Develop a Sample Preparation Procedure for HPLC Analysis of Glucosinolates in
Traditional Chinese Medicines
By
LEE Kim Chung
(02010364)
A thesis submitted in partial fulfillment of the requirements
for the degree of
Bachelor of Science (Honours)
in Applied Chemistry
(Concentration in Environmental Studies)
at
Hong Kong Baptist University
22/04/2005
ACKNOWLEDGEMENT
This is an ongoing project and has been done by graduated students, Miss C.Y. Cheung, Miss
W.M. Au and Mr. C.K. Kwong.
The negative electrospray ionization-quadrupole time-of- flight
mass spectrometry and MS/MS analysis of glucosinolate standards, vegetable and Traditional
Chinese Medicine (TCM) samples were done by Dr. Zongwei Cai’s M. Phil students, Mr. W.T.
Ma and Mr. W. Chan.
All other experiments described in this thesis were my own original work
and were carried out by myself under the supervision of Dr. Zongwei Cai. Thank you for his
valuable advice and guidance for me in this project.
Thank you for Prof. Albert W.M. Lee as my observer and provide me Rorippa indica (Linn.)
Hiern sample [蔊菜].
Thank you for Dr. Zhong-zhen Zhao provide me Leaf of Isatis indigotica Fort. sample [大青葉].
Thank you for Dr. Zongwei Cai’s M. Phil students, Mr. W.T. Ma and Mr. W. Chan as well as his
PhD student, Miss Q. Luo and laboratory technician, Mr. John Ng who have helped me a lot in
this project.
Signature of Student
Student Name
Department of Chemistry
Hong Kong Baptist University
Date:
II
Develop a Sample Preparation Procedure for HPLC Analysis of Glucosinolates in
Traditional Chinese Medicines
By
LEE Kim Chung
(02010364)
Department of Chemistry
ABSTRACT
Glucosinolates which are β-D-thioglucoside-N-hydroxysulfates found in the plant family
Cruciferae, especially in Brassica. A reversed-phase HPLC method using Hypersil BDS C18
column was developed for analyzing twelve intact glucosinolates (glucoiberin, glucocheirolin,
progoitrin,
sinigrin,
epiprogoitrin,
glucoraphenin,
sinalbin,
gluconapin,
glucosibarin,
glucotropaeolin, glucoerucin, gluconasturtiin) in three vegetable and ten Traditional Chinese
Medicine (TCM) samples.
and evaporation.
The samples were extracted with methanol, followed by filtration
Interferences in the organic sample extracts were removed by using activated
Florisil solid-phase extraction.
A gradient program and mobile phases using methanol and
30mM ammonium acetate at pH5.0 allowed sufficient retention and baseline separation of the
glucosinolates in the sample extracts.
at 233nm.
Individual glucosinolates were detected by a UV detector
Further confirmation was done by using liquid chromatography mass spectrometry.
The glucosinolate concentrations in the sample extracts were determined by using an external
calibration method.
Detection limits of the glucosinolates were 25.2µg/g, 7.8µg/g, 1.8µg/g,
5.7µg/g, 8.3µg/g, 10.8µg/g, 24.2µg/g, 19.8µg/g, 3.3µg/g, 14.7µg/g, 4.3µg/g and 17.5µg/g,
respectively, when 5g of dried TCM was analyzed.
The average recovery (accuracy) of the
method was 99.8 % and the precisions were from 5.3% to 14.6% (relative standard derivation,
n=3) respectively.
III
Contents
1. Introduction
P.1-P.6
2. Experimental preparations and procedures
2.1
Chemicals and reagents
P.7
2.1
Vegetable and Traditional Chinese Medicine (TCM) samples
2.1.1
Vegetable samples
P.7
2.1.2
Traditional Chinese Medicine (TCM) samples
P.8
2.3
Preparation of individual intact glucosinolate standard solutions
P.9
2.4
Preparation of intact glucosinolate standard mixture solutions
P.9
2.5
Preparation of vegetable and Traditional Chinese Medicine
(TCM) samples
2.5.1
Sample grinding and extraction
2.5.2
Clean-up process
P.9-P.10
P.10
2.6
Preparation of buffer solution
P.11
2.7
HPLC analysis
P.11-P.12
2.8
Mass spectrometry analysis
P.12-P.13
3. Experimental results and analysis
3.1
Qualitative analysis
3.1.1
3.1.2
3.1.3
3.2
Determination of the retention times for
each intact glucosinolate standard
P.13-P.14
Identification of the glucosinolates in vegetable
and Traditional Chinese Medicine (TCM) samples
by using reversed-phase HPLC analysis
P.15-P.16
Identification of the glucosinolates in vegetable
and Traditional Chinese Medicine (TCM) samples
by using ESI-QTOF-MS and MS/MS analysis
P.16-32
Quantitative analysis
3.2.1
Calibration curves for each individual glucosinolate standard
IV
P.33-P.34
3.2.2
Detection limits of each glucosinolate
P.35
3.2.3
Method recoveries of each glucosinolate
P.36-37
3.2.4
Glucosinolate concentrations in vegetable and
Traditional Chinese Medicine (TCM) samples
P.38-39
4. Discussion
4.1 Extraction
P.40
4.2 Clean-up process
P.40-P.42
4.3 Optimization of buffer system
P.42
4.4 Gradient program
P.42-P.43
4.5 Optimization of glucosinolate concentrations in the sample extracts
P.43-P.44
5. Conclusion
P.45
6. Future plan
P.45-P.46
7. References
P.47-P.48
V
1. Introduction
Glucosinolates are β-D-thioglucoside-N-hydroxysulfates.[1]
More than 120 individual
glucosinolates differing from each other in their structures of their glycon mioties have been
identified: these generally classified as alkyl, aliphatic, alkenyl, hydroxyalkenyl, aromatic, or
indole.[2] The diversity of the R Group leads to a wide variation in the polarity and biological
activity of the natural products.[3] The glucosinolates generally occur in the form of the
sodium or potassium salt, and the general structure is shown in Figure 1.[4,5]
CH2OH
ΟΗ
Ο
R
S
C
NOSO3
OH
OH
Figure 1: General structure of glucosinolates
Glucosinolates are a class of approximately 100 plant secondary metabolites which contained
in the seeds, roots, stems, and leaves of plants belonging to 11 families of dicotyledonous
angiosperms of which the crucifers are certainly the most important.[6] Structural types and
individual concentrations differ according to various factors, for example, species, tissue type,
physiological age, and plant health and nutrition.[2]
Glucosinolate concentrations in the
reproductive tissues (florets/ flowers and seeds) are often as much as 10-40 times higher than
in vegetative tissues. [2] Plant myrosinase is widespread in seeds and tissues of the family
Cruciferae and catalyzes the hydrolysis of glucosinolates which are also contained in plant
vacuoles of the cruciferous plants.[7] This reaction produces goitrogenic and potentially
hepatoxic compounds e.g. isothiocyanates, thiocyanates, nitriles, and thiones,[7] depending on
reaction conditions such as pH, temperature, metal ions, protein cofactors, and the properties
1
of the side chain. [2]
β⎯D⎯glucose
S
R
C
N
OSO3
Myrosinase, H2O
C H
2
O H
S
R
O H
Ο
Ο Η
C
+
+
O H
N
HSO4
O H
unstable aglucon
Glucose
hydrogen suphate ion
pH3, Fe2+
> pH7
R
S
C
Thiocyanate
N
R
S
C
N
R
Isothiocyanate
C
Nitrile
N
S
Sulfur
Figure 2: Degradation of glucosinolate by myrosinase in the presence of water[8]
The great number of the individual glucosinolates produces a large range of flavours as well as
toxic effect upon consumption.[9,10] Glucosinolates have long been known for the fungicidal,
bacteriodical, nematocidal, and allelopathic properties.[1]
The activity of isothiocyanates
such as sulforaphane against numerous human pathogens, for example, Escherichia coli,
Salmonella typhimurium and Candida spp. could even contribute to the medicinal properties
ascribed to cruciferous vegetables, such as cabbage and mustard.[1]
Glucosinolate hydrolysis products, especially the isothiocyanates, were demonstrated that
these molecules affect human health, either beneficially or adversely.[2] Several mechanism
have been proposed to the cancer prevention by breakdown products from cruciferous
vegetables. And they have been proposed to act as blocking agents against carcinogenesis by
2
quinone reductase activity.[11] Moreover, glucosinolates and derived products would prevent
carcinogen molecules from reaching the target site or interacting with the reactive
carcinogenic molecules or activating the important hepatic enzymes for the protection against
several carcinogens.[11] while some glucosinolates and their breakdown products are found to
have anti-nutritional effect in cattle.[12]
For the determination of glucosinolates, it is performed either by indirectly measuring the
enzymatic degradation products or by directly determining the intact glucosinolates.[13]
However, enzymatically or chemically released products such as isothiocyanates,
oxazolidinethiones, thiocyanate ion, sulfate, nitrile or glucose are also measured in the direct
analysis.[13] Since, the direct analysis of the intact glucosinolates can reflect the specificity
for the analysis of each individual glucosinolate. Therefore, the direct analysis of intact
glucosinolates is also used. [13]
Product analyzed
1.Total glucosinolates
Method of dramatization and identification
Main references
I. Palladium chloride for tetrachloropallidate assay Moller et al.(1985); Bennert and Pauling (1988)
II. Thymol assay
Tholen et al.(1989); Bennert and Pauling(1988)
III. Glucose-release enzyme-coupled assay
Heaney et al. (1988)
IV. Sulphate-release assay
Schung (1987, 1988)
V. ELISA
Van Doorn et al. (1998)
VI. Near infra-red reflectance(NIR) spectroscopy
Velasco and Becker (1998)
VII. Alkaline degradation and thioglucose detection Jesek et al. (1999)
2. Individual intact
I. Reverse phase HPLC-MS
Moller et al. (1985); Bjerg and Sorenson (1987);
glucosinolates
Hogge et al. (1988); Kokkonen et al. (1991)
Prestera et al. (1996); Lewkw et al. (1996);
Zrybko et al. (1997); Schutze et al. (1999)
Kaushik and Agnihotri (1999)
II. Thermospray LC with tandem MS
Heeremans et al. (1989)
III. High performance capillary electrophoresis
Arguello et al. (1999)
IV. Capillary GC-MS, GC-MS, GC-MS-MS
Shaw et al. (1987, 1989)
3. Desulphoglucosinolates I. Reverse phase HPLC
Fenwick et al. (1983); Quinsac et al. (1991)
3
Heaney and Fenwick (1993); Bjergegaard et al.
(1995); Hrncirik and Velisek (1997); Robertson
and Botting (1999); Griffiths et al. (2000)
4. Degradation Products
II. X-ray fluorescence spectroscopy (XRF)
Schung and Hane Klaus, (1990)
I. GC or GC-MS
Velisek et al. (1990); Daxenbichler et al. (1991)
II. HPLC (all degradation products)
Matthaus and Fiebig (1996)
II. HPLC (fluorescent labeled products)
Karcher and EI Rassi (1998)
III. HPLC ( 1,2-benzenedithiol derivatives of
Jiao et al. (1998)
isothiocyanates
Table 1: Some of the commonly used methods for the quantitative and qualitative
analysis of the intact glucosinolates, desulphoglucosinolates, and their
breakdown products [14]
Various alternative methods e.g. GC analysis of the trimethsilyl (TMS) derivatization of
glucosinolates
and
high-performance
liquid
chromatography
(HPLC)
of
the
desulfoglucosinolate have been used for direct or indirect determination of total glucosinolate
and individual glucosinolates.
often used. [15]
GC-MS analysis of the glucosinolate breakdown products is
After a simple clean-up process, the hydrolysis products were determined
qualitatively and quantitatively by GC-FID.
[16]
However, some side chains of the
glucosinolates are non-volatile or breakdown products are unstable for the determination. [15]
Therefore, the most suitable technique is to use the HPLC analysis of the enzymatically
desulfated glucosinolates.
Desulfated glucosinolate gives a better separation.
However,
desulfated glucosinolates are often subject to the difficulties in interpreting results of the
individual glucosinolates due to concerns over the effect of pH value, time, and enzyme
concentration on desulfation products.[19]
Therefore, the direct analysis of the intact
glucosinolates is needed for more specific and accurate determination, for better interpretation
of analytical results, for the reduction of analytical time. [13]
It is an ongoing project, three more glucosinolate standards would be analyzed and clean-up
process is also studied.
Twelve intact glucosinolate standards including epiprogoitrin,
4
glucocheirolin,
glucoerucin,
glucoiberin,
gluconapin,
gluconasturtiin,
glucoraphenin,
glucosibarin, glucotropaeolin, progoitrin, sinalbin, sinigrin are determined qualitatively and
quantitatively by using the reversed-phase high-performance liquid chromatography (HPLC).
