Microsoft Word - cover.docDevelop 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)
22/04/2005
II
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 [].
Thank you for Dr. Zhong-zhen Zhao provide me Leaf of Isatis indigotica Fort. sample [].
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.
Date:
III
Develop a Sample Preparation Procedure for HPLC Analysis of Glucosinolates in Traditional Chinese Medicines
By
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. The samples were extracted with methanol, followed by filtration
and evaporation. 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. Individual glucosinolates were detected by a UV detector
at 233nm. 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.
2.1 Vegetable and Traditional Chinese Medicine (TCM) samples
2.1.1 Vegetable samples P.7
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 P.9-P.10
2.5.2 Clean-up process P.10
2.7 HPLC analysis P.11-P.12
3.1 Qualitative analysis
3.1.1 Determination of the retention times for each intact glucosinolate standard P.13-P.14
3.1.2 Identification of the glucosinolates in vegetable and Traditional Chinese Medicine (TCM) samples by using reversed-phase HPLC analysis P.15-P.16
3.1.3 Identification of the glucosinolates in vegetable and Traditional Chinese Medicine (TCM) samples by using ESI-QTOF-MS and MS/MS analysis P.16-32
3.2 Quantitative analysis
V
3.2.4 Glucosinolate concentrations in vegetable and Traditional Chinese Medicine (TCM) samples P.38-39
4. Discussion
4.4 Gradient program P.42-P.43
5. Conclusion P.45
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]
Ο ΟΗ
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
2
R C
O H
HSO4 unstable aglucon Glucose hydrogen suphate ion
R S C N R S C N R C N S
Thiocyanate Isothiocyanate Nitrile 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
+ +
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 Method of dramatization and identification Main references
1.Total glucosinolates 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)
4
(1995); Hrncirik and Velisek (1997); Robertson
and Botting (1999); Griffiths et al. (2000)
II. X-ray fluorescence spectroscopy (XRF) Schung and Hane Klaus, (1990)
4. Degradation Products 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. GC-MS analysis of the glucosinolate breakdown products is
often used. [15] 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,
5
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.
6
Chemical Name of glucosinolate (GS)
Chemical structure of R Group a
Molecular weight b, g/mol
Phenethyl-GS
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
H2 C
H2 C
H2 CS
2.1 Chemicals and reagents
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 [] Raphanus sativus Cruciferae []
II. Cherry Tomato [] Lycopersicon esculentum Solanaceae []
III. Tomato [] Lycopersicon esculentum Solanaceae []
Table 3: Local, Scientific and Family names of the vegetable samples used for analysis
8
2.2.2 Traditional Chinese Medicine (TCM) samples
Ten TCM samples were analyzed and shown in Table 4. Leaf of Isatis indigotica Fort. [
] was a gift from Dr. Zhong-zhen Zhao, School of Chinese Medicine, Hong Kong Baptist
University. Rorippa indica (Linn.) Hiern [] 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. Root of Isatis indigotica Fort. Cruciferae []
2. Root of Baphicacanthus cusia (Nees) Bremek. Acanthaceae []
3. Patrinia scabiosaefolia Fisch. ex Trev. Valerianaceae []
4. Thlaspi arvense L. Cruciferae []
5. Leaf of Isatis indigotica Fort. Cruciferae []
6. Leaf of Baphicacanthus cusia (Nees) Bremek. Acanthaceae []
7. Rorippa indica (Linn.) Hiern Cruciferae []
8. Seed of Sinapis alba L. Cruciferae []
9. Seed of Raphanus sativus L. Cruciferae []
10. Seed of Lepidium apetalum Willd Cruciferae []
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
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. The sample mixture was heated with stirring at 70oC for 15
minutes. 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
10
was centrifuged by cyclone centrifuge (Alltech, Deerfield, U.S.A.) for 15 minutes at
13,000rpm. The supernate was collected for clean-up process.
2.5.2 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. The solid sample extract was then dissolved in
300µL of Milli-Q water. The aqueous sample extract was centrifuged by the cyclone
centrifuge for 15 minutes at 13,000rpm. The supernate was collected for HPLC analysis.
