DPPH·–luminol chemiluminescence system and its application in the determination of scutellarin in pharmaceutical injections and rat plasma with flow injection analysis

Article abstract of DOI:10.1002/bio.3225

In this article, a DPPH·–luminol chemiluminescence (CL) system was reported and the CL mechanism was discussed according to the CL kinetic properties after sequence injecting DPPH· into the DPPH·–luminol reaction mixture. It was observed that scutellarin could inhibit the CL response of the DPPH·–luminol system. Based on this observation, a simple and rapid flow injection CL method was developed for the determination of scutellarin using the inhibition effect in alkaline medium. The optimized chemical conditions for the CL reaction were 5?×?10^?6?mol/L DPPH· and 1.0?×?10^?4?mol/L luminol in 0.01?mol/L NaOH. Under optimized conditions, the CL intensity was inversely proportional to the concentration of scutellarin over the ranges 5–2000 and 40–3200?ng/ml in pharmaceutical injection and rat plasma, respectively. The limits of detection (S/N?=?3) were 5 and 40?ng/ml in preparations and rat plasma, respectively. Furthermore, the precision, recovery and stability of the validated method were acceptable for the determination of scutellarin in both pharmaceutical injections and rat plasma. The presented method was successfully applied in the determination of scutellarin in pharmaceutical injections and real rat plasma samples.

Full text of DOI:10.1002/bio.3225

Received: 20 June 2016
DOI 10.1002/bio.3225
Revised: 26 August 2016
Accepted: 31 August 2016
R E S E A R C H A R T I C L E
DPPH·–luminol chemiluminescence system and its application
in the determination of scutellarin in pharmaceutical injections
and rat plasma with flow injection analysis
Hong Yao1,2† Xiaomei Huang1† Peiying Shi3 Zhen Lin1 Meilan Zhu1 Ailin Liu1
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Xinhua Lin1
Yuhai Tang2
*
*
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1 Department of Pharmaceutical Analysis,
Fujian Medical University, Fuzhou, China
Abstract
2 Institute of Analytical Sciences, Xi’an
Jiaotong University, Xi’an, China
In this article, a DPPH·–luminol chemiluminescence (CL) system was reported and the CL
mechanism was discussed according to the CL kinetic properties after sequence injecting DPPH·
into the DPPH·–luminol reaction mixture. It was observed that scutellarin could inhibit the CL
response of the DPPH·–luminol system. Based on this observation, a simple and rapid flow
injection CL method was developed for the determination of scutellarin using the inhibition effect
in alkaline medium. The optimized chemical conditions for the CL reaction were 5 × 10−6 mol/L
DPPH· and 1.0 × 10−4 mol/L luminol in 0.01 mol/L NaOH. Under optimized conditions, the CL
intensity was inversely proportional to the concentration of scutellarin over the ranges 5–2000
and 40–3200 ng/ml in pharmaceutical injection and rat plasma, respectively. The limits of
detection (S/N = 3) were 5 and 40 ng/ml in preparations and rat plasma, respectively. Further-
more, the precision, recovery and stability of the validated method were acceptable for the
determination of scutellarin in both pharmaceutical injections and rat plasma. The presented
method was successfully applied in the determination of scutellarin in pharmaceutical injections
and real rat plasma samples.
3 Department of Traditional Chinese Medicine
Resource and Bee Products, Fujian Agriculture
and Forestry University, Fuzhou, China
Correspondence
X. H. Lin, Department of Pharmaceutical
Analysis, Faculty of Pharmacy, Fujian Medical
University, Fuzhou 350108, China.
Email: xhl1963@sina.com
Y. H. Tang, Institute of Analytical Sciences,
Xi’an Jiaotong University, Xi’an 710061, China.
Email: tyh57@mail.xjtu.edu.cn
Funding Information
New Century Excellent Talents in Fujian
Province University, JA14128. Fujian Provin-
cial Natural Science Foundation, 2014J07009;
2016J01371. National Nature Science Foun-
dation of China, 81202987; 81303298. The
Medical Elite Cultivation Program of Fujian,
(Nos. 2013‐ZQN‐JC‐25 and 2016‐ZQN‐63).
