BIOCHEXlICAL hIEDICINE 11, 49-59 ( 1974)

A New Fluorometric Analysis of Citric Acid

MASATAKE HORI, TADAHIKO KOMETANI, HAYAO UENO.
AND HIROSHI MORIMOTO
Chemical Research Laboratories, Takeda Chemical Industries, Ltd.,
&so, Higashiyodogawa-ku, Osaka, Japan

Received November 7, 1973

Since the citric acid is an important member of TCA cycle, its content
in bIood is considered to vary with many diseases (1, 2). For instance,
(i) hematic citric acid generally increases in the case of diabetes (3))
and its relation to lipogenesis was examined (4)) (ii) the relationship be-
tween calcium and citric acid in blood has been examined for bone me-
tabolism, and administration of thyrocarcitonine resulted in decrease of
its level (5), ( 111
“‘) some periodontal diseases were thought to be related
to hypercitricemia (6). For the determination of very small amounts of
the citric acid such as that in blood, a high sensitive, specific and simple
method is required, although several quantitative analyses of citric acid
have been reported (7-14). The Auorometric methods already described
( 15, 16) are highly sensitive but not specific, because aconitic acid and
isocitric acid in blood also react to produce fluorescent substances.
Recently Olthoff et aI. (17) reported that D-penicillamine produced on
heating with citric acid the fluorescent substances I, II, and III ( Chart I ) .

\
I In
CHART 1. Fluorescent substances produced by the reaction of citric acid and D-
penicillamine [reported by Olthoff et al. ( 17) 1.

The authors expected that the fluorescence of reaction product(s) bc-

comes more intense by substitution of aromatic ring for alkyl group in
n-penicillamine, hence we studied this reaction for the microanalysis of
49
Copyright @ 1974 by Academic Press, Inc.
All rights of reproduction in any form reserved.
50 HOHI ET AL.

citric acid and established more sensitive and selective method by using
o-aminothiophenol (o-ATH) .

MATERIALS AND METHODS

Citric acid, ethylacetate, sodium chloride, phosphoric acid, and meta-

phosphoric acid were analytical reagent grade and were obtained from
Wako Pure Chemical Industries Co., Ltd.
o-ATH solution. o-ATH was dissolved in 52.5% w/v phosphoric acid to
obtain a 0.75% w/v solution.
Metaphosphoric acid solution. Metaphosphoric acid was dissolved in
water to obtain a 2% w/v solution.
Instruments. Fluorescence intensity was determined with an Aminco-
Bowman spectrophotofluorometer. The reaction mixture was heated in a
Tominage Model S-90N sterilizer.
Procedure. To 0.05 ml of sample (blood, plasma or authentic citric
acid solution), 0.95 ml of water and 1.0 ml of the metaphosphoric acid
solution were added. The mixture was shaken for 5 min and centrifuged
(3000 rpm, 10 min) to remove protein. One milliliter of the supernatant
was taken into a test tube and 2 ml of the o-ATH solution was added.
After replacement with nitrogen gas, the test tube was stoppered and
allowed to react at 125°C for 15 hr in a sterilizer. The reaction mixture
was cooled with water, and sodium chloride (3 g) and ethylacetate (4
ml) were added. Then the test tube was shaken for about 5 min. The
ethylacetate layer was separated and its fluorescence intensity was mea-
sured with excitation at 415 nm and emission at 450 nm. The citric acid
content was calculated from the calibration curve obtained with l-10
mg/dl of authentic citric acid solution,

PH

FIG. 1. Effect of pH on the formation of fluorescence. After heating a solution of

citric acid (8 ag) and o-ATH ( 15 mg) in 3 ml of 0.2 M H8POI-NaOH (pH 0.5-12)
at 125°C for 6 hr, the mixture was extracted with ethylacetate (4 ml) and the
fluorescence of the extract was measured with excitation at 415 nm and emission at
450 nm. In all figures, relative fluorescence is expressed in highest sensitivity of the
spectrophotofluorometer used, and corrected for blank.
 DETERMINATION OF CITRIC ACID 31

0 IO 20 30 40 50
HSPO, CONC. (%W/V)

FIG. 2. Effect of phosphoric acid concentration on the formation of fluorescence.