11
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. HP1100
series degasser was used for the degas process. 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:
12
Time(min)
% B
1
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. 30% mobile phase B was
then kept until the end of the separation. 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.
13
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 Figure 4: Chromatogram of 300ppm glucosinolate standard mixture solution
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
200
400
600
800
1000
1200
4 .7
Sinigrin
14
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
Glucoerucin 16.16 ± 0.03
Gluconasturtiin 19.11 ± 0.03
Table 6: The average and relative retention times for each glucosinolate standard
15
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. [ ] contained glucoiberin, glucocheirolin, progoitrin, sinigrin,
epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucotropaeolin, glucoerucin and
gluconasturtiin.
The chromatograms of Root of Isatis indigotica Fort. [] extract and Root of Isatis
indigotica Fort. [] extract with standards spiked were shown in Figures 5a and 5b
respectively.
16
Figure 5a: Chromatogram of Root of Isatis indigotica Fort. [] extract
Figure 5b: Chromatogram of Root of Isatis indigotica Fort. [] 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.
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE095.D)
4 .7
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE094.D)
4 .8
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. [] 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. [
] extract at 11.883min. The ESI-QTOF-MS spectrums of gluconapin standard and Root
of Isatis indigotica Fort. [] extract at 11.883min were shown in Figure 6a and 6b
respectively.
18
Figure 6a: ESI-QTOF-MS spectrum of gluconapin standard
Figure 6b: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. []
extract at 11.883min
-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.
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 m/z, amu
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
315.0541267.2177 383.0960 440.0099341.0834 455.9759 484.0196393.9971297.2460
-TOF MS: 30 MCA scans from Sample 5 (gluconapin) of Chung.wiff a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 4487.0 counts.
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 m/z, amu
0
500
1000
1500
2000
2500
3000
3500
4000
[M-H]- Ο
CH2
Gluconapin in Root of Isatis indigotica Fort. [] extract at 11.883min M.W. = 373.0501
19
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. []
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. [] at 11.883min.
20
Figure 7a: MS/MS spectrum of gluconapin standard
Figure 7b: MS/MS spectrum of Root of Isatis indigotica Fort. [] at 11.883min
-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
Max. 124.0 counts.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu
0
10
20
30
40
50
60
70
80
90
100
110
85.0283 292.0578145.0374 227.0103 240.9984 300.981556.4612
-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.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu
0
10
20
30
40
50
60
70
80
90
100
110
Ο
(S)(S)
CH2
Root of Isatis indigotica Fort. [] extract at 11.883min M.W. = 373.0501
HSO4 -
SO3 -
progoitrin, sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucotropaeolin,
glucoerucin and gluconasturtiin detected in the Root of Isatis indigotica Fort. []
extract were analyzed and all gave the positive results expect glucoiberin and glucoerucin.
Therefore, the Root of Isatis indigotica Fort. [] contained glucocheirolin, progoitrin,
sinigrin, epiprogoitrin, glucoraphenin, sinalbin, gluconapin, glucotropaeolin and
gluconasturtiin. The other sample extracts were analyzed by the similar methods and the
chromatograms of the sample extracts were shown in the following:
Figure 8a: Chromatogram of Raphanus sativus [(Chinese Radish)] extract
Figure 8b: Chromatogram of Raphanus sativus [(Chinese Radish)] extract with
standards spiked
Progoitrin G
min0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
250
300
4 .7
min0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
250
300
4 .6
with standards spiked
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE058.D)
4 .6
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE056.D)
8 .2
mAU
-50
0
50
100
150
200
250
300
350
5 .8
