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A R T I C L E
I N F O
A B S T R A C T
Article history:
The variety and content of phenolic acids and flavonoids in a Chinese herb Rhinacanthus
detection and tandem-mass-spectrometry. A high yield of phenolic acids and flavonoids was
November 2014
ing a C18 column and gradient mobile phase of 0.1% formic acid-in-water and acetonitrile,
a total of 20 phenolic acids and 3 flavonoids were separated within 45 min with detection
at 280 nm, flow rate at 0.8 mL/min and column temperature at 35 C. Internal standards va-
Keywords:
nillic acid and quercetin were used for quantitation of phenolic acids and flavonoids in
Rhinacanthus nasutus
R. nasutus, respectively, which included caffeic acid derivatives (6.40 mg/g), quercetin de-
Chinese herb
rivatives (4.43 mg/g), ferulic acid derivatives (3.20 mg/g), p-coumaric acid derivatives (1.63 mg/
Phenolic acids
g), sinapic acid hexoside (1.11 mg/g), kaempferol-3-O-rutinoside (1.02 mg/g), hydroxycinnamic
Flavonoids
HPLC-PADMS/MS
1.
Introduction
Hl-60 and oral cancer cell HSC-2, HSC-3 and HSC-4 (Horri et al.,
2011), as well as inflammation (Siriwatanametanon et al., 2010).
Furthermore, the liposomal naphthoquinone esters isolated
from R. nasutus were efficient in retarding tumor growth in
meth-A sarcoma-bearing BALB/c mice at a dose of 5.0 mg/kg
(Siripong et al., 2006). In a later study Puttarak et al. (2010) used
a microdilution assay to determine potent bacteriostatic activity of R. nasutus extract and reported that the minimum
inhibitory concentrations toward Streptococcus mutans, Streptococcus epidermidis, propionibacterium acnes and Staphylococcus
aureus ranged from 4 to 16 g/mL. All these biological activities are believed to be closely associated with the presence of
* Corresponding author. Department of Food Science, Fu Jen University, Taipei 242, Taiwan. Tel.: +886 2 29053626; fax: +886 2 29051215.
E-mail address: 002622@mail.fju.edu.tw (B.H. Chen).
http://dx.doi.org/10.1016/j.jff.2014.12.002
1756-4646/ 2014 Elsevier Ltd. All rights reserved.
various functional components such as naphthoquinones, carotenoids, chlorophylls, flavonoids and phenolic acids (Kao &
Chen, 2011; Wu, Hsu, Wu, Teng, & Wu, 1998). However, the composition of flavonoids and phenolic acids in R. nasutus remains
uncertain.
Phenolic acids are widely present in plant material as secondary metabolites in the form of free, soluble ester and
glucosides, and insoluble bound compounds. The main types
of phenolic acids in plants include hydroxybenzoic acid derivatives such as vanillic acid and gallic acid as well as
hydroxycinnamic acid derivatives such as caffeic acid and
ferulic acid (Chen, Inbaraj, & Chen, 2012). Most importantly,
phenolic acids have been demonstrated to possess vital biological activities including anti-cancer (Spilioti et al., 2014), antibacteria (Sanchez-Maldonado, Schieber, & Ganzle, 2011) and
antioxidation (John & Shahidi, 2010).
Like phenolic acids, flavonoids being a class of polyphenol
compounds, are widely distributed in plants, especially fruits,
vegetables and flowers, among others. More than 6000 flavonoids have been characterized in nature and most are present
in glycosidic form (Chen et al., 2012). From the structural point
of view, flavonoids can be divided into flavones, flavonols, flavanones, flavonols, isoflavones, anthocyanidins, dihydroflavonols
and chalcones (Cook & Samman, 1996). Numerous reports have
shown that flavonoids exhibit important physiological functions such as antioxidation (Chandrasekara & Shahidi, 2011),
anti-cancer (Tsai, Lin, & Chen, 2010), antibacterial (Orhan,
Ozcelik, Ozgen, & Ergun, 2010) and anti-inflammation (Kao, Wu,
Hong, Wu, & Chen, 2007). In addition, the antioxidant activity of flavonoids can be dependent upon the number and
position of sugar moiety, hydroxyl and methoxy groups (John
& Shahidi, 2010).
