35

A method for the simultaneous separation and direct determination

of oxalic acid (OA), tartaric acid (TA), malic acid (MA), vitamin C
(VC), citric acid (CA), and succinic acid (SA) in Fructus mume using
reversed-phase high-performance liquid chromatography with a UV
detector in an acidic medium is presented in this study. In the
experiment, the optimization of chromatographic conditions
(i.e., the pH and flow rate of the mobile phase, the absorption
wavelength, and temperature of column) that affect the separation
degree and peak shape of organic acids has been obtained. The
linear ranges are found to be 0.054.7 g for OA (r = 0.9999),
0.1110.5 g for TA (r = 0.9999), 0.11411.4 g for MA (r =
0.9999), 0.0333.30 g for VC (r = 0.9999), 0.15515.5 g for CA
(r = 0.9998), and 0.19419.4 g for SA (r = 0.9996). For OA, TA,
MA, VC, CA, and SA, the even recovery (n = 3) of six effective
components are 100.9%, 99.97%, 101.2%, 102.1%, 101.1%, and
100.7%, respectively, and the largest relative standard deviation
(n = 11) for the six components is less than 1.7%. The detection
limits are 0.01 g for OA, TA, and VC; 0.05 g for MA; 0.03 g for
CA; and 0.1 g for SA. In a single chromatographic run, OA, TA,
MA, VC, CA, and SA can be determined in less than 7 min. The
method can be used for the purpose of routine analysis and the
quality control of a botanic (Fructus mume) containing these
effective components.
Introduction
Fructus mume is dry and nearly a mature fruit of Prunus
mume (Sieb.) Sieb. et Zucc. It is picked in summer when it has
nearly grown to maturity. As a medico-material, it must become
black by putting it in a container that must be separated from air
after it has dried in a lower temperature (1). It is a useful medico-
botanic that is recorded in the Chinese pharmacopoeia. The
Chinese book, Bencao Gangmu, has recorded it as a drug that can
cure diarrhea (2). Previously, scientists considered that there
were small amounts of tartaric acid (TA), malic acid (MA), and
succinic acid (SA) in Fructus mume besides the large amounts of
citric acid (CA). Presently, results of modern pharmacology
research indicate that the antibacterial, antifiber, and round-
worm-killing actions of Fructus mume are related to its acid con-
stituents such as TA, MA, CA, and SA (3,4).
A number of methods have been reported for the determination
of organic acids in a solution by fluorescence, chemilumines-
cence (58), potential titration (9), high-performance liquid chro-
matography (HPLC) (10), capillary isotachophoresis (11),
capillary zone electrophoresis with electrochemical detection
(12), and thin-layer chromatography (13). Many of these were for
the determination of single organic acids (i.e., chemilumines-
cence), and some of them were for the determination of total or
mixed organic acids (i.e., potential titration and fluorescence).
The normal-phase HPLC method coupled with an electrochem-
ical detector offers advantages of high resolution with low detec-
tion limits. It has therefore been used as a very sensitive method
for the determination of a few organic acids in solution in recent
years. Although octodecylsilyl (ODS) columns (normal phase)
have been frequently used for the separation of the different
organic acids, an important limitation factor with such columns
is that an organic solvent is required, which often leads to com-
plicated electrochemical behaviors when using an electrochem-
ical detector. Besides this limitation, an electrochemical detector
is not so popular in general analytical laboratories because it is
too expensive and responds only to a limited number of compo-
nents.
In this work, a new method was developed for simultaneous
and direct determination above multiple organic acids in the
botanic Fructus mume by reversed-phase (RP) HPLC with UV
detection. This method offers four advantages. First, a commer-
cial HPLC instrument can be directly applied to the simultaneous
Abstract
Simultaneous and Direct Determination of Oxalic Acid,
Tartaric Acid, Malic Acid, Vitamin C, Citric Acid, and
Succinic Acid in Fructus mume by Reversed-Phase
High-Performance Liquid Chromatography
Chen Zhanguo* and Lu Jiuru
Department of Chemistry, School of Chemistry and Material Science, Shaanxi Normal University, Xian, 710062, P.R. China
Reproduction (photocopying) of editorial content of this journal is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, January 2002
* Author to whom correspondence should be addressed.
