Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

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Systematic chemical profiling of Citrus grandis ‘Tomentosa’ by ultra-fast liquid chromatography/diode-array detector/quadrupole time-of-flight tandem mass spectrometry Pan-lin Li a , Meng-hua Liu a , Jie-hui Hu b , Wei-wei Su a,∗ a b

Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, PR China AB SCIEX, Shanghai 200233, PR China

a r t i c l e

i n f o

Article history: Received 25 August 2013 Accepted 27 November 2013 Available online 4 December 2013 Keywords: Citrus grandis ‘Tomentosa’ LC-DAD-Q-TOF-MS/MS Chemical profiling Medicinal part Quality evaluation

a b s t r a c t Citrus grandis ‘Tomentosa’, as the original plant of the traditional Chinese medicine “Huajuhong”, has been used as antitussive and expectorant in clinic for thousands of years. The fruit epicarp and whole fruit of this plant were both literarily recorded and commonly used. In the present study, an ultra-fast liquid chromatography coupled with diode-array detection and quadrupole/time-of-flight mass spectrometry (UFLC–DAD–Q-TOF-MS/MS) based chemical profiling method was developed for rapid holistic quality evaluation of C. grandis ‘Tomentosa’, which laid basis for chemical comparison of two medicinal parts. As a result, forty-eight constituents, mainly belonging to flavonoids and coumarins, were unambiguously identified by comparison with reference standards and/or tentatively characterized by elucidating UV spectra, quasi-molecular ions and fragment ions referring to information available in literature. Both of the epicarp and whole fruit samples were rich in flavonoids and coumarins, but major flavonoids contents in whole fruit were significantly higher than in epicarp (P < 0.5). The proposed method could be useful in quality control and standardization of C. grandis ‘Tomentosa’ raw materials and its products. Results obtained in this study will provide a basis for quality assessment and further study in vivo. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Citrus grandis, with more than 200 cultivars [1], is a commercially important tropical and subtropical fruit crop, well known for its nutritional benefits and pharmaceutical effects [2–5]. C. grandis ‘Tomentosa’ is a cultivar of C. grandis (L.) Osbeck particularly originated from Huazhou town in Guangdong province, southern China. It was recorded officially in the current Chinese Pharmacopoeia (2010 edition) as the original plant of the traditional Chinese medicine “Huajuhong”, and received a China GI protection in 2007. Although extremely bitter and inedible, it has been used as antitussive and expectorant for thousands of years [6], and recently has been confirmed by modern researches on anti-inflammatory [7], anti-microbial [8], anti-oxidant [9], anti-proliferative [10] and anti-atherosclerotic [11] activities. Two different parts of C. grandis ‘Tomentosa’, fruit epicarp and whole fruit, have been both used as medicine according to written records [6,12]. The utilization of epicarp was described in the Chinese Pharmacopoeia officially. But based on our survey results, the

∗ Corresponding author. Tel.: +86 20 84110808; fax: +86 20 84112398. E-mail address: [email protected] (W.-w. Su). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.11.030

whole fruit medicinal materials had a bigger market share. However, there was no definitive data on these equivocal subjects. It is widely accepted that the phytochemicals are the material basis for the efficacy. Therefore, the identification and quantification of complex constituents in C. grandis ‘Tomentosa’ is necessary. And it leads to the chemical comparison of the two parts which is of utmost importance to substantiate their potential health benefits. In the past years, several researches [13–16] were mainly focused on volatile oil and a few active compounds such as naringin and naringenin in C. grandis ‘Tomentosa’. However, to the best of our knowledge, the special investigation on the profile constituents has not been reported yet. Furthermore, exhaustive quantitative data on the contents including major and minor components are still lacking. Due to the limited availability of reference substances, LC–DAD–Q-TOF-MS/MS, combining with the efficient separation capability of LC, ultraviolet absorption features by DAD detector and exact mass measurement for both MS and MS/MS was applied to characterized components in the present study. It provides significant advantages for unequivocal identification and quantification of low levels of ingredients [17–19]. In the current work, we developed a UFLC–DAD–Q-TOF-MS/MS method for analysis of the chemical profile of C. grandis ‘Tomentosa’, and applied this method to analyse crude drug samples of

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two C. grandis ‘Tomentosa’ parts, fruit epicarp and whole fruit. The aims and contents of this study mainly focused on two aspects: (i) identification of the multiple constituents based on accurate mass measurement and subsequent fragment ions in C. grandis ‘Tomentosa’; (ii) a comparison of the chemical differences between two parts of C. grandis ‘Tomentosa’, epicarp and whole fruit, based on the simultaneous quantitative analysis of multiple constituents. The results obtained will provide useful information for the suitable application of C. grandis ‘Tomentosa’ fruit as traditional medicine.

time-of-flight mass spectrometer equipped with ESI source. The system was operated with Analyst® TF 1.6 software (AB SCIEX, Foster City, CA). The conditions of MS/MS detector were as follows: ion source gas 1 60 psi; ion source gas 2 60 psi; curtain gas 30 psi; temperature 550 ◦ C; ion spray voltage floating 4500 V; collision energy 10 V; collision energy spread 20 V; declustering potential 80 V. TOFMS range was set at m/z 100–1000 and product ions mass range was set at m/z 50–1000. Both positive and negative ion modes were used for compounds ionization. Nitrogen was used as nebulizer and auxiliary gas.

2. Experiment 2.1. Reagents and materials

2.4. Establishment of tentative peak assignment

HPLC-grade Methanol was purchased from Fisher Scientific (Pittsburgh, PA, USA) and HPLC-grade Formic acid was purchased from Sigma (St. Louis, USA). All water used was purified by a Simplicity 185 personal water purification system (Millipore, Bedford, MA, USA). The reference standards naringin, naringenin, neoeriocitrin, hesperidin, kaempferol and bergapten were purchased from Sigma–Aldrich (St. Louis, USA). Rhoifolin, protocatechuic acid and apigenin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Meranzin hydrate was purchased from Shanghai Tauto Biotech Co. (Shanghai, China). Isoimperatorin was isolated and purified in our laboratory. The purity of each compound was determined to be higher than 98%, detected by HPLC. Crude drug samples of two C. grandis ‘Tomentosa’ parts, including ten batches fruit epicarp and ten batches whole fruit were supplied from Huazhou Huajuhong medicinal materials development Co., Ltd. (with GMP certificate, Huazhou, Guangdong, China). The samples were authenticated by Prof. Wenbo Liao from Sun Yat-sen University. The voucher specimens were deposited in our laboratory.

The UFLC–DAD–Q-TOF-MS/MS data of samples were extracted and analysed by PeakView software (AB SCIEX, Foster City, CA), mainly with the XIC manager tool which provided the quasimolecular weight, mass errors and isotope pattern fit. The predicted formula with errors less than ±5 ppm was searched against the compounds reported in citrus genus to obtain the tentative identification. This identification was further confirmed by determining the possible elemental compositions of the fragment ions and proposed fragmentation pathways using MS/MS spectrum.