Figure 10b: Chromatogram of Lycopersicon esculentum [(Tomato)] extract with standards spiked
Figure 11a: Chromatogram of Root of Isatis indigotica Fort. [] extract
Figure 11b: Chromatogram of Root of Isatis indigotica Fort. [] extract with
standards spiked
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE095.D)
4 .7
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE147.D)
4 .7
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE094.D)
5 .2
Sinalbin
24
Figure 12a: Chromatogram of Root of Baphicacanthus cusia (Nees) Bremek. [] extract
Figure 12b: Chromatogram of Root of Baphicacanthus cusia (Nees) Bremek. [] extract with standards spiked
Figure 13a: Chromatogram of Patrinia scabiosaefolia Fisch. ex Trev. [] extract
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
50
100
150
200
250
300
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE157.D)
4 .6
mAU
0
100
200
300
400
25
Figure 13b: Chromatogram of Patrinia scabiosaefolia Fisch. ex Trev. [] extract with standards spiked
Figure 14a: Chromatogram of Thlaspi arvense L. [] extract
Figure 14b: Chromatogram of Thlaspi arvense L. [] extract with standards spiked
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
-50
0
50
100
150
200
250
300
350
4 .9
mAU
0
100
200
300
400
4 .6
min0 2 4 6 8 10 12 14 16 18
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE153.D)
4 .6
Sinigrin
26
Figure 15a: Chromatogram of Leaf of Isatis indigotica Fort. [] extract
Figure 15b: Chromatogram of Leaf of Isatis indigotica Fort. [] extract with standards spiked
Figure 16a: Chromatogram of Leaf of Baphicacanthus cusia (Nees) Bremek. []
extract
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE149.D)
4 .7
mAU
5 .5
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE152.D)
27
Figure 16b: Chromatogram of Leaf of Baphicacanthus cusia (Nees) Bremek. []
extract with standards spiked
Figure 17a: Chromatogram of Rorippa indica (Linn.) Hiern [] extract
Figure 17b: Chromatogram of Rorippa indica (Linn.) Hiern [] extract with standards spiked
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE159.D)
4 .6
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE164.D)
6 .2
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE160.D)
4 .6
28
Figure 18a: Chromatogram of Seed of Sinapis alba L. [] extract
Figure 18b: Chromatogram of Seed of Sinapis alba L. [] extract with standards spiked
Figure 18c: Chromatogram of five-fold dilution of Seed of Sinapis alba L. [] extract
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE101.D)
4 .7
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE100.D)
4 .7
mAU
4 .7
29
Figure 18d: Chromatogram of five-fold dilution of Seed of Sinapis alba L. []
extract with standards spiked
Figure 19a: Chromatogram of Seed of Raphanus sativus L. [] extract
Figure 19b: Chromatogram of Seed of Raphanus sativus L. [] extract with
standards spiked
mAU
-25
0
25
50
75
100
125
150
175
4 .7
mAU
-50
0
50
100
150
200
250
300
350
5 .7
mAU
DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE135.D)
4 .6
30
Figure 19c: Chromatogram of ten-fold dilution of Seed of Raphanus sativus L. [] extract
Figure 19d: Chromatogram of ten-fold dilution of Seed of Raphanus sativus L. [] extract with standards spiked
Figure 20a: Chromatogram of Seed of Lepidium apetalum Willd [] extract
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
-25
0
25
50
75
100
125
150
5 .9
mAU
-25
0
25
50
75
100
125
150
4 .6
mAU
-50
0
50
100
150
200
250
300
350
5 .1
31
Figure 20b: Chromatogram of Seed of Lepidium apetalum Willd [] extract with standards spiked
Figure 20c: Chromatogram of twenty-fold dilution of Seed of Lepidium apetalum Willd [] extract
Figure 20d: Chromatogram of twenty-fold dilution of Seed of Lepidium apetalum Willd [] extract with standards spiked
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
4 .7
mAU
0
50
100
150
200
5 .2
45 6
.3 36
1 1.
20 1
1 1.
87 5
1 6.
11 2
G lucocheirolin
mAU
0
50
100
150
200
250
300
4 .6
16 4
.9 97
5 .6
57 6
.0 21
6 .8
Sinigrin
32
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
G lucoiberin
G lucocheirolin
G luconapin
G lucosibarin
G lucotropaeolin
G lucoerucin
G luconasturtiin
Raphanus sativus [(Chinese Radish)] O O X O X X X X O X O X
Lycopersicon esculentum [(Cherry Tomato)] X X X X X O X X O X X O
Lycopersicon esculentum [(Tomato)] X X O X X O X X X X O O
Root of Isatis indigotica Fort. [] X O O O O O` O O X O X O
Root of Baphicacanthus cusia (Nees) Bremek.