KEYWORDS
chemiluminescence, DPPH, flow injection, pharmaceutical injections, rat plasma, scutellarin
the analysis highly empirical.[1] The fact that the CL response of a fresh
luminol solution is lower than that of luminol that has been stored in
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1
INTRODUCTION
Since the chemiluminescence (CL) of luminol was first reported in
1928,[1] luminol‐based CL reactions have been investigated and used
widely in many fields including environmental monitoring, medical
diagnosis and pharmaceutical or biological analysis. It is generally
accepted that luminol can be oxidized by oxidants to generate the
excited 3‐aminophthalate, which then releases energy as light
(λmax = 425 nm). However, to date, there is insufficient evidence to
mechanistically understand the luminol‐based CL system, which makes
the dark for more than 1 week, also has no definite explanation. Thus,
there is a need to explore the reaction mechanism before the mysteries
of the luminol‐based CL system can be fully revealed.
Scutellarin (Figure S1), a flavonoid glycoside, is a main active ingre-
dient of breviscapine, which is a total flavonoid extract from the whole
dried herb of Erigeron breviscapus (vant.) Hand‐Mazz. Because of its
strong antioxidative property,[2,3] scutellarin has favorable pharmaco-
logical effects, such as protecting the cardiovascular and cerebrovascu-
lar systems.[4–7] Accordingly, breviscapine has been produced in
several dosage forms, such as tables, dropping pills and injections, for
the treatment of angina pectoris, coronary artery disease and apoplexy
sequelae. Not unreasonably, scutellarin has often been considered one
of the most important markers of quality control or pharmacokinetic
analysis of E. breviscapus extract preparations, especially breviscapine
preparations.
†Hong Yai and Xiaomei Peiying contributed equally to this work.
Abbreviations used: CL, Chemiluminescence; DPPH, 1,1‐Diphenyl‐2‐
picrylhydrazyl; DPPHH, 1,1‐Diphenyl‐2‐(2,4,6‐trinitrophenyl)hydrazine; FI,
Flow injection; HPLC, High‐performance liquid chromatography; LOD, Limit of
detection; PMT, Photomultiplier tube; QC, Quality control; ROS, Reactive
oxygen species; RSD, Relative standard deviation; S/N, Signal‐to‐noise ratio.
Luminescence 2016; 1–8
wileyonlinelibrary.com/journal/bio
Copyright © 2016 John Wiley & Sons, Ltd.
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YAO ET AL.
Over recent decades, many approaches to the determination of
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2.2
Solutions preparation
scutellarin in pharmaceutical preparations or biological materials have
been developed, for example, high‐performance liquid chromatogra-
phy (HPLC),[8,9] capillary electrophoresis with electrochemical
detection,[10] liquid chromatography‐tandem mass spectrometry (LC–
MS/MS) and ultra performance liquid chromatography‐tandem mass
spectrometry (UPLC‐MS/MS)[11,12] and quantitative nuclear magnetic
resonance.[13] Most of the reported methods had some merits, e.g.
favorable precision, accuracy and sensitivity. However, there exist
some disadvantages, such as reactions being time‐consuming, the
greater consumption of organic solvents, the need for more expensive
facilities and the higher costs of the experiments.
A 1 × 10−2 M luminol stock solution was prepared by dissolving
177.16 mg luminol in 10 ml of 0.1 M NaOH, and then diluting in
90 ml of 0.01 M NaOH. The stock solution was stored in the dark at
4°C. Working solutions of luminol were made by diluting the stock
solution with a suitable concentration of NaOH. The stock solution
of DPPH· 1 × 10−3 M was freshly prepared by dissolving 3.94 mg
DPPH· solid in 10 ml of absolute ethyl alcohol, and was kept in the dark
at −20°C. The working solutions were prepared before analysis by dilut-
ing the stock solution with double‐distilled water. a 1 × 10−3 g/ml
standard solution of scutellarin was prepared by dissolving 1 mg of
scutellarin in 1 ml of methanol in a 1 ml volumetric flask. A series of
standard stock solutions was prepared by diluting standard solution
with methanol to the appropriate concentration. All these standard
stock solutions were stored in a refrigerator at 4°C. Before analysis,
10 μl of the series standard stock solution was diluted with 2 ml of
double‐distilled water to prepare working solutions with a concentra-
tion of 2–6400 ng/ml.
