Fast Separation and Sensitive Quantitation of Polymethoxylated

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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]

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ABSTRACT

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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

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

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

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

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

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INTRODUCTION

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

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

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

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

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

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

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

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

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analysis of PMFs in Citrus fruits.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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separation and identification of 32 chemical compositions within only seven minutes

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in Ponkan peel.

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

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

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

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

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

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

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

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resources in future.

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MATERIALS AND METHODS

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

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including

5,

7,

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

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

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(S-8,

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

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

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SinoStandards

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(S-3,purity≥98.0%),

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

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

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

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

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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%),

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

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

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

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ANPEL Inc. (Shanghai, China).

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Materials

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

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National Citrus Germplasm Repository at the commercial maturity stage and

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

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

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

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

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

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

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

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further use.

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Sample Preparation

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

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

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

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

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

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

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membrane.

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UPLC-PDA-ESI-MSE Analysis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Method Validation

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

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

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

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

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

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

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

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

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further by spiking the authentic standards in samples directly.

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RESULTS AND DISCUSSION

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Optimization of UPLC Analysis Conditions

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To obtain a better separation efficiency, several parameters were investigated

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

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

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

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

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

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

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Identification of PMFs

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

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

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

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

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

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polymethoxylated flavanones and chalcones with the basic structures of flavanone and

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

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

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

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

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

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

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

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

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

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

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

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

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

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PMFs were identified tentatively according to the MSE fragments and references

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

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

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

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

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

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[M+H-n×15]

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

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

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

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

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

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

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

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

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

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

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

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

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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

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As for polymethoxylated flavanones, they preferentially underwent the

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

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position of the C-ring producing the prominent ions

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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

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

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0or1, 2

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

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

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

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

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

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

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

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

205

match indicated that the compound 11 is a pentamethoxyflavanone or a

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

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

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

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

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

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

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spectra.

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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’,

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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basic structures, 33 polymethoxylated flavones and 9 polymethoxylated flavanones

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were identified.

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Validation of Analytical Method

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

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between concentrations and the corresponding MS responses with correlation

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

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

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

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

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precision with interday RSDs< 2.79% and intraday RSDs