Journal of Chromatographic Science, Vol. 40, January 2002
36
direct determination of the six important biological components
(six organic acids) in Fructus mume without any modification.
Second, an RP C18 column is applied for the separation of these
organic acids, which allows an aqueous solution to be used as the
mobile phase. Third, an expensive electrochemical detector
becomes unnecessary. Fourth, extracted solutions of Fructus
mume do not need to be treated with any special chemical pro-
cess before analysis. With this method, six important organic
acids (OA, TA, MA, VC, CA, and SA) can be efficiently separated
using an RP column with a mobile phase of a dilute aqueous solu-
tion of (NH
4
)H
2
PO
4
and can be successfully determined with a UV
detector. No simultaneous and direct determination of OA, TA,
MA, VC, CA, and SA in a Fructus mume study has been previously
reported.
Experiment
Instrument
A Shimadzu (Kyoto, Japan) LC-6A HPLC system, SPD-6AV
detector, CR-3A chromatography data-processing apparatus, and
a Shimadzu UV-260 UV spectrophotometer were used.
Reagents and material
OA (C
2
H
2
O
4
), TA (C
4
H
6
O
6
), VC (C
6
H
8
O
6
), CA (C
6
H
9
O
7
), SA
(C
4
H
6
O
4
), (NH
4
)H
2
PO
4
, and H
3
PO
4
reagents were purchased from
TianJin Chemical Reagent Company (TianJin, China). L-MA
(C
4
H
6
O
4
) was obtained from the Test and Research Institute of
Medicines and Chemical Reagents and Living Things Products
(Beijing, P.R. China). Fructus mume was yielded in Fujian and
Sichua of China and appraised by Prof. Quanhong Liu, who works
at the College of Life Science in Shaanxi Normal University
(Xian, P.R. China). All chemicals were of analytical-reagent grade
or higher and were used without further purification. All solid
regents had been dried until constant weight before applying.
The standard stock solutions were prepared by dissolving
0.0117 g of OA, 0.1645 g of TA, 0.0317 g of MA, 0.0413 g of VC,
0.0646 g of CA, and 0.0486 g of SA in a small amount of distilled
water, respectively, and these solutions were then transferred into
six volumetric bottles (50.00 mL). Some of the volume mobile
phase was added into them until the mark was reached. The
mixed standard stock solution was prepared by transferring the
standard stock solution of 5.00 mL OA, 0.80 mL TA, 4.50 mL MA,
1.00 mL VC, 3.00 mL CA, and 5.00 mL SA into the same volu-
metric bottle (25.00 mL). Some of the volume mobile phase was
added into it until the mark was reached. The concentrations of
each constituent in this mixed standard stock solution were 46.58
g/mL for OA, 105.28 g/mL for TA, 114.00 g/mL for MA, 33.04
g/mL for VC, 155.04 g/mL for CA, and 194.40 g/mL for SA.
The mobile phase of 0.5% (NH
4
)H
2
PO
4
(w/v) was prepared by
dissolving solid (NH
4
)H
2
PO
4
in distilled water. Then, it was
adjusted to pH 2.80 with a solution of 1.0-mol/L H
3
PO
4
, and it was
filtered under vacuum through a 0.45-m membrane (Shanghai,
China) when used.
Experiment method
The chromatographic column was an LC-18 column (150-
4.6-mm i.d, 5-m particle size). The mobile phase was a 0.5%
(NH
4
)H
2
PO
4
(w/v) aqueous solution, and its pH was 2.80. The
optimum flow rate of the mobile phase was set at
1.0 mL/min, and the absorbance units full scale
was 0.16. The detected wavelength was at 214 nm.
