Analysis of Phenolic Compounds in Some Medicinal Herbs by LCMS [602302]

Analysis of Phenolic Compounds in Some Medicinal Herbs by LC–MS
Alina O. Matei1, Florentina Gatea1and G. L. Radu1,2*
1Centre of Bioanalysis, National Institute for Biological Science, Bucharest, Romania, and2Faculty of Applied Chemistry and Material
Science, Politehnica University of Bucharest, Bucharest, Romania
*Author to whom correspondence should be addressed. Email: [anonimizat]
Received 8 April 2014; revised 7 November 2014
In this paper, a liquid chromatography-atmospheric pressure chemi-
cal ionization-mass spectrometry in negative mode method was de-veloped for the identification and quantitative determination of 13individual phenolics (chlorogenic acid, caffeic acid, coumaric acid,ferulic acid, ( 1)-catechin, (–)-epicatechin, rutin, quercitrin, isoquer-
citrin, fisetin, isorhamnetin, hesperidin and chrysin) from ethanolicextracts [30, 50 and 70% (w /v)] of Calendula officinalis ,Hypericum
perforatum ,Galium verum andOriganum vulgare and some commer-
cial extracts of these medicinal herbs. Correlation coefficients ( r
2)
from calibration curves for all the compounds were between0.9971 and 0.9996. Limit of detection was in the range of 0.070–0.280mg/mL and limit of quantification was from 0.233 to
0.932mg/mL. The method was partially validated and the results ob-
tained are: the intra- and interday relative standard deviation valueswere within 0.086 and 2.821% and recovery values vary from 95.84%(coumaric acid) to 103.20% (rutin).
Introduction
In the past few years, researchers have been attracted by the iden-
tification and determination of phenolic compounds in medicinalherbs due to their beneficial effects on health. Some of their dif-ferent biological activities and properties have been described,such as antioxidant, anti-inflammatory, antimicrobial and cardio-protective ( 1–3). The main sources of polyphenols are medicinal
herbs, vegetables and fruits. One of the most important propertiesof these phenolic compounds is linked to their protective roleagainst oxidative damage diseases (coronary heart disease, strokeand cancers) and implicitly against free radicals ( 4).
Numerous analytical procedures (TLC, LC–DAD, LC–FLD, LC–
MS, GC and CE) have been developed for the quantification of phe-
nolic compounds in herbs. High-performance liquid chromatogra-phy method coupled with diode array detection (HPLC–DAD) isone of the most used techniques due to high selectivity, reproduc-ibility and simplicity. There is some published data for identifica-tion and quantitative determination of polyphenols constituentsfound in herbal extracts ( 5–9). According to Bagdonaite et al .,
flavonoids found in Hypericum perforatum are rutin, hypero-
side, quercitrin, quercetin and 3,8
00biapigenin ( 10). Different
amounts of phenolic acid and flavonoids (caffeic acid, rosmarinic
acid, luteolin, apigenin, naringenin and eriodyctiol) found inOriganum vulgare extracts were quantified by Kruma et al .
(11). Chlorogenic acid, coumaric acid and ferulic acid were de-
tected in alcoholic extracts of Calendula officinalis by Khalil
et al .(12). Another technique commonly used for the analysis
of natural extracts is LC–MS which facilitates rapid and accurateconfirmation of chemical compounds and is extensively used forthe analysis of phenolic compounds in herbs ( 13–18).
The main objective of this study was to propose a simple and
efficient extraction procedure described briefly ( 19)a n dt o
develop an innovative and selective liquid chromatography-atmospheric pressure chemical io nization-mass spectrometry
method for separation of the phenolic compounds from herbs
extracts. The new method allowed the identification of four phe-nolic acids (chlorogenic acid, caffeic acid, coumaric acid and fe-rulic acid), two flavanols (catechin and epicatechin), fiveflavonols (rutin, quercetin, isoquercitrin, fisetin and isorhamne-tin), one flavanone (hesperidin) and one flavone (chrysin)(Figure 1). This method was validated in terms of linearity, limits
of detection and quantification (LOD and LOQ), precision and
recovery.
