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The Interference of Putrefactive Bases in the Analysis of Biological Materials for Drugs
J. Forens. Sci. Soc. (1973), 13, 47
The Interference of Putrefactive Bases in the Analysis of Biological Materials for Drugs JOHN S. OLIVER and HAMILTON SMITH The Department of Forensic Medicine, The University of Glasgow, Glasgow G21 8QQ Scotland.
A study has been made of a group of compounds known as putrefactive bases. Data has been collected for them using thin-layer chromatograpty, colour reactions on the silica gel layer, gas-liquid chromatography and ultraviolet spectrography. The results have been used to interpret the analysis o f both blood samples stored at room temperaturefor several months and of cases of medicolegal interest where considerable delay has occurred before death has been discovered. I t was found that P-phenylethylamine was the most frequently encountered putrefactive base. However, it was impossible either to determine the degree to which it occurs with time or predict any pattern of occurrencefor the other compounds. Introduction A large amount of post-mortem material is to some extent decaying or even in a n advanced state of putrefaction when it is received for analysis. The result is that the group of compounds known as "putrefactive bases" require consideration. These are basic materials produced by the decomposition of the normal body matrix. Fulton (1965) lists a number of nitrogenous bases that may be formed by the bacterial decarboxylation of amino acids resulting from protein breakdown. He reports that all of them have been identified in putrefied material. The related materials choline and trimethylamine are also mentioned. Attention is drawn to the similarities between two of them, P-phenylethylamine and isopentylamine and the sympathomimetic drugs amphetamine, methylhexane amine and tuaminoheptane. This can be of great importance when searching for drugs in putrefied materials. Goldbaum and Domanski (1965) also report P-phenylethylamine and pyridine in decomposed tissue. Turner (1965) mentions three compounds which must be considered when searching for drug metabolites. Two of them, P-phenylethylamine and tyramine, are relevant to the analysis of biological materials for basic drugs. The other can affect the analysis for barbituric acid derivatives and is not relevant to this particular paper. Fulton (1966) lists six putrefactive bases that have been detected during analysis of tissue in his laboratory. They are P-phenylethylamine, isopentylamine, tyramine, tryptamine, trimethylamine, and a substance which may be either tetrahydronorharman or norharman. This latter substance was subsequently identified as norharman (Fulton 1967). Corley (1967) reports the use of infra-red spectroscopy to distinguish between amphetamine and p-phenylethylamine in a putrefied liver. Thin-layer chromatography and gas-liquid chromatography both revealed that the liver constituent was not amphetamine. Data from both sources indicated that the compound was probably P-phenylethylamine. The importance of being able to differentiate between these substances can be seen in the report of a case by Goldbaum and others (1963) where P-phenylethylamine had been mistaken for amphetamine. Storage of biological samples at low temperatures does not altogether inhibit the formation of putrefactive bases. Kaempe (1965) demonstrated the
occurrence of tyramine and P-phenylethylamine in liver which was stored a t temperatures of 4-5°C. At - 18°C their formation was appreciably reduced although tyramine could be demonstrated in 25% of the samples analysed after 18 months. The presence of 1-hydroxymethyl-P-carboline (hydroxyharman) in fresh human liver has been demonstrated (Kaempe, 1967). Techniques for the isolation, identification and separation of this substance from other alkaloids are also described. I t was found that heating tryptophane in tartaric acid (IN) produced a compound with similar ultraviolet and infra red absorption spectra. As a result it was found that hydroxyharman was a n artefact produced by the extraction for basic drugs using the method of Stas-Otto on any autopsy material containing tryptophane. A similar substance can be produced by the action of diluted strong acids on liver tissue (Kaempe, 1967). Similarly, harman is a possible source of interference. This can be formed by the combination of tryptophane and acetaldehyde. I n a case reported the harman was isolated in a bottle of home-made mead. The tryptophane was contained in the yeast (Clarke, 1965). This survey has shown that a large number of compounds arising from putrefaction can be encountered in the course of analytical toxicology. I t would appear that P-phenylethylamine is the most commonly encountered base. The following study presents data which has been collected for a number of possible putrefactive bases and other related substances to enable them to be detected, identified and separated from co-extractable drugs using modern analytical techniques. In addition the application of these techniques to some cases of medicolegal interest is described.
