Develop a Sample Preparation Procedure for HPLC Analysis of ...

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. 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. 

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 P.9-P.10 

2.5.2 Clean-up process 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 Determination of the retention times for 

each intact glucosinolate standard 

3.1.2 Identification of the glucosinolates in vegetable 

and Traditional Chinese Medicine (TCM) samples 

by using reversed-phase HPLC analysis 

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.13-P.14 

P.15-P.16 

P.16-32 

3.2 Quantitative analysis 

3.2.1 Calibration curves for each individual glucosinolate standard P.33-P.34 

IV

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] 

CH 2 OH 

R 

ΟΗ 

Ο 

S 

C 

NOSO 3 

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] R C 

S 

β⎯D⎯glucose 

N 

OSO 3 

Myrosinase, H 2 O 

S 

R C 

+ 

OH 

CH 2 OH 

OH 

Ο 

ΟΗ 

+ 

N 

OH 

HSO 4 

unstable aglucon Glucose hydrogen suphate ion 

> pH7 

pH3, Fe 2+ 

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 

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 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) 

3

Heaney and Fenwick (1993); Bjergegaard et al. 

(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, 

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) 

Glucoiberin 

(C 11 H 20 NO 10 S 3 ) 

Glucocheirolin 

(C 11 H 20 NO 11 S 3 ) 

Progoitrin 

(C 11 H 18 NO 10 S 2 ) 

Sinigrin 

(C 10 H 16 NO 9 S 2 ) 

Epiprogoitrin 

(C 11 H 18 NO 10 S 2 ) 

Chemical Name of 

glucosinolate (GS) 

Chemical structure of Molecular weight b , 

R Group a g/mol 

H H 

3-(methylsulfinyl)propyl-GS 2 H 2 2 

423.0327 

H 3 C 

H H 

3-(Methylsulfonyl)propyl-GS 

2 H 2 2 

H 3 C S C C C 439.0277 

(2R)-2-Hydroxybut-3-enyl-GS 

H 2 C C 

H 

C C 

389.0450 

(2R) 

H 

Prop-2-enyl-GS 

2 

H 2 C C C 

359.0345 

H 2 C 

H 

(2S)-2-Hydroxybut-3-enyl-GS 389.0450 

(2S) 

O 

S 

O 

O 

C 

C 

H 

H 

C 

C 

OH 

H 

OH 

H 2 

C 

C 

H 2 

Glucoraphenin 

(C 12 H 20 NO 10 S 3 ) 

4-(methylsulfinyl)but-3-enyl-GS H 2 H 2 435.0327 

H 3 C 

O 

S 

C 

H 

C 

H 

C 

C 

Sinalbin 

(C 14 H 18 NO 10 S 2 ) 

Gluconapin 

(C 11 H 18 NO 9 S 2 ) 

Glucosibarin 

(C 15 H 20 NO 10 S 2 ) 

Glucotropaeolin 

(C 14 H 18 NO 9 S 2 ) 

Glucoerucin 

(C 12 H 22 NO 9 S 3 ) 

Gluconasturtiin 

(C 15 H 20 NO 9 S 2 ) 

p-Hydroxybenzyl-GS HO 

C 425.0450 

But-3-enyl-GS H 2 C C C C 

373.0501 

C C 

(2R)-2-Hydroxy-2-phenethyl-GS 439.0607 

Benzyl-GS C 

409.0501 

H H 

4-(Methylthio)butyl-GS 2 H 2 H 2 2 

421.0535 

H 3 C 

S 

C C 

Phenethyl-GS 423.0658 

H 

C 

H 2 

C 

OH 

H 

H 2 

H 2 

H 2 

C 

H 2 

H 2 

H 2 

C 

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. Valerianaceae [ 敗醬草科 ] 

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 ] 

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. The sample mixture was heated with stirring at 70 o C 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 55 o C 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. 

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 200 o C 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. 

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. 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: 

11

Gradient program 

%B 

35 

30 

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. 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. 

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 o C 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 

DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\KCLEE108.D) 

mAU 

1200 

1000 

800 

600 

400 

Sinigrin 

Progoitrin 


Glucoiberin 

4.772 

5.150 

5.837 

6.244 

Epiprogoitrin 

7.042 

Glucoraphenin 

Sinalbin 

11.234 

11.899 

Gluconapin 

Glucosibarin 

15.084 


15.811 

16.160 

Glucoerucin 

19.101 


200 

8.492 

0 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 4: Chromatogram of 300ppm glucosinolate standard mixture solution 

min 

13

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 


19.11 ± 0.03 

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

mAU 

350 

300 

250 

200 

150 


Glucoiberin 

4.805 


5.834 

Progoitrin 

Sinigrin 

6.971 

Epiprogoitrin 

Glucoraphenin 

Sinalbin 

11.883 

Gluconapin 


Glucoerucin 


100 

50 

5.212 

6.276 

8.548 

11.306 

15.799 

16.026 

19.255 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 5a: Chromatogram of Root of Isatis indigotica Fort. [ 北板藍根 ] extract 

min 

mAU 

350 

300 

250 

200 

150 

100 


Glucoiberin 

4.796 

Progoitrin 


5.168 

5.830 

Sinigrin 

6.272 

6.972 

Epiprogoitrin 

Glucoraphenin 

8.557 

Sinalbin 

11.290 

11.883 

Gluconapin 

Glucosibarin 


15.881 

15.137 

16.210 

Glucoerucin 

19.182 


50 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 5b: Chromatogram of Root of Isatis indigotica Fort. [ 北板藍根 ] extract with 

standards spiked 

min 

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

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. 

4487 

372.0536 

4000 

(S) 

CH 2 OH 

ΟΗ 

Ο 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 2 

C 

H 

CH 2 

[M-H] - 

3500 

3000 

OH (R) 

(S) 

OH 

Gluconapin standard 

M.W. = 373.0501 

2500 

2000 

1500 

1000 

500 

0 

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 

m/z, amu 

Figure 6a: ESI-QTOF-MS spectrum of gluconapin standard 

374.0497 

220.1537 

255.2393 

227.2081 

283.2709 293.1867 325.1875339.2078 358.0353 388.0655 

-TOF MS: 30 MCA scans from Sample 10 (Rt11.955-1) of Chung291104.wiff 

a=3.55978894933761710e-004, t0=5.66641529975095180e+001 


3982 

3800 

3600 

3400 

3200 

3000 

2800 

2600 

2400 

2200 

2000 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 

372.0233 

[M-H] - 

264.1464 374.0192 

267.2177 315.0541 

383.0960 

297.2460 341.0834 393.9971 

440.0099 455.9759 484.0196 

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 

m/z, amu 

Figure 6b: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [ 北板藍根 ] 

extract at 11.883min 

18 

(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 2 

C 

H 

Gluconapin in Root of Isatis 

indigotica Fort. 

