Journal of Chromatographic Science, 2017, 18

doi: 10.1093/chromsci/bmx040
Article

Article

Optimized and Validated HPLC Analysis of

St. Johns Wort Extract and Final Products by
Simultaneous Determination of Major
Ingredients
Konstantina Zeliou1,, Nikos I. Kontaxis1,, Evangelia Margianni1,
Christos Petrou2, and Fotini N. Lamari1,*
1
Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, University of Patras,
26504 Patras, Greece and 2Department of Life and Health Sciences, Pharmacy Program, University of Nicosia, 46
Makedonitissas Ave., Nicosia, CY-1700, Cyprus

Author to whom correspondence should be addressed. Email: am@upatras.gr

*

These authors contributed equally.

Received 9 June 2016; Revised 24 January 2017; Editorial Decision 12 April 2017

Abstract
Aim of this work was to develop a validated high performance liquid chromatography method for
the analysis of extracts and nal products of St. Johns wort, according to international guidelines
for bioanalytical method validation. Chromatographic separation was performed on a C18 column
with a combination of gradient and isocratic steps; the mobile phase composed of ammonium
acetate solution (pH 4.5; 10 mM), acetonitrile and methanol. Quantication and method validation
was performed using extract spiked with external reference standards of chlorogenic acid, rutin,
hyperoside, isoquercitrin, quercetin and hypericin. Validation study revealed that trans-
chlorogenic acid is partially transformed into its cis-isomer during analysis. The method showed
good linearity, precision and accuracy. Hyperforin was completely unstable. All other ingredients
were stable at 18C and after three freezethaw cycles, while stability of most ingredients was
limited at room temperature and 4 8C; quercetin was the most unstable one. The major ingredi-
ents of methanolic extracts, infusions and nal products of Hypericum perforatum were
completely resolved and quantied. Beyond its potential usefulness in the analysis of St. Johns
wort products, this study addresses the issue of validation from the perspective of the eld of
bioanalysis and reveals the wealth of critical information which can be derived.

Introduction including naphthodianthrones (mainly hypericin and pseudohypericin),

Hypericum perforatum L., commonly known as St. Johns wort, is a
herbaceous perennial plant worldwide distributed and is repeatedly
listed as one of top 10 best selling medicinal plants. Herbal teas, liquid
(ethanol or oil) extracts and dry extracts of owering aerial parts of H.
perforatum L (Hyperici herba) have traditional or well established use
for as nervous system disorders, skin problems, gastrointestinal disor-
ders as well as against infections and infestations (1, 2). St. Johns
Wort extracts contain several active compounds of different classes,
avonols (mainly quercetin and their glycosides), biavonoids (I3,II8
biapigenin and amentoavone), phloroglucinols (hyperforin), and other
phenolic acids and their esters (chlorogenic acid, pcoumaric acid) (3).
Standardization and strict quality control of herbal drug or
extracts is necessary in order to ascertain effectiveness and consumer
safety. A number of high performance liquid chromatography
(HPLC) methods for analysis of H. perforatum extracts have been
published employing mainly UVvis detection (420) or mass