Experimental Thin-layer chromatography The TLC plates coated with 0.25mm layers of Kieselgel G were prepared and activated by heating at 1 10°C for 30 minutes. The solvent system used is a 3:l mixture of' chloroform and methanol. This is placed in a chromato-tank and allowed to equilibrate for a t least 1 hour before use. Samples and standards are transferred to the plates by repeated spotting from glass capillaries to form discrete spots. Preparation of standards The standard solutions contained approximately 5mg of base per lml of chloroform. They are prepared by dissolving the free base directly in chloroform or by extraction from a salt. Further dilutions were made as required in order to produce discrete spots. Preparation of spray reagents The spray reagents used are prepared as follows: 1. Iodoplatinate reagent. Chloroplatinic acid (1g) is dissolved in water (1 Oml) . A solution of potassium iodide (log) in water (250ml) is added. The final volume is made up to 500ml with water. 2 . AcidiJied iodoplatinate reagent. This is prepared as above except that the final volume is made up to 500ml with a solution of hydrochloric acid (144ml S.G. 1.18) and water (96ml). 3. Iodine vapour reagent. Iodine contained in a polyethylene bottle is heated on a water bath prior to use. 4. Mandelin's reagent. This is prepared by dissolving ammonium vanadate (lg) in sulphuric acid (100g S.G. 1.98). The reagent is stirred with a glass rod prior to use. 5 . Gold chloride reagent. Using chloroauric acid (lg) to replace chloroplatinic acid (lg), the procedure is that used for the preparation of the acidified iodoplatinate reagent. 6. Tetranitromethane Tetranitromethane (100%) is used.
Cholorojorm Methanol Base (3 :1) R j Ethanolamine 0.89 Harman 0.61 Histamine 0.05 Hypoxanthine 0.00 Isoamylamine 0.07 Isobutylaminc 0.83 str. Norharman 0.72 Pentamethylenediamine 0. l l P-phenylethylamine 0.21 n-propanolamine 0.07 Pyrrolidine 0.9 1 Tetramethylinediamine 0.06 Trimethylamine Diffuses Tryptamine 0.16 Tyramine 0.15 Xanthine 0.00
TABLE 1 ANALYTICAL DATA FOR PUTREFACTIVE BASES THIN-LAYER CHROMATOGRAPHY DATA Iodine .followed b y Acidified acidajed Iodoplatinate iodoplatinate indoplatinate Mandelin's reagent rpagent Iodine reagent reagent Brown Grey Purple-brown Brown Brown Purple Purple Pale blue Brown Brown Brown Purple Purple Brown Blue Purple Brown Purple Purple-brown Brown Brown Purple Purple Blue-grey Blue Brown Brown Purple Brown Brown Purple Brown Grey Purple Purple-blue Brown Purple-brown Blue-grey Blue Brown Brown Blue-grey Purple Brown Blue Pale blue Purple Brown Brown Orange Brown Brown Purple
-
-
-
Gold chloride reagent
-
Tetranitro Methane reagent
-
Brown -
Brown Brown Brown Grey-brown Brown Brown -
-
Pale yellow Pale yellow Pale yellow
-
Pale yellow -
Application of reagents All reagents were sprayed directly on to the TLC plate using a Shandon Laboratory Spray Gun. Iodine vapour was "puffed" from the warm polyethylene bottle. Gas-liquid chromatography The instrument used was a Pye Model (54) Isothermal Gas Chromatograph equipped with a Hydrogen Flame Ionisation Detector. The column used was 5' x 4'' 0.d. glass packed with 5% SE30 on acid washed 60-80 mesh Chromosorb W treated with dimethyldichlorosilane. The column temperatures were 200°C and 125°C. Retention times were measured relative to codeine or ephedrine respectively with a nitrogen flow rate of 60ml per minute. Ultraviolet spectrography The ultraviolet absorption spectra were recorded using a Unicam SP800 instrument and fused silica cells with a lcm path length. Solutions of the putrefactive bases in ethanol, 0.1N hydrochloric acid, 0.1N sulphuric acid and 10% sulphuric acid were used. Analysis procedure The techniques described were applied to extracts of samples received from people found a t a considerable period of time after death occurred. Blood samples containing small amounts of citrates were allowed to stand a t room temperature for various lengths of time after which they were analysed using the described techniques. Extraction procedure 1. Urine Extraction. 25ml of urine is made alkaline with ammonium hydroxide solution (1.5N) and then shaken with 40ml of chloroform. The organic layer is filtered through Whatman No. 90 filter paper to remove excess moisture and suspended urine droplets. The resulting solution is shaken with 5ml of sulphuric acid (0.1N) and the separated acid layer centrifuged to remove any suspended chloroform. The ultraviolet spectrum of the acid solution is then recorded against a n acid blank. Following this, the acid solution is made alkaline with ammonium hydroxide solution (1.5N) and extracted with chloroform as before. The resulting chloroform solution is evaporated carefully to dryness and the residue is dissolved in a few drops of chloroform for transfer to thin-layer chromatography plates. TABLE 2 ANALYTICAL DATA FOR PUTREFACTIVE BASES GAS-LIQUID CHROMATOGRAPHY AND ULTRAVIOLET SPECTROSCOPIC DATA Gas-liquid chromatography Ultraviolet spectroscopy 5% SE30 column Maximum absorption in Retention times relative to 0.I N hydrochloric acid Base Codeine 200°C Ephedrine l25OC nanometers Ethanolamine 0.00 243,298, 355 Harman 0.33 Histamine 0.19 Hypoxanthine 244 Isoamylamine 0.00 Isobutylamine 0.00 244, 298, 355 Norharman 0.32, 0-38 Pentamethylenediamine 0.17 p-phenylethylamine 0.33 257, 246, 251, 261-5, inf. 266 n-propanolamine Pyrrolidine 0.02 00.0, 0.22, 2.33 Tetramethylenediamine 0.12 Trimethylamine 0.00 Tryptamine 0.2 1 278, 22865 Tyramine 0.08 274 inf. 280 261, 225 Xanthine 50
2. Blood and tissue extraction. 5ml of blood or 25g of macerated tissue are pretreated by precipitating proteins using the tungstate method. The resulting filtrate is processed using the same technique as that described for urine. The reagent volumes may require scaling up and while the extraction for the ultraviolet spectrography step in the blood analysis is made with the usual 5ml of acid, lOml is suitable for the tissue analysis. 3. Proteinprecipitation. 5ml of blood or 5g ( x 5) of macerated tissue is mixed with 30ml of distilled water and lml of sodium hydroxide (2.5N) solution. The mixture is shaken and then allowed to stand for 10 minutes. lOml of sodium tungstate (10% w/v) solution is added followed by the dropwise addition of 3.5ml sulphuric acid (3.6N) with shaking. The solution is heated on a water bath for 10 minutes until precipitation is complete. The resulting solution is filtered through Whatman No. 1 filter paper. The residue is washed with further portions of sulphuric acid (3.6N) which is, in turn, filtered and combined with the original filtrate.
