FRIN-06533; No of Pages 9

Food Research International xxx (2016) xxx–xxx

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Food Research International

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Characterization of phenolic and other polar compounds in peel and flesh

of pink guava (Psidium guajava L. cv. ‘Criolla’) by ultra-high performance
liquid chromatography with diode array and mass
spectrometric detection
Carolina Rojas-Garbanzo a,b, Benno F. Zimmermann a,c, Nadine Schulze-Kaysers a,⁎, Andreas Schieber a
a
Institute of Nutritional and Food Sciences, Molecular Food Technology, University of Bonn, Römerstraße 164, D-53117 Bonn, Germany
b
National Center for Food Science and Technology (CITA), University of Costa Rica, 11501-2060 San José, Costa Rica
c
Institut Kurz, Stöckheimer Weg 1, D-50829 Köln, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Pink guava (Psidium guajava L.) is a highly consumed fruit in tropical countries. Despite of interesting research on
Received 26 September 2016 health effects of this fruit, investigations into the profile of secondary plant metabolites are scarce. In this study,
Received in revised form 5 December 2016 the phenolic compounds in the peel and flesh of pink guava were characterized by ultra-high performance liquid
Accepted 10 December 2016
chromatography with diode array and mass spectrometric detection. Sixty phenolic compounds were character-
Available online xxxx
ized by MS2 and classified as ellagitannins, flavones, flavonols, flavanols, proanthocyanidins, dihydrochalcones,
Keywords:
and anthocyanidins, and non-flavonoids such as phenolic acid derivatives, stilbenes, acetophenones, and benzo-
Psidium guajava L. phenones. Forty-two polyphenols are reported for the first time in both peel and flesh, and twenty-four com-
Phenolic compounds pounds were detected for the first time in P. guajava, e.g., phlorizin, nothofagin, astringin, chrysin-C-glucoside,
UHPLC-DAD-MS/MS valoneic acid bilactone, cinnamoyl-glucoside, and two dimethoxycinnamoyl-hexosides.
Peel © 2016 Elsevier Ltd. All rights reserved.
Flesh

1. Introduction Research on health effects of guava has mostly focused on polyphe-

Psidium guajava L. is native to Central America and nowadays it is
grown throughout the tropical and subtropical regions of the world.
Among the numerous cultivars of P. guajava, the pink fruit has an in-
tense aroma and color, making it most popular for consumption as a
fresh fruit or for processing (Mitra, Irenaeus, Gurung, & Pathak, 2012).
This fruit is one of the most important commercial fruit crops in coun-
tries such as Mexico, Brazil, Pakistan, Thailand, China, and India. In
2014, India was considered the largest guava producer worldwide,
followed by China and Thailand (FAOSTAT, 2016).
Leaves of P. guajava have a history of use as a traditional medicine in
countries such as Taiwan, Japan, China and Korea (Chang et al., 2013;
Diaz-de-Cerio, Verardo, Gómez-Caravaca, Fernández-Gutiérrez, &
Segura-Carretero, 2015). The antibacterial, anti-inflammatory,
cicatrizant, and anticarcinogenic properties are some bioactivities re-
ported for this plant, which are mainly attributed to the content of phy-
tochemicals such as vitamin C, carotenoids and polyphenols (Chang et
al., 2013; Fracassetti, Costa, Moulay, & Tomás-Barberán, 2013).
⁎ Corresponding author at: Römerstraße 164, D-53117 Bonn, Germany.
E-mail addresses: carolina.rojasgarbanzo@ucr.ac.cr (C. Rojas-Garbanzo),
benno.zimmermann@uni-bonn.de (B.F. Zimmermann), nschulze@uni-bonn.de
(N. Schulze-Kaysers), schieber@uni-bonn.de (A. Schieber).
nol-containing leaf extracts, e.g., their effect on dipeptidyl-peptidase IV
(DP-IV), a key enzyme of blood glucose homoeostasis (Eidenberger,
Selg, & Krennhuber, 2013), and the impact of quercetin from an aqueous
guava leaf extract on the alleviation of hypoglycemia diabetes (Cheng,
Shen, & Wu, 2009). Moreover, the antimicrobial activity of an extract
from guava leaves has been evaluated against Salmonella enteritidis
and Bacillus cereus (Arima & Danno, 2002), but polyphenols were not
identified.
Studies on the phytochemical composition of guava flesh or peel are
scarce. To our knowledge, the analysis of phenolic compounds in guava
flesh is limited to the total polyphenol content (Chopda & Barrett, 2001;
Kek, Chin, & Yusof, 2013; Kong, Ismail, Tan, Nagendra Prasad, & Ismail,
2010; Musa, Abdullah, & Subramaniam, 2015; Osorio, Forero, &
Carriazo, 2011; Thuaytong & Anprung, 2011), whereas only few studies
reporting the identification of polyphenols were published.
Main phenolic components previously identified in the fruit of
Psidium guajava are glycosides of myricetin, quercetin, kaempferol,
luteolin, apigenin, and isorhamnetin, a B-type proanthocyanidin
(gallocatechin-catechin), and two benzophenone compounds (Flores,
Wu, Negrin, & Kennelly, 2015; Musa et al., 2015; Shu, Chou, & Wang,
2010). Considering other fruits of the family Myrtaceae, another species
of the genus Psidium as well as Eucalyptus sp. and Myrciaria sp. have
been demonstrated to contain flavonoids, triterpenoids and other