[ 北板藍根 ] extract at 

11.883min 

M.W. = 373.0501 

CH 2

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 SO 3 from the [M-H] - ion. 

The peak at m/z 274.9975 corresponded to the molecular ion with the loss of HSO 4 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 HSO - 4 and SO - 3 , 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 


118 

110 

100 

90 

80 

70 

60 

50 

40 

30 

74.9866 

96.9584 

HSO 4 

- 

SO 3 

- 

Figure 7a: MS/MS spectrum of gluconapin standard 

OH 

CH 2 OH 

ΟΗ 

Ο 

OH 

S 

20 

372.0600 

79.9501 

130.0304 

195.0337 

259.0176 

274.9975 

10 

178.9801 

85.0244 128.9378 

59.0074 175.9927 

292.0796 

163.0819 

0 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 

m/z, amu 

(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 2 


M.W. = 373.0501 

[M-HSO 4 -H] - [M-SO 3 -H] - 

C 

H 

[M-H] - 

CH 2 

-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 


124 

120 

110 

100 

90 

80 

70 

96.9548 

HSO 4 

- 

CH 2 OH 

ΟΗ 

Ο 

S 

(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 2 

C 

H 

Root of Isatis indigotica 

Fort. [ 北板藍根 ] extract 

at 11.883min 

M.W. = 373.0501 

CH 2 

[M-H] - 

372.0156 

60 

74.9854 

OH 

OH 

50 

SO 3 

- 

[M-HSO 4 -H] - 

40 

30 

130.0262 

195.0168 

258.9959 

[M-SO 3 -H] - 

20 

274.9716 

79.9515 

178.9716 

10 

128.9233 

85.0283 145.0374 292.0578 

227.0103 240.9984 

56.4612 

300.9815 

0 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 

m/z, amu 

Figure 7b: MS/MS spectrum of Root of Isatis indigotica Fort. [ 北板藍根 ] at 11.883min 

20

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, 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: 


mAU 

300 

250 

200 

Glucoiberin 

Sinigrin 


Glucosibarin 

16.142 

Glucoerucin 

150 

100 

50 

4.675 

5.148 

6.186 

8.709 

15.122 

0 

0 2 4 6 8 10 12 14 16 18 

Figure 8a: Chromatogram of Raphanus sativus [ 蘿蔔 (Chinese Radish)] extract 

min 

mAU 

300 

250 

200 

150 

100 

50 



Glucoiberin 

4.760 

5.178 

Progoitrin 

5.840 

6.242 

Epiprogoitrin 

Sinigrin 

7.083 

Glucoraphenin 

8.616 

Sinalbin 

11.271 

Gluconapin 

11.861 

Glucosibarin 

15.077 


15.834 

16.103 

Glucoerucin 


19.110 

0 

0 2 4 6 8 10 12 14 16 18 

Figure 8b: Chromatogram of Raphanus sativus [ 蘿蔔 (Chinese Radish)] extract with 


min 

21


mAU 

350 

300 

250 

200 

150 

8.267 

Glucoraphenin 

Glucosibarin 

14.950 

18.873 


100 

50 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 9a: Chromatogram of Lycopersicon esculentum [ 車厘茄 (Cherry Tomato)] extract 

min 

mAU 

350 

300 

250 

200 

150 

100 

50 


Glucoiberin 

4.611 

4.971 

Sinigrin 

Progoitrin 


5.631 

6.064 

Epiprogoitrin 

6.838 

8.240 

Glucoraphenin 

Sinalbin 

11.051 

11.718 

Gluconapin 

Glucosibarin 

14.935 

15.656 


Glucoerucin 

15.991 

18.904 


0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 9b: Chromatogram of Lycopersicon esculentum [ 車厘茄 (Cherry Tomato)] extract 

with standards spiked 

min 


mAU 

350 

300 

250 

200 

150 

Progoitrin 

Glucoraphenin 

8.729 

Glucoerucin 


100 

50 

5.853 

16.168 

19.153 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 10a: Chromatogram of Lycopersicon esculentum [ 番茄 (Tomato)] extract 

min 

22


mAU 

350 

300 

250 

200 

150 

100 

Glucoiberin 


Sinigrin 

Progoitrin 

4.778 

5.158 

5.818 

6.257 

7.091 

Epiprogoitrin 

Glucoraphenin 

8.668 

Sinalbin 

11.291 

11.881 

Gluconapin 

Glucosibarin 

15.111 

15.847 

16.159 


Glucoerucin 

19.138 


50 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 10b: Chromatogram of Lycopersicon esculentum [ 番茄 (Tomato)] extract with 


min 

mAU 

350 

300 

250 

200 



5.834 

Progoitrin 

6.971 

Sinigrin 

Epiprogoitrin 

Glucoraphenin 

Sinalbin 

11.883 

Gluconapin 



150 

100 

6.276 

8.548 

11.306 

15.799 

19.255 

50 

5.212 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 11a: Chromatogram of Root of Isatis indigotica Fort. [ 北板藍根 ] extract 

min 


mAU 

350 

300 

250 

200 

150 

100 

Glucoiberin 

4.796 

5.168 

5.830 

Progoitrin 


Sinigrin 

6.272 

6.972 

Epiprogoitrin 

Glucoraphenin 

8.557 

Sinalbin 

11.290 

11.883 

Gluconapin 

Glucosibarin 


15.881 

15.137 

16.210 

Glucoerucin 

19.182 


50 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 11b: Chromatogram of Root of Isatis indigotica Fort. [ 北板藍根 ] extract with 