Due to the highly-polar nature of phenolic acid and flavonoid, both are often extracted with polar solvents including
hot water, methanol, ethanol, acetone, or a combination of these
solvents in different proportions (Bae, Jayaprakasha, Jifong, &
Patil, 2012; Chen et al., 2012). However, due to the formation
of large complexes between polyphenol compounds and proteins or carbohydrates in plant matrices, many impurities such
as wax, lipid and chlorophylls have to be removed during the
extraction (Naczk & Shahidi, 2004). Many recent studies have
shown a combination of ethanol and water in an appropriate
proportion to be the most efficient solvent system for simultaneous extraction of phenolic acids and flavonoids (Bae et al.,
2012; Chen et al., 2012; Qiao et al., 2011). After extraction, both
phenolic acids and flavonoids are often subjected to separation, identification and quantitation by HPLC coupled with
photodiode-array detection (PAD) and mass spectrometry (MS),
with the C-18 column and gradient mobile phase being the most
frequently used (Chen et al., 2012; Kao, Huang, Inbaraj, & Chen,
2008; Qiao et al., 2011). As most published methods suffer a
major drawback of long separation time or inadequate resolution, it is necessary to develop an improved HPLC method
for simultaneous determination of phenolic acids and flavonoids in R. nasutus.
The objectives of this study were to develop an appropriate method for simultaneous extraction, separation,
identification and quantitation of phenolics and flavonoids from
R. nasutus extract by HPLC coupled with a photodiode-array detector and a mass spectrometer.
2.
2.1.
Materials
499
2.2.
Instrumentation
2.3.
Methods
2.3.1.
Extraction
Initially 3 solvent systems of ethanolwater in different proportions were compared with respect to extraction efficiency
of total phenolic acids and total flavonoids. In brief, 0.5 g of
R. nasutus powder were mixed with 30 mL of 30, 50 or 70%
500
2.3.2.
A method described by Kao et al. (2012) was used to determine total phenolic acids in R. nasutus samples. Briefly, 5
concentrations of 50, 100, 200, 300 and 400 g/mL of gallic acid
standard in ethanol were prepared. Then 50 L each was collected and mixed with 200 L FolinCiocalteu reagent, after
which the mixture was stirred homogeneously, followed by
standing in the dark for 5 min, adding 1 mL aqueous solution
of sodium carbonate (15%) for reaction for 1 h at room temperature, and measuring absorbance at 750 nm. The gallic acid
standard curve was thus prepared by plotting concentration
against absorbance, while both the linear regression equation and correlation coefficient were obtained. Then 50 L
sample of R. nasutus extract was collected for absorbance measurement at 750 nm for calculation of gallic acid equivalents
based on the linear regression equation.
2.3.3.
flavonoids in R. nasutus samples, after which some modifications were made so that a satisfactory separation could be
achieved. The peak purity of each phenolic acid and flavonoid on the HPLC chromatogram was automatically determined
by using an Agilent G2180A spectral evaluation software data
management system through comparison of absorption spectra
of unknown peaks with reference standards. However, for
unknown peaks without commercially available standards, the
purity was calculated based on the degree of overlapping
through determination of absorption spectra of left tip, apex
and right tip of the peak.