Experimental
Materials and reagents
Chlorogenic acid ( /C2095%), ( ț)-catechin ( /C2098%), caffeic acid
(/C2098%), rutin ( /C2095%), coumaric acid ( /C2098%), isoquercitrin
(/C2090%), quercetin ( /C2095%), isorhamnetin ( /C2095%) and ferulic
acid ( /C2099%) were purchased from Sigma-Aldrich (Germany).
(–)-Epicatechin ( /C2097%), hesperidin ( /C2097%), fisetin ( /C2098%)
and chrysin ( /C2097%) were purchased from Fluka (Germany).
Chromatographic ultrapure water with Milli Q system (Millipore),
methanol (ULC/ MS Biosolve), acetonitrile (ULC /MS Biosolve)
and analytical grade formic acid (Sigma-Aldrich) were used. Allthe solutions were filtrated before LC–MS analysis using SyringeDriven Filter Unit 0.2 mm (Chromafil, PTFE, Macherey-Nagel).
Preparation of standard solution and samples
To prepare the standard stock solution, accurately weighed
amounts of 1 mg of the standards were dissolved in 1 mL meth-anol (mg /mL). All solutions were diluted in mobile phase and
brought to room temperature prior to each assay.
Dried plant materials (aerial parts) of C. officinalis ,H. perfora-
tum,Galium verum andO. vulgare were obtained from the
Romanian market. Five milligrams of each plant material weremacerated with 50 mL of aqueous solutions of ethanol [30, 50and 70% (w /v)] and were kept for 1 week at 4 8Ci nt h ed a r k .
The extracts were then filtered using a 0.2 mm chromatographic
fi
lter. The filtrate was stored at 4 8C prior to each analysis. The
commercial extracts of C. officinalis ,H. perforatum and
G. verum were bought from Romanian producers found on the
food market. Commercial extracts of C. officinalis were bought
#The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.comJournal of Chromatographic Science 2015;1–8
doi:10.1093 /chromsci /bmu177 Article Journal of Chromatographic Science Advance Access published January 12, 2015

from three different manufacturers (D. Plant, Brasov County; Hof.
Plant, Bucharest and R. Santo, Bucharest). The H. perforatum ex-
tracts were supplied by D. Plant (Brasov County) and Hof. Plant
(Bucharest) and G. verum extracts were provided by D. Plant
(Brasov County) and R. Santo (Bucharest). Manufactures speci-
fied that all extracts have minimum 40% ethanol in their compo-sition. All extracts were stored at 48 C until analysis. Commercial
samples were filtered through a 0.2- mm filter and were directly
injected in the HPLC system.Equipment and procedure of analysis
Analyses were performed on Shimadzu equipment consisting of
two pumps LC20-ADsp, a contr oller SCL-10A, a photodiode-
array detector SPD-M20Avp, an auto sampler SIL-20AC and acolumn oven CTO-20AC. The system was coupled to an MSdetector Shimadzu 2010 EV equipped with an atmospheric
pressure chemical ionization (APCI) interface. UV and MS
data were acquired and processed using LC–MS Solutions oper-ating system.
Figure 1. Structures of identified phenolic compounds: 1—chlorogenic acid, 2—( ț)-catechin, 3—caffeic acid; 4—(–)-epicatechin; 5—rutin; 6—coumaric acid; 7—isoquercitrin;
8—ferulic acid; 9—hesperidin; 10—fisetin; 11—quercetin; 12—isorhamnetin; 13—chrysin.
2Matei et al.