23 

min

mAU 


300 

250 

200 

150 

100 

50 

0 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 12a: Chromatogram of Root of Baphicacanthus cusia (Nees) Bremek. [ 南板藍根 ] 

extract 

min 


mAU 

350 

300 

250 

200 

150 

100 

50 

Glucoiberin 


Sinigrin 

Progoitrin 

4.661 

5.060 

5.698 

6.103 

6.918 

Epiprogoitrin 

Glucoraphenin 

8.401 

Sinalbin 

11.169 

11.741 

Gluconapin 

Glucosibarin 


15.692 

14.944 

16.011 

Glucoerucin 

18.963 


0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 12b: Chromatogram of Root of Baphicacanthus cusia (Nees) Bremek. [ 南板藍根 ] 

extract with standards spiked 

min 

mAU 


400 

300 

200 

100 

0 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 13a: Chromatogram of Patrinia scabiosaefolia Fisch. ex Trev. [ 敗醬草 ] extract 

min 

24

mAU 

400 

300 

200 

100 


Sinigrin 

Progoitrin 


Glucoiberin 

4.657 

5.054 

5.693 

6.099 

Epiprogoitrin 

6.901 

Glucoraphenin 

8.367 

Sinalbin 

11.161 

11.743 

Gluconapin 

Glucosibarin 

14.941 


15.688 

16.006 

Glucoerucin 

18.908 


0 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 13b: Chromatogram of Patrinia scabiosaefolia Fisch. ex Trev. [ 敗醬草 ] extract 

with standards spiked 

min 


mAU 

350 

300 

250 

200 

150 


6.005 

Progoitrin 

Sinigrin 

Glucoraphenin 

8.350 

Glucosibarin 

100 

50 

4.978 

5.568 

14.842 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 14a: Chromatogram of Thlaspi arvense L. [ 菥蓂 ] extract 

min 


mAU 

350 

300 

250 

200 

150 

100 

Glucoiberin 


6.004 

Progoitrin 

Epiprogoitrin 

Sinigrin 

4.643 

5.021 

5.667 

6.875 

Glucoraphenin 

8.347 

Sinalbin 

11.137 

11.724 

Gluconapin 

Glucosibarin 

14.931 


15.684 

16.005 

Glucoerucin 


18.962 

50 

0 

-50 

0 2 4 6 8 10 12 14 16 18 

Figure 14b: Chromatogram of Thlaspi arvense L. [ 菥蓂 ] extract with standards spiked 

min 

25


mAU 

350 

300 

250 

200 

150 

Progoitrin 

8.478 

Glucoraphenin 

Glucosibarin 

100 

50 

5.588 

14.918 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 15a: Chromatogram of Leaf of Isatis indigotica Fort. [ 大青葉 ] extract 

min 

mAU 

350 

300 

250 

200 

150 

100 


Sinigrin 

Progoitrin 


Glucoiberin 

4.763 

5.169 

5.823 

6.238 

Epiprogoitrin 

7.065 

8.668 

Glucoraphenin 

Sinalbin 

11.293 

11.873 

Gluconapin 

Glucosibarin 

15.081 


15.832 

16.141 

Glucoerucin 

19.126 


50 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 15b: Chromatogram of Leaf of Isatis indigotica Fort. [ 大青葉 ] extract with 


min 

mAU 

350 


300 

250 

200 

150 

100 

50 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 16a: Chromatogram of Leaf of Baphicacanthus cusia (Nees) Bremek. [ 廣東大青葉 ] 

extract 

min 

26

mAU 

350 

300 

250 

200 

150 

100 

50 

0 

mAU 

350 

300 

250 

200 

150 


Progoitrin 


Glucoiberin 

4.604 

4.988 

5.602 

Sinigrin Sinigrin 

5.996 

Figure 16b: Chromatogram of Leaf of Baphicacanthus cusia (Nees) Bremek. [ 廣東大青葉 ] 



Epiprogoitrin 

6.777 

Glucoraphenin 

8.220 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Sinalbin 

11.064 

11.629 

Gluconapin 

Glucosibarin 

14.850 


15.611 

15.941 

Glucoerucin 

18.920 


19.122 


min 

100 

50 

6.209 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 17a: Chromatogram of Rorippa indica (Linn.) Hiern [ 蔊菜 ] extract 

min 


mAU 

350 

300 

250 

200 

150 

100 

50 

Sinigrin 

Progoitrin 


Glucoiberin 

4.659 

5.050 

5.694 

6.094 

Epiprogoitrin 

6.901 

Glucoraphenin 

8.364 

Sinalbin 

11.141 

11.736 

Gluconapin 

Glucosibarin 

14.940 


15.708 

16.010 

Glucoerucin 

18.967 


0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 17b: Chromatogram of Rorippa indica (Linn.) Hiern [ 蔊菜 ] extract with 


min 

27


mAU 

350 

300 

250 

200 

150 

100 

50 

Glucoiberin 

4.772 

5.821 

5.999 

Progoitrin 

Epiprogoitrin 

Sinigrin 

7.050 

Sinalbin 

11.138 

11.723 

Gluconapin 

Glucosibarin 

15.177 

16.204 

Glucoerucin 


19.192 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 18a: Chromatogram of Seed of Sinapis alba L. [ 白芥子 ] extract 

min 

mAU 

350 

300 

250 

200 

150 

100 

50 


Progoitrin 


Glucoiberin 

4.769 

5.146 

5.819 

6.006 

Epiprogoitrin 

Sinigrin 

7.049 

Glucoraphenin 

8.526 

Sinalbin 

11.128 

11.722 

Gluconapin 


Glucosibarin 

15.166 

15.899 

16.228 

Glucoerucin 

19.194 


0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 18b: Chromatogram of Seed of Sinapis alba L. [ 白芥子 ] extract with standards 

spiked 

min 


mAU 

175 

150 

125 

100 

75 

Progoitrin 

Glucoiberin 

6.176 

Sinigrin 

Epiprogoitrin 

Sinalbin 

11.212 

11.833 

Gluconapin 

Glucosibarin 

Glucoerucin 


50 

25 

0 

4.753 

5.818 

7.027 

15.119 

16.145 

19.093 

-25 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 18c: Chromatogram of five-fold dilution of Seed of Sinapis alba L. [ 白芥子 ] 

extract 

min 

28


mAU 

175 

150 

125 

100 

75 

50 

4.747 

Progoitrin 


Glucoiberin 

5.130 

5.812 

6.174 

Epiprogoitrin 

Sinigrin 

7.023 

Glucoraphenin 

8.467 

Sinalbin 

11.208 

11.834 

Gluconapin 

Glucosibarin 

15.077 


15.818 

16.140 

Glucoerucin 

19.084 


25 

0 

-25 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 18d: Chromatogram of five-fold dilution of Seed of Sinapis alba L. [ 白芥子 ] 


min 


mAU 

350 

300 

250 

200 

150 

Epiprogoitrin 

Sinigrin 

Progoitrin 

7.953 

Glucoraphenin 

Gluconapin 

100 

50 

5.750 

6.092 

6.929 

11.809 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 19a: Chromatogram of Seed of Raphanus sativus L. [ 萊菔子 ] extract 