http://dx.doi.org/10.1016/j.foodres.2016.12.004
0963-9969/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
2 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx
biologically active secondary metabolites (Flores et al., 2013; Fracassetti
et al., 2013; Gordon, Jungfer, da Silva, Maia, & Marx, 2011; Santos, Vilela,
Freire, Neto, & Silvestre, 2013). Therefore, examination of the phenolic
profile of other edible fruits of the family Myrtaceae or especially of
the genus Psidium would lead to a better understanding of the chemo-
taxonomic relationship.
In view of the evident lack of information about the profile of pheno-
lic compounds in Psidium guajava, the peel and flesh of pink guava fruits
were analyzed using UHPLC-DAD-MS/MS. The peels were examined
separately to assess these by-products as a source of bioactive
components.
2. Materials and methods
2.1. Plant material
Ripe pink guava fruits at usual stage of consumption, with a light
green to light yellow color of the peel and a total soluble solid content
(TSS) of the flesh of 9.07 ± 0.12° Brix were obtained from the agricul-
tural farm “San Vicente” (Turrialba, Costa Rica) in July 2015. The peel
was manually separated and the fresh flesh was cut into small pieces
and homogenized. The seeds were separated. Samples of flesh and
peel were frozen separately, lyophilized (Alpha 2–4 LSC, Christ,
Osterode am Harz, Germany), ground (Clatronic KSW 3306, Kempen,
Germany), packed into brown bottles under nitrogen atmosphere, and
stored at −80 °C (HERAfreeze HFU B Serie, Thermo Scientific, Osterode,
Germany) until used for extraction.
2.2. Chemicals
All reagents and solvents were of analytical or MS grade. Methanol,
acetonitrile and water were obtained from Chemsolute (Renningen,
Germany). Ethyl acetate and n-hexane were purchased from Fischer
Chemical (Loughborough, UK). Formic acid and the standards abscisic
acid, phlorizin, (+)-catechin, (−)-epicatechin, and quercetin were ob-
tained from Sigma-Aldrich (St. Louis, Missouri, USA) at 98% purity. Gallic
acid monohydrate and cinnamic acid at 99% purity were sourced from
Fluka Chemika (Buchs, Switzerland). Cyanidin-3-O-glucoside was ob-
tained from PhytoLab (Vestenbergsgreuth, Germany).
2.3. Total soluble solids
After removing the peel using a knife, ripe fresh guava was cut into
small pieces and homogenized. The total soluble solids (TSS) were de-
termined in triplicate at 20 °C using a handheld refractometer (Krüss,
Hamburg, Germany).
2.4. Extraction of polar compounds
Extraction of polar compounds was carried out in triplicate accord-
ing to Chang et al. (2013) with some modifications in order to improve
the extraction yield. One gram of lyophilized sample was weighed into a
50 mL graduated centrifuge tube and 10 mL of methanol + water
(9 + 1, v + v) containing 1% of formic acid was added. Subsequently,
polyphenols were extracted using an Analog vortex mixer (VWR, Leu-
ven, Belgium) for 1 min followed by 20 min in a sonication bath. The
sample was then centrifuged for 5 min at 10,947g (Megafuge 40R, Ther-
mo Scientific, Osterode, Germany) and the supernatant was collected.
The residue was extracted another two times. The pooled supernatants
were filtered through Whatman no. 4 filter paper. Methanol was re-
moved under a stream of nitrogen. The aqueous solution was
partitioned three times against n-hexane (10 mL) and then three
times against ethyl acetate (10 mL). The ethyl acetate fraction was sep-
arated and dried under nitrogen, whereas the aqueous phase was freeze
dried in vacuo (Alpha 2–4 LSC, Christ, Osterode am Harz, Germany). The
dried extracts were dissolved in 2.5 mL of methanol/bidistilled water
Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
(1 + 1, v + v). Samples were filtered using a 0.22 μm pore size
Chromafil filter made from regenerated cellulose (Macherey-Nagel,
Düren, Germany) before injection in the UHPLC system.
2.5. UHPLC-DAD-MS/MS analysis
The analysis of polar compounds was carried out in an Acquity UPLC
system from Waters (Milford, MA, USA) as used previously (Feuereisen
et al., 2014). The compounds were separated with a BEH Shield RP18
column (150 mm × 2.1 mm, 1.7 μm), protected by a 5 mm × 2.1 mm
guard column (Waters). The mobile phase consisted of water/formic
acid (99.9 + 0.1, v + v, eluent A) and acetonitrile/formic acid
(99.9 + 0.1, v + v, eluent B) using the following gradient elution:
0 min, 2% B; 28 min, 50% B; 28.5 min, 100% B; 30.5 min, 100% B;
32 min, 2% B; 34 min, 2% B. The flow rate was 0.4 mL/min, the column
temperature was 40 °C, and the injection volume was 5 μL. The mass
spectrometer was tuned using a solution of gallic acid, and the following
parameters were applied: capillary voltage −1.0 kV, cone voltage 25 V,
extractor voltage 2.0 V, RF voltage 2 V, source temperature 150 °C,
desolvation temperature 450 °C, cone gas (nitrogen) flow 50 L/h, and
desolvation gas (nitrogen) flow 800 L/h. The collision gas (argon) flow
used in MS/MS experiments was 0.22 mL/min, and a collision energy
of 20, 25 or 40 V was applied. For the analysis of anthocyanidins positive
ionization was applied. Data acquisition and processing were performed
using MassLynx Software (Waters).
Characterization of the analytes was based on retention time and
mass spectrometric data. The compounds were first detected using a
single MS scanning in the m/z range from 180 to 1150 and targeted
search using selected ion recording (SIR), followed by product ion
scans of the peaks showing major signals in the MS or UV–Vis chro-
matograms. To obtain more structural information of several molecules,
a cone voltage of 40 or 60 V was applied for enhanced in-source frag-
mentation. The in-source fragments generated were then subjected to
product ion scans. For the identification of phloretin derivatives, querce-
tin derivatives, and abscisic acid derivatives, precursor ion scans were
conducted. It was not possible to obtain clear UV spectra due to peak
overlapping and/or low concentrations.
3. Results and discussion
All polyphenols were found in both the ethyl acetate and the water
fraction. Because of the higher intensity of the peaks in the ethyl acetate
extract, the results presented in Table 1 are based on this fraction.
3.1. Identification of polar compounds
A total of 74 compounds was detected but only 63 compounds were
successfully identified (Table 1), comprising a cinnamoyl hexoside,
abscisic acid and a derivative of the latter, and 60 phenolic compounds.
The polyphenols were then classified as ellagitannins, flavones,
flavonols, flavanols, proanthocyanidins, dihydrochalcones, and
anthocyanidins, belonging to the flavonoid family, and as phenolic
acid derivatives, stilbenes, acetophenones, and benzophenones, belong-
ing to other sub-classes of phenolic compounds. Their structures are
presented in Fig. 1. Forty-two polyphenols are reported for the first
time in both peel and flesh of P. guajava. The flavonoids kaempferol,
luteolin and apigenin previously reported in pink guava from Malaysia
(Musa et al., 2015) were not found in our samples. This result can be ex-
plained due to differences of cultivars, method of extraction, as well as
differences in the parameters of the chromatographic analysis. Twen-
ty-four compounds are reported for the first time in P. guajava.
3.1.1. Phenolic acid and derivatives
The monomer gallic acid was confirmed for compound 4 by the
same retention time and MS data obtained for the standard, which
gave a [M–H]− ion at m/z 169, as well as daughter ions at m/z 125 via
 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx 3

Table 1
MS data of polar compounds in peel and flesh of ripe pink guava (P. guajava L. cv. ‘Criolla’).

Peak number Retention time m/z UHPLC–ESI−–MSn experimenta,b Tentatively identified as Previously described in guava
(min) [M–H]−

Phenolic acid derivatives

MS2 [331]: 169, 151, 123, 211, 125, 69
1 1.80 331 Galloyl-hexoside –
MS3 [331 → 169]: 125, 79, 69
MS2 [331]: 169, 123, 151, 125, 211, 69
2 2.10 331 Galloyl-hexoside –
MS3 [331 → 169]: 125, 79, 69
4 3.01 169 MS2 [169]: 150, 151, 69, 79, 107 Gallic acidd Leavesi
MS2 [301]: 169, 125, 150, 124, 79, 69
14 5.70 301 Galloyl-pentosidee –
MS3 [301 → 169]: 125, 79, 69
MS2 [453]: 169, 313, 125, 151, 301, 253
24 7.40 453 Hydroxybenzoyl-galloylglucoside –
MS3 [453 → 313]: 169, 125, 151
39 11.25 415c MS2 [415]: 207, 169, 151, 145, 119, 177 Dimethoxycinnamoyl-hexoside –
40 11.30 415c MS2 [415]: 207, 145, 169, 177, 119, 249 Dimethoxycinnamoyl-hexoside –

Flavones
42 11.95 415 MS2 [415]: 267, 295, 325, 149, 162 Chrysin-C-hexoside –
MS3 [415 → 267]: 253, 209, 167

Ellagitannins
37 10.82 473 MS2 [473]: 265, 275, 289 See Section 3.1.3 –
38 11.14 473 MS2 [473]: 265, 267, 289, 385 See Section 3.1.3 –
55 13.75 469 MS2 [469]: 169, 125, 232, 313, 257 Valoneic acid bilactone –