Separations were performed with a Kromasil C18 (5 mm,
150/C22.1 mm i.d.) column. Mobile phases consisted of formic
acid in water, pH ¼4.0 (A) and formic acid in acetonitrile,
pH¼4.0 (B) with the following conditions: 0–20 min from
5 to 50% B; 10 min with 50% B; 30–50 min from 50 to 80% B;
50–60 min from 80 to 5% B. The column was equilibrated for15 min prior to each analysis. Flow rate was 0.2 mL /min and
the volume of the sample solution injected was 10 mL. Analysis
were performed at 35 8C.
The conditions used for mass spectrometry for APCI source
were: nebulizing gas (N
2) flow rate 1.5 L min21, curved desolva-
tion line (CDL) voltage, 25V; CDL temperature, 250 8C; heat
block, 250 8C, detector voltage, 1.5 kV, interface voltage 25k V .
For full scan MS analysis, the spectra were recorded in the nega-tive mode in the range of m/z50–700. Peaks were identified by
comparison of their retention times with those of standards andstandard addition. The constituents were confirmed by MS spec-tra date.
LC–MS method validation
The method was validated in terms of linearity, LODs and LOQs
intra- and interday precision and recovery.
Linearity, LOD and LOQ
Linearity was investigated by replicate analysis of calibration so-
lutions in the concentration range of interest (2.5–50 mg/mL). A
good correlation between peak area and concentration of thecompounds was obtained. LOD was established at signal-to-noiseratio (S /N) of 3. LOQ was calculated at S/ N of 10.
Precision
Intraday precision was determined by extracting and analyzing
six replicates of each of the three different concentration solu-tions of individual standards (5, 10 and 15 mg/mL). The interday
assay precision was determined by analyzing six replicates ofeach concentration solution in three different days.
Recovery
In this study, three different concentrations (2.5, 7.5 and
12.5mg/mL) of the individual standards were added to the dilut-
ed solutions of sample. The mix ture solutions were analyzed
using the developed LC–MS method and the quantity of eachcompound was calculated from the calibration curve.
Results
Assay validation
The method was linear through the concentration range 2.5–
50mg/mL with correlation coefficients ( r
2) between 0.9971–
0.9996. LOD was from 0.070 to 0.280 mg/mL and LOQ was in
the range 0.233–0.932 mg/mL (Table I). The intraday assay pre-
cision (expressed as % RSD) ranged from 0.086% for coumaric
acid to 2.098% for ( ț)-catechin. The interassay precision was
from 0.631% for rutin to 2.821% for ( ț)-catechin (Table II).Recovery values of these 13 phenolic compounds were between95.84% for coumaric acid and 103.20% for rutin (Table III).
Chromatography and mass spectrometry analysis
Two types of detection were used in order to identify the indi-
vidual compounds: DAD and mass spectrometric detection. For agood separation different mobile phases compositions (water /
formic acid: methanol /formic acid at pH ¼2.7; water /formic
Table I
Results of Analysis on Calibration Curves and Limits of Detection ( n¼3)
Compounds Linearity ( mg/mL) Correlation coefficient ( r2) LOD ( mg/mL) LOQ ( mg/mL)
1 2.5–50 0.9971 0.121 0.400
2 2.5–50 0.9994 0.070 0.233
3 2.5–50 0.9993 0.160 0.5324 2.5–50 0.9977 0.054 0.1805 2.5–50 0.9985 0.080 0.2616 2.5–50 0.9980 0.211 0.7007 2.5–50 0.9996 0.086 0.2868 2.5–50 0.9995 0.190 0.6339 2.5–50 0.9994 0.280 0.93210 2.5–50 0.9990 0.110 0.366
11 2.5–50 0.9985 0.080 0.266
12 2.5–50 0.9995 0.250 0.83213 2.5–50 0.9990 0.201 0.669
Table II