The trivial and chemical names, chemical formulas, and chemical structures of side-chains and
molecular weights of intact glucosinolate standards used for analysis were shown in Table 2.
The retention times of each of the intact glucosinolates in the HPLC column depend on the
polarities of the glucosinolates in the differences by the R groups.
Longer side chain or
containing of the aromatic ring in the R group will make it relative non-polar.
On the other
hand, shorter side chain or containing the polar R group will make it relative polar.
Therefore, the HPLC can be used for analysis.
5
Trivial Name and Chemical
formula of glucosinolate (GS)
Chemical Name of
glucosinolate (GS)
Chemical structure of Molecular weight b,
g/mol
R Group a
O
Glucoiberin
(C11H20NO10S3)
Glucocheirolin
(C11H20NO11S3)
3-(methylsulfinyl)propyl-GS
H3C
S
H2
C
H2
C
H2
C
423.0327
H2
C
H2
C
H2
C
439.0277
O
3-(Methylsulfonyl)propyl-GS
H 3C
S
O
OH
Progoitrin
(C11H18NO10S2)
Sinigrin
(C10H16NO9S2)
(2R)-2-Hydroxybut-3-enyl-GS
H 2C
Prop-2-enyl-GS
C
H
H2C
C
H2
C
H
(2R)
H2
C
C
H
389.0450
359.0345
H
Epiprogoitrin
(C11H18NO10S2)
(2S)-2-Hydroxybut-3-enyl-GS
H 2C
C
H
H2
C
C
(2S)
OH
389.0450
O
Glucoraphenin
(C12H20NO10S3)
4-(methylsulfinyl)but-3-enyl-GS
Sinalbin
(C14H18NO10S2)
p-Hydroxybenzyl-GS
Gluconapin
(C11H18NO9S2)
But-3-enyl-GS
H3C
S
C
H
H2
C
C
H
H2
C
H2
C
HO
H2 C
H2
C
H2
C
C
H
435.0327
425.0450
373.0501
OH
Glucosibarin
(C15H20NO10S2)
(2R)-2-Hydroxy-2-phenethyl-GS
H2
C
C
439.0607
H
Glucotropaeolin
(C14H18NO9S2)
Benzyl-GS
Glucoerucin
(C12H22NO9S3)
4-(Methylthio)butyl-GS
Gluconasturtiin
(C15H20NO9S2)
Phenethyl-GS
H2
C
H3C
S
H2
C
H2
C
H2
C
409.0501
H2
C
H2
C
H2
C
421.0535
423.0658
Remarks: a = the side-chain R in the general structures shown in Figure 1
b
= Molecular weights of intact glucosinolates
Table 2: The trivial and chemical names, chemical formulas, and chemical structures of
side-chains and molecular weights of intact glucosinolate standards used for
analysis
6
2. Experimental preparations and procedures
2.1 Chemicals and reagents
Epiprogoitrin,
glucocheirolin,
glucoerucin,
glucoiberin,
gluconapin,
gluconasturtiin,
glucoraphenin, glucosibarin, glucotropaeolin, progoitrin, sinalbin were obtained from KVL
(Frederiksberg C, Denmark).
Sinigrin was obtained from Sigma (St. Louis, U.S.A.).
HPLC-grade hexane and methanol were obtained from Riedel-de Haën® (Hanover, Germany).
HPLC-grade dichloromethane and ethyl acetate were obtained from Tedia (Fairfield, U.S.A.)
Ammonium acetate was obtained from Panreac (Barcelona, Spain) and formic acid was from
Merck (Darmstadt, Germany).
Milli-Q water was produced by using a Milli-Q® Ultrapure
Water Purification Academic System from Millipore (Billerica, U.S.A.).
2.2 Vegetable and Traditional Chinese Medicine (TCM) samples
2.2.1 Vegetable samples
Three vegetable samples were analyzed and shown in Table 3.
They were purchased from
Wellcome supermarket in Hong Kong.
Local name
Scientific name
Family name
I. Chinese Radish [蘿蔔]
Raphanus sativus
Cruciferae [十字花科]
II. Cherry Tomato [車厘茄]
Lycopersicon esculentum
Solanaceae [茄科]
III. Tomato [番茄]
Lycopersicon esculentum
Solanaceae [茄科]
Table 3: Local, Scientific and Family names of the vegetable samples used for analysis
7
2.2.2 Traditional Chinese Medicine (TCM) samples
Ten TCM samples were analyzed and shown in Table 4.
Leaf of Isatis indigotica Fort. [大青
葉] was a gift from Dr. Zhong-zhen Zhao, School of Chinese Medicine, Hong Kong Baptist
University. Rorippa indica (Linn.) Hiern [蔊菜] was a gift from Prof. Albert W.M. Lee,
Department of Chemistry, Hong Kong Baptist University.
The other TCM samples were
purchased from Mr. & Mrs. Chan Hon Yin Chinese Medicine Specialty Clinic & Good
Clinical Practice Centre, Hong Kong Baptist University.
Local name
Scientific name
Family name
1. 北板藍根
Root of Isatis indigotica Fort.
Cruciferae [十字花科]
2. 南板藍根
Root of Baphicacanthus cusia (Nees) Bremek.
Acanthaceae [爵床科]
3. 敗醬草
Patrinia scabiosaefolia Fisch. ex Trev.
4. 菥蓂
Thlaspi arvense L.
Cruciferae [十字花科]
5. 大青葉
Leaf of Isatis indigotica Fort.
Cruciferae [十字花科]
6. 廣東大青葉
Leaf of Baphicacanthus cusia (Nees) Bremek.
Acanthaceae [爵床科]
7. 蔊菜
Rorippa indica (Linn.) Hiern
Cruciferae [十字花科]
8. 白芥子
Seed of Sinapis alba L.
Cruciferae [十字花科]
9. 萊菔子
Seed of Raphanus sativus L.
Cruciferae [十字花科]
10. 葶藶子
Seed of Lepidium apetalum Willd
Cruciferae [十字花科]
Valerianaceae [敗醬草科]
Remarks: 1 is the original TCM and commonly confused by 2 in Hong Kong.
3 is the original TCM and commonly confused by 4 in Hong Kong.
5 is the original TCM and commonly confused by 6 in Hong Kong
Table 4: Local, Scientific and Family names of the Traditional Chinese Medicine (TCM)
samples used for analysis
8
2.3 Preparation of individual intact glucosinolate standard solutions
1,000ppm of individual intact glucosinolate standard solutions were prepared by dissolving
1mg each of intact glucosinolates (glucoiberin, glucocheirolin, progoitrin, sinigrin,
epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin,
gluconasturtiin) in 1mL of Milli-Q water respectively.
2.4 Preparation of intact glucosinolate standard mixture solutions
1mg each of intact glucosinolates (glucoiberin, glucocheirolin, progoitrin, sinigrin,
epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin, glucotropaeolin, glucoerucin,
gluconasturtiin) was weighed by precision weighing balance (Sartorius, Bradford, Germany)
and was dissolved in 1mL of Milli-Q water to prepare stock standard mixture solution with a
concentration of 1,000ppm.
This stock solution was diluted to prepare standard mixture
solutions at 500ppm, 400ppm, 300ppm, 200ppm, 100ppm, 50ppm and 5ppm.
2.5 Preparation of vegetable and Traditional Chinese Medicine (TCM) samples
2.5.1 Sample grinding and extraction
Fresh vegetable or dried TCM sample was submitted to an initial grinding in an Extra Fine
Blade Blender (Hitachi, Ibaraki, Japan) for 2 minutes to form vegetable paste or dried TCM
powder.
50g of the vegetable paste or 5g of the dried TCM powder was weighed and
blended with 100mL methanol.
minutes.
The sample mixture was heated with stirring at 70oC for 15
After cooling to room temperature, the sample mixture was filtered through a
Whatman No. 40 filter paper (Maidstone, England) by suction filtration using water vacuum
pump.
The sample extract residue was washed twice with 50mL methanol.
The collected
sample extract was evaporated to dryness under vacuum using a Rotovap (Caframo, Germany)
at 55oC with 60 rpm.
The solid sample extract was then dissolved in 10mL methanol and
9
was centrifuged by cyclone centrifuge (Alltech, Deerfield, U.S.A.) for 15 minutes at
13,000rpm.
2.5.2
The supernate was collected for clean-up process.
Clean-up process
Non-glucosinolate interferences were eliminated from the organic sample extract by using
activated Florisil solid-phase extraction (SPE) column.
Florisil sorbent (Fisher Certified
ACS, 60-100mesh) (Sigma, St. Louis, U.S.A.) was activated overnight at 200oC before using
for solid-phase extraction procedure.
A 5mL polypropylene syringe barrel was filled with
0.8g of the activated Florisil sorbent in between two 20µm polypropylene frits.
All solvents were kept at a flow rate of 1-2mL/min in a vacuum manifold (Alltech, Deerfield,
U.S.A.) by using water vacuum pump during the clean-up process. The activated Florisil
column was rinsed with 5mL of 30% (v/v) dichloromethane in hexane.
300µL of the organic
sample extract was mixed with 5mL of 30% (v/v) dichloromethane in hexane and then
transferred onto the column.
5mL of 30% (v/v) dichloromethane in hexane was added to
wash the non-polar interferences from the column.
The glucosinolates in the sample extract
were eluted from the column by using 5mL of 30% (v/v) ethyl acetate in methanol.
The
fraction was evaporated to dryness using a TurboVap® LV Evaporator (Zymark, Hopkinton,
U.S.A.) under a slow stream of nitrogen.
300µL of Milli-Q water.
The solid sample extract was then dissolved in
The aqueous sample extract was centrifuged by the cyclone
centrifuge for 15 minutes at 13,000rpm.
The supernate was collected for HPLC analysis.
10
2.6 Preparation of buffer solution
30mM ammonium acetate buffer solution at pH5.0 was prepared by dissolving 2.31g of
ammonium acetate in 1L of Milli-Q water, followed by adding a certain amount of 100%
formic acid until a calibrated Orion Model 420 pH meter (Delhi, India) showed pH5.0 value.
The buffer solution was then filtered through 0.2µm cellulose acetate filter paper (Alltech,
Deerfield, U.S.A.) by suction filtration using water vacuum pump.
The buffer solution was
degassed ultrasonically by a Branson 2510 series Ultrasonic degasser (Danbury, U.S.A.) for 10
minutes and was ready for HPLC analysis.
2.7 HPLC analysis
High-performance liquid chromatography (HPLC) experiments were performed on a Hewlett
Packard HP1100 series HPLC instrument with a diode array detector (DAD) (San Francisco,
U.S.A.).
A reversed-phase Hypersil BDS C18 column (250mm x 4.6mm i.d., 5µ particle size)
(Alltech, Deerfield, U.S.A.) was used for separation of the glucosinolates in three vegetable
and ten TCM sample extracts.
20µL of the aqueous sample extract was injected into the
HPLC system by 100µL HPLC-syringe (Alltech, Deerfield, U.S.A.).
Individual intact
glucosinolates were detected by the DAD detector at a UV wavelength of 233nm.
series degasser was used for the degas process.
HP1100
HP chemstation was used to control the
operation of the system and performed data analysis.
A gradient program was used for
sufficient retention and baseline separation of the glucosinolates, in which mobile phase A
consisted of 30mM ammonium acetate containing formic acid at pH5.0 and mobile phase B
consisted of pure methanol.
The gradient program was shown in Figure 3:
11
Gradient program
35
30
%B
25
20
15
10
5
0
0
5
10
15
1
20
25
30
Time(min)
Figure 3: Gradient program for separation of the glucosinolates
The flow rate was kept at 1mL/min during the HPLC analysis. 100% mobile phase A and 0%
mobile phase B were kept for the first 5 minutes.
Then, 0% mobile phase B was gradually
increased to 30% mobile phase B from 5 minutes to 17 minutes.
then kept until the end of the separation.
30% mobile phase B was
The twelve glucosinolate standards were separated
completely under these conditions and their corresponding retention times were recorded.
By comparing the retention times of the twelve glucosinolate standards with those of the
sample extracts, the presence of the glucosinlates in the sample extracts could be identified.
The peak areas of the identified glucosinoates in the sample extracts were recorded and used
for quantitative analysis.
2.8 Mass spectrometry analysis
HPLC fractions of the twelve intact glucosinolates detected in the vegetable and TCM sample
extracts were collected and analyzed by using electrospray ionzation-quadrupole time-of-flight
mass spectrometry (ESI-QTOF-MS) in negative mode and MS/MS analysis for the
confirmation of the glucosinolates in the sample extracts.
12
The confirmation depended on the masses of the molecular ions and their corresponding
fragment ions.
