Current Drug Metabolism, 2007, 8, 773-777 773

In Vitro Monitoring Chlorogenic Acid in Human Urine and Serum by a Flow Injec-
tion System Exploiting the Luminol-Dissolved Oxygen Chemiluminescence Reaction

Xiaofeng Xie, Xili He, Xiaoxiang Qiu and Zhenghua Song*

Department of Chemistry, Northwest University, Xi’an, 710069, China

Abstract: A sensitive flow injection chemiluminescence method, based on the inhibitory effect of chlorogenic acid on the reaction be-
tween luminol and dissolved oxygen, was presented for the determination of chlorogenic acid. It was found that the decrease of chemilu-
minescence intensity was linear with the logarithm of chlorogenic acid concentration over the range from 1.0 ng·ml-1 to 100 ng·ml-1 (r2 =
0.9978), with the detection limit of 0.3 ng·ml-1 (3). At the flow rate of 2.0 ml·min-1 for each line, a complete analytical process could be
performed within 0.5 min, including sampling and washing, with a relative standard deviation lower than 3.0% (n = 5). The proposed
procedure was applied successfully to determine chlorogenic acid in Flo Lonicerae for different drawn time and monitor the excretion of
chlorogenic acid in human urine. It was found that the excretive amounts of chlorogenic acid in urine reached its maximum in 2 hours af-
ter intake of Flo Lonicerae tea, presenting an excretive ratio of 63.82% in 6 hours. With urinary excretion rate method, the total elimina-
tion rate constant k and half-life time t1/2 of chlorogenic acid was calculated, which were 0.7667 and 0.91 hours, respectively.

Keywords: Chlorogenic acid, chemiluminescence, luminol, dissolved oxygen, flow injection, Flo Lonicerae, human urine, excretive.