Intra- and Interday Assay Precision and Relative Standard Deviation RSD ( n¼6)
Compounds Concentrations
(mg/mL)Intraday
(mg/mL)RDS(%)Interday(mg/mL)RDS(%)
1 5 5.018 +0.010 0.199 5.174 +0.041 0.792
10 10.245 +0.056 0.546 10.165 +0.115 1.131
15 15.036 +0.125 0.831 15.224 +0.169 1.110
2 5 5.525+0.059 1.067 5.307 +0.090 1.696
10 10.103 +0.212 2.098 10.140 +0.286 2.821
15 15.357 +0.298 1.940 15.288 +0.325 2.125
3 5 5.071+0.022 0.433 5.081 +0.098 1.929
10 10.066 +0.026 0.258 10.194 +0.160 1.570
15 15.072 +0.085 0.564 15.282 +0.189 1.236
4 5 5.018+0.048 0.956 5.034 +0.110 2.185
10 10.544 +0.131 1.242 10.091 +0.151 1.496
15 15.036 +0.097 0.645 15.043 +0.157 1.043
5 5 5.072+0.042 0.828 5.242 +0.073 1.392
10 10.677 +0.125 1.171 10.267 +0.173 1.685
15 15.058 +0.050 0.332 15.204 +0.096 0.631
6
5 5.032+0.020 0.397 5.076 +0.039 0.768
10 10.307 +0.124 1.203 10.125 +0.200 1.975
15 15.082 +0.013 0.086 15.479 +0.167 1.078
7 5 5.289+0.010 0.189 5.172 +0.050 0.967
10 9.944+0.198 1.991 10.159 +0.223 2.195
15 15.182 +0.043 0.283 15.297 +0.106 0.692
8 5 5.577+0.061 1.094 5.210 +0.066 1.266
10 9.990+0.019 0.200 10.175 +0.076 0.747
15 15.103 +0.044 0.291 15.213 +0.107 0.703
9 5 5.004+0.014 0.280 5.177 +0.052 1.004
10 10.092 +0.145 1.437 10.167 +0.281 2.764
15 15.072 +0.015 0.100 15.119 +0.072 0.476
10 5 5.024+0.005 0.100 5.124 +0.033 0.644
10 10.027 +0.041 0.409 10.109 +0.108 1.068
15 15.524 +0.158 1.020 15.258 +0.199 1.304
11 5 5.561+0.054 0.842 5.452 +0.067 1.229
10 10.070 +0.085 0.844 10.218 +0.106 1.037
15 15.378 +0.187 1.216 15.508 +0.226 1.457
12 5 5.075+0.044 0.867 5.298 +0.057 1.075
10 10.012 +0.019 0.190 10.206 +0.076 0.745
15 15.180 +0.116 0.764 15.402 +0.195 1.266
13 5 5.089+0.030 0.589 5.368 +0.058 1.080
10 10.149 +0.062 0.611 10.291 +0.098 0.952
15 15.062 +0.076 0.505 15.320 +0.194 1.266
Analysis of Phenolic Compounds 3

acid: methanol /formic acid at pH ¼3.5; 0.5% formic acid /water:
formic acid /acetonitrile /water; 0.5% formic acid in water: 0.5%
formic acid in acetonitrile at pH ¼4.0) were tested and it was
found that acetonitrile: water with formic acid at pH ¼4.0 was
the most suitable eluting system.
It is very important to use mobile phase additives in LC–MS
methods to improve ionization. The addition of acidic modifiers
such as weak acids (formic acid, TFA) is very common in thesemethods. At different concentrations, some additives such as tri-fluoroacetic acid can determine ion suppression and reduce sen-sitivity in some LC–MS methods. Since the properties of thestationary phase can influence the concentration of the additive,the choice of the column is an important tool. Formic acid can
produce protons that facilitate the production of negative charge
in excess by reducing the number of protons to hydrogen gas.These charges in excess increase the local pH value and leadto deprotonation of the analytes. Adding formic acid to the mo-bile phase was the best way to improve peak shape of the analyte,accelerate the analysis time, improve the separation and enhanceMS intensity under the negative ionization mode. All chromato-grams were recorded in the range of wavelength from 270 to360 nm. The chromatogram of 30% diluted ethanolic extract ofC. officinalis and the chromatogram of 50% diluted ethanolic ex-
tract of H. perforatum are presented in Figures 2and3.T h e
chromatogram of 50% diluted ethanolic extract of G. verum is
shown in Figure 4, where all the investigated phenolic com-
pounds were successfully separated in 55 min.