2.2. Sample preparation A mixed standard solution containing protocatechuic acid, neoeriocitrin, naringin, hesperidin, rhoifolin, meranzin hydrate, naringenin, kaempferol, bergapten, apigenin and isoimperatorin was prepared by accurately weighing and dissolving them in methanol. All standard solutions were stored at 4 ◦ C in refrigerator. All the dried crude materials were ground and passed through a 40 mesh screen. 0.5 g of pulverized samples was accurately weighed and ultrasonic extracted with 100 mL methanol for 30 min and prior to use passed through a membrane filter having a 0.22␮m porosity, discarded the first part of the filtrate.

2.5. Validation of the method Eight major chemical markers were selected for simultaneous quantitative determination. The linearity and range was determined on six serial working solutions by plotting the peak areas versus their concentrations. Each solution was tested in duplicate. The limit of detection (LOD) and the limit of quantification (LOQ) were defined as the signal-tonoise ratio (S/N) of about 3 and 10, respectively. To evaluate the repeatability, six replicates of the same sample were prepared and analyzed. Intra- and inter-day variations were performed by analyzing the standard mixture solution six times within a day and three times for three consecutive days. Accuracy was calculated as the percentage of recovery by the assay of the known added amount of reference standards in the sample. Spiked sample solutions were prepared in three different amount levels (80%, 100% and 120%) using the aforementioned method, and triplicate experiments at each level. Recoveries were counted by the formulae: Recovery (%) = 100 × (amount found − original amount)/amount spiked. Variations were indicated by relative standard deviation (RSD) in all tests.

2.3. UFLC–DAD–Q-TOF-MS/MS system and conditions

3. Results and discussion

UFLC analysis was performed on a Shimadzu UFLC XR instrument (Shimadzu Corp., Kyoto, Japan), consisting of a binary pump, an autosampler, a column oven and a diode-array detector. Samples were separated on a Phenomenex Kinetex column (2.1 mm × 100 mm, 2.6 ␮m, Phenomenex, CA, USA). The mobile phase consisted of methanol (A) and 0.1% aqueous formic acid (v/v) (B). The following gradient elution program was used: isocratic 10% B (0–0.5 min), linear gradient from 10% to 25% B (0.5–8 min), 25–60% B (8–20 min), 60–100% B (20–28 min), isocratic 100% B for 8 min. A 8-min post-run time was set to equilibrate the column. The flow rate was kept at 0.3 mL/min. The injected volume was 2 ␮L and the column temperature was set at 40 ◦ C. The DAD detector scanned from 190 nm to 400 nm. Mass spectrometry was performed on the Triple TOFTM 5600 plus (AB SCIEX, Foster City, CA) a hybrid triple quadrupole

3.1. Optimization of sample preparation method In order to achieve optimized extraction efficiency, a comparison of ultrasonic extraction and reflux extraction were carried out. The extraction efficiencies of the major compounds by ultrasonic extraction were higher than reflux, and ultrasonic extraction gave the advantages of shorter extraction time and less solvent than reflux extraction. Other extraction conditions such as extraction solvent (methanol, ethanol, 75%, 50% and 25% aqueous ethanol and methanol (v/v)), extraction solvent volume (50, 75 and 100 mL) and extraction time (15, 30 and 45 min) were investigated. The results indicated that the extract condition of 50 mL methanol sonicated for 30 minutes showed the best extraction efficiency, and it obtained the most comprehensive compound information, covering both water-soluble and weak-polarity constituents.

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

169

Fig. 1. Representative total ion chromatogram (TIC) of fruit epicarp (A) and whole fruit (B) in positive ion mode by UFLC–DAD–Q-TOF-MS/MS.

3.2. Optimization of chromatographic conditions To achieve the higher resolution and better separation, the LC chromatographic conditions, including column and mobile phase, were optimized in the pilot study. Different kinds of mobile phase systems including methanol–water, acetonitrile–water, methanol–water with 0.1% formic acid and acetonitrile–water with 0.1% formic acid were compared to optimize the chromatographic conditions. It was found that methanol–water with 0.1% formic acid on the optimized gradient mode was the optimum choice, for showing a good separation and abundant signal response both in positive and negative ion scan mode. The MS conditions, such as the parameters of gas pressure, ion spray voltage, capillary temperature and voltage of declustering potential were optimized. And both positive and negative ion modes were detected. 3.3. Qualitative analysis of constituents in C. grandis ‘Tomentosa’ UFLC–DAD–Q-TOF-MS/MS analyses were performed in both positive and negative ion modes. The total ion chromatograms (TIC) corresponding to positive and negative signals of all samples were obtained. All compounds could be observed in positive ion mode, the representative positive signals from fruit epicarp and whole fruit was showed in Fig. 1. Accurate molecular weight was determined by the data of [M+H]+ , [M+Na]+ and [M−H]− . When a reference standard was available, the compound was identified by comparing its retention time, UV spectra and MS/MS spectra with those of the standard. While the identification of compound without available standard was mainly based on the MS/MS spectra and literature information. Eleven reference standard compounds were investigated with the established analysis method firstly. In our study, flavonoids and coumarins were found to be the main constituents in C. grandis ‘Tomentosa’. Among 48 major constituents identified or tentatively characterized, include19 flavonoids, 16 coumarins and 13 other compounds (Table 1). Their structures were shown in Fig. 2. 3.3.1. Identification of flavonoids Flavonoids are extensively distributed in citrus genus [20]. The UV spectra of flavonoids exhibit two major absorption peaks, which are commonly referred to as band I (300–380 nm) and band II (240–290 nm). More specifically, Band I lies in the range of 310–350 nm for flavones, 350–385 nm for flavonols, and reduced to little more than a shoulder in 300–330 nm for flavanones. Band II is almost similar and presents a bathochromic shift as the

