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Fast Separation and Sensitive Quantitation of Polymethoxylated Flavonoids in the Peels of Citrus Using UPLC-Q-TOF-MS Tian Tian Xing, Xi Juan Zhao, Yi Dan Zhang, and Yuan Fang Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05821 • Publication Date (Web): 05 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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Journal of Agricultural and Food Chemistry

Fast Separation and Sensitive Quantitation of Polymethoxylated Flavonoids in the Peels of Citrus Using UPLC-Q-TOF-MS Tian Tian Xing,a Xi Juan Zhao,b,* Yi Dan Zhanga and Yuan Fang Li a,* a

Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest

University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P R China b

College of Horticulture and Landscape Architecture, Southwest University, Key

Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education,

Chongqing 400715, P R China Corresponding Author: *

(ZH. X. J.) Phone: 86-23-68250483; Email: [email protected]

1

ACS Paragon Plus Environment


1

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ABSTRACT

2

A rapid, sensitive and efficient ultra-performance liquid chromatography coupled

3

with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method has

4

been developed to analyze polymethoxylated flavonoids (PMFs) in 14 Citrus peels,

5

including 7 Citrus reticulata (C. reticulata) and 7 Citrus Sinensis (C. sinensis). In this

6

study, fast separation can be achieved within 12 minutes and 42 PMFs have been

7

identified including 33 flavones and 9 flavanones. Most C. reticulata were shown to

8

contain more than 20 PMFs, except Guangxihongpisuanju (GX) containing only 12

9

PMFs. While most C. sinensis contained less than 20 PMFs, except Edangan (EG)

10

containing as many as 32 PMFs. To our knowledge, there are few reports about the

11

quantitation of PMFs using the MS response. Here, a MS quantitative method was

12

established and systematically validated in linearity, precision and recovery. The

13

linearity was from 1.25ng/mL to 1.0 µg/mL with the limit of detection (LOD) as low

14

as 75 pg/mL and the limit of quantitation (LOQ) as low as 0.25 ng/mL. Up to 13

15

PMFs more types than ever before were undoubtedly identified and quantitated

16

according to the PMF standards. The results showed that the contents of PMFs in the

17

C. reticulata were generally higher than that in the C. sinensis. This study is

18

systematic for analyzing PMFs and is of great significance because it can provide

19

guidance on utilization of both PMFs and citrus germplasm resources in future.

20

Keywords: PMFs, Citrus, UPLC-Q-TOF-MS

2


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21

INTRODUCTION

22

Polymethoxylated flavonoids (PMFs) refer to a group of natural products of

23

flavones attached with four or more methoxy groups on their basic 2-phenyl

24

chromone skeleton. They exist widely in Citrus fruits particularly in Citrus reticulata

25

(C. reticulata) and Citrus sinensis (C. sinensis) possessing a broad spectrum of

26

beneficial properties such as anticancer,1 anti-inflammatory,2 anti-allergic,3 and

27

antifungal activities.4 However, to the best of our knowledge, there have been few

28

systematic researches on the identification and quantitation of PMFs in the peels of

29

Citrus by MS until now.5-8 Therefore, it is of great importance to perform a systematic

30

analysis of PMFs in Citrus fruits.

31

Early reported methods for study of PMFs mainly used high-performance liquid

32

chromatography (HPLC) and HPLC coupled to photodiode array detection and

33

electrospray tandem mass spectrometry (HPLC-DAD–ESI-MS/MS). Curtis O. Green

34

et al.9 separated 6 major PMFs within 45 minutes in the peels of 20 Citrus cultivars,

35

which was carried out by RP-HPLC and UV detection. Jia-Yu Zhang et al.5

36

established an HPLC-DAD-ESI-MS/MS method to identify 32 PMFs in ‘Shatangju’

37

mandarin within 95 minutes. And Yu-Shan Lin et al.6 developed a method using

38

HPLC coupled with linear ion trap mass spectrometry, and quantitated 6 PMFs and 6

39

hydroxylated polymethoxyflavones (OH-PMFs) within 20 minutes in the peels of

40

Citrus. It is obvious that the previous methods needed a long time to perform the