In order to identify the compounds, APCI in the negative mode
was used. Total ion chromatogram (TIC) of the components fromO. vulgare 70% extract and ion chromatograms of the identified
standards are shown in Figures 5and6. Identification and quan-
tification of the compounds was performed using SIM (selectedion monitoring) detection-ion chromatogram at m/zvalues cor-
responding to the molecular weight of the identified phenolicTable III
Recovery Values of Phenolic Compounds in Herbs Extracts
Phenolic compounds Added ( mg/mL) Found ( mg/mL) Recovery (%, n¼3)
1 2.50 2.43 97.20
7.50 7.38 98.40
12.50 12.42 99.36
2 2.50 2.45 98.00
7.50 7.58 101.06
12.50 12.61 100.88
3 2.50 2.55 102.00
7.50 7.62 101.60
12.50 12.38 99.04
4 2.50 2.46 98.40
7.50 7.34 97.86
12.50 12.78 102.24
5 2.50 2.58 103.20
7.50 7.72 102.93
12.50 12.59 100.72
6 2.50 2.56 102.40
7.50 7.53 100.40
12.50 11.98 95.84
7 2.50 2.55 102.00
7.50 7.68 102.40
12.50 12.32 98.56
8 2.50 2.43 97.20
7.50 7.57 100.93
12.50 12.23 97.84
9 2.50 2.57 102.80
7.50 7.67 102.26
12.50 12.73 101.84
10 2.50 2.53 101.20
7.50 7.66 102.13
12.50 12.80 102.40
11 2.50 2.56 102.40
7.50 7.45 99.33
12.50 12.16 97.28
12 2.50 2.48 99.20
7.50 7.36 98.13
12.50 12.44 99.52
13 2.50 2.46 98.40
7.50 7.54 100.53
12.50 12.23 97.84
Recovery (%) ¼(amount found /amount added) /C2100.
Figure 2. LC–DAD chromatogram of 30% diluted ethanolic extract of C. officinalis .
4Matei et al.

Figure 3. LC–DAD chromatogram of 50% diluted ethanolic extract of H. perforatum .
Figure 4. LC–DAD chromatogram of 50% diluted ethanolic extract of G. verum .
Figure 5. TIC profile of the 70% diluted ethanolic extract of O. vulgare .
Analysis of Phenolic Compounds 5

compounds. MS conditions allowed the detection of the molec-
ular ion for each compound and produced the fragmentation innegative APCI mode (Figure 6). The unprotonated molecular
ions [M 2H]
2were detected at m/z353.31 for chlorogenic acid
(1), 289.20 for ( ț)-catechin (2), 179.16 for caffeic acid (3), 289.27
for (2)-epicatechin (4), 609.52 for rutin (5), 163.16 for coumaric
acid (6), 463.38 for isoquercitrin (7), 193.18 for ferulic acid (8),609.56 for hesperidin (9), 285.23 for fisetin (10), 301.23 for querce-tin (11), 315.26 for isorhamnetin (12) and 253.24 for chrysin (13).
Discussion
The results demonstrated that the developed method is precise,
accurate and sensitive for the quantitative determination of thephenolic compounds in herbs extracts. The method used, coversphenolic compounds which are common to the four herbsanalyzed and not for all the compounds which are found inherbs extracts. The four herbs extracts and commercial extractsstudied showed differences in their composition and content ofphenolic compounds (Table IV).
The major phenolic coumpound found in 50% ethanolic ex-
tract of C. officinalis was ( ț)-catechin (4.667 mg /gD W ) .