The QTOF mass spectrometer was equipped with a turbo ionspray source
(Sciex Q-Star Pulsar i, Applied Biosystem, Canada).
The parameters of the turbo ionspray
were shown in Table 5:
Ion source gas 1
25
Declustering potential 1
-65.0V
Ion source gas 2
8
Focusing potential
-165.0V
Curtain gas
15
Declustering potential 2
-15.0V
Ionspray voltage
-4,000V
Collision gas
3
Temperature of Ion
source gas 2
200 oC
Scan mass mode
50-600 amu
Table 5: Experimental conditions for ESI-QTOF-MS analysis of the glucosinolates
3. Experimental results and analysis
3.1 Qualitative analysis
3.1.1 Determination of the retention times for each intact glucosinolate standard
15.811
19.101
16.160
8.492
7.042
15.084
11.899
6.244
5.837
4.772
5.150
Gluconasturtiin
Glucoerucin
Glucotropaeolin
400
Glucosibarin
600
Gluconapin
800
Sinalbin
1000
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
1200
11.234
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE108.D)
mAU
200
0
0
2.5
5
7.5
10
12.5
15
Figure 4: Chromatogram of 300ppm glucosinolate standard mixture solution
13
17.5
20
min
In the HPLC analysis, the twelve intact glucosinolate standards were baseline separated as
shown in Figure 4.
The most polar glucosinolate standard, glucoiberin was first eluted out.
When the composition of mobile phase B was increased by the gradient program, the
relatively non-polar glucosinolates were eluted out in an order of descending the polarities of
the glucosinolate standards.
The most non-polar glucosinolate standard, gluconasturtiin was
the last one to be eluted out.
Under the chromatographic conditions described in Chapter 2.7, the average and relative
retention times for each glucosinolate standard were determined and shown in Table 6:
Intact Glucosinolate standard
Retention time (min)
Glucoiberin
4.77 ± 0.02
Glucocheirolin
5.14 ± 0.02
Progoitrin
5.83 ± 0.03
Sinigrin
6.25 ± 0.03
Epiprogoitrin
7.04 ± 0.03
Glucoraphenin
8.48 ± 0.04
Sinalbin
11.23 ± 0.03
Gluconapin
11.90 ± 0.04
Glucosibarin
15.08 ± 0.03
Glucotropaeolin
15.82 ± 0.03
16.16 ± 0.03
Glucoerucin
19.11 ± 0.03
Gluconasturtiin
Table 6: The average and relative retention times for each glucosinolate standard
14
3.1.2 Identification of the glucosinolates in vegetable and Traditional Chinese Medicine
(TCM) samples by using reversed-phase HPLC analysis
The glucosinolates in three vegetable and ten TCM samples were analyzed.
By comparing
the retention times of the sample extracts with those of the glucosinolate standards, the
presence of the glucosinolates in the sample extracts could be identified. However, the
retention times of the glucosinolates in the sample extracts varied a little bit due to the
complicated martices in the sample extracts.
Therefore, a small volume of 1,000ppm
glucosinolate mixture standard solution was spiked into the sample extracts for the
identification of the glucosinolates in the sample extracts. By comparing the peak areas of
the corresponding retention times in the chromatogram of original sample extract with the
spiked one, the peak areas of the spiked one were increased.
It identified that the sample
extract contained the glucosinolates being spiked.
By repeating the spiked standard method described above, it identified that Root of Isatis
indigotica Fort. [ 北板藍根 ] contained glucoiberin, glucocheirolin, progoitrin, sinigrin,
epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucotropaeolin, glucoerucin and
gluconasturtiin.
The chromatograms of Root of Isatis indigotica Fort. [北板藍根] extract and Root of Isatis
indigotica Fort. [北板藍根] extract with standards spiked were shown in Figures 5a and 5b
respectively.
15
11.883
19.255
15.799
16.026
6.276
8.548
11.306
6.971
5.834
4.805
5.212
50
Gluconasturtiin
100
Glucoerucin
150
Glucotropaeolin
200
Gluconapin
250
Sinalbin
300
Glucoraphenin
Epiprogoitrin
350
ProgoitrinSinigrin
Glucocheirolin
Glucoiberin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE094.D)
mAU
0
-50
0
5
2.5
7.5
10
12.5
15
17.5
20
min
19.182
15.881
15.137
8.557
16.210
6.972
5.830
6.272
11.883
4.796
5.168
Gluconasturtiin
Glucoerucin
150
100
Glucotropaeolin
200
Glucosibarin
Gluconapin
Sinalbin
250
Glucoraphenin
300
Epiprogoitrin
350
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE095.D)
mAU
11.290
Figure 5a: Chromatogram of Root of Isatis indigotica Fort. [北板藍根] extract
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
Figure 5b: Chromatogram of Root of Isatis indigotica Fort. [北板藍根] extract with
standards spiked
3.1.3 Identification of the glucosinolates in vegetable and Traditional Chinese Medicine
(TCM) samples by using ESI-QTOF-MS and MS/MS analysis
By using the spiked standard method described in Chapter 3.1.2, the glucosinolates in the
sample extracts can be detected. However, the retention times of the spiked glucosinolate
standards might be same as those of the interferences in the sample extracts.
16
20
min
To pinpoint the co-elution problem mentioned above, the electrospray ionzation-quadrupole
time-of-flight mass spectrometry in negative mode was used for further confirmation of the
HPLC fractions of the glucosinolates detected in the sample extracts.
The identification was
based on the molecular ion mass and the pattern of their corresponding fragment ions.
It was
a more powerful method than the spiked standard method and capable of providing the
information on the elemental compositions and the structures of the molecules.
HPLC fractions of the glucosinolates detected in vegetable and TCM sample extract were
collected.
The collected fractions were diluted with methanol, followed by negative
ESI-QTOF-MS analysis.
The deprotoned molecular ion, [M-H]- of the HPLC fraction in ESI-QTOF-MS spectrum was
compared with that of the corresponding glucosinolate standard.
For example, the
deprotonated molecular ion, [M-H]- of gluconapin standard was found to be m/z 372.0536 in
ESI-QTOF-MS spectrum. By comparing the mass of the deprotonated molecular ion, [M-H]of the gluconapin standard with that of the Root of Isatis indigotica Fort. [北板藍根] extract at
11.883min, m/z 372.0233 was found.
It showed a positive result for the further confirmation
of the gluconapin in the HPLC fraction collected from the Root of Isatis indigotica Fort. [北板
藍根] extract at 11.883min.
The ESI-QTOF-MS spectrums of gluconapin standard and Root
of Isatis indigotica Fort. [北板藍根] extract at 11.883min were shown in Figure 6a and 6b
respectively.
17
-TOF MS: 30 MCA scans from Sample 5 (gluconapin) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 4487.0 counts.
372.0536
4487
H2
C
CH2OH
4000
ΟΗ
(S)
3500
S
Ο
C
H2
C
C
H
CH2
[M-H]-
NOSO3H
(S)
(S)
OH (R)
OH
3000
Gluconapin standard
M.W. = 373.0501
2500
2000
1500
1000
374.0497
500
220.1537
0
200
210
220
255.2393
227.2081
230
240
283.2709
250
260
270
280
293.1867
290
300 310
m/z, amu
320
330
340
388.0655
358.0353
325.1875339.2078
350
360
370
380
390
400
Figure 6a: ESI-QTOF-MS spectrum of gluconapin standard
-TOF MS: 30 MCA scans from Sample 10 (Rt11.955-1) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
Max. 3982.0 counts.
372.0233
3982
3800
3600
H2
C
CH 2OH
3400
3200
ΟΗ
(S)
Ο
S
C
(S)
C
H2
C
H
CH2
NOSO3H
3000
(S)
OH (R)
2800
OH
2600
Gluconapin in Root of Isatis
indigotica Fort.
[北板藍根] extract at
11.883min
M.W. = 373.0501
2400
2200
[M-H]-
2000
1800
1600
1400
1200
1000
800
600
264.1464
374.0192
400
200
0
260
267.2177
315.0541
383.0960
297.2460 341.0834
280
300
320
340
360
380
393.9971
400
440.0099
420
440
m/z, amu
455.9759 484.0196
460
480
500
520
540
560
580
600
Figure 6b: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [北板藍根]
extract at 11.883min
18
However, interferences in the sample extracts might have the similar molecular mass.
For
further confirmation of the glucosinolates present in the sample extracts, the MS/MS analysis
with resolution of 10,000 was done.
It provides further confirmation of the glucosinolates
detected and structural elucidation. The pattern of the fragment ions of the glucosinolates is
different for different compounds even they have similar molecular mass.
The MS/MS spectrum of the gluconapin standard was shown in Figure 7a, the peak at m/z
372.0600 corresponded to the deprotoned molecular ion, [M-H]- of the gluconapin. The
observed fragment ion at m/z 292.0796 resulted from the loss of SO3 from the [M-H]- ion.
The peak at m/z 274.9975 corresponded to the molecular ion with the loss of HSO4 from the
[M-H]- ion.
The peak of m/z 195.0337 corresponded to the fragment ion of the glucose
group in gluconapin.
The peaks of m/z 96.9584 and m/z 79.9501 represented the fragment
ions of HSO4- and SO3-, respectively. By comparing the MS/MS spectrum of the gluconapin
with that of HPLC fraction collected from the Root of Isatis indigotica Fort. [北板藍根]
extract at 11.883min, similar fragment ion pattern in the sample extract was shown in Figure
7b. Therefore, the gluconapin was identified in the HPLC fraction collected from the Root of
Isatis indigotica Fort. [北板藍根] at 11.883min.
19
-TOF Product (372.0): 30 MCA scans from Sample 7 (gluconapinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 118.0 counts.
74.9866
118
H2
C
CH2OH
110
ΟΗ
(S)
Ο
90
HSO4
C
H2
C
H
CH2
NOSO3H
(S)
OH (R)
-
C
(S)
100
96.9584
S
OH
Gluconapin standard
80
M.W. = 373.0501
70
CH2OH
60
ΟΗ
50
40
Ο
S
[M-SO3-H]-
OH
SO3-
[M-HSO4-H]-
OH
[M-H]-
30
20
195.0337
259.0176
274.9975
178.9801
10
59.0074
0
372.0600
130.0304
79.9501
85.0244
128.9378
60
80
100
120
140
160
292.0796
175.9927
163.0819
180
200
220
240
m/z, amu
260
280
300
320
340
360
380
400
Figure 7a: MS/MS spectrum of gluconapin standard
-TOF Product (372.0): 59 MCA scans from Sample 14 (Rt11.955-1(MS/MS)-2) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
H2
C
CH2OH
124
120
(S)
ΟΗ
Max. 124.0 counts.
S
Ο
C
H2
C
C
H
372.0156
CH2
NOSO3H
(S)
110
OH (R)
HSO4-
100
90
OH
Root of Isatis indigotica
Fort. [北板藍根] extract
at 11.883min
M.W. = 373.0501
96.9548
CH2OH
80
ΟΗ
70
74.9854
60
(S)
S
Ο
[M-H]-
OH
OH
50
SO3-
[M-HSO4-H]-
40
195.0168
30
[M-SO3-H]-
258.9959
130.0262
20
0
274.9716
178.9716
79.9515
10
85.0283
128.9233
145.0374
227.0103
56.4612
60
80
100
120
140
160
180
200
220
240
m/z, amu
240.9984
260
292.0578
300.9815
280
300
320
340
360
380
Figure 7b: MS/MS spectrum of Root of Isatis indigotica Fort. [北板藍根] at 11.883min
20
400
By using similar confirmation procedures described as above, glucoiberin, glucocheirolin,
progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucotropaeolin,
glucoerucin and gluconasturtiin detected in the Root of Isatis indigotica Fort. [北板藍根]
extract were analyzed and all gave the positive results expect glucoiberin and glucoerucin.