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2 Zeliou et al.
spectrometry (MS) detection (12, 2126). MS offers unequivocal char-
acterization capabilities but is not so common for routine quality con-
trol due to its high cost. In European Pharmacopeia, one HPLC-UV
method is suggested for the determination of naphthodianthrones and
another for avonoids and phloroglucinols. The determination of all
the major ingredients in one chromatographic run is described in a
fraction of these studies; (4, 6, 8, 11, 1820, 24, 26) in the great major-
ity of these studies a C18 column is used.
Over the years, the serious ethical and legal implications of analy-
tical testing have made imperative the need for quality, consistency, reli-
ability and comparability of analytical measurements; thus standards
for laboratory accreditation (e.g., ISO17025) were developed and stric-
ter regulatory requirements for analytical measurements came into
force. However, the situation nowadays in different sectors is far from
harmonized; even the terminology differs. This issue is under careful
consideration in several international unions, e.g., International Union
of Pure and Applied Chemistry, Eurachem, International Council for
Harmonization of Technical Requirements for Pharmaceuticals for
Human Use (ICH) and guides for proper validation are constantly
issued (27, 28). In Europe, the European Medicines Agency (EMA) also
issued guidelines in 2011 (in line with the ICH Q2R1 ones) on bioana-
lytical method validation; those guidelines are stricter than those of the
European Pharmacopoeia for herbal medicines.
Only few of those previous published reports on the simulta-
neous determination of St. Johns wort major components have pre-
sented some aspects of validation (usually linearity and repeatability
using external standards) (4, 6, 8, 19, 20). To the best of our know-
ledge, precision and accuracy have been studied only by Li and
Fitzloff (8) with external standards at three concentration levels,
whereas recovery from fortied plant material was evaluated by
Pellati et al. (20). Stability has been examined in both studies only
within 72 h. Chlorogenic acid was not determined in the Li and
Fitzloff method (8), whereas hyperoside and isoquercitrin were not
resolved in the Pellati et al. (20) study.
Aim of this project was the development and modern valida-
tion of an HPLC method for the analysis of H. perforatum extracts,
and the accurate determination of the main phenolic acids, avo-
noid, naphthodianthrone and phloroglucinol ingredients in different
St. Jonhs wort extracts and products, such as infusions, alcoholic
extracts, tablets and tinctures. To this purpose, we followed the
EMA guidelines for bioanalytical method validation.
Experimental
Chemicals, solvents and standards
Chromatographic grade ultrapure water (18 MOhm*cm) was produced
by a MilliQ system (Merck Millipore, USA). HPLC grade methanol
and acetonitrile were purchased from ChemLab NV (Zedelgem,
Belgium). Analytical grade ammonium acetate, hydrochloric acid 37%
and pyridine were purchased from Merck (Darmstadt, Germany).
Reference HPLC standards of chlorogenic acid (3-O-caffeoylquinic
acid, 99%), quercetin (99%), rutin (quercetin-3-O-rutinoside,
99%), hyperoside (quercetin-3-O-galactoside, 98%), isoquercitrin
(quercetin-3-O-glycoside, 99%) and hypericin (95%) were pur-
chased from Extrasynthese (GenayCedex, France).
Plant material
Aerial parts of owered H. perforatum subsp. veronense (Scrank) A.
Frhl (Family: Hypericaceae) were collected in the greater area of
Patras, Greece, in June 2013 and identied by Prof. Gregorios
Iatrou, Univeristy of Patras. A voucher specimen (1308) is deposited
at the Botanical Museum of the University of Patras (UPA), Greece.
The sample was dried at room temperature and preserved in dark
conditions, until used. Methanol extracts were prepared with macer-
ation from 5 g of dried material of H. perforatum (100 mL for 24 h
twice, under magnetic stirring in dark), and concentrated to dryness
under vacuum at 30C. Infusion was prepared through immersing
2 g of dried material of H. perforatum in deionized water at
95100C for 5 min under stirring, ltered through a uted lter
and lyophilized (Freezone 6, Labconco, MO, USA). The total yield
was 32% (w/w) and 20% (w/w) for methanol and aqueous extrac-
tion, respectively. Tablets containing dry extract (ethanol 68%, v/v)
were purchased from a local pharmacy. A tincture (artisanal; etha-
nol 60%) containing St. Johns wort (H. perforatum L.) and chamo-
mile (Matricaria chamomilla L.) was provided by a local producer.
Dry extracts and tincture were dissolved in appropriate volume of
ammonium acetate (pH 4.5; 10 mM)-acetonitrile 85:15:0, v/v and
ltered through 0.2 m pore size membrane lters (Phenex syringe
lters RC 0.2 m, Phenomenex USA). Samples were kept in 20C.