min 

mAU 

350 

300 

250 

200 

150 

100 


Sinigrin 

Progoitrin 


Glucoiberin 

Epiprogoitrin 

4.633 

5.010 

5.677 

6.070 

6.857 

7.924 

Glucoraphenin 

Sinalbin 

11.128 

11.732 

Gluconapin 

Glucosibarin 


14.924 

15.677 

15.992 

Glucoerucin 


18.929 

50 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 19b: Chromatogram of Seed of Raphanus sativus L. [ 萊菔子 ] extract with 


min 

29


mAU 

150 

125 

100 

75 

Progoitrin 

Sinigrin 

Epiprogoitrin 

8.592 

Glucoraphenin 

50 

25 

0 

5.924 

6.300 

7.180 

-25 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 19c: Chromatogram of ten-fold dilution of Seed of Raphanus sativus L. [ 萊菔子 ] 

extract 

min 

mAU 

150 

125 

100 

75 

50 



Glucoiberin 

4.636 

5.016 

Sinigrin 

Progoitrin 

5.679 

6.088 

Epiprogoitrin 

6.871 

8.216 

Glucoraphenin 

Sinalbin 

11.115 

11.735 

Gluconapin 

Glucosibarin 

14.924 


15.672 

15.990 

Glucoerucin 


18.921 

25 

0 

-25 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 19d: Chromatogram of ten-fold dilution of Seed of Raphanus sativus L. [ 萊菔子 ] 


min 

mAU 

350 


6.202 

11.413 

300 

250 

200 

150 


Sinigrin 

Sinalbin 

Gluconapin 

Glucoerucin 

100 

50 

5.157 

11.110 

16.027 

0 

-50 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 20a: Chromatogram of Seed of Lepidium apetalum Willd [ 葶藶子 ] extract 

min 

30


mAU 

300 

250 

200 

150 

100 

50 


Glucoiberin 

4.616 

4.997 

5.657 

6.021 

Progoitrin 

Epiprogoitrin 

Sinigrin 

6.837 

Glucoraphenin 

8.250 

Sinalbin 

10.946 

11.256 

Gluconapin 


Glucosibarin 

15.177 

15.666 

15.990 

Glucoerucin 


18.917 

0 

0 2.5 5 7.5 10 12.5 15 17.5 20 

Figure 20b: Chromatogram of Seed of Lepidium apetalum Willd [ 葶藶子 ] extract with 


min 

mAU 

200 

150 

100 



Sinigrin 

Sinalbin 

11.875 

Gluconapin 

Glucoerucin 

50 

6.336 

11.201 

0 

5.245 

16.112 

0 2.5 5 7.5 10 12.5 15 17.5 20 

min 

Figure 20c: Chromatogram of twenty-fold dilution of Seed of Lepidium apetalum Willd 

[ 葶藶子 ] extract 

mAU 

175 

150 

125 

100 

75 

50 

25 


Glucoiberin 

4.767 

Progoitrin 


5.147 

5.803 

6.209 

Epiprogoitrin 

Sinigrin 

6.991 

Glucoraphenin 

8.416 

Sinalbin 

11.234 

11.777 

Gluconapin 


Glucosibarin 

15.093 

15.854 

16.186 

Glucoerucin 


19.203 

0 

-25 

0 2.5 5 7.5 10 12.5 15 17.5 20 

min 

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 

Raphanus sativus 

[ 蘿蔔 (Chinese Radish)] 

Lycopersicon esculentum 

[ 車厘茄 (Cherry Tomato)] 


[ 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 ] 

Thlaspi arvense L. 

[ 菥蓂 ] 

Leaf of Isatis indigotica Fort. 

[ 大青葉 ] 

Baphicacanthus cusia (Nees) 

Bremek. 

[ 廣東大青葉 ] 

Rorippa indica (Linn.) Hiern 

[ 蔊菜 ] 

Seed of Sinapis alba L. 

[ 白芥子 ] 

Seed of Raphanus sativus L. 

[ 萊菔子 ] 

Glucoiberin 


Progoitrin 

32 

Sinigrin 

Epiprogoitrin 

Glucoraphenin 

Sinalbin 

Gluconapin 

Glucosibarin 


Glucoerucin 

O O X O X X X X O X O X 

X X X X X O X X O X X O 

X X O X X O X X X X O O 

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 O O O X O X X O X X X 

X X O X X O X X O X X X 


X X X O X X X X X X X O 

O X O O O X O O O X O O 

X X O O O O X O X X X X 

Seed of Lepidium apetalum Willd 

[ 葶藶子 ] 

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 

Gluconasturtiin

3.2 Quantitative analysis 

3.2.1 Calibration curves for each individual glucosinolate standard 

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 (R 2 values) of the calibration curves for each 

glucosinolate were shown in Table 9. 

According to the R 2 values of the calibration curves for 

each glucosinolate, the linearity of all calibration curves were acceptable. 

dddddddddddddddddddddddddddddddd 

. 

33

Calibration curve for Epiprogoitrin 

7000 

6000 

Area 

5000 

4000 

3000 

y = 11.916x + 28.152 

R 2 = 0.9997 

2000 

1000 

0 

0 100 200 300 400 500 600 

Concentration of Epiprogoitrin (ppm) 

Figure 21: Calibration curve for epiprogoitrin 

Glucosinolates Equations of the calibration curves R 2 values 

Glucoiberin y = 11.758x + 27.812 R 2 = 0.9996 

Glucocheirolin y = 10.588x + 25.809 R 2 = 0.9996 

Progoitrin y = 13.687x + 38.843 R 2 = 0.9996 

Sinigrin y = 20.569x + 63.207 R 2 = 0.9996 

Epiprogoitrin y = 11.916x + 28.152 R 2 = 0.9997 

Glucoraphenin y = 9.290x + 17.229 R 2 = 0.9997 

Sinalbin y = 28.268 x+ 39.627 R 2 = 0.9997 

Gluconapin y = 12.954x + 26.35 R 2 = 0.9996 

Glucosibarin y = 13.585x+ 38.123 R 2 = 0.9996 

Glucotropaeolin y = 20.928x + 98.804 R 2 = 0.9993 

Glucoerucin y = 11.927x + 57.822 R 2 = 0.9994 

Gluconasturtiin y = 13.974x + 34.193 R 2 = 0.9996 

Table 9: The summary of the equations and their corresponding R 2 values of the 

calibration curves for each glucosinolate 

34

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: 

C L =kS B /b 

Remarks: C L = Concentration related to the smallest measure of response can be 

detected 

k = 3(based on the confidence interval) 

S B = 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, 

S B 

Instrument Detection 

Limit, ng/µL 

Method Detection 

Limit, µg/g a 

Method Detection 

Limit, µg/g 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 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 

