Flow-injection chemiluminescence determination of dihydralazine sulfate in serum using luminol and diperiodatocuprate (III) system

Article abstract of DOI:10.1016/j.saa.2009.09.044

A novel flow-injection chemiluminescence (CL) method for the determination of dihydralazine sulfate (DHZS) is described. The method is based on the reaction of luminol and diperiodatocuprate (K₂[Cu(H₂IO₆)(OH)₂],

Full text of DOI:10.1016/j.saa.2009.09.044

Spectrochimica Acta Part A 75 (2010) 77–82
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Flow-injection chemiluminescence determination of dihydralazine sulfate in
serum using luminol and diperiodatocuprate (III) system
Chunyan Yang, Zhujun Zhang∗, Jinli Wang
College of Chemistry and Materials Science, Shaanxi Normal University, Xi’an 710062, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel flow-injection chemiluminescence (CL) method for the determination of dihydralazine sul-
fate (DHZS) is described. The method is based on the reaction of luminol and diperiodatocuprate
(K2[Cu(H2IO6)(OH)2], DPC) in alkaline medium to emit CL, which is greatly enhanced by DHZS. The pos-
sible CL mechanism was first proposed based on the kinetic characteristic, CL spectrum and UV spectra.
The optimum condition for the CL reaction was in detail studied using flow-injection system. The experi-
ments indicated that under optimum condition, the CL intensity was linearly related to the concentration
Received 14 January 2009
Received in revised form 23 August 2009
Accepted 25 September 2009
Keywords:
Chemiluminescence
Diperiodatocuprate
Dihydralazine sulfate
Serum
of DHZS in the range of 7.0 × 10−9 to 8.6 × 10−7 g mL−1 with a detection limit (3ꢀ) of 2.1 × 10−9 g mL−1
.
The proposed method had good reproducibility with the relative standard deviation 3.1% (n = 7) for
5.2 × 10−8 g mL−1 of DHZS. This method has the advantages of simple operation, fast response and high
sensitivity. The special advantage of the system is that very low concentration of luminol can react with
DPC catalyzed by DHZS to get excellent experiment results. And CL cannot be observed nearly when lumi-
nol with same concentration reacts with other oxidants, so luminol–DPC system has higher selectivity
than other luminol CL systems. The method has been successfully applied to determine DHZS in serum.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
oxidants. In the experiment, it was found that the CL intensity of
luminol–DPC reaction can be greatly enhanced by DHZS. Under the
Dihydralazine sulfate (DHZS, Fig. 1), which contains two
hydrazine groups, is a well-known antihypertensive compound.
It can induce peripheral vasodilatation, thereby reducing periph-
eral vascular resistance and thus lowering elevated blood pressure.
Several methods have been reported for its determination in phar-
maceutical preparations, including spectrophotometry [1,2], liquid
chromatography [3–5] and also CL method [6–10].
Flow-injection CL method is known to be a powerful analyt-
ical technique that possesses low detection limit, a wide linear
dynamic range and relatively simple and inexpensive instrumenta-
tion [11,12]. Trivalent copper is in the highest oxidation state which
can be stabilized by chelating with suitable polydentate ligands.
Although there are many reports concerning the use of diperi-
odatocuprate (III) (DPC) as oxidant, the use of DPC in CL analysis
is relatively few [13–15]. In recent studies, it was found that CL
phenomena accompanied with reaction between DPC and lumi-
nol in alkaline medium with low concentration of luminol, which
could be significantly enhanced by DHZS. According to previously
study, it was found that the reaction of DPC and luminol can emit
CL with low concentration of luminol, but CL cannot be observed
nearly when luminol with same concentration reacts with other
same condition, the CL intensity of luminol–H2O2–metal ion sys-
tem, which is one of the most important luminol CL systems, was
much lower than luminol–DPC–DHZS. So this method possesses
particular advantage which is less luminol consumption than other
luminol CL systems. So the method holds higher selectivity than
other luminol CL systems because of using low luminol concentra-
tion. Based on these observations, a novel flow-injection CL method
for the determination of DHZS was developed. Under optimum
conditions, the relative CL intensity was linearly related to the con-
centration of DHZS in the range of 7.0 × 10−9 to 8.6 × 10−7 g mL−1
with a detection limit (3ꢀ) of 2.1 × 10−9 g mL−1. The method has
been successfully applied to determine DHZS in serum. The possible
CL mechanism was first proposed briefly.