Therefore, the Root of Isatis indigotica Fort. [北板藍根] contained glucocheirolin, progoitrin,
sinigrin,
epiprogoitrin,
gluconasturtiin.
glucoraphenin,
sinalbin,
gluconapin,
glucotropaeolin
and
The other sample extracts were analyzed by the similar methods and the
chromatograms of the sample extracts were shown in the following:
Glucoerucin
200
Glucosibarin
250
Sinigrin
Glucocheirolin
Glucoiberin
300
16.142
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE143.D)
mAU
50
15.122
8.709
5.148
100
6.186
4.675
150
0
0
2
4
6
8
10
12
14
16
18
min
16.103
15.834
15.077
11.861
19.110
7.083
8.616
6.242
5.840
4.760
5.178
Gluconasturtiin
100
Glucoerucin
150
Glucotropaeolin
200
Glucosibarin
250
Gluconapin
300
Sinalbin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
Glucoraphenin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE144.D)
mAU
11.271
Figure 8a: Chromatogram of Raphanus sativus [蘿蔔(Chinese Radish)] extract
50
0
0
2
4
6
8
10
12
14
16
18
Figure 8b: Chromatogram of Raphanus sativus [蘿蔔(Chinese Radish)] extract with
standards spiked
21
min
18.873
8.267
200
Gluconasturtiin
250
Glucosibarin
300
Glucoraphenin
350
14.950
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE056.D)
mAU
150
100
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
Figure 9a: Chromatogram of Lycopersicon esculentum [車厘茄(Cherry Tomato)] extract
18.904
15.991
15.656
14.935
6.838
11.718
11.051
8.240
6.064
5.631
4.611
4.971
100
Gluconasturtiin
150
Glucosibarin
200
Gluconapin
250
Sinalbin
300
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
350
Glucoerucin
Glucotropaeolin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE058.D)
mAU
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
Figure 9b: Chromatogram of Lycopersicon esculentum [車厘茄(Cherry Tomato)] extract
with standards spiked
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE146.D)
250
200
8.729
Progoitrin
300
Gluconasturtiin
350
Glucoerucin
Glucoraphenin
mAU
150
19.153
50
16.168
5.853
100
0
-50
0
2.5
5
7.5
10
12.5
15
Figure 10a: Chromatogram of Lycopersicon esculentum [番茄(Tomato)] extract
22
17.5
20
min
15.847
19.138
16.159
11.881
15.111
11.291
8.668
7.091
6.257
5.818
4.778
5.158
Gluconasturtiin
Glucoerucin
100
Glucotropaeolin
150
Glucosibarin
200
Gluconapin
250
Sinalbin
300
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
350
Glucoraphenin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE147.D)
mAU
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
11.883
5.834
Gluconasturtiin
Gluconapin
Sinalbin
200
Glucoraphenin
Epiprogoitrin
250
Sinigrin
300
Progoitrin
Glucocheirolin
350
Glucotropaeolin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE094.D)
mAU
6.971
Figure 10b: Chromatogram of Lycopersicon esculentum [番茄(Tomato)] extract with
standards spiked
50
19.255
15.799
8.548
6.276
5.212
100
11.306
150
0
-50
0
5
2.5
7.5
10
12.5
15
17.5
8.557
19.182
16.210
15.137
15.881
11.883
6.972
5.830
6.272
4.796
5.168
Gluconasturtiin
Glucoerucin
150
100
Glucotropaeolin
200
Glucosibarin
Gluconapin
Sinalbin
250
Epiprogoitrin
300
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
350
Glucoraphenin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE095.D)
mAU
11.290
Figure 11a: Chromatogram of Root of Isatis indigotica Fort. [北板藍根] extract
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
Figure 11b: Chromatogram of Root of Isatis indigotica Fort. [北板藍根] extract with
standards spiked
23
20
min
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE161.D)
mAU
300
250
200
150
100
50
0
0
2.5
5
7.5
10
12.5
15
17.5
20
min
Figure 12a: Chromatogram of Root of Baphicacanthus cusia (Nees) Bremek. [南板藍根]
extract
18.963
15.692
16.011
14.944
11.741
8.401
6.918
6.103
5.698
4.661
5.060
Gluconasturtiin
Glucoerucin
100
Glucotropaeolin
150
Glucosibarin
200
Gluconapin
250
Sinalbin
300
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
350
11.169
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE157.D)
mAU
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
Figure 12b: Chromatogram of Root of Baphicacanthus cusia (Nees) Bremek. [南板藍根]
extract with standards spiked
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE165.D)
mAU
400
300
200
100
0
0
2.5
5
7.5
10
12.5
15
17.5
Figure 13a: Chromatogram of Patrinia scabiosaefolia Fisch. ex Trev. [敗醬草] extract
24
8.367
6.901
18.908
15.688
16.006
14.941
11.161
11.743
6.099
5.693
4.657
5.054
Gluconasturtiin
Glucoerucin
Glucotropaeolin
Glucosibarin
Gluconapin
200
Sinalbin
300
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
400
Glucoraphenin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE158.D)
mAU
100
0
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
Figure 13b: Chromatogram of Patrinia scabiosaefolia Fisch. ex Trev. [敗醬草] extract
with standards spiked
8.350
200
Glucosibarin
250
Glucoraphenin
300
Sinigrin
Progoitrin
Glucocheirolin
350
6.005
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE154.D)
mAU
150
14.842
50
5.568
4.978
100
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
Figure 14a: Chromatogram of Thlaspi arvense L. [菥蓂] extract
18.962
15.684
16.005
14.931
11.137
11.724
8.347
6.875
5.667
5.021
4.643
Gluconasturtiin
100
Glucoerucin
150
Glucotropaeolin
200
Glucosibarin
250
Gluconapin
300
Sinalbin
350
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
6.004
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE153.D)
mAU
50
0
-50
0
2
4
6
8
10
12
14
16
Figure 14b: Chromatogram of Thlaspi arvense L. [菥蓂] extract with standards spiked
25
18
min
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE150.D)
300
200
8.478
Progoitrin
250
Glucosibarin
350
Glucoraphenin
mAU
14.918
150
5.588
100
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
Figure 15a: Chromatogram of Leaf of Isatis indigotica Fort. [大青葉] extract
19.126
16.141
15.832
15.081
11.293
11.873
8.668
7.065
6.238
5.823
4.763
5.169
Gluconasturtiin
100
Glucoerucin
150
Glucotropaeolin
200
Glucosibarin
250
Gluconapin
300
Sinalbin
350
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE149.D)
mAU
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
Figure 15b: Chromatogram of Leaf of Isatis indigotica Fort. [大青葉] extract with
standards spiked
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE152.D)
mAU
350
300
250
200
150
100
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
Figure 16a: Chromatogram of Leaf of Baphicacanthus cusia (Nees) Bremek. [廣東大青葉]
extract
26
18.920
15.611
15.941
14.850
11.629
8.220
6.777
5.996
5.602
4.604
4.988
Gluconasturtiin
Glucoerucin
100
Glucotropaeolin
150
Glucosibarin
200
Gluconapin
250
Sinalbin
300
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
350
11.064
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE160.D)
mAU
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
Figure 16b: Chromatogram of Leaf of Baphicacanthus cusia (Nees) Bremek. [廣東大青葉]
extract with standards spiked
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE164.D)
Gluconasturtiin
mAU
350
300
200
150
19.122
Sinigrin
250
6.209
100
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
Figure 17a: Chromatogram of Rorippa indica (Linn.) Hiern [蔊菜] extract
18.967
15.708
16.010
8.364
6.901
11.141
11.736
6.094
5.694
4.659
5.050
Gluconasturtiin
Glucoerucin
Glucotropaeolin
100
Glucosibarin
150
Gluconapin
200
Sinalbin
250
Glucoraphenin
300
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
350
14.940
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE159.D)
mAU
50
0
-50
0
2.5
5
7.5
10
12.5
15
Figure 17b: Chromatogram of Rorippa indica (Linn.) Hiern [蔊菜] extract with
standards spiked
27
17.5
20
min
15
16.204
15.177
50
7.050
5.821
4.772
100
19.192
11.723
11.138
12.5
150
Gluconasturtiin
200
Glucosibarin
250
Gluconapin
300
Sinalbin
Progoitrin
Glucoiberin
Epiprogoitrin
Sinigrin
350
Glucoerucin
5.999
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE100.D)
mAU
0
-50
0
2.5
5
7.5
10
17.5
20
min
19.194
15.899
16.228
8.526
7.049
15.166
11.722
6.006
5.819
5.146
4.769
Gluconasturtiin
100
Glucoerucin
150
Glucotropaeolin
Glucosibarin
200
Gluconapin
250
Sinalbin
300
Glucoraphenin
350
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE101.D)
mAU
11.128
Figure 18a: Chromatogram of Seed of Sinapis alba L. [白芥子] extract
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
Figure 18b: Chromatogram of Seed of Sinapis alba L. [白芥子] extract with standards
spiked
5.818
7.027
11.833
11.212
Glucoerucin
Glucosibarin
75
Epiprogoitrin
100
Sinigrin
125
Progoitrin
Glucoiberin
150
Gluconapin
Sinalbin
175
Gluconasturtiin
6.176
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE120.D)
mAU
19.093
16.145
25
15.119
4.753
50
0
-25
0
2.5
5
7.5
10
12.5
15
17.5
Figure 18c: Chromatogram of five-fold dilution of Seed of Sinapis alba L. [白芥子]
extract
28
19.084
15.818
16.140
15.077
8.467
7.023
11.834
6.174
5.812
4.747
5.130
Gluconasturtiin
Glucoerucin
Glucotropaeolin
75
Glucosibarin
100
Sinalbin
125
Glucoraphenin
150
Gluconapin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
175
11.208
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE121.D)
mAU
50
25
0
-25
0
2.5
5
7.5
10
12.5
15
17.5
20
min
Figure 18d: Chromatogram of five-fold dilution of Seed of Sinapis alba L. [白芥子]
extract with standards spiked
300
200
50
11.809
6.929
5.750
100
6.092
150
Gluconapin
250
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
350
7.953
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE113.D)
mAU
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
Figure 19a: Chromatogram of Seed of Raphanus sativus L. [萊菔子] extract
18.929
15.677
15.992
14.924
11.732
11.128
7.924
6.857
6.070
5.677
5.010
4.633
Gluconasturtiin
100
Glucoerucin
150
Glucosibarin
200
Gluconapin
250
Sinalbin
300
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
350
Glucotropaeolin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE135.D)
mAU
50
0
-50
0
2.5
5
7.5
10
12.5
15
17.5
Figure 19b: Chromatogram of Seed of Raphanus sativus L. [萊菔子] extract with
standards spiked
29
Epiprogoitrin
Sinigrin
Progoitrin
150
125
100
75
Glucoraphenin
8.592
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE126.D)
mAU
7.180
5.924
25
6.300
50
0
-25
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
20
min
15.672
18.921
15.990
14.924
6.871
11.735
8.216
6.088
5.679
4.636
5.016
Gluconasturtiin
Glucoerucin
50
Glucotropaeolin
75
Glucosibarin
100
Gluconapin
125
Sinalbin
150
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE132.D)
mAU
11.115
Figure 19c: Chromatogram of ten-fold dilution of Seed of Raphanus sativus L. [萊菔子]
extract
25
0
-25
0
2.5
5
7.5
10
12.5
15
17.5
Figure 19d: Chromatogram of ten-fold dilution of Seed of Raphanus sativus L. [萊菔子]
extract with standards spiked
6.202
11.413
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE117.D)
350
5.157
11.110
100
50
Glucoerucin
150
Gluconapin
200
Sinalbin
250
Sinigrin
Glucocheirolin
300
16.027
mAU
0
-50
0
2.5
5
7.5
10
12.5
15
Figure 20a: Chromatogram of Seed of Lepidium apetalum Willd [葶藶子] extract
30
17.5
18.917
15.666
15.177
8.250
6.837
50
15.990
10.946
6.021
5.657
4.997
4.616
100
Gluconasturtiin
150
Glucoerucin
Gluconapin
200
Sinalbin
250
Glucoraphenin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
300
Glucotropaeolin
Glucosibarin
11.256
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE134.D)
mAU
0
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
20
min
Figure 20b: Chromatogram of Seed of Lepidium apetalum Willd [葶藶子] extract with
standards spiked
200
11.201
6.336
16.112
5.245
50
Glucoerucin
100
Gluconapin
Sinalbin
Sinigrin
150
11.875
Glucocheirolin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE124.D)
mAU
0
0
2.5
5
7.5
10
12.5
15
17.5
19.203
15.854
16.186
11.234
8.416
6.991
15.093
6.209
5.803
5.147
4.767
50
Gluconasturtiin
75
Glucoerucin
100
Glucotropaeolin
Glucosibarin
125
Gluconapin
150
Sinalbin
Epiprogoitrin
Sinigrin
Progoitrin
Glucocheirolin
Glucoiberin
175
Glucoraphenin
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE128.D)
mAU
11.777
Figure 20c: Chromatogram of twenty-fold dilution of Seed of Lepidium apetalum Willd
[葶藶子] extract
25
0
-25
0
2.5
5
7.5
10
12.5
15
17.5
Figure 20d: Chromatogram of twenty-fold dilution of Seed of Lepidium apetalum Willd
[葶藶子] extract with standards spiked
31
By similar confirmations described in Chapter 3.1, the glucosinolates in the vegetable and
TCM sample extracts were identified and shown in Table 7.