HPLC analysis
The HPLC systems consisted of a Dionex (Thermo Scientic, USA)
model Ultimate 3000 quaternary pump (Pump LPG-3400 A) equipped
with thermostated column compartment (Column Compartment TCC-
3100) and a model Ultimate DAD3000 Photodiode Array Detector
(HPLC-DAD). A 20 L Rheodyne 8125 injector (Rheodyne, Rohnert
Park, CA, USA) was connected with the pump. Data were collected,
stored and integrated with Chromeleon v 6.80 Systems software.
Absorbance was measured at 270 nm (for phloroglucinols), 300 nm (for
phenolic acids), 350 nm (for avonoids) and 590 nm (for naphthodian-
thrones). Analysis was carried out at 30C on a Phenomenex Luna C18
column (250 4.6 mm, i.d.: 5 m; LC Column Phenomenex, USA),
with a coupled guard column cartridge C18 (4 3.0 mm, i.d.: 5 m; LC
Column Phenomenex, USA), using ammonium acetate (pH 4.5;
10 mM)-acetonitrile-methanol in a combination of gradient and iso-
cratic steps. In detail, initial elution was performed with 85:15:0, v/v/v
for 10 min. Then, the organic solvent content was raised in four steps:
(i) up to 70:20:10, v/v/v, in 15 min, (ii) up to 10:75:15, v/v/v, in 10 min,
(iii) up to 5:80:15, v/v/v, in 15 min and to (iv) to 0:90:10 in 1 min. The
mobile phase was kept constant at 0:90:10, v/v/v for 4 min, returned to
the initial 85:15:0, v/v/v in 3 min, and kept constant for 5 min. The ow
rate of the mobile phase was 1 mL/min and the injected volume was
20 L. All the HPLC solvents were ltered carefully through 0.45 m
pore lters (RC & CA type, Macherey-Nagel, Germany) and degassed
in an ultrasonic processor (Ultrasonic cleaner, Banson 2200, USA).
Electrospray Ionization ((ESI)MS, Micromass-Platform LC instrument,
Waters Micromass Technologies, Milford-Massachusets, USA) of iso-
lated pure peaks was in agreement with the expected mass.
Standard solutions and calibration curves
A stock solution of each standard compound was prepared by weigh-
ing an amount of high purity dry standard and by dissolving it in pure
methanol. Concentration of these solutions varied from 1 to 4 mg/mL.
Difculty was encountered in dissolving hypericin, thus hypericin was
dissolved in a solution of ethanol-pyridine (80:20, v/v) at the concen-
tration of 1 mg/mL (29). The solutions were kept at 20C.
Calibration, quality control and stability samples were prepared
by spiking the H. perforatum sample extract (0.5 mg/mL) with stan-
dards. External standard addition calibration curves were estab-
lished on 610 data points (n = 3 on each data point). Calibration
Optimized and Validated HPLC Analysis of St. Johns Wort Extract
standards were spiked with chlorogenic acid at concentrations of
2.0, 5.0, 10.0, 30.0, 50.0 and 75.0 g/mL, rutin at 2.0, 3.0, 4.0 ,
10.0, 20.0, 40.0, 100.0, 150.0 and 200.0 g/mL, hyperoside at 2.0,
10.0, 20.0, 40.0, 100.0, 150.0 and 200.0 g/mL, isoquercitrin at
2.0, 3.0, 4.0, 5.0, 8.0, 20.0, 40.0, 100.0, 150.0 and 200.0 g/mL,
quercetin at 7.2, 9.6, 40.0, 100.0, 150.0 and 200.0 g/mL and hy-
pericin at 3.5, 4.0, 6.0, 10.0, 20.0, 25.0, 50.0 and 80.0 g/mL. Each
calibration curve was obtained by plotting the nal peak area
(derived by subtracting the peak area in blank sample from that in
the spiked sample) versus concentration.
Repeatability and accuracy estimation
Quality control samples were prepared in H. perforatum sample
solution (dry methanolic extract 0.5 mg/mL in dissolving solution)
at four concentration levels [Lower Limit of Quantitation (LLOQ),
low, medium, high]. Chlorogenic acid was spiked at concentrations
of 3.0, 9.1, 20.0 and 45.0 g/mL, rutin at 2.0, 6.0, 80.0 and
120.0 g/mL, hyperoside at 3.0, 9.1, 80.0 and 120.0 g/mL, isoquer-
citrin at 2.0, 6.0, 80.0 and 120.0 g/mL, quercetin at 7.2, 24.0, 60.0
and 90.0 g/mL and hypericin at 3.5, 10.0, 20.0 and 30.0 g/mL.
The intra-day repeatability and accuracy were examined on four
individual samples in 1 day, and inter-day parameters are deter-
mined on 2 different days. The relative standard deviation (RSD)
was calculated as a measurement of method repeatability. The %
recovery, [(measured concentration/spiked (theoretical) concentra-
tion)*100] was calculated as a measurement of method accuracy.
Stability
Compound stability was studied with Low- and High-quality chlorogenic acid, we present the results as the sum of both peaks,
control samples at different conditions and time intervals. Finally,
the stability of a 0.5 mg/mL solution of dry H. perforatum in dissol-
ving buffer was also studied. The stability was expressed as %rela-
tive error (RE) [((Cn C0)/ C0)*100 %, n is number of reference
day] (n = 3 at each concentration level).
Results
Reversed-phase conditions, the mobile phase of phosphoric acid
(0.3%)-acetonitrile-methanol and the elution system earlier suggested by
Brolis et al. (4) provided good separation, but it was difcult to dissolve
the dry extract in the initial elution system and this resulted to failing