Results and discussion Tables 1, 2 and 3 list the analytical data obtained for the putrefactive bases. Using thin-layer chromatography good separations are obtained using the chloroform, methanol solvent system. The spray reagents described readily detect all the materials except the two xanthine derivatives which can be ignored for interference purposes using the TLC and colour reagent systems described. No single colour reagent system can be used for the identification of all the putrefactive bases listed. Iodine is of little value as a screening reagent with biological extracts. I t can be used as a locating reagent to obtain Rf values of pure compounds. Mandelin's reagent and tetranitromethane show limited use in the field, but care should be used in interpreting results when there is a possibility of drugs being present. More information is obtainediby using one of the iodoplatinate screening reagents and interpreting positive reactions with the Rf values. The results obtained can be further confirmed by use of a selection of putrefactive bases run with the unknown materials for reference purposes. The other colour reactions, can, if required, be used for confirmation of results. The retention times given for gas liquid chromatography were obtained under two of the most useful conditions for the detection and identification of basic drugs extracted from biological media. I t can be seen that a minority of the putrefactive bases are successfully chromatographed under these conditions With the exception of /3-phenylethylamine they can be readily identified using thin-layer chromatography and ultraviolet spectroscopy. The similarity of 9-phenylethylamine and amphetamine has already been mentioned. These compounds can be readily differentiated using a carbowax-KOH column such as those described by Beckett and others (1967). To use the SE30 column for differentiation necessitates the formation of acetone derivates. If a few drops of acetone are added to an ethereal solution of the compounds, and the solution is subsequently chromatographed, the derivative peaks obtained can be used to differentiate between the materials. Also, identification can be obtained using a column packed with 5% Carbowax 6000, 5% K O H on Chromosorb G (AW. DMCS) which differentiates the materials. The absorption data for the putrefactive bases was obtained in the solvent commonly used when analysing materials for basic and neutral drugs. Of the techniques described only ultraviolet spectrometry shows interference from the xanthine compounds. The data given can be used as a guide to the identification of any of the putrefactive bases present. When these absorptions do occur further separation is required to ascertain whether a drug is being masked by the interfering material. The analysis of some biological specimens received from postmortems often produce an absorption spectrum characteristic of tyramine. Since subsequent
TABLE 3 ANALYTICAL DATA FOR PUTREFACTIVE BASES ULTRAVIOLET SPECTROSCOPIC DATA Absorption m a x i m Absorption minima Compounds nanometers nanometers 1. In 0. I N sulphuric acid Ethanolamine Harman Histamine Hypoxanthine Isoamylamine Isobutylamine Norharman Pentamethylenediamine P-phenylethylamine n-propanolamine Pyrrolidine Tetramethylenediamine Trimethylamine Tryptamine Tyramine Xanthine
245, 299, 355 249 -
247, 300, 357 -
257, 246, 251, 262.5, 266 (inflexion) 278, 287, 272 (inflexion) 276, 280 (inflexion) 265,225
220, 272, 319 222
221, 273, 322 -
226, 247, 253.5, 261
-
240, 285.5 243 239
2. In dilute sulphuric acid (10% u / v ) Ethanolamine Harman Histamine Hypoxanthine Isoamylamine Isobutylamine Norharman 246, 299, 358 Pentamethylenediamine 8-phenylethylamine 257, 251, 262.5 inf. 246,266 n-propanolamine Pyrrolidine Tetramethylenediamine Trimethylamine Tryptamine 279,287 inf. 272 Tyramine 274 inf. 280 Xanthine 260, 229 3. In ethanol Ethanolamine Harman Histamine Hypoxanthine Isoamylamine Isobutylamine Norharman Pentamethylenediamine P-phenylethylamine n-propanolamine Pyrrolidine Tetramethylenediamine Trimethylamine Tryptamine Tyramine Xanthine
240, 277, 288, 337, 349 inf. 249
-
258, 247, 252.5, 260.5, 264, 268 inf. 242.5