Vegetable and Traditional
Chinese Medicine Samples
Glucoiberin
Glucocheirolin
Progoitrin
Sinigrin
Epiprogoitrin
Glucoraphenin
Sinalbin
Gluconapin
Glucosibarin
Glucotropaeolin
Glucoerucin
Gluconasturtiin
Raphanus sativus
[蘿蔔(Chinese Radish)]
O
O
X
O
X
X
X
X
O
X
O
X
Lycopersicon esculentum
[車厘茄(Cherry Tomato)]
X
X
X
X
X
O
X
X
O
X
X
O
Lycopersicon esculentum
[番茄(Tomato)]
X
X
O
X
X
O
X
X
X
X
O
O
Root of Isatis indigotica Fort.
[北板藍根]
X
O
O
O
O O`
O
O
X
O
X
O
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Thlaspi arvense L.
[菥蓂]
X
O
O
O
X
O
X
X
O
X
X
X
Leaf of Isatis indigotica Fort.
[大青葉]
X
X
O
X
X
O
X
X
O
X
X
X
Baphicacanthus cusia (Nees)
Bremek.
[廣東大青葉]
X
X
X
X
X
X
X
X
X
X
X
X
Rorippa indica (Linn.) Hiern
[蔊菜]
X
X
X
O
X
X
X
X
X
X
X
O
Seed of Sinapis alba L.
[白芥子]
O
X
O
O
O
X
O
O
O
X
O
O
Seed of Raphanus sativus L.
[萊菔子]
X
X
O
O
O
O
X
O
X
X
X
X
Seed of Lepidium apetalum Willd
X
[葶藶子]
O
X
O
X
X
O
O
X
X
O
X
Root of Baphicacanthus cusia
(Nees) Bremek.
[南板藍根]
Patrinia scabiosaefolia Fisch.ex
Trev.
[敗醬草]
Remarks: O = Detected
X = Not Detected
Table 7: Identified glucosinolates in vegetable and Traditional Chinese Medicine samples
32
3.2 Quantitative analysis
3.2.1 Calibration curves for each individual glucosinolate standard
Concentration of
standards
Glucosinolates
Glucoiberin
Glucocheirolin
Progoitrin
Sinigrin
Epiprogoitrin
Glucoraphenin
Sinalbin
Gluconapin
Glucosibarin
Glucotropaeolin
Glucoerucin
Gluconasturtiin
Peak Areas
5ppm
57.50
50.42
66.55
101.99
58.53
46.08
123.42
58.57
67.63
104.51
60.38
67.97
50ppm
595.28
533.42
693.25
1046.88
602.21
466.58
1362.18
649.87
690.12
1080.15
614.48
709.73
100ppm
1284.74
1164.18
1512.60
2268.54
1305.75
1012.19
2970.99
1416.32
1496.50
2355.41
1345.29
1536.00
200ppm
2339.79
2113.01
2743.52
4135.44
2373.71
1848.87
5694.20
2577.45
2718.96
4272.70
2445.27
2783.57
300ppm
3548.86
3200.47
4144.70
6231.07
3604.46
2786.48
8622.51
3911.87
4112.67
6421.64
3660.38
4221.78
400ppm
4774.39
4274.74
5528.09
8345.19
4801.89
3739.58
11414.40
5220.12
5495.41
8542.62
4855.57
5634.59
500ppm
5878.06
5308.13
6866.26
10297.54
5979.22
4667.01
14046.44
6493.76
6809.96
10457.04
5969.82
7015.29
Table 8: Peak areas of the glucosinolates at 5ppm, 50ppm, 100ppm, 200ppm, 300ppm, 400ppm, and
500ppm
Calibration curves for each glucosinolate standard were obtained by plotting the peak areas
against concentrations for each glucosinolate standard respectively.
The graph of the
calibration curve for epiprogoitrin was shown in Figure 21. The summary of the equations
and their corresponding correlation coefficients (R2 values) of the calibration curves for each
glucosinolate were shown in Table 9. According to the R2 values of the calibration curves for
each
glucosinolate,
the
linearity
of
all
calibration
dddddddddddddddddddddddddddddddd
.
33
curves
were
acceptable.
Calibration curve for Epiprogoitrin
7000
6000
Area
5000
y = 11.916x + 28.152
R2 = 0.9997
4000
3000
2000
1000
0
0
100
200
300
400
500
Concentration of Epiprogoitrin (ppm)
Figure 21: Calibration curve for epiprogoitrin
Glucosinolates
Equations of the calibration curves
R2 values
Glucoiberin
y = 11.758x + 27.812
R2 = 0.9996
Glucocheirolin
y = 10.588x + 25.809
R2 = 0.9996
Progoitrin
y = 13.687x + 38.843
R2 = 0.9996
Sinigrin
y = 20.569x + 63.207
R2 = 0.9996
Epiprogoitrin
y = 11.916x + 28.152
R2 = 0.9997
Glucoraphenin
y = 9.290x + 17.229
R2 = 0.9997
Sinalbin
y = 28.268 x+ 39.627
R2 = 0.9997
Gluconapin
y = 12.954x + 26.35
R2 = 0.9996
Glucosibarin
y = 13.585x+ 38.123
R2 = 0.9996
Glucotropaeolin
y = 20.928x + 98.804
R2 = 0.9993
Glucoerucin
y = 11.927x + 57.822
R2 = 0.9994
Gluconasturtiin
y = 13.974x + 34.193
R2 = 0.9996
Table 9: The summary of the equations and their corresponding R2 values of the
calibration curves for each glucosinolate
34
600
3.2.2 Detection limits of each glucosinolate
Both instrument detection limits and method detection limits of each glucosinolate can be
calculated by applying the following equation:
CL=kSB/b
Remarks: CL = Concentration related to the smallest measure of response can be
detected
k = 3(based on the confidence interval)
SB = Standard derivation of the blank of the method
b = Slope of the calibration curve for corresponding glucosinolates
By using the the equations of calibration curves for each glucosinolate and equation above, the
detection limits for each glucosinolate were calculated and shown in Table 10:
Standard deviation, Instrument Detection Method Detection Method Detection
SB
Limit, ng/µL
Limit, µg/g a
Limit, µg/g b
Glucosinolates
Slope, b
Glucoiberin
11.758
49.33
12.59
25.17
2.52
Glucocheirolin
10.588
13.75
3.90
7.79
0.78
Progoitrin
13.687
4.10
0.90
1.80
0.18
Sinigrin
20.569
19.52
2.85
5.69
0.57
Epiprogoitrin
11.916
16.42
4.13
8.27
0.83
Glucoraphenin
9.290
10.02
5.39
10.79
1.08
Sinalbin
28.268
113.80
12.08
24.15
2.42
Gluconapin
12.954
42.72
9.89
19.79
1.98
Glucosibarin
13.585
7.42
1.64
3.28
0.33
Glucotropaeolin
20.928
51.13
7.33
14.66
1.47
Glucoerucin
11.927
8.62
2.17
4.34
0.43
Gluconasturtiin 13.974
40.80
8.76
17.52
Remarks: a = Method Detection Limit when 5g of dried TCM was analyzed
b
= Method Detection Limit when 50g of fresh vegetable was analyzed
1.75
Table 10: Detection limits of each glucosinolate
35
3.2.3 Method recoveries of each glucosinolate
In the sample preparation, some of the glucosinolates might lose in heating, transfer of the
sample extract, filtration, evaporation and clean-up process. Therefore, recovery tests were
done to determine both accuracies of the glucosinolates extracted from those sample
preparation procedures described in Chapter 2.5 and the effects of the interferences on the
glucosinolates in the sample extracts.
A recovery test was carried out by using a dried TCM sample, Root of Belamcanda chinensis
(L.) DC. [射干] that had been repeatedly analyzed and showed no existing glucosinolates.
50µL of 10,000ppm of the glucosinolate mixture standard was spiked into 5 g Root of
Belamcanda chinensis (L.) DC. [ 射干] in 100mL methanol solution.
Then, the spiked
sample was prepared by repeating the same experimental procedures described in Chapter 2.5.
Theoretically, the final concentration of the glucosinolates would be 50ppm. The peak areas
of the glucosinolates determined in the sample extract were compared with those of standard
mixture solutions at 50ppm to obtain the recovery data.
The recovery test was repeated three
times in order to get the average recoveries (accuracies) and precisions [relative standard
derivations, (RSD, n=3)] of each standard.
Method recoveries of the glucosinolates were calculated by applying the following equation:
Peak area of the glucosinolates in the testing solution
Recoveries (%) =
X 100%
Peak area of the glucosinolates in the 50ppm glucosinolate
standard mixture solution
36
By using the similar calculation, both the method recoveries and RSD values for each
glucosinolate were obtained and shown in Table 11. The method recoveries of each
glucosinolate were over 86.10% with average 99.80%. The precisions were from 5.27% to
14.60% (RSD, n=3) respectively.
Glucosinolates
Glucoiberin
Peak areas of
Peak areas of
Peak areas of
Peak areas of
Recoveries,
50ppm standard
RSD, %
testing solution 1 testing solution 2 testing solution 3
%
mixture solution
513.98
534.96
536.40
595.28
88.77
10.24
Glucocheirolin
496.38
501.96
512.35
533.42
94.40
Progoitrin
705.11
712.87
717.92
693.25
102.70
Sinigrin
1025.82
1019.19
1033.70
1046.88
98.03
Epiprogoitrin
540.48
562.65
551.10
602.21
91.56
Glucoraphenin
556.86
563.22
590.52
466.58
122.21
Sinalbin
1269.54
1255.47
1282.99
1362.18
93.18
Gluconapin
776.46
788.20
811.59
649.87
121.88
Glucosibarin
577.80
602.60
604.20
690.12
86.10
Glucotropaeolin
1124.00
1106.50
1119.62
1080.15
103.38
Glucoerucin
604.14
586.20
584.89
614.48
96.30
Gluconasturtiin
688.13
683.23
696.56
709.73
97.12
Average
99.80
Table 11: Summary of method recoveries and RSD values for each glucosinolate
37
6.61
5.27
5.93
9.05
14.60
11.24
14.60
12.09
7.44
8.78
5.51
9.28
3.2.4
Glucosinolate concentrations in vegetable and Traditional Chinese
Medicine (TCM) samples
For sinigrin in Thlaspi arvense L. [菥蓂] extract,
y, Peak area of sinigrin in Thlaspi arvense L. [菥蓂] extract = 9628.09;
y = 20.569x + 63.207 for sinigrin;
By substituting the peak area of sinigrin in Thlaspi arvense L. [菥蓂] extract into the equation
of the calibration curve for sinigrin, the concentration of sinigrin in 10mL sample extract was
found.
Therefore, x = 465.0145ppm (mgL-1)
Weight of sinigrin in 5g Thlaspi arvense L. [菥蓂] extract
= Concentration of sinigrin x diluted factor / recovery of sinigrin
= 465.0145ppm x 10 x 10-3L / 98.03%
= 4.7436mg
Concentration of sinigrin in Thlaspi arvense L. [菥蓂]
= Weight of sinigrin / weight of Thlaspi arvense L. [菥蓂]
= 4.7436mg / 5g
=0.9487mg/g
=948.70µg/g
Therefore, the concentration of sinigrin in Thlaspi arvense L. [ 菥蓂 ] was found to be
948.70µg/g.
38
By similar calculation and compare the concentrations of the identified glucosinolates in the
sample extract with detection limit, the glucosinolate concentrations in three vegetable and ten
TCM samples were obtained and shown in Table 12:
Concentration (µg/g)
Total glucosinolates
Gluconasturtiin
Glucotropaeolin
Glucoraphenin
X
X
X
6.31
X
61.16
X
84.14
Lycopersicon esculentum
[車厘茄(Cherry Tomato)]
X
X
X
X
X
26.63
X
X
5.13
X
X
6.83
38.59
X
X
3.40
X
X
30.41
X
X
X
X
4.13
9.96
47.90
X
9.55
795.32
28.91
2099.66
124.54
24.32
636.09
X
18.58
X
9.56
3746.53
X
X
X
X
X
X
X
X
X
X
X
X
0.00
X
X
X
X
X
X
X
X
X
X
X
X
0.00
X
42.87
16.45
984.70
X
281.05
X
X
28.95
X
X
X
1354.02
X
X
14.32
X
X
488.77
X
X
42.32
X
X
X
545.41
X
X
X
X
X
X
X
X
X
X
X
0.00
Rorippa indica (Linn.) Hiern
[蔊菜]
X
X
X
20.69
X
X
X
X
X
X
X
60.63
81.32
Seed of Sinapis Alba L.
[白芥子]
66.31
X
8.34
4313.82
41.43
X
1591.08
2966.87
26.75
X
17.01
21.56
9053.17
X
X
23.64
13.67
37.68
10919.08
X
20.67
X
X
X
X
11014.74
X
39.92
X
430.70
X
X
66.55
12648.94
X
X
13.71
X
13199.82
Thlaspi arvense L.
[菥蓂]
Leaf of Isatis indigotica Fort.
[大青葉]
Leaf of Baphicacanthus
cusia (Nees) Bremek.