The extraction solution of the sample Fructus
mume was filtered under vacuum through the
0.45-m membrane. The injection volume of the
sample solution was 5.0 L. Organic acid peaks
were identified by comparing the retention time
in the sample solution with that of the standard
solution. The contents of OA, TA, MA, VC, CA, and
SA in the sample solution were quantitated by
comparing peak areas in the sample solution with
that of known standards.
Results and Discussion
Optimization of chromatographic conditions
The key work of this study was to achieve a sep-
aration of the six organic acids by using RP-HPLC
with an aqueous solution as the mobile phase. In
order to achieve baseline separation, a series of
experiments were performed.
Optimization of mobile phase
Because the effective constituents of Fructus
mume are organic acid species, a mobile phase of
Figure 2. Effect of mobile phase pH on the separation degree: (1) between OA and TA, (2) between TA
and MA, (3) between MA and VC, (4) between VC and CA, and (5) between CA and SA.
Figure 1. Effect of mobile phase concentration on the retention time of six constituents: OA, 1; TA, 2;
MA, 3; VC, 4; CA, 5; and SA, 6.
Journal of Chromatographic Science, Vol. 40, January 2002
37
0.5% KH
2
PO
4
(w/v, pH 3.0) was used for the separation of TA and
CA in the preliminary work. This condition was recommended by
reference 14 for the separation of chlorogenic acid, caffeic acid,
and isochlorogenic acid in plants. Under this condition, however,
the six organic acids were not completely separated, especially OA,
TA, and MA. A series of new mobile phases were then selected in
order to obtain a better separation. When the solution of 0.5%
KH
2
PO
4
was replaced by an aqueous solution of (NH
4
)H
2
PO
4
, the
six organic acids were completely separated, especially OA, TA, and
MA. In the experiment, by keeping the pH of the mobile phase at
2.8, the retention time of the six organic acids changed less signif-
icantly when the aqueous concentration of (NH
4
)H
2
PO
4
changed
from 0.1%, 0.25%, 0.5%, 0.75%, to 1.0% (shown in Figure 1). For
convenience purposes in the experiment, we chose the concentra-
tion of the mobile phase to be 0.5% (w/v). Therefore, 0.5% of
(NH
4
)H
2
PO
4
was applied to the subsequent study.
The effect of the pH value of the mobile phase on the retention
time of the separated constituents was also examined. In this
analysis, we observed that when other conditions did not change,
it could greatly affect the separation degree of the six organic
acids (taken from the separation degree between two peaks calcu-
lated by reference 15) and thus cause the pH value of the mobile
phase to change in small ranges. The smaller the pH value was in
the mobile phase, then the smaller would be the separation
degree. The greater the pH value was, then the greater the sepa-
ration degree would be. However, when the pH value of the
mobile phase was more than 3, the separation degrees between
OA and TA, TA and MA, MA and VC, VC and CA, and CA and SA
were reversely smaller. Figure 2 shows the effect of the mobile
phase on the separation degree when pH was changed from 2.0,
2.4, 2.6, 2.8, 3.0, to 3.2. This situation was observed in the exper-
iment. Although the reason was not completely clear to us, we
considered that because the constituents were multiple carboxyl
organic acids, when the pH of the mobile phase becomes large,
the part of the carboxyl group in the molecule can form carboxy-
late. The separated molecular polarity becomes stronger in this
condition. The stronger the molecular polarity is, then the
smaller would be the force of the molecule that is adsorbed on the
column. Therefore, the retention time of the separated molecule
would also be smaller, which is a possible reason when the pH of
the mobile phase was more than 3 and the separation degrees
between OA and TA, TA and MA, MA and VC, VC and CA, and CA
and SA were reversely smaller. The experiment indicated that the
suitable pH value was 2.8. Therefore, the pH value of 2.8 was
applied to the subsequent study.