280, 273,289 277-5 inf. 285 268
241,276,287 242 239 (not very soluble)
analysis by thin-layer chromatography using iodoplatinate reagents will not reveal the presence of this substance, the absorption cannot be assigned to any positive reaction obtained without further separation procedures. Several blood samples were allowed to stand for various periods of time. The results of their analysis by thin-layer chromatography and ultraviolet spectrography are given in Table 4. The results show that P-phenylethylamine can be
Sample
TABLE 4 ANALYSIS O F PUTREFIED BLOOD SAMPLES Ultraviolet spectrum in 0.I N sulphuric Reaction with Chloroform :Methanol Acid Number of iodoplatinate (3:I) absodtion m i m u m months old reagent Rf nanometers
+ + +Strong Reaction + +Weak Reaction *Low Intensity
detected in all the samples to varying degrees. The positive identification was achieved by gas-liquid chromatography. No other reactions were observed for the blood samples. Some Cases of Interest Case 1 Female. Had been dead for 3 weeks. The blood sample was analysed for barbiturates, methaqualone, salicylate and alcohol. I t contained 86mg of alcohol per lOOml of blood. The analysis for basic drugs revealed tyramine (by UV spectroscopy), tryptamine, P-phenylethylamine and possibly a trace of norharman. The relevant TLC data is listed in Table 5. TABLE 5 ANALYSIS OF PUTREFIED BLOOD FOR BASIC DRUGS Reaction with acidtJid Chloroform : iodoplatinate Methanol 3:l R f Other information Spot No. reagent 0.10 No ultraviolet spectrum 1 Purple obtainable. Indistinguishable from tryptamine added to the extract. 2 Purple 0.2 1 Identified by UV and GLC as 8-phenylethylamine. 3 Brown Purple 0.73 This spot was indistinguishable from norharman. However, no ultraviolet spectrum was obtained to enable confirmation
Case 2
Male. Blood was 1-month-old. The blood sample contained 105mg of alcohol per 1OOml. Putrefactive bases identified in the sample were tyramine and p-phenylethylamine. A purple-black spot corresponding to tryptamine, and a purple spot identical to norharman were also obtained. No confirmation of the latter was possible by ultraviolet spectroscopy.
Case 3 Male. He had been dead for 1-2 weeks. The urine sample was found to contain 9-phenylethylamine. The blood sample contained p-phenylethylamine. Also, it gave a positive reaction with acidified iodoplatinate reagent, a purpleblack spot, at Rf 0.72. This compound had a very weak absorption maximum at 246nm. This is similar to norharman. Conclusion A study has been made of a number of materials which may be present in samples as a result of putrefactive changes in the body. The data obtained for these putrefactive bases enables them to be recognized when encountered using the techniques described. In practice it has been shown that P-phenylethylamine is the most comnlonly encountered putrefactive base in blood. Unfortunately, it was not found to be possible to predict the degree to which it occurs. This and the presence of other putrefactive bases appears to be erratic. Acknowledgement J. S. Oliver would like to acknowledge the Instaprint Camera Company, London, for financial support during this work. References G. T., and MOFFAT,A. C., 1967, J. Pharm. Pharmacol. BECKETT, A. H., TUCKER, 19, 273. CLARK,E. G. C., 1965, Chromatography and the classical methods of analysis, in; Symp. Ident. of Drugs and Poisons, p.54. The Pharmaceutical Society of Great Britain. CORLEY,C. A., 1967, Bull. T.I.A.F. T. 4.2. FULTON, C. C., 1965, Bull. T.I.A.F.T. 2.4. FULTON, C. C., 1966, Bull. T.I.A.F. T. 3.3. FULTON, C. C., 1967, Bull. T.I.A.F. T. 4.1. GOLDBAUM, L. R., and DOMANSKI, T. J., 1965, An approach to the analysis of biological specimens for basic drugs, in; Progress in Chemical Toxicology, Vol. 2, Stolman, A., (ed.). New York, London: Academic Press. E. H., and BLUMBERG, J. M., 1963, J. Forens. Sci. GOLDBAUM, L. R., JOHNSTON, 8, 286. KAEMPE, B., 1965, Acta Pharmac. et Toxicol., 23, 15. KAEMPE, B., 1967, Acta Pharmac. et Toxicol., 25, 155. TURNER, L. K., 1965, Searching for drug metabolites in viscera, in; Methods of Forensic Science, Vol. 4. Curry, A. S. (ed.). London, New York, Sidney: Interscience Publishers, John Wiley & Sons Ltd.