[] X X X X X X X X X X X X
Patrinia scabiosaefolia Fisch.ex Trev.
[] X X X X X X X X X X X X
Thlaspi arvense L. [] X O O O X O X X O X X X
Leaf of Isatis indigotica Fort. [] X X O X X O X X O X X X
Baphicacanthus cusia (Nees) Bremek.
[] X X X X X X X X X X X X
Rorippa indica (Linn.) Hiern [] X X X O X X X X X X X O
Seed of Sinapis alba L. [] O X O O O X O O O X O O
Seed of Raphanus sativus L. [] X X O O O O X O X X X X
Seed of Lepidium apetalum Willd [] X O X O X X O O X X O X
Remarks: O = Detected X = Not Detected
Table 7: Identified glucosinolates in vegetable and Traditional Chinese Medicine samples
33
Concentration of standards Peak Areas
Glucosinolates 5ppm 50ppm 100ppm 200ppm 300ppm 400ppm 500ppm Glucoiberin 57.50 595.28 1284.74 2339.79 3548.86 4774.39 5878.06
Glucocheirolin 50.42 533.42 1164.18 2113.01 3200.47 4274.74 5308.13 Progoitrin 66.55 693.25 1512.60 2743.52 4144.70 5528.09 6866.26 Sinigrin 101.99 1046.88 2268.54 4135.44 6231.07 8345.19 10297.54
Epiprogoitrin 58.53 602.21 1305.75 2373.71 3604.46 4801.89 5979.22 Glucoraphenin 46.08 466.58 1012.19 1848.87 2786.48 3739.58 4667.01
Sinalbin 123.42 1362.18 2970.99 5694.20 8622.51 11414.40 14046.44 Gluconapin 58.57 649.87 1416.32 2577.45 3911.87 5220.12 6493.76
Glucosibarin 67.63 690.12 1496.50 2718.96 4112.67 5495.41 6809.96 Glucotropaeolin 104.51 1080.15 2355.41 4272.70 6421.64 8542.62 10457.04
Glucoerucin 60.38 614.48 1345.29 2445.27 3660.38 4855.57 5969.82 Gluconasturtiin 67.97 709.73 1536.00 2783.57 4221.78 5634.59 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 curves were acceptable.
dddddddddddddddddddddddddddddddd
0
1000
2000
3000
4000
5000
6000
7000
Concentration of Epiprogoitrin (ppm)
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
35
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:
Glucosinolates Slope, b Standard deviation, SB
Instrument Detection Limit, ng/µL
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 1.75 Remarks: a = Method Detection Limit when 5g of dried TCM was analyzed
b = Method Detection Limit when 50g of fresh vegetable was analyzed
Table 10: Detection limits of each glucosinolate
36
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. [] 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. [] 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
37
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 Peak areas of testing solution 1
Peak areas of testing solution 2
Peak areas of testing solution 3
Peak areas of 50ppm standard mixture solution
Recoveries, % RSD, %
Glucoiberin 513.98 534.96 536.40 595.28 88.77 10.24
Glucocheirolin 496.38 501.96 512.35 533.42 94.40 6.61
Progoitrin 705.11 712.87 717.92 693.25 102.70 5.27
Sinigrin 1025.82 1019.19 1033.70 1046.88 98.03 5.93
Epiprogoitrin 540.48 562.65 551.10 602.21 91.56 9.05
Glucoraphenin 556.86 563.22 590.52 466.58 122.21 14.60
Sinalbin 1269.54 1255.47 1282.99 1362.18 93.18 11.24
Gluconapin 776.46 788.20 811.59 649.87 121.88 14.60
Glucosibarin 577.80 602.60 604.20 690.12 86.10 12.09
Glucotropaeolin 1124.00 1106.50 1119.62 1080.15 103.38 7.44
Glucoerucin 604.14 586.20 584.89 614.48 96.30 8.78
Gluconasturtiin 688.13 683.23 696.56 709.73 97.12 5.51
Table 11: Summary of method recoveries and RSD values for each glucosinolate
Average 99.80 9.28
For sinigrin in Thlaspi arvense L. [] extract,
y, Peak area of sinigrin in Thlaspi arvense L. [] extract = 9628.09;
y = 20.569x + 63.207 for sinigrin;
By substituting the peak area of sinigrin in Thlaspi arvense L. [] extract into the equation
of the calibration curve for sinigrin, the concentration of sinigrin in 10mL sample extract was
found.