Identification was carried out by comparing retention times,
absorption spectra and mass spectra of various phenolic acid
and flavonoid peaks with reference standards and values reported in the literature. As mentioned in Section 2.3, two HPLC
MS systems were employed for mass spectra determination
for positive identification. One was a single quadrupole mass
spectrometer with ESI in negative mode for detection with the
scanning range of 1001000, drying gas flow 13 L/min, nebulizer pressure 60 psi, drying gas temperature 350 C, vaporizer
temperature 250 C, capillary voltage 2500 V, charging voltage
2000 V, and fragmentor voltage 200 V. The other was a ultrahigh resolution LTQ Orbitrap XL mass spectrometer (MSMS)
with ESI in negative mode for detection with the scanning range
of 1001000, heater temperature 250 C, sheath gas flow rate
30 arbitrary units, auxiliary gas flow rate 5 arbitrary units, spray
voltage 4 kV, capillary temperature 275 C, capillary voltage 35 V,
and tube lens voltage 100 V.
The internal standard, vanillic acid, was subsequently dissolved in acetonitrile/water (1:1, v/v) at a concentration of 20 g/
mL for phenolic acid quantitation, while quercetin dissolved
in methanol at the same concentration for flavonoid
quantitation. For preparation of standard curves, phenolic acid
standards including protocatechuic acid, caffeic acid, ferulic
acid and sinapic acid were dissolved in acetonitrile/water (1:1,
v/v) separately at 6 concentrations of 0.5, 1.0, 5.0, 10, 20 and
40 g/mL. Likewise, 7 concentrations of 0.1, 0.5, 1.0, 5.0, 10, 20
and 40 g/mL were prepared for p-coumaric acid. For flavonoids, both quercetin-3-O-rutinoside and kaempferol-3-Orutinoside were dissolved in methanol to obtain 6
concentrations of 0.5, 1.0, 5.0, 10, 20 and 40 g/mL. Then all the
phenolic acid and flavonoid standard solutions were mixed with
vanillic acid and quercetin, respectively, so that each solution contained internal standard at 20 g/mL. Each standard
concentration was injected into HPLC twice and all the standard curves were prepared by plotting the concentration ratios
(standard versus internal standard) against the area ratios (standard versus internal standard). The linear regression equations
and coefficients of determination (R2) were then obtained automatically for each standard curve.
501
deviation and relative standard deviation (RSD %). For interday variability determination, sample extracts were injected
into HPLC on day 1, day 2 and day 3 with 3 injections each in
the morning, afternoon and evening for calculation of
mean standard deviation and relative standard deviation.
Both limit of detection (LOD) and limit of quantitation (LOQ)
of various phenolic acids and flavonoids were determined based
on the method as described by International Conference of
Harmonization (ICH) (1996). Three concentrations of 200, 300
and 400 ng/mL were prepared for procatechuic acid; 100, 150
and 200 ng/mL for caffeic acid; 20, 50 and 100 ng/mL for
p-coumaric acid and ferulic acid; 100, 200 and 300 ng/mL for
sinapic acid; 100, 200 and 400 ng/mL for quercetin-3-Orutinoside; 50, 100 and 200 ng/mL for kaempferol-3-O-rutinoside.
Each standard concentration was injected into HPLC three times
and the standard curves were prepared by plotting concentration against peak height. Then the linear regression equations
were obtained for calculation of slope (s) and standard deviation () for each standard curve. Both LOD and LOQ of phenolic
acid and flavonoid standards were calculated using the following formula: LOD = 3.3 /s and LOQ = 10 /s.
For recovery study, two concentrations of various phenolic acid and flavonoid standards were added to 0.5 g of R. nasutus
sample extracts: protocatechuic acid (400 and 1000 g), caffeic
acid (500 and 1000 g), p-coumaric acid and ferulic acid (800
and 2000 g each), sinapic acid (500 and 1500 g), quercetin3-O-rutinoside (1500 and 3000 g), kaempferol-3-O-glucoside
(1000 and 2500 g). After extraction and HPLC analysis, the recovery of various phenolic acids and flavonoids were obtained
based on the relative ratio of the amount of each standard after
HPLC to that before HPLC (original amount).
A fixed amount of internal standards vanillic acid and quercetin was added to R. nasutus sample separately for extraction
and HPLC analysis for quantitation of phenolic acid and flavonoid respectively. By using the linear regression equation
derived from each standard curve as shown earlier, the amounts
of various phenolic acids and flavonoids were determined based
on a formula reported by Chen et al. (2012).