elution of some components (hyperforin and hypericin). The direct
injection of a sample dissolved in methanol, as earlier suggested, led to
irreproducible results due to the high HPLC pump pressure observed
after some runs. We used a low pH (2.5 and 3.5) phosphate solution
but the sample was not dissolved. Complete dissolution was achieved in
ammonium acetate in accordance to the earlier study; (30) various con-
centrations (50, 30, 10 and 5 mM) and pH values in the range of 35
were studied and the optimal values of pH and buffer concentration
were 4.5 and 10 mM. The elution system was slightly modied and
optimized (see HPLC Analysis section). The effect of temperature was
also studied, i.e., room temperature, 30C and 40C. At 30C, the opti-
mum separation of all the components and improved shapes of the
chromatographic peaks were observed. The detection wavelengths of
270, 300, 350 and 350 nm were selected after careful examination of
spectra of the peaks and in accordance to the earlier study (4).
As demonstrated in the chromatogram of a methanolic extract of
H. perforatum (Figure 1), good separation was achieved. Eleven peaks
were identied based on the elution time of the reference standards,
the elution order, UV-Vis absorption spectra (Supplementary Data,
3
Figure 3) and ESIMS spectra (Supplementary Data, Figure 4) in
agreement with previous reports (4, 9, 20, 31). In brief, molecular ion
[M H] of compound at Peak 6 had m/z 447, at peak 8 had m/z
537, at peak 9 had m/z 519 and peak 11 had m/z 535 which lead to
the identication of quercitrin, I3,II8-biapigenin, pseudohypericin and
hyperforin, respectively. Chlorogenic acid elutes in two separate peaks,
peaks 1 and 2 (Figure 1) in both the references standard and in the ex-
tracts. Peak 2 is always present in the chromatograph, however, this is
not the case for Peak 1; at low concentrations chlorogenic acid eluted
mainly in Peak 2 but at higher concentrations (>75 g/mL) both peaks
appear. Their MS spectra are the same both in positive (m/z 377
[M + Na]+) and negative ionization (m/z 353 [M H]) (Supplementary
data, Figure 4), although subtle differences in the absorption spectra
are present as expected (Supplementary data, Figure 3). In detail, the
maxima around 326,296 (shoulder) and 218 nm are found in both
peaks, while max around 244 nm is found only in peak 1 (max at
218 nm is lower) which resembles the spectrum differences of 5-O-
caffeoylquinic acid (32). Furthermore, in previous studies (33, 34) it
has been reported that trans- and cis-isomers of 3-O-caffeoylquinic
acid (chlorogenic acid) differ in their elution times and the cis-isomer
elutes earlier. Therefore, Peak 1 is identied as cis-3-O-caffeoylquinic
acid and Peak 2 as trans-3-O-caffeoylquinic acid.
Hyperoside (Peak 4) and isoquercitrin (Peak 5) are completely
resolved, almost at 1 min apart (Figure 1). Rutin (Peak 3), with tR
value 16 min, was not detected in the particular methanolic extract,
albeit it is completely resolved and detected in other products.
All commercially available reference standards, i.e., chlorogenic
acid, rutin, hyperoside, isoquetcitrin, quercetin and hypericin were used
for quantication using the standard addition method. Especially for
although the results for Peak 2 (trans- isomer) are always presented in
parentheses. No standards were available for quercitrin (Peak 6) and I3,
II8-biapigenin (Peak 8), and thus we used the equations of the closest
eluted avonoids, isoquercitrin (Peak 5) and quercetin (Peak 7) respec-
tively. Isoquercetrin (quercetin-3-glucoside) and quercitrin (quercetin-3-
O-rhamnoside) are both glycosides of quercetin and have similar
absorption spectra (Supplementary data, Figure 3), whereas quercetin
and I3,II8-biapigenin are both aglycon avonoids; the spectra of the lat-
ter present minor differences and the expression of I3,II8-biapigenin as
quercetin equivalents is a compromise imposed by the lack of standard.
Pseudohypericin (Peak 9) was quantied with the hypericin calibration
curve, since they have similar spectra. Finally, hyperforin concentration
(Peak 11) was estimated after integration at 270 nm and expressing this
peak area as rutin equivalent (Peak 3) concentration and multiplying by
2.3 according to European Pharmacopeia method.
Calibration curves were based on corrected peak areas in mAU * min,
versus concentration in mg/mL. LLOQ and Upper LOQ values were
determined as the lowest and upper concentrations, respectively, giv-
ing analysis of acceptable accuracy RE <20%. The calibration
curves were linear within the determined concentration range:
chlorogenic acid (3.075.0 g/mL, for the sum of both peaks y =
0.73x 1.00, R2 = 0.9969/only for CA2 y = 0.75x 1.34, R2 =
0.9989), rutin (2.0200.0 g/mL, y = 0.41x +0.15, R2 = 0.9977),
hyperoside (3.0200.0 g/mL, y = 0.77x 0.19, R2 = 0.9985), iso-