CL methods are widely used in the determination of compounds of
interest in pharmaceutical preparations and physiological fluids,[14,15]
especially the detection of small biological molecules, the analysis of
DNA and protein, cell imaging and immunoassays[16,17] due to their sim-
ple, rapid and sensitive properties. Among the reported CL methods,
luminol CL systems, such as luminol–H2O2,[18,19] luminol–KMO4[20] and
[21]
luminol–K3Fe(CN)6
have often been used because of their low cost
and high CL efficiency. Some researchers have used flow‐injection (FI)
CL methods for the detection of flavonoid compounds.[22,23] However,
to date, no reports have referred to the use of CL methods for the deter-
mination of scutellarin in pharmaceutical preparations or biological fluids.
In this paper, it is observed that 1,1‐diphenyl‐2‐picrylhydrazyl
(DPPH·), a stable free radical with a single electron, can trigger the CL
reaction of luminol in alkaline solution. The CL mechanism is discussed
in detail according to the CL kinetic properties after sequence injecting
DPPH· into the DPPH·–luminol reaction mixture. Furthermore, it was
observed that scutellarin could inhibit the CL signal of DPPH·–luminol
in alkaline solution. Based on this observation, a new FI‐CL method is
presented for the determination of scutellarin. Compared with the
above‐mentioned methods for the analysis of scutellarin, the present
method is simple and rapid (complete analysis, including sampling
and washing, could be performed in 50 s, giving a sampling measure-
ment frequency of ~60/h). The method was applied to the determi-
nation of scutellarin in pharmaceutical injections and real rat plasma
with satisfactory results.
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2.3
FI‐CL apparatus and procedure
The FI‐CL system instrument used in this study is shown in Figure S2.
All the CL measurements were carried out on a FI‐CL analyzer with a
multifunction CL detector (IFFA‐S, Xi’an Remex Analysis instruments
Co., Ltd, Xi’an, China). The instrument with one peristaltic pump
(pump 1) was used to deliver the luminol solutions and one deputy
peristaltic pump (pump 2) was used to pump blank solution or samples
and carry DPPH· solutions. Along with the carriers, pumps, the detector
photomultiplier tube (PMT) and all components were connected using a
polytetrafluoroethylene tube (0.8 mm i.d.). The carriers of DPPH· and
blank solutions or samples were added simultaneously to the mixed cell,
and then flowed into the eight‐way valve. At the same time, luminol
solution was injected into the eight‐way valve and mixed with them.
The mixture solutions reacted in the flow cell and through the PMT,
which was in a dark box to generate the CL signal. The CL signal from
the reaction was recorded using the corresponding support software.
The control signal for the system is I0, (b carrier is the blank solution),
the inhibited signal is Is (b carrier is the scutellarin samples), the relative
CL intensity is ΔI (ΔI = I0 – Is).
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EXPERIMENTAL
Reagents
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2.1
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2.4
HPLC conditions
All reagents and chemicals were used of analytical grade. Luminol was
from Shanghai yuanye Bio‐Technology Co. (Shanghai, China); DPPH·
was from Sigma‐Aldrich Co. (St. Louis, MO, USA); scutellarin (batch:
150329) was from Shanghai Ronghe Pharmaceutical Technology
Development Co. (Shanghai, China); HPLC grade methanol was from
Sigma‐Aldrich; ethanol and NaOH were from Sinopharm Chemical
Reagent Co., Ltd. All solutions were prepared using distilled water.
Breviscapine injections (batch: 1011407094, 1011510202,
1011510194, 10115109111, Shiyao Yinghu Pharmaceutical Co., Ltd;
batch: 20141202, Yunnan Yuyao Biopharmaceutical Co., Ltd; batch:
150428C1, Shenwei Pharmaceutical Co., Ltd) were purchased from
local drugstores.
To study the reaction mechanism of the DPPH·–luminol CL system, a
Shimadzu LC‐20 A HPLC system equipped with a UV detector was
used in the chromatographic analysis. All separations were carried
out on an Ultimate XP‐C18 column (250 × 4.6 mm i.d., 5 μm) (Welch
Materials Inc., Ellicott, MD, USA) at 30°C. The mobile phase comprised:
(A) aqueous acetic acid (0.5%, v/v) and (B) acetonitrile using a gradient
elution of 25% to 30% B at 0–6 min, 30% to 45% B at 6–8 min, 45% to
85% B at 8–10 min, 85% to 95% B at 10–13 min and 95% B at
13–14 min. The re‐equilibration time was 6 min, giving a total run time
of 20 min. The flow rate was 1.0 ml/min and the injection volume was
5 μl. The detection wavelength was set at 254 nm for the analytes.