2. Experimental
2.1. Reagents
Potassium persulfate (Shanghai Aijian Chemical Reagent Com-
pany), potassium hydroxide, Hydrogen peroxide, Sodium nitrate,
sodium periodate, nickel sulfate, calcium chloride (Shanghai Chem-
ical Reagent Company). All the reagents were of analytical grade.
Deionized and doubly distilled water was used throughout the
work.
∗
Corresponding author. Tel.: +86 29 85308748.
E-mail address: zhangzhujun18@yahoo.com.cn (Z. Zhang).
1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2009.09.044

78
C. Yang et al. / Spectrochimica Acta Part A 75 (2010) 77–82
Fig. 1. The structure of DHZS.
A stock solution of 1.0 × 10−4 g mL−1 DHZS (Wuhan Yuancheng
Technology Development Co., Ltd.) was prepared by dissolving
appropriate amount of DHZS in warm water and diluting to the
mark with water. The 1.0 × 10−2 mol L−1 luminol stock solution was
prepared by dissolving 1.772 g luminol in 1 L 0.1 mol L−1 carbonate
buffer and left to stand for approximately 24 h before use. The lumi-
nol solution was stable for at least 1 month when stored in dark.
Fig. 3. The UV–vis absorption spectra of DPC.
2.2. Apparatus
boiling was continued for another 20 min for the completion of the
reaction. The mixture was then cooled, filtered through sintered
glass crucible. The solution was cooled in an ice bath to elimi-
nate as much of potassium sulfate as possible and filtered again
while it was cold. The resulting brown clear filtrate was left to
attain room temperature. In order to isolate the complex, 40 mL of
NaNO3 solution (50%, in excess) was added to the solution and the
mixture left to crystallise. Almost immediately dark brown crys-
tals started appearing and crystallisations were complete when
the supernatant liquid was colorless. The crystals were filtered and
washed several times with doubly distilled water until the complex
itself started dissolving, which was indicated by the brown drops
being formed under the crucible. Then the dark brown crystals of
DPC were obtained.
The flow system used in this work is shown in Fig. 2. Two peri-
staltic pumps (HL-2, Shanghai Huxi, China) were used to deliver
all flow streams. Polytetrafluoroethylene (PTFE) tubing (0.8 mm
i.d.) was used as connection tubing in the flow system. Sample
solution was injected by a eight-way injection valve into the car-
rier stream (H2O) and then merged with CL reagents in a spiral
flow cell in the front of a photomultiplier tube (PMT). The CL sig-
nal was monitored by IFFM-A multifunction chemiluminescence
analyzer (Remex Analytical Instrument Co. Ltd., Xian, China). The
UV absorbance was detected by the TU1901 UV–vis spectropho-
tometer (Beijing Purkinje General Instrument Co., Ltd., China). The
chemiluminescence spectrum was monitored using F-4600 fluo-
rescence spectrophotometer (Shimadzu, Japan).
Not less than 20 tablets were weight, ground to a fine pow-
der, and mixed. A sample equivalent to approximately 10 mg of
DHZS was weighed accurately and dissolved with 50 mL of double-
distilled warm water. The resulting mixture was filtered and the
filtrate was transferred into a 100 mL flask and diluted to the mark
with water. This solution was further diluted with water appropri-
ately so that the final DHZS concentration was within the working
range.
The DPC solutions were freshly prepared by dissolving an
amount of complex in 1.0 × 10−2 mol L−1 KOH solution before use.