quercitrin (2.0200.0 g/mL, y = 0.73x0.37, R2 = 0.9994), querce-
tin (7.2200 g/mL, y = 0.85x3.63, R2 = 0.9985) and hypericin
(3.580.0 g/mL, y = 1.63x 2.38, R2 = 0.9949). The respective
correlation coefcients R2 were higher than 0.99 in all cases.
Overall, the results show good linearity for the six analytes.
Accuracy study assesses both systematic and random effects on
single results, i.e., both precision and trueness of the method. The
4 Zeliou et al.
Figure 1. Typical chromatogram of analysis of a methanolic extract of H. perforatum (1 mg of dry extract per mL) at four different detection wavelengths 270,
300, 350 and 590 nm 1: cis-chlorogenic acid, 2: trans-chlorogenic acid, 3: rutin, 4: hyperoside, 5: isoquercitrin, 6: quercitrin, 7: quercetin, 8: I3,II8-biapigenin, 9:
pseudohypercin, 10: hypericin and 11: hyperforin.
mean recoveries were above 85% and below 115% for all compounds
at all levels (Table I), showing the methods satisfactory intra- and inter-
day accuracy. The relatively low %RSD values (in most cases far below
20% at LLOQ, 15% at Low, Intermediate and High) demonstrate
the adequate intra- and inter-day repeatability.
The stability was evaluated by analysis of low- and high-quality
control samples (n = 3) at different time intervals (Table II). At
18C all the tested compounds were stable after a month. At
4 8C chlorogenic acid and hyperoside were stable for a month,
isoquercitrin and hypericin for at least 7 days, and nally quercetin
for only 1 day at high concentration and for 2 days at the low-
quality control sample. At room temperature only hyperoside was
stable for a week; isoquercetin and hypericin were stable for 48 h,
whereas chlorogenic acid and rutin, at the low concentration, were
stable for a week, and completely unstable at the high concentra-
tion. Quercetin was not stable at room temperature for any time
period. Also the freezethaw stability of the stability samples was
evaluated and all those constituents were stable.
The method was directly applied to the analysis of the methano-
lic extract, of an infusion and of two commercially available pro-
ducts (tablets and tincture) of H. perforatum. The results are
presented in Table III. The estimation of the total avonoid content
was made by including all peaks having the UV-vis spectrum charac-
teristic of avonoids.
Discussion
Based on the previous method (4), we changed the acidic aqueous
phase to ammonium acetate, which was also used in other studies
and optimized the gradient. The peaks were sharp, the resolution was
very good and all peaks were resolved but the analysis time was
rather long (~1 h), as also described in other similar studies (4, 8, 31),
however, the time necessary to actually run two different faster meth-
ods with different buffers and/or different columns is far longer.
The validation was performed for chlorogenic acid, rutin,
hyperoside, isoquercitrin, quercitrin, quercetin and hypericin in
methanolic extract and included evaluation of (1) linearity (2), preci-
sion (repeatability and intermediate precision) and accuracy, and (3)
stability according to the European Medicines Agency guidelines
(35). It is the rst time that precision and accuracy of each com-
pound are examined at four concentration levels, while stability of
quality control samples is examined at two concentration levels. The
examination of those parameters at different concentration levels
spanning the whole calibration curve, apart from being explicitly re-
commended in EMA guidelines, seemed necessary due to the fact
that not all ingredients in the extract/nal product are in the same
concentration range and thus even different dilutions of the same
sample may be necessary so as all ingredients are in the working
range of the method.
We validated the developed analytical method using spiked sam-
ples with multiple point standard additions to compensate for
matrix effects. No blank matrix can be attained and the plant
extract is too complex; it consists of numerous secondary meta-
bolites but also primary metabolites e.g., proteins, carbohydrates
which might interact with each other. The lack of appropriate blank
matrix also precludes selectivity studies as usually described.
Instead, DAD detection, isolation of each peak and MS screening re-
vealed the peak purity, the absence of interferences and the high
selectivity of HPLC. Extraction efciency from the plant material
has not been a subject of this investigation since it has been earlier
thoroughly studied (11, 20, 30, 3638).
The transformation of trans-3-CQA to cis-3-CQA, during HPLC
analysis of extracts and of pure external standards, is demonstrated
for the rst time. Caffeoylquinic acids are prone to transformations,
as previous studies have demonstrated that 5-O-caffeoylquinic acid
Optimized and Validated HPLC Analysis of St. Johns Wort Extract 5