OH substituents on A-ring increase [21]. In MS/MS spectra, the notable Ret-Diels-Alder (RDA) fragmentation patterns was found to be common to almost all the flavonoids analyzed [22]. Nineteen flavonoids were identified in C. grandis ‘Tomentosa’, including nine flavanones, nine flavones and one flavonols. Peak 13 was unequivocally identified as naringin by comparing with the reference standard. It showed maximum UV absorption at 282 and 326 nm, quasi-molecular ion [M+H]+ at m/z 581.1860 (C27 H32 O14 ) and major fragment ions at m/z 273.0759 by loss of a rhamnosylglucose (308 Da). Peak 12 showed same [M+H]+ ions at m/z 581.1859 (C27 H32 O14 ), and its fragmentation pathway was very similar to peak 13. Therefore, peak 12 was identified as narirutin, which was an isomeride of naringin and also a known constituent in pummelo [23]. By comparing and analyzing the structures of 12 and 13, it is found that these two compounds only differ on the interglycosideic linkage types between rhamnose and glucose. And there were two other isomeride pairs with the same condition, i.e. peaks 9/11 and peaks 16/18. Peaks 9 and 11 were a pair of isomerides. Peak 11 was identified as neoeriocitrin by comparison with the reference standard. Peak 11 gave the maximum UV absorption at 283 and 329 nm, the [M+H]+ ions at m/z 597.1810 (C27 H32 O15 ), and obtained a high intensity fragment ions at m/z 289.0702 by the loss of a rhamnosylglucose (308 Da) residue. Peak 9 had the UV absorption at 284 and 319 nm, similar [M+H]+ ions at m/z 597.1805 (C27 H32 O15 ) and product ions at m/z 289.0699. Thus, peak f1 was identified as eriocitrin [22]. Peaks 16 and 18 have the same molecular formula of C27 H30 O14 and [M+H]+ ions were m/z 579.1710 and 579.1712, respectively. Both of the two peaks presented a high intensity product ion at m/z 271.0606 and 271.0613, respectively, indicating the loss of a rhamnosylglucose moiety. Peak 18 was undoubtedly identified as rhoifolin by comparing the retention time, UV absorptions and MS/MS spectrum with the reference standard, and peak 16 was identified as isorhoifolin in accordance with the literature data [22]. Peaks 6 and 8 gave same [M+H]+ ions at m/z 743.2394, 162 Da more than that of peaks 12 and 13. And both of them displayed high intensity fragment ions at m/z 273.0748 and 273.0752, respectively, by successive losses of a glucose moiety and a rhamnosylglucose moiety. Therefore, peaks 6 and 8 were tentatively identified as narirutin 4 -glucoside and naringin 4 -glucoside which had been reported in citrus fruit [22,23]. Peak 15 showed the [M+H]+ ions at m/z 595.1652 (C27 H30 O15 ), 16 Da (O) more than that of peak 18. It also had similar fragmentation pathway to peak 18 and obtained an aglycone ion at m/z

170

Table 1 Identification of the chemical constituents of Citrus grandis ‘Tomentosa’ by UFLC–DAD–Q-TOF-MS/MS. TR (min)

Formula

[M+H]+ (error, ppm)

[M−H]− (error, ppm)

Fragment ions in positive (+) ion mode

Fragment ions in negative (−) ion mode

Identification

1

2.430

C7 H6 O4

155.0333 (−3.5)

153.0199 (3.9)

109.0297 [M−H–CO2 ]− , 91.0199 [M−H–CO2 –H2 O]− , 81.0359 [M−H–CO2 –CO]− , 65.0061

Protocatechuic acid

2

3.401

C9 H10 O4

183.0647(−2.9)

181.0505 (−0.9)

137.0269 [M+H–H2 O]− , 109.0311 [M+H–2H2 O]− , 93.0342 [M+H–H2 O–CO2 ]− , 81.0362, 65.0403, 63.0312 165.0531 [M+H–H2 O]+ , 135.0468 [M+H–H2 O–OCH2 ]+ , 123.0427 [M+H–2OCH2 ]+ , 113.9632, 97.9705, 84.9595, 56.9441

136.9201 [M−H–CO2 ]− , 92.9184 [M−H–2CO2 ]− , 78.9606

Veratric acid

3

5.717

C9 H8 O4

181.0492 (−1.9)

179.0346 (−1.9)

Caffeic acid

4

8.436

C9 H8 O3

165.0543 (−1.9)

163.0402 (0.2)

135.0447 [M−H–CO2 ]− , 107.0473 [M−H–C3 H4 O2 ]− 117.0354 [M−H–HCOOH]− , 93.0349 [M−H– C3 H2 O]−

5

9.615

C27 H30 O15

595.1655 (−0.4)

593.1510 (−0.3)

473.1100 [M−H–C4 H8 O4 ]− , 383.0771, 353.0668 [M−H–2C4 H8 O4 ]− , 297.0769

Vicenin-2

6

10.128

C33 H42 O19

743.2394 (0.2)

741.2250 (0.3)

Narirutin 4 -glucoside

7

10.497

C28 H32 O16

625.1760 (−0.5)

623.1612 (−0.9)

579.1731 [M−H–Glc]− , 459.1132 [M−H–Glc–C4 H8 O4 ]− , 433.1117 [M−H–Glc–Rha]− , 271.0586 [M−H–2Glc–Rha]− , 151.0021 533.1302, 503.1224 [M−H–C4 H8 O4 ]− , 413.0871, 383.0766 [M−H–2 C4 H8 O4 ]−

8

10.547

C33 H42 O19

743.2394 (0.1)

741.2252 (0.7)

9

10.622

C27 H32 O15

597.1805 (−1.4)

595.1661 (−1.3)

10

11.415

C21 H20 O11

449.1077 (−0.4)

447.0924 (−2.0)

11

11.728

C27 H32 O15

597.1810 (−0.6)

595.1666 (−0.5)

12

12.861

C27 H32 O14

581.1859 (−1.0)

579.1713 (−1.0)

147.0444 [M+H–H2 O]+ , 119.0485 [M+H–HCOOH]+ , 95.0632 [M+H–C3 H2 O]+ , 77.0394 [M+H–C3 H2 O–H2 O]+ 577.1553 [M+H–H2 O]+ , 559.1451 [M+H–2H2 O]+ , 529.1344 [M+H–2H2 O–CH2 O]+ , 523.1230 [M+H–4H2 O]+ , 457.1127, 409.0922, 379.0818, 325.0713, 295.0607, 401.1229, 339.0848, 273.0748 [M+H–2Glc–Rha]+

607.1637 [M+H–H2 O]+ , 589. 1550 [M+H–2H2 O]+ , 571.1442 [M+H–3H2 O]+ , 541.1361 [M+H–2H2 O–CH2 O]+ , 487.1237, 439.1011, 409.0899, 355.0815, 325.0700, 285.0374, 151.0405 417.1136 [M+H–2Glc]+ , 297.0740, 273.0752 [M+H–2Glc–Rha]+ , 153.0171 451.1222 [M+H–Rha]+ , 289.0699 [M+H–Glc–Rha]+ , 169.0112 [M+H–Glc–Rha–C4 H8 O4 ]+ , 129.0534 395.0718 [M+H–3H2 O]+ , 383.0789 [M+H–2H2 O–CH2 O]+ , 365.0633 [M+H–3H2 O–CH2 O]+ , 353.0639, 329.0633, 299.0549, 283.0674 619.1635 [M+Na]+ , 417.1167 [M+H–Glc–H2 O]+ , 339.0851, 273.0760 [M+H–OGlc–Rha]+ , 153.0175 419.1340 [M+H–Glc]+ , 273.0767 [M+H–Glc–Rha]+ , 153.0179

3-Coumaric acid

Lucenin-2,4 -methyl ether

621.1705 [M−H–C4 H8 O4 ]− , 271.0609 [M−H–2Glc–Rha]− , 151.0033 475.1102 [M−H–C4 H8 O4 ]− , 431.1002 [M−H–Rha–H2 O]− , 287.0552 [M−H–Glc–Rha]− , 181.0129

Naringin 4 -␤-d-glucoside

429.0815 [M−H–H2 O]− , 387.0770, 357.0608, 327.0497, 297.0429, 285.0370 [M−H–Glc]−

