Thidarat Du PDF

QUANTITATIVE ANALYSIS OF CIVETONE AND NORMUSCONE IN SECRETION FROM
VIVERRICULA INDICA AND IN AROMATIC REMEDIES BY

GAS CHROMATOGRAPHY/ MASS SPECTROMETRY
Miss Thidarat Duangyod
A Thesis Submitted in Partial Fulfillment of the Requirements

for the Degree of Master of Science Program in Public Health Sciences
College of Public Health Sciences
Chulalongkorn University
Academic Year 2011
Copyright of Chulalongkorn University
บทคัดย่อและแฟ้มข้ อมูลฉบับเต็มของวิทยานิพนธ์ตงแต่ ั ้ ปีการศึกษา 2554 ที่ให้ บริ การในคลังปั ญญาจุฬาฯ (CUIR)
เป็ นแฟ้มข้ อมูลของนิสติ เจ้ าของวิทยานิพนธ์ที่สง่ ผ่านทางบัณฑิตวิทยาลัย
The abstract and full text of theses from the academic year 2011 in Chulalongkorn University Intellectual Repository(CUIR)
are the thesis authors' files submitted through the Graduate School.
การวิเคราะหหาปริมาณสารซิเวทโทนและสารนอรมัสโคนในสมุนไพรชะมดเช็ดและตํารับยาหอม
โดยแกสโครมาโทกราฟ/แมสสเปกโทรเมทรี
นางสาวธิดารัตน ดวงยอด
วิทยานิพนธนี้เปนสวนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรมหาบัณฑิต
สาขาวิชาวิทยาศาสตรสาธารณสุข
วิทยาลัยวิทยาศาสตรสาธรารณสุข จุฬาลงกรณมหาวิทยาลัย
ปการศึกษา 2554
ลิขสิทธิ์ของจุฬาลงกรณมหาวิทยาลัย
vi
ACKNOWLEDGEMENTS
The author wishes to express her heartily gratitude and appreciation to her
thesis advisor, Dr. Chanida Palanuvej, for her continuous guidance, suggestion, and
support throughout the course of this study.
The author is sincerely grateful to her thesis co-advisor, Associate Professor

Dr. Nijsiri Ruangrungsi, for his kindness and valuable suggestion to complete the
present study.
Gratitude is grateful to the thesis committee members, Professor Dr. Surasak

Taneepanichskul, Dr. Tiwatt Kuljanabhagavad, and Dr. Naowarat Kanchanakhan for
their important and constructive suggestion in finalizing this thesis.
The author is thankful to the Herbal Remedies and Alternative Medicine Task
Force of STAR: Special Task Force for Activating Research under 100 Years
Chulalongkorn University Fund.
Appreciation is also extended to College of Public Health Sciences,

Chulalongkorn University and all the staff members for necessary assistance and
instrumental support.
The author’s final thank goes to her family, especially her parents for their
love, understanding, encourage and support her to concentrate on her study.
CONTENTS
Page
ABSTRACT (THAI) iv
ABSTRACT (ENGLISH) v
ACKNOWLEDGEMENTS vi
CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xii

LIST OF ABBREVIATIONS xviii
CHAPTER
I INTRODUCTION 1
Background and Significance of the Study 1
Objectives of the Study 2
Expected Benefits 2
II LITERATURE REVIEWS 3
Family Viverridae 3
Small Indian Civet (Viverricula indica Desmarest) 7
Small Indian Civet Secretion 10
Macrocyclic Ketones of Fragrance Ingredient 12
Civetone 12
Biological Data of Civetone 13
Usage of Civetone 13
Normuscone 14
Biological Data of Normuscone 14
Usage of Normuscone 15
Aromatic Remedy 15
Effect of Aromatic Remedy 16
viii
Page
CHAPTER
Gas Chromatography/Mass Spectrometry (GC/MS) 16
Full Scan Mode and Selected Ion Monitoring Mode 17
III MATERIALS AND METHODOLOGY 19
Samples 19
Materials 19
Chemicals and Reagents 19
Instrumentations 19
Sample Collection 20
Small Indian Civet Secretion Analysis 20
Small Indian Civet Furs Analysis 20
Small Indian Civet Feces Analysis 21
Aromatic Remedies Analysis 21
Qualitative Analysis of Small Indian Civet Secretion Constituents 21
Quantitative Analysis of Civetone and Normuscone 21
Calibration Curve and Linearity 22
Limit of Detection (LOD) and Limit of Quantitation (LOQ) 22
Precision 22
Recovery of Small Indian Civet Secretion 22
Recovery of Aromatic Remedies 23
Selected Ion Monitoring (SIM) Analysis of Civetone and Normuscone 23
IV RESULTS 24
Small Indian Civet Farm 24
Small Indian Civet Secretion 25
Small Indian Civet Furs 26
Small Indian Civet Feces 26
Small Indian Civet Secretion Constituents 27
ix
Page
CHAPTER
Linearity 29
LOD and LOQ 30
Precision 30
Recovery 31
Civetone and Normuscone Contents in Small Indian Civet Secretion 32
Civetone and Normuscone Contents in Small Indian Civet Furs 33
Civetone and Normuscone Contents in Small Indian Civet Feces 34
Civetone and Normuscone Contents in Aromatic Remedies 35
V DISCUSSION 37
CONCLUTION AND RECOMMENDATION 42
REFERENCES 43
APPENDICES 51
Appendix A 52
Appendix B 67
Appendix C 117
Appendix D 123
VITA 136
x
LIST OF TABLES
Table Page
1 Percentage of relative standard deviation of intra-day and inter-day

analysis 31
2 The percentage recoveries of civetone and normuscone in civet

secretion 31
3 The percentage recoveries of civetone in aromatic remedy 32
4 The percentage recoveries of normuscone in aromatic remedy 32
5 The concentration of civetone and normuscone in small Indian civet

secretion (µg/mg of secretion) 32
6 The concentration of civetone (µg/mg) in aromatic remedies

obtained by GC/MS 36
7 The concentration of normuscone (µg/mg) in aromatic remedies

obtained by GC/MS 36
8 Percent areas of male small Indian civet secretion constituents 54
9 Percent areas of female small Indian civet secretion constituents 55
10 Percent areas of pooled small Indian civet secretion constituents 56
11 Civetone and normuscone contents (µg/mg of secretion) in male

small Indian civet secretion (n=3) 58
12 Civetone and normuscone contents (µg/mg of secretion) in female

small Indian civet secretion (n=3) 59
13 Civetone and normuscone contents (µg/mg of aromatic remedy) in

aromatic remedy A 60

aromatic remedy B 61

aromatic remedy C 62

aromatic remedy D 63
xi
Table Page

aromatic remedy E 64

aromatic remedy F 65

aromatic remedy G 66
xii
LIST OF FIGURES
Figure Page
1 Left footprints of front (F) and hind (H) feet of civets 4
2 Scent gland of Viverricula indica 5
3 Scent gland of Viverra tangalunga 5
4 Asian palm civet (Paradoxurus hermaphroditus) 6
5 Civet coffee (Kopi Luwak) 6
6 Viverricula indica Desmarest 8
7 Small Indian civet in a cage 9
8 Pa Noi’s civet farm 9
9 Small Indian civet secretion 10
10 Small Indian civet secretion which placed on betel vine 11
11 Thai traditional processing for civet secretion cleaning 11
12 Structure of civetone 12
13 Structure of normuscone 14
14 Aromatic remedies 15
15 A small Indian civet secretion in the bamboo cage 24
16 Small Indian civet cages 25
17 Male and female small Indian civet secretion 25
18 Small Indian civet furs 26
19 Male and female small Indian civet feces 26
20 GC chromatogram of male small Indian civet secretion 27
21 GC chromatogram of female small Indian civet secretion 27
22 GC chromatogram of pooled small Indian civet secretion 28
23 Mass spectrum of civetone 29
24 Mass spectrum of normuscone 29

xiii
Figure Page
25 Calibration curve of civetone 30
26 Calibration curve of normuscone 30
27 Full scan and SIM chromatogram of the last aliquot of hexane from
civet furs washing 33
28 GC chromatogram of small Indian civet furs 34
29 GC chromatogram of male small Indian civet feces 34
30 GC chromatogram of female small Indian civet feces 35
31 Calibration curve of civetone determination by GC/MS 57
32 Calibration curve of normuscone determination by GC/MS 57
33 GC chromatogram of male small Indian civet (1) 68
48 GC chromatogram of female small Indian civet (1) 75

xiv
Figure Page
63 GC chromatogram of pooled small Indian civet (1) 83
73 GC chromatogram of small Indian civet furs (1) 88

xv
Figure Page
civet furs washing (1) 88
79 Full scan and SIM chromatogram of male small Indian civet feces (1) 91
94 Full scan and SIM chromatogram of female small Indian civet feces (1) 98
xvi
Figure Page
103 Full scan and SIM chromatogram of female small Indian civet feces
(10) 103
(11) 103
(12) 104
(13) 104
(14) 105
(15) 105
109 Full scan and SIM chromatogram of aromatic remedy A (Lot No. 1) 106
112 Full scan and SIM chromatogram of aromatic remedy B (Lot No. 1) 107
115 Full scan and SIM chromatogram of aromatic remedy C (Lot No. 1) 109
xvii
Figure Page
118 Full scan and SIM chromatogram of aromatic remedy D (Lot No. 1) 110
121 Full scan and SIM chromatogram of aromatic remedy E (Lot No. 1) 112
124 Full scan and SIM chromatogram of aromatic remedy F (Lot No. 1) 113
127 Full scan and SIM chromatogram of aromatic remedy G (Lot No. 1) 115
130 Mass spectrum of cyclohexadecanone 116
131 Mass spectrum of dihydrocivetone 116

xviii
LIST OF ABBREVIATIONS
amu = Atomic Mass Unit
AOAC = Association of Official Analytical Chemists
°C = Degree Celsius
cm = Centimeter
EI = Electron Ionization
ECG = Electrocardiography
g = Gram
GC = Gas chromatography
GC/MS = Gas chromatography/mass spectrometry
HPLC = High performance liquid chromatography
hr = Hour
IUPAC = International Union of Pure and Applied Chemistry
kg = Kilogram
KHz = Kilohertz
LD50 = Lethal dose 50%
LOD = Limit of detection
LOQ = Limit of quantitation
m/z = Mass-to-charge ratio
mg = Milligram
MIM = Multiple ions monitoring
min = Minute
ml = Microlitre
mm = Millimetre
xix
mmol = Millimole
MS = Mass spectrometry
NIST = National Institute of Standard and Technology
nm = Nanometre
PTFE = Polytetrafluoroethylene
r2 = Correlation coefficients
rpm = Revolutions per minute
RSD = Relative standard deviation
SARS = Severe acute respiratory syndrome
SIM = Selected ion monitoring
US$ = United States dollar
UV = Ultraviolet
α = Alpha
β = Beta
1
CHAPTER I
INTRODUCTION
Background and Significance of the Study
Thailand is located in tropical rain forest, so it is suitable a habitat for living

things with high biodiversity. The wild animals are important species in ecology and
food chain. Forest destroying causes wild animals extinction or risk on extinction
including animal in the family Viverridae. A civet is small animal in a group of
carnivores that found inhabiting the tropical forest area. A civet family (Viverridae) is
related to the cat family (Felidae) that why most civets have catlike bodies [1]. Small
Indian civet (Viverricula indica Desmarest) which in subfamily Viverrinae is found
many countries in Southeast Asia, Pakistan, India, Nepal, Bangladesh, and South
China [2]. Although it is a controlled animal that is announced in the Wildlife
Protection Enactment (1992), the ministerial regulation issue 6th (1994) allows that
farmer can keep the small Indian civet for the business [3]. There are several farms in
central Thailand and the numbers of small Indian civets are around 800. The farmer
keeps the small Indian civet for producing and exporting civet secretion to the world
market. This secretion is used in medicinal and perfumeries purposes [4]. Both male
and female civets produce the strong smelling secretion from the perineal gland.
Civetone is the main constituent of the secretion produced from the civet of the genera
Civettictis, Viverra and Viverricula [5, 6]. It is an animalic musk which consists of 17
carbon atoms [7].
Small Indian civet secretion is widely used in perfume industry and in

traditional medicine for a long time. In Ethiopia traditional medicine, the secretion is
used for treatment of headaches, skin discoloration and skin infections, by topical
application. Similarly, some of the indigenous communities in India use the secretion
of the small Indian civet in tribal medicine, particularly to treat respiratory ailments
and skin infections, in addition to its constant use in Ayurvedic drugs which is a
traditional Indian medicine [8-10]. The small Indian civet secretion is also used as an
ingredient in Thai traditional medicine especially in aromatic remedy.
2
Thai traditional medicine uses the small Indian civet secretion for relief of
faint, dizziness, nausea and vomiting; therefore it is an important ingredient in
aromatic remedy. In the local markets, there are many aromatic remedies that claim to
use the secretion as an ingredient in the remedies but it cannot prove that there is the
secretion in the remedies. The civet secretion is often adulterated with Vaseline,
butter, mashed banana, and petrolatum to increase the quantity because of the
expensive secretion (US$6,600/kg) [11, 12]. In Thailand, there has been no report
about the chemical constituents in secretion of small Indian civet. Hence, this present
study is attempted to determine the chemical constituents of secretion from small
Indian civet (V. indica) and investigate the civet secretion ingredient in aromatic
remedies by gas chromatography/mass spectrometry (GC/MS).
Objectives of the Study
1. To determine the chemical constituents of secretion from Viverricula indica

by GC/MS.
2. To investigate the civet secretion ingredient in aromatic remedies by GC/MS.
Expected Benefits
1. This research provides the chemical constituents of secretion from small

Indian civet in Thailand.
2. This research provides the methodology to confirm small Indian civet
secretion in aromatic remedy ingredient.
3
CHAPTER II
LITERATURE REVIEW
Family Viverridae
Animals in family Viverridae from order Canivora occupy the tropical forest
area that can be found in the South-Western Europe, the Southern Asia, the Eastern
Indies, Africa, and Maddascar [13]. The Viverridae family is divided into four
subfamilies, Hemigalinae, Paradoxurinae, Prionodontinae, and Viverrinae, covering
35 species.
- The Hemigalinae includes four genera and four species of palm civet:
Owston’s civet (Chrotogale owstoni), otter civet (Cynogale bennettii),
Hose’s palm civet (Diplogale hosei) and the banded palm civet
(Hemigalus derbyanus).
- The Paradoxurinae includes five genera and seven species: Binturong

(Arctictis binturong), small-toothed palm civet (Arctogalidia trivirgata),
Sulawesi palm civet (Macrogalidia musschenbroekii), masked palm civet
(Paguma larvata) and three Paradoxurus species, the Asian palm civet (P.
hermaphrodites), Jerdon’s palm civet (P. jerdoni), and the golden palm
civet (P. zeylonensis).
- The Prionodontinae includes two Prionodon linsang species: the banded

and spotted linsangs (P. linsang and P. pardicolor respectively).
- The Viverrinae includes five genera and 22 species: African civet

(Civettictis civetta); 14 genet species, Abyssinian genet (Genetta
abysssinica), Angolan genet (G. angolensis), Bourlon’s genet (G.
bourloni), Crested Servaline genet (G. cristata), common or small-spotted
genet (G. genetta), Johnston’s genet (G. johnstoni), panther or rusty-
spotted genet (G. maculate), Pardine genet (G. pardina), aquatic genet (G.
piscivora), king genet (G. poensis), servaline genet (G. servalina), Haussa
genet (G. thierryi), Cape or large spotted genet (G. tigrina), giant forest or
4
giant genet (G. victoriae); two linsang species of the genus Poiana,
Leighton’s linsang (P. leightoni) and the African linsang (P. richardsonii);
four civet species of the genus Viverra, the Malabar or Malabar large-
spotted civet (V. civettina), large spotted civet (V. megaspila), Malayan
civet (V. tangalunga), and large Indian civet (V. zibetha). Last in this
subfamily is the small Indian civet (Viverricula indica) [14].
Viverrids (Viverrdae) are primarily carnivorous and eat small vertebrates or

insects [15]. They are solitary or live in pairs or groups. Breeding may occur
seasonally or throughout the year. Viverrids are small and medium-sized carnivores.
Most of them have a relatively pointed muzzle and long tail (except the otter civet,
Cynogale bennetti, which has a broad muzzle and short tail); a total of 40 teeth (the
linsangs, Prionodon spp., have 38). Head and body length ranges from 350 to 950
mm, tail length 130 to 900 mm, and the adult weight range is 0.6 to 20 kg. There are
various striped, spotted, and uniform color patterns. In some genera, the tail is banded
or ringed. The body is generally long and sinewy, with short legs and typically a long,
bushy tail. Footprints of most civets have five distinct toe impressions, except those of
Viverra and Viverricula, which have only four toes visible and closely resemble the
prints of dogs [2, 13] as shown in Figure 1.
Figure 1 Left footprints of front (F) and hind (H) feet of civets:
Cynogale bennetti (a), Arctogalidia trivirgata (b), Prionodon linsang (c), Viverricula
indica (d), Paradoxurus hermaphroditus (e) and Hemigalus derbyanus (f) [2]
5
Figure 2 Scent gland of Viverricula indica [16]
Figure 3 Scent gland of Viverra tangalunga [17]
Most viverrids (except lingsangs and male small-toothed palm civet,

Arctogalidia trivirgata) have scent gland in the anal region (Figure 2, 3) that secretes
a nauseous-smelling fluid as a defensive measure. The secretion of the scent gland
which obtained from several genera (Civettictis, Viverra, and Viverricula) is used to
perfume and drug ingredients [2, 13]. In Ethiopia, it also takes in tea and coffee [18].
Some of macrocyclic musks such as civetone, dihydrocivetone and normuscone are
important chemical constituents in civet secretion [19]. The secretion of African civet
(Civettictis civetta) and small Indian civet (Viverricula indica) have been traded
commercially for a long time. It is valued more than gold, ivory, or myrrh. Ethiopia
produces 90% of the world’s civet secretion [18]. Nowadays, some viverrids are
tamed and kept to produce the secretion and civet coffee.
6
Civet coffee is one of the world’s most expensive and low-production varieties
of coffee. Asian palm civet (Paradoxurus hermaphroditus) and other related civets eat
the bean of coffee berries (Figure 4), and then passed through its digestive tract. Civet
cannot digest the beans; therefore, the beans can be found on the ground after the
civets have passed them through their digestive tracts. After gathering, thorough
washing, sun drying, light roasting and brewing these beans yield an aromatic coffee
with much less bitterness, widely noted as the most expensive coffee in the world
(Figure 5) [20].
Figure 4 Asian palm civet (Paradoxurus hermaphroditus) [21]
Figure 5 Civet coffee (Kopi Luwak) [22]
Civets are linked to SARS virus that was first recognized in China and killed
nearly 800 people worldwide in 2003. A virus in civets is similar to the virus that
infected in human SARS patients, or severe acute respiratory syndrome [23, 24].
7
Small Indian Civet (Viverricula indica Desmarest)

Taxonomic classification of small Indian civet
Kingdom: Animalia
Phylum: Chordata
Class: Mammalia
Order: Carnivora
Family: Viverridae
Subfamily: Viverrinae
Genus: Viverricula
Species: Viverricula indica
In Thailand, these viverrids were found 11 species, 9 genera and 3 subfamilies

