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Identification of 10-hydroxy-12-octadecenoic acid in adipocere
Forensic
Science
Elsevier
Scientific
Publishers
IDENTIFICATION IN ADIPOCERE
117
Ireland Ltd.
OF lo-IWDROXY-12-OCTADECENOIC
TAKEHIKO TAKATORI,” HIDEMI MATSUMNAb Departments Laboratory
23 (1983)117-122
International,
KOICHI TERAZAWA,”
of Legal Medicine, Medicine, Hokkaido
Hokkaido University
KYOKO
University School Hospitalb Sapporo
NAKANO”
ACID
and
of Medicine” 060 (Japan)
and
Department
of
(Received January 14,1983) (Accepted May 17,1983)
Summary Studies are reported on separation and identification of lo-hydroxy-12-octadecenoic human adipocere. The chemical structure was determined by thin-layer chromatography gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry @C-MS). Key words: Adipocere;
Argentation-TLC;
GC-MS;
lo-Hydroxy-12-octadecenoic
acid in (TLC),
acid
Introduction It is frequently recognized that there are different compositions of fatty acids in adipocere from distinct regions of even the same dead body. For example, an unknown peak is sometimes found after development of methyl lo-hydroxy stearate in gas chromatogram patterns. This appears to be due to differences in adipocere formation in distinct regions of the body rather than to that in compositions of fatty acids which form triglycerides in adipose tissue. In this paper we identify the unknown fatty acid developed posterior to methyl lo-hydroxy stearate in human adipocere by gas-liquid chromatography (GLC). Materials
and methods
Adipoceres Adipocere samples were obtained from three bodies found in the sea. The post-mortem period was estimated to be about Cl0 months. Adipoceres were collected from several regions of the dead body. Lipid extraction and methylation of fatty acid Total lipids in adipocere were extracted with chloroform/methanol 0379-0738/83/$03.00 @ 1983 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland Ltd.
(2: 1,
118
v/v), and the aliquot of the total lipids was methylated methanol, as described previously 11-31.
Thin-layer
with
5% HCl in
chromatography
The total methyl esters of normal and hydroxy fatty acids (OH-FA) in adipocere were separated by thin-layer chromatography (TLC, 20.0 x 20.0 cm, 250-pm thick plate coated with silica gel 60 H). The TLC was developed using a benzene/ethyl ether (9: 1, v/v) mixture as solvent, and each spot visualized by spraying a 1% iodine methanol solution. The fraction of OH-FAs was then eluted with ethyl ether. Subsequently, the OH-FA fraction was again developed by argentation-TLC 141 with the same solvent, and the spots were detected by charring with 50% sulfuric acid. The argentation-TLC was prepared as follows; 3g of silica gel 60 H was slurried with 10ml of distilled water containing 5% silver nitrite, and then coated on a glass-plate (20.0 x 10.0 cm) in the usual way.
Gas-liquid
chromatography
and gas chromatography-mass
spectrometry
Gas-liquid chromatography (GLC) was carried out with a Shimadzu GC6A instrument equipped with a flame ionization detector. The conditions of GLC were followed as described previously [l-3]. Gas chromatography-mass spectrometry (GC-MS) was performed on a high-resolution Hitachi-BMU-6MG instrument. A 0.3 x 200 cm glass column, used for the GC, was packed with 3% EGSS-X on Chromosorb W (SO-60 mesh) treated with the acid washing and dimethyldichlorosilane. The column temperature was set at 190°C; helium was used as the carrier gas at a flow rate of 6Oml/min. The ionizing voltage and ion source temperature were adjusted to 20 eV and 180°C.
Preparation
of IO-hydroxyoctadecanoic
acid
lo-D,L-hydroxyoctadecanoic acid was prepared by reduction of 10-0x0octadecanoic acid in the presence of sodium borohydride in methanol, as described elsewhere [l].
Chemicals Silica gel 60 H was purchased from Merck, Darmstadt, reagents were obtained from Wako Pure Chemical Organic solvents were redistilled before use.
Germany. All other Industries, Japan.
Results Figure 1 shows the gas chromatogram of total methyl fatty acids in adipocere. Peaks A and B had been identified by GC-MS as methyl 10-0x0octadecanoate and lo-hydroxyoctadecanoate, respectively, as described previously [1,2], and peak C (unknown peak) which developed after methyl lo-hydroxyoctadecanoate had the relative retention time of 23.6 min.
t
Fig. 1. Gas chromatogram
of total methyl fatty acids in adipocere.