INTRODUCTION
Chlorogenic acid {1, 3, 4, 5-tetrahydroxycyclohexane carbox-
ylic acid 3-(3, 4-dihydroxycinna-mate)}, one of major bioactive
compounds of some Chinese herbs like Flo Lonicerae [1], is a kind
of polyphenol derivative that widely exists in plants like fruits,
vegetables, black teas, and some traditional Chinese medicines [2].
It not only has the functions of antioxidation, of inhibiting hyper-
tension, and of stimulating the flowering of plants, but also affects
the activity of trypsin, amylase, and other enzymes [3]. Due to its
obvious anti-infectious effects, chlorogenic acid can be used as an
active ingredient with wide anti-virus, anti-bacteria effects, and
relatively lower toxicity and side effects and can be used widely in
many other fields like food, feed additives, cosmetic also [4].
HPLC separation with UV or MS detection was widely used for
the determination of chlorogenic acid (with the lowest detection
limit of 100 ng·ml-1) [5-8]. Spectrophotometry (with the lowest
detection limit of 2
g·ml-1) [9, 10] and capillary electrophoresis
(with the lowest detection limit of 8
g·ml-1) [11, 12] were reported.
The chemiluminescence (CL) owning to its high sensitivity and
rapidity, with luminol-KMnO4 and luminol-K3Fe(CN)6 CL systems
(with the lowest detection limit of 5 ng·ml-1) was also applied for
the determination of chlorogenic acid [13, 14]. However, no report
has been found for in vitro monitoring chlorogenic acid in human
urine to date.
Recently, it was presented that in vitro monitoring roxithromy-
cin in human urine using luminol-hydrogen peroxide CL procedure
was reported by our group [15]. In this paper, it was found that
chlorogenic acid could obviously inhibit the CL reaction between
luminol and dissolved oxygen in alkaline medium. The decrease of
CL intensity was linear with the logarithm of chlorogenic acid con-
centration over the range from 1.0 ng·ml-1 to 100 ng·ml-1 (r2 =
0.9978), with the detection limit of 0.3 ng·ml-1 (3). At the flow
rate of 2.0 ml·min-1 for each line, one analysis cycle including sam-
pling and washing could be accomplished in 30 s. The simple, rapid
and sensitive CL procedure was applied successfully to determine
chlorogenic acid in Flo Lonicerae aqueous for different drawn time-
lag and monitor the excretion of chlorogenic acid in human urine
samples. It was found that the excretive amounts of chlorogenic
acid in urine reached its maximum in 2 hours after intake of the tea,
presenting an excretive ratio of 63.82% in 6 hours.
*Address correspondence to this author at the Department of Chemistry,
Northwest University, Xi’an, 710069, China; Tel: 86-29-88302604; Fax:
+86-29-88303798; E-mail: songzhenghua@hotmail.com
OH COOH
OH OOC
OH
OH
OH
Scheme (1). Chlorogenic acid.
EXPERIMENTAL
Reagents
All chemicals were of analytical grade and the double distilled
water used throughout was purified in a Milli-Q system (Millipore,
Bedford, MA, USA). Chlorogenic acid standard solution (0.20
mg·ml-1) was obtained from Shaanxi Institute for Drug Control.
Working strength solutions were prepared daily from the above
stock standard solutions as required. Luminol (2.510-2 mol·l-1) was
prepared by dissolving 4.40 g luminol (Fluka, Switzerland) in 1
liter of 0.1 mol·l-1 NaOH solution.
Apparatus and Procedures
The flow injection system used in this work is shown in Fig.
(1). A peristaltic pump (Shanghai meter electromotor plant, Model
ND-15, 15 rpm) was utilized to pump each of flow streams at a
flow rate of 2.0 ml·min-1. PTFE tubing (1.0 mm i.d.) was used in
flow system for carrying the CL reagents. A six-way valve with
loop of 100
l was employed for sampling. The CL emission cell is
a spiral glass tube (1.0 mm i.d., 15 cm length) in order to produce a
large surface area exposed to the adjacent photomultiplier tube
(PMT) (Hamamatsu, Model IP28). Extreme precautions were taken
to ensure that the cell compartment and the photomultiplier tube
were light-tight. The CL signal induced in the CL emission cell was
detected without wavelength discrimination and the PMT output
was amplified and quantified by a luminosity meter (Xi’an Keri
Electron Device Ltd., Model GD-1) connected to a recorder
(Shanghai Dahua Instrument Plant, Model XWT-206). The concen-
tration of the sample was quantified by the decrease of CL intensity
(
I = Io - Is), where Io and Is were CL signals in the absence and in
the presence of chlorogenic acid, respectively.

1389-2002/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

774 Current Drug Metabolism, 2007, Vol. 8, No. 8 Xie et al.

centration of about 0.25 mol·l-1. Thus, this concentration was em-

P ployed for the analysis.
Sample Effect of Flow Rate and the Length of Mixing Tubing
The influence of the reactants flow rate on the determination
H 2O chlorogenic acid was examined by investigating the signal-to-noise
V M CE D
Luminol ratio under different flow rates. It was found that the flow rate of
about 2.0 ml·min-1 offered the highest signal-to-noise ratio and,
NaOH R therefore, it was chosen as a suitable condition for the subsequent
measurements. The length of the mixing tube was also adjusted to
W yield the maximum light emission in the CL cell. By comparing the
CL intensities measured in the presence of 10 ng·ml-1 chlorogenic
Fig. (1). Schematic diagram of the flow injection system for determination acid, the most intense signal was detected using tubes with the
of chlorogenic acid length of 5.0 cm. Accordingly, this length was selected for the fol-
P: peristaltic pump; V: valve; M: mixing tube; CE: flow cell; lowing experiments.
W: waste; D: detector; R: recorder
The Operational Stability of the Proposed Flow System
RESULTS AND DISCUSSION 100
l of H2O/sample (10 ng·ml-1) was injected into the flow
CL Intensity-Time Profile system and the ratio of Is/Io was recorded to test the operational
The kinetics of luminol-dissolved oxygen-chlorogenic acid stability of the flow system. The experiment lasted for 10 days and
reaction was tested by a static method. As shown in Fig. (2), the CL the flow system was regularly used 8 - 10 h per day. It was found
intensity reached the maximum 325 at 5 s after luminol and NaOH that the Is/Io, viz. relative
I, kept stable under the fluctuation of Io
were mixed, and then vanished within the following 20 s. When the and the average of Is/Io was calculated in ten spot check determina-
solutions being degassed the CL signal was only 17, which sug- tions with an RSD lower than 3.0% throughout the experiment. In
gested that the dissolved oxygen possessed crucial effect in the general, the flow system showed satisfactory stability in determina-
system. Scintillescent CL signal was detected also in the presence tion.
of chlorogenic acid, the CL intensity reached the maximum at 6 s Performance of Proposed Method for Chlorogenic Acid Meas-
after mixing the reactants and the CL intensity was decreased to urement
221. Under the optimized conditions, a series of standard solutions
were injected into the manifold depicted in Fig. (1) to test the line-
360 arity of chlorogenic acid. It was found that the decrease of CL in-
tensity was proportional with the logarithm of chlorogenic acid
concentration and the calibration graph corresponded to a straight
line in the chlorogenic acid concentration range of 1.0-100 ng·ml-1
270
Relative CL intensity

