Spectrophotometric determination of tiopronin in pharmaceutical samples by phosphorus molybdenum blue

Article abstract of DOI:10.14233/ajchem.2014.15375

A novel method is established for spectrophotometric determination of tiopronin by phosphorus molybdenum blue. It is based on the fact that PO ₄^3- reacts with Mo₇O₂₄^6- in 0.47 mol/L H₂SO₄ solution to form a product of phosphorus-molybdenum heteropoly acid ([H₂PMo₁₂O ₄₀]^-) which is reduced to phosphorus molybdenum blue (H₃PO₄·10MoO₃·Mo ₂O₅) by tiopronin. The absorbance of phosphorus molybdenum blue is measured at the absorption maximum of 730 nm and the amount of tiopronin can be determined based on this absorbance. Absorbance is linear with the concentration of tiopronin in the range of 1.6-40 μg/mL and the regression equation is A = -0.1193 + 0.01927c (μg/mL) with a correlation coefficient of 0.9989 and the apparent molar absorption coefficient of 3.14 × 10³ L/(mol cm). The detection limit and the RSD are 1 μg/mL and 0.92 %, respectively. Thus, the method is validated and commonly available to determine tiopronin in pharmaceutical samples.

Full text of DOI:10.14233/ajchem.2014.15375

Asian Journal of Chemistry; Vol. 26, No. 2 (2014), 331-334
http://dx.doi.org/10.14233/ajchem.2014.15375
Spectrophotometric Determination of Tiopronin in
Pharmaceutical Samples by Phosphorus Molybdenum Blue
2
2
2
1,*
BINGLIN FAN1,2,*, YINGLING WANG , JIE YANG , TIANJUN NI and QUANMIN LI
1College of Chemistry and Environmental Science, Henan Normal University, Key Laboratory of Environmental Pollution Control Technology of
Henan Province, Xinxiang 453007, Henan Province, P.R. China
2School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang 453003, Henan Province, P.R. China
*Corresponding authors: Fax: +86 373 3326336; Tel: +86 373 3326335; E-mail: mercury6068@hotmail.com; binglinfan2006@126.com
Received: 26 February 2013;
Accepted: 3 May 2013;
Published online: 15 January 2014;
AJC-14536
A novel method is established for spectrophotometric determination of tiopronin by phosphorus molybdenum blue. It is based on the fact
that PO43- reacts with Mo7O246- in 0.47 mol/L H2SO4 solution to form a product of phosphorus-molybdenum heteropoly acid ([H2PMo12O40]–)
which is reduced to phosphorus molybdenum blue (H3PO4·10MoO3·Mo2O5) by tiopronin. The absorbance of phosphorus molybdenum
blue is measured at the absorption maximum of 730 nm and the amount of tiopronin can be determined based on this absorbance.
Absorbance is linear with the concentration of tiopronin in the range of 1.6-40 µg/mL and the regression equation is A = -0.1193 +
0.01927c (µg/mL) with a correlation coefficient of 0.9989 and the apparent molar absorption coefficient of 3.14 × 103 L/(mol cm). The
detection limit and the RSD are 1 µg/mL and 0.92 %, respectively. Thus, the method is validated and commonly available to determine
tiopronin in pharmaceutical samples.
Keywords: Phosphorus molybdenum blue, Tiopronin, Spectrophotometry.
From the viewpoint of application, visible spectropho-
tometry has some advantages, for example, its equipment is
simple and its operation is easy. In this paper, we have deve-
loped a novel method named phosphorus molybdenum blue
spectrophotometry for the determination of tiopronin in pharma-
ceutical samples. Phosphorus molybdenum blue, mainly used
for the determination of phosphorus in the sample9-11, has not
been reported for the determination of tiopronin. The experi-
INTRODUCTION
Tiopronin i.e., N-(2-mercaptopropionyl)-glycine (TP), a
synthetic thiol compound, has been used as a hepatoprotective
agent, an antidote to heavy metal poisoning and a radioprotec-
tive agent. It has also been successfully applied to prevent
kidney injury and to treat cystinuria and rheumatoid arthritis1,2.