2.3.6.
Statistical analysis
3.
3.1.
Three solvent systems containing ethanol and water in different proportions as mentioned in Section 2.3 were used for
evaluation of extraction efficiency based on total phenolic acids
and total flavonoids expressed as gallic acid and quercetin
equivalents, respectively, which are shown in Table 1. A solvent
system of 30% ethanol in water was found to obtain the highest
yield (2.16 mg/g) of total phenolic acids, followed by 50% ethanol
in water (1.98 mg/g) and 70% ethanol in water (1.94 mg/g).
However, there was no significant difference (P > 0.05) between
Total phenolicsc
Total flavonoidsd
30%
50%
70%
2.16 0.04A
1.98 0.05B
1.94 0.02B
4.93 0.18A
4.58 0.19B
3.81 0.09C
a
b
c
d
50% ethanol in water and 70% ethanol in water. The same trend
was observed for total flavonoids, with 30% ethanol in water
generating the highest yield (4.93 mg/g), followed by 50% ethanol
in water (4.58 mg/g) and 70% ethanol in water (3.81 mg/g). Apparently these outcomes demonstrated that a solvent system
of 30% ethanol in water should be the most appropriate for
simultaneous extraction of phenolic acids and flavonoids in
R. nasutus.
3.2.
In many published reports the mobile phases used for separation of phenolic acids and flavonoids by HPLC often include
a combination of water and methanol or acetonitrile with acidic
modifiers such as formic acid, acetic acid or phosphoric acid
to retard ionization, reduce interaction between polyphenol
compounds and column stationary phase, and improve peak
tailing as well as broadening (Chen et al., 2012; Inbaraj, Lu, Kao,
& Chen, 2010; Luo et al., 2011). In addition, Wang and Huang
(2004) pointed out that with tetrahydrofuran (THF) as modifier the selectivity of mobile phase toward phenolic compounds
could be enhanced. Nevertheless, THF being an aprotic solvent
would be unable to provide proton for ionization of target compounds during MS analysis of phenolic acids and flavonoids,
which could induce polymerization with APCI mode to contaminate corona needle and thus lower sensitivity. Therefore
in this study we choose a combination of water and methanol or acetonitrile as mobile phase with 0.1% formic acid as
modifier for evaluation of separation efficiency of phenolic acids
and flavonoids.
Prior to mobile phase evaluation, three C18 columns as mentioned in Section 2.3 were compared for separation efficiency
by employing a gradient mobile phase developed by Chen et al.
(2012); 92% A (0.1% formic acid in water) and 8% B (acetonitrile) initially, maintained for 10 min, increased to 14% B in
24 min, 23% B in 35 min, 24% B in 44 min, maintained for 12 min,
32% B in 60 min, 37% B in 66 min and returned to 8% B in
68 min. Of the 3 columns, the Phenomenex Gemini C18 column
showed a better resolution of phenolic acid and flavonoid peaks
than the other two columns, and thus was selected for mobile
phase evaluation. After numerous studies, we found that acetonitrile was superior to methanol in resolving phenolic acid
and flavonoid peaks for R. nasutus samples as the separation
number of both increased substantially. However, the resolution of both phenolic acids and flavonoids has still to be
502
Fig. 1 HPLC-DAD chromatogram of phenolic acids and flavonoids extracted from R. nasutus. Column, Gemini C18; mobile
phase, 0.1% formic acid in water (A) and ACN (B); flow rate, 0.8 mL/min; detection wavelength, 280 nm. Peak identification:
peak 1, hydroxycinnamic acid derivative (1); peak 2, hydroxycinnamic acid derivative (2); peak 3, hydroxyferulic acid
hexoside (1); peak 4, protocatechuic acid hexoside; peak 5, 5-hydroxydihydroferulic acid derivative; peak 6, caffeic acid
hexoside (1); peak 7, dihydrocaffeic acid hexoside; peak 8, sinapic acid hexoside (1); peak 9, hydroxyferulic acid hexoside
(2); peak 10, caffeic acid hexoside pentoside; peak 11, p-coumaric acid hexoside (1); peak 12, caffeic acid hexoside (2); peak
13, hydroxyferulic acid deoxyhexoside; peak 14, p-coumaric acid hexoside (2); peak 15, sinapic acid hexoside (2); peak 16,
caffeic acid hexoside pentoside (2); peak 17, dihydro- p-coumaric acid hexoside; peak 18, p-coumaric acid hexoside (3);
peak 19, p-coumaric acid glucuronide; peak 20, quercetin-3-O-rutinoside; peak 21, kaempferol-3-O-rutinoside; peak 22,
dihydrocaffeic acid hexoside pentoside; peak 23, quercetin pentoside.