The Interference of Putrefactive Bases in the Analysis of Biological Materials for Drugs JOHN S. OLIVER and HAMILTON SMITH The Department of Forensic Medicine, The University of Glasgow, Glasgow G21 8QQ Scotland.
A study has been made of a group of compounds known as putrefactive bases. Data has been collected for them using thin-layer chromatograpty, colour reactions on the silica gel layer, gas-liquid chromatography and ultraviolet spectrography. The results have been used to interpret the analysis o f both blood samples stored at room temperaturefor several months and of cases of medicolegal interest where considerable delay has occurred before death has been discovered. I t was found that P-phenylethylamine was the most frequently encountered putrefactive base. However, it was impossible either to determine the degree to which it occurs with time or predict any pattern of occurrencefor the other compounds. Introduction A large amount of post-mortem material is to some extent decaying or even in a n advanced state of putrefaction when it is received for analysis. The result is that the group of compounds known as "putrefactive bases" require consideration. These are basic materials produced by the decomposition of the normal body matrix. Fulton (1965) lists a number of nitrogenous bases that may be formed by the bacterial decarboxylation of amino acids resulting from protein breakdown. He reports that all of them have been identified in putrefied material. The related materials choline and trimethylamine are also mentioned. Attention is drawn to the similarities between two of them, P-phenylethylamine and isopentylamine and the sympathomimetic drugs amphetamine, methylhexane amine and tuaminoheptane. This can be of great importance when searching for drugs in putrefied materials. Goldbaum and Domanski (1965) also report P-phenylethylamine and pyridine in decomposed tissue. Turner (1965) mentions three compounds which must be considered when searching for drug metabolites. Two of them, P-phenylethylamine and tyramine, are relevant to the analysis of biological materials for basic drugs. The other can affect the analysis for barbituric acid derivatives and is not relevant to this particular paper. Fulton (1966) lists six putrefactive bases that have been detected during analysis of tissue in his laboratory. They are P-phenylethylamine, isopentylamine, tyramine, tryptamine, trimethylamine, and a substance which may be either tetrahydronorharman or norharman. This latter substance was subsequently identified as norharman (Fulton 1967). Corley (1967) reports the use of infra-red spectroscopy to distinguish between amphetamine and p-phenylethylamine in a putrefied liver. Thin-layer chromatography and gas-liquid chromatography both revealed that the liver constituent was not amphetamine. Data from both sources indicated that the compound was probably P-phenylethylamine. The importance of being able to differentiate between these substances can be seen in the report of a case by Goldbaum and others (1963) where P-phenylethylamine had been mistaken for amphetamine. Storage of biological samples at low temperatures does not altogether inhibit the formation of putrefactive bases. Kaempe (1965) demonstrated the
occurrence of tyramine and P-phenylethylamine in liver which was stored a t temperatures of 4-5°C. At - 18°C their formation was appreciably reduced although tyramine could be demonstrated in 25% of the samples analysed after 18 months. The presence of 1-hydroxymethyl-P-carboline (hydroxyharman) in fresh human liver has been demonstrated (Kaempe, 1967). Techniques for the isolation, identification and separation of this substance from other alkaloids are also described. I t was found that heating tryptophane in tartaric acid (IN) produced a compound with similar ultraviolet and infra red absorption spectra. As a result it was found that hydroxyharman was a n artefact produced by the extraction for basic drugs using the method of Stas-Otto on any autopsy material containing tryptophane. A similar substance can be produced by the action of diluted strong acids on liver tissue (Kaempe, 1967). Similarly, harman is a possible source of interference. This can be formed by the combination of tryptophane and acetaldehyde. I n a case reported the harman was isolated in a bottle of home-made mead. The tryptophane was contained in the yeast (Clarke, 1965). This survey has shown that a large number of compounds arising from putrefaction can be encountered in the course of analytical toxicology. I t would appear that P-phenylethylamine is the most commonly encountered base. The following study presents data which has been collected for a number of possible putrefactive bases and other related substances to enable them to be detected, identified and separated from co-extractable drugs using modern analytical techniques. In addition the application of these techniques to some cases of medicolegal interest is described.