with the detection limit of 0.3 ng·ml-1. The regression equation was

ICL = 24.41LnCchlorogenic acid + 204.8, r2 = 0.9978.
The RSDs of seven determinations were 3.41, 2.98 and 1.74%
180 with the chlorogenic acid concentration of 1.0, 10 and 70 ng·ml-1,
respectively. At the flow rate of 2.0 ml·min-1, the determination of
the analyte could be performed in 0.5 min, including sampling and
90 washing, giving a throughput about 120 times·h-1 with an RSD
lower than 5.0%.
Interference Studies
0 Interference foreign species was tested by analyzing a standard
0 5 10 15 20 solution of chlorogenic acid (10 ng·ml-1) to which increasing
amounts of interfering species were added. The tolerable limit of a
T(second) foreign species was taken as a relative error lower than 5.0% and
the results are listed in Table 1.
Fig. (2). Kinetic CL intensity-time profile in static system

: CL intensity of luminol-dissolved oxygen Table 1. Tolerable Concentration with Respect to 10 ng·ml-1
: CL intensity of luminol-dissolved oxygen-chlorogenic acid (10 ng·ml-1) Chlorogenic Acid for Some Interfering Species (<5% Er-
ror)
: CL intensity of luminol after degassing
The concentrations of chlorogenic acid, luminol and NaOH were 10 ng·ml-1,
5.010-5 mol·l-1, and 0.025 mol·l-1, respectively. Species Tolerable Conc. (
g·ml-1)
The high voltage was -750 V.
Glutin, barbiturate, oxalic acid, urea, acetone >5102
Effect of Luminol and NaOH Concentration on CL Intensity Starch, lactose, cellulose, stearic acid, agar, talc,
10
citric acid, fructose, sucrose, phenobarbital
The influence of luminol on the intensity of CL signal was ex-
amined by changing the luminol concentration from 1.0  10-7 Cholesterol, albumin 5
mol·l-1 to 5.0  10-4 mol·l-1. An optimum luminol concentration of Cl-, NO 3-, Ac-, I-, SO 42- , PO 43-, Cr2O 72- , borate,
5.0  105 mol·l-1 was selected on the basis of 10 ng·ml-1 chloro- oxalate, tartrate, citrate, malic acid, globulin
1
genic acid. Selected optimum luminol concentration was used in all NH4+, Mg2+, Ca2+, Ba2+, Ag+, lysozyme, salicylic, L-
subsequent experiments. Owing to the nature of the luminol reac- 0.5
threonine
tion, which is favored in an alkaline medium, a series of NaOH
Uric acid, L-lysine, L-cysteine, myoglobin 0.3
solutions from 0.01 mol·l-1 to 0.25 mol·l-1 were tested in the pres-
ence of 10 ng·ml-1 chlorogenic acid. The plot of CL intensity versus Co2+, Fe3+, Fe 2+, Mn2+ 0.05
the NaOH concentration showed the maximum at the NaOH con- Catechol, hydroquinone, L-histidine 0.01
In Vitro Monitoring Chlorogenic Acid in Human Urine Current Drug Metabolism, 2007, Vol. 8, No. 8 775