The complex was characterized by its UV/visible spectrum, which
exhibits a broad absorption band at 415 nm (Fig. 3). The aqueous
solution of DPC was standardized by a standard procedure [17].
2.4. General procedure
The FIA–CL configuration used is outlined in Fig. 2. In order to
achieve good mechanical and thermal stability, the instruments
were allowed to run for 10 min before the first measurement was
made. The flow line were connected with DHZS standard solu-
tion (a), a carried H2O (b), 6.0 × 10−8 mol L−1 luminol solution in
1.0 × 10−3 mol L−1 potassium hydroxide (c), 1.0 × 10−4 mol L−1 DPC
solution in 4.0 × 10−2 mol L−1 potassium hydroxide (d). Luminol
solution was firstly mixed with DPC solution to give stable base-
line. The 100 ␮L solution of standard DHZS was injected into the
carrier stream via the sample injection valve which pumped con-
tinuously. The mixture passed through the flow cell, while the CL
signal was recorded by IFFS-A multifunction chemiluminescence
analyzer.
2.3. Preparation of DPC
The diperiodatocuprate (III) was prepared by oxidizing Cu (II)
in the alkaline medium [16]. CuSO4·5H2O (1.56 g), NaIO4 (2.67 g),
K2S2O8 (1 g) and KOH (8 g) were added in 100 mL water. The mix-
ture was heated to boiling for about 20 min on a hot plate with
constant stirring. The boiling mixture turned intensely red and the
3. Result and discussion
3.1. Kinetics curve of the CL reaction
In order to determine the life of the CL, the dynamical profile
of the CL reaction was tested with a static system. In static mode,
the experimental parameters were kept constant, the CL intensity-
time profile of luminol (1.0 × 10−7 mol L−1) CL reaction catalyzed
Fig. 2. Schematic diagram of the CL–FIA system a: DHZS solution or samples; b:
distilled water; c: luminol solution; d: DPC solution; V: injection valve; F: spiral
glass flow cell; PMT: photomultiplier tube; pump1, pump2: peristaltic pump.
by DHZS (5.2 × 10−8 g mL−1) in the present DPC (1.0 × 10−4 mol L−1
)

C. Yang et al. / Spectrochimica Acta Part A 75 (2010) 77–82
79
Fig. 4. Kinetics curves of luminol–DPC catalyzed by DHZS luminol:
Fig. 6. CL spectrum of luminol and DPC catalyzed by DHZS in the presence (A) and
absence (B) conditions luminol: 1.0 × 10−4 mol L−1; DPC: 1.0 × 10−4 mol L−1; DHZS:
1.0 × 10−7 mol L−1; DPC: 1 × 10−4 mol L−1; DHZS: 8 × 10−8 g mL−1
.
2.0 × 10−4 g mL−1
.
was recorded to study the kinetic characteristic. Fig. 4 demonstrates
that the CL reaction was very quick. The CL intensity peak appeared
within 3 s since the DHZS solution was injected. The CL signal would
decrease to baseline at within 2 s. The kinetic curve indicated the
CL system is rapid and sensitive enough and suitable to perform
determination of DHZS.
tometer (Shimadzu, Japan) combined with flow-injection system,
whose light entrance slot was shut. Both CL spectras were almost
identical with a maximum wavelength at about 425 nm (Fig. 6). It
is well known that 3-aminophthalate is the luminophor, and the
maximum emission of CL reaction is at 425 nm. This indicated that
the CL spectra were independent of DHZS. So the CL emitter in the
both CL reactions between luminol and DPC with and without DHZS
is 3-aminophthalate, which is the oxidation product of luminol.