[19]. Three subfamilies include subfamily Paradoxurinea, subfamily Viverrinae, and
subfamily Hemigalinae. Small Indian civet is in subfamily Viverrinae. It is native to
Bangladesh, Bhutan, Cambodia, South and East China, India, Indonesia, Laos,
Malaysia, Myanmar, Nepal, Sri Lanka, Thailand and Vietnam [25].
Small Indian civet is a small animal; weigh from 2 to 4 kg. Its body is slim, 54
to 63 cm in length. It has catlike body, long tail, and weasellike face. It has brown or
tawny orange pelage ornamented with black and white rings on its neck, small spots
on the body which converge into 6 to 8 dark stripes on the back toward the tail, and
black and white banded tail. The tip of tail is usually white. The paws are typically
dark brown or black and the breast is lighter brown or gray, with few if any marking.
The small Indian civets are distinguished from closely related civets by their
significantly smaller size, lack of dorsal crest of fur, smaller gap between ears, and
shorter rostra (Figure 6). Male are generally larger than female [26-28]. The small
Indian civet will feed on rats, small birds, lizards, insects, grubs and fruits. It likes to
seek for food on the ground, although it can climb trees with agility. It digs readily
and prefers to sleep in burrow [29].
8
Figure 6 Viverricula indica Desmarest [30]
Small Indian civet is usually solitary but occasionally associates in pairs. The
reproduction of small Indian civet can occur throughout the year in Sri Lanka.
Captive in China has two breeding seasons, the first during February to April and the
second during August to September [31]. In each year it gives birth 2 to 5 young that
is weaned at 2 months old. The civet gland has been shown to be of great importance
to reproduction. It is likely the chemicals emitted by this gland attract mates to each
other or demonstrate which animal is estrus [32, 33]. Civet secretion is produced by
both sexes of civet (Viverridae). It originates from special pouched glands situated in
the male civet between the testicles and the praeputium of the penis and in female
between the vulva and anus [11]. Both male and female deposit the secretion from
their glands on many types of objects.
The diploid chromosome numbers of small Indian civet are 2n=36, the
fundamental numbers (NF) are 60 chromosomes in female and 59 in male. The
autosomes consist of 6 large metacentrics, 8 large submetacentrics, 6 large
acrocentrics, 2 large telocentrics, 2 medium metacentrics, 4 medium submetacentrics,
4 medium acrocentrics and 2 small metacentrics. The X chromosome is the largest
submetacentric and Y chromosome is a medium tetocentric chromosome. The
karyotype formula for the small Indian civet is 2n (36) = Lm6 + Lsm8 + La6 + Lt2 + Mm2
+ Msm4 + Ma4 + Sm2 + sex-chromosome [34].
9
Small Indian civet is one of the controlled animals that were designated in
Wildlife Protection Enactment (1992) but according to the ministerial regulations
issue 6th (1994), farmer can keep and breed the civet for commercial purposes. In
Thailand, there are many civet farms and the number of small Indian civets
throughout Thailand is about 800 including 500 civets of Thai civet club which
consists of the several small civet farms, 100 civets of Tipprasert civet farm and 200
civets of Pa Noi’ s civet farm [12, 35, 36 ]. All of the civets are caught from wild.
They are kept in large cage made of bamboo which one cage for one civet (Figure 7).
All of the civet farms are registered with the Forestry Department while officials of
the department can enter and inspect the farm at any time during day time without
prior notification [4]. Pa Noi’s civet farm is the largest farm that produces the civet
secretion to traditional medicine producers (Figure 8). At another civet farm, the small
Indian civets are fed with coffee beans for civet coffee producing.
Figure 7 Small Indian civet in a cage [4]
Figure 8 Pa Noi’s civet farm

10
Small Indian Civet Secretion
Figure 9 Small Indian civet secretion
The secretion (Figure 9) is almost strong smelling oily substance with a

yellowish color. It darkens when exposed to light. The taste is bitter and the odor is
less agreeable than musk. It is offensive when undiluted but agreeable when a small
portion is mixed with another substance. It contains a volatile oil and free ammonia.
Male civet secretion is dirty than female civet secretion because some male civet
urinates during deposits the secretion on the object. The quantity and quality are not
different between male and female civet secretion [3]. In extreme dilution, the civet
secretion has a pleasant odor. Civetone is the main constituent of civet secretion (2.5-
3.4%). The civet secretion also contains other macrocyclic ketones such as
cyclohexadecanone, cycloheptadecanone, and 6-cis-cycloheptadecenone [37]. It has
powerful holding properties for other scent and is mainly used as fixative in the
manufacture of expensive perfumes. Thai traditional medicine uses the secretion for
heart stimulating effect, therefore it is an important ingredient in aromatic remedy.
Moreover Arabs also use civet secretion for medical purposes and in Japan; it is used
as a preservative of antibiotic [11].
In Thai traditional medicine, there are 115 remedies that used the secretion as
an ingredient [3]. Most of the remedies are aromatic remedies. Before used the
secretion as a crude drug, the secretion had moved the impurity with the traditional
process. The secretion and kaffir lime (Citrus hystrix D.C.) peel are placed on betel
11
vine (Piper betle L.) leave, and then singed to make more cleaning and pleasing smell
as shown in Figure 10 and 11.
Figure 10 Small Indian civet secretion which placed on betel vine
Figure 11 Thai traditional processing for civet secretion cleaning

12
Macrocyclic Ketones of Fragrance Ingredient
The common structure of macrocyclic ketone group of fragrance ingredient is

a keto group, R-C (=O)-R', contained within a macrocyclic ring of C15-C17 carbon
chain length. Macrocyclic ketones fragrance ingredients include 11 structurally
diverse C15, C16, and C17 compounds that consist of three saturated and eight
unsaturated ketones. Naturally occurring macrocyclic ketones are obtain from various
animal rather than plant sources. The molecular weights of the macrocyclic ketones
are range from a high of 250.43 g/mol for the C17 congener civetone to a low of
222.37 g/mol for the C15 4-cyclopentadecen-1-one. All macrocyclic ketones have
similar metabolism and are detoxified in the same manner [38].
Civetone
Formula: C17H30O
IUPAC name: 9-Cycloheptadecen-1-one
Synonyms: civettone, zibeton, cis-civetone, α-trans civettone
Molecular weight: 250.43 g/mol
Civetone is a 17-membered macrocyclic ketone which is a pheromone sourced

from civet (Figure 12). The structure of civetone was elucidated by Ruzicka in 1926
as a macrocyclic unsaturated ketone [39]. It contains one ethylenic double bond
therefore it can exist as either the cis or trans isomer. Naturally occurring civetone has
been identified as the cis isomer [40]. It is semi-solid, yellowish to brown, unctuous
substance, bitter taste, fusible and burns without leaving much residue. It is insoluble
in water but soluble in hot alcohol or in ether [41]. The previous study demonstrated
the synthesis civetone from ethyl oleate which prepared from palm oil [42].
Figure 12 Structure of civetone

13
Civetone can be detected by thin layer chromatographic method as a yellowish

green spot after spraying with mixed solution of 1 ml of 10% methanolic solution of
phosphomolybdic acid and 49 ml of p-anisaldehyde - acetic acid - sulfuric acid -
methanol (1:10:20:200) then heating for 5 min at 105 °C. In addition, civetone is
easily separated from other saturated macrocyclic ketones by GC/MS method using
polyethylene glycol 20 M as stationary phase [43].
Biological Data of Civetone
The acute oral LD50 value in rats is more than 5 g/kg body weight and the
acute dermal LD50 value in rabbits exceeds 2 g/kg. Civetone applied to intact or
abraded rabbit skin for 24 hr under occlusion is moderately irritating. Evaluation of
civetone at dose of 4% in petrolatum in a 48 hr closed path test shows no irritation
and no sensitization reactions. Civetone at dose of 1% and 20% in petrolatum or
ethanol is negative for phototoxicity in guinea pigs. Irradiation for 30, 60, and 120
min at approximate energy level of 1.6-7.6 J/cm2 (UV at 300-430 nm) at 15-20 cm
from the skin do not produce phototoxic reactions in guinea pigs [38, 44]. cis-
Civetone and trans-civetone can exhibit strong type I spectral interactions with
hepatic microsomal cytochrome P-450. The cis isomer is a potent specific inhibitor of
the in vitro aromatization of androgens to estrogens [45].
Usage of Civetone
Many products use civetone as a fragrance ingredient. Civetone may be found

in fragrance used in cosmetic, shampoos, toilet soaps, and non-cosmetic products such
as detergent and household cleaners. The worldwide volume of civetone which used
as fragrance ingredient has been about 0.01-0.1 metric tons per year [38, 44].
14
Normuscone
Formula: C15H28O
IUPAC name: Cyclopentadecanone
Synonyms: Exaltone, normuscon
Molecular weight: 224.39
Normuscone is a macrocyclic ketone with a 15-carbon ring and colorless

crystal needles (Figure 13). It is found in the scent gland of Louisiana muskrat [46]. It
is insoluble in water but soluble in alcohol.
Figure 13 Structure of normuscone
Biological Data of Normuscone
The acute oral LD50 value in rats and acute dermal LD50 value in rabbits are
reported as greater than 5 g/kg body weight. An intraperitoneal dose of 11.25
mmol/kg (~ 2.5 g/kg) causes no deaths. An oral dose of 45 mmol/kg (~10.0 g/kg)
causes no signs of toxicity in mice. Normuscone at 10% in petrolatum produces no
irritation after a 48 hr closed path test on human subjects and produces no
sensitization reactions. Normuscone produces no irritation to rabbit eyes when applies
an instillation of 0.1 g of 100% into the conjunctival sac of rabbits. The 30% dilution
causes transient redness in the conjunctivae that cleared by 24-48 hr. Normuscone at
10% in ethanol causes no phototoxic reaction in rabbits; 1% or 20% of normuscone in
petrolatum or ethanol also do not cause a phototoxic reaction in guinea pigs.
Normuscone has been reported not being mutagenic in the Ames test and not
clastogenic in human lymphocytes in vitro testing [38, 47].
15
Usage of Nomuscone
Normuscone is a fragrance ingredient used in many compounds such as

decorative cosmetics, find fragrances, shampoos, toilet soaps, and non-cosmetic
products. The worldwide volume of use for normuscone has been about 10-100 metric
tons per year. This report volume is for normuscone that used as a fragrance
ingredient [38, 47].
Aromatic Remedy
Aromatic remedy is one of the most Thai ancient remedies. It has been used
for relief of faint, dizziness, nausea and vomiting for a long time. Aromatic remedy is
usually taken by mixing 0.5-1.0 g of aromatic remedy powder with warm water and
drinking the whole preparation as needed. In the local markets, there are several
aromatic remedies which are different trade names and different proportion of the
medicinal plants. Most aromatic remedies contain similar major ingredients such as
civet secretion, clove and agarwood. Moreover, most aromatic remedy ingredients are
aromatic plants. Civet secretion is also used as a fixative in aromatic remedy because
of its strong holding property [11].
Figure 14 Aromatic remedies [47]

16
Effect of Aromatic Remedy
The water extract of aromatic remedy at concentration of 0.83-16.67 mg/ml

can stimulate rat aortic ring contraction which is partially inhibited by phentolamine.
The stimulatory effect of aromatic remedy is partially dependent on α receptor,
similar to norepinephrine but dose not involve β receptor [49].
The major effect of aromatic remedy is to increase the blood pressure. In rat,
the water extract of aromatic remedy at doses of 0.2-0.8 g/kg initially transiently
decreases pressure and over time, increases blood pressure. Aromatic remedy initially
decreases blood pressure for a short period and increases blood pressure over a longer
period. The time to decrease blood pressure is in seconds, whereas the increasing
blood pressure effect is in minutes. However, the duration of action and time to
maximum response are dose dependent. Therefore, the dominant effect of aromatic
remedy on increasing blood pressure supports the use of aromatic remedy for the
treatment of fainting [50].
In human, aromatic remedy increases blood pressure by increasing diastolic

blood pressure and decreasing pulse pressure. Aromatic remedy powder and its water
extract have no effect of ECG, heart rate and peripheral blood flow. An Increasing
blood pressure effect of aromatic remedy supports the use of aromatic remedy for the
treatment of fainting because decreasing blood pressure is a sign of fainting [51].
Gas Chromatography/Mass Spectrometry (GC/MS)
Gas Chromatography/Mass Spectrometry (GC/MS) is one of the most popular

and effective methods for separating, identifying and quantifying compound in
chemical mixtures. It is made up of two parts. The gas chromatography (GC) portion
separates the chemical mixtures and the mass spectrometry (MS) identifies and
quantifies the chemicals. GC is a popular, powerful and easy to use analytical tool.
GC is based on the repeated partition or absorption between a mobile phase and a
stationary phase of the components to be separated. The chemical mixtures are
injected into an inert gas stream and swept into a column with a solid or liquid support
coated [52]. The chemicals in the mixture separate based on their volatility. In
17
general, small molecules travel more quickly than larger molecules. For most GC, the
identification is based on retention time. The GC method is particularly suitable for
quantitative analysis of natural volatile components, and lipophilic compounds with
low boiling point and good thermal stability in medicinal herbs. Although high
boiling point and heat-sensitive compounds are now mostly analyzed by HPLC, some
scientists still like to use GC to quantify them by making them into less polar
derivatives, because GC provides better resolution due to its longer column than
HPLC, and less interference by the mobile phase [53]. Mass spectrometer (MS) is the
most common detector coupled with GC. The MS is used to identify chemicals based
on their structures. There are four steps including ionization of the sample,
acceleration of the ions by an electric field, dispersion of the ions according to their
mass-to-charge ratio and detection of the ions to produce a corresponding electrical
signal [54]. A mass spectrometer is an excellent tool for clear identifying the structure
of a single compound but is less useful when presented with a mixture.
GC/MS is an instrument that uses to separate mixtures into individual

components, identifying and then providing quantitative and qualitative information
on the amounts and chemical structure of each compound [52]. It is very sensitive
and has high resolution.
Full Scan Mode and Selected Ion Monitoring Mode
Full scan Mode
The continuous recording of mass spectra and the simultaneous determination

of the retention time allow the identification of analyses by comparison with libraries
of mass spectra [55]. The full scan mode is used to detect any molecules with a mass-
to-charge ratio (m/z) which a certain range in the analyzed sample. Therefore, the full
scan mode can be used for unambiguous confirmation of the analyst content and
identifying any non-targeted analyst that may be present [56, 57].
18
Selected Ion Monitoring Mode (SIM)
SIM or multiple ions monitoring (MIM) mode refers to the recording of only
ions current with one or multiple selected m/z values [56, 58]. It is used to detect the
content of compounds with a specific m/z and used to unambiguous confirmation of
the analyst from a full scan mode. It is also appropriate for quantitative analysis of
low concentration of an analyzed sample [57, 58].
19
CHAPTER III
MATERIALS AND METHODOLOGY
Samples
1. Secretion of small Indian civets (Viverricula indica)

- Male small Indian civet secretion (n=15)
- Female small Indian civet secretion (n=15)
- Pooled small Indian civet secretion (n = 10)
2. Feces of small Indian civets (n = 15)
3. Furs of small Indian civets (n = 3)
4. Aromatic remedies with civet secretion in ingredient
5. Aromatic remedy without civet secretion in ingredient
Materials
1. Zebron ZB 5 capillary column (30 m x 0.25 mm x 0.25 µm) (Phenomenex®,

CA, USA)
2. Polytetrafluoroethylene (PTFE) syringe membrane filter (0.45 µm) (Chrom
Tech, Inc., USA)
Chemicals and Reagents
1. 9-cyclohetadecon-1-one (Sigma-Aldrich Company Co., St. Louis, MO, USA)

2. Cyclopentadacanone (Sigma-Aldrich Company Co., St. Louis, MO, USA)
3. Hexane AR grade (Lab-Scan Asia Co., LTD, Bangkok, Thailand)
Instrumentations
1. Gas chromatograph (Trace GC Ultra, Thermo Finnigan, USA) equipped with

MS detector (DSQ, Thermo Finnigan, USA)
2. Ultrasonic bath (Analytical Lab Science Co., LTD, Bangkok)
3. Centrifuge (Sorvall® Primo R, UK)
20
Methods
Sample collection
Male small Indian civet (Viverricula indica) secretion (n =15), female small
Indian civet secretion (n =15), pooled small Indian civet secretion (n = 10), small
Indian civet feces (n = 15), and small Indian civet furs (n=3) were collected from Pa
Noi’s civet farm in Petchaburi, Thailand. Each sample was kept in tightly capped vial
and refrigerated until analysis.
Three different lot numbers of aromatic remedies with civet secretion

ingredient in the description and one aromatic remedy not containing civet secretion
ingredient were collected from the local markets. Each aromatic remedy stored at
ambient temperature until analysis.
Small Indian Civet Secretion Analysis
One milligram of each sample was vortically mixed with 1 ml of hexane for 1
min then centrifuged at 10,000 rpm for 10 min at 25°C. One microliter of hexane
supernatant was analyzed by gas chromatography/mass spectrometry (GC/MS). The
analysis was performed using a Finnigan trace GC ultra gas chromatography equipped
with ZB-5 capillary column (30m x 0.25mm x 0.25µm) and interfaced to a Finnigan
trace DSQ MS detector. The oven temperature was ramped from 60°C to 240°C at a
constant rate of 3°C/min. The injection port was held at 180°C throughout the
separation. The carrier gas was helium with a flow rate of 1 ml/min and split ratio of
10:1. MS was performed by electron ionization (EI) mode at 70 electron volts.
Analysis of each sample was performed in triplicates.
Small Indian Civet Furs Analysis
Fifteen milligrams of each sample was washed in aliquots of 2 ml hexane until

exhaustion. Washing hexane aliquots were kept in the refrigerator for further analysis.
The furs were removed, dried and cut into fine pieces. Five milligrams of the washed
fine pieces of small Indian civet furs were mixed with 1 ml of hexane and sonicated at
30°C for 15 min at 53 KHz. Then, it was centrifuged at 10,000 rpm for 10 min at
21
25°C. Hexane extract as well as washing hexane aliquots were analyzed with the
same conditions GC as used for the secretion samples. Analysis of each sample was
performed in triplicates.
Small Indian Civet Feces Analysis
One hundreds milligrams of each sample was dissolved in 1 ml of hexane and

vortex for 1 min. This solution was centrifuged at 10,000 rpm for 10 min at 25°C.
One microliter of the hexane supernatant was analyzed by GC/MS with the same
conditions as used for the secretion samples. Analysis of each sample was performed
in triplicates.
Aromatic Remedies Analysis
One hundreds milligrams of each sample was mixed with 1 ml of hexane and
vortex for 1 min. Then, it was filtrated though 0.45 µm PTFE membrane filters and
evaporated. After this, the extract was adjusted to 250 µl of hexane and then vortex
again. The solution was analyzed within the same conditions GC as used for the
secretion samples. Each sample was performed in triplicates.
Qualitative Analysis of Small Indian Civet Secretion Constituents
The chromatogram of small Indian civet secretion was obtained by full scan
mass spectra with a scan range of 40-650 amu at 500.0 amu/second. The chemical
constituents in the secretion extract were identified by matching their mass spectra
and retention time indices with Adams Essential Oils Mass Spectral library and NIST
05 Mass Spectral library. The percentages of chemical compositions were computed
as the percentage of GC peak areas.
Quantitative Analysis of Civetone and Normuscone
The amount of civetone and normuscone were determined by comparing the

area of the chromatogram with the calibration curve of standard solutions and were
expressed in mean and standard deviation (SD) as µg/mg of samples.
22
Calibration Curve and Linearity
Stock solution of civetone was prepared by dissolving 1.1 µl of civetone