41

analysis and some components could not be detected owing to the low abundance,

42

co-elution and high background of HPLC.10 Recent years, the emergence of UPLC 3



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43

technology and high resolution mass spectrometry (HRMS) has brought great benefits

44

to the analysis work such as the fast separation, high sensitivity, less organic solvent

45

and accurate mass.11-13 Medina-Remon group developed a rapid method using

46

UHPLC-PDA to allow the simultaneous separation and quantitation of 11 selected

47

flavonoids in only 5.5 minutes,14 which displayed the advantages of UHPLC

48

including rapid, solvent-saving and small sample volume. HRMS can give more

49

specific and much exacter MS and MS2 information, making the identification more

50

accurate. UPLC coupled with quadrupole time-of-flight mass spectrometry

51

(UPLC-Q-TOF-MS) combines the advantages of both UPLC and HRMS effectively

52

and is shown to be a powerful tool to identify compounds in botanic extracts and

53

complex matrix. For example, Yang et al.15 used UPLC-Q-TOF-MS to achieve the

54

separation and identification of 32 chemical compositions within only seven minutes

55

in Ponkan peel.

56

The present study focuses on the identification and quantitation of PMFs in the

57

peels of Citrus including 7 C. reticulata and 7 C. sinensis and the comparison of

58

PMFs with respective to types and contents between them. In order to carry out the

59

objective, an effective UPLC-Q-TOF-MS method was established after a series of

60

parameters optimization and method validation in linearity, precision and accuracy.

61

This work is a systematic investigation on PMFs in Citrus peels and is important

62

because it can provide guidance on efficient utilization of PMFs and citrus germplasm

63

resources in future.

4


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64

MATERIALS AND METHODS

65

Chemicals 13 PMFs reference compounds (Table S1 in the Supporting Information),

66 67

including

5,

7,

68

purity≥98.0%),5,6,7,4’-tetramethoxyflavone

69

5,7,4’-trimethoxyflavone (S-5, purity≥95.0%), 3,5,6,7,8,3’,4’-heptamethoxyflavone

70

(S-8,

71

5-hydroxy-6,7,8,3’,4’-pentamethoxyflavone

72

5-hydroxy-7,3’,4’-trimethoxyflavone (S-11, purity≥98.0%) were purchased from

73

SinoStandards

74

(S-3,purity≥98.0%),

75

5,6,7,8,3’,4’-Hexamethoxyflavone (S-6, purity≥98.0%), 5,7,8,4’-tetramethoxyflavone

76

(S-7, purity≥98.0%), 5-Hydroxy-6,7,8,3’,4’,5’-Hexamethoxyflavone (S-12, RG),

77

5-hydroxy-6,7,8,4’- tetramethoxyflavone (S-13, purity≥98.0%) were purchased from

78

ChromaDex Inc. (Santa Ana, CA, USA). The structures of the 13 PMFs reference

79

standards were confirmed by MS (Figure S1-S13 in the Supporting Information).

purity≥98.0%),

8,

3’,

4’-pentamethoxyflavone (S-4,

5,6,7,8,4’-pentamethoxyflavone

(Chengdu,

China),

(S-10,

(S-1,

purity≥98.0%),

(S-9,

purity≥98.0%),

purity≥98.0%)

and

5,6,7,3’,4’-pentamethoxyflavone

5,7,3’,4’-tetramethoxyflavone

(S-2,

purity≥97.0%),

80

HPLC grade acetonitrile and formic acid were purchased from Sigma–Aldrich

81

(St. Louis, MO). Ultrapure water (Milli-Q, 18.2 MΩ) was obtained from a Millipore

82

System (Bedford, MA, USA). The filters (PTFE, 0.22µm) were purchased from

83

ANPEL Inc. (Shanghai, China).

84

Materials

85

The materials used in this study were collected on Nov. 20, 2015 from the 5



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86

National Citrus Germplasm Repository at the commercial maturity stage and

87

identified by Dong Jiang who is a research associate in the Citrus Research Institute

88

of the Chinese Academy of Agricultural Sciences in Chongqing, China. There are 14

89

Citrus, including 7 C. sinensis and 7 C. reticulata (Table S2 in the Supporting

90

Information). Firstly, the peels of the collected citrus fruits were separated from the

91

fruits after washing with the clean water. Secondly, the peels were dried in an

92

electrothermal blowing drier at 40˚C for 48 hours. Then, the dried peels were crushed

93

and screened with a 40 mesh sieve. Finally, the dried powder was stored in a dryer for

94

further use.