Series experiments have been performed to know more details
of luminol–DPC–DHZS CL reaction. Firstly, the UV–vis absorp-
tion spectra of DHZS, DPC and their mixture were made in basic
medium. The result showed (Fig. 7) that the absorption peaks of
DHZS and DPC almost disappeared as they were mixed. Secondly,
when DPC and DHZS were mixed in alkaline medium, the color of
DPC faded immediately. However, the primary color of DPC recov-
ered when strong oxidant (K2S2O8 or HNO3) was added into the
near colorless solution. Considering of above experiment, it is obvi-
ous that a redox reaction took place between DHZS and DPC. What
is more, no CL enhancement could be observed when the mixture of
DHZS and DPC was injected into luminol. The observation implied
the existence of a intermediate product which maybe a free radi-
cal (DHZSox•) [18–20], although more evidences are not available.
Based on above analysis, reactions (1) and (2) were proposed.
3.2. Stability of DPC
Trivalent copper is in the highest oxidation state which should
be stabilized by chelating with suitable polydentate ligands due
to the fact of its limited stability. The stability of DPC in aque-
ous solution was studied by the variety of CL intensity of
luminol–DPC–DHZS within 2 h (Fig. 5). As can be seen, the CL inten-
sity varied little within 2 h when DPC concentration was up to
6.0 × 10−5 mol L−1. The stability can achieve the test requirement.
3.3. Possible reaction mechanism
In order to explain the possible reaction mechanism, the CL spec-
tra of the luminol–DPC reaction in the absence and presence of
DHZS were measured using an F-4600 fluorescence spectropho-
Fig. 5. The stability of DPC luminol: 1.0 × 10−7 mol L−1; DHZS: 4.0 × 10−8 g mL−1
;
DPC: 1.0 × 10−5 mol L−1 (A); 2.0 × 10−5 mol L−1 (B); 6.0 × 10−5 mol L−1 (C);
1.0 × 10−4 mol L−1 (D); 2.0 × 10−4 mol L−1 (E).
Fig. 7. UV–vis absorption spectra.

80
C. Yang et al. / Spectrochimica Acta Part A 75 (2010) 77–82
Fig. 8. The most possible reaction mechanism.
More experiments to evaluate reactions (1) and (2) were car-
It is known luminol forms the dianion of luminol (I) as shown
in reaction (3) in basic aqueous. And in this paper, it is consid-
ered that the dianion (I) can be oxidized by DPC to the semidione
structure shown in one of its resonance form (reaction (4)). The
semidione (II) reacts with oxygen to give the proxy radical, the
proxy radical is subsequently reduced in a fast step to give proxy
anion (III) (reaction (5)). The proxy anion decomposes to electron-
ically excited (IV) with loss of nitrogen and then, contributes CL
emission (reaction (6)) [21]. Based on the CL enhancement of DHZS
on DPC–luminol reaction, it is suggested that DHZSox• could oxi-
dize the luminol dianion (I) to semidione structure (II) as shown in
reaction (7), and this reaction performs as the competing reaction
with reaction (4). It also enabled us to conclude that K1K7 ꢀ K4
and K1 > K2, otherwise the CL enhancement of DHZS should not be
observed. Based on the above discussions, the most possible reac-
tion mechanism is shown in Fig. 8, although more evidences are
not available.
ried out. When DHZS standard solution was firstly mixed with DPC
flow instead of luminol in the present flow system (Fig. 2), the CL
intensity was much lower than that obtained without above flow
system change. And by varying the tube length from DPC–DHZS
confluence point to luminol confluence point, it was found that the
longer the tube length, the weaker CL was observed. The experi-
mentalresults coincide withwhat it has beensuggestedinreactions
(1) and (2). And it can be concluded the CL difference observed
in above experiments is attributed to the instability of proposed
DHZSox•.
Table 1
Result of determination of DHZS in tablet samples.
Sample
Amount labeled (mg)
Amount found (mg)a
Proposed method
HPLC method
3.4. Optimization of CL reaction conditions
Tablet 1
Tablet 2
10
10
9.8 3.2
10.1 2.8
9.9 1.0
10.2 2.1
A series of experiments was conducted to select optimum
a
analytical condition using 3.5 × 10−7 mol L−1 DHZS solution. The
Average of three measurements ( R.S.D.).