Luteolin-6-C-glucoside

475.1140 [M−H–C4 H8 O4 ]− , 271.0610 [M−H–OGlc–Rha]− , 151.0036

Neoeriocitrina

271.0604 [M−H–2Glc]− , 151.0032

Narirutin

Eriocitrin

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

No

13.350

C27 H32 O14

581.1860 (−0.9)

579.1711 (−1.4)

417.1177 [M+H–OGlc]+ , 383.1125, 339.0859, 273.0759[M+H–Glc–Rha]+ , 263.0548, 195.0284, 153.0178

14

13.721

C28 H34 O15

611.1953 (−2.9)

609.1817 (−1.2)

633.1796 [M+Na]+ , 331.0986

15 16

13.827 14.668

C27 H30 O15 C27 H30 O14

595.1652 (−0.9) 579.1710 (0.2)

593.1503 (−1.5) 577.1556 (−1.1)

17

14.788

C11 H6 O4

203.0338 (−0.3)

201.0195 (1.0)

18

15.103

C27 H30 O14

579.1712 (0.6)

577.1565 (0.4)

19

15.441

C28 H32 O15

609.1815 (0.1)

607.1663 (−0.9)

287.0556 [M+H–Glc–Rha]+ 433.1140 [M+H–Rha]+ , 271.0606 [M+H–Glc–Rha]+ 175.0382 [M+H–CO]+ , 159.0437 [M+H–CO2 ]+ , 147.0440 [M+H–2CO]+ , 131.0488 [M+H–CO–CO2 ]+ , 119.0487, 91.0548, 65.0400 601.1529 [M+Na]+ , 271.0613 [M+H–Glc–Rha]+ , 153.0176 301.0710 [M+H–Glc–Rha]+ , 286.0459

20

15.639

C15 H18 O5

279.1230 (0.9)

277.1075 (−2.5)

21

16.078

C28 H34 O14

595.2034 (2.1)

593.1870 (−1.0)

22

16.301

C33 H40 O18

725.2293 (0.8)

723.2149 (1.0)

23

16.338

C16 H16 O6

305.1025 (1.6)

303.0869 (−1.9)

24

16.656

C15 H12 O5

273.0759 (0.7)

271.0612 (−0.1)

25

17.012

C26 H30 O8

471.2018 (0.9)

469.1854 (−2.9)

26

17.563

C15 H10 O6

287.0553 (1.1)

285.0399 (−2.1)

27

17.652

C12 H8 O4

217.0497 (0.8)

215.0345 (−2.2)

28

17.840

C26 H32 O9

489.2123 (0.9)

487.1965 (−1.8)

261.1125 [M+H–H2 O]+ , 243.1015 [M+H–2H2 O]+ , 201.0466, 189.0542 [M+H–H2 O–C4 H8 O]+ , 159.0442 [M+H–H2 O–C4 H8 O–CH2 O]+ , 131.0487, 103.0546, 77.0395 617.1835 [M+Na]+

671.1982 [M+H–HCOOH]+ , 561.1588 [M+H–HCOOH–C4 H8 O4 ]+ , 461.1427, 381.0948, 273.0756 [M+H–C6 H8 O4 –Glc–Rha]+ , 261.0387, 153.0173 203.0352 [M+H–H2 O–C5 H8 O]+ , 119.0459, 91.0554, 59.0515 255.0652 [M+H–H2 O]+ , 153.0189 [M+H–C8 H8 O]+ , 147.0445, 119.0495 425.1970 [M+H–HCOOH]+ , 407.1853 [M+H–HCOOH– H2 O]+ , 367.1908, 339,1959, 279.1373, 213.0907, 161.0597, 133.0644 245.0588, 181.1018, 153.0173 [M+H–C8 H6 O2 ]+ , 131.0492, 124.9977 [M+H–C8 H6 O2 –CO]+ , 93.0745, 55.0593 202.0274 [M+H–CH3 ]+ , 174.0320, 146.0365, 118.0419, 89.0395 471.2062 [M+H–H2 O]+ , 453.1900 [M+H–2H2 O]+ , 443.2032 [M+H–HCOOH]+ , 337.1769, 161.0597, 95.0491

459.1149 [M−H–C4 H8 O4 ]− , 339.0716 [M−H–C8 H16 O8 ]− , 313.0709, 271.0595 [M−H–Glc–Rha]− , 235.0238, 177.0185, 151.0031, 119.0504 459.1168 [M−H–C4 H8 O4 ]− , 301.0705 [M−H–Glc–Rha]− , 151.0028 285.0403 [M−H–Glc–Rha]− 269.0445 [M−H–Glc–Rha]−

Naringina

Hesperidina Veronicastroside Isorhoifolin

173.0241 [M−H–CO]− , 145.0295 [M−H–2CO]− , 129.0348 [M−H–CO–CO2 ]− , 117.0353, 101.0405, 89.0415

Bergaptol

413.0894, 269.0460 [M−H–Glc–Rha]−

Rhoifolina

299.0561 [M−H–Glc–Rha]− , 284.0323 [M−H–Glc–Rha–CH3 ]−

Neodiosmin Meranzin hydratea

547.1855, 491.1106, 449.0954 [M−H–Rha]− , 353.1020, 285.0407 [M−H–Rha–Glc]− , 177.0525, 125.0242 579.1750 [M−H–C6 H8 O]− , 501.1261, 271.0609 [M−H–C6 H8 O4 –Glc–Rha]− , 151.0032

Poncirin

285.0829 [M−H–H2 O]− , 259.0905 [M−H–CO2 ]− , 227.0349 [M−H–H2 O–C3 H6 O]− , 215.0370 177.0191 [M−H–C6 H6 O]− , 151.0038 [M−H–C8 H8 O]− , 119.0511, 107.0151, 65.0079 381.2087, 249.0914, 229.1221, 199.1114, 147.0829

Oxypeucedanin

217.0475, 175.0378, 151.0008 [M−H–C4 H4 O4 –H2 O]− , 133.0274

Melitidin

Naringenina

Limonin

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

13

Kaempferola

Bergaptena 457.1846 [M−H–2CH3 ]− , 369.2081 [M−H–2CH3 –2CO2 ]− , 337.1430, 295.1337

Ichangin

171

172

Table 1 (Continued) TR (min)

Formula

[M+H]+ (error, ppm)

[M−H]− (error, ppm)

Fragment ions in positive (+) ion mode

29

17.851

C16 H20 O6

309.1333 (0.2)

307.1171 (−5.3)

30

18.173

C15 H16 O4

261.1125 (1.3)

259.0982 (2.3)

31

18.373

C15 H16 O5

277.1072 (0.6)

275.0919 (−2.1)

32

18.473

C15 H16 O4

261.1124 (0.9)

259.0962 (−1.1)

33

19.155

C15 H10 O5

271.0602 (0.2)

269.0452 (−1.3)