Effect of wavelength
Upon researching six organic acids by RP-HPLC, we found that
the choice of wavelength was also an important factor. The stan-
dard stock solution of six organic acids was investigated on the
UV-260 UV spectrophotometer. The wavelength ranges for them
were from 180 to 240 nm, but the maximum absorbed wave-
length was approximately 190 nm (shown in Figure 3). However,
the study met a problem of complexity. If the wavelength in ana-
lyzing the sample was chosen in a lower range (i.e., 190 nm), the
chromatogram would become more complex and the separation
efficiency would not be satisfactory. This phenomenon was
observed in this experiment. After many comparisons and consid-
ering other factors that would affect the experiment, the suitable
absorbed wavelength was finally chosen to be 214 nm. At this
wavelength, not only did the peaks of the six organic acids in the
extracted solution of Fructus mume appear, but also many of the
other peaks disappeared. This was an important factor to ensure
accurate determination of the six organic acids in Fructus mume.
Linearity and detection limits
A mixed standard stock solution was prepared as described
Figure 4. RP-HPLC chromatogram of a standard stock solution: OA, 1; TA, 2;
MA, 3; VC, 4; CA, 5; and SA, 6.
Figure 3. UV spectrum of six chemical constituents: OA, 1; TA, 2; MA, 3; VC,
4; CA, 5; and SA, 6.
Journal of Chromatographic Science, Vol. 40, January 2002
38
previously. In this mixed standard stock solution, the concentra-
tion of the six constituents were 46.58 g/mL for OA, 105.28
g/mL for TA, 114.00 g/mL for MA, 33.04 g/mL for VC, 155.04
g/mL for CA, and 194.40 g/mL for SA. The injection volume
range varied from 0.5 to 100 L. The peak areas were assigned to
the longitudinal coordinate (y), and the injection quantities (g)
were assigned to the transverse coordinate (x). Using the opti-
mized conditions as described previously, the detection limit
(signal-to-noise equal to 3) was 0.01 g for OA, 0.01 g for TA, 0.05
g for MA, 0.01 g for VC, 0.03 g for CA, and 0.1 g for SA. The
linear ranges were 0.054.7 g for OA, 0.1110.5 g for TA,
0.11411.4 g for MA, 0.0333.30 g for VC, 0.15515.5 g for CA,
and 0.19419.4 g for SA. The regression coefficient (r) was 0.9999
for OA, 0.9999 for TA, 0.9999 for MA, 0.9999 for VC, 0.9998 for CA,
and 0.9996 for SA. The relative standard deviation (RSD) was
examined, and the RSD values (n = 11) were 0.25% for OA, 0.99%
for TA, 1.68% for MA, 0.99% for VC, 0.61% for CA, and 1.23% for
SA.
The mixed standard stock solution was continuously injected
three times every 3 h during a day. Every injected volume was
15 L and the result was averaged. The intraday RSD values
(n = 5) were 0.45% for OA, 1.1% for TA, 1.69% for MA, 1.23% for
VC, 0.86% for CA, and 1.80% for SA. Also, the solution was only
continuously injected three times every day. The operation was
the same as that previously described. The interday RSD values
(n = 5) were 2.8% for OA, 2.1% for TA, 1.7% for MA, 67.8% for VC,
6.7% for CA, and 2.2% for SA.
Obviously, the variation of VC in the determination was very
great when its kept time was more than 15 h (for example, the
RSD value (n = 5) was 67.8% during 96 h). After 48 h, VC had
been decomposed to approximately 69.9%. This was because of
the VC being a stronger reducer that can easily be oxidized by
oxygen in air when the system pH is less than 4. Thus, one possi-
bility is that VC can be determined timely (time < 15 h) in an anal-
ysis. The results are summarized in Table I, and a chromatogram
is shown in Figure 4.