Weight of sinigrin in 5g Thlaspi arvense L. [] extract
= Concentration of sinigrin x diluted factor / recovery of sinigrin
= 465.0145ppm x 10 x 10-3L / 98.03%
= 4.7436mg
= Weight of sinigrin / weight of Thlaspi arvense L. []
= 4.7436mg / 5g
=0.9487mg/g
=948.70µg/g
Therefore, the concentration of sinigrin in Thlaspi arvense L. [] was found to be
948.70µg/g.
39
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)
G lucoiberin
G lucocheirolin
G luconapin
G lucosibarin
G lucotropaeolin
G lucoerucin
G luconasturtiin
Total glucosinolates
Raphanus sativus [(Chinese Radish)] 13.02 1.00 X 2.65 X X X X 6.31 X 61.16 X 84.14
Lycopersicon esculentum [(Cherry Tomato)] X X X X X 26.63 X X 5.13 X X 6.83 38.59
Lycopersicon esculentum [(Tomato)] X X 3.40 X X 30.41 X X X X 4.13 9.96 47.90
Root of Isatis indigotica Fort.
[] X 9.55 795.32 28.91 2099.66 124.54 24.32 636.09 X 18.58 X 9.56 3746.53 Root of Baphicacanthus
cusia (Nees) Bremek. [] X X X X X X X X X X X X 0.00
Patrinia scabiosaefolia Fisch. ex Trev.
[] X X X X X X X X X X X X 0.00
Thlaspi arvense L. [] X 42.87 16.45 984.70 X 281.05 X X 28.95 X X X 1354.02
Leaf of Isatis indigotica Fort. [] X X 14.32 X X 488.77 X X 42.32 X X X 545.41
Leaf of Baphicacanthus cusia (Nees) Bremek.
[] X X X X X X X X X X X X 0.00
Rorippa indica (Linn.) Hiern [] X X X 20.69 X X X X X X X 60.63 81.32
Seed of Sinapis Alba L. [] 66.31 X 8.34 4313.82 41.43 X 1591.08 2966.87 26.75 X 17.01 21.56 9053.17
Seed of Raphanus sativus L. [] X X 23.64 13.67 37.68 10919.08 X 20.67 X X X X 11014.74
Seed of Lepidium apetalum Willd
[] X 39.92 X 430.70 X X 66.55 12648.94 X X 13.71 X 13199.82
Remark: X = Not Detected
40
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.
41
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. [ ] were eluted together with the glucosinolates. The
chromatograms of the original Root of Isatis indigotica Fort. [] and Root of Isatis
indigotica Fort. [] 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. [] 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.
Figure 21a: Chromatogram of the original Root of Isatis indigotica Fort. []
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
50
100
150
200
250
300
Area: 55.7971 4 .9
42
Figure 21b: Chromatogram of the original Root of Isatis indigotica Fort. [] 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
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
-50
0
50
100
150
200
250
300
350
Area: 5235.53
5 .6
43
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 []
contained 12648.94µg/g of the gluconapin and seed of Raphanus sativus L.[] 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 []
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 [] 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.
44
Figure 22a: Chromatogram of Seed of Lepidium apetalum Willd [] extract
Figure 22b: Chromatogram of twenty-fold dilution of Seed of Lepidium apetalum Willd [] extract
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
200
400
600
800
5. 24
mAU
0
500
1000
1500
2000
2500
5 .1
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 [] contained the highest concentration of total
glucosinolates, 13199.82µg/g. Root of Baphicacanthus cusia (Nees) Bremek.
[], Patrinia scabiosaefolia Fisch. ex Trev. [] and Leaf of Baphicacanthus
cusia (Nees) Bremek. [] 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.