[廣東大青葉]
Seed of Raphanus sativus L.
[萊菔子]
Seed of Lepidium apetalum
Willd
[葶藶子]
Sinigrin
Lycopersicon esculentum
[番茄(Tomato)]
Root of Isatis indigotica
Fort.
[北板藍根]
Root of Baphicacanthus
cusia (Nees) Bremek.
[南板藍根]
Patrinia scabiosaefolia
Fisch. ex Trev.
[敗醬草]
X
Glucoerucin
X
Glucosibarin
2.65
Gluconapin
X
Sinalbin
1.00
Epiprogoitrin
Glucocheirolin
13.02
Progoitrin
Glucoiberin
Vegetable and Traditional
Chinese Medicine Samples
Raphanus sativus
[蘿蔔(Chinese Radish)]
Remark: X = Not Detected
Table 12: Glucosinolate concentrations in vegetable and Traditional Chinese Medicine
(TCM) samples
39
4
Discussion
4.1 Extraction
To inactivate myrosinase to catalyze the hydrolysis of the glucosinolates into glucose and
unstable products that described in Figure 2, 100% methanol was used for extraction of the
glucosinolates from the vegetable and Traditional Chinese Medicine (TCM) samples.
Moreover, less pigment was extracted and all glucosinolates could be extracted with high
recoveries, above 85%. Heating the sample extract at 70oC for 15minutes in extraction
process could denature the myrosinase and increase dissolution of the glucosinolates into
methanol. After cooling to room temperature, the proteins in the organic sample extract were
precipitated out due to the low solubility of proteins in methanol.
4.2 Clean-up process
Both normal-phase solid-phase extraction using activated Florisil column and reversed-phase
solid-phase extraction using C18 column were tried for the clean-up process. The activated
Florisil clean-up process was described in Chapter 2.5. And both C18 and activated Florisil
clean-up processes could achieve more than 88% of method recoveries for all glucosinolates
in testing conditions. They differed from each other by the ability of removal of the
interferences in the sample extracts.
In C18 clean-up process, C18 cartridge (Alltech, Deerfield, U.S.A.) was rinsed with 5mL of
methanol, followed by 5mL of deionized water.
The 5mL aqueous sample extract was
transferred onto the column, followed by collection of the glucosinolates due to their poor
retentions on C18 column. 5mL of the 10% methanol in deionized water was added to elute
the remaining glucosinolates. The final volume of the sample extracts was 10mL.
40
Glucosinolates were very polar that they had poor retention on the C18 column.
And C18
clean-up process was done by retaining of the non-polar interferences on the sorbent.
However, the interferences with the co-retention time of the glucotropaeolin in the Root of
Isatis indigotica Fort. [ 北板藍根 ] were eluted together with the glucosinolates. The
chromatograms of the original Root of Isatis indigotica Fort. [北板藍根] and Root of Isatis
indigotica Fort. [北板藍根] after C18 clean-up process were shown in Figure 21a and 21b
respectively.
Activated Florisil clean-up method could remove the interferences with the co-retention time
of the glucotropaeolin in the Root of Isatis indigotica Fort. [北板藍根] as shown in Figure 11a.
Florisil was activated at 200oC for overnight that could remove volatile organic compounds in
the sorbent. And therefore, the activated Florisil could then be used as adsorption sorbent for
adsorption of slightly polar interferences. By washing the column with 30% dichoromethane
in hexane, the non-polar interferences were removed from the sample extracts. Organic sample
extracts were prepared to load onto the column as water in the aqueous sample extract was too
polar that could inactivate the activated Florisil column.
11.645
6.654
15.480
18.953
8.266
10.990
Area: 237.494
5.919
4.906
50
Gluconasturtiin
100
Area: 14890.5
glucotropaeolin
with
interferences
Area: 1560.09
Area: 5581.52
Gluconapin
Sinalbin
150
Glucoraphenin
200
Epiprogoitrin
Glucocheirolin
250
Area: 6281.9
Area: 13465.5
Sinigrin
Progoitrin
300
5.560
DAD1 A, Sig=233,4 Ref =550,100 (CHUNG2\KCLEE178.D)
mAU
Area: 41.0646
Area: 29.5705
Area: 55.7971
0
0
2.5
5
7.5
10
12.5
15
17.5
Figure 21a: Chromatogram of the original Root of Isatis indigotica Fort. [北板藍根]
41
20
min
15.483
11.660
6.699
Area: 10577.4
Gluconasturtiin
Glucotropaeolin
with
interferences
Area: 4597.09
Gluconapin
Sinalbin
200
Glucoraphenin
250
Epiprogoitrin
300
Area: 5235.53
Area: 10792.9
Sinigrin
Progoitrin
350
5.624
DAD1 A, Sig=233,4 Ref =550,100 (CHUNG2\SUB00006.D)
mAU
50
Area: 848.149
Area: 162.052
18.929
6.040
100
11.013
8.232
150
Area: 88.5345
0
Area: 57.3498
-50
0
2.5
5
7.5
10
12.5
15
17.5
20
Figure 21b: Chromatogram of the original Root of Isatis indigotica Fort. [北板藍根] after
C18 clean-up process
4.3 Optimization of buffer system
Glucosinolates were present in ionic states in aqueous medium.
The retention of the
glucosinolates on the reversed-phase HPLC column was usually poor because of high polarity
of the glucosinolates. Therefore, 30mM ammonium acetate containing formic acid at pH5.0
was used for HPLC analysis. The buffer solution was used to achieve better retention and
good peak shape for the glucosinolates. Formic acid was a strong acid and easily protonate
the glucosinolates in aqueous medium.
Ammonium acetate was completely ionize in the
aqueous medium and suppressed the protonated glucosinolates to ionize.
The protonated
glucosinolates were relatively non-polar and had sufficient retention on the reversed-phase
C18 HPLC column for baseline separation of them.
4.4 Gradient program
When 100% buffer solution was kept at a flow rate of 1mL/min in isocratic condition, some
(relatively non-polar) peaks of the glucosinolates eluted from HPLC column more than one
hour and became flattened in peak shape. When 100% methanol was kept at a flow rate of
1mL/min in isocratic condition, the glucosinolate standards were eluted out together.
Therefore, gradient program was used. The gradient program was used in HPLC system to
42
min
increase the resolutions and lower the detection limits of the glucosinolates.
To get a better
retention of the glucosinolates on the HPLC column, a 100% aqueous phase was used as the
initial condition.
Methanol was used as the organic phase and was increased to 30% from 5
minutes to 17 minutes using the gradient program in order to separate the glucosinolates
completely and elute the relatively non-polar glucosinolates.
4.5 Optimization of glucosinolate concentrationsin the sample extracts
The glucosinolate concentrations in some TCM samples, especially the seed TCM samples
were found to be very high.
For example, seed of Lepidium apetalum Willd [葶藶子]
contained 12648.94µg/g of the gluconapin and seed of Raphanus sativus L.[萊菔子] contained
10919.08µg/g of the glucoraphenin. Therefore, the pre-concentration process was necessary
for quantitative analysis. Otherwise, the glucosinolate concentration in the sample extracts
might be out of the range of the calibration curve or even the HPLC column might be
overloaded. The column overloading could be seen with back tailing of the peak in the
chromatogram.
For example, the gluconapin in seed of Lepidium apetalum Willd [葶藶子]
extract overloaded the column as shown in Figure 22a. And its corresponding peak area,
71780.70 was above the range of the calibration curve, from 58.57 to 6493.76.
After
twenty-fold dilution of the seed of Lepidium apetalum Willd [葶藶子] extract, a good peak
shape of the gluconapin was achieved as shown in Figure 22b. And its corresponding area,
5026.12 was within the range of the calibration curve.
43
11.413
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE117.D)
mAU
2500
16.027
11.110
6.202
5.157
500
Sinalbin
1000
Sinigrin
Glucocheirolin
1500
Glucoerucin
Gluconapin
2000
0
0
2.5
5
7.5
10
12.5
15
17.5
20
min
20
min
Figure 22a: Chromatogram of Seed of Lepidium apetalum Willd [葶藶子] extract
11.875
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE124.D)
mAU
Sinalbin
400
Sinigrin
Glucocheirolin
600
Glucoerucin
Gluconapin
800
0
2.5
5
11.201
7.5
10
16.119
0
6.336
5.245
200
12.5
15
17.5
Figure 22b: Chromatogram of twenty-fold dilution of Seed of Lepidium apetalum Willd
[葶藶子] extract
44
5. Conclusion
The developed reversed-phase HPLC method using C18 column provides sufficient retention
and baseline separation for analyzing twelve intact glucosinolates (glucoiberin, glucocheirolin,
progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucosibarin,
glucotropaeolin, glucoerucin, gluconasturtiin) in three vegetable and twelve Traditional
Chinese Medicine (TCM) samples.
Combined the method with ESI-QTOF-MS in negative
mode and MS/MS analysis, the glucosinolates in the sample extract can be detected and
identified. Glucosinolate concentrations in the sample extract can be successfully determined
by an external calibration method.
At least two glucosinoates were found in the three vegetable and cruciferous TCM samples.
None of the detectable glucosinolates were found in the non-cruciferous TCM samples.
Concentrations of the total glucosinolates were found to be very high in seed TCM samples,
more than 9,000µg/g.
Seed of Lepidium apetalum Willd [葶藶子] contained the highest concentration of total
glucosinolates,
13199.82µg/g.
Root
of
Baphicacanthus
cusia
(Nees)
Bremek.
[南板藍根], Patrinia scabiosaefolia Fisch. ex Trev. [敗醬草] and Leaf of Baphicacanthus
cusia (Nees) Bremek. [廣東大青葉] contained the least concentration of total glucosinolates,
0.00µg/g. The range of the total glucosinolates in the sample extracts was found to be
0.00µg/g to 13199.82µg/g, respectively.
45
6. Future plan
Fresh vegetable samples were analyzed in this project. The water content was different in
different vegetable samples. Therefore, a drying freezer can be used to dry the vegetable
samples before analysis.
Glucosinolate concentrations in the reproductive tissues (florets/ flowers and seeds) are often
as much as 10-40 times higher than those in vegetative tissues.
[2]
And the seed TCM
samples were found with relatively high glucosinolate levels. Therefore, reproductive tissues
of the cruciferous plants can be analyzed and compared with the vegetative tissues.
Different forms of 板藍根 products in the market can be analyzed, for example, 板藍根
extract to investigate whether they are made of Root of Isatis indigotica Fort. [北板藍根] or
Root of Baphicacanthus cusia (Nees) Bremek. [南板藍根].
46
5. References
1. Tsiafoulis, C.G.; Prodromidis, M.I.; Karayannis, M.I. Anal. Chem. 2003, 75, 927-934
2. Bennett, R.N.; Mellon, F.A.; Kroon, P.A. J. Agric. Food Chem. 2004, 52, 428-438
3. Arguello, L.G.; Sensharma, D.K.; Qiu, F.; Nurtaeva, A.; Rassi, Z.E. J. of AOAC int.
1999, 82(5), 1115-1127
4. Warton, B.; Matthiessen, J.N.; Shackleton, M.A. J. Agric. Food Chem. 2001, 49,
5244-5250
5. Szmigielska, A.M.; Schoenau, J.J. J. Agric. Food Chem. 2000, 48, 5190-5194
6. Nastruzzi, C.; Cortesi, R.; Esposito, E.; Menegatti, E.; Leoni, O.; Iori, R.; Palmieri, S.
J. Agric. Food Chem. 1996, 44, 1014-1021
7. Leoni, O.; Iori, R.; Palmieri*, S. J. Agric. Food Chem. 1991, 39, 2322-2326
8. Karcher, A.; Melouk, H.A.; Rassi, Z.E. J. Agric. Food Chem. 1999, 47, 4267-4274
9. Karcher, A.; Melouk, H.A.; Rassi, Z.E. Analytical Biochemistry 1999, 267, 92-99
10. Mellon, F.A.; Bennett, R.N.; Holst, B.; Williamson, G. Analytical Biochemistry 2002,
306, 83-91
11. Nastruzzi, C.; Cortesi, R.; Esposito, E.; Menegatti, E.; Leoni, O.; Iori, R.; Palmieri J.
Agric. Food Chem. 2000, 48, 3572-3575
12. Tolra, R.P.; Alonso, R.; Poschenrieder, C.; Barcelo, D.; Barcelo, J. J. of
Chromatograph A 2000, 889, 75-81
13. Cai, Z.W.; Cheung, C.Y.; Ma, W.T.; Au, W.M.; Zhang, X.Y.; Lee, A. Anal. Bioanal.
Chem. 2004, 378, 827-833
14. Kiddle, G.; Bennett, R.N.; Botting, N.P.; Davidson, N.E.; Robertson, A.A.B.;
Wallsgrove, R.M. Phytochemical Analysis 2001, 12, 226-242
15. Botting, C.H.; Davidson, N.E.; Griffiths, D.W.; Bennett, R.N.; Botting, N.P. J. Agric.
47
Food Chem. 2002, 50, 983-988
16. Warton, B.; Matthiessen, J.N.; Shackleton, M.A. J. Agric. Food Chem. 2001, 49,
5244-5250
48
APPENDIX
-TOF Product (422.0): 30 MCA scans from Sample 6 (glucoriderin-MS2) of chung2.wiff
a=3.56258132228446760e-004, t0=5.70181404275608660e+001
Max. 33.0 counts.