Flavonols
MS2 [615]: 463, 301, 169, 195, 271
44 12.16 615 Quercetin-galloyl-hexoside Leavesi
MS3 [615 → 301]: 179, 151
MS2 [463]: 301, 271, 179, 151, 230,
46 12.30 463 Quercetin-hexoside Fleshh/leavesi
MS3 [463 → 301]: 179, 151, 121
MS2 [463]: 301, 300, 271, 255
47 12.50 463 Quercetin-hexoside Fleshh/leavesi
MS3 [463 → 301]: 179, 151, 121
MS2 [477]: 151, 301, 121, 179, 281
49 12.90 477 Quercetin-glucuronide Leavesi
MS3 [477 → 301]: 151, 179, 121
MS2 [433]: 151, 179, 245, 121, 109, 273
52 13.39 433 Quercetin-pentoside Leavesi
MS3 [433 → 301]: 179, 151, 121
MS2 [433]: 151, 179, 245, 121, 109, 273
54 13.60 433 Quercetin-pentoside Fleshh/leavesi
MS3 [433 → 301]: 179, 151, 121
MS2 [433]: 151, 179, 245, 121, 109, 273
56 13.92 433 Quercetin-pentoside Fleshh/leavesi
MS3 [433 → 301]: 179, 151, 121
MS2 [585]: 433, 301, 255, 169, 454, 295
67 16.67 585 Quercetin-galloyl-pentoside (guavinoside C) Leavesi
MS3 [585 → 301]: 179, 151
MS2 [609]: 463, 301, 345, 317, 433
68 17.95 609 Quercetin-deoxyhexoside-hexoside –
MS3 [609 → 301]: 463, 179, 151
71 18.63 301 MS2 [301]: 151, 179, 107, 121, 273 Quercetind Fleshh/leavesi

Monomeric flavanols
8 4.50 305 MS2 [305]: 125, 167, 179, 219, 164, 221 Gallocatechin Leavesi
17 6.01 305 MS2 [305]: 125, 139, 167, 219, 111, 221 Epigallocatechin Leavesi
19 6.55 289 MS2 [289]: 109, 125, 151, 137, 205, 221 Catechind Leavesi
25 7.81 289 MS2 [289]: 109, 125, 151, 137, 205, 245 Epicatechind Leavesj
30 9.50 457 MS2 [457]: 225, 125, 295, 195, 161, 305 Gallocatechin gallate –
35 10.53 457 MS2 [457]: 169, 193, 213, 125, 305, 368 Epigallocatechin gallate –
41 11.63 441 MS2 [441]: 169, 289, 165, 215, 106, 137 Catechin gallate Leavesj
53 13.47 441 MS2 [441]: 169, 125, 142, 289, 203, 119 Epicatechin gallate –

Proanthocyanidinsk
3 2.71 913 MS2 [913]: 413, 697, 863, 558, 776, 229 PAC B-Type (E)GCg-(E)GCg –
6 3.81 609 MS2 [609]: 305, 423, 177, 125, 179, 441 PAC B-Type (E)GC-(E)GCe,f Fleshh/leavesi
9 4.64 609 MS2 [609]: 305, 423, 125, 177, 179, 441 PAC B-Type (E)GC-(E)GCf Fleshh/leavesi
10 4.93 593 MS2 [593]: 289, 177, 407, 425, 125, 303 PAC B-Type (E)GC-(E)Cf Fleshh/leavesi
11 5.03 593 MS2 [593]: 289, 125, 177, 407, 245, 425 PAC B-Type (E)GC-(E)Cf Fleshh/leavesi
12 5.48 593 MS2 [593]: 305, 423, 125, 289, 179, 441 PAC B-Type (E)C-(E)GCf Leavesi
16 5.97 593 MS2 [593]: 289, 125, 305, 407, 177, 467 PAC B-Type (E)GC-(E)Cf Fleshh/leavesi
20 6.79 609 MS2 [609]: 305, 423, 125, 177, 441, 275 PAC B-Type (E)GC-(E)GCf Fleshh/leavesi
21 6.80 577 MS2 [577]: 289, 125, 407, 245, 273 PAC B-Type (E)C-(E)C Leavesi
22 7.07 897 MS2 [897]: 289, 357. 261. 395. 644. 577 PAC B-Type (E)C-(E)GC-(E)GC –
23 7.26 881 MS2 [881]: 125, 530, 547, 287, 431, 210 PAC B-Type (E)Cg-(E)Cg –
27 8.32 593 MS2 [593]: 305, 423, 289, 125, 137, 467 PAC B-Type (E)C-(E)GCf Leavesi
28 8.44 593 MS2 [593]: 125, 177, 289, 305, 407, 425 PAC B-Type (E)GC-(E)Cf Fleshh/leavesi
29 8.67 865 MS2 [865]: 413, 405, 423, 260, 557, 574 PAC B-Type (E)C-(E)C-(E)C –
34 10.28 577 MS2 [577]: 289, 125, 245, 407, 419, 287 PAC B-Type (E)C-(E)C Leavesi
50 13.10 745 MS2 [745]: 569, 255, 510, 175, 587, 195 PAC B-Type (E)Cg-(E)GC Leavesi
60 14.35 897 MS2 [897]: 670, 569, 125, 356, 529, 460 PAC B-Type (E)Cg-(E)GCg –
73 20.07 593 MS2 [593]: 300, 297, 423, 301, 178, 153 PAC B-Type (E)C-(E)GCf Leavesi

Dihydrochalcones
43 12.00 435 MS2 [435]: 315, 167, 345, 209, 273, 285 Phloretin-C-glucoside (nothofagin) –

(continued on next page)

Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
4 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx

Table 1 (continued)

Peak number Retention time m/z UHPLC–ESI−–MSn experimenta,b Tentatively identified as Previously described in guava
(min) [M–H]−

MS3 [435 → 273]: 167, 125

MS2 [435]: 273, 167, 125, 179, 301, 137
51 13.23 435 Phloretin-O-glucoside (phlorizin)d –
MS3 [435 → 273]: 167, 125

Stilbenes
57 14.04 405 MS2 [405]: 243, 225, 165, 199, 175, 159 Piceatannol-O-Glucoside (astringin) –
MS3 [405 → 243]: 175, 159

Acetophenones
48 12.68 481 MS2 [481]: 313, 169, 151, 137, 125 Myrciaphenone B Leavesi
MS3 [481 → 313]: 169, 125, 151

Benzophenones
MS2 [543]: 313, 229, 169, 211, 253
45 12.29 543 Guavinoside A Leavesi
MS3 [543 → 313]: 169, 125, 151
MS2 [693]: 371, 301, 313, 229, 275
59 14.23 693 Guavin B - isomer Leavesi
MS3 [693 → 313]: 169, 125, 151
MS2 [571]: 313, 257, 169, 271, 153, 281
61 14.64 571 Guavinoside B - isomerg Leavesi
MS3 [571 → 313]: 169, 125, 151
MS2 [571]: 313, 257, 169, 271, 241
62 15.39 571 Guavinoside B - isomerg Leavesi
MS3 [571 → 313]: 169, 125, 151
MS2 [693]: 301, 313, 275, 229
63 15.50 693 Guavin B - isomer Leavesi
MS3 [693 → 313]: 169, 125, 151
MS2 [693]: 301, 313, 275, 229, 161, 336
65 15.62 693 Guavin B - isomer Leavesi
MS3 [693 → 313]: 169, 125, 151
MS2 [571]: 257, 169, 543, 481, 137, 313
66 15.82 571 Guavinoside B isomerg Leavesi
MS3 [571 → 313]: 169, 125, 151
MS2 [557]: 243, 313, 169, 411
69 18.12 557 Glucopyranosyl-benzophenone Leavesi
MS3 [557 → 313]: 169, 125, 151

Other polar compounds

31 9.90 355c MS2 [355]: 147, 103, 165, 172, 337, 322 Cinnamoyl-hexoside –
MS2 [425]: 153, 219, 263, 111, 161, 407
33 10.24 425 Abscisic acid - hexoside –
MS3 [425 → 263]: 153, 219
58 14.10 263 MS2 [263]: 153, 219, 204, 201, 138, 163 Abscisic acidd Fleshh

