). The acid extracts were clear for UV spectrophotometry, but if water only was used for the trituration of the fatty residues, the aqueous extracts were turbid. It is hoped that this work may assist toxicologists in cases where drugs are being sought in tissues undergoing putrefaction, and that information on the stability of any compound not included here may be gained by inference. 588

Acknowledgements The author thanks Drs A R Allan, R E Ardrey and MCH Oon for carrying out the mass spectrometry of the products obtained from various compounds during putrefaction. He also thanks D r A R Allan for his estimation of morphine in a sample by HPLC using electrochemical detection, and Dr R Gill for informative discussions concerning the technique of HPLC.

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