(equivalent to 1 mg, density = 0.917 g/cm3 at 33°C) in 1 ml of hexane. Stock solution
of normuscone was prepared by dissolving 1.0 mg of normuscone in 1 ml of hexane.
The stock solutions were serial diluted for linearity range and calibration curves. The
standards were analyzed under the same conditions of GC as mentioned above. The
calibration curves were obtained according to the linear regression analysis of the
peak areas versus the amount of civetone and normuscone.
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
LOD and LOQ determination were based on the standard deviation of the
blank [59]. The triplicates of 1 mg/ml of blank sample were prepared and analyzed
with the same conditions as used for the samples. The mean and SD of concentration
corresponds to analytical responses were detected and calculated the LOD and LOQ
as follow [59]:
LOD = mean of blank sample + 3SD
LOQ = mean of blank sample + 10SD
Precision
The precision of the method was assessed with intra-day and inter-day
analyses. For repeatability, different concentration levels (3 concentrations / triplicate)
which covered the specified range were analyzed on day 1 and this were repeated on 3
consecutive days. The quantity of each component was determined by the respective
calibration curve. Relative standard deviation (RSD) was used to measure precision
[59].
Recovery of Small Indian Civet Secretion
The extraction efficiency method was used for recovery evaluation of civet
secretion by re-extracting the residue until exhaustion [59] and determining civetone
23
and normuscone by GC/MS. The extraction of civetone and normuscone was

performed at two concentrations of the small Indian civet secretion (1 and 2 mg/ml).
The secretion was dissolved in 1 ml of hexane, vortex for 1 min and

centrifuged at 10,000 rpm for 10 min at 25°C. The filtrate was removed and then
analyzed by GC/MS. The marc was re-dissolved in 1 ml of hexane and performed with
the aforementioned method. The extraction was repeatedly done until exhaustion. The
percentage of recovery was calculated as follow:
Recovery (%) = (Ca x 100)/Cs
Ca: concentration in the first filtrate

Cs: concentration in sum of filtrates
Recovery of Aromatic Remedies
Recovery was carried out by spiking method [60] using three concentrations of
standard solution. The average recoveries of every spiking concentration were
calculated.
Recovery (%) = [(As-A)/Aa] x 100
As: the amount of civetone or normuscone that found after spiking of the
standard solution
A: the amount of those found that before spiking
Aa: the amount of reference standards actually added to the sample
Selected Ion Monitoring (SIM) Analysis of Civetone and Normuscone
For analysis of trace amount of civetone and normuscone, SIM mode was
performed simultaneously with full scan mass spectra monitoring. A selected ion
mornitoring chromatogram at m/z 250 and 224 were measured for representing
civeone and normuscone respectively.
24
CHAPTER IV
RESULTS
Small Indian Civet Farm
Pa Noi’s civet farm is the largest civet farm in Thailand that keeps civets for
over 80 years. The farm is run by 60 years old couple. It is a family business and they
do it for almost a century. There are about 200 small Indian civets in this farm and can
get almost a kilogram of civet secretion monthly. Most of the civets were caught from
wild and some civets were bought from the neighbor who stopped to keep the civets.
Male small Indian civets are more than female civets. The farmers try to breed the
civets but they have never been able to raise civet babies to adult size. Most of them
died before reach the age of 6 weeks. All of the small Indian civets are kept in
individual cages made of bamboo (Figure 15, 16). The size of the cage is 0.90 X 1.0
meter and 0.50 meter high. The cages are under roof to protect the small Indian civets
from direct sun and rain. A timber wood stick about 1.0 inches square and about 0.60
meter long is put in the middle of the cage that used to deposit the civet secretion. The
farmers take the stick out every morning at around 6 a.m. and take the secretion out
with a stainless steel tea spoon. Then, they pool the secretion gathering from all small
Indian civets together. Therefore, the commercial civet secretion from the civet farm
does not separate male and female secretion. The amount of secretion collected from
one civet depends on the health of the civets and the temperature. If the weather is
cold, there is more civet secretion onto the wooden stick. The secretion of each small
Indian civet was of 3-4 g/month. When the civet is too old, farmer will release it back
into the wild or keep it until die. This farm produces the civet secretion to most
traditional medicine producers in Thailand [12].
Figure 15 A small Indian civet in the bamboo cage

25
Figure 16 Small Indian civet cages
Small Indian Civet Secretion
Small Indian civet secretion was an oily substance with yellowish in color. It
had an offensive animal odor. Male small Indian civet secretion was stronger smelling
and darker than female secretion (Figure 17).
Figure 17 Male (left) and female (right) small Indian civet secretion
26
Small Indian Civet Furs
Small Indian civet furs were dark brown and white (Figure 18). It has the same
smell as the secretion.
Figure 18 small Indian civet furs
Small Indian Civet Feces
The small Indian civet feces was a soft and paste substance with foul smell.
It was yellowish in color (Figure 19). There was broken-milled rice in the feces. The
male small Indian civet feces was darker than female feces.
Figure 19 Male (left) and female (right) small Indian civet feces
27
Small Indian Civet Secretion Constituents
Three main chemical constituents of male small Indian civet secretion (Figure
20) were normuscone, dihydrocivetone and civetone with the percent area of 73.4 ±
7.0, 5.1 ± 1.5, and 3.2 ± 1.9 % respectively. The female small Indian civet secretion
exhibited four main chemical constituents (Figure 21) including civetone, dihydro-
civetone, normuscone, and cyclohexadecanone with the percent area of 56.1 ± 5.2,
16.8 ± 2.9, 11.8 ± 2.1, and 3.2 ± 0.6 % respectively.
RT: 9.89 - 59.82
Normuscone 39.90 NL:
100 5.96E6
C15H28O
TIC F: MS
90 m3_1
80
70
Relative Abundance
60
50
40
30
Dihydrocivetone Civetone
C17H32O C17H30O
20
10
46.48
10.23 13.54 19.59 23.17 38.20 42.24 46.79 57.81 59.59
27.14 29.63 32.59 54.64
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 20 GC chromatogram of male small Indian civet secretion

RT: 10.03 - 60.02
Civetone 46.79 NL:
100 3.95E6
C17H30O
TIC F: MS
90 f13_03
80
70
Relative Abundance
60
Dihydrocivetone
50
C17H32O
40
Normuscone 46.48
C15H28O 39.90
30
Cyclohexadecanone
20 C16H30O
43.21
10
45.03
10.95 16.90 20.79 21.99 29.11 32.59 41.76 49.69 52.75 57.35 59.71
35.88
0
15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 21 GC chromatogram of female small Indian civet secretion

28
RT: 10.03 - 59.70

Normuscone 39.93 NL:
100 1.75E6
C15H28O
TIC F: MS
90 mix_secreti
on05
80 Civetone
46.83
C17H30O
70
Relative Abundance
60
50
40 46.52 Dihydrocivetone
C17H32O
30
Cyclohexadecanone
20 C16H30O
10.42
11.93 15.96 43.25
10 17.78 22.26
23.95 29.19 32.61 35.46 40.18 52.49
52.84 58.49
36.64
0
15 20 25 30 35 40 45 50 55
Time (min)
Figure 22 GC chromatogram of pooled small Indian civet secretion
Commercial civet secretion from civet farms did not separate male and female
secretion. The secretion was gathered from all small Indian civets every morning and
pooled together. Ten crops of secretion were analyzed and found that the secretion
consisted of normuscone, civetone, and dihydrocivetone as main components with the
percent area of 45.8 ± 1.3, 34.9 ± 0.7, and 15.7 ± 0.7 % respectively. The chemical
cyclohexadecanone which found only in the secretion of female civet could be
expressed in minor component with the percent area of 3.6 ± 0.3 % (Figure 22).
The mass spectrum of civetone (Figure 23) showed a parent peak at m/z 250
corresponding to the molecular formula C17H30O. The spectrum contained peaks
corresponding to major fragments at m/z value of 250, 96, 95, 82, 81, 80, 69, 55, 54,
and 41. Similarly, the mass spectrum of normuscone (Figure 24) showed a parent
peak at m/z 224 corresponding to the molecular formula C15H28O. The spectrum
contained peaks corresponding to major fragments at m/z value of 96, 83, 82, 71, 69,
58, 55, 43, 42, and 41.
29
55
100
41
O
81
250
50
95
69
29 109
121
135 162
14 32 149 175 189 207 217 233
0
10 30 50 70 90 110 130 150 170 190 210 230 250
(mainlib) 9-Cycloheptadecen-1-one, (Z)-
Figure 23 Mass spectrum of civetone
55
100
41
O
71
58
50
83 96
29
111 125
135 224
149 166 177 191 206
0
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
(mainlib) Cyclopentadecanone
Figure 24 Mass spectrum of normuscone
Linearity
The calibration curves were constructed by plotting the peak area of the
standards against their concentration. The regression equations for the linear portion
of the calibration curves of civetone and normuscone were y = 910946x and
y = -735543 + 354744x respectively (y referred to the peak area; x referred to the
concentration of the unknown).
The correlation coefficients (r2) were 0.9717 and 0.9965 for civetone and
normuscone respectively. Linearity range of civetone was 0-50 µg/mg and of
normuscone was 0-80 µg/mg (Figure 25, 26).
30
civetone
Y = 910946*X R^2 = 0.9717 W: Equal
50000000
40000000
Peak Area 30000000
20000000
10000000
0 µg/mg
0 10 20 30 40 50
Figure 25 Calibration curve of civetone
Normuscone
Y = -1.06378e+006+359534*X R^2 = 0.9965 W: Equal
25000000
20000000
Peak Area
15000000
10000000
5000000
0 µg/mg
0 20 40 60 80
Figure 26 Calibration curve of normuscone
LOD and LOQ
In the present study, LOD and LOQ determination were based on the standard
deviation of the sample blank. LOD and LOQ for civetone were 0.0087 and 0.0165
µg/mg of sample, 0.0596 and 0.1154 µg/mg of sample for normuscone respectively.
Precision
The triplicates of each sample within 3 consecutive days for intra-day and
inter-day precision were determined. The results were presented in Table 1. The intra-
day and inter-day RSDs were less than 8%.
31
Table 1 Percentage of relative standard deviation of intra-day and inter-day analysis
Concentration Intra-day RSD% Inter-day

(mg/ml) Day 1 Day 2 Day 3 RSD%
Civetone 0.025 3.212 0.821 1.276 0.472
0.5 1.008 3.530 5.507 0.139
1 1.660 4.693 3.185 0.180
normuscone 0.025 3.446 4.739 1.889 0.531
0.5 1.117 4.856 7.649 0.929
1 1.114 3.920 0.872 0.464
Recovery
The triplicates of each concentration group were analyzed for the recovery.
This recovery was shown as the percentage amount of civetone and normuscone
which extracted from the sample to validate the method. The average recoveries were
97.3-98.0% in secretion and 91.4-105.7% in aromatic remedy for civetone. For
normuscone, average recoveries in secretion and aromatic remedy were 98.5% and
90.0-103.0% respectively. The percentage recoveries were presented in Table 2-4.
Table 2 The percentage recoveries of civetone and normuscone in civet secretion
% recovery of civetone % recovery of normuscone

Secretion 1 mg/ml Secretion 2 mg/ml Secretion 1 mg/ml Secretion 2 mg/ml
95.7 % 98.7 % 99.4 % 98.3 %
97.9 % 97.3 % 97.6 % 98.9 %
98.3 % 97.9 % 98.5 % 98.3 %
32
Table 3 The percentage recoveries of civetone in aromatic remedy
No. of % recovery of civetone

sample Civetone 10 µg/mg of sample Civetone 20 µg/mg of sample Civetone 30 µg/mg of sample
1 112.6% 96.4% 99.9%

2 66.1% 90.3% 110.4%
3 95.3% 102.2% 105.7%
Table 4 The percentage recoveries of normuscone in aromatic remedy
% recovery of normusone
No. of
sample Normuscone 10 µg/mg of sample Normuscone 20 µg/mg of sample Normuscone 30 µg/mg of sample
1 116.4% 91.9% 103.1%

2 102.8% 85.5% 90.0%
3 95.3% 95.2% 92.6%
Civetone and Normuscone Contents in Small Indian Civet Secretion
The civetone and normuscone were main chemical constituents in the

secretion of small Indian civet. Table 5 demonstrated that the concentration of
chemical constituents were different between secretion of male and female small
Indian civet. Civetone was dominated in female whereas normuscone was dominated
in male small Indian civet secretion. The analysis of secretion crops which containing
both male and female secretion showed higher concentration of normuscone than
civetone.
Table 5 The concentration of civetone and normuscone in small Indian civet secretion
(µg/mg of secretion)
Male Female Crop

civetone (µg/mg) 0.788 ± 0.138 23.614 ± 1.469 5.931 ± 1.728
normuscone (µg/mg) 52.121 ± 5.931 19.218 ± 1.584 22.304 ± 5.162
33
Civetone and Normuscone Contents in Small Indian Civet Furs
The collected civet secretion was frequently mixed up with civet furs. The furs
sticked with civet secretion were one of commercial products form civet farms.
Besides full scan analysis of mass spectrum, selected ion monitoring (SIM) spectra
were analyzed for trace amount of civetone and normuscone. Figure 27 showed
neither normuscone nor civetone in the last aliquot of hexane from civet furs washing
thus represented exhaustively washing. However after successively washing (as
shown in Figure 27), it was still found that the small Indian civet furs presented four
chemical constituents (Figure 28) including civetone, dihydrocivetone, normuscone,
and cyclohexadecanone that related to the small Indian civet secretion. The
concentration of civetone and normuscone in small Indian civet furs were 0.23 ± 0.09
and 1.27 ± 0.17 µg/mg of washed furs respectively.
RT: 10.09 - 59.95

10.75 NL:
100 1.61E5
10.85
12.81 TIC F: MS
80 13.54 hexane_fuf02
Relative Abundance
15.46 _1
17.22
60 18.47
21.12
23.35
40 26.10
28.69 59.60
31.40 54.58 57.53
37.20 39.93 41.34 46.81
20
0
59.15 NL:
100 5.84E2
56.58 m/z=
46.85 48.80
80 30.47 45.19 52.99 223.5-224.5+
37.06 249.5-250.5
40.39 43.60 55.47 F: MS
49.63
60 22.56 41.65 hexane_fuf02
39.91 _1
27.62
40 34.53
10.95 17.74 21.40
13.91 25.92 33.81
20
0
15 20 25 30 35 40 45 50 55
Time (min)
Figure 27 Full scan (upper) and SIM (lower) chromatogram of the last
aliquot of hexane from civet furs washing
34
RT: 9.89 - 59.82

16.93 NL:
100 4.30E5
TIC F: MS
90 fur_4_6mgc
lean03
Normuscone
80 C15H28O
39.91 Civetone
29.19
70
46.83
C17H30O
Relative Abundance
60
10.19
50 10.66
12.81 Dihydrocivetone
40 14.53
Cyclohexadecanone C17H32O
C16H30O
30 17.12 46.52
18.73 59.58
20.16
22.85 58.36
20 24.41 54.28
29.42 35.46 49.75
43.20
38.11
10
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 28 GC chromatogram of small Indian civet furs
Civetone and Normuscone Contents in Small Indian Civet Feces
There was no civetone and normuscone in the chemical constituents of both

male and female small Indian civet feces as shown in figure 29 and figure 30.
RT: 9.89 - 59.80

59.05 NL:
100 1.01E6
58.49
TIC F: MS
57.87 Male_feces_1
80
Relative Abundance
57.33 00_06
56.34
60
55.53
54.70
40 10.08 52.92
11.99 51.31
15.65 49.05
20 17.89 46.52
20.99 24.08 29.15
32.46 35.51 39.98 43.27
0
59.57 NL:
100 3.33E3
57.31 m/z=
80 56.90 223.5-224.5+
249.5-250.5
F: MS
60 Male_feces_1
56.09 00_06
52.63 54.58
40 46.15
51.84
30.04 43.11
20 10.46 13.35 36.88
19.17 33.65 39.94
24.18 26.13
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 29 GC chromatogram of male small Indian civet feces

35
RT: 9.89 - 59.88

59.58 NL:
100 5.15E5
58.69 TIC F: MS
80 58.22 Female_feces
Relative Abundance
_100_03
10.23 56.29
60 10.85 56.06
12.75 55.32
15.73 17.20 54.05
40 51.96
20.22 51.26
23.00 29.17 44.92 49.23
20 29.52 32.50
37.03 42.98
0
58.38 NL:
100 2.07E3
57.28 m/z=
80 223.5-224.5+
249.5-250.5
F: MS
52.05 _100_03
47.95
40 39.25 44.51
20.12 36.58 39.93
20 12.71 19.62 22.44 26.04 30.95 35.05
14.63 26.95
22.79
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 30 GC chromatogram of female small Indian civet feces
Civetone and Normuscone Contents in aromatic remedies
Civetone and normuscone were found in all aromatic remedies which claimed
to use the small Indian civet secretion as an ingredient. The contents of civetone and
normuscone were different in each lot number of each remedy (Table 6, 7). In
addition, the concentration of normuscone was higher than civetone in all remedies.
The average concentration of civetone in aromatic remedies A, B, C, D, E, F,

and G were 3.397 ± 0.531, 4.520 ± 2.085, 4.774 ± 0.663, 8.247 ± 2.778, 12.413 ± 2.869,
1.073 ± 0.213, and 3.029 ± 2.990 µg/mg of aromatic remedies respectively. Similarly,
the average concentration of normuscone in aromatic remedies A, B, C, D, E, F, and
G were 7.753 ± 1.151, 10.238 ± 4.087, 11.687 ± 1.877, 15.865 ± 6.880, 31.028 ±
8.849, 2.759 ± 0.620, and 6.268 ± 6.088 µg/mg of aromatic remedies respectively.
36
Table 6 The concentration of civetone (µg/mg) in aromatic remedies obtained by GC/MS
Aromatic Lot number Mean SD

remedies 1 2 3
A 3.679 ± 0.32 2.785 ± 0.169 3.727 ± 0.773 3.397 0.531
B 2.913 ± 0.263 3.772 ± 0.341 6.876 ± 0.355 4.520 2.085
C 4.259 ± 0.323 4.540 ± 0.141 5.523 ± 0.521 4.774 0.663
D 10.011 ± 1.471 5.045 ± 0.459 9.686 ± 1.502 8.247 2.778
E 15.696 ± 1.601 10.386 ± 0.713 11.158 ± 1.152 12.413 2.869
F 1.233 ± 0.084 0.830 ± 0.520 1.155 ± 0.250 1.073 0.213
G < LOQ 3.108 ± 0.213 5.978 ± 0.321 3.029 2.990
Table 7 The concentration of normuscone (µg/mg) in aromatic remedies obtained by