However, tiopronin has a relatively high frequency of side
effects, such as the loss of taste or stomach upset.
6-
ment indicates that PO43- reacts with Mo7O24 in 0.30 mol/L
The thiol group can be easily oxidized to disulfides either
as a dimer or mixed forms with endogenous thiols. Thus,
quantitative analysis of the tiopronin can be almost impossible,
unless the thiol group can be stabilized in pharmaceutical
sample. Up to now, several methods have been reported for
the determination of tiopronin such as gas chromatography-
mass spectrometry (GC-MS) method3, chemiluminescent
method4, LC-MS-MS methods2, HPLC-ESI-MS methods1,5,
fluorimetric methods6,7 and electrochemical oxidation by
amperometric flow injection analysis8.
H2SO4 solution to form a product with phosphorus-molybdenum
heteropoly acid ([H2PMo12O40]–) which is reduced to phosphorus
molybdenum blue (H3PO4·10MoO3·Mo2O5) by tiopronin. The
absorbance of phosphorus molybdenum blue is measured at
the absorption maximum of 730 nm and the amount of tiopronin
can be calculated based on this absorbance. In contrast with
the above methods, this method is simple, fast and does not
require expensive equipment and facilities. With the accredited
specificity, lower limit of quantification, accuracy and precision,
the method could be used to determine tiopronin in the pharma-
ceutical sample.
However, some laborious sample processing, expensive
apparatus and complex derivation steps are involved in all of
the above mentioned methods. So, it is necessary for laboratory
to generate a commonly available method for the determination
of tiopronin in pharmaceutical sample.
EXPERIMENTAL
The main solutions were prepared as follows. A standard
solution of 1.000 g/L tiopronin (Xinyi Medicine Plant,

332 Fan et al.
Asian J. Chem.
Xinxiang China) was prepared and preserved at 4 ºC without
light. A 14.13 mg/mL stock Mo(VI) solution was prepared by
dissolving 26 g of ammonium molybdate ((NH4)6Mo7O24·
4H2OA.R., Shanghai colloid chemical plant, Shanghai, China)
and mixing with 460 mL of 1:1 (v/v) H2SO4 (Luoyang Haohua
Chemical Reagent Co., Ltd. Luoyang, China), then transferred
into a 1000 mL standard flask and was diluted to the mark by
using distilled water. The concentration of H2SO4 is 4.23 mol/L
in the stock solution. A 1 mg/mL stock PO43- solution was
obtained by dissolving 1.4329 g g of potassium dihydrogen
phosphate (A.R., Beijing red star Chemical Plant, Beijing,
China) in 1000 mL standard flask with distilled water. Unless
specially stated, all reagents used were of analytical grade and
all solutions were prepared with distilled water.
0.24
0.20
0.16
0.12
0.08
0.04
0.0
0.2
0.4
0.6
0.8
1.0
VD/(VR+VD)
Fig. 1. Determination of the complex formation by the continuous variation
method of equivalent mole
A model T6 UV/VIS spectrophotometer (Beijing purkinje
general instrument Co., Beijing, China) was employed for
scanning the absorption spectrum, in addition, a 722 grating
spectrophotometer (Xiamen Analytical Instruments Plant,
Xiamen, China) for photometric measurements and a model
CS501 super constant temperature instrument (Chongqing
Experiment Equipment Plant, Chongqing, China) for tempe-
rature control.
12MO7O + 7H2PO−4 + 72H+ →7[H2PM012O40 ]− + 36H2O
6−
24
O
HS
[H2PMo12O40] -
2
+
HN
Procedure: At first, 3 mL of 1 mg/mL PO43- was added
into a 12.5 mL colour comparison tube and diluted to two-
thirds of the mark by using distilled water. Secondly, it was
transferred by 1.40 mL of 14.13 mg/mL Mo(VI) and 1 mL of
200 µg/mL tiopronin, respectively, then allow to shaking well
after the solution was diluted to the mark with distilled water
and the concentration of H2SO4 is 0.47 mol/L in the reaction
solution. Finally, after standing for 40 min at 40 ºC in water
bath, the absorbance was measured at 730 nm against a reagent
blank prepared in the same way without tiopronin.