Procedure for the assay is the same as the legend for Fig. 1, except using 5-50s
H,PO, in the place of 0.2 M HsP04-NaOH.

RESULTS AND DISCUSSION

The intense fluorescence was observed when the reaction was pro-
ceeded in acidic phosphate buffer (pH 0.5) as shown in Fig. 1, and the
most intense fluorescence was obtained in 35% phosphoric acid solution
(Fig. 2). o-ATH was favorably soluble in aqueous solutions at this pH.
The fluorescence intensity increased in proportion to the o-ATH concen-
tration up to that of about 15 mg/3 ml of reaction mixture, and it tended
to decrease in concentration over that, hence the amount of o-ATH was
decided to be 15 mg/3 ml (Fig. 3). The fluorescence intensities were de-
termined at various reaction temperatures at the reaction time of 6 hr.
The intensity increased evidently at the reaction temperature about
100°C (Fig. 4). But the reaction temperature in this study was decided
to be 125°C which is the maximum temperature of the sterilizer used. At
this reaction temperature, the fluorescence intensity reached a plateau

OO_1,,,,
O-ATH CONC. img/3ml REACT. MIX.)

Frc. 3. Effect of o-ATH concentration on the fluorescence intensity. Procedure for

the assay is the same as the legend for Fig. 1, except that the reaction was performed
with I-25 mg of o-ATH in 35% HIPO,.
52 HORI EX AL.

REACTION TEMP. I’CI

FIG. 4. Effect of reaction temperature on the formation of fluorescence. Procedure

for the assay is the same as the legend for Fig. 1, except that the reaction was per-
formed at 75-150°C in 35% H,PO,.

after 15 hr (Fig. 5). From the result, reaction time was settled to 15 hr
but shorter time also could be used, if the result is needed in haste.
In the case of blood or plasma samples, the blank values of the reaction
mixtures were increased more or less even after deproteinization. To
avoid this defect, the fluorescent substance was attempted to be extracted
with several organic solvents. Although it could be extracted with n-
butanol, diisopropyl ether, ethylacetate, etc., ethylacetate was used since
it can be separated easily from aqueous layer, moreover the intensity in-
creased about four times as much as that before extraction.
The fluorescence of the ethylacetate solution was stable for 24 hr at
room temperature. Excitation and emission spectra of the extracted so-
lution was shown in Fig. 6, and we choose the excitation wave length at

REACTION TlME IHR)

FIG. 5. Time course of the formation of fluorescence at 125°C. Procedure for the
assay is the same as the legend for Fig. 1, except that the reaction was performed for
3-24 hr in 35% H3P0d.
 DETERMINATION OF CITRIC ACID ,i:?J

350 400 450 500 550

WAVELENGTH INMI

FIG. 6. Esc;tafion and emission spectra of the ethylacetate extract.

415 nm, blank value at which was weaker than that at 400 nm, and
emission wave length at 450 nm.
By this method, a linear relationship was obtained between the fluores-
cence intensity and citric acid in concentration ranging from 0.570
mg/dl, but not in higher concentration than that (Fig. 7).
Blood contains various substances, such as organic acids, amino acids,
sugars, in addition to citric acid. These substances listed in Table 1 hardly
make the fluorescences, except aconitic acid. Each trans- and cis-aconitic
acid gave positive error about 9% and 7% of citric acid, but it may not be
concerned because the citric acid predominated over aconitic acid in
blood and serum. The volume of sample blood was changed between
0.01 and 0.2 ml (Table 2). The results indicate that there are remarkable
difference in too small samples (less than 0.02 ml) and too large samples
(more than 0.2 ml). Hence optimum volume of the saple should be 0.05
0.1 ml. The recovery of citric acid added to human blood was between
95 and 112% (Table 3). The precision of this method was 2.51% as the

ClTRlC ACID CONC. IN SAMPLE tmgldll

FIG. 7. Calibration curve for citric acid.