GC/MS
Aromatic Lot number Mean SD

remedies 1 2 3
A 8.225 ± 0.429 6.441 ± 0.552 8.593 ± 0.782 7.753 1.151
B 6.754 ± 0.727 9.219 ± 1.54 14.739 ± 0.799 10.238 4.087
C 10.447 ± 0.979 10.767 ± 1.257 13.846 ± 1.069 11.687 1.877
D 23.696 ± 2.701 10.792 ± 0.167 13.106 ± 2.467 15.865 6.880
E 41.040 ± 0.585 27.792 ± 2.162 24.251 ± 2.140 31.028 8.849
F 2.874 ± 0.884 2.089 ± 0.474 3.313 ± 0.186 2.759 0.620
G < LOQ 6.644 ± 0.520 12.159 ± 0.675 6.268 6.088
37
CHAPTER V
DISCUSSIONS
In Thailand and some countries in Southeast Asia, small Indian civet and
another civet are kept as captive animal for producing and exporting the secretion or
civet coffee to the world market. Pa Noi’s civet farm has been the largest civet farm in
Thailand where produce the secretion to the world market. The civet secretion from
this farm was around US$6,600 per kilogram [12]. It is so expensive secretion that the
aromatic remedies which use the secretion as an ingredient are also high priced. The
farm had an offensive smell as same as the secretion. All of the small Indian civets
were kept in individual cages. The air flowed freely under the cage’s legs. It had a
good atmospheric environment due to healthy small Indian civets. In India, captive
small Indian civets were also kept in individual wooden cages. A reed pole about 65
to 75 cm long was placed vertically in the center of the cage to facilitate secretion by
perineal gland rubbing. The marked secretion from the pole was scraped out by a
piece of coconut leaf. The secretory output was of 2-6 g/month in each civet [9]. From
the results, maintenance and quantity of small Indian civet secretion were not
different between Thai farm and Indian farm but the harvest of secretion was
different. Thai civet farm used a stainless steel tea spoon to scrap the secretion from a
pole while in India used a piece of coconut leaf. The small Indian civets in this farm
were fed rice mixed with milk daily. Chicken, fish and dessert were supplied 2-3
times per week. Puddle frogs and house gecko were also supplied when available. The
regular diet for captive small Indian civets including milk, cooked rice, and banana
are in accordance with the captive small Indian civet in India [61].
Civet secretion is also called civet or civet musk. It is produced by both sexes
of civet (Viverrdae). Two species are well known for civet secretion producing. They
are African civet (Civettictis civetta), which inhabits Ethiopia, and large Indian civet
(Viverra zibetha), found in India and Southeast Asia [11, 62]. The secretion that
obtained from these two species was a semisolid, yellowish to brown, foul smelling
substance and had a consistency of thick grease [11, 40, and 63]. The powerful and
disagreeable of secretion smell remained detectable for at least four months [64]. The
38
African civet yielded more quantity of the secretion than other species of civet. It
produced about 3-4 g of secretion per week [63, 65, 66]. In the present study, the
properties of small Indian civet secretion were related with the secretion that produced
from different species of civet. The characteristic odor of the civet secretion came
from civetone [63, 65]. Both civetone and normuscone were macrocyclic ketones
carrying musky odor [42, 46]. The standard civetone and normuscone which used in
this study had unpleasant odor as same as the secretion. The standard civetone was
more pleasant odor than normuscone. From the results, male small Indian civet
secretion was stronger smell than female secretion might due to normuscone which
was the main component in male secretion. In Ethiopia, African civet secretion from
the male was preferred to the female secretion. The male yielded more the secretion
than female. The female African civet secretion was less productive because the
secretion was washed out or diluted with urine before the civet deposited the secretion
on the wooden pole [11, 67, 68]. In contrast, Thai female small Indian civet secretion
was preferred to the male secretion; the male secretion was darker than the female
secretion because male small Indian civets urinate during deposit the secretion on the
object [3]. The quantity of the small Indian civet secretion was not different between
male and female secretion [12]. The small Indian civet furs sticked with the secretion
were one of commercial products from Pa Noi’s civet farm but there was no report
about its usage. The chemical constituents in small Indian civet furs were related with
the secretion. Therefore, the small Indian civet furs may have holding properties as
same as the secretion. However, the concentration of civetone and normuscone in the
civet furs were considerably low. In Africa, the African civet furs were also used in
some African folk medicine [69]. In Genetta genetta and Genetta tigrina, the feces
have the same smell as their anal gland secretion [70]. Both smell and chemical
constituents of small Indian civet feces was not related with the secretion. The small
Indian civet might not digest rice thus there was broken-milled rice in the feces.
In the present study, hexane was used as solvent because of low noise and
absence of interfering peaks on the chromatogram at the retention time of civetone
and normuscone. External standard method was used for quantifying civetone and
normuscone in the secretion of small Indian civet and in aromatic remedies because
39
the external procedure was simple (single extraction) and extraneous peaks was in
concern [71]. For the external standard method, the peak areas of the analytes in the
samples are compared to the peak area of the standard solution. The calibration curves
are used to determine the concentrations of the analyte in the sample. The calibration
range of the standards should be around the concentration of analytes expected in the
sample [72, 73]. According to AOAC guidelines, the extraction efficiency method
was used for recovery evaluation of small Indian civet secretion by re-extracting the
residue until exhaustion since the matrix was not homogeneous and hardly miscible
with added standard solution. The precision determinations in range of expected
concentrations level should not exceed 15% of RSD [74]. From this study, linear
calibration curves were obtained with good correlation (r2 = 0.9717 and 0.9965) for
civetone and normuscone respectively. The average recoveries were 97.3-98.0% in
secretion and 91.4-105.7% in aromatic remedy for civetone. For normuscone, average
recoveries in secretion and aromatic remedy were 98.5 % and 90.0-103.0%
respectively. The intra-day and inter-day RSDs of the three concentrations were less
than 8%. The results demonstrated that the method was sufficiently accurate and
precise for determination of civetone and normuscone in small Indian civet secretion
and in aromatic remedies. Hence, GC/MS method was beneficially implemented for
qualitative and quantitative analysis of civetone and normuscone in secretion from
small Indian civet and in aromatic remedies.
The qualitative result showed that the secretion of small Indian civet consisted
of civetone, dihydrocivetone and normuscone as main components which were related
to the previous study [43]. Cyclohexadecanone was found only in the female small
Indian civet secretion therefore, the results suggested that the content of chemical
constituents was different between male and female small Indian civet secretion.
African civet secretion contained scatone, indole, ethylamine, propylamine, some free
acid, and civetone [65]. The results indicated that the chemical constituents of the
secretion were different between the African civet and small Indian civet secretion.
The GC analysis of macrocyclic ketones in the odoriferous gland of small Indian civet
in China demonstrated that civetone was identified as the major constituent in the
odoriferous gland of female civet and normuscone in that of male civet [75]. The
40
quantitative results found that civetone was dominated in female (23.6 ± 1.5 µg/mg of
secretion) whereas normuscone was dominated in male small Indian civet secretion
(51.1 ± 5.9 µg/mg of secretion). This finding was in accordance with the civet in
China [75]. In Pa Not’s civet farm, male small Indian civets are more than female
civets therefore; the secretion which obtained from this farm consisted of male
secretion more than female secretion. This farm has produced the civet secretion to
most traditional medicine producers throughout Thailand [12]. The results indicated
that concentration of normuscone was higher than civetone in all remedies. The
quantity of civetone and normuscone in aromatic remedy sample G, lot number 1
were less than LOQ might be owing to the older manufacture date. The difference in
content of civetone and normuscone among each aromatic remedy might be due to
specific formulary. The difference content of civetone and normuscone among each
lot of same remedy might be due to the variety of each crop of civet secretion.
The SIM mode is a mode in which only of a few m/z values are monitored
rather than monitoring all the m/z values in a specified interval. This mode is usually
used when performing target compound analyses [76, 77]. The chromatographic
signal was obtained by the sum of the MS signals due to all ions detected in the
selected mass or due to selected ions when resolution in the m/z domain was utilized
to improve inadequate resolution in the time domain. It should be emphasized that
molecules of different structures exhibited different ionization yield and consequently
the number of ions and the chromatographic peak areas could be different for
different compounds present in equal molar ratio [78]. In present study, the peak at
the retention time around 46.80 min corresponded to civetone, whose EI mass
spectrum was characterized by the presence of high abundant ions at m/z 250. The
abundant ions of civetone was higher than normuscone (m/z = 224) that presented in
the peak at the retention time around 39.90 min. In the SIM mode, the selection of the
ions at m/z 250 and 224 led a chromatogram with only the peaks of civetone and
normuscone. The civetone peak was higher than peak of normuscone in the SIM
mode because of its abundant ions. From this study, the full scan chromatogram of
most aromatic remedies showed the small peaks of civetone and normuscone because
the concentration was too low. The SIM mode was sensitive than full scan mode
41
because all scan time was used to focus on a specific m/z rather than a broad mass
range in full scan mode. Therefore the SIM mode was useful for confirmation of
civetone and normuscone contents in aromatic remedies.
In Ethiopia, African civet secretion intended for the export would be

controlled for the percentage of civetone contents. If civetone contents were less than
40%, export would not be allowed [11]. However, there have been a few studies about
the analysis of the chemical constituents in civet secretion. It is important to point that
this present study represents the first report on the quantitative analysis of civetone
and normuscone in small Indian civet secretion and in aromatic remedies in Thailand
by GC/MS.
42
CONCLUSION AND RECOMMEMDATION
The small Indian civet secretion has been well known as the fragrance
ingredient and the traditional medicine ingredient for a long time. Aromatic remedies
are the Thai ancient remedies that commonly used for relief of faint. Both small
Indian civet secretion and aromatic remedies with civet secretion ingredient were
investigated for quantitation of civetone and normuscone by GC/MS method. There
were three main of macrocyclic ketone constituents in the secretion and furs of Thai
small Indian civets including civetone, normuscone and dihydrocivetone. Sex
dependent difference in chemical constituents of Thai small Indian civet secretion was
demonstrated and should be concerned for their application. The contents of civetone
and normuscone in commercial civet secretion depended on male and female sex ratio
of the small Indian civet. GC/MS method was found to be precise and accurate for
civetone and normuscone determination in small Indian civet secretion as well as in
aromatic remedies. Civetone and normuscone could be used as quantitatively marker
for civet secretion ingredient in aromatic remedies. Further researches are needed to
establish the bioactivity and toxicity of the small Indian civet secretion.
43
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[70] Apps, P. Wild Ways: Field Guide to the Behaviour of Southern African
Mammal. Cape Town: Struik Publishers, 1998.
[71] Center for Drug Evaluation and Research (CDER). Reviewer Guidance
Validation of Chromatographic Methods [Online]. 1994. Available from:
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInform
ation/Guidances/UCM134409.pdf [2011, 18 September]
[72] Sparkman, O., Penton, Z., and Kitson, F. Gas Chromatography and Mass
Spectrometry: A practical Guide. 2nd ed. Massachusetts: Academic Press,
2011.
[73] Stuart, B. and Prichard, E. Gas Chromatography. Cambridge: Athenaeum Press,

2003.
[74] Biopharmaceutics Coordinatinating Committee in the Center for Drug

Evaluation and Research (CDER). Guidance for Industry, Bioanalytical
Method Validation [Online]. 2001. Available from: http://www.fda.gov/
cder/guidance/index.html [2011, 3 August]
[75] Lianghua, H., and Hauchong, L. Gas-liquid chromatography of civetone and its
homologs. Fenxi Huaxue 10 (1983): 781-783.
[76] Watson, T., and Sparkman, D. Introduction to Mass Spectrometry:

Instrumentation, Application and Strategies for Data Interpretation. 4th ed.
Chichester: John Wiley & Sons, 2007.
50
[77] Subramanian, G. Quality Assurance in Environmental Monitoring: Instrumental

Methods. New York: VCH, 1995.
[78] Lavagnini, I., Magno, F., Seraglia, R., and Trildi, P. Quantitative Application of
Mass Spectrometry. Chichester: John Wiley & Sons, 2006.
51
APPENDICES
52
APPENDIX A
Data of Civetone and Normuscone Determination

53
Formulas
- LOD = mean of blank sample + 3SD

- LOQ = mean of blank sample + 10SD
- RSD % = (SD/mean) x 100
- Recovery % of small Indian civet secretion = (Ca x 100)/Cs
Ca: concentration in the first filtrated
Cs: concentration in sum of filtrated
- Recovery % of aromatic remedies = [(As-A)/Aa] x 100
As: amount of civetone or normuscone that found after spiking
of the standard solution
A: amount of those found that before spiking
Aa: amount of reference standards actually added to the sample
54
Table 8 Percent Areas of Male Small Indian Civet Secretion Constituents
No. of Area %
secretion Normucone Dihydrocivetone civetone
Male1 78.20 3.19 2.71
Male2 80.87 4.75 3.44
Male3 83.48 3.24 1.35
Male4 81.19 5.19 1.39
Male5 77.33 5.08 2.17
Male6 69.04 4.44 2.87
Male7 74.70 3.80 2.36
Male8 67.77 4.56 3.14
Male9 77.96 4.92 2.16
Male10 71.28 5.08 1.07
Male11 79.25 3.17 2.49
Male12 58.94 6.92 4.69
Male13 67.52 8.08 4.46
Male14 66.77 7.49 7.62
Male15 67.28 6.27 6.81
Mean 73.44 5.08 3.25
SD 7.0 1.5 1.9
55
Table 9 Percent Areas of Female Small Indian Civet Secretion Constituents
No. of Area %
secretion Normuscone Cyclohexadecanone Dihydrocivetone civetone
Female1 12.07 3.43 12.29 61.90
Female2 10.78 3.23 15.23 58.57
Female3 8.69 2.30 14.12 64.47
Female4 10.11 2.23 18.67 60.45
Female5 12.34 2.62 20.02 58.41
Female6 14.15 4.48 20.59 46.30
Female7 10.30 3.43 14.19 59.66
Female8 12.63 3.14 13.63 57.52
Female9 11.32 3.34 16.53 52.43
Female10 9.40 3.10 16.62 56.02
Female11 15.52 3.09 20.86 49.82
Female12 14.83 4.29 17.16 52.25
Female13 9.49 3.43 17.11 53.46
Female14 11.73 2.64 13.71 60.48
Female15 13.67 3.88 21.16 50.15
Mean 11.80 3.24 16.79 56.13
SD 2.1 0.6 2.9 5.2
56
Table 10 Percent Areas of Pooled Small Indian Civet Secretion Constituents
No. of Area %
secretion Normuscone Cyclohexadecanone Dihydrocivetone civetone
Pooled 1 46.71 3.37 15.31 34.61

Pooled 2 48.10 3.60 14.70 33.60
Pooled 3 44.26 3.93 15.82 35.99
Pooled 4 44.83 3.52 16.32 35.33
Pooled 5 46.28 3.35 16.20 34.17
Pooled 6 45.31 3.26 16.55 34.88
Pooled 7 44.83 3.41 16.08 35.69
Pooled 8 47.43 3.47 14.49 34.62
Pooled 9 45.54 4.08 15.34 35.04
Pooled 10 44.82 3.88 15.77 35.53
Mean 45.81 3.59 15.66 34.95
SD 1.3 0.3 0.7 0.7
57
civetone
Y = 910946*X R^2 = 0.9717 W: Equal
50000000
40000000
Area Ratio
30000000
20000000
10000000
0
0 10 20 30 40 50
Figure 31 Calibration curve for civetone determination by GC/MS
Normuscone
Y = -1.06378e+006+359534*X R^2 = 0.9965 W: Equal
25000000
20000000
Area Ratio
15000000
10000000
5000000
0
0 20 40 60 80
Figure 32 Calibration curve for normuscone determination by GC/MS

58
Table 11 Civetone and Normuscone Contents (µg/mg of secretion) in Male Small

Indian Civet (n = 3)
No. of male secretion Cvetone Normuscone

1 0.589 ± 0.042 55.718 ± 9.393
2 1.344 ± 0.110 70.301 ± 6.064
3 0.771 ± 0.157 73.673 ± 3.129
4 0.500 ± 0.063 41.550 ± 1.863
5 0.809 ± 0.062 65.956 ± 5.200
6 0.928 ± 0.346 56.586 ± 3.338
7 0.636 ± 0.122 72.583 ± 11.657
8 1.350 ± 0.249 64.993 ± 6.787
9 0.430 ± 0.071 48.526 ± 9.286
10 0.303 ± 0.057 33.246 ± 2.677
11 0.489 ± 0.062 44.277 ± 4.856
12 0.941 ± 0.064 36.674 ± 4.139
13 1.031 ± 0.094 68.207 ± 2.185
14 1.194 ± 0.094 31.627 ± 4.729
15 0.504 ± 0.101 17.893 ± 2.839
Grand mean 0.788 ± 0.138 52.121 ± 5.931
59
Table 12 Civetone and Normuscone Contents (µg/mg of secretion) in Female Small

Indian Civet (n = 3)
No. of male secretion Cvetone Normuscone

1 28.584 ± 2.761 21.661 ± 2.102
2 21.364 ± 1.100 16.116 ± 1.297
3 25.781 ± 1.054 13.787 ± 0.891
4 28.817 ± 1.762 20.466 ± 1.880
5 23.744 ± 2.268 20.236 ± 2.461
6 18.262 ± 0.945 20.494 ± 2.179
7 26.246 ± 1.162 18.640 ± 0.483
8 25.090 ± 1.023 20.353 ± 0.875
9 22.788 ± 1.435 18.413 ± 1.306
10 24.858 ± 1.237 16.520 ± 1.458
11 24.659 ± 0.290 27.246 ± 0.958
12 18.999 ± 0.567 19.734 ± 0.219
13 18.101 ± 1.050 13.652 ± 0.667
14 27.850 ± 0.794 22.821 ± 2.052
15 19.067 ± 2.273 18.135 ± 2.444
Grand mean 23.614 ± 1.469 19.218 ±1.584
60
Table 13 Civetone and Normuscone Contents (µg/mg of aromatic remedy) in

Aromatic Remedy A
Normuscone Lot 1 Lot 2 Lot 3

1 8.251 6.092 8.004
2 8.640 6.154 8.294
3 7.784 7.077 9.481
Mean 8.225 6.441 8.593
SD 0.429 0.552 0.782
Civetone Lot 1 Lot 2 Lot 3

1 3.652 2.701 3.305
2 3.714 2.674 3.258
3 3.671 2.979 4.619
Mean 3.679 2.785 3.727
SD 0.032 0.169 0.773
61

Aromatic Remedy B

1 6.218 7.645 15.545
2 6.464 9.349 13.948
3 7.581 10.664 14.725
Mean 6.754 9.219 14.739
SD 0.727 1.514 0.799

1 2.811 4.132 6.482
2 2.717 3.453 6.975
3 3.212 3.732 7.171
Mean 2.913 3.772 6.876
SD 0.263 0.341 0.355
62

Aromatic Remedy C
Normuscone/4 Lot 1 Lot 2 Lot 3

1 10.163 9.482 12.873
2 11.537 10.827 13.674
3 9.641 11.994 14.991
Mean 10.447 10.767 13.846
SD 0.979 1.257 1.069

1 4.336 4.448 4.957
2 4.538 4.702 5.632
3 3.905 4.469 5.981
Mean 4.259 4.540 5.523
SD 0.323 0.141 0.521
63

Aromatic Remedy D

1 20.599 10.628 12.983
2 24.929 10.961 15.632
3 25.561 10.788 10.704
Mean 23.696 10.792 13.106
SD 2.701 0.167 2.467

1 8.313 4.772 10.241
2 10.821 5.575 10.831
3 10.901 4.789 7.985
Mean 10.011 5.045 9.686
SD 1.471 0.459 1.502
64
Table 17Civetone and Normuscone Contents (µg/mg of aromatic remedy) in

Aromatic Remedy E

1 40.675 25.978 21.963
2 41.714 30.184 24.589
3 40.730 27.215 26.202
Mean 41.040 27.792 24.251
SD 0.585 2.162 2.140

1 16.443 9.971 9.930
2 13.858 11.210 11.332
3 16.787 9.979 12.214
Mean 15.696 10.386 11.158
SD 1.601 0.713 1.152
65

Aromatic Remedy F

1 3.040 1.542 3.134
2 3.663 2.385 3.300
3 1.919 2.341 3.506
Mean 2.874 2.089 3.313
SD 0.884 0.474 0.186

1 1.328 0.386 0.866
2 1.169 0.702 1.316
3 1.202 1.403 1.281
Mean 1.233 0.830 1.155
SD 0.084 0.520 0.250
66

Aromatic Remedy G

1 < LOQ 6.161 12.431
2 < LOQ 6.579 11.391
3 < LOQ 7.194 12.657
Mean - 6.644 12.159
SD - 0.520 0.675

1 < LOQ 2.893 5.906
2 < LOQ 3.114 5.699
3 < LOQ 3.318 6.329
Mean - 3.108 5.978
SD - 0.213 0.321
67
APPENDIX B
GC Chromatogram and Mass Spectrum

68
RT: 10.09 - 59.95

39.94 NL:
100 3.86E6
TIC F: MS
90 m1_1
80
70
Relative Abundance
60
50
40
30
20
10 13.54 59.94
46.52 46.83
14.97 20.93 23.19 28.57 29.65 35.92 43.25 53.29 58.05
0
15 20 25 30 35 40 45 50 55
Time (min)
Figure 33 GC chromatogram of male small Indian civet secretion (1)
RT: 10.03 - 59.82