273.1085 [M+H–2H2 O]+ , 219.0642 [M+H–H2 O–C4 H8 O]+ , 161.0588, 133.0632, 118.0411, 79.0560 189.0547 [M+H–C4 H8 O]+ , 159.0437 [M+H–C4 H8 O–CH2 O]+ , 131.0488 [M+H–C4 H8 O–CO–CH2 O]+ , 103.0545 [M+H–C4 H8 O–CO–CH2 O–CO]+ , 77.0395 259.0975 [M+H–H2 O]+ , 205.0484 [M+H–C4 H8 O]+ , 177.0552 [M+H–C4 H8 O–CO]+ , 149.0579 [M+H–C4 H8 O–2CO]+ 189.0543 [M+H–C4 H8 O]+ , 159.0434 [M+H–C4 H8 O–CH2 O]+ , 131.0493 [M+H–C4 H8 O–CO–CH2 O]+ , 103.0549 [M+H–C4 H8 O–CO–CH2 O–CO]+ , 77.0396 153.0180 [M+H–C8 H6 O]+ , 119.0484 [M+H–C7 H4 O4 ]+ , 91.0540

34

19.174

C28 H34 O9

515.2278 (0.5)

513.2124 (−1.2)

469.2221 [M+H–HCOOH]+ , 455.2068, 437.1964

35

19.266

C26 H34 O9

491.2272 (−0.8)

489.2120 (−2.2)

36

19.985

C9 H6 O3

163.0384 (−3.4)

161.0243 (−1.0)

37

20.004

C19 H22 O4

315.1592 (0.4)

313.1427 (−2.3)

38

20.974

C26 H32 O8

473.2174 (0.9)

471.2017 (−1.5)

39

21.154

C28 H36 O10

533.2386 (0.9)

531.2233 (−0.5)

40

22.957

C16 H14 O4

271.0964 (−0.3)

269.0818 (−0.5)

41

23.380

C15 H16 O3

245.1174 (0.9)

243.1010 (−2.3)

473.2178 [M+H–H2 O]+ , 455.2068 [M+H–2H2 O]+ , 429.2303 [M+H–H2 O–CO2 ]+ , 411.2166, 385.2030 [M+H–H2 O–2CO2 ]+ , 369.2059, 161.0593 135.0443 [M+H–CO]+ , 119.0493 [M+H–CO2 ]+ , 107.0497 [M+H–2CO]+ , 77.0394 163.0383 [M+H–C10 H16 O]+ , 153.1276, 135.1159 [M+H–C10 H16 O–CO]+ , 119.0486, 107.0488 [M+H–C10 H16 O–2CO]+ , 81.0704 455.2053 [M+H–H2 O]+ , 427.2117 [M+H–HCOOH]+ , 369.2055 [M+H–CH3 COOH–CO2 ]+ , 341.2109, 95.0131 515.2279 [M+H–H2 O]+ , 473.2166 [M+H–CH3 COOH]+ , 455.2069 [M+H–H2 O–CH3 COOH]+ , 341.2111, 161.0596 215.0346 [M+H–C4 H8 ]+ , 187.0392, 173.0592, 159.0438 [M+H–C5 H10 O–CO]+ , 131.0491 [M+H–C5 H10 O–2CO]+ , 91.0551 189.0550 [M+H–C4 H8 ]+ , 159.0435 [M+H–C4 H8 –OCH2 ]+ , 77.0399, 103.0549, 131.0493,

Fragment ions in negative (−) ion mode

Identification Mexoticin

Meranzin

260.0684, 189.0186 [M−H–C5 H10 O]− , 161.0238 [M−H–C5 H10 O–CO]− , 133.0295 [M−H–C5 H10 O–2CO]− , 77.0409

5-Hydroxyisomeranzin

Isomeranzin

225.0532 [M−H–CH2 CHOH]− , 151.0026 [M−H–C8 H6 O]− , 117.1347 [M−H–C7 H4 O4 ]− , 65.0061 471.2030 [M−CO–CH3 ]− , 453.1939 [M−H–CH3 COOH]− , 391.1963, 307.0865, 205.0528 471.2032 [M−H–H2 O]− , 333.1361 [M−H–C4 H4 O–2CO2 ]− , 289.1441, 261.1485

133.0261 [M−H–CO]−

Apigenina

Nomilin

Deacetylnomilinic acid

7-Hydroxycoumarin

Epoxyaurapten

427.2081 [M−H–CO2 ]− , 369.1690 [M−H–HCOOH–C4 H8 ]− , 325.1798 [M−H–HCOOH–C4 H8 –CO]− , 307.1690 489.2149 [M−H–C2 H2 O]− , 471.2038 [M−H–CH3 COOH]− , 427.2136 [M−H–CH3 COOH–CO2 ]− , 325.1809, 59.0190 225.0552 [M−H–CO2 ]− , 210.0679, 197.0603, 183.0433 [M−H–C5 H10 O]−

Isoobacunoic acid

Nomilinic acid

Imperatorin

Osthol

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

No

23.733

C16 H14 O4

271.0968 (1.0)

269.0818 (−0.5)

43

23.955

C26 H30 O7

455.2086 (4.7)

453.1905 (−3.0)

44

24.279

C21 H22 O5

355.1540 (−0.1)

353.1385 (−2.7)

45

24.430

C15 H22 O

219.1742 (−0.4)

217.1582 (−2.3)

46

26.351

C19 H22 O3

299.1645 (1.2)

297.1490 (1.6)

47

26.832

C21 H22 O4

339.1597 (1.7)

337.1437 (−2.5)

48

28.441

C16 H32 O2

257.2477 (0.8)

255.2329 (−0.1)

a

215.0342 [M+H–C4 H8 ]+ , 203.0345 [M+H–C5 H10 ]+ , 187.0392, 175.0386 [M+H–C5 H10 –CO]+ , 159.0441 [M+H–C5 H10 O–CO]+ , 147.0440 [M+H–C5 H10 –2CO]+ , 131.0492 [M+H–C5 H10 O–2CO]+ , 119.0488, 91.0550, 69.0711 437.2035 [M+H–H2 O]+ , 411.2153 [M+H–CO2 ]+ , 409.2059 [M+H–HCOOH]+ , 383.1710 [M+H–C3 H4 O2 ]+ , 303.1354, 263.1423, 69.0742 203.0338 [M+H–C10 H16 O]+ , 147.0432 [M+H–C10 H16 O–2CO]+ , 131.0481 [M+H–C10 H16 O–CO–CO2 ]+ , 103.0753, 59.0507 201.1661 [M+H–H2 O]+ , 177.1633 [M+H–C3 H6 ]+ , 163.1125 [M+H–C3 H6 –CH3 ]+ , 149.0971 [M+H–C3 H6 –CO]+ , 135.0796 [M+H–C3 H6 –CO–CH3 ]+ , 119.0865, 93.0726, 81.0710, 67.0568 163.0391 [M+H–C10 H16 ]+ , 119.0487 [M+H–C10 H16 –CO2 ]+ , 107.0493 [M+H–C10 H16 –2CO]+ , 91.0545, 81.0704 203.0343 [M+H–C10 H16 ]+ , 147.0436 [M+H–C10 H16 –2CO]+ , 131.0483 [M+H–C10 H16 –CO–CO2 ]+ 279.2303 [M+Na]+

225.0552 [M−H–CO2 ]− ,214.0268, 186.0316

Isoimperatorina

407.1701 [M−H–HCOOH]− , 365.2098 [M−H–2CO2 ]− , 339.2010 [M−H– C3 H4 O2 –C3 H6 ]− , 257.1162, 197.0983, 149.0956

Obacunone

267.0654 [M−H–C5 H10 O]− , 214.0279

Epoxybergamottin

263.1648 [M−H+CH3 COOH]−

Nootkatone

Auraptene

Bergamottin

237.2241 [M−H–H2 O]−

Palmitic acid

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

42

Confirmation in comparison with authentic standards.