addition, the methods with adequate resolution and reasonable separation time often lack simultaneous separation. For
instance, Thabti, Elfalleh, Hannachi, Ferchichi, and Campos
(2012) resolved a total of 12 phenolic acids and flavonoids in
Tunisian Morus species within 45 min, while Materska (2014)
separated only 8 compounds in Capsicum annuum with similar
retention time. Some other studies have also employed two
different HPLC mobile phase systems for separation of phenolic acids and flavonoids. For example, Gutierrez-Uribe,
Romo-Lopez, and Serna-Saldivar (2011) separated 5 phenolic
acids and 3 flavonoids using two different mobile phase systems
within 23 and 32 min, respectively, but the separation was unsatisfactory. Similarly, Andarwulan et al. (2012) separated 5
flavonoids and 3 phenolic acids in 24 medicinal vegetables
from Indonesia within 20 and 10 min, respectively, but the resolution remained insufficient. In addition to mobile phase,
different detection wavelengths are also selected for detection of phenolic acids and flavonoids for maximum quantitation
accuracy. In one study Khanam, Oba, Yanase, and Murakami
(2012) separated 6 hydroxybenzoic acid-type and 7
hydroxycinnamic acid-type phenolic acids from 8 leafy vegetables within 70 min with detection at 254 and 280 nm, while
3 flavonoids were detected at 360 nm. Likewise, two wavelengths of 280 and 360 nm were used for detection of 4 phenolic
acids and 5 flavonoids from Phoenix dactylifera, respectively, with
the separation time being within 65 min (Benmeddour,
Mehinagic, Meurlay, & Louaileche, 2013). Nevertheless, the separation efficiency has to be improved substantially. In our study
503
Table 2 Retention time (tR), retention factor (k), separation factor (), peak purity and content of phenolic acids and
flavonoids extracted from R. nasutus.
Peak
no.
Compound
Retention
time (tR, min)
Retention
factor (k)
Separation
factor ()
Peak
purity (%)
Content
(mg/g)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
9.83
10.60
12.57
14.97
16.46
16.99
18.72
21.12
22.09
25.40
26.01
27.25
28.42
29.35
30.09
30.95
32.23
33.23
35.67
38.97
40.66
42.30
44.21
1.46
1.66
2.16
2.76
3.13
3.26
3.70
4.30
4.55
5.38
5.53
5.84
6.14
6.37
6.56
6.77
7.09
7.34
7.96
8.79
9.21
9.62
10.10
1.13 (1,2)a
1.13 (1,2)a
1.30 (2,3)a
1.28 (3,4)a
1.13 (4,5)a
1.01 (5,6)a
1.13 (6,7)a
1.16 (7,8)a
1.06 (8,9)a
1.18 (9,10)a
1.03 (10,11)a
1.06 (11,12)a
1.05 (12,13)a
1.04 (13,14)a
1.03 (14,15)a
1.03 (15,16)a
1.05 (16,17)a
1.04 (17,18)a
1.06 (18,19)a
1.10 (19,20)a
1.04 (20.21)a
1.04 (21,22)a
1.05 (22,23)a
99.8
99.3
99.4
99.7
99.6
97.1
88.7
96.9
99.6
92.2
98.2
99.6
99.7
99.8
97.0
99.8
84.4
98.7
93.2
99.4
99.7
99.9
97.8
0.403 0.007
0.038 0.002
1.032 0.017
0.389 0.009
0.155 0.002
0.693 0.006
0.287 0.002
0.429 0.012
1.686 0.013
0.115 0.003
0.123 0.009
0.276 0.009
0.323 0.005
0.133 0.006
0.680 0.012
0.096 0.006
0.065 0.005
1.069 0.013
0.244 0.006
1.612 0.113
1.017 0.081
4.933 0.068
2.813 0.049
a
b
a more comprehensive HPLC method was developed for simultaneous separation of 23 phenolic acids and flavonoids
within 45 min and detection at 280 nm. Also, two different internal standards were employed for quantitation of phenolic
acids and flavonoids separately.