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 

Peak areas of Peak areas of 

testing solution 1 testing solution 2 

Peak areas of 

testing solution 3 

Peak areas of 

50ppm standard 

mixture solution 

Recoveries, 

% 

Glucoiberin 513.98 534.96 536.40 595.28 88.77 

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 

RSD, % 

10.24 

6.61 

5.27 

5.93 

9.05 

14.60 

11.24 

14.60 

12.09 

7.44 

8.78 

5.51 

Average 99.80 9.28 

Table 11: Summary of method recoveries and RSD values for each glucosinolate 

37

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 -3 L / 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) 

Vegetable and Traditional 

Chinese Medicine Samples 

Raphanus sativus 

[ 蘿蔔 (Chinese Radish)] 

Glucoiberin 


Progoitrin 

Sinigrin 

Epiprogoitrin 

Glucoraphenin 

13.02 1.00 X 2.65 X X X X 6.31 X 61.16 X 84.14 


[ 車厘茄 (Cherry Tomato)] X X X X X 26.63 X X 5.13 X X 6.83 38.59 


[ 番茄 (Tomato)] X X 3.40 X X 30.41 X X X X 4.13 9.96 47.90 

Root of Isatis indigotica 

Fort. 

[ 北板藍根 ] 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. 

[ 南板藍根 ] X X X X X X X X X X X X 0.00 

Patrinia scabiosaefolia 

Fisch. ex Trev. 

[ 敗醬草 ] X X X X X X X X X X X X 0.00 

Thlaspi arvense L. 

[ 菥蓂 ] X 42.87 16.45 984.70 X 281.05 X X 28.95 X X X 1354.02 

Leaf of Isatis indigotica Fort. 

[ 大青葉 ] X X 14.32 X X 488.77 X X 42.32 X X X 545.41 

Leaf of Baphicacanthus 

cusia (Nees) Bremek. 

[ 廣東大青葉 ] X 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 

Seed of Raphanus sativus L. 

[ 萊菔子 ] X X 23.64 13.67 37.68 10919.08 X 20.67 X X X X 11014.74 

Seed of Lepidium apetalum 

Willd 

[ 葶藶子 ] X 39.92 X 430.70 X X 66.55 12648.94 X X 13.71 X 13199.82 

Remark: X = Not Detected 

Table 12: Glucosinolate concentrations in vegetable and Traditional Chinese Medicine 

(TCM) samples 

Sinalbin 

Gluconapin 

Glucosibarin 


Glucoerucin 


Total glucosinolates 

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 70 o C 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 200 o C 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. 


mA U 

300 

250 

200 

150 

100 

50 


Progoitrin 

4.906 

5.560 

Area: 6281.9 Area: 13465.5 

5.919 

6.654 

Sinigrin 

Area: 55.7971 Area: 29.5705 

Epiprogoitrin 

8.266 

Glucoraphenin 

Area: 1560.09 

Sinalbin 

10.990 

11.645 

Area: 5581.52 

Area: 237.494 

Gluconapin 

15.480 

Area: 14890.5 

glucotropaeolin 

with 

interferences 


18.953 

Area: 41.0646 

0 

0 2.5 5 7.5 10 12.5 15 17.5 20 

min 

Figure 21a: Chromatogram of the original Root of Isatis indigotica Fort. [ 北板藍根 ] 

41

DAD1 A, Sig=233,4 Ref=550,100 (CHUNG2\SUB00006.D) 

mA U 

350 

300 

250 

200 

150 

Progoitrin 

5.624 

Area: 5235.53 Area: 10792.9 

6.699 

Sinigrin 

Epiprogoitrin 

Glucoraphenin 

Sinalbin 

11.660 

Area: 4597.09 

Gluconapin 

15.483 

Area: 10577.4 


with 

interferences 


100 

50 

0 

6.040 

Area: 88.5345 

8.232 

Area: 848.149 

11.013 

Area: 162.052 

18.929 

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 

min 

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

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

mAU 

2500 


11.413 

2000 

1500 

1000 

500 


Sinigrin 

6.202 

Sinalbin 

Gluconapin 

Glucoerucin 

0 

5.157 

11.110 

16.027 

0 2.5 5 7.5 10 12.5 15 17.5 20 

min 

Figure 22a: Chromatogram of Seed of Lepidium apetalum Willd [ 葶藶子 ] extract 

mAU 


11.875 

800 

600 

400 


Sinigrin 

Sinalbin 

Gluconapin 

Glucoerucin 

200 

0 

5.245 

6.336 

11.201 

16.119 

0 2.5 5 7.5 10 12.5 15 17.5 20 

min 

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 


32 

30 

28 

26 

24 

96.9719 

HSO 4 

- 

(S) 

OH (R) 

CH 2OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

S CH 

O 

H 2 

C CH 2 C 

C 

H 2 

3 

NOSO 3H 

Glucoiberin standard 

M.W. = 423.0327 

358.0605 

22 

20 

18 

CH 2 OH 

ΟΗ 

Ο 

S 

195.9942 

422.0670 

[M-H] - 

16 

14 

12 

10 

8 

6 

4 

2 

0 

74.9981 

OH 

SO 3 

- 

OH 

138.9866 

162.0042 

180.0400 

79.9645 228.9958 

135.9759 

119.0577 

259.0409 

275.0237 

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 

342.1137 

407.0218 

-TOF Product (438.0): 30 MCA scans from Sample 32 (GlucocheirolinMS2) of Chung.wiff 

a=3.56036418804506270e-004, t0=5.68918465398965050e+001 


211 

O 

438.0303 

200 

180 

160 

140 

120 

96.9576 

HSO 4 

- 

(S) 

OH (R) 

CH 2OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

C CH 2 S 

H 2 

O 

CH 3 

Glucocheirolin standard 

M.W. = 439.0277 

CH 2 OH 

[M-H] - 

ΟΗ 

Ο 

S 

100 

74.9871 

OH 

OH 

80 

259.0150 

60 

SO 3 

- 

196.0107 

40 

20 

0 

274.9952 

128.9252 244.9655 

138.9692 

79.9536 241.9766 

358.0900 

119.0313 

145.0517 214.9696 

290.9898 

59.0091 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 

m/z, amu 

Figure A.2: MS/MS spectrum of glucocheirolin standard 

49

-TOF Product (388.0): 30 MCA scans from Sample 8 (progoitrin-MS2) of chung2.wiff 

a=3.56258132228446760e-004, t0=5.70181404275608660e+001 


22.0 

20.0 

96.9659 

HSO 4 

- 

(S) 