173

174

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

Fig. 2. Chemical structures of compounds identified in Citrus grandis ‘Tomentosa’.

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

287.0556. Therefore, peak 15 could be tentatively identified as veronicastroside which was reported in citrus species [24]. Peak 19 gave the UV max at 254, 348 nm and [M+H]+ ions at m/z 609.1815 (C28 H32 O15 ), 30 Da (OCH3 ) more than that of peak 18. Similarly, peak 19 produced an aglycone ion at m/z 301.0710. By comparing with reference [25], peak 19 was plausibly characterized as neodiosmin. Peak 14 displayed the maximum UV absorptions at 284 and 324 nm, [M−H]− ions at m/z 609.1817 (C28 H34 O15 ), and yielded an aglycone ion at m/z 301.0705 by the losses of one rhamnosylglucose. Thus, peak 14 was definitely identified as hesperidin by comparing with those of the reference standards. Peak 21 exhibited [M−H]− ions at m/z 593.1870 (C28 H34 O14 ), 16 Da (O) less than that of peak 14. Fragment ions at m/z 449.0954 and 285.0407 (aglycone ion) were obtained by the losses of a rhamnose and a rhamnosylglucose, respectively. By examining the known constituents in citrus fruits [26], peak 21 was tentatively identified as poncirin. Peaks 5, 7 and 10 were flavone-C-glycosides according to their TOF-MS/MS spectra. Flavone-C-glycosides often obtained fragments that due to losses of water ([M + H-nH2 O]+ ), and the loss of glucosidic methylol group as formaldehyde ([M+H–CH2 O–2H2 O]+ ) [27]. Peak 5 showed UV max at 271, 335 nm, [M+H]+ ions at m/z 595.1655 (C27 H30 O15 ), and displayed secondary fragments at m/z 577.1553, 559.1451 and 523.1230 by the loss of one, two and four H2 O molecules, respectively. Another fragment ion at m/z 529.1344 was yielded by the loss of a formaldehyde residue and two H2 O molecules at the same time. Peak 7 showed UV max at 271, 346 nm and [M+H]+ ions at m/z 625.1760 (C28 H32 O16 ). Fragment ions at m/z 607.1637, 589.1550 and 571. 1442 were corresponding to the loss of one, two and three H2 O molecules. And the loss of a formaldehyde residue added two H2 O molecules led to the product ion at m/z 541.1361. Peak 10 gave [M+H]+ ions at m/z 449.1077 (C21 H20 O11 ) and obtained fragment ions at m/z 395.0718 and 365.0633 by the successive losses of three water molecules and a formaldehyde residue. Therefore, on the basis of MS fragmentations and previous detection in literature data [27], peaks 5, 7 and 10 were tentatively identified as vicenin-2 lucenin-2,4 -methyl ether and luteolin-6-Cglucoside, respectively. Peak 22 was identified as 3-hydroxy-3-methylglutaryl (HMG) conjugates of flavanone O-diglycoside according to its MS2 fragments. Its UV ␭max was 283, 327 nm. It gave a positively charged molecular ion [M+H]+ at m/z 725.2293 (C33 H40 O18 ) and fragment ions at m/z 671.1982 by loss of a formic acid molecule. Peak 22 also showed the diagnostic fragment ions at m/z 273.0756 by successive losses of 3-hydroxy-3-methylglutaryl moiety and a rhamnosylglucose. Thus, peak 22 was tentatively identified as melitidin which was reported in citrus [28]. Peaks 24 and 33 were identified as naringenin and apigenin for having the same chromatographic and mass spectral properties with their reference standards. Peak 24 gave the UV max at 289, 326 nm, and [M+H]+ ions at m/z 273.0579 (C15 H12 O5 ), 308 Da less than that of peaks 13. Peak 33 processed the UV max at 268, 336 nm, and [M+H]+ ions at m/z 271.0602 (C15 H10 O5 ), 308 Da less than that of peak 18. Therefore, naringenin and apigenin should be the aglycone moiety of naringin and rhoifolin. Peak 26 displayed the UV max at 269, 367 nm, [M+H]+ ions at m/z 287.0553 (C15 H10 O6 ) and it was identified as kaempferol by comparison the retention time, UV data and MS2 fragmentation patterns with its reference standard. 3.3.2. Identification of coumarins The UV spectra of coumarins exhibited maximum absorption at nearly 270 and 320 nm. Sixteen coumarins were identified. Peaks 20, 29, 30, 31, 32 had the same fragmentation patterns that yielded product ions by series losses of one isobutyraldehyde (72 Da, C4 H8 O) and one methoxy group (30 Da, CH2 O) in