39.87 NL:
100 6.58E6
TIC F: MS
90 m2_03
80
70
Relative Abundance
60
50
40
30
20
10 46.46
43.19 46.77 59.60
10.15 13.54 14.93 21.72 23.19 27.16 29.60 32.59 36.29 53.35 57.04
0
15 20 25 30 35 40 45 50 55
Time (min)

69
RT: 10.03 - 59.82

39.90 NL:
100 5.96E6
TIC F: MS
90 m3_1
80
70
Relative Abundance
60
50
40
30
20
10
46.48
10.23 13.54 19.59 23.17 38.20 42.24 46.79 57.81 59.59
27.14 29.63 32.59 54.64
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.89 - 59.89

39.88 NL:
100 3.36E6
TIC F: MS
90 m4_03
80
70
Relative Abundance
60
50
40
30
20
46.46 59.83
10 13.56 14.96 57.70
20.90 23.17 28.55 32.59 36.31 43.17 46.77 55.17
0
10 15 20 25 30 35 40 45 50 55
Time (min)

70
RT: 10.09 - 59.96

39.88 NL:
100 6.15E6
TIC F: MS
90 m5_02
80
70
Relative Abundance
60
50
40
30
20
10 46.44
59.94
10.21 13.56 14.93 19.58 23.18 38.18 42.22 46.75 55.33 57.02
27.15 29.62 32.57
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.09 - 59.96

39.88 NL:
100 5.18E6
TIC F: MS
90 m6_02
80
70
Relative Abundance
60
50
40
30
20
10 46.44 59.77
13.54 14.93 19.59 23.19 46.75
27.14 29.63 32.57 35.55 43.17 52.55 56.56 57.02
0
15 20 25 30 35 40 45 50 55
Time (min)

71
RT: 10.03 - 59.96

39.91 NL:
100 4.72E6
TIC F: MS
90 m7_2_1
80
70
Relative Abundance
60
50
40
30
20
10 46.46
10.11 13.54 28.54 42.23
14.93 19.37 32.58 57.06
20.98 35.89 45.02 46.77 50.15 56.71
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.03 - 59.83

39.90 NL:
100 4.86E6
TIC F: MS
90 m8_03
80
70
Relative Abundance
60
50
40
30
20
10 40.81 46.46
46.77
10.10 14.97 19.07 23.17 32.57 35.90 43.19 56.71 57.06
27.14
0
15 20 25 30 35 40 45 50 55
Time (min)

72
RT: 10.02 - 59.67

39.90 NL:
100 3.37E6
TIC F: MS
90 m9_03
80
70
Relative Abundance
60
50
40
30
20
10 42.24 46.48 57.08

10.39 12.09 14.95 28.55 32.59 50.75 56.42
17.79 23.17 35.88 45.03
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.03 - 59.91

39.89 NL:
100 2.55E6
TIC F: MS
90 m10_1
80
70
Relative Abundance
60
50
40
30
20
59.85
46.46
10 13.56
14.94 19.58 23.18 40.80
50.22 56.93 59.38
27.16 29.62 32.58 35.90 42.21
0
15 20 25 30 35 40 45 50 55
Time (min)

73
RT: 9.89 - 59.89

39.90 NL:
100 3.24E6
TIC F: MS
90 m11_1
80
70
Relative Abundance
60
50
40
30
20
10 46.48
10.08 13.54 57.60 57.89
14.89 17.89 23.15 28.53 32.59 35.90 40.13 42.24 46.79 53.83
0
10 15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.09 - 59.87

39.88 NL:
100 3.49E6
TIC F: MS
90 m12_03
80
70
Relative Abundance
60
50
40
30
20
19.61 40.79 46.46

10 59.56
13.56 14.97 23.21 29.65 46.77 54.16 57.66
27.16 32.57 35.86 42.22
0
15 20 25 30 35 40 45 50 55
Time (min)

74
RT: 10.02 - 59.87

39.88 NL:
100 5.47E6
TIC F: MS
90 m13_1
80
70
Relative Abundance
60
50
40
30
20
46.44
10
40.77 46.73
10.33 16.90 19.55 23.15 28.51 29.61 32.55 38.18 42.20 53.23 55.34 59.32
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.09 - 59.94

39.90 NL:
100 2.67E6
TIC F: MS
90 m14_03
80
70
Relative Abundance
60
50
40
30
20
59.92
46.48 46.79
10 59.59
10.56 13.54 19.59 21.74
28.99 29.65 32.59 35.88
43.19 50.58 54.16 57.08
0
15 20 25 30 35 40 45 50 55
Time (min)

75
RT: 10.02 - 59.59

39.89 NL:
100 1.44E6
TIC F: MS
90 m15_02
80
70
Relative Abundance
60
50
40
30
20
42.23
10.50 46.48 46.79 57.63 59.58
11.94
15.01 18.49 56.68
10 20.33 25.71 28.55 32.57 35.40 45.05 50.53 53.29
36.62
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.02 - 59.80

46.79 NL:
100 6.19E6
TIC F: MS
90 f1_2_02
80
70
Relative Abundance
60
50
40
30 39.89
46.48
20
10 43.21
45.03 49.71 52.79
10.33 12.63 16.91 20.82 21.42 29.09 32.56 41.82 59.67
35.88
0
15 20 25 30 35 40 45 50 55
Time (min)
Figure 48 GC chromatogram of female small Indian civet secretion (1)

76
RT: 10.02 - 59.47

46.79 NL:
100 4.32E6
TIC F: MS
90 f2_2_1
80
70
Relative Abundance
60
50
40
46.48
30 39.90
20
10 43.21
10.10 12.11 45.03 49.71 52.78 59.38
14.85 20.81 21.22 29.11 41.80
32.57 35.88
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.15 - 59.80

46.79 NL:
100 5.12E6
TIC F: MS
90 f3_2_1
80
70
Relative Abundance
60
50
40
30
46.48
39.90
20
10 43.21
10.79 12.26 15.07 49.71 52.78 59.73
18.14 21.95 28.55 32.59 35.42 41.80
0
15 20 25 30 35 40 45 50 55
Time (min)

77
RT: 10.03 - 59.82

46.79 NL:
100 5.74E6
TIC F: MS
90 f4_02
80
70
Relative Abundance
60
50
40 46.48
30
39.91
20
10
43.21
10.21 12.30 15.26 49.71 52.77 57.87 59.71
18.41 23.98 29.11 32.57 35.57 40.13
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.02 - 59.75

46.80 NL:
100 4.31E6
TIC F: MS
90 f5_1
80
70
Relative Abundance
60
50
46.47
40
39.89
30
20
10 43.20
10.17 11.82 45.04 49.70 52.77
16.92 20.81 23.58 29.10 30.30 42.25 59.68
35.19 38.39
0
15 20 25 30 35 40 45 50 55
Time (min)

78
RT: 9.89 - 59.69

46.79 NL:
100 3.84E6
TIC F: MS
90 f6_1
80
70
Relative Abundance
60
50 39.90
46.46
40
30
20
43.21
10 45.03
10.23 11.95
16.92 20.81 21.22 28.53 32.57 35.59 41.78 49.71 52.78 59.48
0
10 15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.89 - 59.89

46.75 NL:
100 5.66E6
TIC F: MS
90 f7_02
80
70
Relative Abundance
60
50
40
30 46.44
39.86
20
10 43.17
10.10 12.40 15.09 45.01 49.67
20.81 21.70 28.53 29.07 35.40 36.38 41.78 52.78 58.28 59.67
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 54GC chromatogram of female small Indian civet secretion (7)

79
RT: 10.09 - 59.69

46.79 NL:
100 5.21E6
TIC F: MS
90 f8_02
80
70
Relative Abundance
60
50
40
30 39.89
46.48
20
10 43.20
10.25 11.93 16.91 17.88 29.10 49.71 52.79 59.66
21.11 29.37 36.33 41.78
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.89 - 59.82

46.79 NL:
100 4.84E6
TIC F: MS
90 f9_1
80
70
Relative Abundance
60
50
40
46.48
39.89
30
20
43.21
10
10.13 11.80 48.61 52.77
16.89 20.80 21.36 29.11 32.60 35.90 42.23 57.22 59.58
0
10 15 20 25 30 35 40 45 50 55
Time (min)

80
RT: 10.03 - 59.76

46.79 NL:
100 5.57E6
TIC F: MS
90 f10_03
80
70
Relative Abundance
60
50
40
46.48
30
39.90
20
43.21
10
10.33 11.88 45.03 49.71 52.80
15.40 20.83 59.71
28.55 29.11 32.59 35.90 41.80
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.89 - 59.88

46.79 NL:
100 5.36E6
TIC F: MS
90 f11_1
80
70
Relative Abundance
60
50
46.48
39.89
40
30
20
43.21
10
10.13 11.93 16.58 45.05 49.70 52.79 57.96 59.85
20.82 21.47 29.11 32.59 35.88 42.23
0
10 15 20 25 30 35 40 45 50 55
Time (min)

81
RT: 10.02 - 59.80

46.77 NL:
100 4.03E6
TIC F: MS
90 f12_1
80
70
Relative Abundance
60
50
39.89 46.46
40
30
20
43.21
10 10.13
11.86 14.91 45.03 48.59 52.75 58.12 58.45
17.68 22.59 26.93 29.10 32.58 35.58 42.24
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.03 - 59.82

46.79 NL:
100 3.95E6
TIC F: MS
90 f13_03
80
70
Relative Abundance
60
50
40
46.48
30 39.90
20
43.21
10
45.03
10.95 16.90 20.79 21.99 29.11 32.59 35.88 41.76 49.69 52.75 57.35 59.71
0
15 20 25 30 35 40 45 50 55
Time (min)

82
RT: 10.09 - 59.54

46.79 NL:
100 5.77E6
TIC F: MS
90 f14_1
80
70
Relative Abundance
60
50
40
39.90
30 46.48
20
10 43.21
10.29 12.03 16.90 49.71 52.78 57.31
20.79 22.82 29.11 32.59 35.57 41.82
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.02 - 59.81

46.78 NL:
100 3.82E6
TIC F: MS
90 f15_02
80
70
Relative Abundance
60
50 46.47
40
39.89
30
20
43.20
10 45.04 59.68
10.10 11.95
15.75 18.71 21.69 28.54 41.81 52.45 52.79
32.58 35.87
0
15 20 25 30 35 40 45 50 55
Time (min)

83
RT: 9.89 - 59.94

39.94 NL:
100 1.50E6
TIC F: MS
90 mix_secreti
on1
80
46.83
70
Relative Abundance
60
50
40
46.52
30
20
10.50
12.09
15.05 18.41 43.25
10 20.81 24.02 29.19 32.65 35.49 36.44 40.19 49.77 52.84 57.58 59.65
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 63 GC chromatogram of pooled small Indian civet secretion (1)
RT: 9.89 - 59.63

39.94 NL:
100 1.68E6
TIC F: MS
90 mix_secreti
on02
80
46.83
70
Relative Abundance
60
50
40
46.52
30
20
10.64
11.91 43.25
10 15.07
17.74 20.58 29.19
23.89 32.63 35.47 35.94 40.17 45.07 49.75 52.51 57.43 59.13
0
10 15 20 25 30 35 40 45 50 55
Time (min)

84
RT: 10.02 - 59.86

39.94 NL:
100 1.13E6
TIC F: MS
90 mix_secreti
on03
80
46.83
70
Relative Abundance
60
50
40
46.52
30
10.21
20 11.99
14.37 16.92
19.59 43.25
10 22.32 29.17 35.47 58.50
32.63 35.94 40.19 49.75 52.81 58.02
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.89 - 60.01

39.95 NL:
100 2.23E6
TIC F: MS
90 mix_secreti
on04
80
46.83
70
Relative Abundance
60
50
40
46.52
30
20
10.13
12.51 43.25
10 15.14 17.50
20.97 23.56 29.17 32.64 35.47 40.17 49.75 52.83 58.39 59.78
36.65
0
10 15 20 25 30 35 40 45 50 55 60
Time (min)

85
RT: 10.03 - 59.70

39.93 NL:
100 1.75E6
TIC F: MS
90 mix_secreti
on05
80
46.83
70
Relative Abundance
60
50
40 46.52
30
20
10.42
11.93 15.96 43.25
10 17.78 22.26
23.95 29.19 32.61 35.46 40.18 52.49
52.84 58.49
36.64
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.02 - 59.86

39.95 NL:
100 1.52E6
TIC F: MS
90 mix_secreti
on06
80
46.85
70
Relative Abundance
60
50
40
46.51
30
20
10.50
12.01
16.96 43.24 58.52
10 17.72 29.19
20.55 23.65 52.50 52.85 58.23
32.62 35.48 35.96 40.18
0
15 20 25 30 35 40 45 50 55
Time (min)

86
RT: 10.03 - 59.76

39.96 NL:
100 2.49E6
TIC F: MS
90 mix_secreti
on07
80 46.85
70
Relative Abundance
60
50
40
46.52
30
20
10 10.25 43.25
11.97 15.03
18.51 20.77 24.93 29.19 32.63 35.51 40.19 52.53 52.84 59.67
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.02 - 59.74

39.94 NL:
100 1.06E6
TIC F: MS
90 mix_secreti
on08
80
46.83
70
Relative Abundance
60
50
40 46.52
30
20 10.41
11.82
13.75
16.96 43.25
19.28 21.16 23.54 35.49 59.15
10 29.19
32.63 35.92 40.17
45.09 49.75 52.84 57.64
0
15 20 25 30 35 40 45 50 55
Time (min)

87
RT: 9.89 - 59.82

39.93 NL:
100 1.68E6
TIC F: MS
90 mix_secreti
on09
80
46.83
70
Relative Abundance
60
50
40 46.52
30
20
10.44 11.95 43.25
15.05 58.63
10 17.72 20.58 35.48 45.09 49.75 52.83
23.80 29.18 32.60 40.18
36.51
0
10 15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.83 - 59.76

39.96 NL:
100 1.89E6
TIC F: MS
90 mix_secreti
on10
80 46.84
70
Relative Abundance
60
50
40 46.53
30
20
10.27 11.68 43.25
10 14.84 58.53
18.42 20.26 23.23 57.62
29.19 32.61 35.48 40.16 49.78 52.82
0
10 15 20 25 30 35 40 45 50 55
Time (min)

88
RT: 9.89 - 59.80

39.96 NL:
100 3.96E5
TIC F: MS
90 fur_4_6mgc
lean
46.85
80
70
Relative Abundance
60 10.54
10.91 16.92
50 11.88
13.13 59.54
40 17.06 29.19 46.54
17.41
18.88
30 19.75
22.15 34.08 35.49 54.80 58.49
23.77
20 36.87 52.44
43.29 52.11
48.78
10
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 73 GC chromatogram of small Indian civet furs (1)
RT: 9.89 - 60.00

NL:
100 2.88E5
10.13
10.58 TIC F: MS
80 11.41 fur_13mg_4h
Relative Abundance
exane
13.65
14.72
60
17.16
18.88 20.56 57.06 59.15
40 23.56 55.78
35.49 49.75
25.86 29.17
31.47 53.89
35.76 39.96 44.85 46.81
20
0
54.35 57.02 NL:
100 47.35 49.82 9.98E2
m/z=
44.37
80 223.5-224.5+
15.34 39.92 249.5-250.5
F: MS
60 53.15 fur_13mg_4h
34.37 exane
16.56 38.28 40.27
11.86 26.63
40 25.49 30.27
34.49 43.02
18.63 20.64 29.61 34.20 34.85
20 31.00
0
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 74 Full scan (upper) and SIM (lower) chromatogram of the last aliquot of
hexane from civet furs washing (1)
89
RT: 10.02 - 59.94

46.81 NL:
100 2.21E5
TIC F: MS
90 fur_3_9_02
80
10.23
70 10.81
39.92
11.55
Relative Abundance
60
13.23
50 13.69
14.80
40 17.08
17.76
19.42 29.17 46.50
30 21.80
23.15 24.20 59.65
30.25 58.64
20 31.37 35.88 57.23
43.23 48.72 55.94
37.45
10
0
15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.09 - 59.95

10.75 NL:
100 1.61E5
10.85
12.81 TIC F: MS
80 13.54 hexane_fuf02
Relative Abundance
15.46 _1
17.22
60 18.47
21.12
23.35
40 26.10
28.69 59.60
31.40 54.58 57.53
37.20 39.93 41.34 46.81
20
0
59.15 NL:
100 5.84E2
56.58 m/z=
46.85 48.80
80 30.47 45.19 52.99 223.5-224.5+
37.06 249.5-250.5
40.39 43.60 55.47 F: MS
49.63
60 22.56 41.65 hexane_fuf02
39.91 _1
27.62
40 21.40 34.53
10.95 13.91 17.74
25.92 33.81
20
0
15 20 25 30 35 40 45 50 55
Time (min)
90
RT: 9.89 - 59.82

16.93 NL:
100 4.30E5
TIC F: MS
90 fur_4_6mgc
lean03
80
39.91
29.19
70
46.83
Relative Abundance
60
10.19
50 10.66
12.81
40 14.53
17.12 46.52
30
18.73 59.58
20.16
22.85 24.41 58.36
20 54.28
29.42 35.46 49.75
43.20
38.11
10
0
10 15 20 25 30 35 40 45 50 55
Time (min)
RT: 10.16 - 59.76

NL:
100 10.44 1.95E5
11.31
12.69 TIC F: MS
80 last_hexane0
13.35
Relative Abundance
15.43 3
16.90
60 18.51
20.79
21.51
40 24.10
27.10 59.26
57.85
31.26 33.46 36.54 55.20
39.94 46.83 50.91
20
0
46.85 NL:
100 59.50 7.43E2
m/z=
80 223.5-
30.02 56.07
44.89 224.5+
49.86 249.5-250.5
60 F: MS
41.37 50.02 54.78 57.68 last_hexane0
14.80 37.06
39.92 3
40 16.07 42.03
19.55 22.71 27.14
36.81
14.47 16.69
20 26.52 32.75
20.52
0
15 20 25 30 35 40 45 50 55
Time (min)
91
RT: 9.89 - 59.74

59.73 NL:
100 6.24E5
58.90
58.35 TIC F: MS
80 Male_feces_100
Relative Abundance
57.17 _1_1106042110
20
60 55.96
10.21 54.43
11.99
40 13.09 51.97
15.16
18.30 50.71
20.97 29.17 48.95
24.89 40.85
20 32.49 39.94
0
59.65 NL:
100 2.54E3
58.95 m/z=
80 57.48 223.5-224.5+
56.03 249.5-250.5 F:
51.58 MS
60 Male_feces_100
49.17 _1_1106042110
43.71 20
40 24.29
31.39 47.48
39.94
27.37
20
14.82 16.52 20.31 33.75 35.98
12.28 18.47
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 79 Full scan (upper) and SIM (lower) chromatogram of male

small Indian civet feces (1)
RT: 10.03 - 59.89

58.78 NL:
100 1.43E6
58.43 TIC F: MS
80 Male_feces_
Relative Abundance
57.76 100_2
56.87
60 54.80
50.52 52.90
40 49.94
10.25 13.33 48.05
14.85 18.41 44.99
20 22.03 24.97 29.17 40.64 44.33
34.39
0
58.90 NL:
100 3.56E3
58.34
m/z=
80 223.5-224.5+
56.03 249.5-250.5
48.30 50.48 F: MS
55.55
60 46.63 Male_feces_
51.70 100_2
40 45.71
10.46 32.48 38.88 39.69 45.49
20 14.00 21.51 27.85 31.59 32.82 37.02
14.58 18.92 24.84
0
15 20 25 30 35 40 45 50 55
Time (min)