YAO ET AL.
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Intraday precision was tested by assaying quality control (QC)
samples with known low, mid and high concentrations of scutellarin
(50, 500 and 2500 ng/ml) three times within 1 day. Interday precision
was determined over three successive days with three replicate
measurements for each concentration. The RSD of the recorded CL
intensity values were used to evaluate the precision of the assay.
The stability of scutellarin in rat plasma was evaluated by testing the
QC samples at the above‐mentioned three different concentrations
under three conditions: after processing at room temperature for
24 h, post–pretreatment stability after 6 h at 4°C and stability after
three freeze–thaw cycles at −20°C. After analysis, the CL intensities
were recorded and the measured concentrations were calculated
according to the standard curve equation. Finally, the stability was
presented together with the relative error. The extraction recoveries
of scutellarin from rat plasma at QC levels (50, 500 and 2500 ng/ml)
were determined by comparing the relative CL intensity of plasma
samples spiked with analytes before extraction with that of spiked
matrix at the same concentrations. The matrix effects of scutellarin
from rat plasma at QC levels (50, 500 and 2500 ng/ml) were deter-
mined by comparing the relative CL intensity of spiked matrix with that
of standards diluted with solutions at equivalent concentrations.
2.5
Animal and administration
Sprague–Dawley rats were provided by Fujian Medical University
Laboratory Animal Center and were housed under the appropriate con-
trolled conditions. The system was left to acclimatize to the
environmentfor~5daysbeforetheexperiments.Beforetheexperiment,
animals were fasted 12 h; they were given free access to water for the
whole experiment. The rats received a single dose of breviscapine
(2.1 mg/kg) by intravenous injection. After dosing, ~ 300 μl blood
samples were collected into heparinized tubes at 0, 5, 15, 30, 45
and 60 min, and all the samples were immediately centrifuged at
1684 g for 10 min to obtain plasma, followed by storage at −20°C
until analysis.
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2.6
Sample pretreatment
For pharmaceutical analysis, 75 μl breviscapine injections were trans-
ferred into a 5 ml volumetric flask, followed by diluting with distilled
water. Then 10 μl of the dilute solutions were further diluted to 2 ml
with double‐distilled water for FI‐CL analysis; all solutions were
prepared before analysis.
For plasma analysis, 50 μl of rat plasma and 150 μl of methanol
were mixed and vortexed for 2 min, and placed in an ultrasonic cold
bath for 10 min. The mixture was then centrifuged at 11388 g for
~10 min at 4°C. Then, 150 μl of the supernatant was transferred into
a 2 ml volumetric flask, followed by diluting into 2 ml by distilled water
for final analysis.
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3
RESULTS AND DISCUSSION
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3.1
Reaction mechanism of the DPPH–luminol
system
Figure 1(a) shows a typical CL intensity and time curve for the
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2.7
Method validation
DPPH·–luminol system with 5 × 10−6 M DPPH· and 1 × 10−4
M
The method validation tests were divided into two parts, one for
scutellarin in pharmaceutical injections and one for scutellarin in rat
plasma. The validation procedures included linearity, limit of detection
(LOD), precision, stability and recovery.
luminol (in 0.01 M NaOH) as obtained using static injection CL analy-
sis. Two CL response peaks appeared at ~450 ms (peak 1) and 5–25 s
(peak 2) after injection of 25 μl of DPPH· solution into 25 μl of luminol
solution; the response slowly declined to base. The results suggest that
the DPPH·–luminol CL reaction should simultaneously experience a
quick reaction process and a slow reaction process. It has been reported
that luminol can be rapidly oxidized to excited 3‐aminophthalate to
release energy (CL with a maximum wavelength of ~425 nm) by oxi-
dants. Meanwhile luminol via a non‐luminescent reaction can also be
oxidized to an intermediate, diazaquinone, which might transfer slowly
into 3‐aminophthalate in the presence of oxygen.[1] In fact, the CL spec-
trum of the reaction of DPPH· and luminol showed a maximum wave-
length of ~420 nm (Figure 2), suggesting that the luminator might be
the excited 3‐aminophthalate for the DPPH·–luminol CL reaction. From
this, it was deduced that DPPH· might quickly oxidize luminol in alkaline
solution to produce both excited 3‐aminophthalate and the intermedi-
ate diazaquinone. The excited 3‐aminophthalate returned to the
ground state accompanied by light emission, accounting for CL peak 1
in Figure 1. Meanwhile, the intermediate diazaquinone was subse-
quently oxidized by reactive oxygen species (ROS) to generate excited
3‐aminophthalate,[1] which accounted for peak 2 in Figure 1(a).