CH 2OH 

ΟΗ 

Ο 

S 

(S) 

H 

H 2 

C C (R) C 

C 

H 

OH 

NOSO 3 H 

CH 2 

18.0 

16.0 

14.0 

74.9987 

OH (R) 

(S) 

OH 

Progoitrin standard 

M.W. = 389.0450 

CH 2 OH 

Ο 

ΟΗ 

S 

[M-H] - 

12.0 

135.9813 

OH 

10.0 

8.0 

SO 3 

- 

195.0521 

OH 

388.0835 

6.0 

4.0 

259.0350 

275.0274 

2.0 

79.9665 154.0002 192.0159 

0.0 

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.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 


62 

60 

55 

74.9844 

HSO 4 

- 

(S) 

OH (R) 

CH 2 OH 

S 

Ο 

ΟΗ 

(S) 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 

CH 2 

50 

45 

40 

96.9581 

OH 

Sinigrin standard 

M.W. = 359.0345 

CH 2 OH 

ΟΗ 

Ο 

S 

35 

30 

SO 3 

- 

OH 

OH 

[M-H] - 

25 

20 

15 

10 

5 

0 

79.9540 

116.0077 161.9818 195.0323 

259.0135 

164.9700 

101.0191 275.0175 

128.9360 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 

m/z, amu 

Figure A.4: MS/MS spectrum of sinigrin standard 

358.0175 

50

-TOF Product (388.0): 30 MCA scans from Sample 1 (epiprogoitrin-MS2) of chung2.wiff 

a=3.56258132228446760e-004, t0=5.70181404275608660e+001 

OH 

CH CH 74.9995 

2 OH 

H 2 OH 

2 

C C (S) C 

17.0 

S C 

H 

S 

96.9709 

Ο 

Ο 

H 

16.0 

(S) ΟΗ 

ΟΗ 

NOSO 

(S) 

3 H 

15.0 

14.0 

13.0 

12.0 

11.0 

10.0 

9.0 

HSO 4 

- 

146.0472 195.0836 

135.9845 

OH 

OH 

OH (R) 

(S) 

OH 

Epiprogoitrin standard 

M.W. = 389.0450 

CH 2 

388.0763 


[M-H] - 

8.0 

7.0 

SO 3 

- 

259.0273 

275.0241 

6.0 

5.0 

4.0 

3.0 

192.0093 

2.0 

301.0257 

1.0 

79.9678 

332.0249 

0.0 

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.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 


45 

96.9527 

O 

40 

HSO 4 

- 

(S) 

OH 

CH 2OH 

ΟΗ 

(R) 

Ο 

(S) 

S 

(S) 

C 

H 2 

C 

NOSO 3H 

C 

H 2 

C 

H 

C 

H 

S 

CH 3 

35 

OH 

Glucoraphenin standard 

30 

25 

CH 2 OH 

ΟΗ 

Ο 

S 

M.W. = 435.0327 [M-H] - 

434.0301 

20 

15 

74.9846 

SO 3 

- 

OH 

OH 

259.0061 

419.0080 

10 

5 

195.0467 

129.0238 

274.9921 

192.0068 240.9591 

79.9536 138.9877 255.9360 

330.9917 

370.0284 

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.6: MS/MS spectrum of glucoraphenin standard 

51

-TOF Product (424.0): 30 MCA scans from Sample 57 (SinalbinMS2) of Chung.wiff 

a=3.56036418804506270e-004, t0=5.68918465398965050e+001 

493 


424.0434 

450 

400 

350 

300 

(S) 

OH (R) 

CH 2OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

Sinalbin standard 

M.W. = 425.0450 

OH 

CH 2 OH 

Ο 

ΟΗ 

S 

[M-H] - 

OH 

250 

OH 

200 

150 

182.0288 

259.0156 

274.9930 

100 

195.0314 

230.9794 

50 

0 

227.9960 

168.9768 200.9738 241.0054 246.0136 

291.0005 

344.0885 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 

m/z, amu 

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 


110 

74.9853 

100 

90 

96.9586 

HSO 4 

- 

(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

S 

(S) 

H 2 

C 

C 

NOSO 3 H 

C 

H 2 

C 

H 

CH 2 

80 

70 

60 

OH 


M.W. = 373.0501 

CH 2 OH 

ΟΗ 

Ο 

S 

[M-H] - 

50 

40 

SO 3 

- 

OH 

OH 

30 

20 

79.9513 

195.0279 

130.0285 

10 59.0082 119.0339 

178.9952 

138.9607 

0 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 

m/z, amu 

Figure A.8: MS/MS spectrum of gluconapin standard 

259.0185 

275.0045 

292.0821 

372.0505 

52

-TOF Product (438.0): 30 MCA scans from Sample 12 (glucosibarinMS2) of Chung.wiff 

a=3.56036418804506270e-004, t0=5.68918465398965050e+001 


80 

75 

70 

65 

60 

55 

96.9551 

HSO 4 

- 

135.9682 

CH 2 OH 

ΟΗ 

Ο 

S 

(S) 

OH (R) 

CH 2OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

H 

H 2 

C C (R) 

C 

OH 

NOSO 3H 

Glucosibarin standard 

M.W. = 439.0607 

50 

45 

OH 

OH 

[M-H] - 

40 

438.0619 

35 

30 

25 

74.9869 

SO 3 

- 

195.0317 

259.0157 

20 

15 

10 

5 

0 

79.9553 

138.9689 

153.9711 

244.9909 

85.0281 128.9375 

145.0472 

169.9414 

198.9805 215.0118 

275.0003 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 

m/z, amu 

Figure A.9: MS/MS spectrum of glucosibarin standard 

301.0042 

332.0046 

358.1076 

-TOF Product (408.0): 30 MCA scans from Sample 38 (GlucotropaeolinMS2) of Chung.wiff 

a=3.56036418804506270e-004, t0=5.68918465398965050e+001 


524 

500 

450 

400 

350 

300 

74.9859 

96.9588 

HSO 4 

- 

CH 2 OH 

S 

Ο 

ΟΗ 

OH 

OH 

(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

Glucotropaeolin standard 

M.W. = 409.0501 

[M-H] - 

250 

SO 3 

- 

408.0478 

200 

166.0322 

150 

100 

50 

0 

85.0259 

79.9525 

119.0357 

138.9683 

168.9781 

195.0338 

163.0623 

214.9826 

259.0134 

274.9888 

230.0145 328.0864 

241.0019 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 

m/z, amu 

Figure A.10: MS/MS spectrum of glucotropaeolin standard 

53

-TOF Product (420.0): 30 MCA scans from Sample 22 (GlucoerucinMS2) of Chung.wiff 

a=3.56036418804506270e-004, t0=5.68918465398965050e+001 


220 

96.9567 

HSO 4 

- 

(S) 