Table I. Repeatability and Accuracy Data (Intra- and Inter-Day)

Chlorogenic acid Rutin Hyperoside Isoquercitrin Quercetin Hypericin

LLOQ Day 1 RSD% 6.2 (6.1) 7.0 8.7 15.3 7.0 9.3
Recovery% 112.7 (112.2) 86.9 114 102.7 111.4 97.9
Day 2 RSD% 2.3 (2.8) 5.6 7.1 12.2 8.3 7.7
Recovery% 86.8 (86.0) 93.5 115 106.5 101.3 101.4
Inter-day RSD% 18.4 (18.7) 8.2 10.3 17.9 13 9.5
Recovery% 99.8 (99.1) 90.2 114.5 104.6 106.3 99.7
Low Day 1 RSD% 1.6 (0.8) 2.3 6.4 1.6 4.5 4.0
Recovery% 94.9 (37.2) 103.9 109.6 86.2 94.2 86.4
Day 2 RSD% 4.4 (1.4) 4.7 5.6 4.1 3.2 2.8
Recovery% 95.2 (39.6) 91.8 93.8 87.2 95.1 88.4
Inter-day RSD% 3.9 (7.7) 6.2 6.6 7.2 5.8 4.4
Recovery% 106.6 (43.2) 97.8 100.9 86.7 94.7 87.4
Intermediate Day 1 RSD% 1.4 (2.6) 2.7 2.7 1.0 1.8 4.8
Recovery% 95.9 (64.3) 91.3 103.7 113.9 89.8 91.1
Day 2 RSD% 1.7 (3.7) 3.6 1.5 2.7 3.4 4.9
Recovery% 115.2 (84.0) 91.2 101.4 110.4 92.0 89.3
Inter-day RSD% 12.9 (18.7) 4.0 2.6 2.9 3.4 7.2
Recovery% 105.6 (74.2) 91.3 102.8 112.1 90.9 90.2
High Day 1 RSD% 4.7 (0.1) 3.4 4.3 3.7 1.7 5.9
Recovery% 94.7 (50.5) 111.4 103.2 110.6 89.5 91.7
Day 2 RSD% 1.7 (0.5) 3.8 2.6 1.2 2.9 4.7
Recovery% 85.7 (48.5) 107.4 103.5 111.3 91.6 90.4
Inter-day RSD% 8.7 (2.8) 3.8 4.4 4.1 4.1 6.9
Recovery% 93.7 (49.5) 109.4 103.4 110.9 90.5 91.1

Four concentration levels were used: LLOQ, LOW, INTERMEDIATE, HIGH for quality control samples. Accuracy is expressed as % recovery (ratio of mea-
sured to spiked concentration), and precision is expressed as %RSD (n = 3 at each concentration level and on each day). Values in parentheses in Chlorogenic
acid column represent the values calculated based only on Peak 2.