175

positive ion mode. Comparing with both the reference standard and literature data [29] led to the identification of peak 20 as meranzin hydrate. The MS spectrum focused on [M+H]+ m/z 279.1230 (C15 H18 O5 ), and product ions were observed at m/z 261.1125 indicated the progressive loss of one water molecule. Other product ions focused on m/z 189.0542 ([M+H–H2 O–C4 H8 O]+ ) and 159.0442 ([M+H–H2 O–C4 H8 O–CH2 O]+ ). Peaks 30 and 32 have the same [M+H]+ ions at m/z 261.1125 and 261.1124, respectively. The predicted molecular formula was C15 H16 O4 and their molecular weights were 18 Da (H2 O) less than that of peak 20. Peak 30 showed product ions at m/z 189.0547 ([M+H–C4 H8 O]+ ), 159.0437 ([M+H–C4 H8 O–CH2 O]+ ), and peak 32 generated similar product ions at m/z 189.0543 ([M+H–C4 H8 O]+ ), 159.0434 ([M+H–C4 H8 O–CH2 O]+ ). According to their retention time in reverse-phase HPLC reported before [30], peaks 30 and 32 were plausibly identified as meranzin and isomeranzin, respectively. Peak 31 showed the [M+H]+ ions at m/z 277.1072 (C15 H16 O5 ) and was 16 Da (O) more than that of peak 30. Its product ions focused on 259.0975 ([M+H–H2 O]+ ), 205.0484 ([M+H–C4 H8 O]+ ). Therefore, on the basis of occurrence data in Citrus genus [31] peak 31 was tentatively identified as 5-Hydroxyisomeranzin. Peak 29 gave the [M+H]+ ions at m/z 309.1333 (C16 H20 O6 ), 30 Da (CH2 O) more than that of peak 20. And its MS/MS fragmentation patterns were also very similar to peak 20. It produced fragment ions at m/z 219.0642 ([M+H–H2 O–C4 H8 O]+ ) indicating that peak 29 may be mexoticin as long as the literature data reported before [32]. Peak 41 exhibited [M+H]+ ions at m/z 245.1174 (C15 H16 O3 ), 34 Da (H2 O2 ) less than that of peak 20. Peak 41 yielded fragment ions at m/z 189.0550 by the loss of one isobutene (56 Da), and another product ions focused on m/z 159.0435 were produced by losing of a isobutene and a methoxy group at the same time. Thus, peak 41 was tentatively assigned as osthol also according to the literature data [31]. By comparing the retention time, UV data and MS/MS fragmentation pattern with the reference standard, peak 42 was identified as isoimperatorin. Peak 42 showed the UV max at 222, 265 and 311 nm, [M+H]+ ions at m/z 271.0968 (C16 H14 O4 ), and MS/MS spectra showed fragment ions at m/z 215.0342, 187.0392, resulting from the losses of an isobutene unit (56 Da, C4 H8 ) and a 3-methylcrotonaldehyde unit (84 Da, C5 H8 O), respectively. Peak 40 had the similar UV max at 218, 264 and 308 nm, [M+H]+ ions at m/z 271.0964 and fragment ions at m/z 215.0344 and 187.0392. Therefore, peak 40 was tentatively assigned as imperatorin [33]. Peak23 obtained [M+H]+ ions at m/z 305.1025, 34 Da (H2 O2 ) more than that of peak 42. And produced a high intensity [M+H-102]+ at m/z 203.0352. Peak 23 was tentatively identified as oxypeucedaninhydrate which was reported in C. grandis Osbeck [29]. Peak 36 showed [M+H]+ ions at m/z 163.0384 (C9 H6 O3 ) and obtained product ions at m/z 135.0443, 119.0493 and 107.0497 by the progressive loss of a carbon monoxide, a carbon dioxide and two carbon monoxides, respectively. Thus, peak 36 was identified as 7-hydroxycoumarin which has been reported in citrus fruits [34]. Peak 46 displayed an [M+H]+ ion at m/z 299.1645 (C19 H22 O3 ) and a major fragment ion at m/z 163.0391 by loss of a decadiene residue (136 Da, C10 H16 ). Peak 46 was tentatively assigned as auraptene [35]. Peak 37 generated [M+H]+ ions at m/z 315.1592, 16 Da (O) more than that of peak 46. And it produced similar fragment ions at m/z 163.0383 ([M+H–C10 H16 O]+ ). Refer to the constituents reported in citrus essential oils [30], peak 37 was tentatively characterized as epoxyaurapten. Peak 27 exhibited the UV max at 221, 267 and 310 nm, [M+H]+ ions at m/z 217.0497 and [M+H–CH3 ]+ ions at m/z 202.0274. By comparing its retention time, UV absorptions and MS spectrum with the authentic standard, confirmed its identification as bergapten [36]. Peak 17 gave [M+H]+ ions at m/z 203.0338, 14 Da

The established method was subsequently applied to analyze each 10 batches of epicarp and whole fruit samples. Results showed that the chemical composition between the two parts was

RSD (%) Mean (%)

98.89 99.33 100.75 101.79 101.47 98.61 100.21 98.48 1.22 2.14 2.31 2.76 2.06 2.34 1.63 2.88 0.73 0.98 0.93 1.39 1.20 0.86 0.63 0.77 0.39 0.51 0.87 0.67 0.79 0.13 0.58 0.69 2.61 3.17 2.77 4.87 4.29 2.57 3.76 5.21 0.79 0.96 0.84 1.48 1.30 0.78 1.14 1.58 2.99–119.70 0.91–18.10 0.93–18.60 0.82–16.30 0.30–6.00 0.12–1.20 0.20–4.00 0.09–1.80

Recovery Repeatability (n = 5) Inter-day (n = 3) Intra-day (n = 6) LOQ (ng/mL) LOD (ng/mL) Linear range (␮g/mL) R2 (n = 6)

y = 2.0934.83x + 2.61 y = 8.51e4x + 3.99e4 y = 23669.93x + 4.08 y = 21236.08x + 4128.32 y = 1.86e6x + 7.52e5 y = 1.33e7x + 7304.22 y = 3.15e6x + 5.63e5 y = 4.93e5x − 2199.99

3.5. Chemical comparison of the two kinds of commercial C. grandis ‘Tomentosa’

Naringin Rhoifolin Meranzin hydrate Neoeriocitrin Isoimperatorin Bergapten Naringenin Apigenin

The method validation data were shown in Table 2. The linearity of 8 selected compounds showed good correlation coefficients (R2 : 0.9993–0.9999), and the sensitivity was high (LOD: 0.78–1.58 ng; LOQ: 2.61–5.21 ng). The intra-day and inter-day precisions showed RSD within 0.13–0.87% and 0.73–1.39%, respectively. The RSD of repeatability was within 1.22–2.88%. The mean recoveries were from 98.48 to 101.79% with RSD less than 2.96%. These results indicated that the developed UFLC–DAD–Q-TOF-MS/MS method was a reliable and useful method for quality assessment of C. grandis ‘Tomentosa’.

Linearity

3.4. Validation of the quantitative method

Table 2 Method validation for the determination of selected chemical markers.

3.3.3. Identification of other compounds Peaks 1, 2, 3 and 4 showed [M−H]− ions at m/z 153.0199, 181.0505, 179.0346 and 163.0402 in the negative ion mode, respectively. And these peaks often produced fragment ions by losses of H2 O, CO2 , CO or HCOOH residues suggesting the presence of –OH and –COOH group. In accordance with existing study, peaks 1, 2, 3 and 4 were tentatively identified as protocatechuic acid [38], veratric acid [31], caffeic acid and 3-coumaric acid [39], respectively. Peaks 25, 28, 34 and 43 exhibited [M+H]+ ions at m/z 471.2018 (C26 H30 O8 ), 489.2123 (C26 H32 O9 ), 515.2278 (C28 H34 O9 ) and 455.2086 (C26 H30 O), respectively. And all of their MS/MS spectra obtained fragment ions by losses of formic acid, H2 O and CO2 moieties. By comparing their quasi-molecular weights and chromatographic properties with the reported compounds in citrus fruits [40–43], peaks 25, 28, 34 and 43 were tentatively identified as limonin, ichangin, nomilin and obacunone. Peaks 39 and 38 were tentatively assigned as nomilinic acid and isoobacunoic acid due to the protonated molecular ions [M+H]+ at m/z 533.2386 (C28 H36 O10 ) and 473.2174 (C26 H32 O8 ), respectively. And both of them produced fragment ions by losses of H2 O and acetic acid radicals in MS2 spectra. Peak 35 gave [M+H]+ ions at m/z 491.2272 (C26 H34 O9 ), 42 Da (C2 H2 O) less than that of peak 39. Peak 35 also formed fragments by successive losses of H2 O and CO2 radicals. Therefore, it was tentatively identified as deacetylnomilinic acid which has been reported in Citrus species [42]. And these limonoids have been rarely reported in C. grandis ‘Tomentosa’. Two compounds reported in C. grandis ‘Tomentosa’ essential oil [44] was also detected in the present study. Peak 45 displayed [M+H]+ ions at m/z 219.1741 and produced fragment ions by losses of H2 O and propylene molecules. Peak 45 was tentatively identified as nootkatone. Peak 48 showed the protonated molecule [M+H]+ and sodic adduct ion [M+Na]+ at m/z 257.2477 and 279.2303. It was tentatively identified as palmitic acid.