CH 2OH 

ΟΗ 

Ο 

S 

(S) 

C 

H 2 

C CH C 2 

H 2 

NOSO 3H 

C 

H 2 

S CH 3 

200 

OH 

(R) 

(S) 

180 

160 

140 

74.9853 

CH 2 OH 

ΟΗ 

Ο 

S 

OH 

Glucoerucin standard 

M.W. = 421.0535 

[M-H] - 

420.0475 

120 

SO 3 

- 

OH 

OH 

100 

80 

60 

40 

20 

0 

259.0160 

79.9525 178.0352 

195.0335 

274.9891 

226.9948 

85.0231 119.0323 

138.9692 

163.0610 224.0099 242.0073 

131.0417 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 

m/z, amu 

Figure A.11: MS/MS spectrum of glucoerucin standard 

340.0940 

-TOF Product (422.0): 30 MCA scans from Sample 27 (GluconasturtiinMS2) of Chung.wiff 

a=3.56036418804506270e-004, t0=5.68918465398965050e+001 


686 

650 

600 

550 

500 

450 

400 

74.9855 

96.9574 

HSO 4 

- 

CH 2 OH 

ΟΗ 

Ο 

S 

(S) 

OH (R) 

CH 2OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

C 

H 2 

Gluconasturtiin standard 

M.W. = 423.0658 

[M-H] - 

350 

300 

SO 3 

- 

OH 

OH 

422.0648 

250 

200 

150 

180.0497 

259.0141 

100 

50 

0 

79.9522 

195.0340 

228.9978 

274.9890 

138.9682 

85.0259 

119.0323 

165.0357 240.9993 342.1033 

131.0277 301.0072 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 

m/z, amu 

Figure A.12: MS/MS spectrum of gluconasturtiin standard 

54

-TOF MS: 0.050 min from Sample 1 (t-rt-6.94) of chung1.wiff 

a=3.56264499558618230e-004, t0=5.71842229067915470e+001 


55 

50 

(S) 

CH 2OH 

ΟΗ 

Ο 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

C 

H 2 

C 

H 

C 

H 

O 

S 

CH 3 

434.0255 

45 

40 

35 

30 

OH (R) 

(S) 

OH 

Glucoraphenin in 


[ 番茄 (Tomato)] extract at 

8.729min 

M.W. = 435.0327 

[M-H] - 

25 

20 

15 

10 

152.9173 

183.0124 

265.1454 

293.1837 325.1753 

418.9928 

5 

136.9300 

216.9001 

255.2330 270.8373 311.1668 

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.13a: ESI-QTOF-MS spectrum of Lycopersicon esculentum [ 番茄 (Tomato)] 


-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 

O 


80 

75 

70 

65 

60 

55 

50 

96.9605 

HSO 4 

- 

CH 2 OH 

S 

Ο 

ΟΗ 

OH 

OH 

(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

C 

H 2 

C 

H 

C 

H 

Glucoraphenin in Lcopersicon 

esculentum [ 番茄 (Tomato)] 


M.W. = 435.0327 

S 

CH 3 

434.0272 

[M-H] - 

45 

40 

259.0183 

419.0099 

35 

30 

74.9894 

195.0322 

25 

20 

15 

10 

5 

129.0232 

168.9535 

192.0163 

240.9715 

274.9914 

93.5864 135.9696 

145.0446 

255.9464 

203.2529 297.5118 

339.4955 

393.7627 

427.7920 

488.2259 

113.4158 153.4012 186.4013 

234.2286 

331.7443 

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.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 


2087 

2000 

1900 

1800 

1700 

1600 

1500 

1400 

1300 

1200 

1100 

1000 

900 

800 

700 

600 

500 

400 

300 

200 

100 

0 

264.1433 

281.2313 

[M-H] - 

388.0120 

283.2469 341.0869 

503.1292 

389.0183 

549.1344 

279.2185 

325.0969 

368.9610 

306.1862 431.1085 455.9921 539.0968 

297.1118 339.3029 377.0592 

471.9644 

593.1555 

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 

m/z, amu 

Figure A.14a: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [ 北板藍根 ] 


(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

H 

H 2 

C C (R) C 

C 

H 

OH 

NOSO 3 H 

CH 2 

Progoitrin in Root of Isatis 

indigotica Fort. [ 北板藍根 ] 


M.W. = 389.0450 

-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 

H 

74.9863 

47 

CH 2 OH 

H 2 

C C (R) C 

45 

S C 

H 

Ο 

OH 

(S) ΟΗ 

NOSO 

(S) 

3 H 

CH 

40 

2 OH 

(S) 

OH (R) 

96.9559 

S 

- 

Ο 

OH 

HSO ΟΗ 

35 

4 

30 

25 

OH 

OH 

CH 2 

Progoitrin in Root of Isatis 

indigotica Fort. [ 北板藍根 ] 


M.W. = 389.0450 


20 

135.9633 

[M-H] - 

- 

15 

SO 3 195.0270 

146.0211 

388.0060 

258.9906 

10 

138.9644 

79.9524 

119.0309 

274.9692 

5 

153.9726 

128.9249 191.9868 300.9982 

57.4090 161.0347 

0 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 

m/z, amu 

Figure A.14b: MS/MS spectrum of Root of Isatis indigotica Fort. [ 北板藍根 ] extract at 

5.834min 

56

-TOF MS: 60 MCA scans from Sample 15 (Rt5.801-1) of Chung291104.wiff 

a=3.55978894933761710e-004, t0=5.66641529975095180e+001 


900 

850 

800 

750 

700 

650 

600 

550 

500 

450 

400 

350 

300 

250 

200 

264.1433 

281.2339 

283.2468 

267.2170 

279.2168 

358.0053 

[M-H] - 

503.1270 

150 

341.0884 

270.2344 

284.2501 

100 

368.9543 

388.0106 

50 293.1611298.9842 359.0127 367.3363 412.9449 549.1374 

381.3464 439.0693 

465.0872 505.1302 

593.1540 

0 

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 

m/z, amu 

Figure A.15: ESI-QTOF-MS spectrum of Root of Isatis indigotica Fort. [ 北板藍根 ] 


(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 

Sinigrin in Root of 

Isatis indigotica Fort. 