Determination of sample
Cores of Fructus mume were removed. At 50C, the cored
Fructus mume was sufficiently dried. After that, cored Fructus
mume was ground to powder and the powder was filtered
through a screen (40 hole/inch). The treated powder was dried
again until constant weight at 50C. The powder (1.0000 g) was
accurately weighed, and it was extracted with approximately
50 mL water for 2 h according to reference 11. The extracted solu-
tion was cooled and filtered. The sediment was washed three
times, and each time approximately 5 mL distilled
water was used. The eluted and filtered solutions
were mixed together and completely transferred
into a volumetric bottle (100 mL), and some of the
mobile phase was added to the bottle until the
mark was reached. Thus, the analytical solution of
the sample was obtained.
The sample solution was injected into the chro-
matographic column until it reached 5 L.
Organic acid peaks were identified by comparing
their retention times in the sample solution with
that of the standard solution, and the contents
were quantitated by comparing their peak areas in
Table I. Linearity, Detection Limit, and RSD
%Intraday %Interday
%RSD RSD RSD
Constituents Equations r Linear ranges Limits (n = 11) (n = 5) (n = 5)
OA y = 314004x + 2041 0.9999 0.054.7 g 0.01 g 0.25 0.45 2.80
TA y = 93616x 1027 0.9999 0.1110.5 g 0.01 g 0.99 1.10 2.10
MA y = 23076x + 2028 0.9999 0.11411.4 g 0.05 g 1.68 1.69 1.70
VC y = 83833x + 5832 0.9999 0.0333.30 g 0.01 g 0.99 1.23 67.8
CA y = 43549x 33 0.9998 0.15515.5 g 0.03 g 0.61 0.86 6.70
SA y = 18779x 6209 0.9996 0.19419.4 g 0.10 g 1.23 1.80 2.20
Table II. Determination Results of Effective Components in the Botanic Fructus mume
Sample %OA %TA %MA %VC %CA %SA %Total acid OA/TA/MA/VC/CA/SA
Contents of components in
Fructus mume (Fujian) 0.554 0.023 6.44 0.84 16.66 4.75 29.27 1:0.04:11.63:1.56:30.07:8.57
Contents of components in
Fructus mume (Sichuan) 0.098 0.243 3.53 0.012 6.99 4.3 15.17 1:2.48:36.02:0.12:71.33:43.88
Figure 5. RP-HPLC chromatogram of the extracted solution of Fructus mume:
OA, 1; TA, 2; MA, 3; VC, 4; unknown substances, 5 and 6; CA, 7; and SA, 8.
Journal of Chromatographic Science, Vol. 40, January 2002
39
the sample solution with that of known standards. This process
was repeated three times with each sample. The results are shown
in Table II, and a chromatogram is shown in Figure 5.
Interference
For the previously described chromatographic conditions, the
smallest separation degree between two peaks was greater than
1.5 (according to the calculation procedure in reference 15). This
conclusion indicated that the impurity in the extracted solution
of the sample did not interfere in the determination of the six
organic acids component (shown in Figure 5).
Investigation of recovery
In order to evaluate the accuracy of this method for the deter-
mination of the six compounds in Fructus mume, recovery
studies were carried out. The recoveries of six constituents were
99.8% to 102.5% for OA, 99.6% to 101.1% for TA, 99.8% to
103.2% for MA, 99.9% to 105.3% for VC, 99.8% to 102.3% for CA,
and 100.1% to 101.5% for SA (shown in Table III). These results
indicated that the accuracy of the method is higher for the deter-
mination of OA, TA, MA, VC, CA, and SA in Fructus mume.
Conclusion
This study demonstrated that the use of RP-HPLC for the deter-
mination of six organic acids in Fructus mume can be success-
fully applied to the determination of these organic acids in other
botanics. Although a UV conductor suffers poor detection limits
for the determination of the six effective compounds in Fructus
mume, it offers the advantages of simple procedures. Therefore, it
is a suitable method for the routine quality-control analysis of
botanics containing the organic acids.
Acknowledgments
The authors gratefully acknowledge Prof.
Wan Xiuqin for providing the HPLC appa-
ratus.
References
1. Great Dictionary of Chinese Traditional and
Herbal Drugs, 2nd ed. Shanghai People Press,
Shanghai, 1977, Vol. 1, p 464.