92
RT: 10.02 - 59.47

58.76 NL:
100 1.70E6
TIC F: MS
80 Male_feces_1
Relative Abundance
58.16 00_03
57.54
60
56.36
55.03
40 53.02
51.16
10.17 11.99 48.08
20 14.89 17.99 44.93
21.06 24.00 29.17 29.65 38.90 42.46
0
58.43 NL:
100 57.31 3.89E3
m/z=
56.98 223.5-224.5+
80
56.52 249.5-250.5
55.03 F: MS
60 52.40 Male_feces_1
00_03
47.68 52.11
40
48.14
39.21 46.63
14.04 35.34 45.22
20 26.48
16.58 17.68 23.38 27.33 32.42
0
15 20 25 30 35 40 45 50 55
Time (min)

RT: 9.83 - 59.89

59.32 NL:
100 1.30E6
58.76
TIC F: MS
Relative Abundance
00_04
57.00
60 56.36
52.44
51.93
40 50.89
10.10 11.99 49.73
47.91
20 16.25 17.89
21.64 29.17 30.62 45.30
40.35 43.36
0
58.82 NL:
100 3.46E3
58.72 m/z=
80 223.5-224.5+
57.91 249.5-250.5
F: MS
60 54.70 56.34 Male_feces_1
51.33 00_04
40 41.84 46.79 49.69
40.68 43.17
30.58
20 14.29 19.42 21.74 24.85 32.69 39.94
38.57
0
10 15 20 25 30 35 40 45 50 55
Time (min)

93
RT: 9.89 - 59.94

NL:
100 59.44 1.20E6
58.84 TIC F: MS
Relative Abundance
00_05
57.37
60 56.73
55.76
53.44
40 51.53
10.08
11.91 49.75
15.90 48.24
20 17.95 22.05 46.05
24.62 29.17
37.45 40.89 44.23
0
59.05 NL:
100 4.09E3
m/z=
80 223.5-224.5+
58.72 249.5-250.5
F: MS
57.08 00_05
54.37
40 52.05
47.43 51.49
42.09 47.00
20 32.92 39.90
12.57 14.97 21.91
16.83 26.54 28.72 36.61
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 9.89 - 59.94

59.86 NL:
100 1.04E6
58.70
TIC F: MS
58.12
80 Male_feces_1
Relative Abundance
57.54 00_06
57.33
60 56.34
55.53
54.54
40 52.92
10.08
11.99 51.31
15.65 49.05
20 17.89 46.52
20.99 24.08 29.15
35.51 39.98 43.27
0
59.57 NL:
100 3.33E3
57.31 m/z=
80 56.90 223.5-224.5+
249.5-250.5
F: MS
60 Male_feces_1
56.09 00_06
52.63 54.58
40 46.15
51.84
30.04 43.11
20 10.46 13.35 36.88
19.17 33.65 39.94
24.18 26.13
0
10 15 20 25 30 35 40 45 50 55
Time (min)

94
RT: 10.03 - 59.82

58.88 NL:
100 9.22E5
58.63
TIC F: MS
Relative Abundance
57.10 00_07
56.42
60 55.43
54.58
40 52.49
10.23
12.26 50.99
16.94 48.90
20 18.30 21.02 45.94
29.17
30.50 35.43 43.23
0
58.86 NL:
100 58.76 2.62E3
55.47 m/z=
80 58.18 223.5-224.5+
249.5-250.5
F: MS
60 52.32 54.85 Male_feces_1
00_07
47.29 51.35
40 43.54
11.86 12.44 40.27 45.38 48.90
20.00 23.11 33.15
20 28.05 31.28 39.46
19.11
26.32 30.29
0
15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.03 - 59.96

59.94 NL:
100
59.30 1.09E6
TIC F: MS
58.99
80 Male_feces_1
Relative Abundance
58.06 00_08
60 57.37
56.67
56.03
40 54.35
10.44 53.29
11.97 16.94 51.51
20 17.10 18.88 48.86
21.88 25.24 29.17 47.06
32.13 39.90 42.57
0
59.92 NL:
100
59.17 3.70E3
m/z=
80 223.5-224.5+
249.5-250.5
F: MS
60 57.33
Male_feces_1
00_08
53.56 56.05
40
52.28
32.84 47.62 51.04
44.93
20 13.29 15.65 21.49 23.31 26.36 32.32
34.33
39.96
27.06
0
15 20 25 30 35 40 45 50 55
Time (min)

95
RT: 10.02 - 59.74

59.36 NL:
100 7.66E5
58.95
58.53 TIC F: MS
80 Male_feces_1
57.97
Relative Abundance
00_09
56.83
60 56.11
54.95
10.54 53.81
40
12.30 51.31
14.06 49.88
16.94
20.04 48.10
20 22.53 29.17 46.40
32.49 35.32 42.82
0
59.59 NL:
100 2.76E3
m/z=
80 52.51 223.5-224.5+
57.83 249.5-250.5
F: MS
00_09
38.51 49.42 50.83
40 39.92 47.27
33.75
11.80 29.48 37.95 44.74 46.50
20 15.90 20.93 34.99 43.91
17.45 22.11 27.87 30.31
0
15 20 25 30 35 40 45 50 55
Time (min)

RT: 9.89 - 59.80

59.50 NL:
100 9.09E5
58.78
TIC F: MS
58.14
80 Male_feces_1
Relative Abundance
57.81 00_10
56.63
60
55.82
54.80
40 10.35 53.04
11.91
14.85 51.64
17.91 49.24
20 21.08 24.66 46.15
29.15 29.40 32.49 40.77 44.14
0
59.09 NL:
100 3.80E3
m/z=
80 223.5-224.5+
58.61
249.5-250.5
57.99 F: MS
56.07 00_10
40 55.67
49.15 53.50
34.60 39.92 45.07
20 24.64 25.34
10.62 12.53 19.98 34.35 39.32
0
10 15 20 25 30 35 40 45 50 55
Time (min)

96
RT: 9.89 - 59.80

59.48 NL:
100 5.91E5
58.47
TIC F: MS
56.40 Male_feces_1
80
Relative Abundance
55.72 00_11
54.43
60 10.15
54.00
11.68
13.19 52.36
40 16.94 50.17
19.01 21.35 47.66
29.17 44.91
20 30.00 32.49 40.85
0
58.63 NL:
100 2.01E3
57.85 m/z=
80 56.65 223.5-224.5+
55.51 249.5-250.5
44.62
F: MS
48.88
00_11
40 10.64 30.83 39.86
14.41
28.82 42.09
22.84
13.98 17.70 19.13 35.18 39.11
20 23.54
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.02 - 59.94

59.80 NL:
100 58.10 7.27E5
57.36 TIC F: MS
80 Male_feces_1
Relative Abundance
56.70 00_12
55.95
60 55.20
10.23 54.13
40 12.08 13.40 52.80
17.09 51.10
19.86 22.62 49.30
20 26.74 29.20 34.48 46.57
39.57
0
58.95 NL:
100 3.47E3
58.41
57.75 m/z=
80 223.5-224.5+
57.11 249.5-250.5
F: MS
00_12
40 51.37
49.49
46.80
14.69 39.63 43.90
20 17.54 26.51 30.26 32.76 36.20
13.67 21.89 24.60
0
15 20 25 30 35 40 45 50 55
Time (min)

97
RT: 9.89 - 60.02

59.90 NL:
100
58.78 6.05E5
58.22 TIC F: MS
80 Male_feces_1
Relative Abundance
56.81 00_13
55.96
60 10.54 55.34
11.78 54.04
12.69 15.30 53.13
40
17.27 51.72
20.50 49.67
24.93 29.17 29.55 47.19
20 32.49 40.85 43.81
0
57.33 NL:
100 3.21E3
m/z=
80 223.5-224.5+
249.5-250.5
F: MS
00_13
53.71 56.42
50.37
40
52.42
46.79
46.05
21.97 23.65 33.46 37.37 42.61
20
11.06 14.31 18.01 27.56 40.35
31.12 35.38
0
10 15 20 25 30 35 40 45 50 55 60
Time (min)

RT: 9.89 - 59.74

59.42 NL:
100 5.52E5
11.16 58.95 TIC F: MS
80 Male_feces_1
Relative Abundance
57.68 00_14
56.63
60 55.72
11.37
13.21 54.43
40 15.07 53.09
18.28 51.70
20.10 22.49 48.18 49.86
24.93 29.15
20 30.60 35.30 37.21 44.93
0
59.32 NL:
100 2.52E3
m/z=
80 223.5-224.5+
249.5-250.5
58.68 F: MS
55.88
00_14
49.77 53.83
40 17.58
52.80
25.26 46.61
15.49 38.95 42.11 45.22
20 11.20 20.68 22.71 36.92
25.55 31.04
0
10 15 20 25 30 35 40 45 50 55
Time (min)

98
RT: 9.89 - 59.83

59.67 NL:
100 5.01E5
58.81
58.35 TIC F: MS
Relative Abundance
56.96 00_15
10.23
12.52 56.47
60
13.08 55.53
15.63
53.84
40 17.76 19.15 52.57
21.84 24.22 27.09
30.05 48.56 50.40
20 32.99 36.05 44.44
0
59.24 NL:
100 57.56 1.77E3
55.20 m/z=
80 223.5-224.5+
249.5-250.5
49.34 51.97
F: MS
00_15
46.84
40 12.77 45.76
14.57 14.95 42.28
25.95 40.01 44.79
29.96
15.73 20.68 25.13 37.50
20
17.95
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 9.89 - 59.69

58.74 NL:
100 1.22E6
58.39 TIC F: MS
80 Female_feces
Relative Abundance
57.93 _100_1
55.45
60
52.51 54.54
40 51.64
48.68
10.12 42.63
12.09 47.41
15.16 17.74 42.05
20
20.62 23.50 29.17 41.33
35.34 38.53
0
57.14 NL:
100 3.14E3
m/z=
80 56.98 223.5-224.5+
249.5-250.5
51.82 55.51 F: MS
60 Female_feces
49.23 _100_1
40 48.74
44.43
13.81 40.95
20 20.64 25.16 37.41 39.96
11.70 14.74 29.38
0
10 15 20 25 30 35 40 45 50 55
Time (min)
Figure 94 Full scan (upper) and SIM (lower) chromatogram of female

99
RT: 9.93 - 60.00

NL:
100 59.62 5.30E5
59.37 TIC F: MS
Relative Abundance
57.63 _100_2
10.12 56.64
60
12.09 55.25
13.64
16.93 53.56
40 51.94
19.17 21.07
29.17 50.43
34.45 35.23 39.52
44.94
20 44.74
0
NL:
100 59.25 3.02E3
m/z=
80 46.77 58.19 223.5-224.5+
56.79 249.5-250.5
F: MS
46.54 54.51 _100_2
40 51.63 54.18
36.62 39.70
34.22 44.94
20.80 23.95 40.70
20 10.31 15.67 30.51 33.72
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 9.89 - 59.95

59.58 NL:
100 5.15E5
58.69 TIC F: MS
80 Female_feces
58.05
Relative Abundance
_100_03
10.23 56.29
60 10.85 56.06
12.75 55.32
15.73 54.05
40 17.20 51.96
20.22 51.26
23.00 29.17
29.44 44.92 49.23
20 32.50 37.03 42.98
0
58.38 NL:
100 2.07E3
57.28 m/z=
80 223.5-224.5+
249.5-250.5
F: MS
52.05 _100_03
47.95
40 39.25 44.51
20.12 36.58 39.93
20 12.71 19.62 22.44 26.04 30.95
35.05
14.63 26.95
22.79
0
10 15 20 25 30 35 40 45 50 55
Time (min)

100
RT: 9.89 - 59.82

59.19 NL:
100 4.07E5
11.16 58.61
11.97 TIC F: MS
57.41 Female_feces
80
Relative Abundance
56.52 _100_04
12.11
13.17 15.90 56.05
60 55.22
51.96
17.50
19.03
40 22.11 24.02 51.82
49.67
29.15 29.69 33.83 44.91
35.34 39.94
20
0
58.32 NL:
100 56.67 1.58E3
55.47 m/z=
80 223.5-224.5+
51.84 249.5-250.5
51.17 F: MS
60 Female_feces
_100_04
50.56
40 25.34 37.39 39.92 40.37 45.86
11.31 18.76
11.91 35.65
24.47 26.03 30.33 35.36
20 16.71 21.12
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 9.89 - 59.41

59.22 NL:
100 5.26E5
58.90
TIC F: MS
57.95
80 Female_feces
Relative Abundance
57.37 _100_05
56.38
60 10.15
11.28 54.33
13.35 46.83 53.00
40 15.59 17.89 51.93
20.35 46.52 50.89
23.75 26.30 40.87
29.17 32.51 39.94
20
0
46.83 NL:
100 5.63E3
m/z=
80 223.5-224.5+
249.5-250.5
F: MS
60 Female_feces
_100_05
59.13
40 55.34
54.41
52.51
20 46.28 52.05
11.45 34.76 35.78 39.90 43.33
12.75 17.76 21.12 24.80 29.46
0
10 15 20 25 30 35 40 45 50 55
Time (min)

101
RT: 9.89 - 59.89

59.57 NL:
100 4.82E5
59.01
TIC F: MS
57.87 Female_feces
80
Relative Abundance
10.19 _100_06
56.34
11.47
60 16.94
55.63
14.04 53.87
17.27 52.98
40 19.42 51.78
22.65
29.17
29.48 32.49 47.64 48.47
20 41.06 44.91
0
59.79 NL:
100 2.83E3
59.07 m/z=
80 223.5-224.5+
249.5-250.5
F: MS
58.99
60 Female_feces
56.71 _100_06
54.97
40
48.84 51.80
10.79 42.82 44.08
20 30.02 32.82 38.45
15.28 19.21 24.43 39.50
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 9.89 - 59.89

59.88 NL:
100 5.32E5
59.22
58.49 TIC F: MS
80 Female_feces
Relative Abundance
58.12 _100_07
56.69
10.10
60 55.76
11.89
13.60 54.78
40 15.72 53.00
18.39 21.45 29.17
23.38 48.68 51.53
20 29.77 32.51 35.30 39.94 44.93
0
59.53 NL:
100 1.87E3
56.38 58.66 m/z=
80 223.5-224.5+
56.21
249.5-250.5
F: MS
46.83 52.36 _100_07
36.19 40.02 44.35 47.12
40 19.44 21.20
15.57 51.93
38.10
25.22 33.65 43.44
20 11.86 30.10
17.19
0
10 15 20 25 30 35 40 45 50 55
Time (min)
102
RT: 10.02 - 60.00

59.63 NL:
100 3.90E5
59.36
58.45 TIC F: MS
10.23 Female_feces
80 11.91 16.92 58.12
Relative Abundance
_100_08
12.46 57.31
60 55.99
17.25 51.97 55.07
19.09
40 29.15
22.34 51.43
26.77 49.07
29.59 31.10 35.34 44.93
37.00
20
0
59.57 NL:
100 1.79E3
59.32
m/z=
80 59.01 223.5-224.5+
51.97 56.57 249.5-250.5
F: MS
55.96 _100_08
14.56
50.97
40
20.85 42.65 45.67 48.30
32.22 37.06 38.08
18.82 27.74
10.17 25.45
20 33.00
0
15 20 25 30 35 40 45 50 55 60
Time (min)
RT: 9.89 - 59.82

NL:
100 3.52E5
10.23 59.59 TIC F: MS
80 11.66 58.49 Female_feces
Relative Abundance
13.38 58.32 _100_09

14.68 57.29
60 16.92 56.40
18.28
19.63 54.91
40 23.38 52.71
25.53 29.17
51.62
30.75 33.29 36.15 44.93 49.03
20
0
58.95 NL:
100 1.27E3
58.22 m/z=
80 223.5-224.5+
46.73 53.54 55.09 249.5-250.5
F: MS
41.91 46.36 51.76 _100_09
35.30 43.60
40 10.44 30.06 50.21
22.03 25.05 41.43
17.23 35.86
33.09
20 25.22
0
10 15 20 25 30 35 40 45 50 55
Time (min)
103
RT: 9.90 - 59.77

59.47 NL:
100 3.70E5
10.17 59.35
58.62 TIC F: MS
10.68 Female_feces
80 57.96
Relative Abundance
11.22 _100_10
12.61 14.81 29.16 56.72
60 55.66
17.24 53.72
19.68 51.98
40 22.35 23.74 51.52
49.66
29.76 31.38
35.31 37.69 44.94 47.44
20
0
57.96 NL:
100 55.37 1.41E3
m/z=
53.03 223.5-224.5+
80
249.5-250.5
40.63 50.63 F: MS
45.04 _100_10
40 11.88
18.13 21.79 29.24 38.98
12.90 28.62 38.00
20.40 32.39
20 28.11 30.07
0
10 15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.89 - 59.89

59.85 NL:
100 3.79E5
58.74 TIC F: MS
11.89 16.94 58.34
80 Female_feces
Relative Abundance
12.26 57.53 _100_11

56.33
60 29.17 55.26
17.74 53.77
18.95
21.26 51.53
40 22.88 50.02
29.31 31.41 48.26
34.88 44.95
20
0
59.85 NL:
100 1.86E3
m/z=
59.13
80 223.5-224.5+
57.22 249.5-250.5
56.02
53.08 F: MS
60 Female_feces
46.83 51.53 _100_11
50.37
40
12.11
12.80 19.17 30.49 40.58 42.40 46.46
33.19 38.86
20 14.87 20.29 24.70 27.43 35.01
0
10 15 20 25 30 35 40 45 50 55
Time (min)
104
RT: 9.89 - 59.82

58.84 NL:
100 58.55 3.32E5
10.08
TIC F: MS
11.16
80 Female_feces
57.99
Relative Abundance
12.71 _100_12
14.30 56.44
15.40 16.91 55.86
60
17.06 55.26
19.00 29.17
53.08
40 21.61 22.96 51.53
32.50 35.36 44.92 49.40
40.87
20
0
58.94 NL:
100 1.71E3
m/z=
80 223.5-224.5+
56.87 249.5-250.5
54.51
F: MS
60 Female_feces
15.38 _100_12
53.50
47.04 47.53
40
11.04 39.95 43.25
23.68 34.70 38.26
28.65
20 18.15 25.34 33.25
21.88
0
10 15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.83 - 60.02

NL:
100 59.90 3.23E5
10.08
59.59 TIC F: MS
10.95
Relative Abundance
11.80 _100_13
13.50 58.10
29.15 56.88
60 14.74
16.92 56.30
17.99 54.83
20.48
40 26.19 52.82
51.51
29.59 31.62 48.88
35.38 40.64 44.93
20
0
59.92 NL:
100 57.52 1.37E3
55.07 m/z=
80 223.5-224.5+
52.63 249.5-250.5
51.89
F: MS
12.13 _100_13
47.02
40
12.65 16.87 28.18 37.70 40.52 41.31
23.52 35.40
20 21.43 29.61
23.96
0
10 15 20 25 30 35 40 45 50 55 60
Time (min)
105
RT: 9.89 - 59.95

NL:
100 3.50E5
10.17
10.95 TIC F: MS
80 Female_feces
Relative Abundance
12.24 _100_14
16.94 59.83
60 58.57
17.23 57.88
19.05 55.81
40 22.09 53.66
23.73 29.17 51.96
29.46 32.32 35.30 49.24 50.25
20 39.96
0
58.96 NL:
100 1.38E3
m/z=
80 58.07 223.5-224.5+
51.74 249.5-250.5
40.60 54.82 56.97 F: MS
11.06 _100_14
28.55 46.81
18.55 48.64
40 12.94 31.86 36.81 42.82
14.35 19.98 22.73 24.10
17.41 36.61
29.34
20
0
10 15 20 25 30 35 40 45 50 55
Time (min)
RT: 9.89 - 59.67