OH
O
O
O
S S
HN
O
NH
O
OH
HO
+ H3PO4·10MoO3·Mo2O5 + OH−
Absorption spectrum: According to the procedure, the
absorption spectrum of phosphorus molybdenum blues formed
from the reaction of PO43- and Mo(VI) against the reagent blank
and the reagent blank and tiopronin against distilled water are
shown in Fig. 2. It can be seen that the product of phosphorus
molybdenum blue has an absorption peak at 730 nm (a). In
comparison, the absorbance of tiopronin (b) and the reagent
blank (c) are almost zero in the range of 450-800 nm. In order
to obtain higher sensitivity, all the following measurements
were carried out at 730 nm against the reagent blank.
Interference of PO43- and Mo(VI): In order to study the
influence of PO43- on absorbance, 14.13 mg/mL Mo(VI) and
200 µg/mL tiopronin were kept as 1.4 mL and 1 mL, respec-
tively. The amount of 1.00 mg/mL PO43- ranging from 1 to 5
mL was studied. It can be seen from Fig. 3 that the absorbance
increased with the amount of PO43- until reaching maximum
at 3 mL. When the amount of PO43- exceeded 3 mL, the absor-
bance almost did not change. This result clearly showed that
the formed [H2PMo12O40]– in the solution reached a maximum
and all tiopronin was completely oxidized. Meanwhile, the
formed phosphorus molybdenum blue reached its maximum.
So 3 mL of 1 mg/mL PO43- was selected for further work.
Keeping the amount of 1 mg/mL PO43- at 3 mL and 200
µg/mL tiopronin at 1 mL, the influence of Mo(VI) on the
absorbance is presented in Fig. 4. The amount of 14.13 mg/mL
Mo(VI) ranging from 0.4-2 mL was studied. It can be seen
RESULTS AND DISCUSSION
Discussion of reaction mechanism: PO43- reacts with
Mo7O246- to form a product with [H2PMo12O40]–. Subsequently,
[H2PMo12O40]– is reduced to phosphorus molybdenum blue
(H3PO4·10MoO3·Mo2O5) by tiopronin due to the reducibility
of the sulfhydryl group and tiopronin is oxidized to form
disulfide12.
The continuous variation method of equivalent mole
was used to determine the reaction stoichiometric ratio of
[H2PMo12O40]– and tiopronin. Keeping the total amount of
tiopronin (VD) and [H2PMo12O40]-–(VR) constant (VR + VD =
5 mL), whose concentration were both 1.23 × 10-3 mol/L, diffe-
rent amounts of tiopronin and [H2PMo12O40]– were transferred
into a 12.5 mL colour comparison tube and diluted to the mark
with distilled water. Then the absorbance of every solution
was measured. Absorbance had been plotted as function of
the VD/(VR + VD) ratio (i.e., mole fraction) (Fig. 1). According
to the intersection of two tangents, 2:1 of the reaction stoichio-
metric ratio of tiopronin and [H2PMo12O40]– was obtained.
Based on the continuous variation method of equivalent
mole, the reaction stoichiometric ratio of tiopronin and
[H2PMo12O40]– is 2:1, which is consistent with the number of
electronic transfer from the oxidation-reduction reaction of
tiopronin and [H2PMo12O40]–. Therefore, it seems to be
reasonable that reaction mechanism is as follow:

Vol. 26, No. 2 (2014)
Spectrophotometric Determination of Tiopronin in Samples by Phosphorus Molybdenum Blue 333
0.20
0.20
a
0.16
0.12
0.08
0.04
0.16
0.12
b
0.08
c
0.00
500
0.4
0.8
1.2
1.6
2.0
600
700
(nm)
800
V (mL)
Fig. 4. Influence of Mo(VI) on absorbance
λ
Fig. 2. Absorption spectrum of phosphorus molybdenum black
that the absorbance began to increase and became stable after
40 min. Furthermore, the absorbance can remain constant for
at least 1 h. Therefore, 40 min of reaction time has been
selected as the optimum.