3.3.
Table 3 shows the absorption and mass spectral data for phenolic acids and flavonoids in R. nasutus. Based on the major
absorption peak obtained at 310328 nm, peaks 119 and 22
were tentatively identified as phenolic acids, while peaks 20,
21 and 23 as flavonoids based on a relatively higher absorption maximum ranging from 344 to 356 nm (Chen et al., 2012;
Inbaraj et al., 2010). However, further identification of phenolic acids and flavonoids was based on the [M-H] value and the
corresponding fragment ions obtained by tandem MS/MS. Peaks
1, 2, 3, 5, 9 and 13 were tentatively identified as derivatives of
ferulic acid based on the [M-H] value of 389, 389, 371, 255, 371,
355, respectively, all of which yielding the same fragment ions
at m/z 209211 and 191193 with the latter being consistent
with the MW of ferulic acid. Furthermore, both peaks 3 and 9
were identified as hydroxyferulic acid hexoside as the fragment ions are formed due to the elimination of hexose and
water molecule, while peak 13 was identified as hydroxyferulic
acid deoxyhexoside because of the fragment ions formed after
removal of deoxyhexose and water molecule (Simirgiotis,
Caligari, & Schmeda-Hirschmann, 2009). Based on the spectral data reported by Narvaez-Cuenca, Vincken, and Gruppen
(2012), peak 5 was identified as 5-OH-dihydroferulic acid owing
to the formation of fragment ions at m/z 211 and 193 after the
removal of carbon dioxide and water molecule. Though the
same fragment ions were obtained for peaks 1 and 2, they were
categorized as hydroxycinnamic acid derivative because of their
relatively higher [M-H] value at 389. Peaks 6, 7, 10, 12, 16 and
22 were tentatively identified as derivatives of caffeic acid based
on the [M-H] values of 341, 343, 473, 341, 473 and 475, all of
which yielding the same fragments ions at m/z 179181 and
135137 with the former corresponding to the MW of caffeic
acid and the latter resulting from the loss of carbon dioxide.
More specifically, peaks 6, 7 and 12 were identified as caffeic
acid hexoside (1), dihydrocaffeic acid hexoside and caffeic acid
hexoside (2), respectively, based on the fragment ion at m/z
179181 obtained due to loss of hexose (Chen et al., 2012). Likewise, peaks 10, 16 and 22 were identified as caffeic acid hexoside
pentoside (1), caffeic acid hexoside pentoside (2) and
dihydrocaffeic acid hexoside pentoside, respectively, because
of the formation of a fragment ion at m/z 341343 after the
removal of hexose and pentose. Peaks 11, 14, 17, 18 and 19 were
tentatively identified as derivatives of p-coumaric acid based
on the same molecular ion peak ranging from 325 to 339 and
fragments ions at m/z 163165 and 119121, with the former
obtained after the removal of hexose conforming with the MW
of p-coumaric acid and the latter resulting from the loss of
carbon dioxide (Gavrilova, Kajdzanoska, Gjamovski, & Stefova,
2011). Based on these fragmentation patterns, peaks 11, 14 and
18 were identified as p-coumaric acid hexoside, while the peak
17 as dihydro-p-coumaric acid hexoside. However, peak 19 was
identified as p-coumaric acid glucuronide as the fragment ion
at m/z 163 was formed after the removal of a glucuronide
504
Table 3 Ultraviolet and mass spectral data for tentative identification of phenolic acids and flavonoids in R. nasutus.