59.55 NL:
100 3.47E5
10.56 59.15
10.66 TIC F: MS
57.56
80 Female_feces
Relative Abundance
12.13 57.19 _100_15

13.35 55.18
60 15.11 16.94 54.93
53.02
20.25
21.88 51.68
40 25.03 28.63
32.51 44.95 49.98
35.80
37.50 40.85
20
0
58.78 NL:
100 53.23 56.85 1.38E3
47.37
54.41 m/z=
80 223.5-224.5+
249.5-250.5
11.37 F: MS
33.96 48.32
49.61 _100_15
19.77 30.77 34.49 38.30 43.87 46.75
40 14.00
20.70 26.65
18.61 22.80 27.12 32.84
20
0
10 15 20 25 30 35 40 45 50 55
Time (min)
106
RT: 10.06 - 59.89

14.93
100
50.81
80
Relative Abundance
60 14.53 38.08
40
34.72
45.01 51.14 57.56
20.15 28.66
20 37.39 46.77
33.42 39.90 52.07
14.02 16.03 24.80 27.25 40.77
0
46.77
100
80
60
40
39.90
20
50.77 55.70 57.56
12.55 16.90 19.01 21.20 28.57 32.75 33.40 35.22 40.77 46.54
0
15 20 25 30 35 40 45 50 55
Time (min)
Figure 109 Full scan (upper) and SIM (lower) chromatogram of

aromatic remedy A (Lot No. 1)
RT: 10.02 - 59.80

14.97
100
80 58.64
Relative Abundance
14.58
60
50.79
38.10
40
34.74
20 52.05 57.60
50.25
28.68 37.39 39.92 45.01 46.81
14.04 33.44 49.19
16.05 19.94 23.75 27.29 43.21
0
46.81
100
80
60
40
39.92
20
51.80 52.11 55.76
11.62 17.43 19.15 24.74 27.72 33.42 34.74 35.18 44.45 46.59
0
15 20 25 30 35 40 45 50 55
Time (min)

107
RT: 10.09 - 59.59

15.04
100
14.63
80
Relative Abundance
60
58.66
40 38.19 50.88
55.72
14.07 34.81
20 52.18
45.14 50.32
27.34 28.73 33.51
37.48 40.01 48.06
12.80 16.10 21.91 24.89 44.46
0
46.88
100
80
60
40 39.99
20 45.79 52.16 55.83

50.82 54.54
34.81 35.66
13.53 18.89 23.01 25.25 27.79 33.51 45.58
0
15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.00 - 59.78

14.99
100
80 14.58
Relative Abundance
60
22.94
40 53.23
20 38.59 51.08
55.45 57.58
16.03 19.21 25.55 29.63 31.95 35.67 39.92 44.93 46.81
14.02
0
46.81
100
80
60
40
39.92
20
57.56
12.24 17.25 20.91 25.47 29.17 33.19 37.81 38.61 43.58 46.50 50.13 53.23 54.85
0
10 15 20 25 30 35 40 45 50 55
Time (min)

aromatic remedy B (Lot No. 1)
108
RT: 10.09 - 59.74

58.72
100
80
Relative Abundance
14.95
60
40 22.95 38.79
14.58 55.61
53.27
20 51.15
19.26 39.97 46.88
25.51 26.11 35.74 36.18 45.04
12.61
0
46.86
100
80
60
40 39.97
20
54.95 57.62
13.87 16.42 20.15 23.96 25.89 32.04 33.84 38.58 40.28 46.55 51.15 52.14
0
15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.02 - 59.81

14.95 55.77
100
58.60
38.94
80
Relative Abundance
53.24
60 22.93
14.55 51.11
40 46.84
39.93 45.08
20 48.09
19.23 29.64 31.96 34.77 43.24
10.27 20.74 25.48
0
46.82
100
80
60
40
39.93
20
57.61
13.38 15.46 17.99 21.09 24.22 26.60 30.24 36.33 38.56 42.54 46.49 49.20 53.22 54.90
0
15 20 25 30 35 40 45 50 55
Time (min)

109
RT: 10.06 - 59.81

15.20
100
14.72
80
Relative Abundance
60
40
22.98
20
50.58 53.25 58.47
38.63 45.18
12.77 16.09 25.57 30.85 37.41 39.94 46.83
20.04
0
46.81
100
80
60
40 39.94
20
55.74
50.35 51.95
10.85 15.26 19.13 25.92 27.31 31.64 36.27 38.65 44.60 45.18
0
15 20 25 30 35 40 45 50 55
Time (min)

aromatic remedy C (Lot No. 1)
RT: 10.00 - 59.90

15.11
100
80 14.66
Relative Abundance
60
40
22.96
53.27
20 55.76
45.09 46.83 50.54
12.78 16.07 20.04 25.49 30.85 35.69 38.49 39.94
0
46.81
100
80
60
40 39.94
20
58.61
52.15 57.62
12.84 15.18 20.21 22.24 27.66 32.57 37.19 38.66 42.59 46.50 50.52
0
10 15 20 25 30 35 40 45 50 55
Time (min)

110
RT: 10.00 - 59.82

15.26
100
14.76
80
Relative Abundance
60
40
22.98 53.29
20 55.90
45.22 50.64
16.11 25.55 30.87 36.92 38.61 40.93
12.80 20.06
0
46.85
100
80
60
40
39.94
20
57.62 58.61
32.59 36.25 38.68 44.93 46.61 50.40 52.18
10.73 13.62 21.28 22.92 25.53 28.59
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.06 - 59.96

51.19
100
80
Relative Abundance
60
14.93
40 58.47
45.31
38.71 55.55
22.90 50.24
20 14.53 38.05 39.91
20.15 25.47 28.66 33.41 37.36 44.32 49.14
10.21 16.03
0
46.81
100
80
60
40 39.91
20
51.15 54.87 58.47
12.94 19.38 21.43 26.00 31.22 33.41 38.61 43.95
0
15 20 25 30 35 40 45 50 55
Time (min)

aromatic remedy D (Lot No. 1)
111
RT: 10.00 - 59.97

15.01
100
80
Relative Abundance
14.60
60 51.02
40
22.92
58.51
45.22 55.59
20 38.59 50.25
38.08 39.92
16.32 20.17 25.55 28.66 33.42 49.17
12.78 43.23
0
46.81
100
80
60
40 39.92
20
50.87 54.89 58.53
11.97 14.06 19.44 20.97 24.60 29.75 33.42 38.51 44.00 46.59
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.00 - 59.71

15.03
100
80 51.20
Relative Abundance
14.60
60
40 22.94
45.34 58.59
38.72 46.83 52.11 57.60
20 39.94
38.08 47.08
20.19 25.55 28.66
12.78 16.32 33.44 37.39
0
46.83
100
80
60
40 39.94
20
51.10 57.60 58.59
13.40 14.91 20.04 22.63 27.29 28.97 33.44 38.63 43.07 46.48
0
10 15 20 25 30 35 40 45 50 55
Time (min)

112
RT: 10.11 - 59.89

15.09
100
80 14.64
Relative Abundance
60
40
14.06
20 58.61
38.10 57.60
15.88 39.94 44.93 46.83 49.67 54.91
12.78 19.96 24.54 27.29 29.81 34.43 37.08
0
55.76
100
80
60 46.83
40
39.94
20
57.60
13.29 18.76 24.35 27.37 29.09 34.37 38.66 42.28 46.48 49.88 52.15
0
15 20 25 30 35 40 45 50 55
Time (min)

aromatic remedy E (Lot No. 1)
RT: 10.00 - 60.00

15.30
100
14.78
80
Relative Abundance
60
40
20
12.77 16.13 57.58
17.54 24.53 28.65 34.82 38.08 39.92 42.26 46.81 52.07 54.89
0
46.81
100
80
60
_
39.92
40
20
57.58
12.90 15.38 18.80 22.15 25.28 28.20 34.78 36.19 42.26 46.46 47.87 52.09 54.87
0
10 15 20 25 30 35 40 45 50 55 6
Time (min)

113
RT: 10.00 - 59.71

15.34
100
14.83
80
Relative Abundance
60
40
20
12.78 16.15
17.56 24.58 28.68 35.47 38.12
39.96 46.54 46.85 53.25 54.93
57.62
0
46.85
100
80
60
_
40 39.96
20
10.77 14.51 15.98 20.62 23.58 27.33 31.51 36.58 38.65 42.34 46.60 48.07 52.09 54.95 57.62
0
10 15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.03 - 59.96

58.61
100
80
Relative Abundance
60
14.91
40
20 14.53 52.01
54.31
35.34 46.50 46.81 50.52
14.04 16.03 22.90 24.84 29.11 34.12 39.94
0
46.81
100
80
60
40
39.92 58.59
51.99
20
46.38 54.29
41.78 51.86
12.03 14.56 18.05 23.25 30.50 31.99 36.46 38.68
0
15 20 25 30 35 40 45 50 55
Time (min)

aromatic remedy F (Lot No. 1)
114
RT: 10.07 - 59.90

58.65
100
80
Relative Abundance
60
40
55.57
53.21
20
51.08
14.54 14.91 22.88 27.66 29.61 36.46 36.81 38.28 44.89 46.79
22.53
0
46.79
100
80
60
40
39.90 46.54
20 55.70
32.38 35.16 38.39 41.12 45.84 50.48 53.21
13.17 14.83 16.30 23.02 24.60 27.89
0
15 20 25 30 35 40 45 50 55
Time (min)

RT: 10.05 - 59.84

53.27
100
58.61
50.70
80
Relative Abundance
38.82 55.71
45.28
60
40
20 22.90
14.91 39.92 46.81
14.56 27.41 29.63 34.12 35.67
19.23
0
46.81
100
80
60
55.73
40 39.92
20 50.56 54.28 58.59

35.24 44.41 44.91
11.55 12.88 18.36 20.62 25.61 30.37 31.43
0
15 20 25 30 35 40 45 50 55
Time (min)

115
RT: 10.07 - 59.90

58.78
100
80
Relative Abundance
60
40 55.65
20 31.35 31.76
30.85 38.38 50.36 52.36
14.54 14.92 16.03 25.00 27.80 36.81 42.68 45.89
0
55.05
100
52.07
58.59
80
60
50.48
40 46.76 48.75
20 29.67 31.18 37.29 38.65 43.32 44.94

25.13 26.82
10.50 17.33 17.97
0
15 20 25 30 35 40 45 50 55
Time (min)

aromatic remedy G (Lot No. 1)
RT: 10.03 - 59.89

53.23 58.59
100 14.91
80
Relative Abundance
55.65
60
38.55 50.48
45.07
40
14.54 46.81
22.90 39.92
20
16.03 19.21 25.47 29.63 32.28 35.67 48.08
10.41 43.21
0
46.81
100
80
60
40
39.92
20 55.72
10.70 15.92 20.39 22.20 25.24 29.73 34.78 38.53 44.47 46.48 50.35 53.23 57.08
0
15 20 25 30 35 40 45 50 55
Time (min)

116
RT: 10.06 - 59.85

15.01
100
80
Relative Abundance
14.60
60
53.27
40 55.76
51.12
20 22.92 38.63 45.20
39.94 46.83
16.05
19.23 26.09 29.63 32.30 35.70 43.23 48.10
12.78
0
46.83
100
80
60
40
39.94
20
44.93 46.59 50.58 54.80 55.76

11.91 16.07 21.35 27.02 32.30 35.16 37.91
0
15 20 25 30 35 40 45 50 55
Time (min)

41
100 55 71
50 29
82 96 O
111 125 238
63 135 149 163 180 191 203 220
0
20 40 60 80 100 120 140 160 180 200 220 240
(mainlib) Cyclohexadecanone
Figure 130 Mass spectrum of cyclohexadecanone
55
100 41
71
O
50
83 98
29 252
111 125
13 63 135 149 163 176 194 210 223 234
0
10 30 50 70 90 110 130 150 170 190 210 230 250
(mainlib) Cycloheptadecanone
Figure 131 Mass spectrum of dihydrocivetone

117
APPENDIX C
Aromatic Remedies
118
Aromatic Remedy A
Important Ingredients
- โสมเกาหลี, Korean ginseng (Panax ginseng C.A. Mayer)

- พิมเสนเกล็ด, Borneo camphor (Dryobalanops aromatica Gaerth)
- ชะมดเช็ด, Small Indian civet secretion (Viverricula indica Desmarest)
- ใบพิมเสน, Patchouli (Pogostemon cablin (Blanco) Benth.)
- หญาฝรั่น, Saffron (Crocus sativus Linn.)
Use: relief of nausea, dizziness and fever

Direction: 5-7 tablets taken with water or stir one teaspoonful with warm water, when
needed.
Aromatic Remedy B
- รากแฝกหอม, Vetiver root (Vetiveria zizanioides Linn.)

- อบเชยญวณ, Saigon cinnamon (Cinamomum loureirii Nees)
- เห็ดนมเสือ, (Polyporus sacer Fr.)
- โคโรค, Cow bezeoar (Calculas bovis)
Use: relief of tiredness, cough and phlegm

needed.
119
Aromatic Remedy C

- อําพันทอง, Ambergris (Physeter macrocephalus Linn.)
- คุลิกา, Ku-li-ka
Use: for heart stimulating effect and relief of palpitate

Direction: one tablets taken with water or stir one teaspoonful with warm water, when
needed.
Aromatic Remedy D

- เหงาขิงแหง, Ginger (Zingiber officinale Roscoe)
Use: relief of nausea, vomiting and flatulence

needed.
120
Aromatic Remedy E
- กฤษณา, Agarwood (Aquilaria agallocha Roxb.)

- พิกุล, Bullet wood flower (Mimusops elengi Linn.)
Use: for heart stimulating effect, relief of nausea, vomiting, dizziness, tiredness,
flatulence, and nuturing of pregnancy
Direction: 0.25 g stir one teaspoonful with warm water, 3 times a day.
Aromatic Remedy F
Important Ingredient
- จันทนแดง, Red sandal wood (Pterocarpus santalinus Linn.)

- ชะเอมเทศ, Licorice (Glycyrrhiza glabra Linn.)
- ลูกจันทน, Nutmeg (Myristica fragrans Houtt.)
- กานพลู, Clove (Eugenia caryophyllata Thunb.)
- อบเชย, Cinnamon (Cinnamomum cassia J. S. Presl)
- พิมเสน, Borneo camphor (Dryobalanops aromatica Gaerth)
Use: for heart stimulating effect, relief of nausea, dizziness, tiredness, and nuturing of
pregnancy
Direction: 2-3 tablets taken with water, when needed.
121
Aromatic Remedy G

- พิมเสน, Borneo camphor (Dryobalanops aromatica Gaerth)
- กระลําพัก, Triangular Spurge (Euphorbia antiquorum Linn.)
- ขอนดอก, Bullet wood (Mimusops elengi Linn.)
- จันทนแดง, Red sandal wood (Pterocarpus santalinus L.f.)
- แฝกหอม, Vetiver (Vetiveria zizanioides)
- ชะเอม, Licorice (Glycyrrhiza glabra Linn.)
- ทองคําเปลว, Gold leaf
Use: for heart stimulating effect, relief of nausea, dizziness, tiredness, thirst, and
phlegm, and nuturing of pregnancy
Direction: 1-3 tablets taken with water, when needed.
122
Aromatic Remedy without Small Indian Civet secretion
- ดักเฮียง, Korean Mint (Agastache rugosa Fisch. et Mey.) O. Kuntze.

- โสยเซ็ง, Soie-cheng (Asarum sieboldii Mig.)
- ชะเอม, Licorice (Glycyrrhiza glabra Linn.)
- บักเฮียง, โกฐกระดูก, Kote-kra-dook (Saussurea lappa Clarke)
- โกฐสอ, Angelica root (Angelica anomala Lallem)
Use: relief of nausea, dizziness, faint, tiredness, and stomach troubles

Direction: (For adult) Stir one teaspoonful with warm water, 3-4 times a day.
(For Children) one half of the adult dose.
123
APPENDIX D
Publications
124
125
126
Available online www.jocpr.com
Journal of Chemical and Pharmaceutical Research

__________________________________________________
J. Chem. Pharm. Res., 2011, 3(5):196-204

ISSN No: 0975-7384
CODEN(USA): JCPRC5
Quantitative analysis of civetone and normuscone in secretion from

Viverricula indica and in aromatic remedies by gas
chromatography-mass spectrometry
Thidarat Duangyod1, Chanida Palanuvej1*and Nijsiri Ruangrungsi1, 2
1
College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand
2
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical
Sciences, Chulalongkorn University, Bangkok, Thailand
______________________________________________________________________________
ABSTRACT
The quantitation of civetone and normuscone in small Indian civet (Viverricula indica Desmarest)
secretion cultivated in Thailand was analyzed by gas chromatography- mass spectrometry (GC-MS).
Three main chemical constituents of small Indian civet secretion were civetone, dihydrocivetone and
normuscone. The contents of chemical constituents were different between male and female secretion.
Civetone (23.6 ± 1.5 µg/mg of secretion) was a major chemical constituent in the female secretion while
normuscone (52.1 ± 5.9 µg/mg of secretion) was a major in the male secretion. Both civetone and
normuscone were found in civet fur but not in the feces. Aromatic remedies which claimed to use the
small Indian civet secretion as an ingredient showed both civetone and normuscone. Linearity range of
civetone was 0-50 µg/ml with a correlation coefficient of 0.9717, and of normuscone was 0-80 µg/ml with
a correlation coefficient of 0.9965. The average recoveries were 97.3-98.0% in secretion and 91.4-
105.7% in aromatic remedy for civetone. For normuscone, average recoveries in secretion and aromatic
remedy were 98.5 % and 90.0-103.0% respectively. The intra-day and inter-day RSDs of the three
components were less than 8%. Civetone and normuscone could be used as quantitatively marker for
civet secretion ingredient in aromatic remedies. Civetone and normuscone contents in commercial civet
secretion varied crop by crop and depended on male to female sex ratio of the small Indian civets.
Keywords: small Indian civet secretion, aromatic remedy, civetone, normuscone, gas
chromatography-mass spectrometry
______________________________________________________________________________
INTRODUCTION
Small Indian civet is a mammal in a group of carnivores. It is found in Southeast Asia, Pakistan,
India, Nepal, Bangladesh and South China [1]. Both male and female produce the strong
smelling secretion from the perineal gland. Civetone is the main constituent of the secretion that
produced from the civet of the genera Civettictis, Viverra and Viverricula [2]. Moreover, the
196
Chanida Palanuvej et al J. Chem. Pharm. Res., 2011, 3(5):196-204
_____________________________________________________________________________
civet secretion contains other macrocyclic ketones such as cyclohexadecanone,
cycloheptadecanone, and 6-cis-cycloheptadecenone [3]. This secretion is widely used in perfume
industry and in traditional medicine for a long time. Thai traditional medicine uses the secretion
as an ingredient in aromatic remedy for relief of faint, dizziness, nausea and vomiting. In the
local market, there are many aromatic remedies that claim to use the secretion as an ingredient in
the remedies but it cannot prove that there is the secretion in the remedies. Nowadays, the
secretion is adulterated with vaseline and petrolatum to increase the quantity because of the
expensive secretion. In Thailand, there has been no report about the chemical constituents in
secretion of small Indian civet. GC-MS is useful and applicable for qualitative and quantitative
investigation of the chemical composition in complex mixtures for example the essential oil
components as well as phytoconstituent analysis [4-7]. Hence, this study attempted to determine
the chemical constituents of secretion from V. indica cultivated in Thailand and investigate V.
indica secretion ingredient in aromatic remedies by GC-MS analysis.
EXPERIMENTAL SECTION
Chemicals and materials