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 products in the market can be analyzed, for example,
extract to investigate whether they are made of Root of Isatis indigotica Fort. [] or
Root of Baphicacanthus cusia (Nees) Bremek. [].
47
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.
48
Food Chem. 2002, 50, 983-988
16. Warton, B.; Matthiessen, J.N.; Shackleton, M.A. J. Agric. Food Chem. 2001, 49,
5244-5250
49
APPENDIX
Figure A.1: MS/MS spectrum of glucoiberin standard Figure A.2: MS/MS spectrum of glucocheirolin standard
-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.
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
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
OH
OH
CH2OH
S
SO3 -
HSO4 -
-TOF Product (438.0): 30 MCA scans from Sample 32 (GlucocheirolinMS2) of Chung.wiff a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 211.0 counts.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, amu
0
20
40
60
80
100
120
140
160
180
200
Ο ΟΗ
Figure A.3: MS/MS spectrum of progoitrin standard
Figure A.4: MS/MS spectrum of sinigrin standard
-TOF Product (388.0): 30 MCA scans from Sample 8 (progoitrin-MS2) of chung2.wiff a=3.56258132228446760e-004, t0=5.70181404275608660e+001
Max. 22.0 counts.
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
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
OH
OH
CH2OH
S
SO3 -
HSO4 -
-TOF Product (358.0): 30 MCA scans from Sample 4 (sinigrinMS2) of Chung.wiff a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 62.0 counts.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
Figure A.5: MS/MS spectrum of epiprogoitrin standard
Figure A.6: MS/MS spectrum of glucoraphenin standard
-TOF Product (388.0): 30 MCA scans from Sample 1 (epiprogoitrin-MS2) of chung2.wiff a=3.56258132228446760e-004, t0=5.70181404275608660e+001
Max. 17.0 counts.
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
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
OH
OH
CH2OH
S
SO3 -
HSO4 -
-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.
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
0
5
10
15
20
25
30
35
40
330.9917 370.0284
Figure A.7: MS/MS spectrum of sinalbin standard
Figure A.8: MS/MS spectrum of gluconapin standard
-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.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, amu
0
50
100
150
200
250
300
350
400
450
OH
OH
CH2OH
S
-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.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu
0
10
20
30
40
50
60
70
80
90
100
OH
OH
CH2OH
S
SO3 -
HSO4 -
53
Figure A.9: MS/MS spectrum of glucosibarin standard Figure A.10: MS/MS spectrum of glucotropaeolin standard
-TOF Product (408.0): 30 MCA scans from Sample 38 (GlucotropaeolinMS2) of Chung.wiff a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 524.0 counts.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, amu
0
50
100
150
200
250
300
350
400
450
500
Glucotropaeolin standard M.W. = 409.0501
-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.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
198.9805 215.0118169.9414
Figure A.11: MS/MS spectrum of glucoerucin standard
Figure A.12: MS/MS spectrum of gluconasturtiin standard
-TOF Product (422.0): 30 MCA scans from Sample 27 (GluconasturtiinMS2) of Chung.wiff a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 686.0 counts.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, amu
0
50
100
150
200
250
300
350
400
450
500
550
600
650
Ο
(S)(S)
OH
OH
CH2OH
S
SO3 -
HSO4 -
-TOF Product (420.0): 30 MCA scans from Sample 22 (GlucoerucinMS2) of Chung.wiff a=3.56036418804506270e-004, t0=5.68918465398965050e+001
Max. 227.0 counts.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, amu
0
20
40
60
80
100
120
140
160
180
200
220
96.9567
74.9853
420.0475
259.0160
Ο
(S)(S)
extract at 8.729min Figure A.13b: MS/MS spectrum of Lycopersicon esculentum [(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.
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
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
Glucoraphenin in Lcopersicon esculentum [(Tomato)] extract at 8.729min M.W. = 435.0327
-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.
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
0
5
10
15
20
25
30
35
40
45
50
[M-H]-
56
Figure A.14a: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. []
extract at 5.834min Figure A.14b: MS/MS spectrum of Root of Isatis indigotica Fort. [] extract at
5.834min
-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.