0.21
0.20
0.19
0.18
0.17
0.16
Interference of the sulfuric acid: According to the
proposed procedure, the influence of the additional H2SO4 (0.50
mol/L) on the determination of tiopronin was studied. It can
be seen from Fig. 5 that the absorbance (A) decreased with
increasing the amount (vol) of H2SO4.A maximum absorbance
was obtained when H2SO4 was not added. The reason was that
oxidation potential of tiopronin increased with the increase of
acidity, which resulted in reducing the amount of phosphorus
molybdenum blue. It agreed with the phenomenon in Fig. 4
that the absorbance significantly decreased, when the amount
of Mo(VI) was above 1.4 mL. On the basis of the fact, a
conclusion can be drawn that the acidity played an important
role in the reducibility of tiopronin reducing the [H2PMo12O40]-
when the concentration of [H2PMo12O40]– was constant in the
solution.
1
2
3
4
5
V (mL)
Fig. 3. Influence of PO43- on absorbance
from Fig. 4 that the absorbance increased with the amount of
Mo(VI) until reaching maximum at 1.4 mL. As the amount of
Mo(VI) increased from 1.4 mL to 2 mL, the concentration of
H2SO4 in the solution increased from 0.47-0.68 mol/L,
increased by 0.21 mol/L, while the absorbance decreased from
0.206 to 0.148.According to “Discussion of reaction mechanism”,
increasing the concentration of H+ and Mo(VI) was of greatly
benefit to increasing the amount of [H2PMo12O40]– and the
oxidbillity of [H2PMo12O40]– was enhanced at the same time,
so the absorbance should increase with increasing the amount
of Mo(VI). But the experiment had indicated that the absorbance
significantly decreased when the amount of Mo(VI) was above
1.4 mL. This is attributed to the fact that oxidation potential of
sulfhydryl-containing compounds increased with the increase
of acidity, which made the reducibility of tiopronin reducing
the [H2PMo12O40]– in the solution decreased and the amount
of phosphorus molybdenum blue reduced. Thus, 1.4 mL of
14.13 mg/mL Mo(VI) was selected for the rest of this work.
Interference of temperature and reaction time: Keeping
other conditions constant, the effect of temperature on absorbance
was studied. The absorbance of product was determined at
different temperature (25, 30, 35, 40, 45 and 50 ºC). It was
found that the absorbance of product was greatly affected by
temperature and reached the top when the temperature was
40 ºC. So 40 ºC in water bath had been chosen for the optimal
experimental conditions.
0.20
0.19
0.18
0.17
0.0
0.5
1.0
1.5
2.0
V (mL)
Fig. 5. Influence of H2SO4 on absorbance
Interference of coexisting components: A systematic
study on the influence of excipients, carbohydrate, amino acids
and minerals was carried out for the determination of tiopronin.
The criterion for interference was a relative error of less than
5 % within analytical determination. The experimental results
indicated that 450 µg/mL sucrose and dextrose, 350 µg/mL
glutamic acid, 20.00 µg/mLAl3+, 32.00 µg/mL Mn2+ and Ca2+,
10.00 µg/mL Cd2+, 6.00 µg/mL Zn2+, 4.00 µg/mL Co2+, 0.30
mg/mL Na+, K+ and Cl–, 0.70 mg/mL Br–, 0.72 mg/mL NO3–
had no interference on the determination of tiopronin.
The absorbance of product was measured after standing
for different times in minutes at 40 ºC water bath. It was found

334 Fan et al.
Asian J. Chem.