95

Sample Preparation

96

0.5g of the powder was weighed accurately, and extracted using 7mL methanol

97

in 300W ultrasonic bath for 30minutes. Later, the extracts were centrifuged at 5000

98

rpm for 15minutes at room temperature and the supernatants were placed in a brown

99

volumetric flask. The above operation was repeated 3 times, and then the supernatants

100

were merged and constant-volumed to 25mL. The extracts were diluted with methanol

101

to a proper concentration and filtered with 13mm syringe filters with 0.22µm PTFE

102

membrane.

103

UPLC-PDA-ESI-MSE Analysis

104

UPLC analysis was performed on an ACQUITY UPLC I-Class (Waters, Milford,

105

MA), including the following components: Binary Solvent Manager (BSM), Sample

106

Manager-FTN (FTN), Photo-Diode Array Detector (PDA Detector) and column heater.

107

Samples were separated on an ACQUITY UPLC BEH C18 column (2.1х100 mm, 1.7 6


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108

mm, UK). The PDA detector scanned from 240 nm to 400 nm, and the peaks were

109

detected at 330 nm. To obtain the optimal condition, several parameters were

110

investigated such as the organic mobile phase of methanol or acetonitrile, the

111

concentration of formic acid in water including 0, 0.01%, 0.05%, 0.1% and 0.2%, the

112

flow rate of 0.2mL/min, 0.3mL/min, 0.4mL/min and 0.5mL/min and the

113

elution gradient. The optimal conditions are as follows. The binary mobile phases are

114

consisted of solvent A (water containing 0.01% formic acid) and solvent B

115

(acetonitrile), with the following gradient elution: 0-1 min, 25-30% B; 1-9 min, 30-45%

116

B; 9-11 min, 45-60 B; 11-12 min, 60-90% B; 12-14 min; 90% B; 14-16 min; 90-25%

117

B. The data acquisition was finished in the first 12 minutes, and the following 4

118

minutes were used to wash and balance the column. The column temperature was

119

settled at 40˚C. The flowing rate was 0.4mL/min and the injection volume was 1.0µL.

120

Xevo G2-S Q-TOF (Waters MS Technologies, Manchester, UK) equipped with

121

electrospray ionization ion (ESI) source was applied for mass spectrometry data’s

122

acquisition in positive ionization mode ranged from m/z 100 to m/z 1200. The

123

LockSpray™ dual electrospray ion source was used to insure the authenticated exact

124

mass measurement. The lock mass compound is Leucine-enkephalin (m/z 556.2766,

125

concentration: 200 ng/mL, flow rate: 10 µL/min) as the reference. The ion scan mode

126

adopted the MSE methodology to obtain alternating MS and MS/MS spectra. The

127

parameters of the source were set as follows: electrospray capillary voltage 1.0 kV for

128

positive ionization mode; cone voltage 40 V; source temperature 120˚C; desolvation

129

temperature 400˚C; low energy 6V, high energy ramp from 20V to 40V; nitrogen and 7



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130

argon were used as the nebulizer and the collision gas, respectively; cone gas flow 50

131

L/h and desolvation gas flow 800 L/h, respectively.

132

Method Validation

133

The 13 reference standards were accurately weighed, dissolved in methanol and

134

dimethyl sulphoxide (DMSO), and then diluted to a proper concentration. Calibration

135

curves were fitted by the MS response at least 9 appropriate concentrations in

136

triplicate of each compounds. The limit of detection (LOD) and limit of quantitation

137

(LOQ) were determined at 3 and 10 times of the basic of signal-to-noise (S/N) ratios,

138

respectively. In addition, the inter- and intraday precision were investigated by

139

analyzing a proper calibration sample during a single day and on three successive

140

days, respectively. The recovery experiments were carried out to insure the precision

141

further by spiking the authentic standards in samples directly.