The fraction of methyl hydroxy fatty acid (OH-FA) in adipocere was first separated by TLC and eluted with ethyl ether. The argentation-thin-layer chromatogram of the fraction of OH-FA is shown in Fig. 2. Two kinds of OH-FAs were found in the OH-FA fraction of adipocere; one (spot-A) is hydroxy saturated fatty acids (RF = 0.44) and the other (spot-B) is the unknown spot (RF = 0.13). The latter spot is indicated to be hydroxy unsaturated fatty acids because of the fact that difference in unsaturated degrees of fatty acids in molecules is separable by argentation-TLC and the Rp value of unsaturated fatty acid is low compared to that of the corresponding saturated fatty acid 143. When the spot-B separated by argentation-TLC was subjected to GLC, its relative retention time coincided with that of peak C. These findings indicate that the fatty acid developed as peak C possesses double bond(s) as well as hydroxy group(s) in molecules. When the constituent of peak C in Fig. 1 was subjected to GC-MS, it gave the mass spectrum shown in Fig. 3. In the high mass range, a small ion peak was found at m/e 294. This peak is not a molecular ion peak but may be due to the loss of one water molecule (M-18) because methyl esters of hydroxy fatty acids generally do not show a molecular ion peak [1,5]. The prominent peak at m/e 201 is due to -CHOH-(CH&CO-O-CH3, and the base peak at m/e 169 also due to the loss of one water molecule from m/e 201, as described previously [l].
A
Fig. 2. Thin-layer chromatogram of the fraction authentic methyl lo-hydroxystearate; 2: sample.
of methyl hydroxy
fatty acids in adipocere.
1:
In the low mass range, a middle ion fragment was found at m/e 112, which is not due to a series of hydrocarbon peaks of the fatty acid, but may be due to a fragment formed through the cleavage of the lO,ll-bond (the carbonylcarbon is numbered one) with rearrangement of one hydrogen atom, [CH&CHACH=CH-CH.J +H. From these findings of the mass fragments and the property of TLC, the peak C was identified as methyl lo-hydroxy-12-octadecenoate, CH3-(CH2)4-CH=CH-CH~HOH_08COOCH~. Discussion In this paper evidence was presented that lo-hydroxy-12-octadecenoic acid is found in human adipocere. This methyl monohydroxy-monoenoate was developed posterior to methyl lo-hydroxy stearate by GLC and its RF value by argentation-TLC was 0.13. By the mass spectrum, the specific ion fragments were found at m/e 294,201,
121
201
(H,
O~CO
Wtl,t8
(‘II
/
OH
M-18 294 I
I
I,, ,
,
.
,
,
200
Fig. 3. Mass spectrum of methyl
lo-hydroxy-12-octadecenoate
,
‘,I,.
,
,
,
300
in adipocere.
169 and 112 as shown in Fig. 3. The last ion peak appeared to be unstable because it was reduced or disappeared if the ionizing voltage was increased. Since the monohydroxy-monoenoic acid is liquidized at an ordinary temperature similar to ricinoleic acid (Cd)-lZhydroxy-cis-9-octadecenoic acid), it may flow away into the sea water from the surface of adipocere. The property of the compound will never contribute to formation and stabilization of adipocere. This may also reflect that there are different compositions of fatty acid in adipocere from distinct regions of even the same body. Linoleic acid (cis-9-cis-12-octadecadienoic acid) contained 7-11% of total fatty acids in normal adipose tissue 161 appears to become the substrate of lo-hydroxy-12-octadecenoic acid. It is worth noting that all of the specific fatty acids already found in human adipocere are stereospecific, and hydroxy and keto groups are located only in the carbon-10 position of fatty acid. Acknowledgement This work was supported in part by Scientific Research Grants from the Ministry of Education, Japan. The Hitachi RMU-6MG instrument in the
122
General used.
Research
Institute,
Hokkaido
University
School
of Medicine,
was
References 1 T. Takatori and A. Yamaoka, The mechanism of adipocere formation, I. Identification and chemical properties of hydroxy fatty acid in adipocere. Forensic Sci., 9 (1977) 63-73. 2 T. Takatori and A. Yamaoka, The mechanism of adipocere formation, II. Separation and identification of 0x0 fatty acids in adipocere. Forensic Sci., 10 (1977) 117-125. 3 T. Takatori and A. Yamaoka, Separation and identification of 9-chloro-lo-methoxy (9-methoxylo-chloro) hexadecanoic and octadecanoic acids in adipocere. Forensic Sci. Znt., 14 (1979) 63-73. 4 H.K. Mangold, M. aliphatic lipids, In E. Stahl (ed.), Thin-Layer Chromatography, 2nd edn., Springer-Verlag, Berlin, Heidelberg, New York, 1969, p. 363. 5 R. Ryhage and E. Stenhagen, Mass spectrum in lipid research, J. Lipid Res., l(1960) 361390. 6 J. Hirsch, Fatty acid patterns in human adipose tissue, In A.E. Renold and G.F. Cahill, Jr. (eds.), Handbook of Physiology, Section 5, Adipose Tissue, American Physiology Society, Washington, D.C., 1965, p. 181.