It should be noticed that the interference of some biospecies
often found in serum and urine was studied. The total urinary pro-
tein in a healthy adult is about 100
g·ml-1 and the total serum pro-
teins are about 67-83 mg ml-1 [18] which showed no interference in
the determination of chlorogenic acid after diluting by 104 to 105
with water. Other compounds abundant in human urine and serum
such as salt and lipid also caused no obvious interference for the
determination of chlorogenic acid.
APPLICATIONS
Determination of Chlorogenic Acid in Flo Lonicerae for Differ-
ent Drawn Time
Triplet Flo Lonicerae teas about 2.0 g were weighed and put
into three beakers respectively, to which 100 ml boiling water was
added. After drawing for 30, 60, 90 min, respectively, the filtrate
was diluted to appropriate concentration, and then determined by
the proposed procedure. The results of determination for drawn
time of 30, 60 and 90 min are listed in Table 2, which are 6.18%,
5.66% and 5.58%, respectively, with recoveries from 95.8% to
109.9%. The results are in a good agreement with the results ob-
tained by HPLC.
In Vitro Monitoring of Excretive Chlorogenic Acid in Human
Urine
Following the procedure described in the experimental section,
the proposed method was applied to the determination of chloro-
genic acid in human urine samples. 1.0 g Flo Lonicerae was

Table 2. Results of Chlorogenic acid in Flo Lonicerae for Different Drawn Timea

Content/%
Sampleb
Added/ ng·ml-1 Found/ ng·ml-1 RSD/% Recovery/% The proposed
No. HPLC
method

0 7.4 1.09
1 110.0 6.17 5.91
3.0 10.7 1.47
0 9.9 2.26
2 98.7 6.19 6.07
30.0 39.5 2.06
0 3.3 1.68
3 94.0 5.65 5.78
5.0 8.0 1.43
0 13.5 2.77
4 101.0 5.67 5.75
10 23.6 3.17
0 1.6 1.78
5 96.0 5.61 5.65
5.0 6.4 2.61
0 33.6 3.81
6 106.0 5.54 5.58
10.0 44.2 1.93
a
The average of five determinations.
b
Drawn time: No.1-2, 30 min; No.3-4, 60 min; No.5-6, 90 min.

Table 3. Determination of Excreted Chlorogenic Acid in Human Urinea

Chlorogenic acid in Chlorogenic acid

Time Added Found RSD Recovery
urine excreted ratio
hours ng·ml-1 ng·ml-1 % %
M(mg)/V(ml) in urine %

0


0.0 101.0
/210

10.0 10.1 3.63
0 17.3 1.16
0.5 100.0 3.62/145 5.86
10.0 27.3 2.70
0 17.5 1.41
1.0 111.0 4.38/125 7.09
10.0 28.6 2.86
0 22.0 1.89
2.0 98.0 16.25/75 26.29
10.0 31.8 1.73
0 19.3 2.76
3.0 107.0 12.50/100 20.23
10.0 30.0 1.77
0 5.2 2.12
4.0 108.0 2.48/75 4.01
10.0 16.0 1.04
0 4.26 3.06
5.0 106.4 0.21/110 0.34
10.0 14.9 2.62
0 — 1.58
6.0 95.0 —/125 —
10.0 9.5 1.22

Total
39.44 mg/965 ml, Excretion ration: 63.82%
a
The average of six determinations.
776 Current Drug Metabolism, 2007, Vol. 8, No. 8 Xie et al.

Table 4. Results of Chlorogenic Acid in Spiked Human Seruma

Sample
Added/ ng·ml-1 Found/ ng·ml-1 RSD/% Recovery/ % Content/ mg·ml-1
No.