142

RESULTS AND DISCUSSION

143

Optimization of UPLC Analysis Conditions

144

To obtain a better separation efficiency, several parameters were investigated

145

such as the types of mobile phase, the concentration of formic acid in aqueous

146

solution, the flow rate of the mobile phase and the elution gradient. The optimized

147

aqueous phase A is water containing 0.01% formic acid, and the organic phase B is

148

acetonitrile with the flow rate of 0.4mL/min. An optimal gradient program was

149

applied in the elution: 0-1 min, 25-30% B; 1-9 min, 30-45% B; 9-11 min, 45-60 B;

150

11-12 min, 60-90% B; 12-14 min; 90% B; 14-16 min; 90-25% B.

8


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151

Identification of PMFs

152

PMFs are based on a core aglycone structure that is modified by different numbers

153

and/or positions of methoxyl/hydroxyl substituents. For polymethoxylated flavones,

154

they have the same basic flavone structure as shown in Figure 1 with the molecular

155

weight of 222 Da. And the molecular weights of polymethoxylated flavones can be

156

calculated in advance by adding n×30 and/or n×16. The same principle also applies to

157

polymethoxylated flavanones and chalcones with the basic structures of flavanone and

158

chalcones, respectively (Figure 1). According to the numbers and types of substituent

159

groups, either methoxyl groups or hydroxyl groups, we can designate the chemical

160

formula of every possible PMF isomer and know the corresponding molecular weight,

161

which is very helpful to identify the structures of PMFs in the complex extracts of

162

Citrus. Here, with the optimized UPLC condition, the PMFs in methanolic extracts of

163

14 Citrus peels are well separated in a short analytical time within 12 minutes (Figure 2

164

and Figure S14-S25 in the Supporting Information). Figure 2A shows the base peak

165

ion (BPI) chromatogram of methanolic extracts of Jiaogan (JG), which is a C. reticulata,

166

and 30 PMFs have been identified. Figure 2B displays the BPI chromatogram of

167

8045Tiancheng (8045), a C. sinensis, and 20 PMFs have been identified in it. Actually,

168

there are up to 42 PMFs that have been identified in the peels of 14 Citrus, including

169

33 flavones and 9 flavanones as shown in Table 1. Among them, 13 PMFs were

170

identified undoubtedly compared with the standards (Figure 2C), and the other 29

171

PMFs were identified tentatively according to the MSE fragments and references

172

(Table 1 and Figure S26-S67 in the Supporting Information). 9



173

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For polymethoxylated flavones, they tend to lose nCH3• preferentially, yielding +

174

the characteristic fragments of [M+H-n×15]

175

fragments such as neutral loss of CH4(16), H2O(18), CO(28), CH4+CH3•(31),

176

H2O+CH3•(33), CO+CH3•(43), CO2(44), H2O+CO(44), and CO+H2O+CH3•(59) from

177

[M+H-n×15]

178

the quasi-molecular ion at m/z 403.1390 was observed by HRMS at the low energy

179

mode (Figure 3A), and then the molecular formula C21H22O8 was obtained by element

180

matching. Next, the compound was identified as a hexamethoxyflavone through

181

comparing with the basic flavone structure (Figure 1). In addition, prominent ions at

182

m/z 373.0919 [M+H-2CH3] + and m/z 388.1156 [M+H-CH3] + by loss of 30 Da (CH3)

183

and 15 Da (2CH3) from the quasi-molecular ion [M+H]

184

characteristic ions of polymethoxylated flavones at the high energy mode as shown in

185

Figure 3B. Further, turn attention to the relative low abundance ions which underwent

186

the fragmentation pathways of Retro-Diels-Alder (RDA) cleavage of C-ring. The ions

187

at m/z 211.0240 [1,3A+-2CH3] and m/z 163.0755 [1,3B+] indicated that there were three

188

methoxy substituents at the ring A and two at the ring B. Finally, compare the

189

retention time, UV absorption and MSE spectra with that of the standard. Based on the

190

above analysis, the compound 27 was identified as nobiletin (Table 1).