Peak
no.
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
a
b
c
d
e
f
g
h
[M-H]
max (nm)
Online
Reported
Online
Reported
Online
Reported
242, 316
226, 294sh, 318
234, 293sh, 324
242, 316e
234, 293sh, 322
242, 316e
356g
389
389
371
315
255
341
343
385
371
473
325
341
355
325
385
473
327
325
339
609
593
475
433
b
b
371c
315d
b
341d
b
385c
371c
b
325e
341d
355c
325e
385c
b
b
325e
b
609e
593f
b
433e
b
b
209, 191c
153d
b
179, 135d
b
223, 205, 191c
209, 191c
b
163, 119e
179, 135d
209, 191c
163, 119
223, 205, 191c
b
b
163, 119e
b
301e
285f
b
301
3.4.
Method validation
505
Table 4 Quality control data of phenolic acids and flavonoids extracted from R. nasutus.
Intra-day variabilitya
Peak
no.
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Inter-day variabilitya
Mean SD (mg/g)
RSD (%)
Mean SD (mg/g)
RSD (%)
0.408 0.027
0.037 0.002
1.022 0.017
0.393 0.009
0.159 0.006
0.693 0.006
0.297 0.012
0.424 0.020
1.676 0.013
0.105 0.007
0.117 0.005
0.276 0.014
0.301 0.024
0.123 0.006
0.700 0.037
0.099 0.006
0.065 0.005
1.080 0.015
0.234 0.008
1.633 0.143
1.057 0.081
4.994 0.063
2.883 0.061
6.6
6.5
1.7
2.3
4.0
0.8
4.1
4.8
0.8
7.0
4.1
5.1
7.8
5.0
5.3
6.0
7.5
1.4
3.2
8.7
7.7
1.3
2.1
0.385 0.003
0.034 0.002
1.002 0.005
0.408 0.003
0.160 0.003
0.702 0.024
0.321 0.019
0.416 0.016
1.663 0.011
0.109 0.005
0.117 0.003
0.273 0.002
0.283 0.030
0.152 0.011
0.711 0.003
0.101 0.003
0.058 0.003
1.151 0.029
0.264 0.016
1.683 0.095
1.273 0.040
5.092 0.015
3.155 0.106
0.7
6.3
0.5
0.7
2.0
3.4
5.8
3.8
0.6
4.8
2.4
0.6
10.6
7.2
0.4
2.6
5.2
2.5
6.0
5.6
3.2
0.3
3.4
Compound
Caffeic acid
Protocatechuic acid
p-Coumaric acid
Ferulic acid
Sinapic acid
Quercetin-3-O-rutinoside
Kaempferol-3-O-rutinoside
a
b
c
Original (g)
c
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1486
1486
1011
1011
Spiked (g)
Found (g)
Recovery (%)a
Mean SD (%)
RSD (%)b
500
1000
500
1000
800
2000
800
2000
500
1500
1500
3000
1000
2500
464
967
452
935
734
1721
689
1653
399
1309
2792
4188
1942
3328
92.8
96.7
90.6
93.5
91.8
86.1
86.1
82.7
79.8
87.3
87.1
90.0
93.1
92.7
94.8 2.8
3.0
92.1 2.1
2.3
89.0 4.0
4.5
84.4 2.4
2.8
83.6 5.3
6.3
88.6 2.1
2.4
92.9 0.3
0.3
506
3.5.
4.
Conclusion
507
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