0 1.0 1.91
1 110.0 5.60
1.0 2.1 1.25
0 4.0 2.62
2 105.0 6.02
4.0 8.2 3.25
0 6.0 2.68
3 104.0 5.30
5.0 11.2 2.06
0 3.6 2.46
4 98.6 6.20
7.0 10.5 3.67

a
The average of five determinations

Table 5. Results of Different Reaction Systems by UVa and CL b

Types of reaction system A324 nm Aa221 nm ICL

luminol 0 0.3573 325

chlorogenic acid 0.0955 0 0
luminol + chlorogenic acid 0 0.4359 221
[luminol + chlorogenic acid]/degassed 0 0.4361 17
a
The concentrations of chlorogenic acid and luminol were 5.0
10-6 mol·l-1 and 2.0
10-5 mol·l-1, respectively.
b
The concentrations of chlorogenic acid, luminol and NaOH were 5.0
10-8 mol·l-1, 5.0
10-5 mol·l-1 and 0.025 mol·l-1, respectively.

weighed exactly and put into a beaker, to which 100 ml boiling
water was added, and then drawn for 30 min. A healthy male volun-
teer took it in the morning and the first-voided urine samples were
collected in brown calibrated flasks after 0.5, 1, 2, 3, 4, 5 and 6
hours, respectively. The collected urine samples were diluted with
water directly and sometimes supplemented with chlorogenic acid
to test the recovery of the method. Thus, excretive chlorogenic acid
could be determined simply by FI-CL. The results of these trial
determinations are summarized in Table 3. It can be seen that the
total chlorogenic acid excreted through urine was 39.44 mg in a
total volume of 0.965 liter in 6 hours. The concentration of chloro-
genic acid reached its maximum in 2 hours after drinking and
dropped sharply in succedent 4 hours. According to the determined
results (6.18%) of chlorogenic acid in Flo Lonicerae for drawing 30
min, the chlorogenic acid excretive ratio in 6 hours was calculated,
yielding 63.82% in the body of volunteer. With urinary excretion
rate method [19], the total elimination rate constant k and half-life
time t1/2 of chlorogenic acid in body were calculated which were
0.7667 and 0.91 hours, respectively.
Determination of Chlorogenic Acid in Spiked Human Serum
Samples
The samples of human serum supplied by the Hospital of
Northwest University were spiked before determination. In order to
prepare spiked samples, known amount of chlorogenic acid was
mixed with 0.1 ml serum. After homogenization, the mixture was
directly diluted to the concentration in the working range of deter-
mination. The results of determination of chlorogenic acid are listed
in Table 4. The method was verified by determination of recoveries,
which varied from 97.7% to 105.5%. It was obvious that the pro-
posed method was very sensitive for the determination of chloro-
genic acid in biological fluids, especially in serum sample.
Possible Mechanism of the CL Reaction
The possible mechanism of luminol-dissolved oxygen-
chlorogenic acid CL reaction was studied by UV and CL means,
and the results are listed in Table 5. In Table 5, It can be seen that
the CL intensity for luminol-dissolved oxygen is 325, and for lumi-
nol- dissolved oxygen-chlorogenic acid is 221. When the luminol
solution is gassed the CL signal is 17, which suggests that the dis-
solved oxygen possesses crucial effect in the system. It is very
clear that in the presence of luminol the A324nm of chlorogenic acid
is vanished from 0.0955; and the A221nm of luminol is increased
from 0.3573 to 0.4359 in the presence of chlorogenic acid, which
suggests that luminol does react with chlorogenci acid. It is shown
that the reaction between luminol and chlorogenci acid results in
the decrease of luminol concentration, and then, the CL signal of
luminol-dissolved oxygen are decreased.
CONCLUSIONS
The results obtained clearly demonstrate that the proposed
method offers the advantages of simplicity, rapidity, widely linear
range as well as high sensitivity for the determination of chloro-
genic acid.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support from
Shaanxi Province Nature Science Foundation and Ministry of Edu-
cation, China, Grant No. 2006B05 and No. 07Jk395.
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Received: January 09, 2007 Revised: June 20, 2007 Accepted: June 21, 2007