6.276min 

CH 2 


a=3.55978894933761710e-004, t0=5.66641529975095180e+001 


388.0270 

2800 

OH 

2600 

2400 

(S) 

CH 2 OH 

ΟΗ 

Ο 

S 

(S) 

H 2 

C C (S) C 

C 

H 

H 

NOSO 3 H 

CH 2 

2200 

2000 

1800 

1600 

1400 

1200 

[M-H] - 

OH (R) 

(S) 

OH 

Epiprogoitrin in Root of 



6.971min 

M.W. = 389.0450 

1000 

800 

600 

400 

264.1544 

390.0253 

200 267.2234 

341.1002 368.9640 456.0104 

445.9772 503.1513 549.1584 

563.1551 

0 

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 



57

-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 


207 

CH 2 OH 

200 

CH 2 OH 

S C 

Ο 

190 

H 

S 

(S) ΟΗ 

NOSO 

(S) 

3 H 

Ο 

180 

ΟΗ 

(S) 

OH (R) 

170 74.9891 

160 

96.9602 

- 

HSO 4 

OH 

OH 

150 

OH 

140 

130 

120 

110 

135.9680 

100 

90 

195.0321 

80 

70 

- 

259.0038 

SO 3 

60 

146.0223 

50 

40 

274.9864 

30 

138.9633 

20 79.9575 119.0328 

191.9884 

210.0027 

10 

128.9266 

85.0345 

59.0129 164.9880 

198.9876 225.9676 

0 

240.9916 308.0766 

H 2 

C C (S) C 

H 

332.0057 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 

m/z, amu 


6.971min 

OH 

CH 2 

Epiprogoitrin in Root of 



6.971min 

M.W. = 389.0450 

[M-H] - 

388.0269 


a=3.55978894933761710e-004, t0=5.66641529975095180e+001 


534 

500 

264.1495 

(S) 

CH 2OH 

ΟΗ 

Ο 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

OH 

450 

400 

350 

300 

250 

281.2373 

283.2526 

267.2217 

[M-H] - 

OH (R) 

(S) 

OH 

Sinalbin in Root of 



11.290min 

M.W. = 425.0450 

200 

150 

100 

270.2400 

50 

0 

279.2205 

284.2546 368.9629 

424.0150 

277.1950 306.1949 341.0987 

362.2543 412.9511 426.0113 

451.4223 487.3240 550.1018 

564.0580 

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 

m/z, amu 



58

-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 


9.0 

8.5 

8.0 

7.5 

7.0 

6.5 

6.0 

5.5 

5.0 

4.5 

4.0 

74.9881 

96.9522 

HSO 4 

- 

182.0260 

(S) 

OH (R) 

CH 2OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

Sinalbin in Root of 



11.290min 

M.W. = 425.0450 

OH 

[M-H] - 

424.0178 

3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 

m/z, amu 


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 


75 

70 

65 

60 

55 

50 

45 

40 

35 

(S) 

OH (R) 

CH 2 OH 

ΟΗ 

Ο 

(S) 

OH 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 

Sinigrin in Thlaspi 

arvensis L. [ 菥蓂 ] 


M.W. = 359.0345 

CH 2 

183.0122 

265.1545 

293.1855 

309.1856 

358.0384 

[M-H] - 

30 

25 

20 

15 

281.2549 

353.2094 

325.1939 

337.2132 

397.2451 

10 

5 

255.2342 

184.0176 241.2168 297.1492 

321.2279 

381.2446 

441.2700 

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.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 

60 

74.9926 

(S) 

CH 2 OH 

ΟΗ 

Ο 

S 

(S) 

C 

H 2 

C 

NOSO 3 H 

C 

H 

CH 2 

55 

50 

45 

40 

35 

30 

96.9621 

HSO 4 

- 

CH 2 OH 

S 

Ο 

ΟΗ 

OH 

OH 

[M-H] - 

OH (R) 

(S) 

OH 

Sinigrin in Thlaspi 

arvensis L. [ 菥蓂 ] 


M.W. = 359.0345 

25 

20 

95.9562 

SO 3 

- 

15 

195.0349 

358.0394 

10 

161.9916 

275.0004 

79.9581 116.0176 

259.0274 

138.9804 

5 

134.9781 

63.8038 177.4611 262.9085 314.9930 376.8085 432.4078 447.5264 

211.1616 460.1431 

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.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 

O 


38 

36 

34 

32 

30 

28 

26 

24 

22 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

148.0700 

178.0723 

205.1455 

220.1677 

250.1725 

233.1742 

223.0486 

265.1727 

255.2594 

325.2181 

311.1937 

183.0295 

126.9162 206.1480 

281.2803 

186.9412194.9564 

293.1983 

241.2383 259.0410 

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 

(S) 

OH 

CH 2OH 

ΟΗ 

(R) 

Ο 

(S) 

OH 

S 

(S) 

C 

H 2 

C 

NOSO 3H 

C 

H 2 

C 

H 

C 

H 

Glucoraphenin in 

Thlaspi arvensis L. 

[ 菥蓂 ] extract at 

434.0624 

8.347min 

M.W. = 435.0327 [M-H] - 

S 

CH 3 

60

-TOF MS: 0.367 min from Sample 6 (t-rt-11.8) of chung1.wiff 

a=3.56264499558618230e-004, t0=5.71842229067915470e+001 


471 

450 

(S) 

CH 2OH 

ΟΗ 

Ο 

S 

(S) 

H 2 

C 

C 

NOSO 3 H 

C 

H 2 

C 

H 

CH 2 

372.0464 

400 

350 

300 

250 

OH (R) 

(S) 

OH 

Gluconapin in Seed of 

Lepidium apetalum 

Willd [ 葶藶子 ] extract 

at 11.875min 

M.W. = 373.0501 

[M-H] - 

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 ] 


-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 

74.9918 

(S) 

CH 2OH 

ΟΗ 

Ο 

S 

(S) 

H 2 

C 

C 

NOSO 3H 

C 

H 2 

C 

H 

CH 2 

140 

OH (R) 

(S) 

130 

120 

110 

100 

90 

96.9639 

HSO 4 

- 

CH 2 OH 

ΟΗ 

Ο 

S 

OH 

Gluconapin in Seed of 

Lepidium apetalum 

Willd [ 葶藶子 ] extract 

at 11.875min 

M.W. = 373.0501 

80 

OH 

70 

60 

OH 

[M-H] - 

50 

40 

30 

20 

79.9621 

- 

SO 3 

130.0357 259.0226 

195.0410 241.0128 

275.0018 

372.0598 

10 

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.20b: MS/MS spectrum of Seed of Lepidium apetalum Willd [ 葶藶子 ] extract at 

11.875min 

61

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