O
H2
C
CH2OH
32
HSO4
-
Ο
ΟΗ
(S)
S
358.0605
CH3
NOSO3H
OH
96.9719
Glucoiberin standard
M.W. = 423.0327
26
24
22
CH2OH
20
ΟΗ
18
16
Ο
422.0670
S
195.9942
[M-H]-
OH
74.9981
OH
259.0409
12
180.0400
SO3
10
162.0042
8
275.0237
407.0218
6
138.9866
4
79.9645
228.9958
135.9759
2
0
C
H2
(S)
OH (R)
14
C
(S)
30
28
S
CH2
342.1137
119.0577
60
80
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z, amu
Figure A.1: MS/MS spectrum of glucoiberin standard
-TOF Product (438.0): 30 MCA scans from Sample 32 (GlucocheirolinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
211
Max. 211.0 counts.
438.0303
O
H2
C
CH2OH
200
HSO4
180
-
Ο
ΟΗ
(S)
C
CH2
S
CH3
O
(S)
NOSO3H
(S)
OH (R)
96.9576
S
C
H2
OH
Glucocheirolin standard
M.W. = 439.0277
160
140
[M-H]-
CH2OH
ΟΗ
Ο
S
120
OH
100
74.9871
OH
80
259.0150
SO3-
60
40
196.0107
274.9952
128.9252
244.9655
138.9692
20
79.9536
0
60
80
100
120
358.0900
241.9766
119.0313
290.9898
145.0517 214.9696
59.0091
140
160
180
200
220
240 260
m/z, amu
280
Figure A.2: MS/MS spectrum of glucocheirolin standard
49
300
320
340
360
380
400
420
440
-TOF Product (388.0): 30 MCA scans from Sample 8 (progoitrin-MS2) of chung2.wiff
a=3.56258132228446760e-004, t0=5.70181404275608660e+001
H
96.9659
22.0
H2
C
CH2OH
20.0
18.0
Max. 22.0 counts.
HSO4
-
74.9987
Ο
ΟΗ
(S)
S
(R)
C
C
C
H
CH2
OH
NOSO3H
(S)
(S)
OH (R)
OH
Progoitrin standard
M.W. = 389.0450
16.0
CH2OH
14.0
135.9813
12.0
S
Ο
ΟΗ
[M-H]-
OH
OH
195.0521
10.0
SO3
388.0835
-
8.0
259.0350
6.0
275.0274
4.0
79.9665
2.0
0.0
60
80
154.0002
100
120
140
160
192.0159
180
200
220
240
260 280 300
m/z, amu
320
340
360
380
400
420
440
460
480
500
Figure A.3: MS/MS spectrum of progoitrin standard
-TOF Product (358.0): 30 MCA scans from Sample 4 (sinigrinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
H2
C
CH2OH
74.9844
62
60
HSO4-
ΟΗ
(S)
55
Ο
S
C
(S)
Max. 62.0 counts.
C
H
CH2
NOSO3H
(S)
OH (R)
OH
50
45
Sinigrin standard
M.W. = 359.0345
96.9581
CH2OH
S
Ο
ΟΗ
40
OH
35
[M-H]-
OH
30
SO3-
25
20
15
10
79.9540
116.0077
161.9818
195.0323
358.0175
259.0135
5
164.9700
101.0191
0
60
80
100
275.0175
128.9360
120
140
160
180
200
220
240
m/z, amu
Figure A.4: MS/MS spectrum of sinigrin standard
50
260
280
300
320
340
360
380
400
-TOF Product (388.0): 30 MCA scans from Sample 1 (epiprogoitrin-MS2) of chung2.wiff
a=3.56258132228446760e-004, t0=5.70181404275608660e+001
17.0
CH2OH
74.9995
96.9709
16.0
15.0
ΟΗ
HSO4-
14.0
Ο
Max. 17.0 counts.
OH
H2
C
CH2OH
S
Ο
ΟΗ
(S)
S
(S)
C
C
H
C
CH2
H
NOSO3H
(S)
(S)
OH (R)
OH
OH
Epiprogoitrin standard
M.W. = 389.0450
OH
13.0
12.0
388.0763
11.0
146.0472
10.0
[M-H]-
195.0836
135.9845
9.0
8.0
SO3
7.0
259.0273
275.0241
6.0
5.0
4.0
192.0093
3.0
301.0257
2.0
79.9678
1.0
0.0
60
80
332.0249
100
120
140
160
180
200
220
240
260 280 300
m/z, amu
320
340
360
380
400
420
440
460
480
500
Figure A.5: MS/MS spectrum of epiprogoitrin standard
-TOF Product (434.0): 30 MCA scans from Sample 18 (GlucorapheninMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 45.0 counts.
96.9527
45
O
H2
C
CH2OH
40
HSO4
-
ΟΗ
(S)
Ο
S
C
(S)
OH (R)
C
H2
C
H
C
H
S
CH3
NOSO3H
(S)
OH
Glucoraphenin standard
35
M.W. = 435.0327
CH2OH
30
ΟΗ
25
Ο
[M-H]-
S
434.0301
OH
419.0080
20
OH
SO3
15
74.9846
10
195.0467
129.0238
192.0068
5
0
259.0061
-
79.9536
60
80
274.9921
240.9591
138.9877
100
120
140
160
255.9360
180
200
220
240
260 280 300
m/z, amu
330.9917
320
Figure A.6: MS/MS spectrum of glucoraphenin standard
51
340
370.0284
360
380
400
420
440
460
480
500
-TOF Product (424.0): 30 MCA scans from Sample 57 (SinalbinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 493.0 counts.
424.0434
493
H2
C
CH2OH
450
ΟΗ
(S)
Ο
S
NOSO3H
(S)
400
OH
C
(S)
OH (R)
Sinalbin standard
M.W. = 425.0450
350
[M-H]-
CH2OH
OH
Ο
ΟΗ
S
300
OH
OH
250
259.0156
200
182.0288
274.9930
150
195.0314
100
230.9794
50
227.9960
168.9768 200.9738
0
60
80
100
120
140
160
180
246.0136
241.0054
200
220
240 260
m/z, amu
291.0005
280
300
320
344.0885
340
360
380
400
420
440
Figure A.7: MS/MS spectrum of sinalbin standard
-TOF Product (372.0): 30 MCA scans from Sample 6 (gluconapinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 110.0 counts.
74.9853
110
H2
C
CH2OH
100
(S)
90
96.9586
HSO4
Ο
ΟΗ
S
C
(S)
-
C
H2
C
H
CH2
NOSO3H
(S)
OH (R)
OH
80
Gluconapin standard
M.W. = 373.0501
70
CH2OH
60
ΟΗ
50
SO3
-
Ο
[M-H]-
S
OH
OH
40
30
372.0505
20
79.9513
195.0279
130.0285
259.0185
119.0339
10 59.0082
138.9607
0
275.0045
178.9952
292.0821
60
80
100
120
140
160
180
200
220
240
m/z, amu
Figure A.8: MS/MS spectrum of gluconapin standard
52
260
280
300
320
340
360
380
400
-TOF Product (438.0): 30 MCA scans from Sample 12 (glucosibarinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 80.0 counts.
96.9551
80
H
HSO4
75
-
H2
C
CH2OH
ΟΗ
(S)
70
Ο
(S)
135.9682
50
Glucosibarin standard
M.W. = 439.0607
S
Ο
ΟΗ
55
OH
NOSO3H
OH
CH2OH
60
(R)
C
(S)
OH (R)
65
S
C
OH
[M-H]-
OH
45
40
35
438.0619
74.9869
SO3
30
195.0317
259.0157
25
20
275.0003
15
332.0046
79.9553
10
85.0281
5
0
138.9689
60
80
100
153.9711
128.9375
120
244.9909
301.0042
145.0472
169.9414 198.9805
140
160
180
200
358.1076
215.0118
220
240 260
m/z, amu
280
300
320
340
360
380
400
420
440
Figure A.9: MS/MS spectrum of glucosibarin standard
-TOF Product (408.0): 30 MCA scans from Sample 38 (GlucotropaeolinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
HSO4-
96.9588
524
Max. 524.0 counts.
H2
C
CH2OH
500
(S)
450
400
74.9859
Ο
ΟΗ
C
(S)
NOSO3H
[M-H]-
(S)
OH (R)
CH2OH
S
Ο
ΟΗ
OH
S
Glucotropaeolin standard
M.W. = 409.0501
350
OH
300
OH
250
SO3
408.0478
166.0322
200
150
259.0134
195.0338
100
79.9525
274.9888
214.9826
50
85.0259 119.0357
138.9683
168.9781
0
60
80
100
120
140
160
163.0623
180
200
230.0145 241.0019
220
240 260
m/z, amu
280
328.0864
300
Figure A.10: MS/MS spectrum of glucotropaeolin standard
53
320
340
360
380
400
420
440
-TOF Product (420.0): 30 MCA scans from Sample 22 (GlucoerucinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
96.9567
Max. 227.0 counts.
H2
C
CH2OH
220
HSO4
-
ΟΗ
(S)
CH2
C
H2
C
C
H2
S
CH3
NOSO3H
(S)
(S)
OH (R)
200
S
Ο
OH
Glucoerucin standard
M.W. = 421.0535
180
160
CH2OH
74.9853
Ο
ΟΗ
S
[M-H]-
140
420.0475
OH
120
SO3
-
OH
100
80
259.0160
60
79.9525
178.0352
195.0335
40
274.9891
226.9948
20
0
85.0231 119.0323
138.9692
163.0610
224.0099
242.0073
340.0940
131.0417
60
80
100
120
140
160
180
200
220
240 260
m/z, amu
280
300
320
340
360
380
400
420
440
Figure A.11: MS/MS spectrum of glucoerucin standard
-TOF Product (422.0): 30 MCA scans from Sample 27 (GluconasturtiinMS2) of Chung.wiff
a=3.56036418804506270e-004, t0=5.68918465398965050e+001
96.9574
686
H2
C
CH2OH
650
HSO4-
600
ΟΗ
(S)
Ο
S
C
550
C
H2
NOSO3H
(S)
OH (R)
(S)
OH
Gluconasturtiin standard
M.W. = 423.0658
500
CH2OH
450
400
Max. 686.0 counts.
74.9855
Ο
ΟΗ
350
S
[M-H]422.0648
OH
SO3
300
-
OH
250
200
259.0141
180.0497
150
50
0
195.0340
79.9522
100
228.9978
85.0259 119.0323
60
80
100
274.9890
138.9682
165.0357
131.0277
120
140
160
180
200
240.9993
220
240 260
m/z, amu
301.0072
280
300
Figure A.12: MS/MS spectrum of gluconasturtiin standard
54
320
342.1033
340
360
380
400
420
440
-TOF MS: 0.050 min from Sample 1 (t-rt-6.94) of chung1.wiff
a=3.56264499558618230e-004, t0=5.71842229067915470e+001
Max. 56.0 counts.
434.0255
O
55
H2
C
CH2OH
50
ΟΗ
(S)
S
Ο
C
(S)
OH (R)
45
C
H2
C
H
C
H
S
CH3
NOSO3H
(S)
[M-H]-
OH
Glucoraphenin in
Lycopersicon esculentum
[番茄(Tomato)] extract at
8.729min
M.W. = 435.0327
40
35
30
25
20
15
265.1454
183.0124
152.9173
10
293.1837
255.2330
5
0
136.9300
60
80
100
120
140
418.9928
325.1753
270.8373
311.1668
216.9001
160
180
200
220
240
260 280 300
m/z, amu
320
340
360
380
400
420
440
460
480
500
Figure A.13a: ESI-QTOF-MS spectrum of Lycopersicon esculentum [番茄(Tomato)]
extract at 8.729min
-TOF Product (434.0): 30 MCA scans from Sample 2 (t-rt-6.94-MS2) of chung1.wiff
a=3.56264499558618230e-004, t0=5.71842229067915470e+001
Max. 82.0 counts.