Experimental Thin-layer chromatography The TLC plates coated with 0.25mm layers of Kieselgel G were prepared and activated by heating at 1 10°C for 30 minutes. The solvent system used is a 3:l mixture of' chloroform and methanol. This is placed in a chromato-tank and allowed to equilibrate for a t least 1 hour before use. Samples and standards are transferred to the plates by repeated spotting from glass capillaries to form discrete spots. Preparation of standards The standard solutions contained approximately 5mg of base per lml of chloroform. They are prepared by dissolving the free base directly in chloroform or by extraction from a salt. Further dilutions were made as required in order to produce discrete spots. Preparation of spray reagents The spray reagents used are prepared as follows: 1. Iodoplatinate reagent. Chloroplatinic acid (1g) is dissolved in water (1 Oml) . A solution of potassium iodide (log) in water (250ml) is added. The final volume is made up to 500ml with water. 2 . AcidiJied iodoplatinate reagent. This is prepared as above except that the final volume is made up to 500ml with a solution of hydrochloric acid (144ml S.G. 1.18) and water (96ml). 3. Iodine vapour reagent. Iodine contained in a polyethylene bottle is heated on a water bath prior to use. 4. Mandelin's reagent. This is prepared by dissolving ammonium vanadate (lg) in sulphuric acid (100g S.G. 1.98). The reagent is stirred with a glass rod prior to use. 5 . Gold chloride reagent. Using chloroauric acid (lg) to replace chloroplatinic acid (lg), the procedure is that used for the preparation of the acidified iodoplatinate reagent. 6. Tetranitromethane Tetranitromethane (100%) is used.
Cholorojorm Methanol Base (3 :1) R j Ethanolamine 0.89 Harman 0.61 Histamine 0.05 Hypoxanthine 0.00 Isoamylamine 0.07 Isobutylaminc 0.83 str. Norharman 0.72 Pentamethylenediamine 0. l l P-phenylethylamine 0.21 n-propanolamine 0.07 Pyrrolidine 0.9 1 Tetramethylinediamine 0.06 Trimethylamine Diffuses Tryptamine 0.16 Tyramine 0.15 Xanthine 0.00
TABLE 1 ANALYTICAL DATA FOR PUTREFACTIVE BASES THIN-LAYER CHROMATOGRAPHY DATA Iodine .followed b y Acidified acidajed Iodoplatinate iodoplatinate indoplatinate Mandelin's reagent rpagent Iodine reagent reagent Brown Grey Purple-brown Brown Brown Purple Purple Pale blue Brown Brown Brown Purple Purple Brown Blue Purple Brown Purple Purple-brown Brown Brown Purple Purple Blue-grey Blue Brown Brown Purple Brown Brown Purple Brown Grey Purple Purple-blue Brown Purple-brown Blue-grey Blue Brown Brown Blue-grey Purple Brown Blue Pale blue Purple Brown Brown Orange Brown Brown Purple
-
-
-
Gold chloride reagent
-
Tetranitro Methane reagent
-
Brown -
Brown Brown Brown Grey-brown Brown Brown -
-
Pale yellow Pale yellow Pale yellow
-
Pale yellow -
Application of reagents All reagents were sprayed directly on to the TLC plate using a Shandon Laboratory Spray Gun. Iodine vapour was "puffed" from the warm polyethylene bottle. Gas-liquid chromatography The instrument used was a Pye Model (54) Isothermal Gas Chromatograph equipped with a Hydrogen Flame Ionisation Detector. The column used was 5' x 4'' 0.d. glass packed with 5% SE30 on acid washed 60-80 mesh Chromosorb W treated with dimethyldichlorosilane. The column temperatures were 200°C and 125°C. Retention times were measured relative to codeine or ephedrine respectively with a nitrogen flow rate of 60ml per minute. Ultraviolet spectrography The ultraviolet absorption spectra were recorded using a Unicam SP800 instrument and fused silica cells with a lcm path length. Solutions of the putrefactive bases in ethanol, 0.1N hydrochloric acid, 0.1N sulphuric acid and 10% sulphuric acid were used. Analysis procedure The techniques described were applied to extracts of samples received from people found a t a considerable period of time after death occurred. Blood samples containing small amounts of citrates were allowed to stand a t room temperature for various lengths of time after which they were analysed using the described techniques. Extraction procedure 1. Urine Extraction. 25ml of urine is made alkaline with ammonium hydroxide solution (1.5N) and then shaken with 40ml of chloroform. The organic layer is filtered through Whatman No. 90 filter paper to remove excess moisture and suspended urine droplets. The resulting solution is shaken with 5ml of sulphuric acid (0.1N) and the separated acid layer centrifuged to remove any suspended chloroform. The ultraviolet spectrum of the acid solution is then recorded against a n acid blank. Following this, the acid solution is made alkaline with ammonium hydroxide solution (1.5N) and extracted with chloroform as before. The resulting chloroform solution is evaporated carefully to dryness and the residue is dissolved in a few drops of chloroform for transfer to thin-layer chromatography plates. TABLE 2 ANALYTICAL DATA FOR PUTREFACTIVE BASES GAS-LIQUID CHROMATOGRAPHY AND ULTRAVIOLET SPECTROSCOPIC DATA Gas-liquid chromatography Ultraviolet spectroscopy 5% SE30 column Maximum absorption in Retention times relative to 0.I N hydrochloric acid Base Codeine 200°C Ephedrine l25OC nanometers Ethanolamine 0.00 243,298, 355 Harman 0.33 Histamine 0.19 Hypoxanthine 244 Isoamylamine 0.00 Isobutylamine 0.00 244, 298, 355 Norharman 0.32, 0-38 Pentamethylenediamine 0.17 p-phenylethylamine 0.33 257, 246, 251, 261-5, inf. 266 n-propanolamine Pyrrolidine 0.02 00.0, 0.22, 2.33 Tetramethylenediamine 0.12 Trimethylamine 0.00 Tryptamine 0.2 1 278, 22865 Tyramine 0.08 274 inf. 280 261, 225 Xanthine 50
2. Blood and tissue extraction. 5ml of blood or 25g of macerated tissue are pretreated by precipitating proteins using the tungstate method. The resulting filtrate is processed using the same technique as that described for urine. The reagent volumes may require scaling up and while the extraction for the ultraviolet spectrography step in the blood analysis is made with the usual 5ml of acid, lOml is suitable for the tissue analysis. 3. Proteinprecipitation. 5ml of blood or 5g ( x 5) of macerated tissue is mixed with 30ml of distilled water and lml of sodium hydroxide (2.5N) solution. The mixture is shaken and then allowed to stand for 10 minutes. lOml of sodium tungstate (10% w/v) solution is added followed by the dropwise addition of 3.5ml sulphuric acid (3.6N) with shaking. The solution is heated on a water bath for 10 minutes until precipitation is complete. The resulting solution is filtered through Whatman No. 1 filter paper. The residue is washed with further portions of sulphuric acid (3.6N) which is, in turn, filtered and combined with the original filtrate.