54 HORI ET AL.

TABLF: 1
COMPARISON OF RI~:L.ITIVE FLUOKESCI~N~W OIST.IINED IVY THE REACTIOS OF
o-ATH W-ITH V.i~rous COMPOUNDS
-
Compound R.F. Compound R.F.

Cikic acid 100.00 Ribose 0.28

t,rans-Aconitic acid 8.94 Rhamnose 0.04
cis-Aconitic acid 6.36 Xylose 0.25
iso-Citric acid 0.93 Arabinose 0 25
Succinic acid 0.00 Fucose 0. on
Maleic acid 0.00 Maltose 0.13
Fumaric acid 0. 00 Trehalose 0.15
a-Ketoglutaric acid 0.00 Cellobiose 0.11
Pyruvic acid 0.00 Glucosamine 0.00
Oxalacetic acid 0.56 Galact.osamine 0.04
Oxalsuccinic acid 0 00 ‘v-Aret,ylglucosamine 0.17
Malic acid 0.00 (:lucuronic acid 0.48
a-Hydroxybutyric acid 0.00 Galacturonic acid 0.45
P-Hydroxybutyric acid 0.06 Idonic acid 0.22
Ascorbic acid 0.23 Gluconic acid 0 .20
Lactic acid 0.00 Glu tamine 0.00
Acet,ic acid 0.00 Glutamic acid 0.02
Uric acid 0.00 Asparagine 0.01
Ethyl acetoacet’ate 0.00 Asparl ic acid 0.04
Glucose 0.20 Serine 0 03
Mannose 0.09 Glutat.hione (red.) 0.41
Galactose 0.10 Glutathione (ox.) 0.1”
Fruct,ose 0 09

The relat,ive fluorescence per mole was compared with c.it,ric arid.

coefficient of variation, which was calculated from 10 repetitive data of

definite human blood (Table 4). Citric acid levels in plasma and blood
of various animals were determined by both the author’s method and the
pentabromoacetone calorimetry (8), which is one of the most useful

TABLE 2
I<FFMZT OF S.~MPLING VOI.UMX OF HUMAN BLOOD

Citric acid levels determined (mg,/dl)

Blood vol
ml Exp. I Exp. 2

0.01 1.2 3.6

0 .02 1.5 2 0
0.05 1.16 1.12
0.1 1.16 1.14
0. 2 I) 07 1 0.5

From 0.01 lo 0.2 ml of definite hlunan blood samples were determined in every thrice
al~d the repr~,Jl~cihilities were compared.
 DETERMINATION OF CITRIC ACID

TABLE 3
I~IGCUV~:RY OF CITRIC ACID ADDED TO HUMAN BLOOD

Amolmt of citric acid (mg,‘dl)

Sample -
?;o. Added Fo\md

(‘on tro1 0.00 1.13

I 0.50 1 .69
2 1.00 2.15
3 2.00 3.lh
4 5.00 6 0.5
5 10.00 10.67

Different concentration of citric acid was added in blood sample (0.05 ml) and it was
determined by the analytical procedure presented. The recovery was calcnlated after
sttbtra&)n of control value.

quantitative methods for citric acid (Table 5). The levels determined
with the author’s method were slightly high. The reason for this dis-
crepancy is not clear, but it can be assumed that the inhibitors for these
methods are different and affect their precision.
The authors isolated the fluorescent substances from a reaction mixture
according to the procedure presented in Chart 2. The yields, fluorescence
spectra and thin-layer chromatograms showed that the main fluorescent
product is IV, and small amount of by-product V is also produced in the
reaction mixture. IV was yellow needles and sublimated at about 250°C.

TABLE 4
Ib3PRODUCIBILITY OF THE PKESENT ~IE:THoD

Citric. acid levels determined

Exp. ivo. Cmg/dl j

1 I. 10
2 1.1:;
3 1,lR
4 1.E
-5 1.19
6 1. l!)
7 1 2
s 1.1s
0 I .I4
10 1.22

Average (?) I .lS6

$I) (‘u) 0.030
Coefficient of variation 2 *51’;;

Definite human blood sample was determined 10 times.