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 m/z, amu
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
Ο
(S)(S)
CH2
H
OH
Progoitrin in Root of Isatis indigotica Fort. [] extract at 5.834min M.W. = 389.0450
[M-H]-
-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.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu
0
5
10
15
20
25
30
35
40
CH2
H
OH
Progoitrin in Root of Isatis indigotica Fort. [] extract at 5.834min M.W. = 389.0450
[M-H]-
OH
OH
CH2OH
S
HSO4 -
SO3 -
57
Figure A.15: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. []
extract at 6.276min Figure A.16a: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. []
extract at 6.971min
-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.
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 m/z, amu
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
388.0270
267.2234 456.0104341.1002 549.1584368.9640 503.1513445.9772 563.1551
-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.
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 m/z, amu
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
Ο
(S)(S)
H CH2
Sinigrin in Root of Isatis indigotica Fort. [] extract at 6.276min
[M-H]-
Ο
(S)(S)
CH2
OH
H
Epiprogoitrin in Root of Isatis indigotica Fort. [] extract at 6.971min M.W. = 389.0450
[M-H]-
58
Figure A.16b: MS/MS spectrum of Root of Isatis indigotica Fort. [] extract at
6.971min Figure A.17a: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. []
extract at 11.290min
-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.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
Ο
(S)(S)
CH2
OH
H
Epiprogoitrin in Root of Isatis indigotica Fort. [] extract at 6.971min M.W. = 389.0450 [M-H]-
Ο ΟΗ
OH
OH
CH2OH
S
HSO4 -
SO3 -
-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.
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 m/z, amu
0
50
100
150
200
250
300
350
400
450
500
Ο
(S)(S)
H2 C OH
Sinalbin in Root of Isatis indigotica Fort. [] extract at 11.290min M.W. = 425.0450
[M-H]-
59
Figure A.17b: MS/MS spectrum of Root of Isatis indigotica Fort. [] extract at
11.290min
Figure A.18a: ESI-QTOF-MS spectrum of Thlaspi arvense L. [] extract at 6.005min
-TOF MS: 0.400 min from Sample 3 (z-rt-6.15) of chung1.wiff a=3.56264499558618230e-004, t0=5.71842229067915470e+001
Max. 76.0 counts.
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
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
255.2342 441.2700381.2446 321.2279241.2168184.0176 297.1492
-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
Max. 9.0 counts.
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z, amu
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
H2 C OH
Sinalbin in Root of Isatis indigotica Fort. [] extract at 11.290min M.W. = 425.0450 [M-H]-
HSO4 -
Ο
(S)(S)
H CH2
Sinigrin in Thlaspi arvensis L. [] extract at 6.005min M.W. = 359.0345
[M-H]-
60
Figure A.18b: MS/MS spectrum of Thlaspi arvense L. [] extract at 6.005min
Figure A.19a: ESI-QTOF-MS spectrum of Thlaspi arvense L. [] extract at 8.347min
-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.
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
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
126.9162 206.1480 281.2803
293.1983194.9564186.9412 241.2383 259.0410
-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
Max. 65.0 counts.
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
0
5
10
15
20
25
30
35
40
45
50
55
60
H CH2
Sinigrin in Thlaspi arvensis L. [] extract at 6.005min M.W. = 359.0345
[M-H]-
S
O
CH3
Glucoraphenin in Thlaspi arvensis L. [] extract at 8.347min M.W. = 435.0327 [M-H]-
61
Figure A.20a: ESI-QTOF-MS spectrum of Seed of Lepidium apetalum Willd []
extract at 11.875min
Figure A.20b: MS/MS spectrum of Seed of Lepidium apetalum Willd [] 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
Max. 156.0 counts.
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
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
372.0598130.0357 259.0226
241.0128195.0410 275.001879.9621
-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.
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
0
50
100
150
200
250
300
350
400
CH2
Gluconapin in Seed of Lepidium apetalum Willd [] extract at 11.875min M.W. = 373.0501
[M-H]-
Ο
(S)(S)
CH2
Gluconapin in Seed of Lepidium apetalum Willd [] extract at 11.875min M.W. = 373.0501
[M-H]-