Table II. Stability of Low- and High-Quality Control Samples at Different Time Points and Temperature of Storage

Stability%

Chlorogenic acid Rutin Hyperoside Isoquercitrin Quercetin Hypericin

Low 18C 1 month 10.8 (35.4) 3.2 9.7 0.1 2.4 2.2
48C 1 day 9.5 (46.2) 2.1 0.6 4.6 7.3 2.0
2 days 6.1 (15.5) 5.2 11.2 0.9 7.7 6.0
7 days 3.7 (42.2) 2.0 2.2 0.6 37.5 4.6
1 month 9.6 (45.3) 1.0 10.2 3.3 43.6 21.4
2025C 1 day 8.7 (39.5) 3.4 5.8 0.3 24.1 4.7
2 days 7.0 (41.8) 3.0 3.8 0.6 33.1 9.6
7 days 6.5 (37.1) 4.5 1.1 2.8 52.4 19.4
High 18C 1 month 3.9 (44.3) 14.3 2.1 5.9 0.2 7.8
48C 1 day 5.9 (48.6) 12.8 3.9 0.2 11.9 1.1
2 days 5.3 (46.0) 5.8 12.2 4.4 27.9 4.1
7 days 5.2 (61.8) 15.8 1.1 2.2 33.3 2.1
1 month 7.9 (52.8) 15.3 1.7 20.8 94.4 15.8
2025C 1 day 22.9 (70.5) 15.6 1.1 8.4 94.0 6.2
2 days 21.8 (75.1) 19.4 3.5 13.1 94.4 9.9
7 days 39.0 (82.6) 23.7 7.9 63.7 94.1 21.2

Stability is expressed as % ratio of relative change in concentration levels ((Cn C0)/C0), n = 3 for each concentration level. Values in parentheses in
Chlorogenic acid represent the values calculated based only on Peak 2. Values in bold are those that deviate >15%.

is subject to isomerization during its buffered water extraction under
short heating or alcoholic extraction at high temperature (39, 40).
These transformations also depend on the concentration of the analyte
(the higher the concentration the greater the degree of isomerization).
In the current assay, quercetin is relatively unstable (Table II), which
in agreement with other studies that have also shown that its instability
in aqueous solutions especially with pH <5 (41). Contrariwise, hypericin
was stable for at least 48 h at room temperature (Table II), even though
it has been reported that it is relatively unstable due to its high sensitivity
to oxidation (42). With regard to hyperforin, since no HPLC standard
was commercially available, its stability was assessed only in the 0.5
mg/mL H. perforatum methanol extract (Supplementary data, Table V).
At room temperature hyperforin was completely unstable, while at
4 8C the compounds concentration declined by ~40% on Day 7.
6 Zeliou et al.

Table III. Concentration of Main Compounds in the Methanolic Extract (Concentration 3.5 mg/mL) and an Infusion (Concentration
3.5 mg/mL) are Expressed in g/mg of Dry Extract

Methanolic extract (g/mg) Infusion (g/mg) Tablet (g per tablet) Tincture (g/mL)

Chlorogenic acid (1, 2) 37.3 0.1 8.9 0.3 1174.6 10.2 198.3 28.7
Rutin (3) n.q. n.q. 126.5 5.7 325.1 29.2
Hyperoside (4) 42.6 0.7 33.2 0.1 483.2 5.0 445.2 22.4
Isoquercitrin (5) 21.6 0.3 11.0 0.1 564.0 2.6 149.2 5.4
Quercitrin (6) 5.6 0.2 2.0 0.1 513.4 5.8 674.2 29.6
Quercetin (7) 3.0 0.2 3.8 0.2 432.7 10.0 792.3 33.3
I3,II8-Biapigenin (8) 4.8 0.5 1.7 0.1 325.3 7.9 352.8 11.4
Pseudohypericin (9) 1.6 0.1 0.7 0.0 45.9 0.3 98.6 1.1
Hypericin (10) 1.8 0.1 1.3 0.1 28.7 0.1 101.4 3.5
Hyperforin (11) 11.0 0.6 n.q. n.d. 735.0 2.3
Total avonoids 94.6 2.7 67.9 3.3 3769.7 80.1 3023.8 188.2
Total naphthodianthrones 3.4 0.3 2.0 0.2 117.2 1.3 266.4 5.9

n.d., not detected; n.q., not quantied.