For pharmaceutical injections, standard solutions within a concen-
tration range of 5–2000 ng/ml were examined using the FI‐CL method
under optimized conditions. Calibration curves were prepared by
plotting relative CL intensity (ΔI) against the concentration of
scutellarin or the logarithm of the concentration of scutellarin. The
LOD for scutellarin was obtained as the concentration required to
generate a signal‐to‐noise ratio (S/N) of 3. Relative standard deviations
(RSD) were used to evaluate the precision of the method with 10
replicates detecting 200 ng/ml scutellarin. Recovery was examined
by spiking samples with known low, mid and high amounts of standard
solutions.
For rat plasma analysis, the calibration samples were prepared by
adding 10 μl standard stock solutions to 50 μl black plasma and
vortexing for ~30 s. Then, 140 μl of methanol was added and the
mixtures were treated as described in the section ‘Sample
pretreatment’ to give working solutions with concentrations in the
range of 40–3200 ng/ml. The calibration graphs were prepared by
plotting the relative CL intensity (ΔI) against the concentration of
scutellarin or the logarithm of the concentration of scutellarin. The
LOD values for scutellarin were obtained as the concentration
required to generate a S/N of 3.
The deduction is supported because the secondary CL emission
was observed when another 25 μl DPPH· (5 × 10−6 M) was injected
into the reaction mixture of luminol and DPPH·. As shown in
Figure 1(b), in the secondary CL emission, peaks 1 and 2 (at ~25 s)

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YAO ET AL.
FIGURE 1 CL kinetic graphs for: (a) DPPH·–luminol system with 5 × 10−6 M DPPH· and 1 × 10−4 M luminol (first CL reaction or the first addition of
DPPH· in luminol); (b) the secondary CL reaction (secondary addition of DPPH· after the first CL reaction); (c) the third addition of DPPH· after the
secondary CL reaction in the reaction mixture; (d) the addition of 50 μl of 0.1 M NaOH after the third addition of DPPH· in the reaction
mixture; the continuous fourth (e), fifth (f), sixth (g), seventh (h) and eighth (i) additions of 25 μl DPPH· (5 × 10−6 M) in the reaction mixture; and
(j) mixing the oxygen‐rich DPPH· and luminol after both of the de‐aerated DPPH· and luminol were purged again with oxygen for 5 min
could still be observed. However, the intensity of peak 1 was lower
than that of peak 2 in the secondary CL emission. This is because
DPPH· oxidizing luminol consumed a large amount of OH− in the first
CL reaction such that the secondary CL reaction was influenced nega-
tively by the decreasing concentration of OH− (Figure S3) and the CL
reaction of ROS oxidizing diazaquinone did not require the participa-
tion of OH−. The amount of intermediate diazaquinone generated in
the secondary CL reaction was accordingly decreased, which accounts
for the decreased intensity of peak 2 in the secondary reaction
compared with peak 2 in the first CL reaction. The third 25 μl DPPH·
solution (5 × 10−6 M) was injected into the reaction mixture after
the secondary reaction, and did not result in the third CL emission
(Figure 1c). It is suggested that following the secondary CL reaction,
the amount of OH− in the reaction mixture was so low that no luminol
was oxidized to release light and produce the intermediate
diazaquinone on addition of the third 25 μl DPPH· solution (5 × 10
−6
M). After that, there was a certain amount of unreacted DPPH· in
the mixture. Increasing the concentration of OH− should increase CL
emission. Indeed, as shown in Figure 1(d), on addition of 50 μl of
0.01 M NaOH after the third reaction, a strong CL signal could be
recorded in the fourth reaction and peaks 1 and 2 were naturally
observed. It was predicted that further continuous addition of DPPH·
(5 × 10−6 M) in the reaction mixture after the fourth reaction would still
lead to a CL response and peak 2 would tend to disappear because the
ROS in the reaction mixture would be consumed. In fact, after the
continuous fourth (Figure 1e), fifth (Figure 1f), sixth (Figure 1g) and
seventh (Figure 1h) additions of 25 μl of DPPH· (5 × 10−6 M) to the
reaction mixture, decreased CL responses were observed and peak 2
tended to disappear. After the eighth addition of 25 μl of DPPH·
(5 × 10−6 M) to the reaction mixture, peak 2 could be no longer be

YAO ET AL.