56 HORI ET AL.

TABLE 5
CITRIC ACID LEVELS IN PLASMA AND BLOOD SSMPLES

Cit,ric acid levels determined (mg/dl)

Present Pentabromoacetone
&Sample Race method method

Human 2.51 3.33

Plasma Rabbit 5 54 -5 .05
Rat 6.20 ,569

Human 1.57 1.43

Blood Rabbit 3.53 3.23
Rat 4.74 4.39

Each measurement was performed twice, by present met,hod and Pentabromoacetone

method (8). Each datum is average of two determinations.

CHART 2. Isolation process of the fluorescent substances IV and V from a reaction

mixture.
 DETERMINATION OF CITRIC ACID .57

COOH

\\ ’
+ PO-ATH ’
\
- 6 H,O \
\
\
+ o-ATH

V
CHART 3. Presumablereaction processof citric acid and o-ATH.

The structure was assigned to be I-oxopyrido[2,1-b]-benzothiazole-3-car-

boxylic acid (Chart 3) from the results of elemental analysis, MS,l IR,’
UV1 and NMR’ (Table 6). V was yellow-green scales and melted at
245°C. The structure was assigned to be 1-0x0-3-( 2’-benzothiazole)-
pyrido[2,1-b]b enzothiazole (Chart 3) from the results of the physico-
chemical data (Table 6), and a synthesis from the IV through a
chlorination of carboxyl group by thionylchloride and a reaction with
an additional o-ATH, but IV did not react directly with o-ATH to
produce V.
From the excitation and fluorescence spectra, and Rf value on thin-layer
chromatograms the aconitic acid seems to produce the same fluorescent
substance as that of the citric acid, but their intensities are extremely
weak. The reason that isocitric acid hardly give the fluorescent substance
is ascribed to its easy transformation in a stable fivemembered lactone
under this acidic condition,
Specificity of the conventional fluorometric methods is insufficient for
citric acid in blood because aconitic acid and isocitric acid give the flu-
orescent substancesof the same intensity as those of citric acid (1,15, 16).

’ Abbreviation: MS: massspectrum,IR: infrared spectrum,WV: ultraviolet spec-

trum, NMR: nuclear magneticresonancespectrum.
 TABLE 6
PHYSICOCHIGMICAI, l).\~.\ OF THE FLTJORWCENT SUFGWANCES IV .INI) 1’

Sample MS (m/eja Ilt icm-l)u uv @rn,x )‘I NMIZ (6)”

-. ~~ ..~.. -
(methyl ester)
245 (la+) 3300-2500 230 3.9 (s)b(methyl)
217 (>\I+-CO) 1730 (COOH i 248 6.9 (d)(H4)
IV 200 (M+-COOH) 1640 (CO) 392 7.3 (d)(W)
172 {IM*-(CO + OH)\ 412 (shoulder) 7.6 (m)WJ)
S. 0 (d-d)(H6)
0.4 (d-d)(H9)

334 (M+) 3060 25s 7.0 (dF

:m (M+-(x)) 1670 (CO) 320 7.5 (d)
V 405 7.7 (m)
S.2 (m)
9.2 (mi

11Instruments \lsed are: MS: HITBCHI RMS-4, IR: HITACHI 215, UV: HITACHI EPS-2, N&11{: VARIAX H,4-100.
h Abbreviation: Y: singlet, d: doublet, d-d: donble-doublet, m: multiplet, >I+: molecular ion.
1 Not assigtled because the peaks overlapped.
 DETERMINATION OF CITRIC ACID 59

The fluorescent substance formed
rived from other acids as shown in
The long reaction time (15 hr)
cause the reaction mixture is only
lizer and the reaction time can
strictly required.
by the present method was hardly dc-
Table 1.
required is not practically trouble be-
allowed to stand overnight in a steri-
be shortened if the sensitivity is not

SUMMARY

A new fluorometric method for analysis of citric acid in blood and

plasma was established, where the citric acid react with o-aminothio-
phenol in 35% phosphoric acid solution, at 125°C for 15 hr. The fluores-
cent substance was extracted by ethylacetate and measured with excita-
tion at 415 nm and emission at 450 nm. Only 0.05 ml of human blood or
plasma is required.

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