Results and discussion Tables 1, 2 and 3 list the analytical data obtained for the putrefactive bases. Using thin-layer chromatography good separations are obtained using the chloroform, methanol solvent system. The spray reagents described readily detect all the materials except the two xanthine derivatives which can be ignored for interference purposes using the TLC and colour reagent systems described. No single colour reagent system can be used for the identification of all the putrefactive bases listed. Iodine is of little value as a screening reagent with biological extracts. I t can be used as a locating reagent to obtain Rf values of pure compounds. Mandelin's reagent and tetranitromethane show limited use in the field, but care should be used in interpreting results when there is a possibility of drugs being present. More information is obtainediby using one of the iodoplatinate screening reagents and interpreting positive reactions with the Rf values. The results obtained can be further confirmed by use of a selection of putrefactive bases run with the unknown materials for reference purposes. The other colour reactions, can, if required, be used for confirmation of results. The retention times given for gas liquid chromatography were obtained under two of the most useful conditions for the detection and identification of basic drugs extracted from biological media. I t can be seen that a minority of the putrefactive bases are successfully chromatographed under these conditions With the exception of /3-phenylethylamine they can be readily identified using thin-layer chromatography and ultraviolet spectroscopy. The similarity of 9-phenylethylamine and amphetamine has already been mentioned. These compounds can be readily differentiated using a carbowax-KOH column such as those described by Beckett and others (1967). To use the SE30 column for differentiation necessitates the formation of acetone derivates. If a few drops of acetone are added to an ethereal solution of the compounds, and the solution is subsequently chromatographed, the derivative peaks obtained can be used to differentiate between the materials. Also, identification can be obtained using a column packed with 5% Carbowax 6000, 5% K O H on Chromosorb G (AW. DMCS) which differentiates the materials. The absorption data for the putrefactive bases was obtained in the solvent commonly used when analysing materials for basic and neutral drugs. Of the techniques described only ultraviolet spectrometry shows interference from the xanthine compounds. The data given can be used as a guide to the identification of any of the putrefactive bases present. When these absorptions do occur further separation is required to ascertain whether a drug is being masked by the interfering material. The analysis of some biological specimens received from postmortems often produce an absorption spectrum characteristic of tyramine. Since subsequent
TABLE 3 ANALYTICAL DATA FOR PUTREFACTIVE BASES ULTRAVIOLET SPECTROSCOPIC DATA Absorption m a x i m Absorption minima Compounds nanometers nanometers 1. In 0. I N sulphuric acid Ethanolamine Harman Histamine Hypoxanthine Isoamylamine Isobutylamine Norharman Pentamethylenediamine P-phenylethylamine n-propanolamine Pyrrolidine Tetramethylenediamine Trimethylamine Tryptamine Tyramine Xanthine
245, 299, 355 249 -
247, 300, 357 -
257, 246, 251, 262.5, 266 (inflexion) 278, 287, 272 (inflexion) 276, 280 (inflexion) 265,225
220, 272, 319 222
221, 273, 322 -
226, 247, 253.5, 261
-
240, 285.5 243 239
2. In dilute sulphuric acid (10% u / v ) Ethanolamine Harman Histamine Hypoxanthine Isoamylamine Isobutylamine Norharman 246, 299, 358 Pentamethylenediamine 8-phenylethylamine 257, 251, 262.5 inf. 246,266 n-propanolamine Pyrrolidine Tetramethylenediamine Trimethylamine Tryptamine 279,287 inf. 272 Tyramine 274 inf. 280 Xanthine 260, 229 3. In ethanol Ethanolamine Harman Histamine Hypoxanthine Isoamylamine Isobutylamine Norharman Pentamethylenediamine P-phenylethylamine n-propanolamine Pyrrolidine Tetramethylenediamine Trimethylamine Tryptamine Tyramine Xanthine
240, 277, 288, 337, 349 inf. 249
-
258, 247, 252.5, 260.5, 264, 268 inf. 242.5