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In order to clarify the reaction mechanism of scutellarin inhibiting
DPPH·–luminol CL, the effluent liquid of the DPPH·–luminol reaction
mixture from the FI‐CL instrument was collected and analyzed by
HPLC after 1 × 10−4 M luminol (in 0.01 M NaOH; stream a), 1 μg/ml
scutellarin (stream b) and 5 × 10−6 M DPPH· (stream c) solutions were
simultaneously injected into the flow system to detect the resulting CL
signal. Meanwhile, the effluent liquids of the luminol–DPPH·‐
scutellarin CL reaction with one or two of the three streams replaced
by their solvents (e.g. luminol was replaced by 0.01 M NaOH, or/
and DPPH· or scutellarin were replaced by double‐distilled water),
were also collected and comparatively analyzed by HPLC. As shown
in Figure 4(a), when streams a, b and c were 1 × 10−4 M luminol
(in 0.01 M NaOH), water and water, respectively, the collected
effluent showed a retention time for luminol of 5.14 min and a peak
of 88 736. When streams a, b and c were 0.01 M NaOH, water and
5 × 10−6 M DPPH·, respectively, the chromatogram (Figure 4b)
shows the DPPH· peak at 17.35 min with a peak area of 30 230.
When streams a, b and c were 0.01 M NaOH, 1 μg/ml scutellarin
and water, respectively, the chromatogram (Figure 4c) shows the
scutellarin peak at 7.62 min with a peak area of 18 731. However,
when streams a, b and c were 1 × 10−4 M luminol (in 0.01 M NaOH),
water and 5 × 10−6 M DPPH·, respectively, Figure 4(d) shows that
the peak areas for luminol and DPPH· decreased 3.7% and 47.6%,
respectively (85 432 and 15 849 for luminol and DPPH·, respectively)
compared with Figure 4(a,b). This suggests that DPPH· indeed reacts
with luminol and the reduction product of DPPH· should be DPPHH
(1,1‐diphenyl‐2‐(2,4,6‐trinitrophenyl)hydrazine) because only the
structure of DPPHH can explain why there was still a peak at the
retention time of 17.35 min in Figure 4(d). As shown in Figure 4(e),
the reductant scutellarin could react spontaneously with DPPH· lead-
ing to the peak of scutellarin tending to disappear. According to the
above discussion, the proposed reaction for scutellarin with DPPH,
which inhibits the CL of DPPH·–luminol system, is shown in
Figure 5.
FIGURE 2 CL spectrum of the reaction of DPPH· and luminol by
static injection CL analysis. Conditions: 50 μl of 1 × 10−4 M luminol
in 0.01 M NaOH and 50 μl of 5 × 10−6 M DPPH; using fluorescence
filters (cut‐off wavelength: 400, 420, 450, 470, 490 or 505 nm) to
shade the PMT window
observed in the CL profile (Figure 1i) due to the exhaustion of ROS in
reaction mixture. Furthermore, it was deduced that ROS mainly
originated from the alkaline luminol solution and not the added DPPH·
solution, because the addition of 25 μl of DPPH· solution (undeaerated)
in the 1 × 10−4 M luminol (in 0.01 M NaOH) (undeaerated) could
produce a CL profile similar to that obtained with 25 μl DPPH· solution
deaerated (by purging with nitrogen for 5 min) in the 1 × 10−4 M luminol
(in 0.01 M NaOH) (undeaerated). However, when 1 × 10−4 M luminol
(in 0.01 M NaOH) solution was purged with nitrogen for 5 min, regard-
less of adding undeaerated or deaerated DPPH·, the CL intensity of
peak 1 decreased ~80% and peak 2 could not be observed. It is
suggested that dissolved oxygen or ROS played a crucial role in gener-
ating CL in the DPPH·–luminol system. After both of the deaerated
DPPH· and luminol were purged again with oxygen for 5 min, mixing
the oxygen‐rich DPPH· and luminol produced resilient CL intensity for
peak 1, but the CL intensity for peak 2 was very low (Figure 1j). This
confirmed that the dissolved oxygen was an important enhancer of
the CL reaction for peak 1, and it was the ROS, and not the dissolved
oxygen, that played a crucial role in the CL reaction for peak 2. In partic-
ular, it also suggested that purging with nitrogen could remove ROS in
solution and regenerating ROS was a very slow process in the alkaline
medium, which might be a reasonable explanation for why the CL inten-
sity of fresh luminol solution is lower than that of the luminol stored in
the dark for 1 week.