Civetone (9-cyclohetadecon-1-one, CAS no 542-46-1) and normuscone (cyclopentadecanone,
CAS no 502-72-7) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hexane was of
AR grade (Lab-Scan Asia Co., LTD, Bangkok, Thailand). Polytetrafluoroethylene (PTFE)
syringe membrane filter (0.45 µm) was from Chrom Tech, Inc (USA).
Sample collection
Male small Indian civet (V. indica) secretion (n =15), female small Indian civet secretion (n
=15), pooled small Indian civet secretion (n = 10), small Indian civet feces (n = 15), and small
Indian civet fur (n = 3) were collected from a civet farm in Petchaburi, Thailand. Each sample
was kept in tightly capped vial and refrigerated until analysis.
Three different lot numbers of aromatic remedies with civet secretion ingredient in the label and
one aromatic remedy not containing civet secretion ingredient were collected from the local
markets. Each aromatic remedy was stored at ambient temperature until analysis.
Sample preparation
One milligram of secretion was mixed with 1 ml of hexane, vortex for 1 min, centrifuged at
10,000 rpm for 10 min at 25°C. One microliter of hexane supernatant was analyzed by GC-MS.
One hundreds milligrams of each feces was dissolved in 1 ml of hexane and vortex for 1 min.
This solution was centrifuged at 10,000 rpm for 10 min at 25°C. One microliter of the hexane
supernatant was analyzed by GC-MS.
Fifteen milligrams of small Indian civet fur was washed in aliquots of 2 ml hexane until
exhaustion. Washing hexane aliquots were kept for further analysis. The fur was removed, dried
and cut into fine pieces. Five milligrams of the washed fine pieces of small Indian civet fur was
mixed with 1 ml of hexane and sonicated at 30°C for 15 min at 53 KHz. Then, it was centrifuged
at 10,000 rpm for 10 min at 25°C. Hexane extract as well as washing hexane aliquots were
analyzed by GC-MS.
One hundreds milligrams of each aromatic remedy was mixed with 1 ml of hexane and vortex
for 1 min. Then, it was filtered through 0.45 µm PTFE membrane filter and evaporated. After
this, the extract was adjusted to 250 µl of hexane and vortex again. The solution was analyzed by
GC-MS.
197
_____________________________________________________________________________
Each sample was performed in triplicates.
Instruments and chromatographic conditions

The analysis was performed using a Finnigan trace GC ultra gas chromatography (Thermo Fisher
Scientific Inc., USA) equipped with ZB-5 capillary column (30m x 0.25mm x 0.25µm) and
interfaced to a Finnigan trace DSQ MS detector. The oven temperature was ramped from 60°C to
240°C at a constant rate of 3°C/min. The injection port was held at 180°C throughout the
separation. The carrier gas was helium with a flow rate of 1ml/min and split ratio of 10:1. MS
was performed by electron ionization (EI) mode at 70 electron volts.
Identification and determination of compounds

The chemical constituents in the secretion extract were identified by matching their mass spectra
and retention time indicated with Adams Essential Oils Mass Spectral library and NIST 05 Mass
Spectral library. The contents of civetone and normuscone in secretion were determined by
comparing the area under peak with the calibration. The average contents were expressed as
grand mean ± pooled standard deviation in µg / mg of secretion.
Method validation
- Calibration curve and linearity
Stock solution of civetone (1mg/ml) was prepared by dissolving 1.1 µl of civetone (density =
0.917 at 33°C) in 1 ml of hexane. Stock solution of normuscone (1mg/ml) was prepared by
dissolving 1 mg of normuscone in 1 ml of hexane. The stock solutions were diluted at various
concentrations for calibration curves and linearity range.
- Limit of detection (LOD) and limit of quantitation (LOQ)

LOD and LOQ determination were based on the standard deviation of the blank. The triplicates
of 1 mg/ml of aromatic remedy without civet secretion ingredient (blank sample) were prepared
and analyzed. LOD and LOQ were calculated as follow [8]:
LOD = mean of blank sample + 3SD
LOQ = mean of blank sample + 10SD
- Precision
The precision of the method was assessed with intra-day and inter-day analyses. For
repeatability, different concentration levels (3 concentrations / triplicate) which covered the
specified range were analyzed on day 1 and this were repeated on 3 consecutive days. Relative
standard deviation (RSD) was used to measure precision [8].
- Recovery
The extraction efficiency method was used for recovery evaluation of civet secretion by re-
extracting the residue until exhaustion [9] and determining civetone and normuscone by GC-MS.
The extraction of civetone and normuscone was performed at two concentrations of the secretion
(1 and 2 mg/ml). The percentage of recovery was calculated as follow: % recovery =
(Concentration in the first filtrated x 100)/ Concentration in sum of filtrated.
Recovery of aromatic remedy was carried out by spiking three concentrations of standard
solution. Recovery (%) = (As-A)/Aa x 100. As refers to the amount of civetone or normuscone
that found after spiking of the standard solution whereas A refers to the amount of those found
198
_____________________________________________________________________________
that before spiking and Aa refers to the amount of reference standards actually added to the
sample. The average recoveries of every spiking concentration were calculated.
RESULTS AND DISCUSSION
Small Indian civet secretion constituents

Three main chemical constituents of male small Indian civet secretion (Figure 1) were
normuscone, dihydrocivetone and civetone with the percent area of 73.4 ± 7.0, 5.1 ± 1.5, and 3.2
± 1.9 % respectively. The female small Indian civet secretion exhibited four main chemical
constituents (Figure 2) including civetone, dihydrocivetone, normuscone, and
cyclohexadecanone with the percent area of 56.1 ± 5.2, 16.8 ± 2.9, 11.8 ± 2.1, and 3.2 ± 0.6 %
respectively.
Figure 1 GC chromatogram of male small Indian civet secretion
Figure 2 GC chromatogram of female small Indian civet secretion
199
_____________________________________________________________________________
Figure 3 GC chromatogram of pooled small Indian civet secretion

55
100
41
O
81
250
50
95
69
29 109
121
135 162
14 32 149 175 189 207 217 233
0
10 30 50 70 90 110 130 150 170 190 210 230 250
(ma inlib ) 9-Cyc lohep ta d ecen-1-one, (Z)-
Figure 4 Mass spectrum of civetone

55
100
41
O
71
58
50
83 96
29
111 125
135 224
149 166 177 191 206
0
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
(ma inlib ) Cyc lop enta d eca none
Figure 5 Mass spectrum of normuscone
Commercial civet secretion from civet farms did not divide male and female secretion. The
secretion was gathered from all small Indian civets every morning and pooled together. Ten
crops of secretion were analyzed and found that the secretion consisted of civetone,
dihydrocivetone and normuscone as main components which were related to the previous study
[10]. Cyclohexadecanone which found only in the secretion of female civet could be expressed
in minor component (Figure 3).
Linearity
The calibration curves were constructed by plotting the peak area of the standards against their
concentration. The regression equations for the linear portion of the standard curves of civetone
and normuscone were y = 910946x and y = -735543 + 354744x respectively. Linear calibration
200
_____________________________________________________________________________
curves were obtained with good correlation (r2 = 0.9717 and 0.9965) for civetone and
normuscone respectively. Linearity range of civetone was 0-50 µl/ml and of normuscone was 0-80
µl/ml (Figure 6, 7).
civetone
Y = 910946*X R^2 = 0.9717 W: Equal
50000000
40000000
Area Ratio
30000000
20000000
10000000
0
0 10 20 30 40 50
Figure 6 Calibration curve of civetone
Normuscone
Y = -1.06378e+006+359534*X R^2 = 0.9965 W: Equal
25000000
20000000
Area Ratio
15000000
10000000
5000000
0
0 20 40 60 80
Figure 7 Calibration curve of normuscone
LOD and LOQ

LOD and LOQ for civetone were 0.0087 and 0.0165 µg/mg of secretion and for normuscone
were 0.0596 and 0.1154 µg/mg of secretion respectively.
Recovery
The triplicates of each concentration group were analyzed for the recovery. This recovery was
shown in percentage amount of civetone and normuscone which extracted from the sample to
validate the method. The average recoveries were 97.3-98.0% in secretion and 91.4-105.7% in
aromatic remedy for civetone. For normuscone, average recoveries in secretion and aromatic
remedy were 98.5% and 90.0-103.0% respectively. The results demonstrated that the method
was sufficiently accurate for determination.
Precision
The intra-day and inter-day precision of civetone and normuscone quantitation were determined.
The results were presented in Table 1. The intra-day and inter-day RSDs were less than 8%
which shown that the method was precise.
201
_____________________________________________________________________________
Table 1 Percentage of relative standard deviation of intra-day and inter-day analysis
Concentration (mg/ml) Intra-day RSD% Inter-day RSD% (n=3)

Day 1 (n=3) Day 2 (n=3) Day 3 (n=3)
Civetone 0.025 3.212 0.821 1.276 0.472
0.5 1.008 3.530 5.507 0.139
1 1.660 4.693 3.185 0.180
normuscone 0.025 3.446 4.739 1.889 0.531
0.5 1.117 4.856 7.649 0.929
1 1.114 3.920 0.872 0.464
Civetone and normuscone contents in small Indian civet secretion

Table 2 demonstrated the concentration of chemical constituents which was different between
secretion of male and female small Indian civet. Civetone was dominated in female whereas
normuscone was dominated in male small Indian civet secretion. This finding was in accordance
with the civet in China [11]. Analysis of secretion crops which containing both male and female
secretion showed higher concentration of normuscone than civetone. This was in accordance
with higher male small Indian civets in the farm.
Table 2 The concentration of civetone and normuscone in small Indian civet secretion obtained by GC-MS
Male Female Crop

Civetone 0.788±0.138 23.614±1.469 5.931 ± 1.728
Normuscone 52.121±5.931 19.218±1.584 22.304 ± 5.162
Determination of civetone and normuscone in small Indian civet feces

There was no civetone and normuscone in the chemical constituents of small Indian civet feces.
Figure 8 GC chromatogram of small Indian civet fur
Determination of civetone and normuscone in small Indian civet fur

The collected civet secretion was frequently mixed up with civet fur. The fur sticked with civet
was one of commercial products form civet farms. However after exhausted washing, it was still
found that the small Indian civet fur presented four chemical constituents (Figure 8) including
civetone, dihydrocivetone, normuscone, and cyclohexadecanone that related to the small Indian
civet secretion. The concentration of civetone and normuscone in small Indian civet fur were
202
_____________________________________________________________________________
0.23 ± 0.09 and 1.27 ± 0.17 µg/mg of washed fur respectively. Besides full scan analysis of mass
spectrum, selected ion monitoring (SIM) of civetone and normuscone were analyzed for
confirmation.
Table 3 The concentration of civetone (µg/mg of sample) in aromatic remedies obtained by GC-MS
No. of aromatic remedies Lot number Mean SD

1 2 3
A 3.679 ± 0.32 2.785 ± 0.169 3.727 ± 0.773 3.397 0.531
B 2.913 ± 0.263 3.772 ± 0.341 6.876 ± 0.355 4.520 2.085
C 4.259 ± 0.323 4.540 ± 0.141 5.523 ± 0.521 4.774 0.663
D 10.011 ± 1.471 5.045 ± 0.459 9.686 ± 1.502 8.247 2.778
E 15.696 ± 1.601 10.386 ± 0.713 11.158 ± 1.152 12.413 2.869
F 1.233 ± 0.084 0.830 ± 0.520 1.155 ± 0.250 1.073 0.213
G < LOQ 3.108 ± 0.213 5.978 ± 0.321 3.029 2.990
Table 4 The concentration of normuscone (µg/mg of sample) in aromatic remedies obtained by GC-MS
No. of aromatic remedies Lot number Mean SD

1 2 3
A 8.225 ± 0.429 6.441 ± 0.552 8.593 ± 0.782 7.753 1.151
B 6.754 ± 0.727 9.219 ± 1.54 14.739 ± 0.799 10.238 4.087
C 10.447 ± 0.979 10.767 ± 1.257 13.846 ± 1.069 11.687 1.877
D 23.696 ± 2.701 10.792 ± 0.167 13.106 ± 2.467 15.865 6.880
E 41.040 ± 0.585 27.792 ± 2.162 24.251 ± 2.140 31.028 8.849
F 2.874 ± 0.884 2.089 ± 0.474 3.313 ± 0.186 2.759 0.620
G < LOQ 6.644 ± 0.520 12.159 ± 0.675 6.268 6.088
RT: 10.09 - 60.00

15.38 NL:
100 1.62E8
14.82 TIC F: MS
80 Pulprasit_lot2
Relative Abundance
_02
60
40
20
16.17 52.05 57.58
12.78 17.56 24.56 28.68 36.13 38.08 39.94 42.26 46.81
0
46.81 NL:
100 1.75E5
m/z=
80 223.5-224.5+
249.5-250.5
F: MS
60 Pulprasit_lot2
_02
39.92
40
20
52.07 57.56
11.43 16.65 18.61 21.16 25.49 32.30 34.12 38.63 42.26 48.66
0
15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 9 Full scan (upper) and SIM (lower) chromatogram of aromatic remedy
Determination of civetone and normuscone in aromatic remedies

Civetone and normuscone were found in all aromatic remedies which claimed to use the small
Indian civet secretion as an ingredient. The concentration of normuscone was higher than
civetone in all remedies. The different in content of civetone and normuscone among each
aromatic remedy might be due to specific formulary. The difference content of civetone and
normuscone among each lot of same remedy might be due to the variety of each crop of civet
secretion.
203
_____________________________________________________________________________
CONCLUSION
The chemical constituents of Thai V. indica secretion were different between male and female.
Either civetone or normuscone could be used as marker for civet secretion ingredient in Thai
traditional medicine products including aromatic remedies. The quality control of civet secretion
on crude drug should be concern for sex dependent chemical compositions. The GC-MS method
is precise and accurate for civetone and normuscone determination in small Indian civet
secretion as well as in aromatic remedies.
Acknowledgements
The authors are thankful to the Herbal Remedies and Alternative Medicine Task Force of STAR:
Special Task Force for Activating Research under 100 Years Chulalongkorn University Fund.
The authors are also grateful to College of Public Health Sciences, Chulalongkorn University
and all the staff members for necessary assistance and instrumental support.
REFERENCES
[1] CM Francis. Field guide to the mammals of South-East Asia, Princeton University Press,
New Jersey, 2008; 289.
[2] JW Wheeler; DM Lay; MS Bulm; PJ Weldon. Biochemical systematics and ecology, 1998, 26, 457-458.
[3] H Surburg; J Panten. Fragrance and flavor materials, 5th Edition, Wiley – VHC, Germany, 2006,
185.
[4] RK Jananie; V Priya; K Vijiyalakshmi. Journal of Chemical and Pharmaceutical Research,
2011, 3(4), 460-466.
[5] S Gopalakrishnan; K Saroja; JD Elizabeth. Journal of Chemical and Pharmaceutical
Research, 2011, 3(3), 477-480.
[6] N Rajeswari; S Lakshmi; K Muthuchelian. Journal of Chemical and Pharmaceutical
Research, 2011, 3(3), 792-798.
[7] FS Sharopov; IS Gulmurodov; WN Setzer. Journal of Chemical and Pharmaceutical
Research, 2010, 2(6), 284-290.
[8] http://www.ich.org/products/guideline/quality/article/quality-guideline.html
[9] http://www.aoac.org/Official_Methods/slv_guidelines.pdf
[10] O Yukio; T Shigeyuki. Japan Analyst, 1977, 26(4), 232-236.
[11] H Lianghua; L Hauchong. Fenxi Huaxue, 1983, 10(11), 781-783.
204
136
VITA
Miss Thidarat Duangyod was born on January 30, 1987 in Songkhla, Thailand.
She receives her Bachelor’s degree of Applied Thai Traditional Medicine with first class
honor from School of Health Sciences, Mae Fah Luang University, Thailand in 2009.
Publications
1. Duangyod, T., Palanuvej, C., and Ruangrungsi, N. Quantitative Analysis of

Civetone and Normuscone in Secretion from Viverricula indica and in Aromatic
Remedies by Gas Chromatography-Mass Spectrometry. Journal of Chemical and
Pharmaceutical Research 3 (2011): 196-204.
2. Duangyod, T., Palanuvej, C., and Ruangrungsi, N. Chemical Constituents of

Viverricula indica Secretion. Proceedings of the 9th Joint Seminar Natural Medicine
Research for the Next Decade: New Challenges and Future Collaboration, pp. 85-86.
Bangkok, 2010.

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QUANTITATIVE ANALYSIS OF CIVETONE AND NORMUSCONE IN SECRETION FROM

VIVERRICULA INDICA AND IN AROMATIC REMEDIES BY

Miss Thidarat Duangyod

A Thesis Submitted in Partial Fulfillment of the Requirements

The author is sincerely grateful to her thesis co-advisor, Associate Professor

Gratitude is grateful to the thesis committee members, Professor Dr. Surasak

Appreciation is also extended to College of Public Health Sciences,

LIST OF FIGURES xii

1 Percentage of relative standard deviation of intra-day and inter-day

2 The percentage recoveries of civetone and normuscone in civet

5 The concentration of civetone and normuscone in small Indian civet

6 The concentration of civetone (µg/mg) in aromatic remedies

7 The concentration of normuscone (µg/mg) in aromatic remedies

11 Civetone and normuscone contents (µg/mg of secretion) in male

12 Civetone and normuscone contents (µg/mg of secretion) in female

13 Civetone and normuscone contents (µg/mg of aromatic remedy) in

14 Civetone and normuscone contents (µg/mg of aromatic remedy) in

15 Civetone and normuscone contents (µg/mg of aromatic remedy) in

16 Civetone and normuscone contents (µg/mg of aromatic remedy) in

17 Civetone and normuscone contents (µg/mg of aromatic remedy) in

18 Civetone and normuscone contents (µg/mg of aromatic remedy) in

19 Civetone and normuscone contents (µg/mg of aromatic remedy) in

1 Left footprints of front (F) and hind (H) feet of civets 4

2 Scent gland of Viverricula indica 5

3 Scent gland of Viverra tangalunga 5

4 Asian palm civet (Paradoxurus hermaphroditus) 6

5 Civet coffee (Kopi Luwak) 6

6 Viverricula indica Desmarest 8

7 Small Indian civet in a cage 9

8 Pa Noi’s civet farm 9

9 Small Indian civet secretion 10

10 Small Indian civet secretion which placed on betel vine 11

11 Thai traditional processing for civet secretion cleaning 11

15 A small Indian civet secretion in the bamboo cage 24

16 Small Indian civet cages 25

17 Male and female small Indian civet secretion 25

18 Small Indian civet furs 26

19 Male and female small Indian civet feces 26

20 GC chromatogram of male small Indian civet secretion 27

21 GC chromatogram of female small Indian civet secretion 27

22 GC chromatogram of pooled small Indian civet secretion 28

23 Mass spectrum of civetone 29

24 Mass spectrum of normuscone 29

25 Calibration curve of civetone 30

26 Calibration curve of normuscone 30

28 GC chromatogram of small Indian civet furs 34

29 GC chromatogram of male small Indian civet feces 34

30 GC chromatogram of female small Indian civet feces 35

31 Calibration curve of civetone determination by GC/MS 57

32 Calibration curve of normuscone determination by GC/MS 57

33 GC chromatogram of male small Indian civet (1) 68

34 GC chromatogram of male small Indian civet (2) 68

35 GC chromatogram of male small Indian civet (3) 69

36 GC chromatogram of male small Indian civet (4) 69

37 GC chromatogram of male small Indian civet (5) 70

38 GC chromatogram of male small Indian civet (6) 70

39 GC chromatogram of male small Indian civet (7) 71

40 GC chromatogram of male small Indian civet (8) 71

41 GC chromatogram of male small Indian civet (9) 72

42 GC chromatogram of male small Indian civet (10) 72

43 GC chromatogram of male small Indian civet (11) 73

44 GC chromatogram of male small Indian civet (12) 73

45 GC chromatogram of male small Indian civet (13) 74