TABLE-1
DETERMINATION RESULTS OF SAMPLES AND RECOVERY (n = 5, t0.05, 4 = 2.78)
Sample content
(µg/mL)
Proposed method
(µg/mL)
HPLC method
(µg/mL)
Added
(µg/mL)
Found
(µg/mL)
Recovery
(%)
RSD (%)
(n = 5)
Sample
1
2
3
4
5
5.00
5.08
9.86
4.97
9.98
5.00
5.00
5.07
4.85
9.87
9.67
15.14
101.4
97.0
98.7
1.6
1.4
1.0
0.8
1.0
10.00
10.00
15.00
20.00
10.12
15.09
19.88
10.05
15.07
15.03
10.00
10.00
15.00
96.7
100.9
Calibration curve: According to the proposed procedure,
a series of standard solutions of tiopronin was prepared.
Absorbance has been plotted as function of the concentration
of tiopronin.A linear relationship between the absorbance (A)
and the concentration (c) of tiopronin is obtained in the range
of 1.6-40 µg/mL. The equation of the linear regression is A =
-0.1193 + 0.01927c (µg/mL) with a linear correlation coeffi-
cient of 0.9989 and the apparent molar absorption coefficient
of indirect determination of tiopronin is 3.14 × 103 L/(mol cm).
Determination of reproducibility and limit of detection:
According to the procedure, the product was determined 11
times (n = 11) with a RSD of 0.92 %. Then, a reagent blank is
measured 11 times (n = 11) and a detection limit has been
obtained from three-time standard deviation of the reagent
blank divided by the slope of the linear regression equation,
the result is 1 µg/mL.
mance liquid chromatography method (HPLC). At the same
time, Low RSD for precision and high recoveries has been
obtained. As the sample in the experiment is real tablet, it also
shows that other components of the sample (starch, dextrin
and calcium stearate) do not affect the determination of
tiopronin with [H2PMo12O40]– and the results are satisfactory.
Conclusion
A novel method for the spectrophotometric determination
of tiopronin by phosphorus molybdenum blue has been
proposed. The method has been successfully applied to the
determination of tiopronin in pharmaceutical samples and
average recoveries are in the range of 96.7-101.4 % with
satisfactory results. In a comparison with previous methods,
the presented analysis procedure does not require any sophis-
ticated instruments and the method is simple, sensitive and
rapid. These merits make it applicable for common laboratories.
Apparent rate constant and activation energy: Under
the optimized experimental conditions, keeping the tempe-
rature at 40 ºC water bath, the absorbance of the solution was
measured under different reaction time in initial rate method.
-ln (Amax –A)/Amax was plotted as function of time (min) and a
line was obtained as -ln (Amax – A)/Amax = 0.05519 + 0.07208t
(min), with a linear correlation coefficient of 0.9969. Since
the quantity of PO43- and Mo(VI) was much more excessive
than that of tiopronin in the solution, whose concentration
variation was relatively small, the reaction could be regarded
as a pseudo first-order reaction. Therefore, the reaction rate
equation was d[product]/dt = k' [tiopronin], the apparent rate
constant (k'40 ºC = k'1 = 7.208 × 10-2 min) was obtained. Similarly,
under the selected conditions, keeping the temperature at 40ºC,
the apparent rate constant (k'35 ºC) was 6.086 × 10-2 min-1.
Then, in light of the Arrhenius formula (Ea = RT1T2/
(T2-T1)·ln k'2 /k'1) and different pairs of K-T data, the apparent
activation energy (Ea) of the indirect determination of tiopronin
was calculated to obtain the result of 27.13 kJ/mol, which was
less than 40 kJ/mol. It indicated that the reaction could take
place easily13.
ACKNOWLEDGEMENTS
The authors are grateful for the financial support from
the Basic and Front-line Technological Research Project of
Henan Province (122300410412, 132300410205).
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elaborated method and determine tiopronin in pharmaceuticals,
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