O
H2
C
CH2OH
CH2OH
80
HSO4
75
70
ΟΗ
S
Ο
ΟΗ
(S)
Ο
S
C
(S)
C
H2
C
H
C
H
S
CH3
434.0272
NOSO3H
(S)
OH (R)
OH
96.9605
Glucoraphenin in Lcopersicon
esculentum [番茄(Tomato)]
extract at 8.729min
M.W. = 435.0327
OH
65
OH
60
55
[M-H]-
50
259.0183
45
419.0099
40
35
74.9894
195.0322
30
25
192.0163
20
129.0232
240.9715
168.9535
274.9914
15
10
93.5864
5
0
60
135.9696 145.0446
113.4158
153.4012 186.4013
80
120
100
140
160
255.9464
203.2529
180
297.5118
234.2286
200
220
240
339.4955
331.7443
260 280 300
m/z, amu
320
340
360
393.7627 427.7920 488.2259
380
400
420
440
460
480
500
Figure A.13b: MS/MS spectrum of Lycopersicon esculentum [番茄(Tomato)] extract at
8.729min
55
-TOF MS: 40 MCA scans from Sample 21 (Rt5.435) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
Max. 2087.0 counts.
388.0120
2087
H
2000
H2
C
CH 2OH
1900
1800
ΟΗ
(S)
1700
Ο
C
H
CH 2
OH
NOSO 3H
(S)
OH
[M-H]-
1500
(R)
C
C
(S)
OH (R)
1600
S
Progoitrin in Root of Isatis
indigotica Fort. [北板藍根]
extract at 5.834min
M.W. = 389.0450
1400
1300
1200
1100
1000
900
800
264.1433
700
600
281.2313
500
283.2469
400
341.0869
300
503.1292
389.0183
279.2185
200
100
368.9610
306.1862 297.1118 339.3029
0
260
280
549.1344
325.0969
300
320
340
360
431.1085 455.9921
377.0592
380
400
420
440
m/z, amu
539.0968
471.9644
460
480
500
520
540
593.1555
560
580
600
Figure A.14a: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [北板藍根]
extract at 5.834min
-TOF Product (388.0): 60 MCA scans from Sample 22 (Rt5.435(MS/MS)-388.01-1) of Chung291104.wi...
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
Max. 47.0 counts.
H
74.9863
47
H2
C
CH 2OH
45
ΟΗ
(S)
CH2OH
40
96.9559
HSO4
35
-
ΟΗ
Ο
(R)
C
C
H
C
(S)
CH 2
OH
NOSO 3H
(S)
OH (R)
S
OH
Progoitrin in Root of Isatis
indigotica Fort. [北板藍根]
extract at 5.834min
M.W. = 389.0450
OH
30
Ο
S
OH
25
20
135.9633
SO3
15
[M-H]-
-
195.0270
146.0211
388.0060
258.9906
10
138.9644
79.9524
119.0309
5
128.9249
57.4090
0
274.9692
153.9726
60
80
100
120
140
161.0347
160
191.9868
180
200
300.9982
220
240
m/z, amu
260
280
300
320
340
360
380
Figure A.14b: MS/MS spectrum of Root of Isatis indigotica Fort. [北板藍根] extract at
5.834min
56
400
-TOF MS: 60 MCA scans from Sample 15 (Rt5.801-1) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
Max. 918.0 counts.
H2
C
CH2OH
264.1433
900
850
ΟΗ
(S)
800
S
Ο
C
H
C
CH2
NOSO3H
(S)
(S)
OH (R)
750
OH
Sinigrin in Root of
Isatis indigotica Fort.
[北板藍根] extract at
6.276min
700
650
281.2339
600
283.2468
550
500
450
[M-H]-
267.2170
400
350
300
250
150
270.2344
100
50
341.0884
284.2501
368.9543
293.1611298.9842
0
260
503.1270
358.0053
279.2168
200
388.0106
359.0127 367.3363
412.9449
381.3464
280
300
320
340
360
380
400
439.0693
420
440
m/z, amu
460
549.1374
465.0872505.1302
480
500
593.1540
520
540
560
580
600
Figure A.15: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [北板藍根]
extract at 6.276min
-TOF MS: 33 MCA scans from Sample 46 (Rt6.369) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
Max. 2890.0 counts.
388.0270
OH
2800
H2
C
CH2OH
2600
(S)
2400
ΟΗ
(S)
C
C
C
H
CH2
H
NOSO3H
(S)
OH (R)
2200
Ο
S
(S)
OH
2000
Epiprogoitrin in Root of
Isatis indigotica Fort.
[北板藍根] extract at
6.971min
M.W. = 389.0450
[M-H]-
1800
1600
1400
1200
1000
800
600
400
200
264.1544
390.0253
267.2234
341.1002
0
260
280
300
320
340
360
368.9640 445.9772 456.0104
380
400
420
440
503.1513
460 480 500
m/z, amu
520
549.1584 563.1551
540
560
580
600
620
640
660
680
Figure A.16a: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [北板藍根]
extract at 6.971min
57
700
-TOF Product (388.0): 100 MCA scans from Sample 47 (Rt6.369(MS/MS)-388) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
Max. 207.0 counts.
OH
207
200
H2
C
CH2OH
CH2OH
190
180
ΟΗ
S
Ο
160
150
C
CH2
H
NOSO3H
(S)
Epiprogoitrin in Root of
Isatis indigotica Fort.
[北板藍根] extract at
6.971min
M.W. = 389.0450
[M-H]-
OH
OH
140
130
120
135.9680
110
S
C
H
OH
HSO4-
96.9602
Ο
(S)
OH (R)
74.9891
170
ΟΗ
(S)
388.0269
(S)
C
100
90
195.0321
80
70
SO3-
60
259.0038
146.0223
50
274.9864
40
30
20
10
0
59.0129
138.9633
119.0328
79.9575
191.9884
128.9266
85.0345
60
80
100
120
140
164.9880
160
180
210.0027
198.9876 225.9676
200
220
240
m/z, amu
260
332.0057
308.0766
240.9916
280
300
320
340
360
380
400
Figure A.16b: MS/MS spectrum of Root of Isatis indigotica Fort. [北板藍根] extract at
6.971min
-TOF MS: 39 MCA scans from Sample 32 (Rt11.258) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
Max. 534.0 counts.
264.1495
534
H2
C
CH2OH
500
(S)
ΟΗ
S
Ο
(S)
OH (R)
450
OH
C
NOSO3H
(S)
OH
Sinalbin in Root of
Isatis indigotica Fort.
[北板藍根] extract at
11.290min
M.W. = 425.0450
281.2373
400
350
283.2526
300
[M-H]-
267.2217
250
200
150
424.0150
279.2205
100
270.2400
284.2546
368.9629
50
277.1950
0
260
280
306.1949
300
320
341.0987
340
362.2543
360
380
412.9511
400
426.0113
420
440
m/z, amu
451.4223
460
480
487.3240
500
520
550.1018
540
560
564.0580
580
Figure A.17a: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [北板藍根]
extract at 11.290min
58
600
-TOF Product (424.0): 60 MCA scans from Sample 34 (Rt11.258(MS/MS)-424-1) of Chung291104.wiff
a=3.55978894933761710e-004, t0=5.66641529975095180e+001
96.9522
9.0
H2
C
CH2OH
8.5
ΟΗ
(S)
8.0
Ο
S
OH
C
(S)
NOSO3H
(S)
OH (R)
HSO4-
7.5
Max. 9.0 counts.
OH
Sinalbin in Root of
Isatis indigotica Fort.
[北板藍根] extract at
11.290min
M.W. = 425.0450
7.0
6.5
6.0
5.5
5.0
424.0178
[M-H]-
4.5
74.9881
4.0
182.0260
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
60
80
100
120
140
160
180
200
220
240 260
m/z, amu
280
300
320
340
360
380
400
420
440
Figure A.17b: MS/MS spectrum of Root of Isatis indigotica Fort. [北板藍根] extract at
11.290min
-TOF MS: 0.400 min from Sample 3 (z-rt-6.15) of chung1.wiff
a=3.56264499558618230e-004, t0=5.71842229067915470e+001
70
(S)
ΟΗ
OH (R)
60
50
45
40
Ο
S
C
(S)
65
55
H2
C
CH2OH
75
Max. 76.0 counts.
265.1545
C
H
CH2
[M-H]-
NOSO3H
(S)
OH
Sinigrin in Thlaspi
arvensis L. [菥蓂]
extract at 6.005min
M.W. = 359.0345
358.0384
293.1855
183.0122
309.1856
35
30
353.2094
25
325.1939
397.2451
20
337.2132
281.2549
15
255.2342
10
184.0176
5
0
60
80
100
120
140
160
180
200
241.2168 297.1492
220
240
260 280 300
m/z, amu
381.2446
441.2700
321.2279
320
340
360
380
400
420
440
460
480
500
Figure A.18a: ESI-QTOF-MS spectrum of Thlaspi arvense L. [菥蓂] extract at 6.005min
59
-TOF Product (358.0): 30 MCA scans from Sample 4 (z-rt-6.15-MS2) of chung1.wiff
a=3.56264499558618230e-004, t0=5.71842229067915470e+001
65
Max. 65.0 counts.
H2
C
CH2OH
74.9926
60
ΟΗ
(S)
HSO4
50
CH2
NOSO3H
(S)
-
OH
Sinigrin in Thlaspi
arvensis L. [菥蓂]
extract at 6.005min
M.W. = 359.0345
CH2OH
45
96.9621
ΟΗ
S
Ο
40
OH
35
C
H
C
(S)
OH (R)
55
S
Ο
OH
[M-H]-
30
25
95.9562
SO3-
20
15
358.0394
195.0349
10
5
0
79.9581
63.8038
60
80
161.9916
116.0176
138.9804
134.9781
100
120
275.0004
259.0274
177.4611
140
160
180
200
262.9085
211.1616
220
240
314.9930
260 280 300
m/z, amu
320
340
376.8085 432.4078
360
380
400
420
447.5264
440
460
460.1431
480
500
Figure A.18b: MS/MS spectrum of Thlaspi arvense L. [菥蓂] extract at 6.005min
-TOF MS: 30 MCA scans from Sample 11 (z8.5) of chung2.wiff
a=3.56258132228446760e-004, t0=5.70181404275608660e+001
Max. 39.0 counts.
O
H2
C
CH2OH
205.1455
38
(S)
36
220.1677
28
233.1742
24
(S)
C
H2
C
H
C
H
S
CH3
NOSO3H
(S)
OH
32
26
C
Glucoraphenin in
Thlaspi arvensis L.
[菥蓂] extract at
8.347min
M.W. = 435.0327
34
30
ΟΗ
OH (R)
250.1725
S
Ο
434.0624
[M-H]-
223.0486
22
265.1727
20
18
16
148.0700
255.2594
14
178.0723
12
325.2181
10
311.1937
183.0295
8
126.9162
206.1480
281.2803
6
186.9412194.9564
293.1983
241.2383
4
259.0410
2
0
60
80
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z, amu
Figure A.19a: ESI-QTOF-MS spectrum of Thlaspi arvense L. [菥蓂] extract at 8.347min
60
-TOF MS: 0.367 min from Sample 6 (t-rt-11.8) of chung1.wiff
a=3.56264499558618230e-004, t0=5.71842229067915470e+001
Max. 471.0 counts.
372.0464
471
H2
C
CH2OH
450
(S)
400
ΟΗ
Ο
S
C
(S)
OH (R)
C
H2
C
H
CH2
NOSO3H
(S)
[M-H]-
OH
Gluconapin in Seed of
Lepidium apetalum
Willd [葶藶子] extract
at 11.875min
M.W. = 373.0501
350
300
250
200
150
100
50
0
60
80
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z, amu
Figure A.20a: ESI-QTOF-MS spectrum of Seed of Lepidium apetalum Willd [葶藶子]
extract at 11.875min
-TOF Product (372.0): 22 MCA scans from Sample 5 (t-rt-11.8-ms2) of chung1.wiff
a=3.56264499558618230e-004, t0=5.71842229067915470e+001
156
150
Max. 156.0 counts.
H2
C
CH2OH
74.9918
ΟΗ
(S)
140
S
Ο
C
(S)
OH (R)
C
H2
C
H
CH2
NOSO3H
(S)
OH
130
120
Gluconapin in Seed of
Lepidium apetalum
Willd [葶藶子] extract
at 11.875min
M.W. = 373.0501
HSO4-
110
CH2OH
100
ΟΗ
96.9639
90
80
S
Ο
OH
OH
70
[M-H]-
60
SO3-
50
40
130.0357
372.0598
259.0226
30
195.0410
20
241.0128
275.0018
79.9621
10
0
60
80
100
120
140
160
180
200
220
240
260 280 300
m/z, amu
320
340
360
380
400
420 440
460 480
500
Figure A.20b: MS/MS spectrum of Seed of Lepidium apetalum Willd [葶藶子] extract at
11.875min
61
62