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3.2
Condition optimization of DPPH·–luminol CL
reaction
The concentrations of DPPH·, luminol and NaOH greatly affected the
CL intensity of the DPPH·–luminol reaction. When keeping the luminol
concentration at 1.0 × 10−4 M in 0.02 M NaOH solution and using
water as a blank sample, the CL intensity increased continuously with
increasing concentrations of DPPH· over a range of 1 × 10−6 to
According to the above discussion, the proposed CL reaction
mechanism for the DPPH–luminol system is as shown in Figure 3.
FIGURE 3 The proposed reaction mechanism for the DPPH·–luminol system

6
YAO ET AL.
DPPH·–luminol system. To obtain favorable detection sensitivity,
5 × 10−6 M was selected for further studies.
The effect of luminol within the concentration range of 1 × 10−5 to
5 × 10−3 M in 0.01 M NaOH was examined when the concentration of
DPPH was kept at 5 × 10−6 M. The maximum CL intensity was
obtained when the concentration of luminol was 5 × 10−4
M
(Figure S5). When the concentration of luminol was >1 × 10−4 M,
5 ng/ml of scutellarin in water or 40 ng/ml of scutellarin in rat plasma
could no longer inhibit the CL signal of the DPPH·–luminol system. To
obtain favorable detection sensitivity, 1 × 10−4 M luminol in 0.02 M
NaOH was selected for further studies.
The concentration of medium NaOH was also examined over a
range of 0.005–0.2 M. Increasing the concentration of NaOH can
enhance the CL intensity (Figure S6). To obtain favorable detection
sensitivity, 0.02 M NaOH was selected as a medium for further
studies.
The flow rate of DPPH and luminol can also significantly affect the
CL intensity. The optimized flow rates for both of pumps 1 and 2 were
30 revolutions per minute (r/min).
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3.3
Interference test
In order to examine the selectivity of the developed FI‐CL method for
the detection of scutellarin in real formulations, a mixed solution of
100 ng/ml scutellarin with each possible coexisting compound was
prepared. The tolerance of foreign species was taken as the largest
concentration producing an error of less than 5% the CL signal of
100 ng/ml scutellarin. No interference was found with up to 1000‐fold
Na+, K+, Mg2+, Ba2+, Ca2+, Cl−,SO42−,700‐fold HCO3−, 10‐fold glucose
and sucrose, equivalent‐ fold EDTA‐2Na and 0.5‐fold ascorbic acid.
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3.4
Method validation results
For pharmaceutical injection analysis, Table 1 lists the calibration
parameters for linearity, range and LOD of scutellarin. Typical flow
injection signals for the different concentrations of scutellarin are
shown in Figure 6. The assay precision of the method for 200 ng/ml
of scutellarin after 10 replicates showed a RSD of 16.33%. Table S1
gives the results of the recovery. These results demonstrate that the
presented method is suitable for the rapid detection of scutellarin in
a breviscapine injection.
For rat plasma analysis, Table 2 presents the calibration curves,
linear range, R2 and LOD in spiked rat plasma. Tables S2 and S3 give
the results for the precision, sample stability and recovery tests. The
RSDs for all the tests were <20%, suggesting that the presented
method is acceptable for the determination of scutellarin in a rat
plasma sample.