C. Yang et al. / Spectrochimica Acta Part A 75 (2010) 77–82
81
Table 2
Results of recovery tests on human serum.
Sample
No. 1
Found (10−8 g mL−1
)
Added (10−8 g mL−1
)
Total found (10−8 g mL−1
)
Recovery/%
R.S.D./%
1.8
5.1
19
2.0
4.0
6.0
3.9
5.7
7.9
105
97.5
102
2.7
1.2
3.7
No. 2
No. 3
6.0
8.0
10.0
11.0
13.4
15.0
98.3
104
99
1.2
0.3
2.0
20.0
40.0
60.0
40.0
56.9
80.0
105
94.8
102
3.0
0.8
1.1
optimized conditions included flow system, flow rate and CL
reagents concentration.
3.5. Analytical parameters
Several flow systems were designed in order to obtain the maxi-
mal CL signal. When employing the system shown schematically in
Fig. 2 and water as carrying stream, the reagent solution could mix
adequately with sample solution as soon as they met and a strong
CL signal with good repeatability was obtained. So, flow system in
Fig. 2 was selected.
The flow rate is an important parameter in the experiment,
which influences the analytical sensitivity. The effect of the
flow rate of pump on CL intensity was examined in the range
of 0.5–1.5 mL min−1. The results showed that the CL intensity
increased with increasing of flow rate, because the CL reaction is
rapid. So, a flow rate of 1.5 mL min−1 which was the maximal flow
rate under present conditions was selected.
Under the optimum condition given above and using the
flow-injection system described in Fig. 2, the relative CL inten-
sity was linearly proportional to the DHZS concentration in
the range of 7.0 × 10−9 to 8.6 × 10−7 g mL−1 and the detection
limit was 2.1 × 10−9 g mL−1 (3ꢀ). The regression equation was
ꢁI = 7.57C − 98.83 (ꢁI being the relative height of CL intensity and C
being the DHZS concentration (ng mL−1)) with R2 = 0.9933 (n = 12).
The relative standard deviation (R.S.D.) (n = 7) for 5.2 × 10−8 g mL−1
DHZS standard solution was 3.1%.
3.6. Interference studies
The interference of foreign species was studied by analyzing
a standard solution 6.0 × 10−8 g mL−1 DHZS to which increasing
amounts of foreign species was added. A substance was consid-
ered no interference if the variation of the CL intensity was within
5%. The influence of some inorganic ions and organic compounds
DPC is the oxidant in the CL reaction whose concentration
influences the CL intensity greatly. The effect of DPC concentra-
tion on the CL intensity was examined in the range of 6.0 × 10−5
to 4.0 × 10−4 mol L−1. The experiments showed the maximal CL
intensity could be obtained when the concentration of DPC was
were studied. The tolerable concentration ratios of foreign species
1.0 × 10−4 mol L−1
.
to 6.0 × 10−8 g mL−1 DHZS was over 1000-fold Na+, Ca2+, Cl−, SO4
,
2−
NO3−, CO32−, pyruvic acid, 500-fold C2O42−, creatinine, carbamide,
starch, 100-fold Fe3+, 50-fold citrate, 10-fold lactic acid, 5-fold lac-
tose, glucose, Co2+, Cu2+, Cr3+, 1-fold Mn2+, 0.1-fold ascorbic acid,
uric acid.
DPC should be used as oxidant in alkaline medium. The effect
of potassium hydroxide in DPC solution on the CL intensity was
examined. The concentration of potassium hydroxide was stud-
ied in a concentration range 2.0 × 10−2 to 2.0 × 10−1 mol L−1. It
was found that the CL intensity increased with potassium hydrox-
ide concentration up to 1.2 × 10−1 mol L−1, then increased little.
For obtaining the highest sensitivity, 1.2 × 10−1 mol L−1 potassium
hydroxide was selected as an optimum concentration.