Anthocyanidinsl
74 6.51 449l MS2 [449]: 287 Cyanidin-3-O-glucoside Leavesi

Unknown compounds
5 3.95 897 MS2 [897]: 125, 161, 179, 567, 729, 561 – –
7 3.96 881 MS2 [881]: 125, 341, 403, 485, 510, 551 – –
13 5.67 451 MS2 [451]: 167, 439, 305, 389, 206, 273 – –
15 5.86 913 MS2 [913]: 337, 360, 404, 318, 483 – –
18 6.21 897 MS2 [897]: 727, 534, 182, 824 – –
26 7.90 467 MS2 [467]: 301, 123, 275, 169, 305, 315 – –
32 9.98 865 MS2 [865]: 134, 391, 481, 122 – –
36 10.67 467 MS2 [467]: 301, 169, 125, 276, 315, 229 – –
64 15.52 593 MS2 [593]: 257, 313, 169, 293, 401, 233 – –
70 18.18 567 MS2 [467]: 229, 300, 433, 441, 189, 243 – –
72 19.59 643 MS2 [643]: 520, 488, 444, 611, 152, 567 – –
a
Fragments are written in order of intensity.
b
MS3 is the fragmentation of an ion-source fragment.
c
Formic acid adducts [M–H + HCOOH]−.
d
Compared with standard.
e
In peel only.
f
Just one isomer found by Flores et al. (2015).
g
Just one isomer found by Shu et al. (2010)
h
Flores et al. (2015).
i
Diaz-de-Cerio et al. (2015).
j
Chang et al. (2013).
k
PAC B-Type: proanthocyanidin with a B-type linkage.
l
Positive ionization: [M+H]+.

loss of CO2 (Rockenbach et al., 2012), and at m/z 69, resulting from the
loss of a C4H4O3 group.
Three compounds (1, 2, and 14) were found to be gallotannins. Com-
pounds 1 and 2 presented a [M–H]− ion at m/z 331 and compound 14 at
m/z 301. The presence of the in source fragment at m/z 169, as well as
fragments at m/z 151, 125, and 69 were indicative of the galloyl group.
Compounds 1 and 2 showed a main loss of 162 Da, corresponding to a
hexoside moiety. These compounds were identified as galloyl-O-
hexosides, corresponding possibly to two different sugar moieties or
different positions of the sugar moiety. In the case of compound 14,
MS2 experiment gave a loss of 132 Da corresponding to a pentose. For
these three compounds, the bond between the sugar moiety and the
gallic acid moiety was supposed to be via an ester link rather than an
ether link as no loss of CO2 was observed (Feuereisen et al., 2014).
These three compounds are reported for the first time in peel and
flesh of guava.
The identification of compound 24 was based on the [M–H]− ion at
m/z 453 as well as on the in-source fragments at m/z 313. The results in-
dicated the presence of a galloyl-hexoside moiety (Table 1). The frag-
ment at m/z 313 corresponded to the galloyl-hexoside group minus

Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx 5

ll

Fig. 1. Chemical structures of polar compounds found in peel and flesh of ripe pink guava (P. guajava L. cv. ‘Criolla’). For some structures, the exact substitution pattern is not known, i.e., the
formula shown here is an example of a possible isomer.

H2O. A loss of 122 Da corresponded to a hydroxybenzoyl moiety. This
compound was, therefore, tentatively identified as hydroxybenzoyl-
galloylglucoside.
Two compounds (39 and 40) were characterized as formic acid ad-
ducts [M–H + HCOOH]− at m/z 415. In the MS2 experiment a main frag-
ment at m/z 207 was obtained for both compounds. This can be
explained by a formula similar to a cinnamoyl moiety with two addi-
tional methoxy moieties, corresponding to the dimethoxycinnamic
moiety. The ions at m/z 177 and 145 coming from [M–H–HCOOH–
2CH3]− and [M–H–HCOOH–2CH2OH]−, respectively, confirmed the
presence of the two methoxy groups in the cinnamoyl moiety.
Based on the main loss of 162 Da both compounds were identified
as dimethoxycinnamoyl-hexosides, presumably with two different
sugar moieties or different positions of the ester bond. Compounds
39, and 40 are reported for the first time in peel and flesh of pink
guava.

Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
6 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx
3.1.2. Flavones
Another ion at m/z 415 was found (compound 42). The losses of 90
and 120 Da suggested a C-glucoside link, as a result of a cross-ring cleav-
age of the sugar moiety. The loss of 148 Da (0,1X) at the C-C link gave a
main fragment at m/z 267, corresponding to a chrysin moiety together
with a methyl moiety. Based on the fragmentation pathway obtained,
compound 42 was tentatively identified as a chrysin-C-glycoside
(Brazier-Hicks et al., 2009). To our knowledge, chrysin-C-glycoside has
been previously reported in cereals (Brazier-Hicks et al., 2009), its pres-
ence in fruits has not been reported. Nevertheless, the chrysin was pre-
viously identified in passion fruit (Brown, Hurd, McCall, & Ceremuga,
2007), and as C-glycoside, compound 42 is reported for the first time
in the genus Psidium.
3.1.3. Ellagitannins
Two compounds (37 and 38) presented a [M–H]− at m/z of 473. The
fragment ions at m/z 265 were obtained as a result of the fission of the C-
C linkage between the two dihydroxy-carboxyphenyl moieties and the
loss of CO2 ([M–H–C8H5O4–CO2]−). The loss of two CO2 moieties result-
ed in the ion at m/z 385 and was observed for compound 38 only. Based
on the molecular weight reported in the database PubChem (2016)
and the fragmentation pathway obtained, these compounds were
tentatively identified as isomers of 2-[5-carboxy-4-(2-carboxy-4,5-
dihydroxyphenyl)-2-hydroxyphenoxy]-3,4,5-trihydroxybenzoic acid,
also referred to as valoneic acid (CID: 71308296; PubChem, 2016).
Interestingly, valoneic acid bilactone is described as ellagic acid attached
to a gallic acid via an ether bond with a molecular weight of 470 g/mol
(CID: 10151874; PubChem, 2016). If this compound is the bilactone of
valoneic acid, then valoneic acid must have a molecular weight of
506 g/mol, i.e., two oxygen atoms more than the above mentioned 2-[5-
carboxy-4-(2-carboxy-4,5-dihydroxyphenyl)-2-hydroxyphenoxy]-3,4,5-
trihydroxybenzoic acid. In PubChem a second bilactone called “valoneaic
acid bilactone” (CID: 101156920) can be found with the same formu-
la as valoneic acid bilactone, suggesting that the compound with the
molecular weight of 506 g/mol should be named valoneaic acid.
Because the use of the trivial names “valoneic acid” and “valoneaic
acid” is confusing, we preferred describing compounds 37 and 38
as isomers of 2-[5-carboxy-4-(2-carboxy-4,5-dihydroxyphenyl)-2-
hydroxyphenoxy]-3,4,5-trihydroxybenzoic acid instead of using the
trivial names.
Compound 55 with [M–H]− 469 presented a main fragment at m/z
169, i.e., a galloyl moiety. The loss of 300 Da corresponds to an ellagic
acid moiety, confirmed by the presence of its typical fragment ion at
m/z 257 (Seeram, Lee, Scheuller, & Heber, 2006). Compound 55 was
therefore tentatively identified as the ellagitannin valoneic acid
bilactone (Wyrepkowski et al., 2014). These ellagitannins have previ-
ously been isolated from camu-camu (Myrciaria dubia), another plant
of the Myrtaceae family (Fracassetti et al., 2013), but for the first time
in P. guajava.
3.1.4. Flavonols
Quercetin with a [M–H]− ion at m/z 301 was confirmed for com-
pound 71 by co-elution of the standard and coinciding MS spectra;
nine quercetin derivatives were found. They showed the m/z 301 of
the aglycone as the main fragment, as well as the typical fragments of
quercetin at m/z 179 and 151 (Seeram et al., 2006). Because the main
fragment was the aglycone, all of them were categorized as O-
glycosides.
Compound 44 showed a [M–H]− ion at m/z 615. Based on its molec-
ular weight and the presence of the fragments at m/z 463, 301 and 169,
it was identified as quercetin-galloyl-hexoside. The galloyl moiety was
confirmed by the presence of fragments at m/z 79 and 69, obtained
from the in-source fragment of m/z 169 (Table 1). With the same
criteria, a quercetin-galloyl-pentoside was assigned to compound 67.
It showed a [M–H]− ion at m/z 585 and a main fragment at m/z 301.
Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
An ion with similar fragmentation pathway was previously reported
in leaves of P. guajava as guavinoside C (Diaz-de-Cerio et al., 2015).
Two peaks were detected with m/z 463 (46 and 47), showing the
same main fragment at m/z 301. Further fragments were also the
same, but had different intensities. In both compounds, a loss of
162 Da was observed, corresponding to a hexoside moiety. Based on
the results previously reported (Flores et al., 2015), these polyphenols
were identified as quercetin-3-O-galactoside (hyperin) and quercetin-
3-O-glucoside (isoquercitrin), respectively. Two quercetin-pentosides
were previously described in guava fruit, but in our study three com-
pounds (peaks 52, 54, and 56) corresponding to m/z 433 showed the
fragments at m/z 301, 151 and 179. Based on studies on polyphenols in
guava leaves (Diaz-de-Cerio et al., 2015) and on the order of elution for
quercetin glycosides previously reported (Schieber, Hilt, Conrad, Beifuss,
& Carle, 2002), these three quercetin-pentosides were tentatively
identified as quercetin-3-O-xyloside (reynoutrin), quercetin-3-O-
arabinopyranoside (guajaverin), and quercetin-3-O-arabinofuranoside
(avicularin), respectively.
A precursor ion scan allowed to assign the fragment ions with m/z
301 at 12.90 min (49) and 17.95 min (68) to the precursor ions with
m/z 477 and 609, respectively. By analysis of the fragmentation pathway
and comparison of the ions obtained with those previously reported by
Chang et al. (2013) and Diaz-de-Cerio et al. (2015a), compound 49 was
identified as quercetin glucuronide. Fragmentation of compound 68
showed the fragment ions at m/z 463, and 301, i.e., losses of 146 Da (a
deoxyhexosyl moiety), and 308 Da (a deoxyhexosyl and a hexosyl moi-
ety). The presence of fragments at 151 and 179 obtained by product ion
scan of the in-source fragment with m/z 301 confirmed quercetin as the
aglycone.
The presence of the fragment ion at m/z 463 as the main fragment
suggested that the deoxyhexosyl moiety and the hexosyl moiety are
linked to the aglycone at different positions, presumably at C3 and C7,
respectively, because the glycosidic bond at C7 has been reported to
be more stable (Bonaccorsi, Caristi, Gargiulli, & Leuzzi, 2008). Therefore,
compound 68 was tentatively identified as a 3-O-deoxyhexosyl-7-O-
hexosyl quercetin.
3.1.5. Monomeric flavanols
The monomeric flavanols such as catechin (19) and epicatechin (25)
were identified based on the retention time, order of elution, [M–H]− at
m/z 289, as well as the main fragments at m/z 109 compared to the re-
spective standards. The presence of another hydroxyl group resulted in
gallocatechin and epigallocatechin (8 and 17, respectively), giving a
precursor ion [M–H]− at m/z 305 and a main fragment at m/z 125. The
esterification of catechin or epicatechin with gallic acid explained the
presence of catechin gallate and its isomer epicatechin gallate (39 and
51, respectively) (Motilva, Serra, & Macia, 2013). These compounds pre-
sented a precursor ion at m/z 441 and a main fragment at m/z 169, cor-
responding to the loss of a galloyl moiety. Two compounds (30 and 35)
presented a molecular ion at m/z 457. They were identified as
gallocatechin gallate (30) and its isomer epigallocatechin gallate (35).
The presence of gallocatechin gallate isomers in pink guava could be ex-
plained as a result of the esterification of compound 8 and 17 (Motilva et
al., 2013). All monomeric catechin derivatives were confirmed by com-
parison of the extract of pink guava with a polyphenol extract from
green tee, which is well known to contain these flavanols (Amarowicz
& Shahidi, 1996; Zimmermann & Gleichenhagen, 2011).
From this group, compounds 8, 17, 19, 25, and 41 were previously
reported in leaves of guava (Chang et al., 2013; Diaz-de-Cerio et al.,
2015). Catechin and its derivatives are reported for the first time in
peel and flesh of pink guava.
3.1.6. Proanthocyanidins
In pink guava, 18 proanthocyanidins were detected (Table 1). Com-
pounds 21 and 34 showed a molecular ion [M–H]− at m/z 577 and a
main fragment at m/z 289, corresponding to (epi)catechin. These
 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx
compounds were, therefore, identified as dimers of (epi)catechin. The
presence of the fragment at m/z 407 in both compounds suggested a
4β-6 link between the two subunits (Papagiannopoulos, 2007).
Proanthocyanidins with a molecular ion [M–H]− at m/z 593 were
most frequent, corresponding to (epi)catechin linked to (epi)gallocate-
chin. After comparison of the fragment ions obtained with the fragmen-
tation pathway previously described by Friedrich, Eberhardt, and
Galensa (2000), compounds 10, 11, 16, and 28 were identified as iso-
mers of (epi)gallocatechin-(epi)catechin, i.e., (epi)gallocatechin is the
T-unit (top unit) and (epi)catechin the B-unit (base unit). The loss of
168 Da is caused by fragmentation of the B-ring in gallocatechin togeth-
er with a C2H3O group resulting from a Retro-Diels-Alder fragmentation
in the heterocyclic ring leading to the 1,3A− ion at m/z 425. A subse-
quent loss of H2O yielded the fragment at m/z 407. Both ions are present
when gallocatechin is the T-unit (Friedrich et al., 2000). When (epi)cat-
echin is the T-unit, the typical ions resulting from the MS2 experiment
are at m/z 423 and 405. Therefore, compounds 12, 27 and 70 were iden-
tified as isomers of (epi)catechin-(epi)gallocatechin.
Three compounds (6, 9, and 20) with a molecular ion at m/z 609 and
fragments at m/z 305, 425, 125, and 177 were detected and identified as
isomers of the dimer of (epi)gallocatechin. Based on a [M–H]− at m/z
745 reported previously by Diaz-de-Cerio et al. (2015) in leaves of
guava, compound 50 was identified as a dimer of (epi)catechin gal-
late-(epi)gallocatechin. Three more dimers were detected at m/z 881,
897, and 913 (23, 57, and 3, respectively), corresponding to (epi)cate-
chin gallate-(epi)catechin gallate, (epi)catechin gallate-(epi)gallocate-
chin gallate, and (epi)gallocatechin gallate-(epi)gallocatechin gallate,
respectively. Two trimers (22 and 29) were found in both peel and
flesh. Compound 22 showed a [M–H]− ion at m/z 897, a main fragment
at m/z 289 (a (epi)catechin moiety), and a loss of 608 Da (a (epi)gallo-
catechin-(epi)gallocatechin moiety), which differentiated this molecule
from a dimer such as compound 60. This molecule was, therefore, built
from a catechin linked to two gallocatechin units, but the T-units and B-
units could not be identified. Proanthocyanidins 29 showed a [M–H]−
ion at m/z 865, corresponding to a molecule built from three (epi)cate-
chin units. Flores et al. (2015) found just one isomer of the
proanthocyanidins (E)GC-(E)C and (E)GC-(E)GC, while seven and
three isomers, respectively, were detected in pink guava cv. ‘Criolla’.
3.1.7. Dihydrochalcones
In pink guava cv. ‘Criolla’, two compounds with dihydrochalcone
structure were found (43 and 51), but differences in the fragments sug-
gested that the glycosylation position was different. Such as flavonols,
dihydrochalcones can be present in O- or C-glycosylated form.
Both compounds presented a [M–H]− ion at m/z 435. In the case of
compound 43, a main fragment at m/z 315 was found, while compound
51 showed a fragment at m/z 273, the latter corresponding to the
deprotonated phloretin. Based on the main loss of 162 Da, ions at m/z
167 and 125, as well as co-injection of the standard, compound 51
was identified as phloretin-3-O-glucoside, also known as phlorizin.
Phlorizin has been identified previously in apples (Schieber, Keller, &
Carle, 2001), strawberries (Hilt et al., 2003), and Eucalyptus wood
(Santos et al., 2013), the latter being another genus of the family
Myrtaceae.
In the case of compound 43, typical losses of 90, 120, and 150 Da, i.e.,
ions at m/z 345, 315, and 285, suggested the C-glycoside character of the
molecule. By analysis of the fragmentation pathway, compound 43 was
identified as nothofagin a C-glycoside of phloretin which is commonly
found in rooibos tea (Kazuno, Yanagida, Shindo, & Murayama, 2005).
These two dihydrochalcones are reported for the first time in the
genus Psidium.
3.1.8. Stilbenes
One compound (57) belonging to the stilbenes was found in pink
guava for the first time. It showed a [M–H]− ion at m/z 405, which
was fragmented into ions at m/z 243, 225, 175, and 159. The loss of
Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
7
162 Da corresponded to a hexoside moiety and the other fragments in-
dicated a piceatannol as the aglycone. The analysis of the fragmentation
pathway with the fragments obtained in this study, as well as compari-
son with previous reports (Lai et al., 2013), allowed the identification of
this compound as astringin, which is typically found in grapes (Ribeiro
de Lima et al., 1999).
3.1.9. Acetophenone and benzophenone
Benzophenone has been reported in both O- and C-glycosylated
forms (Berardini, Carle, & Schieber, 2004). In the case of pink guava cv.
‘Criolla’, compounds 45, 61, 62, 66, and 69 showed an in source frag-
ment at m/z 313 which gave the fragments 169, 125, and 151. This is
the same behavior as compound 24 indicating a galloyl-hexoside moie-
ty. The neutral losses leading to the fragment 313 can be explained by
the different benzophenone or acetophenon moieties.
Compound 48 showed a [M–H]− ion at m/z 481, the main fragment
[M−H−331]− leading to the neutral loss of 150 Da, was identified
as a dihydroxy-acetophenone group. As a result, compound 48 was
characterized as dihydroxy-4-O-(6-O-galloyl-glucopyranosyl)-
phenone, also found in guava leaves and reported as myrciaphenone B
by Diaz-de-Cerio et al. (2015). Compound 45 showed a [M–H]− at m/z
543, i.e., 62 Da heavier than compound 48. The fragment ion at m/z
212 corresponded to a dihydroxybenzophenone group linked to a
galloyl moiety. This is in accordance with dihydroxy-4-O-(6-O-galloyl-
glucopyranosyl)-benzophenone, commonly known as guavinoside A
as reported by Diaz-de-Cerio et al. (2015). Compound 69 showed a
[M–H]− ion at m/z 557, 14 Da heavier than guavinoside A. This differ-
ence corresponded to a methyl group linked to the dihydroxybenzene
part of the molecule. Therefore, compound 69 was identified as 2,6-di-
hydroxy-3-methyl-4-O-(6-O-galloylglucopyranosyl)-benzophenone. A
[M–H]− ion at m/z 571 was found for the compounds 61, 62, and 66.
Again, an increment of 14 compared to the benzophenone moiety of
compound 69 suggested the presence of a methyl group in the benzo-
phenone group. These compounds were identified, therefore, as isomers
of 2,6-dihydroxy-3,5-dimethyl-4-O-(6-O-galloylglucopyranosyl)-ben-
zophenone, which is well-known in guava leaves as guavinoside B
(Diaz-de-Cerio et al., 2015).
In the case of compounds 59, 63, and 65, the fragment at m/z 313
corresponding to the galloyl-glucoside moiety was not found. After
comparison of the fragments with literature reports, as well as analysis
of the obtained fragmentation pathway, these compounds were identi-
fied as guavin B and isomers (Seeram et al., 2006). The loss of 392 Da
was the result of the fragmentation at 1,4A− of the sugar ring attached
to the benzophenone group. The ion at m/z 229 was the result of the
cleavage of the benzophenone group.
All compounds were previously reported for leaves of P. guajava
(Diaz-de-Cerio et al., 2015; Shu, Chou, & Wang, 2012), and except for
guavinoside B (Shu et al., 2010), this is the first report of these polyphe-
nols in peel and flesh of pink guava.
3.1.10. Anthocyanidins
One compound (74) with anthocyanidin character was detected
when positive ionization was performed. It gave a [M]+ ion at m/z 449
and a fragment at m/z 287. The loss of 162 Da suggested a hexoside moi-
ety. Cyanidin-3-O-glucoside was confirmed for compound 74 by co-elu-
tion of the standard. This polyphenol was previously reported in the
fruit (Flores et al., 2015) and leaves of P. guajava (Diaz-de-Cerio et al.,
2015).
3.1.11. Other polar compounds
The ion at m/z 355 of compound 31 was in accordance with the
formic acid adduct [M–H + HCOOH]− of cinnamoyl-glucoside as de-
scribed by Flores et al. (2013). The observed loss of 46 + 162 Da leading
to m/z 147 suggested the presence of a hexose moiety. The MS spectra
of compound 31 and a cinnamic acid standard had identical frag-
ments at m/z 147 and 103, suggesting the presence of cinnamic
8 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx

A.1 Peel 1.0e-1 B.1 Peel

2.0e-1 UV at 280 nm UV at 350 nm

8.0e-2 52
63
39
1.5e-1
6.0e-2

AU
AU

58 48 54
1.0e-1 4.0e-2
45 44
48 66
43
5.0e-2
19 33 43 53 2.0e-2 63 65
42 56
4 89 24 30 67 68
70 72 36 58 71
30
0.0
0.0
-0.00 5.00 10.00 15.00 20.00 -0.00 5.00 10.00 15.00 20.00
201409Guava217 (1) PDA Ch1 280nm@1.2nm 201409Guava217
Range: 1e-1

A.2 Fruit flesh 1.0e-1

B.2 Fruit flesh
2.0e-1
UV at 280 nm 8.0e-2 UV at 350 nm

1.5e-1
6.0e-2
41

AU
AU

31
58
1.0e-1 4.0e-2 39
37 43

25 51
5.0e-2 24 30 33 48 2.0e-2
11 50 54 61
4 30 36 67
0.0
0.0
Time Time
-0.00 5.00 10.00 15.00 20.00 -0.00 5.00 10.00 15.00 20.00

Fig. 2. HPLC chromatograms of polar compounds from peel and flesh of ripe pink guava (P. guajava L. cv. ‘Criolla’). A: UV chromatogram at 280 nm, B: UV chromatogram at 350 nm.

acid as the aglycone. Therefore, compound 31 was tentatively identi-
fied as cinnamoyl-glucoside. This compound was previously found in
another fruit of the same genus, that is, P. friedrichsthalianum (Flores
et al., 2013).
Based on comparison of the molecular ion [M–H]− at m/z 263, as
well as the retention time and fragments of the MS2 experiment with
those of the standard, compound 58 was identified as abscisic acid. A
precursor ion scan allowed to assign the fragment ion with m/z 263 at
10.24 min (33) to the precursor ion with m/z 425. The presence of the
[M–H]− at m/z 425, the main fragment ion at m/z 263, as well as the
ions at m/z 153 and 219 suggested that compound 33 was a derivative
of abscisic acid. The loss of 162 Da indicated the presence of a hexosyl
moiety. Therefore, compound 33 was identified as abscisic acid-O-
hexoside.
3.2. Comparison of peel and fruit flesh
Diverse groups of polyphenols constitute the polyphenol profile of
extracts from peel and flesh of pink guava. These compounds varied
from simple phenolic acids such as gallic acid to oligomers such as
proanthocyanidins and other condensed tannins. However, it was not
possible to obtain clear UV spectra due to peak overlapping and low in-
tensities. Therefore, quantification of individual compounds was not
accomplished.
Despite separation difficulties, some differences between peel and
flesh were observed (Fig. 2). Cinnamoyl-O-hexoside (compound 31)
was the main polar compound found in peel of pink guava, followed
by the benzophenones a guavin B isomer (63), and abscisic acid (58).
The main polar compound in the flesh was compound 41, catechin gal-
late, followed by cinnamoyl-O-hexoside. Based on the height of the
peaks (Fig. 2), the level of compound 31 in peel is more than twice its
level in the flesh.
Among the flavonoids, a quercetin-O-hexoside (47) was the main
compound in the peel, followed by a quercetin-O-pentoside (52) and
a dimethoxycinnamoyl-O-hexoside (39). The latter was the main
polyphenol in the flesh followed by nothofagin (43), and quercetin glu-
curonide (49). Two compounds, proanthocyanidins B-Type (E)GC-
(E)GC (6) and galloyl-O-pentoside (14), were not found in the flesh.
The lower intensity of polyphenols in the flesh could be explained by
the higher exposure of the peel to the sunlight, which generates higher
concentrations in the outer parts than in the inner parts of a fruit
(Manach, Scalbert, Morond, Rémésy, & Jiménez, 2004).
4. Conclusions
The major phenolic compounds present in ripe pink guava were
characterized using UHPLC-DAD-MS/MS. Polyphenols such as querce-
tin, catechin and their derivatives, as well as some proanthocyanidins
had previously been reported in flesh or leaves of guava, but more iso-
mers were found in our study. Phenolic compounds such as galloyl-
pentoside, galloyl-hexosides, cinnamoyl derivatives, chrysin-C-
hexoside, valoneic acid bilactone, nothofagin, phlorizin, and astringin
are reported for the first time in the genus Psidium. The presence of
phlorizin in pink guava is of special interest due to its role as a marker
for adulteration of other juices with apple juice. Besides, the presence
of valoneic acid bilactone and phlorizin in pink guava suggested the che-
motaxonomic relationship of the genus Psidium to other genera of
Myrtaceae such as Myrciaria dubia and Eucalyptus, respectively. This
work is an extensive study of the phenolic and other phytochemicals
from the fruit of P. guajava, indicating that this fruit is a source of bioac-
tive compounds. These findings contribute to a better understanding of
guava being considered as a natural medicine.
Acknowledgements
Joany González from the Farm San Vicente (Turrialba, Costa Rica) is
acknowledged due to his support donating the fruits. Also University of
Costa Rica, San José, Costa Rica, and the German Academy Exchange
Service (DAAD), Bonn, Germany, are acknowledged for conceding the
scholarship to the Ph.D. student (C.R.G).

Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
 C. Rojas-Garbanzo et al. / Food Research International xxx (2016) xxx–xxx
References
Amarowicz, R., & Shahidi, F. (1996). A rapid chromatographic method for separation of in-
dividual catechins from green tea. Food Research International, 29, 71–76.
Arima, H., & Danno, G. -i. (2002). Isolation of antimicrobial compounds from guava
(Psidium guajava L.) and their structural elucidation. Bioscience, Biotechnology and
Biochemistry, 66, 1727–1730.
Berardini, N., Carle, R., & Schieber, A. (2004). Characterization of gallotannins and benzo-
phenone derivatives from mango (Mangifera indica L. cv. ‘Tommy Atkins’) peels, pulp
and kernels by high-performance liquid chromatography/electrospray ionization
mass spectrometry. Rapid Communications in Mass Spectrometry, 18(19), 2208–2216.
Bonaccorsi, P., Caristi, C., Gargiulli, C., & Leuzzi, U. (2008). Flavonol glucosides in Allium
species: A comparative study by means of HPLC–DAD–ESI-MS–MS. Food Chemistry,
107(4), 1668–1673.
Brazier-Hicks, M., Evans, K. M., Gershater, M. C., Puschmann, H., Steel, P. G., & Edwards, R.
(2009). The C-glycosylation of flavonoids in cereals. Journal of Biological Chemistry,
284(27), 17926–17934.
Brown, E., Hurd, N. S., McCall, S., & Ceremuga, T. E. (2007). Evaluation of the anxiolytic ef-
fects of chrysin, a Passiflora incarnate extract, in the laboratory rat. AANA Journal,
75(5), 333–337.
Chang, C. -H., Hsieh, C. -L., Wang, H. -E., Peng, C. -C., Chyau, C. -C., & Peng, R. (2013).
Unique bioactive polyphenolic profile of guava (Psidium guajava) budding leaf tea
in related to plant biochemistry of budding leaves in early dawn. Journal of the
Science and Food Agriculture, 93, 944–954.
Cheng, F. -C., Shen, S. -C., & Wu, J. S. -B. (2009). Effect of guava (Psidium guajava L.) leaf
extract on glucose uptake in rat hepatocytes. Journal of Food Science, 74, H132–H138.
Chopda, C. A., & Barrett, D. M. (2001). Optimization of guava juice and powder produc-
tion. Journal of Food Processing Preservation, 25, 411–430.
Diaz-de-Cerio, E., Verardo, V., Gómez-Caravaca, A. M., Fernández-Gutiérrez, A., & Segura-
Carretero, A. (2015). HPLC-qTOF-MS platform as valuable tool for the exploratory
characterization of phenolic compounds of guava leaves at different oxidation states.
International Conference on Multidisciplinary Science, 1–8 Section A.
Eidenberger, T., Selg, M., & Krennhuber, K. (2013). Inhibition of dipeptidyl peptidase ac-
tivity by flavonol glycosides of guava (Psidium guajava L.): a key to the beneficial ef-
fects of guava in type II diabetes mellitus. Fitoterapia, 89, 74–79.
Food and Agricultural Organization of the United Nations - FAOSTAT (2016). Production
of mangoes, mangosteen and guava for the year 2014. http://faostat.fao.org/site/
567/DesktopDefault.aspx?PageID=567#ancor (Accessed 27.05.16)
Feuereisen, M., Hoppe, J., Zimmermann, B., Weber, F., Schulze-Kaysers, N., & Schieber, A.
(2014). Characterization of phenolic compounds in Brazilian pepper (Schinus
terebinthifolius Raddi) Exocarp. Journal of Agricultural and Food Chemistry, 62,
6219–6226.
Flores, G., Dastmalchi, K., Wu, S. -B., Whalen, K., Dabo, A. J., Reynertson, K. A., & Kennelly, E. J.
(2013). Phenolic-rich extract from Costa Rican guava (Psidium friedrichsthalianum)
pulp with antioxidant and anti-inflammatory activity. Potential for COPD therapy.
Food Chemistry, 141, 889–895.
Flores, G., Wu, S. -B., Negrin, A., & Kennelly, E. J. (2015). Chemical composition and anti-
oxidant activity of seven cultivars of guava (Psidium guajava L.) fruits. Food
Chemistry, 170, 327–335.
Fracassetti, D., Costa, C., Moulay, L., & Tomás-Barberán, F. A. (2013). Ellagic acid deriva-
tives, ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxi-
dant capacity of two powder products from camu-camu fruit (Myrciaria dubia).
Food Chemistry, 139, 578–588.
Friedrich, W., Eberhardt, A., & Galensa, R. (2000). Investiagtion of proanthocyanidins by
HPLC with electrospray ionization mass spectrometry. European Food Research and
Technology, 211, 56–64.
Gordon, A., Jungfer, E., da Silva, B. A., Maia, J. G., & Marx, F. (2011). Phenolic constituents
and antioxidant capacity of four underutilized fruits from the Amazon region. Journal
of Agricultural and food Chemistry, 59, 7688–7699.
Hilt, P., Schieber, A., Yildirim, C., Arnold, G., Klaiber, I., Conrad, J., Beifuss, U., & Carle, R.
(2003). Detection of phloridzin in strawberries (Fragaria x ananassa Duch.) by
HPLC–PDA–MS/MS and NMR spectroscopy. Journal of Agricultural and Food
Chemistry, 51(10), 2896–2899.
Kazuno, S., Yanagida, M., Shindo, N., & Murayama, K. (2005). Mass spectrometric identifi-
cation and quantification of glycosyl flavonoids, including dihydrochalcones with
neutral loss scan mode. Analytical Biochemistry, 347(2), 182–192.
Please cite this article as: Rojas-Garbanzo, C., et al., Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium
guajava L. cv. ‘Criolla’) by ultra-high performance liquid..., Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.12.004
9
Kek, S., Chin, N., & Yusof, Y. (2013). Direct and indirect power ultrasound assisted pre-os-
motic treatments in convective drying of guava slices. Food and Bioproducts
Processing, 91, 491–506.
Kong, K. -W., Ismail, A. R., Tan, S. -T., Nagendra Prasad, K. M., & Ismail, A. (2010). Response
surface optimisation for the extraction of phenolics and flavonoids from pink guava
puree industrial by-product. International Journal of Food Science & Technology, 45,
1739–1745.
Lai, T. N. H., Herent, M. -F., Quetin-Leclercq, J., Nguyen, T. B. T., Rogez, H., Larondelle, Y., &
André, C. M. (2013). Piceatannol, a potent bioactive stilbene, as major phenolic com-
ponent in Rhodomyrtus tomentosa. Food Chemistry, 138(2–3), 1421–1430.
Manach, C., Scalbert, A., Morond, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols: food
sources and bioavailability. American Journal of Chemical Nutrition, 79, 727–747.
Mitra, S., Irenaeus, T., Gurung, M., & Pathak, P. (2012). Taxonomy and importance of
Myrtaceae. Acta Horticulturae, 959, 23–24.
Motilva, M. -J., Serra, A., & Macia, A. (2013). Analysis of polyphenols by ultra high-perfor-
mance liquid chromatography coupled to mass spectrometry: An overview. Journal of
Chromatography A, 1292, 66–82.
Musa, K. H., Abdullah, A., & Subramaniam, V. (2015). Flavonoid profile and antioxidant ac-
tivity of ping guava. Science Asia, 15, 149–154.
Osorio, C., Forero, D. P., & Carriazo, J. G. (2011). Characterisation and performance assess-
ment of guava (Psidium guajava L.) microencapsulates obtained by spray drying. Food
Research International, 44, 1174–1181.
Open Chemistry Database – PubChem (2016). Valoneic acid. https://pubchem.ncbi.nlm.
nih.gov/compound/71308296#section=Top (Accessed 22.11.16)
Papagiannopoulos, M. (2007). Optimierte Analyse von Flavonoiden mit HPLC-MS. Disserta-
tion Bonn Germany: Universität Bonn.
Ribeiro de Lima, M., Waffo-Téguo, P., Teissedre, P. L., Pujolas, A., Vercauteren, J., Cabanis, J.
C., & Mérillon, J. M. (1999). Determination of stilbenes (trans-astringin, cis- and
trans-piceid and cis- and trans-resveratrol) in Portuguese wines. Journal of
Agricultural and Food Chemistry, 47, 2666–2670.
Rockenbach, I. I., Jungfer, E., Ritter, C., Santiago-Schübel, B., Thiele, B., Fett, R., & Galensa, R.
(2012). Characterization of flavan-3-ols in seeds of grape pomace by CE, HPLC-DAD-
MSn and LC-ESI-FTICR-MS. Food Research International, 48(2), 848–855.
Santos, S. A., Vilela, C., Freire, C. S., Neto, C. P., & Silvestre, A. J. (2013). Ultra-high perfor-
mance liquid chromatography coupled to mass spectrometry applied to the identifi-
cation of valuable phenolic compounds from Eucalyptus wood. Journal of
Chromatography B, 938, 65–74.
Schieber, A., Keller, P., & Carle, R. (2001). Determination of phenolic acids and flavonoids
of apple and pear by high-performance liquid chromatography. Journal of
Chromatography A, 910, 265–273.
Schieber, A., Hilt, P., Conrad, J., Beifuss, U., & Carle, R. (2002). Elution order of quercetin
glycosides from apple pomace extracts on a new HPLC stationary phase with hydro-
philic endcapping. Journal of Separation Science, 25, 361–365.
Seeram, N. P., Lee, R., Scheuller, H. S., & Heber, D. (2006). Identification of phenolic com-
pounds in strawberries by liquid chromatography electrospray ionization mass spec-
troscopy. Food Chemistry, 97(1), 1–11.
Shu, J., Chou, G., & Wang, Z. (2010). Two new benzophenone glycosides from the fruits of
Psidium guajava L. Fitoterapia, 81, 532–535.
Shu, J. -C., Chou, G. -X., & Wang, Z. -T. (2012). One new diphenylmethane glycoside from
the leaves of Psidium guajava L. Natural Product Research, 26(21), 1971–1975.
Thuaytong, W., & Anprung, P. (2011). Bioactive compounds and prebiotic activity in
Thailand-grown red and white guava fruit (Psidium guajava L.). Food Science and
Technology International, 17, 205–212.
Wyrepkowski, C. C., Maldonado Gomes da Costa, D. L., Sinhorin, A. P., Vilegas, W.,
Aparecido de Grandis, R., Aparecida Resende, F., ... Campaner dos Santos, L. (2014).
Characterization and quantification of the compounds of the ethanolic extract from
Caesalpinia ferrea stem bark and evaluation of their mutagenic activity. International
Journal of Molecular Sciences, 19, 16039–16057.
Zimmermann, B. F., & Gleichenhagen, M. (2011). The effect of ascorbic acid, citric acid and
low pH on the extraction of green tea: How to get most out of it. Food Chemistry,
124(4), 1543–1548.