FIGURE 4 HPLC chromatograms of the collected effluent liquid from
FI‐CL system, (a) when streams a, b and c were 1 × 10−4 M luminol (in
0.01 M NaOH), water and water, respectively; (b) when streams a, b
and c were 0.01 M NaOH, water and 5 × 10−6 M DPPH·, respectively;
(c) when streams a, b and c were 0.01 M NaOH, 1 μg/ml scutellarin and
water, respectively; (d) when streams a, b and c were 1 × 10−4
M
luminol (in 0.01 M NaOH), water and 5 × 10−6 M DPPH·, respectively;
and (e) when streams a, b and c were 1 × 10−4 M luminol (in 0.01 M
NaOH), 1 μg/ml scutellarin and 5 × 10−6 M DPPH·, respectively
3.5
Applications
|
|
3.5.1
Determination of scutellarin in pharmaceutical
injections
5 × 10−5 M; above 5 × 10−5 M, the CL intensity decreased
gradually (Figure S4). However, when the concentration of DPPH·was
>5 × 10−6 M, 5 ng/ml of scutellarin in water or 40 ng/ml of scutellarin
in rat plasma could no longer inhibit the CL signal of the
Six batches of breviscapine injections were analyzed using the
presented FI‐CL method. The results are shown in Table 3. The
scutellarin contents determined in the breviscapine injections were in
accordance with the labeled amount for the preparations (4 mg/ml).

YAO ET AL.
7
FIGURE 5 The reaction diagram of DPPH· with scutellarin
TABLE 1 Calibration Curves and Limit of Detection (LOD)
Analyte
Linear equation
R2
Linear range (ng/ml)
LOD (ng/ml)
Scutellarin
y = 10.171× + 230.48
0.9912
0.9129
5–800
5
y = 56.094ln(x) + 8358.4
800–2000
FIGURE 6 Typical flow injection signals for the different concentration of scutellarin by the DPPH·–luminol system
TABLE 2 Calibration Curves and Limit of Detection (LOD) in Rat Plasma
Analyte
Linear equation
R2
Linear range (ng/ml)
LOD (ng/ml)
Scutellarin
y = 4.4393× + 1110.6
y = 633.44ln(x) + 863.3
0.979
40–800
40
0.9808
800–3200
TABLE 3 The Determination Results of Scutellarin in Breviscapine
Injection (n = 6)
|
4
CONCLUSIONS
Labeled
(mg/ml)
Detected (mean SD,
mg/ml)
Content
(%)
Here,
a DPPH·–luminol CL system was reported and the CL
Batches
mechanism discussed according to the CL kinetic properties after
sequence injecting DPPH· into the DPPH·–luminol reaction mixture.
A reasonable explanation for why the CL intensity of a fresh luminol
solution is lower than that of a luminol solution stored in the dark for
1 week was that ROS, whose generation equilibrium needs more time
in the alkaline luminol solution, plays a crucial role in generating CL for
the luminol‐based system. This study provides new insights into the
luminol‐based CL process. Furthermore, a simple and rapid FI‐CL
method has been developed based on the inhibition effect of
scutellarin on the CL response of the DPPH·–luminol system. The
method was simple, rapid and sensitive, and could be successfully
applied to the determination of scutellarin in pharmaceutical injections
and real rat plasma samples.
Shiyao202
Shiyao194
Shiyao111
Shenwei28C1
Yuyao202
4
4
4
4
4
4.18 0.29
4.37 0.66
4.65 0.42
4.48 0.59
5.04 0.14
104.58
109.17
116.20
112.03
125.88
TABLE 4 Determination of Scutellarin in Blood Plasma (n = 3)
Time points
(min)
Detected (mean SD,
ng/ml)
RSD
(%)
Analyte
Scutellarin
5
15
30
45
456.65 70.77
363.58 78.08
208.80 62.41
266.43 96.44
15.5
21.47
29.89
36.2
ACKNOWLEDGMENTS
This work was supported by the National Nature Science Foundation
of China (Nos. 81303298 and 81202987), the Fujian Provincial Natural
Science Foundation (Nos. 2014J07009 and 2016J01371), the Medical
Elite Cultivation Program of Fujian (Nos. 2013‐ZQN‐JC‐25 and 2016‐
ZQN‐63) and Program for New Century Excellent Talents in Fujian
|
3.5.2
Determination of scutellarin in blood plasma
Real rat plasma samples were examined using the developed FI‐CL
method. The results are shown in Table 4, and are in accordance with
previous reports.[9,24,25]

8
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