3.7. Sample analysis
3.7.1. Determination of DHZS in pharmaceutical preparations
Following the procedure detailed in Section 2.4, the proposed
method was applied for the determination of DHZS in commercially
available compound antihypertensive, Compound DHZS Tablets
(10 mg DHZS, 12.5 mg hydrochlorothiazide, 0.1 mg reserpine). The
results are listed in Table 1, which agreed well with those obtained
by the HPLC method [5]. The HPLC procedure used as follows,
using ODS column, the mobile phase consisted of 0.06 mol L−1
potassium phosphate monobasic solution–methanol–acetonitrile
(86:9:5), the flow rate was 1 mL min−1 and detecting wavelength
was 240 nm.
The effect of luminol concentration on the signal/noise (S/N)
ratio was investigated in the range 5.0 × 10−9 to 2.0 × 10−7 mol L−1
.
It was found that S/N ratio reached the maximum value when
the concentration of luminol was 1.0 × 10−7 mol L−1. Therefore,
1.0 × 10−7 mol L−1 luminol was used as an optimum concentra-
tion.
The luminol CL reaction should occur in alkaline solution. In the
experiments, the alkalinity of the reaction medium was adjusted
by preparing luminol with suitable concentration of potassium
hydroxide. The effect of potassium hydroxide concentration in
the range of 4.0 × 10−4 to 4.0 × 10−3 mol L−1 was examined. When
1.0 × 10−3 mol L−1 potassium hydroxide was used, the CL reaction
had the maximum CL intensity.
3.7.2. Determination of recovery of DHZS in human serum
The proposed method was attempted to determine DHZS in
serum which was provided by Shaanxi normal university hospi-
tal. 2 mL blank serum sample was collected and transferred into
ultra-filtration tube, then centrifuged at 10,000 rpm for 10 min at
4 ◦C to eliminate the potential influence of proteins. The 1 mL fil-
trate was transferred into a 100 mL volumetric flask and diluted
to the mark with doubly distilled water. The blank serum was
injected to the CL system, and then recorded the blank signal. A
known amount of DHZS standard solution was added to 10 mL
diluted serum. The DHZS concentration of serum samples were
determined by the procedure detailed in Section 2.4. The amount
Table 3
Figures of merit of comparable methods of CL determination of DHZS.
CL system
Analytical range
Detection limit
References
(ng mL−1
)
(ng mL−1
)
Luminol–DPC
7–860
5–800
20–2800
20–4000
10–3000
2.1
1.9
12
1.2
3.6
This work
[7]
[8]
[9]
[10]
MnO4− + RhB + H+
Fe(CN)63− + eosin Y + OH−
Peracetic acid + OH−
2−
ONOO− + CO3

82
C. Yang et al. / Spectrochimica Acta Part A 75 (2010) 77–82
of DHZS in human serum, and the recovery obtained are shown in
Table 2.
cal applications. The method is simple, fast, and needs little luminol
consumption and inexpensive instrumentation. The possible CL
reaction mechanism was investigated.
3.7.3. Comparison with other CL systems
Some reported CL methods for the determination of DHZS are
compared with the proposed CL method in this paper and the
results are shown in Table 3. From Table 3, it can be seen that
under the optimum conditions, the proposed CL method for the
determination of DHZS has considerable or lower detection limit
comparable with other CL methods.
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The potential application of DPC as inorganic CL oxidizing agents
has been demonstrated in the work. The important advantage of
the oxidant over other common oxidants is that very low concen-
tration of luminol can react with DPC catalyzed by DHZS to get
excellent experimental results. So the method holds higher selec-
tivity than other luminol CL systems because of using low luminol
concentration.
The work has shown that the proposed FIA–CL method based on
DPC as oxidants can be successfully used for the determination of
DHZS. The analytical performance in assay of DHZS in pharmaceuti-
cal preparation and human serum demonstrates that the proposed
method can be utilized in routine determination of DHZS and clini-