2. Shi-Zhen Li. Bencao Gangmu, 2nd ed. People
Health Press, Beijing, China, 1982, Vol. 1, p 1736.
3. Jing-Tang Sun. The action of Fructus mume for the
roundworm and gallbladder. Chin. Trad. Herb.
Drugs 18(4): 28 (1987).
4. Hong-Mei Shen, C. Qiao, and Z. Su. The research
and development of Fructus mume in chemistry,
pharmacology and clinical medicine. Chin. Trad.
Pat. Med. 15(7): 35 (1993).
5. Jian-Xiu Du, Yin-Huan Li, and Jiu-Ru Lu.
Determination of ascorbic acid by on-line cou-
pling chemiluminescence. J. Shaanxi Normal Univ. 28(4): 8183
(2000).
6. R.L. Veuzey and T.A. Nieman. Chemiluminescent determination of
clinically important organic reductants. Anal Chem. 51: 2092
(1979).
7. Man-Liang Feng and Jiu-Ru Lu. Determination of ascorbic acid by
chemiluminescence method with copperlominol. Chin. J. Anal.
Chem. 23(1): 7072 (1995).
8. Fu-Chang Wang, Wei Qin, and Zhu-Jun Zhang. Flow-
injection/chemiluminescence sensor for the determination of
ascorbic acid. Chin. J. Anal. Chem. 25(11): 125558 (1997).
9. Zhan-Guo Chen and Jiu-Ru Lu. Determination for free total content
of organic acid by direct potential titration in compound Chinese
herbal medicine containing Fructus mume. J. Shaanxi Normal Univ.
28(4): 7880 (2000).
10. Geng-Qing Yang, Yan Zhu, and Xiao-Ping Zhuang. Simultaneous
determination of ascorbic acid, saccharine, benzoic acid and
ascorbic acid in beverages by high performance liquid chromatog-
raphy. Chin. J. Chromatogr. 7(5): 31821 (1989).
11. Hong-Mei Shen, C. Qiao, and C. Su. Quantitative dynamic analysis
of organic acid in Fructus mume by isotachophoresis. Chin. Pharm.
J. 30(3): 133 (1995).
12. Gang Chen, Xiang-Huan Ding, and Jan-Nong Ye. Determination of
rutin and L-ascorbic acid in pharmaceutical preparations and fruit
juices by capillary zone electrophoresis with electrochemical detec-
tion. Chem. J. Chin. Univ. 21(9): 136468 (2000).
13. Guang-Zhong Wang, Yang Qiu, Jing-Bing Chen, Xiao-Chuan Ye,
and Dan Li. Determination of succinic acid in Pheretima
aspergillum (E. Perrier) by TLC-scanner. Chin. J. Pharm. Anal. 17(4):
274 (1997).
14. Wang Tianzhi, Li Yongmei, and Wang Zhixiao. Determination of
three kinds of organic acid in honeysuckle flower by RP-HPLC.
Chin. J. Pharm. Anal. 20(5): 29396 (2000).
15. Chinese Pharmacopoeia, 6th ed. Guangdong Science and
Technology Press, Guangzhou, China, 1995, Vol. 1, p 38 and
appendix.
Manuscript accepted September 10, 2001.
Table III. Recovery of Organic Acids Added to the Botanic Fructus mume
Components Added (g) Recovered (g) %Recovery (n = 3)
OA 0.233 0.239 102.5
0.466 0.468 100.5
2.330 2.325 99.8
TA 0.526 0.524 99.6
1.053 1.055 100.2
5.260 5.260 101.1
MA 0.570 0.588 103.2
1.140 1.146 100.5
5.700 5.689 99.8
VC 0.165 0.174 105.3
0.330 0.333 101.0
1.650 1.648 99.9
CA 0.775 0.774 99.8
1.550 1.569 101.2
7.750 7.928 102.3
SA 0.972 0.987 101.5
1.944 1.954 100.5
9.720 9.730 100.1