Science
Elsevier
Scientific
Publishers
IDENTIFICATION IN ADIPOCERE
117
Ireland Ltd.
OF lo-IWDROXY-12-OCTADECENOIC
TAKEHIKO TAKATORI,” HIDEMI MATSUMNAb Departments Laboratory
23 (1983)117-122
International,
KOICHI TERAZAWA,”
of Legal Medicine, Medicine, Hokkaido
Hokkaido University
KYOKO
University School Hospitalb Sapporo
NAKANO”
ACID
and
of Medicine” 060 (Japan)
and
Department
of
(Received January 14,1983) (Accepted May 17,1983)
Summary Studies are reported on separation and identification of lo-hydroxy-12-octadecenoic human adipocere. The chemical structure was determined by thin-layer chromatography gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry @C-MS). Key words: Adipocere;
Argentation-TLC;
GC-MS;
lo-Hydroxy-12-octadecenoic
acid in (TLC),
acid
Introduction It is frequently recognized that there are different compositions of fatty acids in adipocere from distinct regions of even the same dead body. For example, an unknown peak is sometimes found after development of methyl lo-hydroxy stearate in gas chromatogram patterns. This appears to be due to differences in adipocere formation in distinct regions of the body rather than to that in compositions of fatty acids which form triglycerides in adipose tissue. In this paper we identify the unknown fatty acid developed posterior to methyl lo-hydroxy stearate in human adipocere by gas-liquid chromatography (GLC). Materials
and methods
Adipoceres Adipocere samples were obtained from three bodies found in the sea. The post-mortem period was estimated to be about Cl0 months. Adipoceres were collected from several regions of the dead body. Lipid extraction and methylation of fatty acid Total lipids in adipocere were extracted with chloroform/methanol 0379-0738/83/$03.00 @ 1983 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland Ltd.
(2: 1,
118
v/v), and the aliquot of the total lipids was methylated methanol, as described previously 11-31.
Thin-layer
with
5% HCl in
chromatography
The total methyl esters of normal and hydroxy fatty acids (OH-FA) in adipocere were separated by thin-layer chromatography (TLC, 20.0 x 20.0 cm, 250-pm thick plate coated with silica gel 60 H). The TLC was developed using a benzene/ethyl ether (9: 1, v/v) mixture as solvent, and each spot visualized by spraying a 1% iodine methanol solution. The fraction of OH-FAs was then eluted with ethyl ether. Subsequently, the OH-FA fraction was again developed by argentation-TLC 141 with the same solvent, and the spots were detected by charring with 50% sulfuric acid. The argentation-TLC was prepared as follows; 3g of silica gel 60 H was slurried with 10ml of distilled water containing 5% silver nitrite, and then coated on a glass-plate (20.0 x 10.0 cm) in the usual way.
Gas-liquid
chromatography
and gas chromatography-mass
spectrometry
Gas-liquid chromatography (GLC) was carried out with a Shimadzu GC6A instrument equipped with a flame ionization detector. The conditions of GLC were followed as described previously [l-3]. Gas chromatography-mass spectrometry (GC-MS) was performed on a high-resolution Hitachi-BMU-6MG instrument. A 0.3 x 200 cm glass column, used for the GC, was packed with 3% EGSS-X on Chromosorb W (SO-60 mesh) treated with the acid washing and dimethyldichlorosilane. The column temperature was set at 190°C; helium was used as the carrier gas at a flow rate of 6Oml/min. The ionizing voltage and ion source temperature were adjusted to 20 eV and 180°C.
Preparation
of IO-hydroxyoctadecanoic
acid
lo-D,L-hydroxyoctadecanoic acid was prepared by reduction of 10-0x0octadecanoic acid in the presence of sodium borohydride in methanol, as described elsewhere [l].
Chemicals Silica gel 60 H was purchased from Merck, Darmstadt, reagents were obtained from Wako Pure Chemical Organic solvents were redistilled before use.
Germany. All other Industries, Japan.
Results Figure 1 shows the gas chromatogram of total methyl fatty acids in adipocere. Peaks A and B had been identified by GC-MS as methyl 10-0x0octadecanoate and lo-hydroxyoctadecanoate, respectively, as described previously [1,2], and peak C (unknown peak) which developed after methyl lo-hydroxyoctadecanoate had the relative retention time of 23.6 min.
t
Fig. 1. Gas chromatogram
of total methyl fatty acids in adipocere.