Concentration of analytes in a tablet is expressed in g per tablet (275 mg dissolved in 10 mL of dissolving solution) and in a tincture as mg/mL (dilution 1:40).
Values are mean standard error (n = 3).

Figure 2. Chromatogram of two St. Johns wort preparations: a tincture (black upper line) of H. perforatum and M. chamomilla, and a tablet (grey lower line).
Detection wavelength A. 270 nm, B. 300 nm, C. 350 nm and D. 590 nm, for the quantication of phloroglucinols, chlorogenic acid, avonoids and naphthodian-
thrones, respectively.

Finally, the concentration also declined by ~100% after three freeze
thaw cycles. These results are in agreement with the reports that hyper-
forin is extremely unstable and suffers degradation when exposed to
air and light; (22, 43, 44) thus estimation had better be performed in
fresh samples kept in dark. This study shows the limitations for its
accurate HPLC determination; however, this might be achieved by
other techniques. The ndings of this study, along with those of Bilia
et al. (45), concerning the limited stability of St. Johns wort nal pro-
ducts in different storage conditions stress the need for careful consid-
eration of the quality control of complex and sensitive herbal extracts,
like St. Johns wort. Method application to the methanolic extract
revealed that rutin was present in minute concentrations (Table III), in
contrast to the tablets and earlier reports. Chemotypes of H. perfora-
tum without rutin have also been identied in Italy (11, 46). The infu-
sion (Table III) contains all bioactive components albeit in lower
concentrations; when the results are expressed as mg/g of dry plant
material it can be deduced that nearly 6-fold more chlorogenic acid,
140% more avonoids and 2-fold more naphthodianthrones
(5673%) are extracted with methanol. Hyperforin is nearly not de-
tected in the infusion.
The tincture was prepared not only from H. perforatum but also
M. chamomilla, so although more peaks appeared, the separation
Optimized and Validated HPLC Analysis of St. Johns Wort Extract
and the determination of the specic analytes was not compromised
(Figure 2), conrming the selectivity of the method. In tincture,
organic acids and avonoids are also possibly extracted from M. cha-
momilla, which is rich in chlorogenic acid, rutin and quercetin (47).
Conclusion
In conclusion, an HPLC method for the analysis of H. perforatum
extracts was developed allowing the simultaneous quantication of
chlorogenic acid, avonoids, naphthodianthrones and a rough esti-
mation of phloroglucinols. The method was validated in terms of
linearity, intra- and inter-day accuracy and precision, and stability
using methanolic samples spiked with standard compounds accord-
ing to ofcial guidelines of EMA applicable to bioanalytical meth-
ods. Trans-chlorogenic acid is partially transformed into its cis-
isomer during analysis and acceptable accuracy is achieved only
when both peaks were taken into account. The evaluation of stabil-
ity showed that hyperforin is extremely unstable, albeit an estima-
tion of its quantity in relation to rutin may be achieved in fresh
samples in dark. Storage temperature has a high impact on the sta-
bility of tested compounds. The method was applied to dry and liq-
uid alcoholic extracts, infusion and nal products of St. John wort.
Its applicability in various products suggests that it could be of value
in the quality control of St. Johns wort preparations. The validation
protocol revealed many critical points that need to be taken into
consideration during the implementation of the method.
Supplementary data
System-suitability report for the separation of the major compounds
of H. perforatum (Table IV), stability of hyperforin (Table V) in
methanolic extract of H. perforatum and UV-vis absorption
(Figure 3) and mass spectra (Figure 4) of detected peaks are avail-
able as Supplementary data. Supplementary data are available at
Journal of Chromatographic Science online.
Acknowledgments
The authors thank Professor Gregorios Iatrou, Department of Biology,
University of Patras for identication of plant material.
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