+

usually as the base peaks. And other

can also be detected frequently.16 Take nobiletin as an example. First,

+

were regarded as the

191

As for polymethoxylated flavanones, they preferentially underwent the

192

fragmentation pathways of Retro-Diels-Alder (RDA) cleavage from the 1, 3 or 1, 4

193

position of the C-ring producing the prominent ions

194

loss of 15 Da (CH3), 28 Da (CO), 30 Da (2CH3) and 33 Da (CH3+H2O) from the base

1,3or4

A+ and

1,3or4 +

B . And the ions

10


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195

peak 1,3A+ or 1,3B+ can be pointed as the characteristic peaks of flavanones.10 The ions

196

0or1, 2

197

replaced, although the abundance of them is very low. Similarly, the ions loss of 15

198

Da (CH3), 28 Da (CO), 30 Da (2CH3) from the base peak xB+ and yA+ can be pointed

199

as the characteristic peaks of chalcones. It is noteworthy that chalcones tend to retain

200

the ring-B as the base peak while flavanones retain the ring-A, and the maximum

201

absorption wavelength of flavanones is centered at about 320 nm, while that of

202

chalcones ranges from 330 to 370 nm.5, 10, 17, 18 Take compound 11 as an example.

203

Firstly, the quasi-molecular ion at m/z 375.1436 was obtained from the low energy

204

mode (Figure 4A). And the molecular formula C20H22O7 obtained through elements

205

match indicated that the compound 11 is a pentamethoxyflavanone or a

206

pentamethoxychalcones according to Figure 1. Then, the abundances of the fragment

207

at m/z 211.0601 (1,3A+), m/z 196.0366 (1,3A+-CH3) were 100% and 26.42% which

208

were regarded as the diagnostic ions of flavanones at the high energy mode (Figure

209

4B). Furthermore, the existence of m/z 211.0601(1,3A+) and m/z 191.0705 (1,4B+-H2O)

210

indicated that there are three methoxy substituents at the ring A and two at the ring B.

211

Finally, make the comparison with literatures about their UV absorption and MS

212

spectra.

213

4’-pentamethoxyflavanone (Table 1).

A+ and

0or1, 2

B+ are the key fragments to determine whether the position-3 is

Therefore,

compound

11

was

identified

as

5,

6,

7,

3’,

214

According to the method stated above, the identification work was carried out

215

between C. sinensis and C. reticulata and a total of 42 PMFs were detected. Among

216

them, the structures of 35 PMFs can be identified in comparison with reported 11



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217

research and compounds 1, 4, 6, 8, 9, 10, 13, and 37 were detected in Citrus for the

218

first time (Figure S68 in the Supporting Information). According to the qualitative

219

analysis of the 14 Citrus, more types of PMFs in C. reticulata were identified than

220

that in C. sinensis. The base peak ion (BPI) currents of the 7 C. reticulata were almost

221

similar expect GX, and the BPI of all the 7 C. sinensis were almost similar expect EG.

222

Most of C. reticulata contained more than 20 PMFs, and among them, JG, NF and EJ

223

contained many types of PMFs up to 30 while GX contains the least types of 12 PMFs.

224

By contrast, most of C. sinensis contained less than 20 PMFs, except EG containing

225

as many as 32 PMFs. In addition, the structures of all the possible PMFs that have

226

been detected were summarized (Table S3 and Figure S68 in the Supporting

227

Information). In brief, a total of 24 OH-PMFs and 18 PMFs were identified according

228

to whether containing hydroxyl substituents or not. And through classification of the

229

basic structures, 33 polymethoxylated flavones and 9 polymethoxylated flavanones

230

were identified.

231

Validation of Analytical Method

232

As displayed in Table 2 and 3, all of 13 standards behaved good linearity

233

between concentrations and the corresponding MS responses with correlation

234

coefficients > 0.99. The linear range is from 1.25ng/mL to 1.0µg/mL with the LOD of

235

75pg/mL and the LOQ of 0.25ng/mL. The developed method showed good

236

repeatability with interday relative standard deviations (RSDs) < 2.79%, and intraday

237

RSDs 0.99, excellent LOD of 75pg/mL and LOQ of 0.25ng/mL, good

267

precision with interday RSDs< 2.79% and intraday RSDs

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