0.9993 0.9995 0.9998 0.9996 0.9993 0.9998 0.9999 0.9993

(CH2 ) less than that of peak 27. And it produced product ions at m/z 175.0382, 159.0437, 147.0440, and 131.0488 by successive losses of one or two CO and CO2 molecules. Peak 17 was tentatively identified as bergaptol which has been reported in literature [37]. In positive ion mode, peak 47 obtained [M+H]+ at m/z 339.1597 (C21 H22 O4 ) and [M+H–C10 H16 ]+ at m/z 203.0343, corresponding to the loss of a decadiene residue. Peak 44 showed [M+H]+ ions and [M+H–C10 H16 O]+ ions at m/z 355.1540 and 203.0338, respectively. Its predicted molecular formula was C21 H22 O5 . Comparing with the literature information reported [30], peaks 47 and 44 were tentatively identified as bergamottin and epoxybergamottin.

1.79 1.95 2.96 1.23 2.51 1.11 2.34 1.30

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

Investigated compound

176

Table 3 Contents of selected compounds in the epicarp and whole fruit samples. Sample no.

Content of each compound in 20 samples Naringina (mg/g)

Rhoifolina (mg/g)

Meranzin hydrate (mg/g)

Neoeriocitrina (mg/g)

Isoimperatorin (mg/g)

Bergapten (␮g/g)

Naringenin (␮g/g)

Apigenin (␮g/g)

Epicarp

1 2 3 4 5 6 7 8 9 10 Average RSD (%)

48.70 47.66 44.84 43.34 53.86 48.36 43.08 47.20 43.82 39.94 46.08 8.47

3.59 5.76 5.74 4.69 5.97 5.88 5.10 5.15 4.47 5.73 5.21 14.79

3.09 2.11 3.24 2.59 3.27 2.72 3.56 2.18 2.67 3.07 2.85 16.75

0.77 0.86 1.19 0.80 0.92 0.87 1.06 0.87 0.79 0.92 0.91 14.39

0.80 1.28 0.81 1.01 0.92 1.06 1.44 0.61 1.39 1.48 1.08 28.10

83.11 94.57 107.00 104.02 95.20 85.14 117.82 103.32 77.60 100.02 96.78 12.66

44.72 51.95 34.00 34.66 73.90 75.62 29.56 56.18 35.23 54.02 48.98 33.55

11.06 21.52 15.59 12.04 16.34 21.42 10.82 16.04 13.51 15.90 15.42 24.64

Whole fruit

11 12 13 14 15 16 17 18 19 20 Average RSD (%)

91.66 89.40 88.08 75.76 82.00 85.68 77.78 78.96 73.80 79.42 82.25 7.45

6.14 8.70 7.03 6.83 7.94 8.32 7.04 6.08 6.62 6.68 7.14 12.52

2.69 2.84 2.62 2.91 2.23 2.65 2.77 2.21 2.12 2.09 2.51 12.57

1.26 1.04 1.81 1.13 1.28 1.19 1.24 1.16 1.03 1.50 1.26 18.53

0.50 0.78 1.31 0.64 0.89 0.82 0.84 0.57 1.17 0.84 0.84 29.96

71.62 74.78 100.88 106.46 68.28 71.88 75.14 101.66 96.66 76.04 84.34 17.83

40.22 49.86 56.32 62.70 48.94 47.22 41.70 56.48 46.40 45.04 49.49 14.31

11.54 17.32 15.72 12.53 14.96 11.89 15.76 15.20 13.43 17.37 14.57 14.56

a

P.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 90 (2014) 167–179

Part

Contents between epicarp and whole fruit are significantly different at P = 0.5 level.

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consistent. While the quantitative analytical results (Table 3) indicated that their contents of 8 major compounds varied in some degree. All samples examined were rich in flavonoids and coumarins, and naringin was found to be the predominant components whose content was much higher than other compounds (Table 3) which was in line with previous observations [45]. The other two major flavonoids were rhoifolin and neoeriocitrin. In recent studies, naringin evidenced profound antitussive and anti-inflammatory activities, such as the antitussive effect on experimentally induced cough [46], the attenuation of LPS-induced acute lung injury [47], and the prevention of cigarette smoke induced chronic bronchitis [48]. Based on these considerations, the determination of naringin and analogous components was of great importance for the medicinal use of C. grandis ‘Tomentosa’. Meranzin hydrate followed by isoimperatorin was found to be the major coumarins components. The contents of naringenin, apigenin and bergaptene were lower by one to two orders of magnitude than the other chemical markers. A two independent samples t-test was performed to test whether there were significant differences in the contents between epicarp and whole fruit for each of the eight marker components. The statistical analysis was performed with PASW Statistics (SPSS) software (version 18.0). Results showed that the contents of three major flavonoids, namely naringin, rhoifolin and neoeriocitrin, were significantly higher (P < 0.5) in whole fruit than in epicarp. While no statistically significant differences were observed in the content of other components. According to the current Chinese Pharmacopoeia (2010 edition), the described medicinal part of C. grandis ‘Tomentosa’ was the fruit epicarp. However, on the basis of our investigation results, the C. grandis ‘Tomentosa’ sold in medicine markets and drug stores was basically whole fruit. It was probably because the fruit of C. grandis ‘Tomentosa’ was inedible and barely used as food substances, due to its characteristics of thick pericarp and bitter flavor. The utilization of the whole fruit seemed more beneficial and convenient. Nevertheless, the medicinal parts of C. grandis ‘Tomentosa’ needed uniform stipulation and was still worthy of further discussion.

4. Conclusion A rapid UFLC–DAD–Q-TOF-MS/MS method was established for the systematic analysis of constituents in C. grandis ‘Tomentosa’. A total of forty-eight compounds were identified or tentatively characterized on the basis of their retention times, UV spectra, exact mass measurement for molecular ions and subsequent product ions. Eight major components of these compounds were simultaneously quantified. Chemical comparison of two medicinal parts, fruit epicarp and whole fruit, were carried out. And results demonstrated that the contents of three major flavonoids, naringin, rhoifolin and neoeriocitrin in whole fruit were significantly higher than in epicarp (P < 0.5). To a certain extent, differences in chemical constituents might lead to possible differences in their biological functions. Since they were all used as Huajuhong in Chinese medicine, further comparative scientific investigations of the two parts were therefore suggested to verify whether they are actually equivalent. The established method could be helpful in quality assessment and standardization of C. grandis ‘Tomentosa’ raw materials and its products.

Acknowledgements The raw materials of Citrus grandis ‘Tomentosa’ were provided by Huazhou Huajuhong medicinal materials development Co., Ltd.

and this study was supported by grants from the National Key Technology R&D Program of PR China (2012BAT29B09).

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