Previously, experiment results have shown that in methanolic ex-tracts of C. officinalis the concentration of the chlorogenic acid
(0.0625 mg /g DW), coumaric acid (0.118 mg /g DW) and ferulic
acid (0.1058 mg /g DW) are lower than in our ethanolic extracts
(7,15). In our ethanolic extracts, the amount of chlorogenic acid
ranged from 0.78 mg /g DW for 30% alcoholic extract to
3.423 mg/ g DW for 50% alcoholic extract. Other phenolics
indentified and quantified in C. officinalis extracts were: caffeic
acid, (–)-epicatechin, rutin, iso quercitrin, hespe ridin, fisetin,
quercetin, isorhamnetin and chrysin.
Figure 6. TIC profile of the standards: 1—chlorogenic acid; 2—( ț)-catechin; 3—caffeic acid; 4—(–)-epicatechin; 5—rutin; 6—coumaric acid; 7—isoquercitrin; 8—ferulic acid;
9—hesperidin; 10—fisetin; 11—quercetin; 12—isorhamnetin; 13—chrysin.
Table IV
Contents of Phenolic Compounds in Samples (mg /g DW)
Herbs Samples 1 2 3 4 5 6 7 8 9 10 11 12 13
C. officinalis 30%alc.extr. 0.78 0.498 0.322 1.811 0.226 0.113 0.522 0.329 0.395 0.102 0.049 0.086 0.007
50%alc.extr. 3.423 4.667 0.418 0.576 0.638 1.442 0.135 0.468 1.296 1.986 0.056 0.039 0.004
70%alc.extr. 1.874 2.798 0.226 6.509 1.287 1.873 0.605 0.652 1.622 1.145 0.061 0.052 0.020
H. perforatum 30%alc.extr. 1.41 0.616 0.774 0.991 0.399 0.107 0.202 0.774 1.288 0.050 0.774 0.053 0.006
50%alc.extr. 2.945 8.106 1.051 1.319 0.375 0.678 1.956 2.261 1.895 2.610 11.42 1.246 0.00570%alc.extr. 1.116 7.179 0.034 8.233 0.685 0.913 1.363 3.642 2.271 0.836 0.937 0.553 0.051
G. verum 30%alc.extr. 3.217 1.024 2.967 1.313 1.550 1.339 0.150 4.286 0.836 0.060 0.503 0.040 0.00250%alc.extr. 2.056 6.688 1.354 2.291 1.402 1.790 2.135 7.646 1.395 3.983 0.575 0.764 0.03770%alc.extr. 1.430 9.426 0.314 9.726 1.464 2.73 2.467 3.616 1.270 0.427 0.402 0.532 0.072
O. vulgare 30%alc.extr. 0.632 2.776 1.219 1.863 2.029 1.794 0.789 3.607 2.982 0.085 0.045 0.204 0.10950%alc.extr. 3.549 4.375 0.822 3.279 2.613 2.645 0.544 1.768 1.124 5.357 0.213 0.751 0.044
70%alc.extr. 0.107 7.464 0.825 7.681 3.562 1.433 3.528 0.175 3.173 6.232 0.317 0.741 0.033
A1 0.350 1.705 0.187 0.641 0.019 0.046 0.528 0.225 0.078 0.689 0.043 0.020 0.022
A2 1.735 0.628 0.183 2.204 0.016 0.033 0.543 0.367 0.628 2.329 0.05 0.053 0.029
A3 0.228 0.433 0.244 2.785 0.150 0.016 0.478 0.278 0.114 1.885 0.079 0.201 0.142
B1 1.308 1.397 0.094 1.155 0.270 0.405 1.497 0.337 1.394 1.137 0.063 0.192 0.410
B2 1.237 0.539 0.069 4.105 0.084 0.518 0.076 0.069 0.698 0.848 10.28 0.356 0.046
C1 1.462 0.996 0.327 3.948 0.362 0.637 0.405 1.525 0.441 3.016 0.301 0.052 0.038
C2 0.396 0.893 0.217 1.071 0.046 0.146 0.043 0.247 2.646 0.472 0.123 0.357 0.038
A1, A2, A3—commercial C. officinalis extracts (D. Plant; Hof. Plant; R. Santo); B1, B2—commercial H. perforatum extracts (D. Plant; Hof. Plant); C1, C2—commercial G. verum extracts (D. Plant; R. Santo).