280, 273,289 277-5 inf. 285 268
241,276,287 242 239 (not very soluble)
analysis by thin-layer chromatography using iodoplatinate reagents will not reveal the presence of this substance, the absorption cannot be assigned to any positive reaction obtained without further separation procedures. Several blood samples were allowed to stand for various periods of time. The results of their analysis by thin-layer chromatography and ultraviolet spectrography are given in Table 4. The results show that P-phenylethylamine can be
Sample
TABLE 4 ANALYSIS O F PUTREFIED BLOOD SAMPLES Ultraviolet spectrum in 0.I N sulphuric Reaction with Chloroform :Methanol Acid Number of iodoplatinate (3:I) absodtion m i m u m months old reagent Rf nanometers
+ + +Strong Reaction + +Weak Reaction *Low Intensity
detected in all the samples to varying degrees. The positive identification was achieved by gas-liquid chromatography. No other reactions were observed for the blood samples. Some Cases of Interest Case 1 Female. Had been dead for 3 weeks. The blood sample was analysed for barbiturates, methaqualone, salicylate and alcohol. I t contained 86mg of alcohol per lOOml of blood. The analysis for basic drugs revealed tyramine (by UV spectroscopy), tryptamine, P-phenylethylamine and possibly a trace of norharman. The relevant TLC data is listed in Table 5. TABLE 5 ANALYSIS OF PUTREFIED BLOOD FOR BASIC DRUGS Reaction with acidtJid Chloroform : iodoplatinate Methanol 3:l R f Other information Spot No. reagent 0.10 No ultraviolet spectrum 1 Purple obtainable. Indistinguishable from tryptamine added to the extract. 2 Purple 0.2 1 Identified by UV and GLC as 8-phenylethylamine. 3 Brown Purple 0.73 This spot was indistinguishable from norharman. However, no ultraviolet spectrum was obtained to enable confirmation
Case 2
Male. Blood was 1-month-old. The blood sample contained 105mg of alcohol per 1OOml. Putrefactive bases identified in the sample were tyramine and p-phenylethylamine. A purple-black spot corresponding to tryptamine, and a purple spot identical to norharman were also obtained. No confirmation of the latter was possible by ultraviolet spectroscopy.
Case 3 Male. He had been dead for 1-2 weeks. The urine sample was found to contain 9-phenylethylamine. The blood sample contained p-phenylethylamine. Also, it gave a positive reaction with acidified iodoplatinate reagent, a purpleblack spot, at Rf 0.72. This compound had a very weak absorption maximum at 246nm. This is similar to norharman. Conclusion A study has been made of a number of materials which may be present in samples as a result of putrefactive changes in the body. The data obtained for these putrefactive bases enables them to be recognized when encountered using the techniques described. In practice it has been shown that P-phenylethylamine is the most comnlonly encountered putrefactive base in blood. Unfortunately, it was not found to be possible to predict the degree to which it occurs. This and the presence of other putrefactive bases appears to be erratic. Acknowledgement J. S. Oliver would like to acknowledge the Instaprint Camera Company, London, for financial support during this work. References G. T., and MOFFAT,A. C., 1967, J. Pharm. Pharmacol. BECKETT, A. H., TUCKER, 19, 273. CLARK,E. G. C., 1965, Chromatography and the classical methods of analysis, in; Symp. Ident. of Drugs and Poisons, p.54. The Pharmaceutical Society of Great Britain. CORLEY,C. A., 1967, Bull. T.I.A.F. T. 4.2. FULTON, C. C., 1965, Bull. T.I.A.F.T. 2.4. FULTON, C. C., 1966, Bull. T.I.A.F. T. 3.3. FULTON, C. C., 1967, Bull. T.I.A.F. T. 4.1. GOLDBAUM, L. R., and DOMANSKI, T. J., 1965, An approach to the analysis of biological specimens for basic drugs, in; Progress in Chemical Toxicology, Vol. 2, Stolman, A., (ed.). New York, London: Academic Press. E. H., and BLUMBERG, J. M., 1963, J. Forens. Sci. GOLDBAUM, L. R., JOHNSTON, 8, 286. KAEMPE, B., 1965, Acta Pharmac. et Toxicol., 23, 15. KAEMPE, B., 1967, Acta Pharmac. et Toxicol., 25, 155. TURNER, L. K., 1965, Searching for drug metabolites in viscera, in; Methods of Forensic Science, Vol. 4. Curry, A. S. (ed.). London, New York, Sidney: Interscience Publishers, John Wiley & Sons Ltd.