The fraction of methyl hydroxy fatty acid (OH-FA) in adipocere was first separated by TLC and eluted with ethyl ether. The argentation-thin-layer chromatogram of the fraction of OH-FA is shown in Fig. 2. Two kinds of OH-FAs were found in the OH-FA fraction of adipocere; one (spot-A) is hydroxy saturated fatty acids (RF = 0.44) and the other (spot-B) is the unknown spot (RF = 0.13). The latter spot is indicated to be hydroxy unsaturated fatty acids because of the fact that difference in unsaturated degrees of fatty acids in molecules is separable by argentation-TLC and the Rp value of unsaturated fatty acid is low compared to that of the corresponding saturated fatty acid 143. When the spot-B separated by argentation-TLC was subjected to GLC, its relative retention time coincided with that of peak C. These findings indicate that the fatty acid developed as peak C possesses double bond(s) as well as hydroxy group(s) in molecules. When the constituent of peak C in Fig. 1 was subjected to GC-MS, it gave the mass spectrum shown in Fig. 3. In the high mass range, a small ion peak was found at m/e 294. This peak is not a molecular ion peak but may be due to the loss of one water molecule (M-18) because methyl esters of hydroxy fatty acids generally do not show a molecular ion peak [1,5]. The prominent peak at m/e 201 is due to -CHOH-(CH&CO-O-CH3, and the base peak at m/e 169 also due to the loss of one water molecule from m/e 201, as described previously [l].
A
Fig. 2. Thin-layer chromatogram of the fraction authentic methyl lo-hydroxystearate; 2: sample.
of methyl hydroxy
fatty acids in adipocere.
1:
In the low mass range, a middle ion fragment was found at m/e 112, which is not due to a series of hydrocarbon peaks of the fatty acid, but may be due to a fragment formed through the cleavage of the lO,ll-bond (the carbonylcarbon is numbered one) with rearrangement of one hydrogen atom, [CH&CHACH=CH-CH.J +H. From these findings of the mass fragments and the property of TLC, the peak C was identified as methyl lo-hydroxy-12-octadecenoate, CH3-(CH2)4-CH=CH-CH~HOH_08COOCH~. Discussion In this paper evidence was presented that lo-hydroxy-12-octadecenoic acid is found in human adipocere. This methyl monohydroxy-monoenoate was developed posterior to methyl lo-hydroxy stearate by GLC and its RF value by argentation-TLC was 0.13. By the mass spectrum, the specific ion fragments were found at m/e 294,201,
121
201
(H,
O~CO
Wtl,t8
(‘II
/
OH
M-18 294 I
I
I,, ,
,
.
,
,
200
Fig. 3. Mass spectrum of methyl
lo-hydroxy-12-octadecenoate
,
‘,I,.
,
,
,
300
in adipocere.
169 and 112 as shown in Fig. 3. The last ion peak appeared to be unstable because it was reduced or disappeared if the ionizing voltage was increased. Since the monohydroxy-monoenoic acid is liquidized at an ordinary temperature similar to ricinoleic acid (Cd)-lZhydroxy-cis-9-octadecenoic acid), it may flow away into the sea water from the surface of adipocere. The property of the compound will never contribute to formation and stabilization of adipocere. This may also reflect that there are different compositions of fatty acid in adipocere from distinct regions of even the same body. Linoleic acid (cis-9-cis-12-octadecadienoic acid) contained 7-11% of total fatty acids in normal adipose tissue 161 appears to become the substrate of lo-hydroxy-12-octadecenoic acid. It is worth noting that all of the specific fatty acids already found in human adipocere are stereospecific, and hydroxy and keto groups are located only in the carbon-10 position of fatty acid. Acknowledgement This work was supported in part by Scientific Research Grants from the Ministry of Education, Japan. The Hitachi RMU-6MG instrument in the
122
General used.
Research
Institute,
Hokkaido
University
School
of Medicine,
was
References 1 T. Takatori and A. Yamaoka, The mechanism of adipocere formation, I. Identification and chemical properties of hydroxy fatty acid in adipocere. Forensic Sci., 9 (1977) 63-73. 2 T. Takatori and A. Yamaoka, The mechanism of adipocere formation, II. Separation and identification of 0x0 fatty acids in adipocere. Forensic Sci., 10 (1977) 117-125. 3 T. Takatori and A. Yamaoka, Separation and identification of 9-chloro-lo-methoxy (9-methoxylo-chloro) hexadecanoic and octadecanoic acids in adipocere. Forensic Sci. Znt., 14 (1979) 63-73. 4 H.K. Mangold, M. aliphatic lipids, In E. Stahl (ed.), Thin-Layer Chromatography, 2nd edn., Springer-Verlag, Berlin, Heidelberg, New York, 1969, p. 363. 5 R. Ryhage and E. Stenhagen, Mass spectrum in lipid research, J. Lipid Res., l(1960) 361390. 6 J. Hirsch, Fatty acid patterns in human adipose tissue, In A.E. Renold and G.F. Cahill, Jr. (eds.), Handbook of Physiology, Section 5, Adipose Tissue, American Physiology Society, Washington, D.C., 1965, p. 181.