6
Matei et al.

InH. perforatum extracts, the compounds founded in the
smallest quantities were isorhamnetin and chrysin. For these ex-
tracts, the highest content of quercetin was found in 50% etha-nolic extract (11.42 mg /g DW) while in 30 and 70% alcoholic
extracts of H. perforatum the amount of quercetin was very
low (0.774 and 0.937 mg /g DW). We also found that the amounts
of (ț)-catechin (8.106 mg /g DW) and (–)-epicatechin
(8.233 mg /g DW) were higher in 50 and 70% alcoholic extract
than in 30% alcoholic extract.
From our knowledge, these are the first results of research in
which the 30, 50 and 70% ethanolic extracts of G. verum were
characterized in order to identify and quantify this type of indi-vidual phenolic compounds using this method. Compared with
the 50 and 70% G. verum extracts, in 30% alcoholic extract
lower concentrations of fisetin (0.060 mg /g DW), isorhamnetin
(0.040 mg /g DW) and chrysin (0.002 mg /g DW) were found.
The highest concentrations of ( ț)-catechin (9.426 mg /gDW),
(–)-epicatechin (9.726 mg /gD W )a n dc o u m a r i ca c i d
(2.73 mg /g DW) were quantified in 70% ethanolic extract of
G. verum while the highest amount of ferulic acid (7.646 mg /g
DW) was found in 50% alcholic extracts of G. verum . Other com-
pounds identified in small quantities in G. verum extracts were:
quercetin and hesperidin.
Rutin, isoquercitrin, hesperidin and fisetin were the major
compounds quantified in 70% ethanolic extract of O. vulgare
while in 30% ethanolic extract the predominat phenolic com-
pound was ferulic acid (3.607 mg /g DW). The highest content
of chlorogenic acid (3.549 mg /g DW) was detected in 50%
O. vulgare alcoholic extract while the lowest amount was
found in 70% (0.107 mg /g DW).
The qualitative data obtained for the commercial extracts of
C. officinalis ,H. perforatum ,G. verum reveal that individual
compounds profile is similar to that of extracts obtained inour laboratory. The predominan t phenolic constituents for
Hypericum commercial samples were quercetin (10.28 mg /g
DW), (–)-epicatechin (4.105 mg /g DW) and isoquercitrin
(1.497 mg /gD W ) .F o r C. officinalis extracts, the majority
of compounds were chlorogenic acid (1.735 mg /gD W )a n d
(ț)-catechin (1.705 mg/ g DW). There are major differences
b
etween the commercial samples of G. verum regarding the
contents of fisetin, chlorogenic acid, ferulic acid (–)-epicatechinand hesperidin. The concentrations of (–)-epicatechin andhesperidin detected in commercial extracts of G. verum were
higher than those found in our G. verum alcoholic extracts
(Table IV).
Conclusion
In this study, a new LC–MS method to determine the 13 phe-
nolic compounds from alcoholic extracts of C. officinalis ,
H. perforatum ,G. verum ,O. vulgare and commercial extracts
found on the Romanian market was developed. The chromato-graphic method was simple, sensitive and reproducible. Ourethanolic extracts have higher concentrations of phenolic com-pounds compared with the commercial samples extracts andother samples mentioned in previous data ( 19–24). From our
knowledge, considering the developed method, this is thefirst time when data regarding the LC–MS analysis of theG. verum ethanolic extracts is reported. The quantitative data
indicate that the amount of phenolic compounds depends onethanol concentration used for samples extraction. The pro-
posed analytical method may be used successfully in the futurefor the analysis of other herbs extracts.
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